1: Oncogene. 2005 Oct 3; [Epub ahead of print] Requirement of STAT3 activation for maximal collagenase-1 (MMP-1) induction by epidermal growth factor and malignant characteristics in T24 bladder cancer cells. Itoh M, Murata T, Suzuki T, Shindoh M, Nakajima K, Imai K, Yoshida K. [1] 1Department of Biology, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo, Japan [2] 4First Department of Internal Medicine, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo, Japan. Signal transducers and activators of transcription (STATs) are latent transcription factors that mediate cytokine- and growth factor-induced transcription. Constitutive activation of STAT3 has been shown in human cancers and transformed cell lines. We report that STAT3, but not STAT1 and STAT5, becomes phosphorylated in response to epidermal growth factor (EGF) and achieves maximal induction of collagenase-1 (MMP-1) transcription by interacting with c-JUN. Phosphorylation of STAT3 protein is biphasic: the first peak within 30 min and the second peak between 4 and 8 h. Association of STAT3 with c-JUN is detected and its constituting STAT3 is increasingly phosphorylated. The STAT and AP-1 elements are necessary for effective induction of MMP-1 promoter by EGF. Mutation of AP-1 element closely located at the STAT site abolishes the binding not only of c-JUN but also of STAT3 to MMP-1 promoter, resulting in the loss of the responsiveness to EGF. By blocking STAT3 activity with the dominant-negative form, we show the requirement of STAT3 for EGF induction of MMP-1 and MMP-10 (stromelysin-2). Furthermore, expression of the dominant-negative STAT3 is sufficient to inhibit the constitutive and EGF-inducible cell migration and invasion and the tumor formation in nude mice. These results demonstrate that STAT3 phosphorylation and its possible interaction with c-JUN are required for the strong responsiveness of MMP-1 to EGF, and STAT3 activation is crucial for exhibition of malignant characteristics in T24 bladder cancer cells.Oncogene advance online publication, 3 October 2005; doi:10.1038/sj.onc.1209149. PMID: 16205632 [PubMed - as supplied by publisher] --------------------------------------------------------------- 2: Cell Signal. 2005 Jun 23; [Epub ahead of print] Prostacyclin receptor induces STAT1 and STAT3 phosphorylations in human erythroleukemia cells: A mechanism requiring PTX-insensitive G proteins, ERK and JNK. Lo RK, Wise H, Wong YH. Department of Biochemistry, Molecular Neuroscience Center, and Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China. The ability of the human prostacyclin receptor (hIP) to regulate the activities of signal transducers and activators of transcription (STATs) has not yet been documented. In the present study, we have delineated the mechanism by which hIP induces STAT3 phosphorylations in human erythroleukemia (HEL) cells. Stimulation of endogenous hIP by its specific agonist, cicaprost, resulted in STAT3 Tyr(705) and Ser(727) phosphorylations in a time- and concentration-dependent manner. Cicaprost-induced STAT3 Tyr(705) and Ser(727) phosphorylations were resistant to pertussis toxin (PTX) treatment, suggesting that these responses were mediated through PTX-insensitive G proteins. In addition, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), but not p38 MAPK, were shown to be phosphorylated by cicaprost in a time- and concentration-dependent manner via PTX-insensitive G proteins. The levels of the interaction between STAT3, ERK and JNK were enhanced by cicaprost treatment. The involvement of Raf-1, MEK1/2 and JNK in cicaprost-induced phosphorylations of STAT3 was illustrated by the use of their selective inhibitors. In contrast, p38 MAPK did not appear to be required. Similar observations were obtained with STAT1 upon stimulation by cicaprost. Taken together, these results demonstrate for the first time that hIP activation by cicaprost can lead to STAT1 and STAT3 phosphorylations via signaling pathways involving PTX-insensitive G proteins, ERK and JNK. PMID: 15979846 [PubMed - as supplied by publisher] --------------------------------------------------------------- 3: Cell Signal. 2005 Jun 19; [Epub ahead of print] CD40 mediated human cholangiocyte apoptosis requires JAK2 dependent activation of STAT3 in addition to activation of JNK1/2 and ERK1/2. Ahmed-Choudhury J, Williams KT, Young LS, Adams DH, Afford SC. Liver Research Group, MRC Centre for Immune Regulation, Institute of Biomedical Research, The University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom. CD40 is critically involved in Fas-mediated cholangiocyte apoptosis during liver inflammation, but the underlying signalling events are poorly understood. Our recent work implicated AP-1 in CD40-induced cholangiocyte apoptosis, but suggested involvement of other signalling pathways. Because STAT3 has been implicated in liver regeneration we investigated this signalling pathway during CD40 mediated cholangiocyte apoptosis. Western immunoblotting, electrophoretic mobility gel shift assays, In situ DNA end labelling and caspase-3 activity were used to investigate intracellular signalling and apoptosis in primary human cholangiocytes following CD40 activation. CD40-activation induced caspase-3 dependent cholangiocyte apoptosis and 3-fold increases in JNK/ERK phosphorylation (concomitant with increased AP-1 binding activity) and 4-fold increases in pSTAT3, which were sustained for up to 24 h. Protein levels of c-Jun, c-Fos and pSTAT3 confirmed the upregulation. Phosphorylation of p38 remained unchanged suggesting that this MAP kinase was not involved in CD40 mediated apoptosis. Increased JAK2 phosphorylation accompanied increased STAT3 phosphorylation after CD40 ligation. Cholangiocytes were also shown to express JAK1 and 3 which was phosphorylated following control stimulation with TNFalpha or IL2 respectively but not after CD40 ligation. JNK, ERK and JAK2 inhibitors partially abrogated apoptosis and when used in combination reduced it to basal levels. In conclusion, induction of CD40-mediated cholangiocyte apoptosis requires JAK2-mediated phosphorylation of STAT3 as well as sustained JNK1/2, ERK1/2 activation. This study demonstrates that STAT3 can function as a proapoptotic factor in primary human liver epithelial cells. PMID: 15970430 [PubMed - as supplied by publisher] --------------------------------------------------------------- 4: Clin Cancer Res. 2005 Jun 15;11(12):4589-600. The farnesyltransferase inhibitor L744832 potentiates UCN-01-induced apoptosis in human multiple myeloma cells. Pei XY, Dai Y, Rahmani M, Li W, Dent P, Grant S. Department of Medicine, Virginia Commonwealth University/Medical College of Virginia, Richmond, Virginia 23298, USA. PURPOSE: The purpose of this study was to characterize interactions between the farnesyltransferase inhibitor L744832 and the checkpoint abrogator UCN-01 in drug-sensitive and drug-resistant human myeloma cell lines and primary CD138+ multiple myeloma cells. EXPERIMENTAL DESIGN: Wild-type and drug-resistant myeloma cell lines were exposed to UCN-01 +/- L744832 for 24 hours, after which mitochondrial injury, caspase activation, apoptosis, and various perturbations in signaling and survival pathways were monitored. RESULTS: Simultaneous exposure of myeloma cells to marginally toxic concentrations of L744832 and UCN-01 resulted in a synergistic induction of mitochondrial damage, caspase activation, and apoptosis, associated with activation of p34cdc2 and c-Jun-NH2-kinase and inactivation of extracellular signal-regulated kinase, Akt, GSK-3, p70(S6K), and signal transducers and activators of transcription 3 (STAT3). Enhanced lethality for the combination was also observed in primary CD138+ myeloma cells, but not in their CD138- counterparts. L744832/UCN-01-mediated lethality was not attenuated by conventional resistance mechanisms to cytotoxic drugs (e.g., melphalan or dexamethasone), addition of exogenous interleukin-6 or insulin-like growth factor-I, or the presence of stromal cells. In contrast, enforced activation of STAT3 significantly protected myeloma cells from L744832/UCN-01-induced apoptosis. CONCLUSIONS: Coadministration of the farnesyltransferase inhibitor L744832 promotes UCN-01-induced apoptosis in human multiple myeloma cells through a process that may involve perturbations in various survival signaling pathways, including extracellular signal-regulated kinase, Akt, and STAT3, and through a process capable of circumventing conventional modes of myeloma cell resistance, including growth factor- and stromal cell-related mechanisms. They also raise the possibility that combined treatment with farnesyltransferase inhibitors and UCN-01 could represent a novel therapeutic strategy in multiple myeloma. PMID: 15958645 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Biochem Biophys Res Commun. 2005 Jul 22;333(1):79-87. Adiponectin activates c-Jun NH2-terminal kinase and inhibits signal transducer and activator of transcription 3. Miyazaki T, Bub JD, Uzuki M, Iwamoto Y. Department of Urology, Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, 53226-0509, USA. Adiponectin, a major adipose cytokine, plays a crucial role in the inhibition of metabolic syndrome by acting on such cell types as muscle cells and hepatocytes. Furthermore, evidence suggests that adiponectin may influence cancer pathogenesis. Adiponectin occurs in non-proteolytic (full-length adiponectin: f-adiponectin) and proteolytic (globular adiponectin: g-adiponectin) forms in various oligomeric states. Different forms of adiponectin show distinct biological effects through differential activation of downstream signaling pathways. Here we identify c-Jun NH(2)-terminal kinase (JNK), and signal transducer and activator of transcription 3 (STAT3) as common downstream effectors of f- and g-adiponectin. f- and g-adiponectin both stimulate JNK activation in prostate cancer DU145, PC-3, and LNCaP-FGC cells, hepatocellular carcinoma HepG2 cells, and C2C12 myoblasts. Furthermore, both f- and g-adiponectin drastically suppress constitutive STAT3 activation in DU145 and HepG2 cells. These suggest that JNK and STAT3 may constitute a universal signaling pathway to mediate adiponectin's pathophysiological effects on metabolic syndrome and cancer. PMID: 15936715 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Infect Immun. 2005 May;73(5):2628-43. Microarray and proteomics analyses of human intestinal epithelial cells treated with the Aeromonas hydrophila cytotoxic enterotoxin. Galindo CL, Fadl AA, Sha J, Pillai L, Gutierrez C Jr, Chopra AK. Department of Microbiology and Immunology, Medical Research Building, 301 University Blvd., University of Texas Medical Branch, Galveston, TX 77555-1070, USA. We performed microarray analyses on RNA from human intestinal epithelial (HT-29) cells treated with the cytotoxic enterotoxin (Act) of Aeromonas hydrophila to examine global cellular transcriptional responses. Based on three independent experiments, Act upregulated the expression of 34 genes involved in cell growth, adhesion, signaling, immune responses (including interleukin-8 [IL-8] production), and apoptosis. We verified the upregulation of 14 genes by real-time reverse transcriptase-PCR and confirmed Act-induced production of IL-8 by enzyme-linked immunosorbent assay on supernatants from nonpolarized and polarized HT-29 cells. Maximal production of IL-8 in response to Act required the presence of intracellular calcium, since chelation of calcium with BAPTA-AM significantly reduced Act-induced IL-8 production in HT-29 cells. We also examined activation of mitogen-activated protein kinases and, as demonstrated by Western blot analysis of apical side-treated polarized HT-29 cells, Act induced phosphorylation of p38, c-Jun NH(2)-terminal kinase, and extracellular signal-regulated kinase 1/2. In addition, KinetWorks proteomics screening of whole-cell lysates revealed Act-induced phosphorylation of cyclic AMP-response element binding protein (CREB), c-Jun, adducin, protein kinase C, and signal transducer and activator of transcription 3 (STAT3) and decreased phosphorylation of protein kinase Balpha, v-raf-1 murine leukemia viral oncogene homolog 1 (i.e., Raf1), and STAT1. We verified activation of CREB and activator protein 1 in polarized cells by gel shift assay. This is the first description of human intestinal epithelial cell transcriptional alterations, phosphorylation or activation of signaling molecules, cytokine production, and calcium mobilization in response to this toxin. PMID: 15845465 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Zhonghua Nei Ke Za Zhi. 2005 Jan;44(1):42-5. [Effects of cytokine signaling 3 suppressors gene on c-fos and c-jun mRNA expression and proliferation of pulmonary arterial smooth muscle cells in rat under hypoxia] [Article in Chinese] Bai L, Yu ZB, Qian P, Qian GS, Guan S, Li SP. Institute of Respiratory Diseases, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China. OBJECTIVE: To explore the effects of suppressors of cytokine signaling (SOCS)3 gene on expression of c-fos, c-jun mRNA and proliferation of rat pulmonary arterial smooth muscle cells(PASMCs) under hypoxia. METHODS: PASMCs were co-transfected with pEFSOCS3 and pSV2neo by liposome, and then expression of SOCS3 protein was detected by immunocytochemistry. After PASMCs were exposed to normoxic and hypoxia at various time points respectively, expression of c-fos and c-jun mRNA was assessed by semi-quantitive RT-PCR. Flow cytometric DNA analysis was used to detect cell cycles. RESULTS: Expression of SOCS3 protein was confirmed by Western blot in PASMCs transfected with SOCS3 gene. The c-fos mRNA level in control cells peaked at 2 h of hypoxia and declined at 4 h, then peaked at 8 h secondly and declined at 12 h. C-fos mRNA level in SOCS3 gene-transfected cells at 2 h and 8 h exposed to hypoxia was lower than that in control cells at the same time points respectively (P < 0.01). The c-jun mRNA level increased at 2 h after exposure of control cells to hypoxia, peaked at 6 h of hypoxia and declined to the basal levels at 12 h. C-jun mRNA level of SOCS3 gene-transfected cells at 2 h, 4 h, 6 h, 8 h under hypoxia was lower than that in controls cells at the same time points. Compared with control PASMCs, cells in transfected with SOCS3 gene at G(1)/G(0) phase increased and those at S + G(2)/M phase decreased under normoxic and hypoxia (P < 0.01). CONCLUSION: Hypoxia induced expression of c-fos and c-jun genes, which might play an vital role in the early stage of PASMCs proliferation. SOCS3 protein may inhibit proliferation of PASMCs by lowering the tyrosine-phosphorylated level of STAT3 protein under hypoxia. PMID: 15769397 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Eur J Clin Invest. 2005 Feb;35(2):140-7. Molecular events associated with accelerated proliferative response in rat livers when partial hepatectomy is preceded by a sham operation. Laurent S, Starkel P, Leclercq IA, Lambotte L, Maiter D, Horsmans Y. Department of Gastroenterology, Universite Catholique de Louvain, 1200 Brussels, Belgium. BACKGROUND: When a sham operation is performed 6 h before partial hepatectomy (PH), the regenerative response is accelerated suggesting that sham operation itself contributes to cellular events leading to proliferation. MATERIALS AND METHODS: In order to examine the mechanisms implicated in this acceleration, we compared the activation of several factors associated with the progression through the cell cycle at various times after PH and after PH preceded by sham operation (S6 h + PH). The effect of a single sham (S) and two combined sham operations (S6 h + S) was also examined. Nonoperated rats were used as controls (C). RESULTS: The early factors NF-kappaB and Stat3 were activated after S6 h + PH and S6 h + S. C-jun expression was increased 0.5 h and 2 h after PH and 6 h after sham. There was no further increase in S6 h + PH and S6 h + S. In contrast, c-myc expression returned to baseline levels after S6 h and a new increase was observed 2 h after S6 h + PH but not after S6 h + S. P53 mRNA was significantly expressed 6 h after S6 h + PH, but at a level similar than that observed 6 and 12 h after PH alone. An earlier increase in c-Ha-ras mRNA and cyclin E protein was found in S6 h + PH, in comparison with PH alone. CONCLUSIONS: The first divergent response between the two combined models involved c-myc expression. However, major differences related to the accelerated liver regenerative response observed after S6 h + PH were found at late time points associating an earlier expression of c-Ha-ras and nuclear cyclin E. PMID: 15667586 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Int J Mol Med. 2005 Feb;15(2):269-75. Functional analysis of the effect of forced activation of STAT3 on M1 mouse leukemia cells. Yoshida T, Iwamoto T, Adachi K, Yokota T, Miyake Y, Hamaguchi M. Department of Ophthalmology, Nagoya University School of Medicine, Showa-ku, Nagoya 466-8550, Japan. M1 mouse myeloid leukemia cells exhibit growth arrest and differentiation to monocytes/macrophages in response to leukemia inhibitory factor (LIF) stimulation. Although recent studies have demonstrated that STAT3 plays a central role in this process, it is unknown whether STAT3 activation alone is sufficient. To address this issue, we have established M1/STAT3ER cells, where STAT3 is selectively activated by 4-hydroxytamoxifen (4HT). 4HT stimulation did not have any effect on growth and morphology of M1/ STAT3ER cells, and did not induce the down-regulation of mRNA of c-myc and c-myb, which is necessary for M1 cell differentiation. On the other hand, mRNA of jun-B, IRF1 and p19 was increased by 4HT. DNA precipitation assay indicated that both stimulation of LIF and 4HT similarly activated STAT3ER. Introduction of a constitutive active MAP kinase kinase (MEK1) into M1/STAT3ER cells did not induce differentiation either. Together, our present data suggest that signaling other than the activation of STAT3 and MEK1 may be necessary for M1 cell-growth arrest and differentiation, while a set of early genes of LIF are induced by only STAT3 activation. PMID: 15647843 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Biochem Biophys Res Commun. 2004 Dec 10;325(2):541-8. Region 752-761 of STAT3 is critical for SRC-1 recruitment and Ser727 phosphorylation. Zhao H, Nakajima R, Kunimoto H, Sasaki T, Kojima H, Nakajima K. Department of Immunology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. STAT3 regulates many target genes in response to cytokines and growth factors. To study the mechanisms of STAT3-dependent transcription, we established several cell lines in which HepG2-STAT3-knockdown cells were reconstituted with a variety of STAT3 mutants. Using these cell lines, we found that truncated STAT3(1-750), but not STAT3(1-761), could not recruit SRC-1/NcoA-1 and was not phosphorylated on Ser727. Furthermore, mutation of STAT3 L755 and F757 to alanines caused the loss of STAT3-dependent SRC-1 recruitment, leaving Ser727 phosphorylation intact. Consistent with this, the STAT3-L755A/F757A mutant showed no increase in acetylated histone H3 at Lys14 and a decreased level of RNA polymerase II recruited to the target gene promoter, although p300 recruitment and histone H4 acetylation were intact. This mutant also lost responsiveness to co-expressed SRC-1. Thus, the conserved STAT3 region from 752 to 761, called STAT3 CR2, plays critical roles in STAT3-dependent transcription by recruiting SRC-1 and allowing Ser727 phosphorylation. PMID: 15530426 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: FEBS Lett. 2004 Nov 5;577(1-2):187-92. Tyrosine dephosphorylation of STAT3 in SARS coronavirus-infected Vero E6 cells. Mizutani T, Fukushi S, Murakami M, Hirano T, Saijo M, Kurane I, Morikawa S. Special Pathogens Laboratory, Department of Virology 1, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashimurayama, Tokyo 208-0011, Japan. tmizutan@nih.go.jp Severe acute respiratory syndrome (SARS) has become a global public health emergency. p38 mitogen-activated protein kinase (MAPK) and its downstream targets are activated in SARS coronavirus (SARS-CoV)-infected Vero E6 cells and activation of p38 MAPK enhances the cytopathic effects of SARS-CoV infection. In addition, weak activation of Akt cannot prevent SARS-CoV infection-induced apoptosis in Vero E6 cells. In the present study, we demonstrated that signal transducer and activator of transcription (STAT) 3, which is constitutively phosphorylated at tyrosine (Tyr)-705 and slightly phosphorylated at serine (Ser)-727 in Vero E6 cells, was dephosphorylated at Tyr-705 on SARS-CoV infection. In addition to phosphorylation of p38 MAPK in virus-infected cells, other MAPKs, i.e., extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK), were phosphorylated. Although inhibitors of ERK1/2 and JNK (PD98059 and SP600125) had no effect on phosphorylation status of STAT3, inhibitors of p38 MAPK (SB203580 and SB202190) partially inhibited dephosphorylation of STAT3 at Tyr-705. Tyr-705-phosphorylated STAT3 was localized mainly in the nucleus in mock infected cells, whereas STAT3 disappeared from the nucleus in virus-infected cells. As STAT3 acts as an activator of transcription in the nucleus, these results suggest that STAT3 lacks its activity on transcription in SARS-CoV-infected Vero E6 cells. PMID: 15527783 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: J Biol Chem. 2005 Jan 14;280(2):1037-43. Epub 2004 Nov 1. Interleukin-12-induced interferon-gamma production by human peripheral blood T cells is regulated by mammalian target of rapamycin (mTOR). Kusaba H, Ghosh P, Derin R, Buchholz M, Sasaki C, Madara K, Longo DL. Lymphocyte Cell Biology Unit, Laboratory of Immunology, Gerontology Research Center, NIA, National Institutes of Health, Baltimore, Maryland 21224, USA. Depending on the type of external signals, T cells can initiate multiple intracellular signaling pathways that can be broadly classified into two groups based on their sensitivity to the immunosuppressive drug cyclosporin A (CsA). Interleukin (IL)-12-mediated interferon (IFN)-gamma production by activated T cells has been shown to be CsA-insensitive. In this report, we demonstrate that the IL-12-induced CsA-resistant pathway of IFN-gamma production is sensitive to rapamycin. Rapamycin treatment resulted in the aberrant recruitment of Stat3, Stat4, and phospho-c-Jun to the genomic promoter region resulting in decreased IFN-gamma transcription. IL-12-induced phosphorylation of Stat3 on Ser-727 was affected by rapamycin, which may be due to the effect of rapamycin on the IL-12-induced interaction between mammalian target of rapamycin (mTOR) and Stat3. In accordance with this, reduction in the mTOR protein level by small interfering RNA resulted in suppression of Stat3 phosphorylation and decreased production of IFN-gamma after IL-12 stimulation. These results suggest that mTOR may play a major role in IL-12-induced IFN-gamma production by activated T cells. PMID: 15522880 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Endocrinology. 2005 Jan;146(1):175-85. Epub 2004 Sep 30. Silymarin protects pancreatic beta-cells against cytokine-mediated toxicity: implication of c-Jun NH2-terminal kinase and janus kinase/signal transducer and activator of transcription pathways. Matsuda T, Ferreri K, Todorov I, Kuroda Y, Smith CV, Kandeel F, Mullen Y. Southern California Islet Cell Resources Center, Department of Diabetes, Endocrinology and Metabolism, City of Hope National Medical Center/Beckman Research Institute, Duarte, California 91010, USA. Silymarin is a polyphenolic flavonoid that has a strong antioxidant activity and exhibits anticarcinogenic, antiinflammatory, and cytoprotective effects. Although its hepatoprotective effect has been well documented, the effect of silymarin on pancreatic beta-cells is largely unknown. In this study, the effect of silymarin on IL-1beta and/or interferon (IFN)-gamma-induced beta-cell damage was investigated using RINm5F cells and human islets. IL-1beta and/or IFN-gamma induced cell death in a time-dependent manner in RINm5F cells. The time-dependent increase in cytokine-induced cell death appeared to correlate with the time-dependent nitric oxide (NO) production. Silymarin dose-dependently inhibited both cytokine-induced NO production and cell death in RINm5F cells. Treatment of human islets with a combination of IL-1beta and IFN-gamma (IL-1beta+IFN-gamma), for 48 h and 5 d, resulted in an increase of NO production and the impairment of glucose-stimulated insulin secretion, respectively. Silymarin prevented IL-1beta+IFN-gamma-induced NO production and beta-cell dysfunction in human islets. These cytoprotective effects of silymarin appeared to be mediated through the suppression of c-Jun NH2-terminal kinase and Janus kinase/signal transducer and activator of transcription pathways. Our data show a direct cytoprotective effect of silymarin in pancreatic beta-cells and suggest that silymarin may be therapeutically beneficial for type 1 diabetes. PMID: 15459112 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Mol Cell Biol. 2004 Aug;24(15):6676-89. Drosophila C-terminal Src kinase negatively regulates organ growth and cell proliferation through inhibition of the Src, Jun N-terminal kinase, and STAT pathways. Read RD, Bach EA, Cagan RL. Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA. Src family kinases regulate multiple cellular processes including proliferation and oncogenesis. C-terminal Src kinase (Csk) encodes a critical negative regulator of Src family kinases. We demonstrate that the Drosophila melanogaster Csk ortholog, dCsk, functions as a tumor suppressor: dCsk mutants display organ overgrowth and excess cellular proliferation. Genetic analysis indicates that the dCsk(-/-) overgrowth phenotype results from activation of Src, Jun kinase, and STAT signal transduction pathways. In particular, blockade of STAT function in dCsk mutants severely reduced Src-dependent overgrowth and activated apoptosis of mutant tissue. Our data provide in vivo evidence that Src activity requires JNK and STAT function. PMID: 15254235 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Cell Signal. 2004 Oct;16(10):1123-32. Mitogen-activated protein kinases Erk1/2 and p38 are required for maximal regulation of TIMP-1 by oncostatin M in murine fibroblasts. Tong L, Smyth D, Kerr C, Catterall J, Richards CD. Department of Pathology and Molecular Medicine, Faculty of Health Sciences, McMaster University, HSC-4H17, 1200 Main Stree West, Hamilton, ON, Canada L8S 3Z5. Oncostatin M (OSM) regulates expression of various genes in connective tissue (CT) cells, including tissue inhibitor of metalloproteinases-1 (TIMP-1). In mouse fibroblast cell lines MLg, NIH 3T3 and primary mouse lung fibroblasts (MLF), murine OSM (muOSM) stimulated high TIMP-1 mRNA expression in comparison to leukemia inhibitory factor (LIF), epidermal growth factor (EGF), interleukin (IL)-1beta and transforming growth factor (TGF)beta. In cell signaling, muOSM induced strong phosphorylation of extracellular-signal regulated protein kinase (Erk) 1/2, p38 and Akt in addition to phosphorylation of signal transducer and activator of transcription (STAT) 1, STAT3 and STAT5 within 15 min. LIF and TGFbeta had no such effects. EGF stimulated comparable or lower Erk1/2, p38 and Akt phosphorylation while IL-1beta induced p38 phosphorylation in the fibroblast cell lines. The Erk1/2 inhibitor PD98059 and the p38 inhibitor SB203580 inhibited TIMP-1 mRNA response to muOSM, whereas the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 enhanced the TIMP-1 mRNA response in NIH 3T3 and MLg cells. PD98059 and SB203580, but not LY294002, also inhibited fold induction of a chloramphenicol acetyltransferase (CAT) reporter gene driven by a minimal TIMP-1 promoter that contained a proximal activator protein-1 (AP-1) site. Co-transfection with JunB or c-Jun expression vector in NIH 3T3 cells caused marked transactivation of the TIMP-1 promoter/CAT reporter gene. muOSM caused a rapid increase of JunB and c-Jun protein in NIH 3T3 cells. PD98059 partially inhibited the increase of JunB, but not c-Jun, whereas SB203580 did not induce detectable changes in expression of either AP-1 factor in response to muOSM. These results demonstrate that Erk1/2 and p38 contribute to the elevation of muOSM induced TIMP-1 expression, but PI3K does not, and suggest that Erk1/2 does so by enhancing JunB expression. PMID: 15240007 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: Mol Pharmacol. 2004 Jun;65(6):1427-39. Signal transducer and activator of transcription 3 activation by the delta-opioid receptor via Galpha14 involves multiple intermediates. Lo RK, Wong YH. Department of Biochemistry, Molecular Neuroscience Center, and Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China. The hematopoietic-specific Galpha14 links a variety of G protein-coupled receptors to phospholipase Cbeta (PLCbeta) stimulation. Recent studies reveal that several Galpha subunits are capable of activating signal transducer and activator of transcription (STAT) proteins. In the present study, we investigated the mechanism by which Galpha14 mediates receptor-induced stimulation of STAT3. In human embryonic kidney 293 cells, coexpression of Galpha14 with delta-opioid receptor supported [D-Pen2, D-Pen5]enkephalin (DPDPE)-induced STAT3 phosphorylations at both Tyr705 and Ser727 in a pertussis toxin-insensitive manner. The constitutively active Galpha4QL mutant also induced STAT3 phosphorylations at these sites and promoted STAT3-dependent luciferase activity. Requirements for PLCbeta, protein kinase C (PKC), and calmodulin-dependent kinase II (CaMKII) in Galpha14QL-induced STAT3 activation were demonstrated by their respective inhibitors as well as by coexpression of their dominant-negative mutants. Inhibition of c-Src and Janus kinase 2 and 3 activities abolished STAT3 activation induced by Galpha14QL, but no physical association between Galpha14QL and c-Src could be detected by coimmunoprecipitation. Various intermediates along the extracellular signal-regulated kinase signaling cascade were apparently required for Galpha14QL-induced STAT3 activation; they included Ras/Rac1, Raf-1, and mitogen-activated protein kinase kinase-1/2. In contrast, functional blockade of c-Jun N-terminal kinase, p38 mitogen-activated protein kinase, and phosphatidylinositol-3 kinase had no effect on Galpha14QL-induced responses. PLCbeta, PKC, and CaMKII were shown to be involved in Galpha14QL-mediated c-Src phosphorylation. Similar results were obtained with human erythro-leukemia cells upon DPDPE treatment. These results demonstrate for the first time that Galpha14 activation can lead to STAT3 stimulation via a complex signaling network involving multiple intermediates. PMID: 15155836 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: Infect Immun. 2004 May;72(5):2864-71. Borrelia burgdorferi-induced expression of matrix metalloproteinases from human chondrocytes requires mitogen-activated protein kinase and Janus kinase/signal transducer and activator of transcription signaling pathways. Behera AK, Thorpe CM, Kidder JM, Smith W, Hildebrand E, Hu LT. New England Medical Center, Tufts University School of Medicine, Department of Infectious Disease, Tupper Research Institute, Boston, Massachusetts 02111, USA. Elevations in matrix metalloproteinase 1 (MMP-1) and MMP-3 have been found in patients with Lyme arthritis and in in vitro models of Lyme arthritis using cartilage explants and chondrocytes. The pathways by which B. burgdorferi, the causative agent of Lyme disease, induces the production of MMP-1 and MMP-3 have not been elucidated. We examined the role of the extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways in MMP induction by B. burgdorferi. Infection with B. burgdorferi results in rapid phosphorylation of p38 and JNK within 15 to 30 min. Inhibition of JNK and p38 MAPK significantly reduced B. burgdorferi-induced MMP-1 and MMP-3 expression. Inhibition of ERK1/2 completely inhibited the expression of MMP-3 in human chondrocytes following B. burgdorferi infection but had little effect on the expression of MMP-1. B. burgdorferi infection also induced phosphorylation and nuclear translocation of STAT-3 and STAT-6 in primary human chondrocytes. Expression of MMP-1 and MMP-3 was significantly inhibited by inhibition of JAK3 activity. Induction of MMP-1 and -3 following MAPK and JAK/STAT activation was cycloheximide sensitive, suggesting synthesis of intermediary proteins is required. Inhibition of tumor necrosis factor alpha (TNF-alpha) significantly reduced MMP-1 but not MMP-3 expression from B. burgdorferi-infected cells; inhibition of interleukin-1beta (IL-1beta) had no effect. Treatment of B. burgdorferi-infected cells with JAK and MAPK inhibitors significantly inhibited TNF-alpha induction, consistent with at least a partial role for TNF-alpha in B. burgdorferi-induced MMP-1 expression in chondrocytes. PMID: 15102798 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: Blood. 2004 Jul 15;104(2):509-18. Epub 2004 Mar 23. Bortezomib and flavopiridol interact synergistically to induce apoptosis in chronic myeloid leukemia cells resistant to imatinib mesylate through both Bcr/Abl-dependent and -independent mechanisms. Dai Y, Rahmani M, Pei XY, Dent P, Grant S. Division of Hematology/Oncology, Virginia Commonwealth University/Medical College of Virginia, MCV Station Box 230, Richmond, VA 23298, USA. Interactions between the cyclin-dependent kinase (CDK) inhibitor flavopiridol and the proteasome inhibitor bortezomib were examined in Bcr/Abl(+) human leukemia cells. Coexposure of K562 or LAMA84 cells to subtoxic concentration of flavopiridol (150-200 nM) and bortezomib (5-8 nM) resulted in a synergistic increase in mitochondrial dysfunction and apoptosis. These events were associated with a marked diminution in nuclear factor kappaB (NF-kappaB)/DNA binding activity; enhanced phosphorylation of SEK1/MKK4 (stress-activated protein kinase/extracellular signal-related kinase 1/mitogen-activated protein kinase kinase 4), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK); down-regulation of Bcr/Abl; and a marked reduction in signal transducer and activator of transcription 3 (STAT3) and STAT5 activity. In imatinib mesylate-resistant K562 cells displaying increased Bcr/Abl expression, bortezomib/flavopiridol treatment markedly increased apoptosis in association with down-regulation of Bcr/Abl and BclxL, and diminished phosphorylation of Lyn, Hck, CrkL, and Akt. Parallel studies were performed in imatinib mesylate-resistant LAMA84 cells exhibiting reduced expression of Bcr/Abl but a marked increase in expression/activation of Lyn and Hck. Flavopiridol/bortezomib effectively induced apoptosis in these cells in association with Lyn and Hck inactivation. The capacity of flavopiridol to promote bortezomib-mediated Bcr/Abl down-regulation and apoptosis was mimicked by the positive transcription elongation factor-b (P-TEFb) inhibitor DRB (5,6-dichloro 1-beta-d-ribofuranosylbenzinida-sole). Finally, the bortezomib/flavopiridol regimen also potently induced apoptosis in Bcr/Abl(-) human leukemia cells. Collectively, these findings suggest that a strategy combining flavopiridol and bortezomib warrants further examination in chronic myelogenous leukemia and related hematologic malignancies. PMID: 15039284 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: FASEB J. 2004 May;18(7):914-6. Epub 2004 Mar 19. Impaired liver regeneration in mice lacking methionine adenosyltransferase 1A. Chen L, Zeng Y, Yang H, Lee TD, French SW, Corrales FJ, Garcia-Trevijano ER, Avila MA, Mato JM, Lu SC. Division of Gastroenterology and Liver Diseases, USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases, USC Liver Disease Research Center, USC School of Medicine, Los Angeles, California 90033, USA. Methionine adenosyltransferase (MAT) is an essential enzyme because it catalyzes the formation of S-adenosylmethionine (SAMe), the principal biological methyl donor. Of the two genes that encode MAT, MAT1A is mainly expressed in adult liver and MAT2A is expressed in all extrahepatic tissues. Mice lacking MAT1A have reduced hepatic SAMe content and spontaneously develop hepatocellular carcinoma. The current study examined the influence of chronic hepatic SAMe deficiency on liver regeneration. Despite having higher baseline hepatic staining for proliferating cell nuclear antigen, MAT1A knockout mice had impaired liver regeneration after partial hepatectomy (PH) as determined by bromodeoxyuridine incorporation. This can be explained by an inability to up-regulate cyclin D1 after PH in the knockout mice. Upstream signaling pathways involved in cyclin D1 activation include nuclear factor kappaB (NFkappaB), the c-Jun-N-terminal kinase (JNK), extracellular signal-regulated kinases (ERKs), and signal transducer and activator of transcription-3 (STAT-3). At baseline, JNK and ERK are more activated in the knockouts whereas NFkappaB and STAT-3 are similar to wild-type mice. Following PH, early activation of these pathways occurred, but although they remained increased in wild-type mice, c-jun and ERK phosphorylation fell progressively in the knockouts. Hepatic SAMe levels fell progressively following PH in wild-type mice but remained unchanged in the knockouts. In culture, MAT1A knockout hepatocytes have higher baseline DNA synthesis but failed to respond to the mitogenic effect of hepatocyte growth factor. Taken together, our findings define a critical role for SAMe in ERK signaling and cyclin D1 regulation during regeneration and suggest chronic hepatic SAMe depletion results in loss of responsiveness to mitogenic signals. PMID: 15033934 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: Biochem Pharmacol. 2004 Apr 1;67(7):1389-97. Hepatic expression of multiple acute phase proteins and down-regulation of nuclear receptors after acute endotoxin exposure. Fang C, Yoon S, Tindberg N, Jarvelainen HA, Lindros KO, Ingelman-Sundberg M. Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm 17177, Sweden. Acute systemic lipopolysaccharide (endotoxin, LPS) exposure, which can lead to septic shock, enhances the hepatic expression of inflammatory and acute-phase proteins (APPs). To better understand how LPS aggravates damage, changes in hepatic gene expression after a single LPS dose was screened by using microarrays for 1176 rat genes. We detected more than 20 new potential LPS-induced APPs. Following acute LPS challenge, significant up-regulation of the steady-state mRNA levels of several important early transcription factors, such as c-jun and STAT3, and cytokine-associated genes, was observed. In contrast, RT-PCR analysis revealed marked down-regulation of the nuclear receptors RXRalpha, PXR, FXR, LXR, PPARalpha and CAR. Also genes encoding lipolytic, antioxidant as well as drug- and alcohol-metabolizing enzymes were down-regulated. These data suggest that acute LPS treatment induces important early transcription factors and co-ordinately down-regulates nuclear receptors, and that this results in altered expression of a large number of downstream genes. PMID: 15013855 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: Genes Cells. 2004 Mar;9(3):233-42. Cytoplasmic c-Fos induced by the YXXQ-derived STAT3 signal requires the co-operative MEK/ERK signal for its nuclear translocation. Higashi N, Kunimoto H, Kaneko S, Sasaki T, Ishii M, Kojima H, Nakajima K. Department of Immunology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. A STAT3 (signal transducer and activator of transcription 3)- and a MEK/Erk-mediated signal can be activated by cytokines, including IL-6 (interleukin-6), PDGF, and EGF. Recently, STAT3 and an ERK-signal were shown to co-operatively activate the c-fos gene. Activation of a truncated form of the IL-6 receptor subunit, gp130, that had only one YXXQ motif, induced both c-Fos and JunB in NIH3T3 cells through STAT3 without an apparent increase in the AP-1 (activator protein-1) activity. In contrast, concomitant stimulation of the STAT3 signal and a MEK/Erk-signal markedly increased AP-1 activity with enhanced c-Fos expression. Surprisingly, the c-Fos induced by the YXXQ-signal alone was localized to the cytoplasm, from which it translocated into the nucleus following TPA (12-O-tetradecanoyl-phorbol 13-acetate) treatment in a MEK/Erk-dependent manner. c-Fos that was expressed from a constitutive promoter localized to the nucleus and did not move into the cytoplasm in response to the YXXQ-signal. Rather, the YXXQ-signal was required during c-Fos production for it to be retained in the cytoplasm. Thus, the YXXQ-signal induces c-Fos expression through STAT3 and anchors the new c-Fos in the cytoplasm. In addition, the YXXQ-signal and an Erk signal co-operatively cause c-Fos activation in the nucleus. PMID: 15005710 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: J Biol Chem. 2004 Apr 16;279(16):16154-60. Epub 2004 Feb 5. Global suppression of IL-6-induced acute phase response gene expression after chronic in vivo treatment with the peroxisome proliferator-activated receptor-alpha activator fenofibrate. Gervois P, Kleemann R, Pilon A, Percevault F, Koenig W, Staels B, Kooistra T. Gaubius Laboratory, TNO-Prevention and Health, Zernikedreef 9, P. O. Box 2215, 2301 CE Leiden, The Netherlands. pgervois@pharma.univ-lille2.fr The peroxisome proliferator-activated receptor alpha (PPARalpha), which is highly expressed in liver, plays key roles in lipid metabolism and inflammation. Interleukin-6 (IL-6) is the principal inducer of acute phase response (APR) gene expression. In the present study, we demonstrate that chronic treatment with the PPARalpha agonist fenofibrate fully prevents the IL-6-induced APR gene expression in wild-type but not in PPARalpha-deficient mice. PPARalpha prevents the IL-6-induced expression of the positive APR genes fibrinogen-alpha, -beta, -gamma, haptoglobulin, and serum amyloid A and the IL-6-induced suppression of the negative APR gene, major urinary protein. Furthermore, the effect of PPARalpha on the APR gene expression does not simply consist in a delayed systemic response to IL-6 but occurs directly at the transcriptional level. This global suppression of acute phase gene transcription may be explained by two PPARalpha-dependent in vivo effects: 1) PPARalpha activation results in the down-regulation of the IL-6 receptor components gp80 and gp130 in the liver, thereby reducing the phosphorylation and activation of the downstream transcription factors STAT3 and c-Jun that transduce the IL-6 signal; and 2) PPARalpha reduces the basal expression of the transcription factors CCAAT enhancer-binding protein-alpha, -beta, -delta, which are responsible for immediate and maintained transcription of APR genes. A similar global effect of fenofibrate on acute phase protein expression is observed in hyperlipidemic patients chronically treated with fenofibrate, which displayed decreased plasma concentrations of the positive APR proteins fibrinogen, C-reactive protein, serum amyloid A, plasminogen, and alpha2-macroglobulin and increased plasma concentrations of the negative APR albumin, underlining the clinical significance of our findings. PMID: 14764586 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: Am J Physiol Cell Physiol. 2004 Jun;286(6):C1302-11. Epub 2004 Jan 21. Selective activation of STAT3 in human monocytes stimulated by G-CSF: implication in inhibition of LPS-induced TNF-alpha production. Nishiki S, Hato F, Kamata N, Sakamoto E, Hasegawa T, Kimura-Eto A, Hino M, Kitagawa S. Department of Physiology, Osaka City University Medical School, Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. Lipopolysaccharide (LPS) induced tumor necrosis factor (TNF)-alpha production in human monocytes, which was dependent on activation of extracellular signal-regulated kinase (ERK), p38, c-Jun NH(2)-terminal kinase (JNK), and nuclear factor (NF)-kappa B. LPS-induced TNF-alpha production was inhibited by granulocyte colony-stimulating factor (G-CSF) and interleukin (IL)-10. G-CSF, like IL-10, exerted the inhibitory effect even when simultaneously added with LPS. Among the signaling pathways, signal transducer and activator of transcription 3 (STAT3) was selectively activated in monocytes stimulated by G-CSF or IL-10. G-CSF-mediated inhibition of LPS-induced TNF-alpha production as well as G-CSF-induced STAT3 phosphorylation and suppressor of cytokine signaling 3 mRNA expression were prevented by pretreatment of monocytes with AG-490, an inhibitor of Janus kinase 2. G-CSF did not affect LPS-induced activation of ERK, p38, JNK, and NF-kappa B, indicating that G-CSF affects the pathway downstream or independently of these signaling molecules. G-CSF-induced, but not IL-10-induced, STAT3 phosphorylation was attenuated in the presence of LPS. These findings suggest that G-CSF, like IL-10, inhibits LPS-induced TNF-alpha production in human monocytes through selective activation of STAT3, and the immunomodulation observed in vivo by G-CSF administration may be partly ascribed to the direct effect of G-CSF on monocyte functions. PMID: 14736711 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: Int J Colorectal Dis. 2004 Sep;19(5):414-20. Epub 2004 Jan 15. Involvement of AP-1 proteins in pancreatic stellate cell activation in vitro. Fitzner B, Sparmann G, Emmrich J, Liebe S, Jaster R. Department of Medicine, Division of Gastroenterology, Medical Faculty, University of Rostock, E.-Heydemann-Strasse 6, Rostock, Germany. BACKGROUND AND AIMS: Pancreatic stellate cells (PSCs) play a key role in the development of pancreatic fibrosis. The molecular mechanisms underlying their activation in response to profibrogenic mediators, however, are largely unknown. Extending previous studies on the transcriptional regulation of PSC activation, we have now focused on the involvement of activator protein (AP)-1. MATERIALS AND METHODS: Using cultured rat PSCs, phenotypic transition of PSCs towards activated myofibroblasts was monitored by an immunoblot analysis of alpha-smooth muscle actin (alpha-SMA) expression. Transcription factor activation profiles were studied by electrophoretic mobility shift assays. DNA synthesis in PSCs was assessed through the quantification of 5-bromo-2'-deoxyuridine incorporation. RESULTS: Activated AP-1 complexes were detectable already before high levels of alpha-SMA were expressed. Maximal DNA binding activity of AP-1, as well as of NF-kappaB, was observed early in the course of PSC culture, while the strongest activation of STAT3 was observed much later. A detailed analysis of AP-1 complex composition revealed that phenotypic transition of PSCs towards myofibroblasts was accompanied by an increase of the JunD content relative to the one of JunB. Studies on the role of JunB and JunD in PSC activation indicated an inhibition of platelet-derived growth factor-induced DNA synthesis by antisense oligonucleotides to JunB but not JunD. CONCLUSIONS: The results of this study implicate AP-1 in PSC activation and suggest distinct roles of individual Jun proteins in the regulation of PSC function. In further studies, it should be analyzed whether signaling pathways involved in PSC activation might be suitable targets for antifibrotic therapies. Copyright 2004 Springer-Verlag PMID: 14727130 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 25: Oncogene. 2003 Nov 27;22(54):8797-801. Biologic sequelae of c-Jun NH(2)-terminal kinase (JNK) activation in multiple myeloma cell lines. Hideshima T, Hayashi T, Chauhan D, Akiyama M, Richardson P, Anderson K. Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA. Although c-Jun NH(2)-terminal kinase (JNK) is activated by treatment with therapeutic agents, the biologic sequelae of inhibiting constitutive activation of JNK has not yet been clarified. In this study, we examine the biologic effect of JNK inhibition in multiple myeloma (MM) cell lines. JNK-specific inhibitor SP600125 induces growth inhibition via induction of G1 or G2/M arrest in U266 and MM.1S multiple myeloma cell lines, respectively. Neither exogenous IL-6 nor insulin-like growth factor-1 (IGF-1) overcome SP600125-induced growth inhibition, and IL-6 enhances SP600125-induced G2/M phase in MM.1S cells. Induction of growth arrest is mediated by upregulation of p27(Kip1), without alteration of p53 and JNK protein expression. Importantly, SP600125 inhibits growth of MM cells adherent to bone marrow stromal cells (BMSCs). SP600125 induces NF-kappaB activation in a dose-dependent fashion, associated with phosphorylation of IkappaB kinase alpha (IKKalpha) and degradation of IkappaBalpha. In contrast, SP600125 does not affect phosphorylation of STAT3, Akt, and/or ERK. IKK-specific inhibitor PS-1145 inhibits SP600125-induced NF-kappaB activation and blocks the protective effect of SP600125 against apoptosis. Our data therefore demonstrate for the first time that inhibiting JNK activity induces growth arrest and activates NF-kappaB in MM cells. PMID: 14647474 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: J Biol Chem. 2003 Dec 26;278(52):52154-65. Epub 2003 Oct 9. Constitutively active Galpha16 stimulates STAT3 via a c-Src/JAK- and ERK-dependent mechanism. Lo RK, Cheung H, Wong YH. Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China. The hematopoietic-specific Galpha16 protein has recently been shown to mediate receptor-induced activation of the signal transducer and activator of transcription 3 (STAT3). In the present study, we have delineated the mechanism by which Galpha16 stimulates STAT3 in human embryonic kidney 293 cells. A constitutively active Galpha16 mutant, Galpha16QL, stimulated STAT3-dependent luciferase activity as well as the phosphorylation of STAT3 at both Tyr705 and Ser727. Galpha16QL-induced STAT3 activation was enhanced by overexpression of extracellular signal-regulated kinase 1 (ERK1), but was inhibited by U0126, a Raf-1 inhibitor, and coexpression of the dominant negative mutants of Ras and Rac1. Inhibition of phospholipase Cbeta, protein kinase C, and calmodulin-dependent kinase II by their respective inhibitors also suppressed Galpha16QL-induced STAT3 activation. The involvement of tyrosine kinases such as c-Src and Janus kinase 2 and 3 (JAK2 and JAK3) in Galpha16QL-induced activation of STAT3 was illustrated by the combined use of selective inhibitors and dominant negative mutants. In contrast, c-Jun N-terminal kinase, p38 MAPK, RhoA, Cdc42, phosphatidylinositol 3-kinase, and the epidermal growth factor receptor did not appear to be required. Similar observations were obtained with human erythroleukemia cells, where STAT3 phosphorylation was stimulated by C5a in a PTX-insensitive manner. Collectively, these results highlight the important regulatory roles of the Ras/Raf/MEK/ERK and c-Src/JAK pathways on the stimulation of STAT3 by activated Galpha16. Demonstration of the involvement of different kinases in Galpha16QL-induced STAT3 activation supports the involvement of multiple signaling pathways in the regulation of transcription by G proteins. PMID: 14551213 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: Genes Dev. 2003 Oct 15;17(20):2564-77. Epub 2003 Oct 1. STAT3-dependent enhanceosome assembly and disassembly: synergy with GR for full transcriptional increase of the alpha 2-macroglobulin gene. Lerner L, Henriksen MA, Zhang X, Darnell JE Jr. Laboratory of Molecular Cell Biology, The Rockefeller University, New York, New York 10021, USA. We describe a detailed time course of the assembly and disassembly of a STAT3-dependent, glucocorticoid-supplemented enhanceosome for the alpha2-macroglobulin (alpha2-M) gene and compare this with a detailed time course of transcription of the gene by run-on analysis. The glucocorticoid receptor (GR) can associate with the enhanceosome without STAT3. Furthermore, the enhanceosome contains c-Jun/c-Fos and OCT-1 constitutively. All of these factors (GR, c-Jun, OCT-1) have transcription activation domains, but STAT3 is required for the observed transcriptional increase. The time course of enhanceosome occupation by GR and tyrosine-phosphorylated STAT3 shows that these transcription factors precede by approximately 5-10 min the arrival of RNA polymerase II (Pol II). The enhanceosome remains assembled for approximately 90 min in the continued presence of both inducers. When IL-6 and Dex are removed (after 30 min of treatment), the disappearance within an additional 30 min of the established enhanceosome indicates that renewal of STAT3 and GR binding must occur in the continued presence of IL-6+Dex. Compared with the total nuclear tyrosine-phosphorylated STAT3 capable of binding DNA, the chromatin-associated STAT3 resists dephosphorylation and appears to recycle to maintain the enhanceosome. Run-on transcription shows a lag after full enhanceosome occupation that can be largely but not completely explained by the approximately 30 min transit time of Pol II across the alpha2-Mlocus. PMID: 14522952 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: Cancer Res. 2003 Sep 15;63(18):5745-53. Inhibition of induced chemoresistance by cotreatment with (E)-5-(2-bromovinyl)-2'-deoxyuridine (RP101). Fahrig R, Heinrich JC, Nickel B, Wilfert F, Leisser C, Krupitza G, Praha C, Sonntag D, Fiedler B, Scherthan H, Ernst H. RESprotect, Dresden, Germany. fahrig@resprotect.de Induced chemoresistance leads to the reduction of apoptotic responses. Although several drugs are in development that circumvent or decrease existing chemoresistance, none has the potential to prevent or reduce its induction. Here, we present data from a drug that could perhaps fill this gap. Cotreatment of chemotherapy with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU, RP101) prevented the decrease of apoptotic effects during the course of chemotherapy and reduced nonspecific toxicity. Amplification of chemoresistance genes (Mdr1 and Dhfr) and overexpression of gene products involved in proliferation (DDX1) or DNA repair (UBE2N and APEX) were inhibited, whereas activity of NAD(P)H: quinone oxidoreductase 1 (NQO1) was enhanced. During recovery, when treatment was with BVDU only, microfilamental proteins were up-regulated, and proteins involved in ATP generation or cell survival (STAT3 and JUN-D) were down-regulated. That way, in three different rat tumor models, the antitumor efficiency of chemotherapy was optimized, and toxic side effects were reduced. Because of these beneficial properties of BVDU, a clinical pilot Phase I/II study with five human tumor entities has been started at the University of Dresden (Dresden, Germany). So far, no unwanted side effects have been observed. PMID: 14522895 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: J Appl Physiol. 2004 Jan;96(1):398-404. Epub 2003 Sep 26. Responsiveness of cell signaling pathways during the failed 15-day regrowth of aged skeletal muscle. Morris RT, Spangenburg EE, Booth FW. Department Medical Pharmacology and physiology, University of Missouri, Columbia, MO 65211, USA. boothf@missouri.edu Various cellular signaling pathways, such as phosphatidylinositol 3-kinase, calcineurin, Janus kinase 2/signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinase (MAPK) have been suggested to play an important role in skeletal muscle growth. Old muscle, compared with young muscle, lacks the ability to completely regrow its muscle mass after an atrophy-induced stimulus. it is hypothesized that defects and/or delays in the activation of specific cell signaling pathways of aged soleus muscle limit the potential for growth. To test this, 42 male Fischer 344 x Brown Norway rats, 30 mo old, were hindlimb immobilized for 10 days, and their muscle samples were compared with muscle samples analyzed from 3- to 4-mo-old rats in a previous report (Childs TE, Spangenburg EE, Vyas DR, and Booth FW. Am J Physiol Cell Physiol: 285: C391-C398, 2003). After 10 days, the immobilization was removed and rats were allowed to ambulate for a series of days. Alterations in the activation or deactivation status of specific signaling pathways were determined by comparing the phosphorylation (phos) and total concentration of specific signaling proteins (pan) through Western blotting with the 10-day immobilization group. Various cell signals and their respective time groups of the old rats were shown to be significantly different compared with the 10-day immobilization group. For example, peak increases during recovery from the immobilization were observed at 1) the third recovery day for calcineurin B-pan and 2) the sixth recovery day for glycogen synthase kinase-3beta-phos, p70 S6 kinase (p70S6k) -phos and -pan, calcineurin A-pan, STAT3-phos and -pan, p44 MAPK-pan, and p42 MAPK-pan. In contrast, Akt-pan, c-Jun NH2-terminal kinase-phos, and p38 MAPK-phos were observed to decrease from 10-day immobilization values to control levels. Also, Aktphos was unchanged among all groups. In a follow-up experiment in which muscle samples from both the present study and a previous study (Childs TE, Spangenburg EE, Vyas DR, and Booth FW. Am J Physiol Cell Physiol: 285: C391-C398, 2003) were reanalyzed together, the recovery-induced increase in p70S6k-phos from immobilization-atrophy was significantly attenuated in soleus muscles of the old group. PMID: 14514701 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: Oncogene. 2003 May 19;22(20):3152-61. Death receptors and melanoma resistance to apoptosis. Ivanov VN, Bhoumik A, Ronai Z. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, NY 10029, USA. Impaired ability to undergo programmed cell death in response to a wide range of external stimuli acquires melanomas a selective advantage for progression and metastasis as well as their notorious resistance to therapy. Better understanding of mechanisms that govern apoptosis has enabled identification of diverse routes by which melanomas manage to escape stimuli of apoptosis. Changes at genomic, transcriptional and post-translational levels of G-proteins and protein kinases (Ras, B-Raf) and their transcription factor effectors (c-Jun, ATF2, Stat3 and NF-kappaB) affects TNF, Fas and TRAIL receptors, which play important roles in acquiring melanoma's resistance to apoptosis. Here, we summarize our current understanding of changes that alters the regulation of death receptors during melanoma development. Publication Types: Review Review, Tutorial PMID: 12789291 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 31: Cancer Res. 2003 May 1;63(9):2206-15. Constitutive activation of nuclear factor kappaB p50/p65 and Fra-1 and JunD is essential for deregulated interleukin 6 expression in prostate cancer. Zerbini LF, Wang Y, Cho JY, Libermann TA. BIDMC Genomics Center and New England Baptist Bone and Joint Institute, Boston, Massachusetts 02115, USA. To date, no effective treatment for patients with advanced androgen-independent prostate cancer is available, whereas androgen ablation therapy, surgery, and radiation therapy are effective in treating local, androgen-dependent tumors. The mechanisms underlying the differences between androgen-dependent and -independent prostate cancer remain elusive. Interleukin (IL)-6 is a pleiotropic cytokine whose expression under normal physiological conditions is tightly controlled. However, aberrant constitutive IL-6 gene expression has been implicated in prostate cancer progression and resistance to chemotherapy and has been directly linked to prostate cancer morbidity and mortality. Particularly striking is the large increase in the expression of IL-6 in hormone-refractory prostate cancer. IL-6, in addition to its role as an immunomodulatory cytokine, functions as a growth and differentiation factor for prostate cancer cells. To determine the molecular mechanisms that lead to deregulated IL-6 expression in advanced prostate cancer, we examined the regulatory elements involved in IL-6 gene expression in androgen-independent prostate cancer cells. We demonstrate that, in contrast to the androgen-sensitive LNCaP cells, androgen-insensitive PC-3 and DU145 cells express high levels of IL-6 protein and mRNA due to enhanced promoter activity. Deregulated activation of the IL-6 promoter is for the most part mediated by a combined constitutive activation of the nuclear factor (NF)-kappaB p50 and p65 and the activator protein 1 (AP-1) JunD and Fra-1 family members as demonstrated by electrophoretic mobility shift assays, site-directed mutagenesis, and transfection experiments. Mutation of the NF-kappaB and AP-1 sites drastically reduces IL-6 promoter activity in both androgen-independent prostate cancer cell lines. Additionally, inhibition of these transcription factors using adenovirus vectors encoding either the IkappaBalpha repressor gene or a dominant negative JunD mutant leads to a strong down-regulation of IL-6 gene expression at the mRNA and protein level as measured by real-time PCR and ELISA, respectively. Furthermore, the blockade of IL-6 gene expression results in drastic inhibition of the constitutively activated signal transducers and activators of transcription 3 signaling pathway in DU145 cells. Our data demonstrate for the first time that a combined aberrant activation of NF-kappaB p50 and p65 and AP-1 JunD and Fra-1 in androgen-independent prostate cancer cells results in deregulated IL-6 expression, suggesting a novel potential entry point for therapeutic intervention in prostate cancer. PMID: 12727841 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 32: Biol Chem. 2003 Mar;384(3):409-21. Interleukin-6 N-terminal peptides modulate the expression of junB protooncogene and the production of fibrinogen in HepG2 cells. Bosze S, Hudecz F, Igaz P, Ortutay Z, Csik G, Falus A, Toth S. Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eotvos L. University, P.O. Box 32, H-1518 Budapest 112, Hungary Interleukin-6 (IL-6) is a helical cytokine exerting pleiotropic activities including the regulation of hematopoiesis, B cell activation and acute-phase reaction. The structure-function relationship of the molecule is the subject of intensive investigation using point and deletion mutants. Our objective was to analyse the role of the N-terminal 18-46 region in IL-6-mediated expression of junB protooncogene and fibrinogen production, reflecting the acute phase response, with synthetic overlapping peptides. mRNA expression of junB was monitored by competitive RT-PCR, while sandwich ELISA was used for the detection of fibrinogen in the supernatant of HepG2 human hepatoma cells. We found that even short synthetic octapeptides can be stimulatory (in the absence of IL-6) or inhibitory (in the presence of IL-6) in both assays. To establish the molecular mechanism by which synthetic peptides exert their biological effects electromobility shift assay was carried out using HepG2 nuclear extracts. Peptides inducing junB expression initiate gel shifts of STAT3/DNA complexes, which may indicate the involvement of this signal transduction pathway. Circular dicroism spectroscopy data suggest that 8-11-mer peptides representing different parts of the 18-46 region have a marked tendency to adopt ordered conformations in a water/trifluoroethanol (1:1 v/v) mixture. Competition studies with rhIL-6 and selected fluorophore-labelled peptides indicate the presence of more than one binding site on soluble IL-6 receptor. Considering the possible multiple etiologic role of IL-6 in the pathogenesis of various diseases, these peptides could be useful for dissection of IL-6 related biological effects. PMID: 12715892 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 33: Endocrinology. 2003 May;144(5):1770-9. Tumor necrosis factor-alpha decreases insulin-like growth factor-I messenger ribonucleic acid expression in C2C12 myoblasts via a Jun N-terminal kinase pathway. Frost RA, Nystrom GJ, Lang CH. Department of Cellular and Molecular Physiology, Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033, USA. rfrost@psu.edu IGF-I is a major anabolic hormone for skeletal muscle in vivo. Yet the mechanisms by which GH and cytokines regulate IGF-I expression remain obscure. Lipopolysaccharide (LPS) dramatically alters the circulating concentration of both TNF alpha and IGF-I, and TNF alpha in part mediates the cachectic activity of LPS. Little is known about the local synthesis of IGF-I and TNF alpha in skeletal muscle per se. The purpose of the present study was to determine whether LPS alters the expression of TNF alpha and IGF-I in mouse skeletal muscle and whether TNF alpha directly inhibits IGF-I mRNA expression in C2C12 myoblasts. Intraperitoneal injection of LPS in C3H/SnJ mice increased the expression of TNF alpha protein in plasma (16-fold) and TNF alpha mRNA in skeletal muscle (8-fold). LPS also decreased the plasma concentration of IGF-I (30%) and IGF-I mRNA in skeletal muscle (50%, between 6 and 18 h after LPS administration). Addition of LPS or TNF alpha directly to C2C12 myoblasts decreased IGF-I mRNA by 50-80%. The TNF alpha-induced decrease in IGF-I mRNA was both dose and time dependent and occurred in both myoblasts and differentiated myotubes. TNF alpha selectively decreased IGF-I but not IGF-II mRNA levels, and the effect of TNF alpha was blocked by a specific TNF-binding protein. TNF alpha did not alter IGF-I mRNA levels in the presence of the protein synthesis inhibitor cycloheximide. TNF alpha did not change the half-life of IGF-I mRNA. TNF alpha completely prevented GH-inducible IGF-I mRNA expression, but this GH resistance was not attributable to impairment in signal transducer and activator of transcription-3 or -5 phosphorylation. TNF alpha increased both nitric oxide synthase-II mRNA and protein, and the nitric oxide donor sodium nitroprusside decreased IGF-I mRNA levels in C2C12 cells. Yet inhibitor studies indicate that nitric oxide did not mediate the effect of TNF alpha on IGF-I mRNA expression. TNF alpha stimulated the phosphorylation of c-Jun and specific inhibition of the Jun N-terminal kinase pathway, but not other MAPK pathways, completely prevented the TNF alpha-induced drop in IGF-I mRNA. These data suggest that LPS stimulates TNF alpha expression in mouse skeletal muscle and autocrine-derived cytokines may contribute to the reduced expression of IGF-I in this tissue. PMID: 12697682 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 34: Cancer Res. 2003 Mar 15;63(6):1270-9. Discovery of JSI-124 (cucurbitacin I), a selective Janus kinase/signal transducer and activator of transcription 3 signaling pathway inhibitor with potent antitumor activity against human and murine cancer cells in mice. Blaskovich MA, Sun J, Cantor A, Turkson J, Jove R, Sebti SM. Drug Discovery Program, H Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612, USA. Constitutively activated, tyrosine-phosphorylated signal transducer and activator of transcription (STAT) 3 plays a pivotal role in human tumor malignancy. To discover disrupters of aberrant STAT3 signaling pathways as novel anticancer drugs, we developed a phosphotyrosine STAT3 cytoblot. Using this high throughput 96-well plate assay, we identified JSI-124 (cucurbitacin I) from the National Cancer Institute Diversity Set. JSI-124 suppressed the levels of phosphotyrosine STAT3 in v-Src-transformed NIH 3T3 cells and human cancer cells potently (IC(50) value of 500 nM in the human lung adenocarcinoma A549) and rapidly (complete inhibition within 1-2 h). The suppression of phosphotyrosine STAT3 levels resulted in the inhibition of STAT3 DNA binding and STAT3-mediated but not serum response element-mediated gene transcription. JSI-124 also decreased the levels of tyrosine-phosphorylated Janus kinase (JAK) but not those of Src. JSI-124 was highly selective for JAK/STAT3 and did not inhibit other oncogenic and tumor survival pathways such as those mediated by Akt, extracellular signal-regulated kinase 1/2, or c-Jun NH(2)-terminal kinase. Finally, JSI-124 (1 mg/kg/day) potently inhibited the growth in nude mice of A549 tumors, v-Src-transformed NIH 3T3 tumors, and the human breast carcinoma MDA-MB-468, all of which express high levels of constitutively activated STAT3, but it did not affect the growth of oncogenic Ras-transformed NIH 3T3 tumors that are STAT3 independent or of the human lung adenocarcinoma Calu-1, which has barely detectable levels of phosphotyrosine STAT3. JSI-124 also inhibited tumor growth and significantly increased survival of immunologically competent mice bearing murine melanoma with constitutively activated STAT3. These results give strong support for pharmacologically targeting the JAK/STAT3 signaling pathway for anticancer drug discovery. PMID: 12649187 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 35: Blood. 2003 Jun 15;101(12):4958-65. Epub 2003 Feb 27. Induction of apoptosis in IL-3-dependent hematopoietic cell lines by guanine nucleotide depletion. Gu JJ, Gathy K, Santiago L, Chen E, Huang M, Graves LM, Mitchell BS. Lineberger Comprehensive Cancer Center, Department of Pharmacology, University of North Carolina at Chapel Hill, 27599, USA. Inosine 5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme that catalyzes the conversion of IMP to xanthosine monophosphate (XMP) at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides. Inhibition of IMPDH results in the depletion of guanine nucleotides, prevents cell growth by G1 arrest, and induces cell differentiation in a cell-type-specific manner. The molecular and sensing mechanisms underlying these effects are not clear. We have examined the induction of apoptosis by mycophenolic acid (MPA), a specific IMPDH inhibitor, in interleukin-3 (IL-3)-dependent murine hematopoietic cell lines. MPA treatment, at clinically relevant doses, caused apoptosis in 32D myeloid cells and in FL5.12 and BaF3 pre-B cells in the ongoing presence of IL-3. Apoptosis was completely prevented by the addition of guanosine at time points up to 12 hours, after which caspase 3 activity increased and apoptosis was not reversible. MPA treatment caused marked down-regulation of the MAP kinase kinase/extracellular regulatory kinase (MEK/Erk) pathway at 3 hours while simultaneously increasing the phosphorylation of c-Jun kinase. In addition, MPA strongly down-regulated the mammalian target of rapamcyin (mTOR) pathway, as indicated by the decreased phosphorylation of p70 S6 kinase and of 4EBP1. Inhibition of either the mitogen-activated protein kinase (MAPK) or the mTOR pathway alone by standard pharmacologic inhibitors did not induce apoptosis in IL-3-dependent cells, whereas inhibition of both pathways simulated the effects of MPA treatment. These results indicate that IMPDH inhibitors may be effective in modulating signal transduction pathways in hematopoietic cells, suggesting their usefulness in chemotherapeutic regimens for hematologic malignancies. PMID: 12609835 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 36: Cardiovasc Res. 2003 Feb;57(2):333-46. Comment in: Cardiovasc Res. 2003 Feb;57(2):294-7. Activation of signal transducer and activator of transcription (STAT) pathways in failing human hearts. Ng DC, Court NW, dos Remedios CG, Bogoyevitch MA. Cell Signalling Laboratory, Biochemistry and Molecular Biology in the School of Biomedical and Chemical Sciences, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. OBJECTIVES: The signal transduction pathways mediating the progression to failure have been intensively studied in a variety of in vitro and in vivo animal models. Recently, acute activation of the Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) has been observed in the heart, but whether this is sustained in ischemic heart disease (IHD) or dilated cardiomyopathy (DCM) has not been previously addressed. METHODS: We assessed the tyrosine phosphorylation of STAT1, 3, 5 and 6 in ventricular samples of explanted human hearts with IHD (n=11) and DCM (n=9) as an indication of STAT activation. Samples from normal donor hearts (n=9) acted as controls. In parallel, we also assessed protein expression and phosphorylation of three major families of mitogen-activated protein kinases (MAPKs); ERK, p38 MAPK and c-Jun NH(2)-terminal kinase (JNK). RESULTS: All STAT isoforms were significantly phosphorylated in DCM. In contrast, only the phosphorylation of STATs 1 and 5 were significantly enhanced in IHD. Expression of total STAT protein remained unchanged. For the MAPKs, significant phosphorylation of p38(MAPK) was only observed in IHD. In contrast, there was no change in ERK or JNK activation despite abundant protein expression. CONCLUSIONS: We have shown that different members of the STAT transcription factor family are chronically phosphorylated in the failing heart as a result of IHD (STAT1 and 5) or DCM (STAT1, 3, 5 and 6). In contrast, IHD but not DCM showed significant p38(MAPK) phosphorylation. Whilst the differences noted between IHD and DCM may reflect different initiating events, the common activation of STATs 1 and 5 suggests that these transcription factors may play a common role regulating the progression of heart failure. PMID: 12566106 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 37: J Biol Chem. 2003 Apr 11;278(15):12650-9. Epub 2003 Jan 31. Ataxia telangiectasia mutated proteins, MAPKs, and RSK2 are involved in the phosphorylation of STAT3. Zhang Y, Cho YY, Petersen BL, Bode AM, Zhu F, Dong Z. Hormel Institute, University of Minnesota, Austin, Minnesota 55912, USA. Phosphorylation at Ser(727) is known to be required for complete activation of STAT3 by diverse stimuli including UV irradiation, but the kinase(s) responsible for phosphorylating STAT3 (Ser(727)) is still not well discerned. In the present study, we observed that activation of ATM is required for a UVA-stimulated increase in Ser(727) phosphorylation of STAT3 as well as in activation and phosphorylation of p90 ribosomal protein S6 kinases (RSKs). Moreover, UVA-stimulated activation of upstream kinases, such as c-Jun N-terminal kinases (JNKs) and ERKs, involved in mediating phosphorylation of RSKs and STAT3 was defective or delayed in ATM-deficient cells. Furthermore, we provide evidence that RSK2-deficient cells were defective for UV-induced Ser(727) phosphorylation of STAT3, and the defect was restored after ectopic expression of transfected full-length RSK2. In vitro experiments showed that active RSK2 and JNK1 induce the phosphorylation of STAT3 precipitates from immunoprecipitation but not from glutathione S-transferase (GST) pull-down. Interestingly, the GST fusion STAT3 proteins mixed together with STAT3 immunoprecipitates can be phosphorylated by JNK. However, the in vitro phosphorylation of STAT3 was reduced by the GST-STAT3 beta protein, a dominant negative form of STAT3. Taken together, our results demonstrate that the STAT3 phosphorylation at Ser(727) is triggered by active RSK2 or JNK1 in the presence of a downstream kinase or a cofactor, and thereby the intracellular phosphorylation process is stimulated through a signaling pathway involving ATM, MAPKs, RSK2, and an as yet unidentified kinase or cofactor. Additionally, RSK2-mediated phosphorylation of STAT3 (Ser(727)) was further determined to be required for basal and UVA-stimulated STAT3 transcriptional activities. PMID: 12562765 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 38: Hunan Yi Ke Da Xue Xue Bao. 2001 Feb 28;26(1):17-9. [Effects of Stat3 phosphorylation and expression of c-fos and c-jun proteins on hepatocarcinogenesis] [Article in Chinese] Feng DY, Zheng H, Jiang HY. Department of Pathology, Xiangya Medical Couege, Central South University, Changsha 410078. Expression of phosphorylated Stat3 (p-Stat3), c-fos and c-jun proteins was detected by immunohistochemical technique in 55 hepatocellular carcinomas (HCC) and their surrounding liver tissues. The results showed that the positive rates and signal intensity of p-Stat3, c-fos and c-jun protein in HCCs were significantly higher than those in pericarcinomatous tissues. The expressive intensity of c-fos and c-jun proteins was positively related to p-Stat3 expression in HCCs, whereas the positive correlation of expressive intensity was only observed between c-jun protein and p-Stat3 in pericarcinomatous tissues. The data suggest that the phosphorylation of Stat3 may be an early event in hepatocarcinogenesis; the overexpression of p-Stat3 protein, which activates c-fos and c-jun genes, may contribute to malignant transformation of hepatocytes; hepatocytes which expressed p-Stat3, c-fos or c-jun proteins may be potentially malignant pre-cancer cells in pericarcinomatous liver tissues. PMID: 12536605 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 39: Am J Physiol Cell Physiol. 2003 Feb;284(2):C316-30. PDGF stimulates pulmonary vascular smooth muscle cell proliferation by upregulating TRPC6 expression. Yu Y, Sweeney M, Zhang S, Platoshyn O, Landsberg J, Rothman A, Yuan JX. Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California, San Diego, California 92103, USA. Capacitative Ca(2+) entry (CCE) through store-operated Ca(2+) (SOC) channels plays an important role in returning Ca(2+) to the sarcoplasmic reticulum (SR) and regulating cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)). A rise in [Ca(2+)](cyt) and sufficient Ca(2+) in the SR are required for pulmonary artery smooth muscle cell (PASMC) proliferation. We tested the hypothesis that platelet-derived growth factor (PDGF)-mediated PASMC growth involves upregulation of c-Jun and TRPC6, a transient receptor potential cation channel. In rat PASMC, PDGF (10 ng/ml for 0.5-48 h) phosphorylated signal transducer and activator of transcription (STAT3), increased mRNA and protein levels of c-Jun, and stimulated cell proliferation. PDGF treatment also upregulated TRPC6 expression and augmented CCE, elicited by passive depletion of Ca(2+) from the SR using cyclopiazonic acid. Furthermore, overexpression of c-Jun stimulated TRPC6 expression and CCE amplitude in PASMC. Downregulation of TRPC6 using an antisense oligonucleotide specifically for human TRPC6 decreased CCE and inhibited PDGF-mediated PASMC proliferation. These results suggest that PDGF-mediated PASMC proliferation is associated with c-Jun/STAT3-induced upregulation of TRPC6 expression. The resultant increase in CCE raises [Ca(2+)](cyt), facilitates return of Ca(2+) to the SR, and enhances PASMC growth. PMID: 12529250 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 40: J Biol Chem. 2003 Mar 28;278(13):11281-8. Epub 2002 Dec 30. Interleukin-6/glycoprotein 130-dependent pathways are protective during liver regeneration. Wuestefeld T, Klein C, Streetz KL, Betz U, Lauber J, Buer J, Manns MP, Muller W, Trautwein C. Department of Gastroenterology, Hepatology, and Endocrinology, Medizinische Hochschule Hannover, 30625 Hannover, Germany. After tissue loss the liver has the unique capacity to restore its mass by hepatocyte proliferation. Interleukin-6 (IL6)-deficient mice show a lack in DNA synthesis after partial hepatectomy (PH). To define better the role of IL6 and its family members for liver regeneration after PH, we used conditional knockout mice for glycoprotein 130 (gp130), the common signal transducer of all IL6 family members. We show that gp130-dependent pathways control Stat3 activation after PH. By using gene array analysis, we demonstrate that c-jun, NF-kappa B, c-myc, and tumor necrosis factor receptor expression is gp130-dependent. However, in gp130-deleted mice only minor effects on cell cycle and on the maximum of DNA synthesis after PH were found compared with controls. As in conditional gp130 animals, the acute phase response was completely abolished, we considered that other means are essential to define the role of gp130-dependent pathways for liver regeneration. LPS stimulation in gp130-deleted and also IL6 -/- animals after PH leads to a significant reduction in survival and DNA synthesis, which was associated with decreased Bcl-xL expression and higher apoptosis in the liver. These results indicate that the phenotype concerning the reduction in DNA synthesis might be linked to the degree of infection after PH. Thus our results suggest that the role of gp130-dependent signaling is not a direct influence on cell cycle progression after partial hepatectomy but is to activate protective pathways important to enable hepatocyte proliferation. PMID: 12509437 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 41: Endocrinology. 2003 Jan;144(1):110-20. Prolactin regulation of neonatal ovine uterine gland morphogenesis. Carpenter KD, Gray CA, Noel S, Gertler A, Bazer FW, Spencer TE. Center for Animal Biotechnology and Genomics, Department of Animal Science, Texas A&M University, College Station, Texas 77843-2471, USA. Uterine gland development or adenogenesis in the neonatal ovine uterus involves budding, proliferation, and branching morphogenesis of the glandular epithelium (GE) from the luminal epithelium (LE) between birth (postnatal day or PND 0) and PND 56. This critical developmental event is coincident with increases in serum PRL and expression of long and short PRL receptors specifically in the nascent and proliferating GE. In study one, ewes were treated with a placebo pellet as a control (CX) or a bromocryptine mesylate pellet from PNDs 0-56. On PND 56, the endometrium of bromocryptine mesylate ewes contained fewer glands, particularly in the stratum spongiosum that contained numerous coiled and branched glands in CX uteri. In study two, ewes were treated with saline as a CX or recombinant ovine PRL from PNDs 0-56. Treatment with PRL increased gland number and density on PND 14 and PND 56. In study three, expression of signal transducers and activators of transcription (STAT) 1, 3, and 5 proteins was detected in the developing glands from PNDs 7-56. In study four, Western blot analyses indicated that PRL increased levels of phosphorylated STATs 1 and 5, but not STAT 3, and phosphorylated ERK 1 and 2 MAPKs and c-Jun N-terminal kinase/stress-activated protein kinase proteins in explanted PND 28 ovine uteri. Collectively, results indicate that PRL regulates endometrial adenogenesis in the neonatal ovine uterus. PMID: 12488336 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 42: Oncogene. 2002 Nov 21;21(53):8186-91. Stat3-dependent induction of BATF in M1 mouse myeloid leukemia cells. Senga T, Iwamoto T, Humphrey SE, Yokota T, Taparowsky EJ, Hamaguchi M. Department of Molecular Pathogenesis, Nagoya University School of Medicine, 65 Tsurumai Showa Nagoya 466-8550, Japan. Stat3 mediates cellular responses associated with proliferation, survival and differentiation, but the mechanisms underlying the diverse effects of this signaling molecule remain unknown. M1 mouse myeloid leukemia cells arrest growth and differentiate into macrophages following treatment with interleukin 6 (IL-6) or leukemia inhibitory factor (LIF), and recent studies have shown that Stat3 plays a central role in this process. Utilizing representational difference analysis, we demonstrate that expression of the mouse BATF gene is upregulated as an early response to IL-6/LIF stimulation and Stat3 activation in this cell system. Immunoblots using antibodies to BATF detected an increase in BATF protein in response to LIF/IL-6 stimulation. BATF is a member of the AP-1 family of basic leucine zipper transcription factors and functions to inhibit the transcriptional and biological functions of AP-1 activity in mammalian cells. BATF forms complexes with c-Jun in M1 cells and forced expression of BATF in the absence of Stat3 signaling results in a reduced rate of cellular growth. These results indicate that Stat3 mediates cellular growth by modulating AP-1 activity through the induction of BATF. PMID: 12444555 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 43: J Biol Chem. 2003 Jan 17;278(3):1450-6. Epub 2002 Oct 25. BMS-345541 is a highly selective inhibitor of I kappa B kinase that binds at an allosteric site of the enzyme and blocks NF-kappa B-dependent transcription in mice. Burke JR, Pattoli MA, Gregor KR, Brassil PJ, MacMaster JF, McIntyre KW, Yang X, Iotzova VS, Clarke W, Strnad J, Qiu Y, Zusi FC. Department of Immunology, Inflammation and Pulmonary Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543, USA. james.burke@bms.com The signal-inducible phosphorylation of serines 32 and 36 of I kappa B alpha is critical in regulating the subsequent ubiquitination and proteolysis of I kappa B alpha, which then releases NF-kappa B to promote gene transcription. The multisubunit I kappa B kinase responsible for this phosphorylation contains two catalytic subunits, termed I kappa B kinase (IKK)-1 and IKK-2. BMS-345541 (4(2'-aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline) was identified as a selective inhibitor of the catalytic subunits of IKK (IKK-2 IC(50) = 0.3 microm, IKK-1 IC(50) = 4 microm). The compound failed to inhibit a panel of 15 other kinases and selectively inhibited the stimulated phosphorylation of I kappa B alpha in cells (IC(50) = 4 microm) while failing to affect c-Jun and STAT3 phosphorylation, as well as mitogen-activated protein kinase-activated protein kinase 2 activation in cells. Consistent with the role of IKK/NF-kappa B in the regulation of cytokine transcription, BMS-345541 inhibited lipopolysaccharide-stimulated tumor necrosis factor alpha, interleukin-1 beta, interleukin-8, and interleukin-6 in THP-1 cells with IC(50) values in the 1- to 5-microm range. Although a Dixon plot of the inhibition of IKK-2 by BMS-345541 showed a non-linear relationship indicating non-Michaelis-Menten kinetic binding, the use of multiple inhibition analyses indicated that BMS-345541 binds in a mutually exclusive manner with respect to a peptide inhibitor corresponding to amino acids 26-42 of I kappa B alpha with Ser-32 and Ser-36 changed to aspartates and in a non-mutually exclusive manner with respect to ADP. The opposite results were obtained when studying the binding to IKK-1. A binding model is proposed in which BMS-345541 binds to similar allosteric sites on IKK-1 and IKK-2, which then affects the active sites of the subunits differently. BMS-345541 was also shown to have excellent pharmacokinetics in mice, and peroral administration showed the compound to dose-dependently inhibit the production of serum tumor necrosis factor alpha following intraperitoneal challenge with lipopolysaccharide. Thus, the compound is effective against NF-kappa B activation in mice and represents an important tool for investigating the role of IKK in disease models. PMID: 12403772 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 44: J Neurochem. 2002 Nov;83(3):696-703. Mechanism of plasminogen activator inhibitor-1 regulation by oncostatin M and interleukin-1 in human astrocytes. Kasza A, Kiss DL, Gopalan S, Xu W, Rydel RE, Koj A, Kordula T. Department of Biological, Geological and Environmental Sciences, Cleveland State University, Cleveland, Ohio 44115, USA. Glial cells that produce and respond to various cytokines mediate inflammatory processes in the brain. Here, we show that oncostatin M (OSM) and interleukin-1 (IL-1) regulate the expression of plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA) in human astrocytes. Using the PAI-1 reporter constructs we show that the -58 to -51 proximal element mediates activation by both cytokines. This element is already bound by c-fos/c-jun heterodimers in unstimulated astrocytes, and treatment with cytokine strongly stimulates both expression of c-fos and binding of c-fos/c-jun heterodimers. In addition, IL-1 activates an inhibitory mechanism that down-regulates PAI-1 expression after longer exposure to this cytokine. Overexpression of dominant-negative signal transducer and activator of transcription-1 (STAT1), STAT3, STAT5 and inhibitor of nuclear factor-kappaB (IkappaB) suppressed OSM/IL-1-induced expression of the PAI-1 reporter construct. We conclude that OSM and IL-1 regulate the PAI-1 gene expression via up-regulating c-fos levels and subsequent binding of c-fos/c-jun heterodimers to the proximal element of the PAI-1 gene. PMID: 12390531 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 45: J Neurosci Res. 2002 Oct 1;70(1):82-9. Methamphetamine activates DNA binding of specific redox-responsive transcription factors in mouse brain. Lee YW, Son KW, Flora G, Hennig B, Nath A, Toborek M. Division of Neurosurgery, Department of Surgery, University of Kentucky Medical Center, Lexington, Kentucky 40536, USA. Cellular oxidative stress and alterations in redox status can be implicated in methamphetamine (METH)-induced neurotoxicity. To elucidate the molecular signaling pathways of METH-induced neurotoxicity, we investigated the effects of a single intraperitoneal injection of METH (1.0, 10, or 20 mg/kg) on DNA-binding activity of specific redox-sensitive transcription factors in mouse brain. Transcription factors studied included activator protein-1 (AP-1), nuclear factor-kappaB (NF-kappaB), cAMP-responsive element-binding protein (CREB), SP-1, and signal transducers and activators of transcription (STAT1 and STAT3). Significant and dose-dependent inductions of AP-1 and CREB DNA-binding activities were observed in four different regions (striatum, frontal cortex, hippocampus, and cerebellum) isolated from the brains of mice injected with METH. However, injections with METH did not affect DNA binding activities of NF-kappaB, SP-1, STAT1, and STAT3. These results suggest that METH-induced oxidative stress may trigger the molecular signaling pathways via specific and selective activation of AP-1 and CREB. Copyright 2002 Wiley-Liss, Inc. PMID: 12237866 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 46: FASEB J. 2002 Oct;16(12):1642-4. Epub 2002 Aug 7. Codon 12 and codon 13 mutations at the K-ras gene induce different soft tissue sarcoma types in nude mice. Guerrero S, Figueras A, Casanova I, Farre L, Lloveras B, Capella G, Trias M, Mangues R. Laboratori d'Investigacio Gastrointestinal, Institut de Recerca, Hospital de Sant Pau, Barcelona, Spain. K-ras codon 12 mutation is more oncogenic in in vitro and in vivo experimental systems than K-ras codon 13 mutation. Moreover, human colorectal tumors bearing a codon 12 mutation are more aggressive, invasive, and metastatic than the same tumor types carrying a codon 13 mutation. However, despite the association between specific sarcoma types and codon 12 or codon 13 mutations, the relationship between the position of the mutated codon at ras genes and tumor aggressiveness has not been studied in this tumor type. Here, we used a nude mice model to evaluate the tumorogenic capacity of stable transfectants of NIH3T3 fibroblasts, expressing K-ras mutated at codon 12 (K12) or 13 (K13), and morphologically, functionally, and molecularly compared these tumors. We found histopathological differences between them, K12-derived tumors showing fibrosarcoma-like features, whereas K13-derived tumors resembled malignant fibrous histiocytomas. Moreover, K12 tumors showed shorter latency of appearance, lower apoptotic and mitotic rates, and higher expression of markers for sarcoma aggressiveness (Ki67, p53 and c-myc) than K13 tumors. They also showed differences in the expression or activation of Ras, Ras downstream pathways [c-Jun N-terminal kinase (JNK), MAPK and AKT], and apoptotic [AKT, Bcl-2, Focal adhesion kinase (FAK)] and mitotic (cyclin B1) regulators, which could explain their functional differences. Most remarkably, the significantly diminished apoptotic rate observed in K12-derived tumors was associated with enhanced antiapoptotic signaling through the AKT pathway. These morphological, functional, and molecular differences demonstrate that codon 12 and codon 13 mutations in the K-ras oncogene can induce two different soft tissue sarcoma types in our in vivo model. PMID: 12207005 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 47: J Biol Chem. 2002 Nov 22;277(47):45129-40. Epub 2002 Sep 6. IKKalpha, IKKbeta, and NEMO/IKKgamma are each required for the NF-kappa B-mediated inflammatory response program. Li X, Massa PE, Hanidu A, Peet GW, Aro P, Savitt A, Mische S, Li J, Marcu KB. Department of Biology, Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut 06877-0368, USA. The IKKbeta and NEMO/IKKgamma subunits of the NF-kappaB-activating signalsome complex are known to be essential for activating NF-kappaB by inflammatory and other stress-like stimuli. However, the IKKalpha subunit is believed to be dispensable for the latter responses and instead functions as an in vivo mediator of other novel NF-kappaB-dependent and -independent functions. In contrast to this generally accepted view of IKKalpha's physiological functions, we demonstrate in mouse embryonic fibroblasts (MEFs) that, akin to IKKbeta and NEMO/IKKgamma, IKKalpha is also a global regulator of tumor necrosis factor alpha- and IL-1-responsive IKK signalsome-dependent target genes including many known NF-kappaB targets such as serum amyloid A3, C3, interleukin (IL)-6, IL-11, IL-1 receptor antagonist, vascular endothelial growth factor, Ptx3, beta(2)-microglobulin, IL-1alpha, Mcp-1 and -3, RANTES (regulated on activation normal T cell expressed and secreted), Fas antigen, Jun-B, c-Fos, macrophage colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. Only a small number of NF-kappaB-dependent target genes were preferentially dependent on IKKalpha or IKKbeta. Constitutive expression of a trans-dominant IkappaBalpha superrepressor (IkappaBalphaSR) in wild type MEFs confirmed that these signalsome-dependent target genes were also dependent on NF-kappaB. A subset of NF-kappaB target genes were IKK-dependent in the absence of exogenous stimuli, suggesting that the signalsome was also required to regulate basal levels of activated NF-kappaB in established MEFs. Overall, a sizable number of novel NF-kappaB/IKK-dependent genes were identified including Secreted Frizzled, cadherin 13, protocadherin 7, CCAAT/enhancer-binding protein-beta and -delta, osteoprotegerin, FOXC2 and FOXF2, BMP-2, p75 neurotrophin receptor, caspase-11, guanylate-binding proteins 1 and 2, ApoJ/clusterin, interferon (alpha and beta) receptor 2, decorin, osteoglycin, epiregulin, proliferins 2 and 3, stromal cell-derived factor, and cathepsins B, F, and Z. SOCS-3, a negative effector of STAT3 signaling, was found to be an NF-kappaB/IKK-induced gene, suggesting that IKK-mediated NF-kappaB activation can coordinately illicit negative effects on STAT signaling. PMID: 12221085 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 48: Blood. 2002 Aug 15;100(4):1438-48. TEL-JAK2 constitutively activates the extracellular signal-regulated kinase (ERK), stress-activated protein/Jun kinase (SAPK/JNK), and p38 signaling pathways. Ho JM, Nguyen MH, Dierov JK, Badger KM, Beattie BK, Tartaro P, Haq R, Zanke BW, Carroll MP, Barber DL. Division of Cellular and Molecular Biology, Ontario Cancer Institute, University Health Network, University of Toronto, Ontario, Canada. The ets transcription factor, TEL, undergoes chromosomal rearrangements with the tyrosine kinase JAK2. TEL-JAK2 is constitutively active, confers cell line factor independence, and activates signal transducer and activator of transcription-1 (STAT1), STAT3, and STAT5. Data from bone marrow transplantation models suggest that STAT5 activation does not account for the entire disease phenotype induced by TEL-JAK2. This study examined additional signaling pathways that are activated by TEL-JAK2. TEL-JAK2 expression in Ba/F3 cells results in constitutive association and tyrosine phosphorylation of Shc and Ship-1 and, consequently, recruitment of Grb2 to TEL-JAK2. Direct Grb2 recruitment is also possible because a putative Grb2 binding site, Tyr314, is present on TEL-JAK2(5-19) and TEL-JAK2(5-12). Studies with a TEL-JAK2(5-19)Tyr314Phe mutant support a role for Tyr314 in Grb2 recruitment, because Grb2 association with TEL-JAK2(5-19)Tyr314Phe is significantly reduced. Interestingly, TEL-JAK2(5-19)Tyr314Phe shows reduced Ras activation when compared with TEL-JAK2(4-17), TEL-JAK2(5-12), and TEL-JAK2(5-19). Analysis of extracellular signal-regulated kinase-1/2 (ERK1/2), stress-activated protein/Jun kinase (SAPK/JNK), and p38 demonstrates the activation of SAPK/JNK and phosphorylation of p38 by all TEL-JAK2 isoforms. TEL-JAK2(5-12) and TEL-JAK2(5-19) preferentially phosphorylate ERK2, whereas TEL-JAK2(4-17) phosphorylated ERK2 at lower levels. Inhibition studies demonstrated that ERK1/2 activation was necessary for Ba/F3 factor independence mediated by TEL-JAK2(5-19), while inhibition of SAPK/JNK or p38 activity had no effect. Our data reveal the requirement of ERK activation by TEL-JAK2(5-19) in Ba/F3 cells and suggest that TEL-JAK2 leukemogenic potential may be mediated in part through ERK1/2. PMID: 12149229 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 49: Br J Pharmacol. 2002 Aug;136(7):1005-14. TNF-alpha, inefficient by itself, potentiates IL-1beta-induced PGHS-2 expression in human pulmonary microvascular endothelial cells: requirement of NF-kappaB and p38 MAPK pathways. Said FA, Werts C, Elalamy I, Couetil JP, Jacquemin C, Hatmi M. Unite de Pharmacologie Cellulaire, Unite Associee, Institut Pasteur-INSERM U 485, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France. 1: Prostaglandin H synthase-2 (PGHS-2), is an inducible enzyme involved in various inflammatory responses. We established here that interleukin-1beta (IL-1beta) but not tumour necrosis factor-alpha (TNF-alpha) increased its expression in human pulmonary microvascular endothelial cells (HPMEC). However, associated with IL-1beta, TNF-alpha greatly potentiated this enzyme induction. 2: Although unable to induce PGHS-2 expression by itself, TNF-alpha promoted a similar transcription nuclear factor-kappaB (NF-kappaB) activation to IL-1beta. This effect was more pronounced when cells were co-exposed to both cytokines. HPMEC pre-treatment with MG-132, a proteasome inhibitor, prevented NF-kappaB activation as well as more distal signalling response, indicating that NF-kappaB activation is required but not sufficient for PGHS-2 expression. 3: Both IL-1beta and TNF-alpha failed to activate c-Jun NH2-terminal kinase (JNK). In addition, PD98059, a p42/44 mitogen-activated protein kinase (MAPK) phosphorylation inhibitor, did not decrease PGHS-2 expression. However, SB 203580, a p38 MAPK inhibitor, suppressed PGHS-2 induction by IL-1beta alone or combined with TNF-alpha, demonstrating that p38 MAPK but not p42/44 MAPK or JNK cascades are required for PGHS-2 up-regulation. 4: Finally, TNF-alpha, unlike IL-1beta, was unable to promote p38 MAPK phosphorylation, indicating that the failure of TNF-alpha to induce PGHS-2 expression is linked, at least in part, to its inability to activate p38 MAPK signalling pathway. Altogether, these data enhanced our understanding of PGHS-2 regulation in HPMEC and emphasize the heterogeneity of cellular responses to proinflammatory cytokines. PMID: 12145100 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 50: J Biol Chem. 2002 Sep 6;277(36):33509-17. Epub 2002 Jul 1. The serine protease plasmin triggers expression of MCP-1 and CD40 in human primary monocytes via activation of p38 MAPK and janus kinase (JAK)/STAT signaling pathways. Burysek L, Syrovets T, Simmet T. Department of Pharmacology of Natural Products and Clinical Pharmacology, University of Ulm, D-89081 Ulm, Germany. The mechanism of proinflammatory activation of human monocytes by plasmin is unknown. Here we demonstrate that in human primary monocytes, plasmin stimulates mitogen-activated protein kinase (MAPK) signaling via phosphorylation of MAPK kinase 3/6 (MKK3/6) and p38 MAPK that triggers subsequent DNA binding of transcription factor activator protein-1 (AP-1). The AP-1 complex contained phosphorylated c-Jun and ATF2, and its DNA binding activity was blocked by the p38 MAPK inhibitor SB203580. In addition, plasmin elicits Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling, as detected by phosphorylation of JAK1 tyrosine kinase and STAT1 and STAT3 proteins. Plasmin-induced DNA binding of STAT1 and STAT3 was blocked by SB203580 and AG490, inhibitors of p38 MAPK and JAK, respectively, but not by U0126, an inhibitor of MKK1/2. DNA binding of NF-kappaB remained unaffected by any of these inhibitors. The plasmin-induced signaling led to expression of monocyte chemoattractant protein-1 (MCP-1) and CD40, which required activation of both p38 MAPK and JAK/STAT signaling pathways. Additionally, signaling through both p38 MAPK and JAK is involved in the plasmin-mediated monocyte migration, whereas the formylmethionylleucylphenylalanine-induced chemotaxis remained unaffected. Taken together, our data demonstrate a novel function of the serine protease plasmin in a proinflammatory signaling network. PMID: 12093796 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 51: Jpn J Cancer Res. 2002 Mar;93(3):329-39. The roles of JNK1 and Stat3 in the response of head and neck cancer cell lines to combined treatment with all-trans-retinoic acid and 5-fluorouracil. Masuda M, Toh S, Koike K, Kuratomi Y, Suzui M, Deguchi A, Komiyama S, Weinstein IB. Department of Otorhinolaryngology, Head and Neck Surgery, Faculty of Medicine, Kyushu University, Fukuoka 812-0052, Japan. masuda@cuccfa.ccc.columbia.edu We have used a combination of vitamin A (all-trans-retinyl palmitate), 5-fluorouracil (5-FU) and radiation to treat human head and neck squamous cell carcinoma (HNSCC). This chemoradiotherapy is called "FAR therapy." In this study we examined the effects of all-trans-retinoic acid (ATRA), the active metabolite of vitamin A, and ATRA plus 5-FU on two HNSCC cell lines (YCU-N861 and YCU-H891) to gain insight into the molecular mechanisms of FAR therapy. ATRA at 1 mM (the order of concentration found in HNSCC tumors treated with FAR therapy) inhibited cell proliferation and caused G1 cell cycle arrest in both cell lines. This was associated with a decrease in cyclin D1, an increase in p27(Kip1) and a reduction in the hyperphosphorylated form of retinoblastoma protein (pRB). With YCU-N861 cells, ATRA also caused a decrease in Bcl-2 and Bcl-X(L) and an increase in Bax. Both ATRA and 5-FU activated c-Jun N-terminal kinase (JNK) 1 and the combination of both agents resulted in additive or synergistic activation of JNK1, and also enhanced the induction of apoptosis. The YCU-H891 cells, in which the epidermal growth factor receptor (EGFR)-signal transducer and activator of transcription 3 (Stat3) pathway is constitutively activated, were more resistant to treatments with ATRA, 5-FU and the combination of both agents than YCU-N861 cells. A dominant negative Stat3 construct strongly enhanced the cellular sensitivity of this cell line to 5-FU but not to ATRA. In addition there is evidence that activation of Stat3 is associated with cellular resistance to radiation in HNSCC. Therefore, the addition to FAR therapy of agents that inhibit activation of the Stat3 pathway may enhance the clinical response of patients with HNSCC to FAR therapy. PMID: 11927016 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 52: World J Gastroenterol. 2001 Feb;7(1):33-6. Effect of phosphorylation of MAPK and Stat3 and expression of c-fos and c-jun proteins on hepatocarcinogenesis and their clinical significance. Feng DY, Zheng H, Tan Y, Cheng RX. Department of Pathology, Hunan Medical University, Changsha 410078, Hunan Province, China. cslj4841401@public.cs.hn.cn AIM: To study the effect of phosphorylation of MAPK and Stat3 and the expression of c-fos and c-jun proteins on hepatocellular carcinogenesis and their clinical significance. METHODS: SP immunohistochemistry was used to detect the expression of p42/44(MAPK), p-Stat3, c-fos and c-jun proteins in 55 hepatocellular carcinomas (HCC) and their surrounding liver tissues. RESULTS: The positive rates and expression levels of p42/44(MAPK), p-Stat3, c-fos and c-jun proteins in HCCs were significantly higher than those in pericarcinomatous liver tissues (PCLT). A positive correlation was observed between the expression of p42/44(MAPK) and c-fos proteins,and between p-Stat3 and c-jun but there was no significant correlation between p42/44(MAPK) and p-Stat3 in HCCs and their surrounding liver tissues. CONCLUSION: The abnormalities of Ras/Raf/MAPK and JAKs/Stat3 cascade reaction may contribute to malignant transformation of hepatocytes. Hepatocytes which are positive for p42/44(MAPK), c-fos or c-jun proteins may be potential malignant pre-cancerous cells. Activation of MAPK and Stat3 proteins may be an early event in hepatocellular carcinogenesis. PMID: 11819729 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 53: J Biol Chem. 2002 Feb 15;277(7):4932-44. Epub 2001 Dec 3. Regulation of Fas expression by STAT3 and c-Jun is mediated by phosphatidylinositol 3-kinase-AKT signaling. Ivanov VN, Krasilnikov M, Ronai Z. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029, USA. Cooperation between STAT3 and c-Jun results in suppression of Fas Receptor (FasR) transcription, which is often seen in advanced human tumors. To identify requirements for STAT3-Jun cooperation, we elucidated the role of protein kinases that affect both transcription factors. The phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway was found capable of down-regulating both STAT3- and c-Jun-dependent transcription, resulting in derepression of FasR transcription. Conversely, inhibition of PI3K-AKT signaling via the specific pharmacological inhibitor LY294002 up-regulated AP1/Jun- and STAT-dependent transcriptional activities, resulting in suppression of the FasR promoter activities and decreased FasR surface expression. PI3K-AKT's ability to affect FasR transcription was not observed in c-jun null fibroblasts, suggesting that c-Jun is required for PI3K/AKT-mediated regulation of FasR transcription. Interestingly, the dominant negative form of Rac1 (RacN17) was also efficient in relieving FasR expression, suggesting that the increase in FasR expression following AKT stimuli could be mediated via AKT ability to elicit suppression of Rac1, which in turn decreases JNK activities and c-Jun phosphorylation. Overall, our findings demonstrate that through its negative effects on both c-Jun and STAT3, the PI3K-AKT pathway disrupts cooperation between c-Jun and STAT3, which is required for silencing the FasR promoter, resulting in increased expression of surface FasR and concomitant sensitization to FasL-mediated programmed cell death. PMID: 11733515 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 54: Transplantation. 2001 Oct 27;72(8):1416-22. Alterations in transcription factor binding at the IL-2 promoter region in anergized human CD4+ T lymphocytes. Heisel O, Keown P. Department of Medicine, Vancouver General Hospital, 910 West 10th Avenue, Vancouver, B.C., Canada. BACKGROUND: The mechanisms responsible for the induction of clonal anergy are not well understood. We have utilized an in vitro model of human T cell anergy to explore the perturbations in cell signaling at the level of interleukin (IL)-2 gene transcription and to define the contribution of other cytokines to this effect. METHODS: An in vitro model of clonal anergy was established by using CD4+ T lymphocytes from healthy human donors. Cells were anergized by prestimulation with an anti-CD3 monoclonal antibody (mAb) followed by restimulation 72 hr later with anti-CD3 mAb with or without anti-CD28. RESULTS: CD4+ T cells, anergized with anti-CD3 monoclonal antibody (OKT3) prestimulation, displayed a marked reduction in proliferation (P=0.0036) and IL-2 production (P<0.0001). Co-incubation with IL-10 reduced cellular proliferation in OKT3/CD28 pretreated cells by 19% (P=NS) and reduced IL-2 production by 40% (P=0.0024). Anergized T cells demonstrated a reduced binding activity of the AP-1 complex to the IL-2 promoter. Supershift experiments and Western blots confirmed that the binding of c-Fos, JunB, and JunD, but not of FosB, was reduced in anergized cells. At the sis-inducible element (SIE)-binding region of the c-Fos promoter, Stat3 binding was reduced. CONCLUSIONS: T cell anergy, induced by prestimulation with OKT3, is characterized by reduced proliferation and a profound decrease in IL-2 production. Anergy can be prevented by co-incubation with anti-CD28 and partially re-established by IL-10. Anergy is accompanied by a reduction in AP-1 binding to the IL-2 promoter, with selective reduction in binding of c-Fos, JunB, and JunD. Defective binding for Stat3 at the c-Fos promoter suggests an involvement of the Jak-Stat pathway. PMID: 11685114 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 55: J Hypertens. 2001 Nov;19(11):1991-9. Angiotensin subtype-2 receptor (AT2 ) negatively regulates subtype-1 receptor (AT1 ) in signal transduction pathways in cultured porcine adrenal medullary chromaffin cells. Ishii K, Takekoshi K, Shibuya S, Kawakami Y, Isobe K, Nakai T. Department of Clinical Pathology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan. BACKGROUND: Two distinct types of angiotensin II (AngII) receptors, AT1 and AT2, have been cloned. We have shown previously that stimulation of AT2 reduces intracellular cyclic guanosine monophosphate (cGMP) levels in cultured porcine chromaffin cells in which AT2 is the predominantly expressed receptor. However, it has not been determined whether AT1 or AT2 affects signal transduction pathways involving mitogen-activated protein kinases (MAPKs) and signal transducers and activators of transcription (STATs) in chromaffin cells. Also, it is unclear whether cGMP/protein kinase G (PKG) is involved in the regulation of MAPKs and STATs in these cells. DESIGN: Chromaffin cells were derived from porcine adrenal medulla. The effects of AngII alone (representing physiological conditions), AngII plus CV-11974 (an AT1 antagonist, which simulates specific AT2 stimulation), AngII plus PD 123319 (an AT2 antagonist, which simulates specific AT1 stimulation), and 8-Br-cGMP (a membrane-permeable cGMP analogue) alone on MAPKs (ERKs, JNK, p-38 MAPK) and STATs (STATs 1, 3 and 5) activity were measured. METHODS: Phosphorylated MAPKs (extracellular signal-related kinases (ERKs), c-jun N-terminal kinase (JNK) and p38 MAPK) and STATs (STATs 1, 3 and 5) were measured by immunoprecipitation-Western blot analysis (IP-Western blot). RESULTS: AT1 stimulation markedly increased expression of ERKs, JNK, p38 MAPK via Ca2+-dependent protein kinase C (PKC) isoforms (cPKC), as well as STATs 1, 3 and 5 in cultured porcine chromaffin cells. In contrast, AT2 stimulation markedly decreased the expression of these signaling molecules. Also, 8-Br-cGMP alone induced increases in ERKs, JNK, p38 MAPK, and STATs 1, 3 and 5. Because AT2 inhibits cGMP production, we speculate that AT2 may act to suppress cGMP production, which in turn reduces the activity of both MAPKs and STATs in chromaffin cells. CONCLUSION: AT2 negatively regulates AT1 in signal transduction pathways in chromaffin cells. PMID: 11677364 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 56: Am J Physiol Lung Cell Mol Physiol. 2001 Oct;281(4):L816-23. Signal transduction pathways of IL-1beta-mediated iNOS in pulmonary vascular smooth muscle cells. Finder JD, Petrus JL, Hamilton A, Villavicencio RT, Pitt BR, Sebti SM. Department of Pediatrics, University of Pittsburgh School of Medicine and Graduate School of Public Health, Pittsburgh, Pennsylvania 15261, USA. finder@pitt.edu Interleukin (IL)-1beta is an important early mediator of inflammation in pulmonary artery smooth muscle cells. We previously reported that a geranylgeranyltransferase inhibitor elevated basal levels of inducible nitric oxide synthase (iNOS) and enhanced IL-1beta-mediated induction, suggesting that Rac or Rho small G proteins are candidates for antagonism of such induction. In this study, overexpression of constitutively active Rac1 or its dominant negative mutant did not affect IL-1beta induction of iNOS. Alternatively, treatment with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates Rho, was associated with superinduction of iNOS, suggesting an inhibitory role for Rho. IL-1beta activated the three mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2, c-Jun NH2-terminal kinase/stress-activated protein kinase, and p38) and the Janus kinase (JAK)-signal transducer and activator of transcription pathways. The former two pathways were not associated with IL-1beta-mediated iNOS induction, whereas the latter two appeared to have inhibitory roles in iNOS expression. These data suggest that a broad intracellular signaling response to IL-1beta in rat pulmonary artery smooth muscle cells results in elevated levels of iNOS that is opposed by the geranylgeranylated small G protein Rho as well as the p38 and JAK2 pathways. PMID: 11557585 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 57: Lab Invest. 2001 Sep;81(9):1299-307. Expression of presumed specific early and late factors associated with liver regeneration in different rat surgical models. Laurent S, Otsuka M, De Saeger C, Maiter D, Lambotte L, Horsmans Y. Gastroenterology Laboratories, Universite Catholique de Louvain, Brussels, Belgium. Experiments performed on the portal branch ligation (PBL) model indicate that early changes observed after surgery are not related to the regenerative process because they also occur in atrophying lobes. To further confirm the lack of specificity of the early events and to exclude the influence of circulatory factors released by proliferating lobes on their occurrence, we investigated this response after sham operation (SO) and portacaval shunt (PCS), a model characterized by liver atrophy. We also attempted to determine expression of later events associated specifically with regeneration, ie, expression of p53 or c-Ha-ras, or inhibition of proliferation, ie, interleukin-1beta (IL-1beta) and transforming growth factor-beta1 (TGF-beta1) after partial (PH) and temporary partial (TPH) hepatectomy, SO and PCS. Nuclear factor-kappaB (NF-kappaB) and signal transducer and activator of transcription 3 (STAT3) DNA binding were assessed by electrophoretic mobility shift assay (EMSA), interleukin-6 (IL-6) mRNA by reverse transcription-polymerase chain reaction (RT-PCR), c-myc and c-jun mRNAs by Northern blot analysis at 0.5 and 2 hours, p53 and c-Ha-ras mRNAs by Northern blot analysis at 8 and 24 hours, and IL-1beta and TGF-beta1 by RT-PCR at 24 hours. The early response including an increase of NF-kappaB, STAT3, IL-6, and immediate-early genes expression was present after PH, PCS, and SO. In SO, slight differences were observed in comparison with PH: no NF-kappaB p65/p50 DNA binding was observed, only three of six SO rats were positive for IL-6, and immediate-early genes induction showed differences in the intensity of the response. At later times, p53 mRNA increased at 8 hours after PH and TPH, c-Ha-ras mRNA at 24 hours after PH, and IL-1beta mRNA at 24 hours after PCS. Early events are not specifically associated with the reduction of liver mass or with the regenerative process, are not predictive of future cell fate, and are most likely related to surgical stress. p53 and c-Ha-ras induction is closely associated with cell cycle progression whereas IL-1beta, but not TGF-beta1, appears to be one of the negative growth regulators that might play an important role in atrophy. PMID: 11555677 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 58: J Biol Chem. 2001 Nov 9;276(45):42534-42. Epub 2001 Sep 11. MSK1 and JNKs mediate phosphorylation of STAT3 in UVA-irradiated mouse epidermal JB6 cells. Zhang Y, Liu G, Dong Z. Hormel Institute, University of Minnesota, Austin, Minnesota 55912, USA. Phosphorylation of Tyr(705) and Ser(727) of signal transducer and activator of transcription 3 (STAT3) are known to be required for maximal activation by diverse stimuli. Tyr(705) phosphorylation is generally accepted to be mediated by the Janus kinase family. But the mechanism for STAT3 (Ser(727)) phosphorylation is not well understood. Here, we provide evidence that UVA-induced phosphorylation of STAT3 at Ser(727) is inhibited by pretreatment of JB6 cells with PD98059 or SB202190. Phosphorylation of STAT3 (Ser(727)) is also markedly prevented by a dominant negative mutant of ERK2, c-Jun N-terminal kinase 1 (JNK1), or p38 kinase and in knockout Jnk1(-/-) or Jnk2(-/-) cells. Furthermore, STAT3 (Ser(727)) phosphorylation is suppressed by C- or N-terminal "kinase-dead" mutants of mitogen- and stress-activated protein kinase 1 (MSK1), a downstream kinase of ERKs and p38 kinase, and H89, a potential MSK1 inhibitor. In vitro experiments showed that active MSK1 and JNKs, but not ERKs or p38 kinase, phosphorylate STAT3 (Ser(727)). Additionally, the role of MAPKs in mediating UVA-stimulated DNA binding activity of STAT3 was investigated. Overall, these results suggest that UVA-induced Ser(727) phosphorylation of STAT3 may occur through MSK1 and JNKs. PMID: 11553624 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 59: Mol Cell. 2001 Mar;7(3):517-28. Cooperation between STAT3 and c-jun suppresses Fas transcription. Ivanov VN, Bhoumik A, Krasilnikov M, Raz R, Owen-Schaub LB, Levy D, Horvath CM, Ronai Z. The Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029, USA. Decreased Fas expression during tumor progression often results in a loss of Fas-ligand (FasL)-mediated apoptosis. Human and mouse melanoma exhibit an inverse correlation between the degree of Fas cell surface expression, tumorigenicity, and metastatic capacity. The expression of dominant negative Stat3 or c-Jun in melanoma cells efficiently increased Fas expression and sensitized cells to FasL-induced apoptosis. Stat3+/- as well as c-Jun-/- cells exhibited increased Fas cell surface expression and higher sensitivity to FasL-mediated apoptosis. Suppression of Fas expression by Stat3 and c-Jun is uncoupled from Stat3-mediated transcriptional activation. Our findings indicate that Stat3 oncogenic activities could also be mediated through its cooperation with c-Jun, resulting in downregulation of Fas surface expression, which is implicated in the tumor's ability to resist therapy and metastasize. PMID: 11463377 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 60: Eur J Immunol. 2001 Jul;31(7):1962-71. Macrophage stimulation with Murabutide, an HIV-suppressive muramyl peptide derivative, selectively activates extracellular signal-regulated kinases 1 and 2, C/EBPbeta and STAT1: role of CD14 and Toll-like receptors 2 and 4. Vidal VF, Casteran N, Riendeau CJ, Kornfeld H, Darcissac EC, Capron A, Bahr GM. Laboratory of Molecular Immunology of infection and Inflammation, Institut Pasteur de Lille, Lille, France. The smallest unit of bacterial peptidoglycans known to be endowed with biological activities is muramyl dipeptide (MDP). A clinically acceptable synthetic derivative of MDP, namely murabutide (MB), has been found to present interesting pharmacological properties and to suppress HIV-1 replication in monocyte-derived macrophages (MDM). We have addressed the signaling events activated in MDM following stimulation with either MB or the potent immunostimulant LPS. We also examined whether signaling by muramyl peptides involves the use of cell surface receptors, including CD14 and Toll-like receptor 2 (TLR2) or TLR4 that are known to be signal-transducing receptors for other bacterial cell wall components. We demonstrate that, unlike LPS, the safe immunomodulator MB selectively activates extracellular signal-regulated kinases (Erk) 1/2, in the absence of detectable Jun N-terminal kinase (JNK) or p38 mitogen-activated kinase activation. Furthermore, STAT1 activation but weak or no activation of STAT3 or STAT5 respectively, could be detected in MB-stimulated MDM. Using MonoMac6 cells, we observed high C/EBPbeta and AP-1 but weaker and transient NF-kappaB activation by MB.Moreover, the truncated form of C/EBPbeta, known to repress HIV-1 transcription, was detected in extracts from MB-treated THP-1 cells. Surprisingly, neither MB nor MDP were able to transduce signals via CD14 and TLR2 or 4. These findings present major differences in the early cell activation process between LPS and muramyl peptides, and strongly argue for the implication of co-receptors other than TLR2 and TLR4 in mediating the signaling events induced by defined subunits of bacterial peptidoglycans. PMID: 11449348 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 61: J Biol Chem. 2001 Sep 7;276(36):33576-81. Epub 2001 Jul 3. Functional importance of Stat3 tetramerization in activation of the alpha 2-macroglobulin gene. Zhang X, Darnell JE Jr. Laboratory of Molecular Cell Biology, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA. A tetrameric Stat3 complex was found to be essential in transfection experiments for maximal interleukin-6-inducible activation of alpha2-macroglobulin gene promoter. Stable tetramer formation of purified phosphorylated Stat3 was dependent on protein.protein interaction involving the N-terminal domain of Stat3. The functional importance of tetramer formation was shown by the decreased levels of transcriptional activation associated with hypomorphic mutations in N-terminal residues. PMID: 11438543 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 62: J Biol Chem. 2001 Aug 3;276(31):29490-8. Epub 2001 May 29. A role for the extracellular signal-regulated kinase and p38 mitogen-activated protein kinases in interleukin-1 beta-stimulated delayed signal tranducer and activator of transcription 3 activation, atrial natriuretic factor expression, and cardiac myocyte morphology. Ng DC, Long CS, Bogoyevitch MA. Department of Biochemistry, University of Western Australia, Crawley 6009, Australia. We have demonstrated that two hypertrophic agents, interleukin-1 beta (IL-1 beta) and leukemic inhibitory factor (LIF), altered cardiac myocyte morphology with striking similarity and prompted us to investigate the common actions of these cytokines. We compared the phosphorylation/activation of signal tranducer and activator of transcription 3 (STAT3), extracellular signal-regulated kinase (ERK), p38(MAPK), and c-Jun N-terminal kinase mitogen-activated protein kinases (MAPKs). The phosphorylation of STAT3 by IL-1 beta was delayed (>60 min), whereas the response to LIF was rapid (<10 min) and transient. We confirmed that IL-1 beta potently stimulated all three MAPK subfamilies. In contrast, LIF promoted strong activation of ERKs, marginal activation of p38(MAPK), and no c-Jun N-terminal kinase activation. To test the roles of ERKs and p38(MAPK), myocytes were pretreated with PD98059 and SB203580. Either inhibitor alone prevented STAT3 phosphorylation, implicating ERKs and p38(MAPK) in the delayed STAT3 response to IL-1 beta. The interplay of MAPKs and STAT3 phosphorylation in regulating IL-1 beta-stimulated hypertrophy was investigated by evaluating the effect of MAPK inhibitors on atrial natriuretic factor (ANF) expression and myocyte morphology. The specific inhibition of either ERK or p38(MAPK) attenuated the IL-1 beta- or LIF-stimulated ANF expression by up to 70%. Inhibition was not further increased in the presence of both inhibitors. Furthermore, although individual inhibition of ERK or p38(MAPK) did not affect morphology, co-treatment with both inhibitors abrogated the hypertrophic morphology stimulated by IL-1 beta but not by LIF. Taken together, our data indicate that the activation of ERK and p38(MAPK) is essential in regulating a delayed STAT3 phosphorylation as well as changes in ANF expression and morphology that follow IL-1 beta treatment. Thus, the role of MAPKs in the hypertrophic response can be dictated at least partly by the nature of the hypertrophic agent employed. PMID: 11382751 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 63: Cytokine. 2001 Apr 21;14(2):78-87. c-Jun and c-Fos cooperate with STAT3 in IL-6-induced transactivation of the IL-6 respone element (IRE). Schuringa JJ, Timmer H, Luttickhuizen D, Vellenga E, Kruijer W. Department of Genetics, Biological Center, Kerklaan 30, Haren, 9751 NN, The Netherlands. Transcriptional activation of eukaryotic genes often requires the cooperative action of many proteins. The interleukin 6 (IL-6) response element (IRE) is activated by signal transducer and activator of transcription 3 (STAT3), and stimulation with IL-6 leads to STAT3 tyr705 phosphorylation, dimerization, translocation to the nucleus and transactivation of target gene promoters containing IREs. Here, we report that IL-6 and 12-O-tetradecanoylphorbol-13-acetate (TPA) synergistically transactivate the IRE in HepG2 cells, which is coupled to a strong upregulation of c-Jun and c-Fos expression by TPA via the mitogen-activated protein kinase (MAPK) pathway. Overexpression of c-Jun and c-Fos strongly enhanced STAT3-driven IRE transactivation as well as transactivation of the human intercellular adhesion molecule (ICAM)-1 promoter. In contrast, c-Jun mutants lacking the transactivation domain, the DNA-binding domain, or mutants in which the serine residues 63 and 73 were replaced by alanine, did not cooperate with STAT3. In immunoprecipitation experiments, a direct association of STAT3 with c-Jun and c-Fos was observed in response to IL-6. Furthermore, c-Jun/STAT3 and c-Fos/STAT3 complexes were detected on IRE probes in electrophoretic mobility shift assay (EMSA) experiments, but did not bind nor transactivate the TPA response element (TRE). These results demonstrate that activator protein-1 (AP-1) transcription factors can cooperate with STAT3 in IRE transactivation in the absence of direct AP-1 DNA binding. Copyright 2001 Academic Press. PMID: 11356008 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 64: Invest Ophthalmol Vis Sci. 2001 May;42(6):1363-9. FGFR1, signaling, and AP-1 expression after retinal detachment: reactive Muller and RPE cells. Geller SF, Lewis GP, Fisher SK. Neuroscience Research Institute, University of California, Santa Barbara, CA, USA. PURPOSE. To identify changes in cellular signaling pathways and AP-1 expression in retina and retinal pigmented epithelium (RPE) after experimental retinal detachment (RD). METHODS. Cat and rabbit neural retinas were separated from the RPE in vivo for 5 minutes to 28 days. Tissues were removed and processed for Western blotting, immunohistochemistry, in situ hybridization, and immunoprecipitation experiments. RESULTS. An ordered sequence of events occurs after RD: (1) fibroblast growth factor (FGF) receptor 1 (FGFR1, flg) is phosphorylated in the retina within 15 minutes and dephosphorylated 2 hours after RD; (2) The extracellular signal-regulated kinase (ERK) is phosphorylated in both Muller and RPE cells within 15 minutes and remains so for several days; (3) De novo expression of c-fos mRNA coincides with increased c-Fos and c-Jun immunoreactivity in both Muller and RPE cells; (4) CREB is phosphorylated in a subpopulation of photoreceptors; and (5) STAT3 and NF-kappaB are activated in inner nuclear layer cells by 1 day of RD. CONCLUSIONS. These data suggest that nonneuronal cells (RPE and Muller cells) respond to RD very rapidly by stimulating ERK signaling and AP-1 transcription factor expression. Furthermore, these data suggest that basic fibroblast growth factor (FGF-2, bFGF) is involved in initiating the retina's earliest responses to RD. The events described here precede changes in gene expression and morphology that can have serious effects on visual outcome in humans treated for retinal detachment or other retinal injuries. PMID: 11328752 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 65: J Biol Chem. 2001 Jul 13;276(28):26421-9. Epub 2001 Apr 23. Synergistic activity of STAT3 and c-Jun at a specific array of DNA elements in the alpha 2-macroglobulin promoter. Yoo JY, Wang W, Desiderio S, Nathans D. Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. The transcriptional activity of natural promoters is sensitive to the precise spatial arrangement of DNA elements and their incorporation into higher order DNA-protein complexes. STAT3 and c-Jun form a specific ternary complex in vitro with a synthetic DNA element containing AP1 and SIE sites. These associations are critical for synergistic activation of transcription from a synthetic promoter by STAT3 and c-Jun. Expression of the acute phase protein alpha(2)-macroglobulin is induced in vivo by interleukin-6 (IL-6)-related cytokines; we demonstrate that coordinate interactions among STAT3, c-Jun, and a specific array of DNA elements contribute to activation of the alpha(2)-macroglobulin promoter in response to IL-6 family members. At least five promoter elements are involved in activation: two AP1 sites at -113 to -107 and -152 to -140, an acute phase response element (APRE (SIE)) at -171 to -163, and two AT-rich regions at -143 to -138 and -128 to -123. Synergism between STAT3 or STAT3-C and c-Jun is impaired by mutation of the APRE (SIE) or either AP1 site, as well as by mutations that alter the AT-rich regions or their phasing. Mutations of STAT3 previously shown to disrupt physical and functional interactions with c-Jun do not impair synergy between STAT3-C and c-Jun at the alpha(2)-macroglobulin promoter in HepG2 cells, suggesting that STAT3-C and STAT3 differ with respect to their precise contacts with c-Jun. PMID: 11319221 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 66: FASEB J. 2001 Apr;15(6):1006-13. Cyclin D1 is an early target in hepatocyte proliferation induced by thyroid hormone (T3). Pibiri M, Ledda-Columbano GM, Cossu C, Simbula G, Menegazzi M, Shinozuka H, Columbano A. Department of Toxicology, Oncology and Molecular Pathology Unit, University of Cagliari, Italy. The thyroid hormone (T3) affects cell growth, differentiation, and regulates metabolic functions via its interaction with the thyroid hormone nuclear receptors (TRs). The mechanism by which TRs mediate cell growth is unknown. To investigate the mechanisms responsible for the mitogenic effect of T3, we have determined changes in activation of transcription factors, mRNA levels of immediate early genes, and levels of proteins involved in the progression from G1 to S phase of the cell cycle. We show that hepatocyte proliferation induced by a single administration of T3 to Wistar rats occurred in the absence of activation of AP-1, NF-kappa B, and STAT3 or changes in the mRNA levels of the immediate early genes c-fos, c-jun, and c-myc. These genes are considered to be essential for liver regeneration after partial hepatectomy (PH). On the other hand, T3 treatment caused an increase in cyclin D1 mRNA and protein levels that occurred much more rapidly compared to liver regeneration after 2/3 PH. The early increase in cyclin D1 expression was associated with accelerated onset of DNA synthesis, as demonstrated by a 20-fold increase of bromodeoxyuridine-positive hepatocytes at 12 h after T3 treatment and by a 20-fold increase in mitotic activity at 18 h. An early increase of cyclin D1 expression was also observed after treatment with nafenopin, a ligand of a nuclear receptor (peroxisome proliferator-activated receptor alpha) of the same superfamily of steroid/thyroid receptors. T3 treatment also resulted in increased expression of cyclin E, E2F, and p107 and enhanced phosphorylation of pRb, the ultimate substrate in the pathway leading to transition from G1 to S phase. The results demonstrate that cyclin D1 induction is one of the earlier events in hepatocyte proliferation induced by T3 and suggest that this cyclin might be a common target responsible for the mitogenic activity of ligands of nuclear receptors. PMID: 11292661 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 67: J Biol Chem. 2001 May 25;276(21):17800-7. Epub 2001 Feb 15. Bcl-XL expression correlates with primary macrophage differentiation, activation of functional competence, and survival and results from synergistic transcriptional activation by Ets2 and PU.1. Sevilla L, Zaldumbide A, Carlotti F, Dayem MA, Pognonec P, Boulukos KE. Institute of Signalisation, Developmental Biology and Cancer, INSERM 470, Centre de Biochimie, Universite de Nice, Faculte des Sciences, 06108 Nice, France. Depriving primary bone marrow-derived macrophages of colony-stimulating factor-1 (CSF-1) induces programmed cell death by apoptosis. We show that cell death is accompanied by decreases in the expression of anti-apoptotic Bcl-x(L) protein and the Ets2 and PU.1 proteins of the Ets transcription factor family. Macrophages require both priming and triggering signals independent of CSF-1 to kill neoplastic cells or microorganisms, and this activation of macrophage competence is accompanied by increased expression of bcl-x(L), ets2, and PU.1. Furthermore, we show that only Ets2 and PU.1, but not Ets1, function in a synergistic manner to transactivate the bcl-x promoter. The synergy observed between PU.1 and Ets2 is dependent on the transactivation domains of both proteins. Although other transcription factors like Fos, c-Jun, Myc, STAT3, and STAT5a are implicated in the activation of macrophage competence or in CSF-1 signaling, no synergy was observed between Ets2 and these transcription factors on the bcl-x promoter. We demonstrate that the exogenous expression of both Ets2 and PU.1 in macrophages increases the number of viable cells upon CSF-1 depletion and that Ets2 and PU.1 can functionally replace Bcl-x(L) in inhibiting Bax-induced apoptosis. Together, these results demonstrate that PU.1 and Ets2 dramatically increase bcl-x activation, which is necessary for the cytocidal function and survival of macrophages. PMID: 11278399 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 68: J Biol Chem. 2001 Jun 15;276(24):21004-11. Epub 2001 Mar 16. Regulation of Stat3 activation by MEK kinase 1. Lim CP, Cao X. Institute of Molecular and Cell Biology, Singapore 117609, Singapore. Stat3 is a latent transcription factor activated by various cytokines and growth factors. Phosphorylation on Tyr-705 is a prerequisite for dimer formation, nuclear translocation, binding to its cognate DNA sequences, and regulation of the target gene transcription. Ser-727 phosphorylation of Stat3 plays an additional role in the regulation of transcription. MEK kinase 1 (MEKK1) is a mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK) that activates the c-Jun NH(2)-terminal kinase signaling pathway. Here we report that MEKK1 is involved in the regulation of Stat3 activation by growth factors. Kinase-inactive MEKK1 inhibits Stat3 phosphorylation on tyrosine and serine, and its transcriptional activity stimulated by epidermal growth factor and platelet-derived growth factor in different cell types. In contrast, active MEKK1 induces Stat3 tyrosine and serine phosphorylation leading to a functionally active Stat3 capable of binding DNA and enhancing transcription. Ser-727 is phosphorylated by MEKK1 in vitro, whereas Tyr-705 phosphorylation induced by MEKK1 involves Src and Janus kinases in vivo. These data demonstrate for the first time a novel role of MEKK1 to modulate tyrosine kinases that results in the activation of specific members of STAT family. PMID: 11278353 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 69: J Surg Res. 2001 Apr;96(2):289-95. Activation of interleukin-6/STAT3 and liver regeneration following transplantation. Debonera F, Aldeguer X, Shen X, Gelman AE, Gao F, Que X, Greenbaum LE, Furth EE, Taub R, Olthoff KM. Department of Surgery, University of Pennsylvania, Philadelphia 19104, USA. BACKGROUND: Every liver that is procured, stored, and transplanted experiences injury from cold ischemia and reperfusion. Most recover quickly, but some grafts sustain enough injury to result in prolonged organ dysfunction or require retransplantation. The molecular mechanisms involved in early graft function and recovery following cold ischemia and reperfusion (I/R) after liver transplantation have not been well defined. Interleukin (IL)-6 is a critical factor in the mitogenic response within the liver, and is important for cell cycle progression and protection from injury. Activation of the latent transcription factor, STAT3, is dependent on IL-6 release. The role of the IL-6/STAT3 pathway and hepatocellular regeneration in graft recovery and cell cycle progression following cold ischemia and reperfusion was studied in a rat liver transplant orthotopic (OLT) model. Methods. Rat OLT was performed in a syngeneic model. The presence, time course, and magnitude of expression of IL-6, STAT3 activation, and upregulation of target immediate early genes were determined in liver grafts with minimal (<1 h) and prolonged (12 h) cold preservation times followed by transplantation. Progression of the cell cycle and replication was confirmed by BrdU uptake. RESULTS: Prolonged cold ischemia resulted in increased IL-6 expression and STAT3 activation. This correlated with upregulation of junB, c-fos, c-myc, and c-jun, immediate early genes associated with hepatic regeneration. Extensive DNA replication was present in livers with 12-h ischemia, demonstrating successful completion of the cell cycle. CONCLUSIONS: The participation of the IL-6/STAT3 pathway leading to cell cycle progression and regeneration is an important component in the recovery of organs immediately following cold preservation and transplantation. Copyright 2001 Academic Press. PMID: 11266286 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 70: EMBO J. 2001 Jan 15;20(1-2):91-100. Specificity of signaling by STAT1 depends on SH2 and C-terminal domains that regulate Ser727 phosphorylation, differentially affecting specific target gene expression. Kovarik P, Mangold M, Ramsauer K, Heidari H, Steinborn R, Zotter A, Levy DE, Muller M, Decker T. Vienna Biocenter, Institute of Microbiology and Genetics, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria. pavel@gem.univie.ac.at Complete activation of signal transducer and activator of transcription 1 (STAT1) requires phosphorylation at both Y701 and a conserved PMS(727)P sequence. S727 phosphorylation of STAT1 in interferon-gamma (IFN-gamma)-treated mouse fibroblasts occurred without a need for p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases 1 and 2 or c-Jun kinases, and required both an intact SH2 domain and phosphorylation of Y701. In contrast, UV irradiation-induced STAT1 phosphorylation on S727 required p38MAPK, but no SH2 domain- phosphotyrosine interactions. Mutation of S727 differentially affected IFN-gamma target genes, at the level of both basal and induced expression. Particularly strong effects were noted for the GBP1 and TAP1 genes. The PMS(727)P motif of STAT3 was phosphorylated by stimuli and signaling pathways different from those for STAT1 S727. Transfer of the STAT3 C-terminus to STAT1 changed the stimulus and pathway specificity of STAT1 S727 phosphorylation to that of STAT3. Our data suggest that STAT C-termini contribute to the specificity of cellular responses by linking individual STATs to different serine kinase pathways and through an intrinsically different requirement for serine phosphorylation at different target gene promoters. PMID: 11226159 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 71: Mol Cell Biol. 2001 Jan;21(2):414-24. Interleukin-6-induced STAT3 and AP-1 amplify hepatocyte nuclear factor 1-mediated transactivation of hepatic genes, an adaptive response to liver injury. Leu JI, Crissey MA, Leu JP, Ciliberto G, Taub R. Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA. Following hepatic injury or stress, gluconeogenic and acute-phase response genes are rapidly upregulated to restore metabolic homeostasis and limit tissue damage. Regulation of the liver-restricted insulin-like growth factor binding protein 1 (IGFBP-1) gene is dramatically altered by changes in the metabolic state and hepatectomy, and thus it provided an appropriate reporter to assess the transcriptional milieu in the liver during repair and regeneration. The cytokine interleukin-6 (IL-6) is required for liver regeneration and repair, and it transcriptionally upregulates a vast array of genes during liver growth by unknown mechanisms. Evidence for a biologic role of IL-6 in IGFBP-1 upregulation was demonstrated by increased expression of hepatic IGFBP-1 in IL-6 transgenic and following injection of IL-6 into nonfasting animals and its reduced expression in IL-6(-/-) livers posthepatectomy. In both hepatic and nonhepatic cells, IL-6 -mediated IGFBP-1 promoter activation was via an intact hepatocyte nuclear factor 1 (HNF-1) site and was dependent on the presence of endogenous liver factor HNF-1 and induced factors STAT3 and AP-1 (c-Fos/c-Jun). IL-6 acted through the STAT3 pathway, as dominant negative STAT3 completely blocked IL-6-mediated stimulation of the IGFBP-1 promoter via the HNF-1 site. HNF-1/c-Fos and HNF-1/STAT3 protein complexes were detected in mouse livers and in hepatic and nonhepatic cell lines overexpressing STAT3/c-Fos/HNF-1. Similar regulation was demonstrated using glucose-6-phosphatase and alpha-fibrinogen promoters, indicating that HNF-1/IL-6/STAT3/AP-1-mediated transactivation of hepatic gene expression is a general phenomenon after liver injury. These results demonstrate that the two classes of transcription factors, growth induced (STAT3 and AP-1) and tissue specific (HNF-1), can interact as an adaptive response to liver injury to amplify expression of hepatic genes important for the homeostatic response during organ repair. PMID: 11134330 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 72: J Clin Invest. 2000 Dec;106(11):1417-25. Direct regulation of pituitary proopiomelanocortin by STAT3 provides a novel mechanism for immuno-neuroendocrine interfacing. Bousquet C, Zatelli MC, Melmed S. Department of Medicine, Cedars-Sinai Research Institute, University of California-Los Angeles School of Medicine, Los Angeles, California, USA. Neuroendocrine ACTH secretion responds to peripheral inflammatory and stress signals. We previously demonstrated that the proinflammatory cytokine, leukemia inhibitory factor (LIF), affects the hypothalamo-pituitary-adrenal axis (HPA) by stimulating in vitro and in vivo pituitary proopiomelanocortin (POMC) gene expression and ACTH secretion and by potentiating the action of hypothalamic corticotropin releasing hormone (CRH). Whereas pathways shown thus far to regulate POMC expression exclusively involve cAMP or calcium, we here describe a direct and indirect STAT3-dependent regulation of POMC transcription by LIF. Using progressive 5'-deletions of POMC promoter, we identified a LIF-responsive -407/-301 region that contains two juxtaposed sequences within -399/-379 related to a STAT3 DNA-binding motif. Each sequence within -399/-379 separately corresponds to a low-affinity and direct binding site for STAT3, but, in combination, these sequences bind STAT3 cooperatively and with high affinity. Moreover, LIF-activated STAT3 indirectly mediates LIF corticotroph action by inducing and potentiating CRH-induced c-fos and JunB expression and binding to the POMC AP-1 element. We therefore conclude that both a direct and indirect route mediate LIF-induced STAT3 activation of POMC transcription. Demonstration of STAT3-dependent regulation of the POMC gene represents a powerful mechanism for immuno-neuroendocrine interfacing and implies a direct stimulation of ACTH secretion by inflammatory and stress-derived STAT3-inducing cytokines. PMID: 11104795 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 73: Brain Res. 2000 Dec 8;885(2):303-21. Gene expression in the brain across the sleep-waking cycle. Cirelli C, Tononi G. The Neurosciences Institute, 10640 John J. Hopkins Drive, San Diego, CA 92121, USA. Sleep and waking differ significantly in terms of behavior, metabolism, and neuronal activity. Recent evidence indicates that sleep and waking also differ with respect to the expression of certain genes. To systematically investigate such changes, we used mRNA differential display and cDNA microarrays to screen approximately 10000 transcripts expressed in the cerebral cortex of rats after 8 h of sleep, spontaneous waking, or sleep deprivation. We found that 44 genes had higher mRNA levels after waking and/or sleep deprivation relative to sleep, while 10 were upregulated after sleep. Known genes that were upregulated in waking and sleep deprivation can be grouped into the following categories: immediate early genes/transcription factors (Arc, CHOP, IER5, NGFI-A, NGFI-B, N-Ras, Stat3), genes related to energy metabolism (glucose type I transporter Glut1, Vgf), growth factors/adhesion molecules (BDNF, TrkB, F3 adhesion molecule), chaperones/heat shock proteins (BiP, ERP72, GRP75, HSP60, HSP70), vesicle- and synapse-related genes (chromogranin C, synaptotagmin IV), neurotransmitter/hormone receptors (adrenergic receptor alpha(1A) and beta(2), GABA(A) receptor beta(3), glutamate NMDA receptor 2A, glutamate AMPA receptor GluR2 and GluR3, nicotinic acetylcholine receptor beta(2), thyroid hormone receptor TRbeta), neurotransmitter transporters (glutamate/aspartate transporter GLAST, Na(+)/Cl(-) transporter NTT4/Rxt1), enzymes (aryl sulfotransferase, c-jun N-terminal kinase 1, serum/glucocorticoid-induced serine/threonine kinase), and a miscellaneous group (calmodulin, cyclin D2, LMO-4, metallothionein 3). Several other genes that were upregulated in waking and all the genes upregulated in sleep, with the exception of the one coding for membrane protein E25, did not match any known sequence. Thus, significant changes in gene expression occur across behavioral states, which are likely to affect basic cellular functions such as RNA and protein synthesis, neural plasticity, neurotransmission, and metabolism. PMID: 11102586 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 74: Ann N Y Acad Sci. 2000 Sep;914:238-62. Protein phosphorylation cascades associated with methamphetamine-induced glial activation. Hebert MA, O'Callaghan JP. Department of Health & Human Services, Public Health Service, Centers for Disease Control and Prevention, Morgantown, West Virginia 26505-2888, USA. Reactive gliosis is the most prominent response to diverse forms of central nervous system (CNS) injury. The signaling events that mediate this characteristic response to neural injury are under intense investigation. Several studies have demonstrated the activation of phosphoproteins within the mitogen-activated protein kinase (MAPK) and Janus kinase (JAK) pathways following neural insult. These signaling pathways may be involved or responsible for the glial response following injury, by virtue of their ability to phosphorylate and dynamically regulate the activity of various transcription factors. This study sought to delineate, in vivo, the relative contribution of MAPK- and JAK-signaling components to reactive gliosis as measured by induction of glial-fibrillary acidic protein (GFAP), following chemical-induced neural damage. At time points (6, 24, and 48 h) following methamphetamine (METH, 10 mg/kg x 4, s.c.) administration, female C57BL/6J mice were sacrificed by focused microwave irradiation, a technique that preserves steady-state phosphorylation. Striatal (target) and nontarget (hippocampus) homogenates were assayed for METH-induced changes in markers of dopamine (DA) neuron integrity as well as differences in the levels of activated phosphoproteins. GFAP upregulation occurred as early as 6 h, reaching a threefold induction 48 h following METH exposure. Neurotoxicant-induced reductions in striatal levels of DA and tyrosine hydroxylase (TH) paralleled the temporal profile of GFAP induction. Blots of striatal homogenates, probed with phosphorylation-state specific antibodies, demonstrated significant changes in activated forms of extracellular-regulated kinase 1/2 (ERK 1/2), c-jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), MAPK/ERK kinase (MEK1/2), 70-kDa ribosomal S6 kinase (p70 S6), cAMP responsive element binding protein (CREB), and signal transducer and activator of transcription 3 (STAT3). MAPK-related phosphoproteins exhibited an activation profile that peaked at 6 h, remained significantly increased at 24, and fell to baseline levels 48 h following neurotoxicant treatment. The ribosomal S6 kinase was enhanced over 60% for all time points examined. Immunoreactivity profiles for the transcription factors CREB and STAT3 indicated maximal increases in phosphorylation occurring at 24 h, and measuring greater than 2- or 17-fold, respectively. Specific signaling events were found to occur with a time course suggestive of their involvement in the gliotic response. The toxicant-induced activation of these growth-associated signaling cascades suggests that these pathways could be obligatory for the triggering and/or persistence of reactive gliosis and may therefore serve as potential targets for modulation of glial response to neural damage. PMID: 11085325 [PubMed - in process] --------------------------------------------------------------- 75: J Cell Physiol. 2000 Sep;184(3):326-33. Platelet-derived growth factor induces collagenase 3 transcription in osteoblasts through the activator protein 1 complex. Rydziel S, Durant D, Canalis E. Department of Research, Saint Francis Hospital and Medical Center, Hartford, Connecticut 06105-1299, USA. Platelet-derived growth factor (PDGF) BB is a mitogen that stimulates bone resorption and increases collagenase 3 transcription in osteoblasts, although the mechanisms involved are as yet unknown. We examined the effect of PDGF BB on collagenase 3 transcription in cultures of osteoblasts from fetal rat calvariae (Ob cells). PDGF BB increased the activity of collagenase 3 promoter fragments transiently transfected into Ob cells. Deletion analysis of the collagenase promoter revealed three regions that impaired the induction of collagenase 3 by PDGF BB. A construct spanning base pair -53 to +28 collagenase 3 sequences, in relation to the start site of transcription +1, was fully responsive to PDGF BB and was studied in detail. Targeted mutations of an AP-1 site in this fragment decreased basal collagenase promoter activity and the responsiveness to PDGF BB, whereas mutations of Stat3 and Ets binding sites did not alter the response to PDGF. Electrophoretic mobility shift assay, using nuclear extracts from control and treated cells, revealed AP-1 nuclear protein complexes that were enhanced in extracts from PDGF BB-treated Ob cells. Supershift assays revealed that antibodies to c-Fos, Fos B, Fra-2, c-Jun, Jun B, and Jun D shifted the binding of nuclear extracts from cells treated with PDGF BB to AP-1 sequences. In conclusion, PDGF BB induces collagenase 3 transcription in osteoblasts by regulating nuclear proteins interacting with AP-1 sequences. Copyright 2000 Wiley-Liss, Inc. PMID: 10911363 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 76: J Neurochem. 2000 Aug;75(2):634-43. The inhibitory effects of interleukin-6 on synaptic plasticity in the rat hippocampus are associated with an inhibition of mitogen-activated protein kinase ERK. Tancredi V, D'Antuono M, Cafe C, Giovedi S, Bue MC, D'Arcangelo G, Onofri F, Benfenati F. Department of Neuroscience, University of Roma Tor Vergata, Roma, Italy. Several cytokines have short-term effects on synaptic transmission and plasticity that are thought to be mediated by the activation of intracellular protein kinases. We have studied the effects of interleukin-6 (IL-6) on the expression of paired pulse facilitation (PPF), posttetanic potentiation (PTP), and long-term potentiation (LTP) in the CA1 region of the hippocampus as well as on the activation of the signal transducer and activator of transcription-3 (STAT3), the mitogen-activated protein kinase ERK (MAPK/ERK), and the stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK). IL-6 induced a marked and dose-dependent decrease in the expression of PTP and LTP that could be counteracted by the simultaneous treatment with the tyrosine kinase inhibitor lavendustin A (LavA) but did not significantly affect PPF. The IL-6-induced inhibition of PTP and LTP was accompanied by a simulation of STAT3 tyrosine phosphorylation and an inhibition of MAPK/ERK dual phosphorylation, in the absence of changes in the state of activation of SAPK/JNK. Both effects of IL-6 on STAT3 and MAPK/ERK activation were effectively counteracted by LavA treatment. The results indicate the tyrosine kinases and MAPK/ERK are involved in hippocampal synaptic plasticity and may represent preferential intracellular targets for the actions of IL-6 in the adult nervous system. PMID: 10899938 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 77: Cell Immunol. 2000 Jun 15;202(2):124-35. STAT3 regulates the growth and immunoglobulin production of BCL(1) B cell lymphoma through control of cell cycle progression. Karras JG, McKay RA, Lu T, Pych J, Frank DA, Rothstein TL, Monia BP. Department of Molecular and Cellular Pharmacology, Isis Pharmaceuticals, Inc., 2292 Faraday Avenue, Carlsbad, California 92008, USA. STAT3 is constitutively phosphorylated on tyrosine(705) in self-renewing, CD5(+) murine B-1 lymphocytes. Nuclear extracts from untreated primary B-1 or CD5(+) BCL(1) B lymphoma cells were found to contain immunoreactive STAT3 protein that binds to a sis-inducible element present in the promoter of the p21(waf1/cip1) tumor suppressor gene and is constitutively phosphorylated on serine(727). To determine the functional significance of constitutive STAT3 activation in B lymphoma cells, a specific STAT3 antisense oligonucleotide was developed and used to examine basal BCL(1) cell growth and IgM production. Abrogating STAT3 expression in BCL(1) cells inhibited their proliferative capacity and induced a corresponding decrease in secretion of IgM. Cell cycle analysis showed a block in progression through G1 in BCL(1) cells treated with the STAT3 antisense oligonucleotide. These results indicate that STAT3 controls cell growth and immunoglobulin secretion by enhancing progression through the G1 phase of the cell cycle in BCL(1) B cell lymphoma. Copyright 2000 Academic Press. PMID: 10896772 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 78: Eur J Neurosci. 2000 Apr;12(4):1165-76. Peripheral but not central axotomy induces changes in Janus kinases (JAK) and signal transducers and activators of transcription (STAT). Schwaiger FW, Hager G, Schmitt AB, Horvat A, Hager G, Streif R, Spitzer C, Gamal S, Breuer S, Brook GA, Nacimiento W, Kreutzberg GW. Department of Neuromorphology, Max-Planck Institute of Neurobiology, D-82152 Martinsried, Germany. schwaige@neuro.mpg.de Nerve injury leads to the release of a number of cytokines which have been shown to play an important role in cellular activation after peripheral nerve injury. The members of the signal transducer and activator of transcription (STAT) gene family are the main mediators in the signal transduction pathway of cytokines. After phosphorylation, STAT proteins are transported into the nucleus and exhibit transcriptional activity. Following axotomy in rat regenerating facial and hypoglossal neurons, a transient increase of mRNA for JAK2, JAK3, STAT1, STAT3 and STAT5 was detected using in situ hybridization and semi-quantitative polymerase chain reaction (PCR). Of the investigated STAT molecules, only STAT3 protein was significantly increased. In addition, activation of STAT3 by phosphorylation on position Tyr705 and enhanced nuclear translocation was found within 3 h in neurons and after 1 day in astrocytes. Unexpectedly, STAT3 tyrosine phosphorylation was obvious for more than 3 months. In contrast, none of these changes was found in response to axotomy of non-regenerating Clarke's nucleus neurons, although all the investigated models express c-Jun and growth-associated protein-43 (GAP-43) in response to axonal injury. Increased expression of Janus kinase (JAK) and STAT molecules after peripheral nerve transection suggests changes in the responsiveness of the neurons to signalling molecules. STAT3 as a transcription factor, which is expressed early and is activated persistently until the time of reinnervation, might be involved in the switch from the physiological gene expression to an 'alternative program' activated only after peripheral nerve injury. PMID: 10762348 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 79: J Mol Cell Cardiol. 2000 Apr;32(4):599-610. Epidermal growth factor induces hypertrophic responses and Stat5 activation in rat ventricular cardiomyocytes. Rebsamen MC, Arrighi JF, Juge-Aubry CE, Vallotton MB, Lang U. Division of Endocrinology and Diabetology, Division of Immunology and Allergy, Geneva 14, CH-1211, Switzerland. Epidermal growth factor (EGF) was tested for its ability to promote hypertrophic responses in neonatal rat ventricular cardiomyocytes. Exposure of these cells to 100 n m EGF for 2-18 h resulted in a time-dependent increase in protein synthesis reaching 174+/-18% of control values at 18 h. After 30 min stimulation, the mRNA levels of c-jun and c-fos were also increased 20- and 36-fold, respectively. We also investigated EGF-induced activation of Stat (signal transducers and activators of transcription) proteins as well as the possible interactions of this signaling pathway with the p38 and p42/44 MAP kinases cascades. EGF did not activate Stat1 and Stat3, but did induce a rapid and transient activation of Stat5, which corresponded mainly to Stat5b DNA-binding. The EGF-promoted Stat5 DNA-binding was decreased in a concentration-dependent manner by the p38 MAPK inhibitor SB 203580 (IC(50)=1.2 microm), whereas it was tripled by 50 micro m PD 98059, an inhibitor of the p42/44 MAPK cascade. This is the first demonstration that EGF increases protein synthesis and early response gene expression in cardiomyocytes, responses considered as markers of hypertrophy in these cells. The results further show that EGF activates Stat5, that this response requires p38 MAPK stimulation, and it is negatively modulated by p42/44 MAPK. Copyright 2000 Academic Press. PMID: 10756117 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 80: Biochem J. 2000 Apr 1;347 Pt 1:89-96. Interleukin-6-induced STAT3 transactivation and Ser727 phosphorylation involves Vav, Rac-1 and the kinase SEK-1/MKK-4 as signal transduction components. Schuringa JJ, Jonk LJ, Dokter WH, Vellenga E, Kruijer W. Department of Genetics, Biological Center, Kerklaan 30, 9751 NN, Haren, The Netherlands. In the present study, signal transducer and activator of transcription 3 (STAT3) Ser(727) phosphorylation and transactivation was investigated in relation to activation of mitogen-activated protein (MAP) kinase family members including extracellular-signal-regulated protein kinase (ERK)-1, c-Jun N-terminal kinase (JNK)-1 and p38 ('reactivating kinase') in response to interleukin (IL)-6 stimulation. Although IL-6 can activate ERK-1 in HepG2 cells, STAT3 transactivation and Ser(727) phosphorylation were not reduced by using the MAP kinase/ERK kinase (MEK) inhibitor PD98059 or by overexpression of dominant-negative Raf. IL-6 did not activate JNK-1 in HepG2 cells and STAT3 was a poor substrate for JNK-1 activated by anisomycin, excluding a role for JNK1 in IL-6-induced STAT3 activation. However, SEK-1/MKK-4 [where SEK-1 stands for stress-activated protein kinase (SAPK)/ERK kinase 1, and MKK-4 stands for MAP kinase kinase 4] was activated in response to IL-6 and overexpression of dominant-negative SEK-1/MKK-4(A-L) reduced both IL-6-induced STAT3 Ser(727) phosphorylation as well as STAT3 transactivation. Subsequently, the SEK-1/MKK-4 upstream components Vav, Rac-1 and MEKK were identified as components of a signal transduction cascade that leads to STAT3 transactivation in response to IL-6 stimulation. Furthermore, inhibition of p38 kinase activity with the inhibitor SB203580 did not block STAT3 Ser(727) phosphorylation but rather increased both basal as well as IL-6-induced STAT3 transactivation, indicating that p38 may act as a negative regulator of IL-6-induced STAT3 transactivation through a presently unknown mechanism. In conclusion, these data indicate that IL-6-induced STAT3 transactivation and Ser(727) phosphorylation is independent of ERK-1 or JNK-1 activity, but involves a gp130 receptor-signalling cascade that includes Vav, Rac-1, MEKK and SEK-1/MKK-4 as signal transduction components. PMID: 10727406 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 81: Mol Immunol. 1999 Sep-Oct;36(13-14):949-55. Complement activation and atherosclerosis. Niculescu F, Rus H. Department of Pathology, University of Maryland, School of Medicine, Baltimore 21201, USA. fnicules@umaryland.edu Atherosclerosis is an inflammatory disease mediated through the action of monocyte/macrophages, complement and T-lymphocytes. C5a and monocyte chemotactic factor released during complement activation in the arterial wall may participate in the initial monocyte recruitment. Assembly of C5b-9 on cells of the arterial wall may also induce cell lysis. On the other hand, sublytic assembly of C5b-9 on smooth muscle cells (SMC) and endothelial cells (EC) induces cell activation and proliferation. Analysis of mitogen activated protein kinases (MAPK) pathways induced by C5b-9 in aortic SMC revealed that extracellular signal regulated kinase (ERK) 1, c-jun NH2-terminal kinase (JNK) 1, and p38 MAPK are all activated by C5b-9. ERK1 activity was inhibited by wortmannin suggesting that ERK1 pathway is activated through phosphatidyl inositol -3 (PI 3-) kinase. Sublytic C5b-9 assembly on the plasma membrane was also able to activate Janus kinase (JAK) 1, signal transducer and activator (STAT) 3 and STAT4 in EC. JAK1 but not STAT3 activation induced by C5b-9 is dependent on Gi protein activation. New evidence accumulated during the last decade support the role of complement activation in both initiation and progression of the atherosclerotic lesions. Complement system activation is a major component of the chronic inflammatory process associated with atherosclerosis. PMID: 10698349 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 82: Am J Physiol Cell Physiol. 2000 Feb;278(2):C331-5. Inhibition of ornithine decarboxylase induces STAT3 tyrosine phosphorylation and DNA binding in IEC-6 cells. Pfeffer LM, Yang CH, Pfeffer SR, Murti A, McCormack SA, Johnson LR. Department of Pathology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA. lpfeffer@utmem.edu Polyamines are required for the proliferation of the rat intestinal mucosal IEC-6 cell line. Ornithine decarboxylase (ODC) is the enzyme that catalyzes the first step in polyamine synthesis. ODC inhibition not only leads to polyamine depletion but also leads to inhibition of cell proliferation and regulates the expression of the immediate-early genes c-fos, c-myc, and c-jun. Members of the signal transducers and activators of transcription (STAT) transcription factor family bind to the sis-inducible element (SIE) present in the promoters to regulate the expression of a variety of important genes. In the present study, we tested the hypothesis that the STAT3 transcription factor, which is responsible for activation of the acute phase response genes, is activated after inhibition of ODC. We found that inhibition of ODC rapidly induces STAT3 activation as determined by STAT3 tyrosine phosphorylation, translocation of STAT3 from the cytoplasm into the nucleus, and the presence of STAT3 in SIE-dependent DNA-protein complexes. STAT3 activation upon inhibition of ODC was accompanied by the activation of a STAT3-dependent reporter construct. Moreover, prolonged polyamine depletion resulted in downregulation of cellular STAT3 levels. PMID: 10666028 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 83: Biochem Biophys Res Commun. 1999 Dec 20;266(2):481-7. c-Src activates the DNA binding and transcriptional activity of Stat3 molecules: serine 727 is not required for transcriptional activation under certain circumstances. Schaefer LK, Wang S, Schaefer TS. Department of Neurosurgery, Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA. Stat3 proteins are constitutively activated in cells transformed by v-Src and the proteins have been shown to interact directly. Subsequent studies have shown that Stat3 is required for cellular transformation of NIH fibroblasts by v-Src, suggesting a potential role for Stat3 in aberrant cell growth. Stat3 is phosphorylated on a single tyrosine (tyrosine 705) which is required for effective dimer formation. An additional phosphorylation event (serine 727) is believed to be required for full transcriptional activity of Stat1 and Stat3 molecules. Here we report that c-Src activates the DNA binding activity of Stat3alpha, Stat3beta, and three Stat3 mutants, one in which serine 727 was replaced by alanine (Stat3alphaS727A) and C-terminal truncated molecules Delta48 and Delta55. Consistent with this finding is a general increase in the tyrosine 705-phosphorylated Stat3 in cells cotransfected with c-Src. Furthermore, transcription from an alpha-2 macroglobulin reporter gene is activated by Stat3alphaS727A to the same magnitude as compared to Stat3alpha and Stat3beta in the presence of c-Src. These results suggest that serine 727, contained in a consensus MAP kinase recognition site and shown to be the only serine in Stat3 phosphorylated in epidermal growth factor (EGF) treated cells, is not necessary for transcriptional activity comparable to wild-type Stat3alpha or Stat3beta when activated by c-Src in COS-7 cells. Copyright 1999 Academic Press. PMID: 10600528 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 84: Hepatology. 1999 Dec;30(6):1405-16. Lipopolysaccharide potentiates the effect of hepatocyte growth factor on hepatocyte replication in rats by augmenting AP-1 activity. Gao C, Jokerst R, Gondipalli P, Cai SR, Kennedy S, Flye MW, Ponder KP. Departments of Internal Medicine and Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, USA. The liver regenerates by replication of differentiated hepatocytes after damage or removal of part of the liver. Although several growth factors and signaling pathways are activated during regeneration, it is unclear as to which of these are essential for hepatocyte replication. We show here that low- (1 mg/kg) and high- (10 mg/kg) dose hepatocyte growth factor (HGF) induced replication of 2.1% and 11.1% of hepatocytes in rats, respectively. Lipopolysaccharide (LPS), an inducer of the acute phase response, augmented hepatocyte replication in response to low- and high-dose HGF by 4- and 2-fold, respectively. HGF alone induced moderate levels of c-Jun-N-terminal kinase (JNK) and p44/p42 mitogen-activated protein kinase (MAPK), resulting in moderate levels of AP-1-DNA binding activity. The combination of LPS + HGF increased JNK and AP-1-DNA binding activity more than levels seen with LPS or HGF alone. The activation of Stat3 that was observed after administration of LPS + HGF, but not HGF alone, could contribute to increased transcription of AP-1 components. Because phosphorylation of the c-Jun component of AP-1 by JNK increases its ability to activate transcription, the AP-1 in hepatocytes from animals treated with LPS + HGF may be more active than in rats treated with LPS or HGF alone. LPS may contribute to hepatocyte replication by potentiating the effect of HGF on the activation of both AP-1-DNA binding and transcriptional activity. PMID: 10573519 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 85: Mol Cell Biol. 1999 Nov;19(11):7519-28. Requirement for Ras/Rac1-mediated p38 and c-Jun N-terminal kinase signaling in Stat3 transcriptional activity induced by the Src oncoprotein. Turkson J, Bowman T, Adnane J, Zhang Y, Djeu JY, Sekharam M, Frank DA, Holzman LB, Wu J, Sebti S, Jove R. Molecular Oncology, Moffitt Cancer Center, University of South Florida College of Medicine, Tampa, Florida 33612, USA. Signal transducers and activators of transcription (STATs) are transcription factors that mediate normal biologic responses to cytokines and growth factors. However, abnormal activation of certain STAT family members, including Stat3, is increasingly associated with oncogenesis. In fibroblasts expressing the Src oncoprotein, activation of Stat3 induces specific gene expression and is required for cell transformation. Although the Src tyrosine kinase induces constitutive Stat3 phosphorylation on tyrosine, activation of Stat3-mediated gene regulation requires both tyrosine and serine phosphorylation of Stat3. We investigated the signaling pathways underlying the constitutive Stat3 activation in Src oncogenesis. Expression of Ras or Rac1 dominant negative protein blocks Stat3-mediated gene regulation induced by Src in a manner consistent with dependence on p38 and c-Jun N-terminal kinase (JNK). Both of these serine/threonine kinases and Stat3 serine phosphorylation are constitutively induced in Src-transformed fibroblasts. Furthermore, inhibition of p38 and JNK activities suppresses constitutive Stat3 serine phosphorylation and Stat3-mediated gene regulation. In vitro kinase assays with purified full-length Stat3 as the substrate show that both JNK and p38 can phosphorylate Stat3 on serine. Moreover, inhibition of p38 activity and thus of Stat3 serine phosphorylation results in suppression of transformation by v-Src but not v-Ras, consistent with a requirement for Stat3 serine phosphorylation in Src transformation. Our results demonstrate that Ras- and Rac1-mediated p38 and JNK signals are required for Stat3 transcriptional activity induced by the Src oncoprotein. These findings delineate a network of tyrosine and serine/threonine kinase signaling pathways that converge on Stat3 in the context of oncogenesis. PMID: 10523640 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 86: J Biol Chem. 1999 Oct 1;274(40):28697-707. Interleukin-6 and leukemia inhibitory factor induction of JunB is regulated by distinct cell type-specific cis-acting elements. Sjin RM, Lord KA, Abdollahi A, Hoffman B, Liebermann DA. Fels Institute for Cancer Research, Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA. Interleukin (IL)-6 plays an important role in a wide range of biological activities, including differentiation of murine M1 myeloid leukemic cells into mature macrophages. At the onset of M1 differentiation, a set of myeloid differentiation primary response (MyD) genes are induced, including the proto-oncogene for JunB. In order to examine the molecular nature of the mechanisms by which IL-6 activates the immediate early expression of MyD genes, JunB was used as a paradigm. A novel IL-6 response element, -65/-52 IL-6RE, to which a 100-kDa protein complex is bound, has been identified on the JunB promoter. Leukemia inhibitory factor (LIF)-induced activation of JunB in M1 cells was also mediated via the -65/-52 IL-6RE. The STAT3 and CRE-like binding sites of the JunB promoter, identified as IL-6-responsive elements in HepG2 liver cells were found, however, to play no role in JunB inducibility by IL-6 in M1 myeloid cells. Conversely, the -65/-52 IL-6RE is shown not to be necessary for JunB inducibility by IL-6 or LIF in liver cells. It appears, therefore, that immediate early activation of JunB is regulated differently in M1 myeloid cells than in HepG2 liver cells. This indicates that distinct cis-acting control elements participate in cell type-specific induction of JunB by members of the IL-6 cytokine superfamily. PMID: 10497240 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 87: Mol Cell Biol. 1999 Oct;19(10):7138-46. Interacting regions in Stat3 and c-Jun that participate in cooperative transcriptional activation. Zhang X, Wrzeszczynska MH, Horvath CM, Darnell JE Jr. Laboratory of Molecular Cell Biology, The Rockefeller University, New York, New York 10021, USA. Independent but closely spaced DNA binding sites for Stat3 and c-Jun are required for maximal enhancer function in a number of genes, including the gene encoding the interleukin-6 (IL-6)-induced acute-phase response protein, alpha(2)-macroglobulin. In addition, a physical interaction of Stat3 with c-Jun, based on yeast two-hybrid interaction experiments, has been reported. Here we confirm the existence of an interaction between Stat3 and c-Jun both in vitro, with recombinant proteins, and in vivo, during transient transfection. Using fragments of both proteins, we mapped the interactive sites to the C-terminal region of c-Jun and to two regions in Stat3, within the coiled-coil domain and in a portion of the DNA binding domain distant from DNA contact sites. In transient-transfection experiments with the alpha(2)-macroglobulin enhancer, Stat3 and c-Jun cooperated to yield maximal enhancer function. Point mutations of Stat3 within the interacting domains blocked both physical interaction of Stat3 with c-Jun and their cooperation in IL-6-induced transcription directed by the alpha(2)-macroglobulin enhancer. While the amino acid sequences and the three-dimensional structures of Stat3 and Stat1 cores are very similar, fragments of Stat1 failed to bind c-Jun in vitro. Although Stat1 binds in vitro to the gamma interferon gene response (GAS) element in the alpha(2)-macroglobulin enhancer, Stat1 did not stimulate transcription, nor did Stat1 and c-Jun cooperate in driving transcription controlled by the alpha(2)-macroglobulin enhancer. PMID: 10490649 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 88: Oncogene. 1999 Jul 1;18(26):3886-93. Stress activated protein kinase p38 is involved in IL-6 induced transcriptional activation of STAT3. Zauberman A, Zipori D, Krupsky M, Ben-Levy R. Department of Molecular Cell Biology, the Weizmann Institute of Science, Rehovot, Israel. The pleiotropic cytokine interleukin-6 (IL-6) induces acute phase protein expression in HepG2 human hepatoma cells and promotes the growth of mouse B9 hybridoma. The signaling cascades leading to these biological functions are only partially known. We analysed the involvement of MAPK homologues in IL-6 transduction pathways and found that interleukin-6 triggered activation of p38 stress-activated protein kinase (p38) but not of jun kinase. p38 activity was required for biological functions including acute phase protein secretion from HepG2 hepatoma and proliferation of B9 hybridoma cells. Using a reporter gene construct containing a 190 bp promoter fragment of the acute phase protein haptoglobin we found that p38 is involved in transcriptional activation of the haptoglobin promoter by STAT3 but not by NF-IL6. Thus, we present evidence for a role of p38 in IL-6 induced functions and a possible cross-talk between this MAPK homologue and the STAT pathway. PMID: 10445852 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 89: Mol Cell Biol. 1999 Aug;19(8):5326-38. Dual signaling role of the protein tyrosine phosphatase SHP-2 in regulating expression of acute-phase plasma proteins by interleukin-6 cytokine receptors in hepatic cells. Kim H, Baumann H. Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. One of the major actions of interleukin-6 (IL-6) is the transcriptional activation of acute-phase plasma proteins (APP) genes in liver cells. Signaling by the IL-6 receptor is mediated through the signal transducing subunit gp130 and involves the activation of Janus-associated kinases (JAKs), signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein (MAP) kinase. Functional analysis of gp130 in rat hepatoma cells by using transduced chimeric G-CSFR-gp130 receptor constructs demonstrates that SHP-2, the Src homology 2 (SH2) domain-containing protein tyrosine phosphatase, acts as a negative regulator of the JAK/STAT signaling in part by downregulating JAK activity, thereby indirectly moderating the induction of STAT3-dependent APP genes. This study shows that in hepatoma cells, the recruitment and tyrosine phosphorylation of SHP-2, but not SHC, is the primary signaling event associated with the activation of MAP kinases (ERK1/2) by gp130. Overexpression of truncated SHP-2 that lacks Grb2-interacting sites, but not the full-length catalytically inactive SHP-2, reduces ERK activation by IL-6, confirming the signal-mediating role of SHP-2. Activation of ERK1/2 is correlated with induction of the immediate-early response genes. Stimulation of the c-fos, c-jun, and egr-1 genes is essentially absent in cells expressing gp130 with a Y759F mutation, which is unable to recruit SHP-2. Interestingly, both JAK/STAT and SHP-2 pathways regulate the induction of the junB gene. Moreover, disengagement of SHP-2 from gp130 signaling not only enhances APP gene induction but also further reduces cell proliferation, in part correlated with the attenuated expression of immediate-early response genes. These results suggest that IL-6 regulation of APP genes is affected by SHP-2 in two ways: SHP-2 acts as a phosphatase on the JAK/STAT pathway and serves as linker to the MAP kinase pathway, which in turn moderates APP production. PMID: 10409724 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 90: Hepatology. 1999 May;29(5):1463-70. After portal branch ligation in rat, nuclear factor kappaB, interleukin-6, signal transducers and activators of transcription 3, c-fos, c-myc, and c-jun are similarly induced in the ligated and nonligated lobes. Starkel P, Horsmans Y, Sempoux C, De Saeger C, Wary J, Lause P, Maiter D, Lambotte L. Laboratories of Gastroenterology, Catholic University of Louvain Medical School, Brussels, Belgium. Several studies have emphasized the involvement of transcription factors, cytokines, and proto-oncogenes in initiating the regenerative process after partial hepatectomy. To assess whether these events do specifically occur in a cellular system undergoing regeneration, we studied the induction of nuclear factor kappaB (NFkappaB), interleukin-6 (IL-6), signal transducers and activators of transcription 3 (Stat3), c-fos, c-myc, c-jun, after portal branch ligation (PBL), which produces atrophy of the deprived lobes (70% of the liver parenchyma), whereas the perfused lobes undergo compensatory regeneration. Nuclear extracts and total RNA were prepared from control livers as well as from atrophying and regenerating lobes at 0.5, 1, 2, 5, and 8 after PBL. NFkappaB and Stat3 induction were studied by electrophoretic mobility shift assays and Western blotting. IL-6 and proto-oncogenes expressions were assessed by reverse transcription polymerase chain reaction and Northern blotting, respectively. Assays were also performed after a sham operation. NFkappaB and Stat3 protein expression and DNA binding were rapidly and similarly induced in nuclear extracts from the atrophying and regenerating lobes. IL-6 was elevated in both lobes from 1 to 8 hours after PBL as well as c-fos, c-myc, and c-jun during the first 2 hours. IL-6 and Stat3 but not NFkappaB were also elevated after a sham operation. These findings suggest that the cellular and molecular changes occurring early in a regenerating liver are nonspecific, possibly stress-induced, cellular responses. They do not indicate the future evolution towards atrophy or regeneration. PMID: 10216130 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 91: J Immunol. 1999 Apr 15;162(8):4472-81. The functional synergy between IL-12 and IL-2 involves p38 mitogen-activated protein kinase and is associated with the augmentation of STAT serine phosphorylation. Gollob JA, Schnipper CP, Murphy EA, Ritz J, Frank DA. Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA. jgoll@bidmc.harvard.edu IL-12 and IL-2 can stimulate mitogen- or CD3-activated T cells to proliferate, produce IFN-gamma, and kill tumor cells. The magnitude of these functional responses is greatly augmented when T cells are activated by the combination of IL-12 and IL-2. Although peripheral blood T cells are largely unresponsive to these cytokines without prior activation, a small subset of CD8+ T cells (CD8+CD18bright) is strongly activated by the combination of IL-12 and IL-2. In this report we show that the functional synergy between IL-12 and IL-2 in CD8+CD18bright T cells correlates with the activation of the stress kinases, p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase (SAPK)/Jun N-terminal kinase, but not with the activation of the extracellular signal-regulated kinases. The functional synergy between IL-2 and IL-12 is also associated with a prominent increase in STAT1 and STAT3 serine phosphorylation over that observed with IL-12 or IL-2 alone. By contrast, STAT tyrosine phosphorylation is not augmented over that seen with either cytokine alone. A specific inhibitor of p38 MAP kinase completely inhibits the serine phosphorylation of STAT1 and STAT3 induced by IL-12 and IL-2 and abrogates the functional synergy between IL-12 and IL-2 without affecting STAT tyrosine phosphorylation. This suggests that p38 MAP kinase may play an important role in regulating STAT serine phosphorylation in response to the combination of IL-12 and IL-2. Furthermore, these findings indicate that the optimal activation of T cells by IL-12 and IL-2 may depend on an interaction between the p38 MAP kinase and Janus kinase/STAT signaling pathways. PMID: 10201984 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 92: J Biol Chem. 1999 Feb 5;274(6):3522-30. Distinct and common pathways in the regulation of insulin-like growth factor-1 receptor gene expression by angiotensin II and basic fibroblast growth factor. Scheidegger KJ, Du J, Delafontaine P. Division of Cardiology, University Hospital of Geneva, Switzerland. Angiotensin II (Ang II) and basic fibroblast growth factor (bFGF) are important modulators of cell growth under physiological and pathophysiological conditions. We and others have previously shown that these growth factors increase insulin-like growth factor-1 receptor (IGF-1R) number and mRNA in vascular smooth muscle cells and that this effect is transcriptionally regulated. To study the mechanisms and the signaling pathways involved, IGF-1R promoter reporter constructs were transiently transfected in CHO-AT1 cells that overexpress angiotensin AT1 receptors. Our findings indicate that Ang II and bFGF significantly increased IGF-1R promoter activity up to 7- and 3-fold, respectively. The effect induced by Ang II was mediated via a tyrosine kinase-dependent mechanism, since tyrphostin A25 largely inhibited the Ang II-induced increase in promoter activity. In addition, co-transfection of dominant negative Ras, Raf, and MEK1 or pretreatment with the MEK inhibitor PD 98059 dose-dependently decreased both the Ang II- and bFGF-induced increase in IGF-1R transcription and protein expression, suggesting that the Ras-Raf-mitogen-activated protein kinase kinase pathway is required for both growth factors. Reactive oxygen species have been shown to act as second messengers in Ang II-induced signaling, and activation of the transcription factor NF-kappaB is redox-sensitive. While co-transfection of dominant negative IkappaBalpha mutant completely inhibited the Ang II-induced increase in transcription, it had no effect on the bFGF signaling. In contrast, co-transfection studies indicated that the transcription factors STAT1, STAT3, and c-Jun and the Janus kinase 2 kinase are required in the signaling pathway of bFGF, whereas only dominant c-Jun inhibited the Ang II-induced effect. In summary, these data demonstrate that Ang II and bFGF increase IGF-1R gene transcription via distinct as well as shared pathways and have important implications for understanding growth-stimulatory effects of these growth factors on vascular cells. PMID: 9920898 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 93: J Endocrinol. 1998 Nov;159(2):323-30. Crosstalk between the interleukin-6 (IL-6)-JAK-STAT and the glucocorticoid-nuclear receptor pathway: synergistic activation of IL-6 response element by IL-6 and glucocorticoid. Takeda T, Kurachi H, Yamamoto T, Nishio Y, Nakatsuji Y, Morishige K, Miyake A, Murata Y. Department of Obstetrics and Gynecology, Osaka University Medical School, Osaka 565-0871, Japan. Cytokines and steroid hormones use different sets of signal transduction pathways, which seem to be unrelated. Interleukin-6 (IL-6) uses JAK tyrosine kinase and STAT (signal transducer and activator of transcription) transcription factor. Glucocorticoid binds glucocorticoid receptor (GR), which is a member of the steroid receptor superfamily. We have studied the crosstalk between the IL-6-JAK-STAT and glucocorticoid-nuclear receptor pathways. IL-6 and glucocorticoid synergistically activated the IL-6 response element on the rat alpha2-macroglobulin promoter (APRE)-driven luciferase gene. The exogenous expression of GR enhanced the synergism. The exogenous expression of dominant negative STAT3 completely abolished the IL-6 plus glucocorticoid-induced activation of the APRE-luciferase gene. Tyrosine phosphorylation of STAT3 stimulated by IL-6 alone was not different from that by IL-6 plus glucocorticoid. The protein level of STAT3 was also not increased by glucocorticoid stimulation. The time course of STAT3 tyrosine phosphorylation by IL-6 plus glucocorticoid was not different from that by IL-6 alone. The synergism was studied on the two other IL-6 response elements, the junB promoter (JRE-IL-6) and the interferon regulatory factor-1 (IRF-1) promoter (IRF-GAS) which could be activated by STAT3. The synergistic activation by glucocorticoid on the IL-6-activated JRE-IL-6 and the IRF-GAS-driven luciferase gene was not detected. Glucocorticoid did not change the mobility of IL-6-induced APRE-binding proteins in a gel shift assay. These results suggest that the synergism was through the GR and STAT3, and the coactivation pathway which was specific for APRE was the target of glucocorticoid. PMID: 9795374 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 94: Hepatology. 1998 Oct;28(4):959-70. Comment on: Hepatology. 1998 Oct;28(4):906-13. Analysis of liver regeneration in mice lacking type 1 or type 2 tumor necrosis factor receptor: requirement for type 1 but not type 2 receptor. Yamada Y, Webber EM, Kirillova I, Peschon JJ, Fausto N. Department of Pathology, University of Washington School of Medicine, Seattle, WA 98195-7705, USA. We used KO mice lacking either TNF receptor 1 (TNFR-1) or receptor 2 (TNFR-2) to determine whether signaling at the start of liver regeneration after partial hepatectomy (PH) involves only one or both TNF receptors and to analyze in more detail the abnormalities caused by lack of TNFR-1 receptor, which is required for the initiation of liver regeneration. Lack of TNFR-2 had little effect on NF-kappaB and STAT3 binding, and no effect in interleukin-6 production after PH, but caused a delay in AP-1 and C/EBP binding and in the expression of c-jun and c-myc messenger RNA (mRNA). In contrast to mice lacking TNFR-1, which had deficient hepatocyte DNA synthesis and massive lipid accumulation in hepatocytes, TNFR-2 KO mice had normal liver structure and similar levels of hepatocyte DNA replication as those of wild type mice. We conclude that TNFR-1, but not TNFR-2, is necessary for liver regeneration, and that NF-kappaB and STAT3 binding are activated by signals transduced by TNFR-1. Inhibition of AP-1 and C/EBP binding and in the expression of c-jun and c-myc mRNA in the first 4 hours after PH, as well as the apparent lack of Fos in AP-1 complexes, had no effect on the timing or extent of DNA replication. Publication Types: Comment PMID: 9755232 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 95: Leuk Lymphoma. 1998 Aug;30(5-6):433-42. Stat3 and G-CSF-induced myeloid differentiation. Chakraborty A, Tweardy DJ. Department of Medicine, University of Pittsburgh School of Medicine, University of Pittsburgh Cancer Institute, PA 15213, USA. Granulocyte colony-stimulating factor (G-CSF) is the cytokine critical for directing neutrophilic granulocyte differentiation. Early G-CSF signaling events in myeloid cells involves activation of STATs, proteins that serve the dual function of signal transduction and activation of transcription, especially the activation of Stat3. A dominant-negative mutant construct of Stat3 inhibited G-CSF-mediated neutrophilic differentiation indicating that Stat3 is a essential component for driving the G-CSF-mediated differentiation program in myeloid cells. Three isoforms of Stat3 have been identified, alpha(p92), beta(p83) and gamma(p72) each derived from a single gene. Stat3alpha is the predominant isoform expressed in most cells. Stat3beta is derived from Stat3alpha by alternative RNA splicing. Stat3gamma is derived from Stat3alpha by limited proteolysis. Mapping of Stat3alpha and Stat3beta activation in M1 murine myeloid leukemia cells revealed that their optimal activation required G-CSFR constructs containing both Y704 and Y744. These amino acid residues has previously been demonstrated to be essential for G-CSF-induced differentiation in this cells. Phosphopeptide affinity and phosphopeptide inhibition studies indicate that Stat3alpha and Stat3beta are recruited to the G-CSF receptor complex through their interaction with the receptor at phosphotyrosines Y704 and Y744. Y744 is followed at the +3 position by Cys (C). This sequence YXXC, represents a novel motif implicated in the recruitment and activation of Stat3alpha, Stat3beta and Stat3gamma by the hG-CSFR. Structurally, Stat3alpha, Stat3beta and Stat3gamma differ from each other in their C-terminal transactivation domain. In the beta isoform, the Stat3alpha transactivation domain is replaced by 7 amino acid residues which enable Stat3beta to interact with c-Jun. In the gamma isoform, the Stat3alpha transactivation domain is removed by limited proteolysis creating a dominant negative isoform. In immature human myeloid cells capable of differentiating into neutrophils in response to G-CSF, G-CSF did not activate Stat3alpha; rather. it activated predominantly Stat3beta. These findings combined with recent reports linking Stat3alpha with proliferation and transformation suggest that the beta isoform of Stat3 may be more critical for G-CSF-mediated differentiation. Activation of Stat3gamma occurred predominantly in terminally differentiated neutrophils suggesting that it may be part of a controlled proteolytic mechanism modulating pro-proliferative protein(s) in mature myeloid cells. Publication Types: Review Review, Tutorial PMID: 9711905 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 96: J Biol Chem. 1998 Mar 13;273(11):6233-41. Coordinate regulation of STAT signaling and c-fos expression by the tyrosine phosphatase SHP-2. Servidei T, Aoki Y, Lewis SE, Symes A, Fink JS, Reeves SA. Neurosurgical Service, Molecular Neuro-Oncology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02129, USA. The src homology 2 (SH2) domain-containing protein-tyrosine phosphatase SHP-2 has been implicated as an important positive regulator of several mitogenic signaling pathways. SHP-2 has more recently been shown to be tyrosine phosphorylated and recruited to the gp130 component of the ciliary neurotrophic factor (CNTF) receptor complex upon stimulation with CNTF. CNTF does not, however, have a proliferative effect on responsive cells, but rather enhances the survival and differentiation of sympathetic, motor, and sensory neurons. In this study, expression of an interfering mutant of SHP-2 in the neuroblastoma cell line NBFL increased CNTF induction of a vasoactive intestinal peptide (VIP) reporter gene, and in cultures of sympathetic neurons, it resulted in an up-regulation of endogenous VIP and substance P (SP) gene expression. Members of the CNTF family of cytokines transmit their signal by activating signaling pathways involving both STAT and Fos-Jun transcription factors. In CNTF-stimulated NBFL cells that constitutively express the SHP-2 interfering mutant, there was increased and prolonged formation of STAT/DNA complexes, but decreased AP-1 binding activity, that mirrored a down-regulation of c-fos expression both at the mRNA and protein level. Taken together, these data indicate that SHP-2 has dual and opposing roles in a signaling cascade triggered by the same ligand, as illustrated by its ability to differentially regulate the levels of activity of both STAT and AP-1 transcription factors. PMID: 9497348 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 97: J Biol Chem. 1998 Mar 13;273(11):6132-8. Autoregulation of the Stat3 gene through cooperation with a cAMP-responsive element-binding protein. Ichiba M, Nakajima K, Yamanaka Y, Kiuchi N, Hirano T. Division of Molecular Oncology, Department of Oncology, Biomedical Research Center, Osaka University Medical School, 2-2 Yamadaoka, Suita City, Osaka 565, Japan. STAT3 (signal transducer and activator of transcription 3) is a key transcription factor mediating the signals for a variety of cytokines, including interleukin-6 (IL-6). The Stat3 gene itself is activated by IL-6 signals. We show that the region of the signal-transducing subunit, gp130, essential for STAT3 activation, is also required for activation of the Stat3 gene. To elucidate the mechanisms activating the Stat3 gene, we identified an IL-6 response element (IL-6RE) in the Stat3 gene promoter containing both a low affinity STAT3-binding element and a cAMP-responsive element (CRE). Electrophoretic mobility shift assays showed that IL-6 induced a slowly migrating complex on the IL-6RE containing a STAT3 homodimer and an unidentified CRE-binding protein. With the combination of transient transfection assays using mutant Stat3 promoter-reporter constructs and electrophoretic mobility shift assays, we found that the formation of a slowly migrating complex was required for full activation of the Stat3 gene. Thus, STAT3 activates the Stat3 gene in cooperation with an unidentified CRE-binding protein. This regulatory mechanism is similar to that of the junB gene, which is activated by IL-6 through the junB IL-6RE, which contains a low affinity STAT3-binding site and a CRE-like site. PMID: 9497331 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 98: J Biol Chem. 1997 Aug 22;272(34):21334-40. Critical cytoplasmic domains of human interleukin-9 receptor alpha chain in interleukin-9-mediated cell proliferation and signal transduction. Zhu YX, Sun HB, Tsang ML, McMahel J, Grigsby S, Yin T, Yang YC. Department of Medicine (Hematology/Oncology), Indianapolis, Indiana 46202, USA. Interleukin-9 receptor (IL-9R) complex consists of a ligand-specific alpha chain and IL-2R gamma chain. In this study, two regions in the cytoplasmic domain of human IL-9Ralpha were found to be important for IL-9-mediated cell growth. A membrane-proximal region that contains the BOX1 consensus sequence is required for IL-9-induced cell proliferation and tyrosine phosphorylation of Janus kinases (JAKs). Deletion of this region or internal deletion of the BOX1 motif abrogated IL-9-induced cell proliferation and signal transduction. However, substitution of the Pro-X-Pro in the BOX1 motif with Ala-X-Ala failed to abolish IL-9-induced cell proliferation but decreased IL-9-mediated tyrosine phosphorylation of JAK kinases, insulin receptor substrate-2, and signal transducer and activator of transcription 3 (STAT3) and expression of c-myc and junB. Another important region is downstream of the BOX1 motif and contains a STAT3 binding motif YLPQ. Deletion of this region significantly impaired IL-9-induced cell growth, activation of JAK kinases, insulin receptor substrate-2, and STAT3 and expression of early response genes. A point mutation changing YLPQ into YLPA greatly reduced IL-9-induced activation of STAT3 and expression of c-myc but did not affect cell proliferation. These results suggest that cooperation or cross-talk of signaling molecules associated with different domains of IL-9Ralpha other than STAT3 is essential for IL-9-mediated cell growth. PMID: 9261146 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 99: Endocrinology. 1997 Jul;138(7):2740-6. Transcriptional regulation of Sertoli cell immediate early genes by interleukin-6 and interferon-gamma is mediated through phosphorylation of STAT-3 and STAT-1 proteins. Jenab S, Morris PL. The Population Council, New York, New York 10021, USA. The immediate early genes are regulated by a variety of extracellular signals, including pleiotropic cytokines. The effects of the testicular cytokines, interleukin-6 (IL-6) and interferon-gamma (IFN-gamma), on signal transducers and activators of transcription 3 and 1 (STAT-3 and STAT-1) and on c-fos gene expression in primary Sertoli cells are suggestive of their roles in differential function. Using the tyrosine phosphorylation inhibitor, genistein, and electrophoretic mobility shift assay, we show that IL-6 and IFN-gamma induce nuclear factor STAT-3 and STAT-1 DNA-binding activity to the sis-inducible element of c-fos in a genistein-dependent pathway. Quantitative solution hybridization, Northern blot, and nuclear run-on analysis show that differential induction of c-fos, junB, and c-myc messenger RNA (mRNA) by these cytokines occur at transcriptional levels. IL-6 stimulates c-fos mRNA levels by 6-fold while increasing junB levels by 2-fold. IFN-gamma increases c-fos message 2-fold, but has no effect on junB mRNA levels. Furthermore, genistein treatment blocks the induction of c-fos and junB gene expression, demonstrating that tyrosine phosphorylation of STAT proteins is involved in the cytokine regulation of the Sertoli immediate early genes. H7, a serine/threonine phosphorylation inhibitor, also blocks c-fos gene induction by IL-6 and IFN-gamma, but does not affect the DNA-binding activities of STAT-3 and STAT-1. Finally, IL-6 treatment of Sertoli cells (3-6 h) increases the amounts of activating protein-1 binding to activating protein-1 element and c-myc transcription. PMID: 9202212 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 100: J Autoimmun. 1997 Jun;10(3):299-307. Molecular role of TGF-beta, secreted from a new type of CD4+ suppressor T cell, NY4.2, in the prevention of autoimmune IDDM in NOD mice. Han HS, Jun HS, Utsugi T, Yoon JW. Department of Microbiology and Infectious Diseases, Julia McFarlane Diabetes Research Centre, Faculty of Medicine, The University of Calgary, Alberta, Canada. A new type of CD4+ T cell clone (NY4.2) isolated from pancreatic islet-infiltrated lymphocytes of acutely diabetic non-obese diabetic (NOD) mice prevents the development of insulin-dependent diabetes mellitus (IDDM) in NOD mice, as well as the recurrence of autoimmune diabetes in syngeneic islet-transplanted NOD mice. It has been demonstrated that the cytokine TGF-beta, secreted from the cells of this clone, is the substance which prevents autoimmune IDDM. This investigation was initiated to determine the molecular role TGF-beta plays in the prevention of autoimmune IDDM by determining its effect on IL-2-induced signal transduction in Con A-activated NOD mouse splenocytes and HT-2 cells. First, we determined whether TGF-beta, secreted from NY4.2 T cells, inhibits IL-2-dependent T cell proliferation in HT-2 cells (IL-2-dependent T cell line) and NOD splenocytes. We found that TGF-beta suppresses IL-2-dependent T cell proliferation. Second, we determined whether TGF-beta inhibits the activation of Janus kinases (JAKs), as well as signal transducers and activators of transcription (STAT) proteins, involved in an IL-2-induced signalling pathway that normally leads to the proliferation of T cells. We found that TGF-beta inhibited tyrosine phosphorylation of JAK1, JAK3, STAT3 and STAT5 in Con A blasts from NOD splenocytes and HT-2 cells. Third, we examined whether TGF-beta inhibits the cooperation between STAT proteins and mitogen-activated protein kinase (MAPK), especially extracellular signal-regulated kinase 2 (ERK2). We found that TGF-beta inhibited the association of STAT3 and STAT5 with ERK2 in Con A blasts from NOD splenocytes and HT-2 cells. On the basis of these observations, we conclude that TGF-beta may interfere with signal transduction via inhibition of the IL-2-induced JAK/STAT pathway and inhibition of the association of STAT proteins with ERK2 in T cells from NOD splenocytes, resulting in the inhibition of IL-2-dependent T cell proliferation. TGF-beta-mediated suppression of T cell activation may be responsible for the prevention of effector T cell-mediated autoimmune IDDM in NOD mice by TGF-beta-producing CD4+ suppressor T cells. PMID: 9218758 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 101: J Biol Chem. 1997 Feb 28;272(9):5783-91. Cardiotrophin 1 (CT-1) inhibition of cardiac myocyte apoptosis via a mitogen-activated protein kinase-dependent pathway. Divergence from downstream CT-1 signals for myocardial cell hypertrophy. Sheng Z, Knowlton K, Chen J, Hoshijima M, Brown JH, Chien KR. Department of Medicine, University of California, San Diego, School of Medicine, La Jolla, California 92093, USA. Cardiac myocyte survival is of central importance in the maintenance of the function of heart, as well as in the development of a variety of cardiac diseases. To understand the molecular mechanisms that govern this function, we characterized apoptosis in cardiac muscle cells following serum deprivation. Cardiotrophin 1 (CT-1), a potent cardiac survival factor (Sheng, Z., Pennica, D., Wood, W. I., and Chien, K. R. (1996) Development (Camb.) 122, 419-428), is capable of inhibiting apoptosis in cardiac myocytes. To explore the potential downstream pathways that might be responsible for this effect, we documented that CT-1 activated both signal transducer and activator of transcription 3 (STAT3)- and mitogen-activated protein (MAP) kinase-dependent pathways. The transfection of a MAP kinase kinase 1 (MEK1) dominant negative mutant cDNA into myocardial cells blocked the antiapoptotic effects of CT-1, indicating a requirement of the MAP kinase pathway for the survival effect of CT-1. A MEK-specific inhibitor (PD098059) (Dudley, D. T., Pang, L., Decker, S.-J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. USA 92, 7686-7689) is capable of blocking the activation of MAP kinase, as well as the survival effect of CT-1. In contrast, this inhibitor did not block the activation of STAT3, nor did it have any effect on the hypertrophic response elicited following stimulation of CT-1. Therefore, CT-1 promotes cardiac myocyte survival via the activation of an antiapoptotic signaling pathway that requires MAP kinases, whereas the hypertrophy induced by CT-1 may be mediated by alternative pathways, e.g. Janus kinase/STAT or MEK kinase/c-Jun NH2-terminal protein kinase. PMID: 9038192 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 102: AIDS. 1996 Apr;10(4):369-78. Differential activation of the extracellular signal-regulated kinase, Jun kinase and Janus kinase-Stat pathways by oncostatin M and basic fibroblast growth factor in AIDS-derived Kaposi's sarcoma cells. Faris M, Ensoli B, Stahl N, Yancopoulos G, Nguyen A, Wang S, Nel AE. Department of Medicine, University of California Los Angles School of Medicine 90024, USA. OBJECTIVES: To determine the integration of signalling pathways associated with two recognized Kaposi's sarcoma (KS) growth factors, oncostatin M (OSM) and basic fibroblast growth factor (bFGF), in the induction of KS cell proliferation. DESIGN AND METHODS: We used protein kinase assays, protein-DNA interactions and AP-1 luciferase assays to study the extracellular signal-regulated kinase (ERK), Janus kinase (JAK)-Stat and Jun kinase (JNK) pathways in AIDS-derived KS cells during stimulation with OSM and bFGF. RESULTS: Treatment with OSM-induced activation of receptor-associated JAK and phosphorylation of Stat1 and Stat3. Stat1/Stat3 heterodimers interacted with known gamma-interferon-activated sites like elements such as the sis-inducible element (SIE) in the C-fos promoter. In contrast, ligation of the bFGF receptor induced Stat3 phosphorylation and its association with the bFGF receptor, but failed to induce JAK activity or protein complexes which interact with GAS-like oligonucleotides. OSM also induced the activation of ERK2 by activating the serine/threonine kinases Raf-1 and [mitogenactivated protein kinase (MAPK) ERK kinase (MEK1)]-1, while bFGF failed to activate any of the above components. Both OSM and bFGF activated the JNK pathway, along with the activation of MEKkinase (MEKK)-1. JNK control the transcriptional activation of c-Jun. Because the above pathways exert an effect on the expression or activation of activation protein (AP)-1 components, we confirm that OSM and bFGF induce TPA response element (TRE)-luciferase activity synergistically. CONCLUSION: We demonstrate that OSM and bFGF activate distinct as well as shared signalling cascades in KS cells, which integrate to provide a synergistic AP-1 response by which OSM and bFGF may sustain KS cell growth. PMID: 8728040 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 103: FASEB J. 1996 Mar;10(4):413-27. Liver regeneration 4: transcriptional control of liver regeneration. Taub R. Department of Genetics and Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104-6145, USA. Determining what factors are responsible for initiating regeneration following partial hepatectomy or toxic damage, and how the liver maintains differentiated functions while the hepatocytes are undergoing cellular proliferation are central issues in understanding the molecular bases of liver regeneration. Examination of the transcriptional milieu in the regenerating liver provides clues to the answers to these questions. Growth factor-generated intracellular signals that trigger liver regeneration result in activation via posttranslational modifications of latent, normally inactive transcription factors that preexist in the liver. Two transcription factors that are activated by this mechanism include posthepatectomy factor/nuclear factor-kappa B) and Stat3. Because cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-l (IL-1), and IL-6 can induce these factors in the liver, the finding of activated Stat3 and PHF/NF-kappa B suggests that these cytokines may play a role in some aspects of growth regulation during liver regeneration. Rapidly induced transcription factors, Stat3, PHF/NF-kappa B, and others are responsible for activation of the primary growth response or immediate-early genes, which play a role in regulating later phases of cell growth in regenerating liver and other mitogen-activated cells. Immediate-early genes encode many members of diverse transcription factor families including the Jun-Fos-LRF-1, nuclear receptor, and myc families to name a few. In this way a transcriptional cascade is established during the G1 phase of liver regeneration. Coexisting with these induced factors are liver-specific transcription factors such as the CAAT enhancer binding proteins and hepatocyte nuclear factors, which may interact with growth-induced factors to help the liver maintain metabolic homeostasis during regeneration. As a result the liver is able to accomplish the goals of reestablishing its mass while it maintains its functional capacity during regeneration. Publication Types: Review Review, Tutorial PMID: 8647340 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 104: Oncogene. 1996 Feb 1;12(3):547-54. IL-6-inducible complexes on an IL-6 response element of the junB promoter contain Stat3 and 36 kDa CRE-like site binding protein(s). Kojima H, Nakajima K, Hirano T. Department of Molecular Oncology, Osaka University Medical School, Japan. The junB gene is one of immediate-early genes whose expression are regulated by a variety of extracellular stimuli and play important roles in cellular responses to the given stimuli. Interleukin-6 (IL-6) activates the junB promoter through an IL-6 response element, JRE-IL6, that is composed of two cooperative DNA motifs, a low affinity Stat-binding site overlapping with an Ets-binding site (JEBS) and a cAMP responsive element (CRE)-like site. This element is a target for the Jak-Stat signal transduction pathway. We showed that IL-6 induced novel complexes on JRE-IL6, termed JRE-IL6-BC1 and 2, which contained Stat3 but migrated more slowly than the complexes containing homo- or heterodimer of Stat3 and Stat1 in gel shift assays. These slow-migrating JRE-IL6-BCs appeared to contain CRE-like site binding proteins besides Stat3, since the formation of JRE-IL6-BCs required both the JEBS and CRE-like site of JRE-IL6 and oligonucleotides containing the CRE-like site or somatostatin CRE efficiently competed with JRE-IL6 for making JRE-IL6-BCs. The formation of the complexes correlated well with the responsiveness of JRE-IL6 to IL-6 signals. U.v.-cross linking study revealed that JRE-IL6 bound a 90 kDa protein, corresponding to Stat3, and a 36 kDa protein, most likely a CRE-like site binding protein(s). Furthermore, we showed that the IL-6/interferon gamma (IFN gamma) response element in the IRF-1 promoter (IR/IRF-1), which contains a Stat-binding site and an adjacent CRE-like site, also makes IL-6-induced binding complexes similar to JRE-IL6-BCs. PMID: 8637711 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 105: Endocrinology. 1996 Jan;137(1):55-64. Acute nuclear actions of growth hormone (GH): cycloheximide inhibits inducible activator protein-1 activity, but does not block GH-regulated signal transducer and activator of transcription activation or gene expression. Gronowski AM, Le Stunff C, Rotwein P. Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA. The mechanisms by which GH regulates gene expression to stimulate somatic growth and alter intermediary metabolism are unknown. We have shown previously that in vivo GH administration rapidly modifies the tyrosine phosphorylation of multiple hepatic nuclear proteins, including the inducible transcription factors, Stat1, Stat3, and (in this report) Stat5, and have found that hormone treatment also rapidly alters gene transcription in the liver. In this study, we have used the protein synthesis inhibitor, cycloheximide (CHX), to investigate which of the acute actions of GH are primary hormonal responses and which require concurrent protein synthesis. We found that many of the early changes in nuclear protein tyrosine phosphorylation and in nuclear protein-DNA binding after GH are not blunted by CHX. The activation of insulin-like growth factor I and Spi 2.1 gene expression and the inhibition of albumin transcription also are not blocked by CHX, suggesting that these effects are primary consequences of GH-activated signal transduction pathways. By contrast, CHX completely inhibits the induction of activator protein-1 DNA-binding activity by GH, indicating that this action is secondary to the stimulation of Fos and/or Jun protein biosynthesis. Our results support the idea that multiple primary and secondary signaling pathways contribute to the pleiotropic effects of GH on gene expression and provide a framework for delineating the mechanisms controlling the acute actions of GH. PMID: 8536642 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 106: J Biol Chem. 1995 Dec 29;270(52):31129-35. An acute phase response factor/NF-kappa B site downstream of the junB gene that mediates responsiveness to interleukin-6 in a murine plasmacytoma. Brown RT, Ades IZ, Nordan RP. Division of Monoclonal Antibodies, Center for Biologics Evaluation and Research, Bethesda, Maryland 20892, USA. The immediate early gene, junB, is induced by interleukin-6 (IL-6) in plasmacytomas. In order to identify enhancers that mediate this effect, we cloned upstream and downstream sequences flanking the gene into a luciferase reporter gene vector containing the junB promoter and evaluated the IL-6 inducibility of these sequences by transient expression in an IL-6-dependent plasmacytoma cell line. Although a 6.5-kilobase fragment of upstream flanking sequence did not increase the IL-6 inducibility of the junB promoter, a 222-base pair fragment was identified in 2.1 kilobases of down-stream flanking sequence that both up-regulates the promoter and confers inducibility by IL-6. Point mutation of an acute phase response factor (APRF) site within this region significantly reduced up-regulation of the promoter in cells grown continuously in IL-6, as well as inducibility upon restimulation of cells with IL-6 after withdrawal from the growth factor. Point mutation of an NF-kappa B site sharing five nucleotides with the APRF site reduced up-regulation of the promoter but not inducibility by IL-6, whereas mutation of two other NF-kappa B sites in the 222-base pair fragment had no effect on expression. Western blotting of nuclear proteins purified by DNA affinity chromatography revealed inducible binding of Stat3 and constitutive binding of NF-kappa B p65 to the APRF/NF-kappa B site. PMID: 8537375 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 107: Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9097-101. Cooperative transcriptional activity of Jun and Stat3 beta, a short form of Stat3. Schaefer TS, Sanders LK, Nathans D. Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205-2185, USA. To identify proteins that regulate the transcriptional activity of c-Jun, we have used the yeast two-hybrid screen to detect mammalian polypeptides that might interact functionally with the N-terminal segment of c-Jun, a known regulatory region. Among the proteins identified is a short form of Stat3 (designated Stat3 beta). Stat3 beta is missing the 55 C-terminal amino acid residues of the long form (Stat3 alpha) and has 7 additional amino acid residues at its C terminus. In the absence of added cytokines, expression of Stat3 beta (but not Stat3 alpha) in transfected cells activated a promoter containing the interleukin 6 responsive element of the rat alpha 2-macroglobulin gene; coexpression of Stat3 beta and c-Jun led to enhanced cooperative activation of the promoter. Nuclear extracts of cells transfected with a Stat3 beta expression plasmid formed a complex with an oligonucleotide containing a Stat3 binding site, whereas extracts of cells transfected with a Stat3 alpha plasmid did not. We conclude that there is a short form of Stat3 (Stat3 beta), that Stat3 beta is transcriptionally active under conditions where Stat3 alpha is not, and that Stat3 beta and c-Jun are capable of cooperative activation of certain promoters. PMID: 7568080 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 108: Oncogene. 1995 Mar 2;10(5):985-94. Transcriptional regulation of the junB promoter: analysis of STAT-mediated signal transduction. Coffer P, Lutticken C, van Puijenbroek A, Klop-de Jonge M, Horn F, Kruijer W. Hubrecht Laboratory for Developmental Biology, Utrecht, The Netherlands. The product of the junB gene is a member of the AP-1 family of transcription factors that activate transcription by binding to TPA-responsive elements (TREs) within the promoters of target genes. Components of AP-1 are immediate-early genes whose expression is upregulated by a plethora of extracellular stimuli and are important in mediating cellular proliferation and differentiation. Such stimuli include the pleiotropic cytokine interleukin-6 (IL-6) which plays a role in immune and inflammatory responses and ciliary neurotrophic factor (CNTF) which enhances survival and differentiation of neurons and glia. We have analysed expression from junB promoter-CAT reporter constructs in HepG2 cells and found that a region between -196 and -91 can mediate response to IL-6 and CNTF and was able to confer responsiveness to a heterologous promoter. We further show by gel retardation analysis that distinct nuclear factors induced by IL-6 specifically bind to this interleukin-6 response element (IRE). This region contains both a putative ETS- and a STAT-transcription factor binding site. We show by mutational analysis and supershift data that the IL-6 induced complex indeed contains the transcription factor APRF/Stat3 that is both necessary and sufficient for activation. Interestingly this site does not appear to bind Stat1 itself, as shown by supershift analysis and a lack of response to IFN-gamma both at the DNA-binding and transcriptional level. Furthermore, we demonstrate that the junB IRE-binding activity induced by IL-6 requires tyrosine kinase activity, whereas induced transactivation of IRE-constructs additionally occurs through an H7-sensitive pathway that is p21ras-independent, implicating serine/threonine kinases in the transactivation of IRE-binding factors. PMID: 7898939 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 109: FEBS Lett. 1995 Feb 27;360(2):137-43. Interleukin-6-induced serine phosphorylation of transcription factor APRF: evidence for a role in interleukin-6 target gene induction. Lutticken C, Coffer P, Yuan J, Schwartz C, Caldenhoven E, Schindler C, Kruijer W, Heinrich PC, Horn F. Institut fur Biochemie der Rheinisch-Westfalischen Technischen Hochschule Aachen, Germany. The cytokine interleukin-6 (IL-6) rapidly activates a latent cytoplasmic transcription factor, acute-phase response factor (APRF), by tyrosine phosphorylation. Activation and DNA binding of APRF are inhibited by inhibitors of protein tyrosine kinases but not serine/threonine kinases. However, immediate-early gene induction by IL-6 and, as we show here, stimulation of the promoters of the genes for alpha 2-macroglobulin, Jun-B, and intercellular adhesion molecule-1 (ICAM-1) are blocked by the serine/threonine kinase inhibitor H7. We now show that IL-6 triggers a delayed phosphorylation of APRF at serine resudues which can be reversed in vitro by protein phosphatase 2A and is also inhibited by H7. Therefore, APRF serine phosphorylation is likely to represent a crucial event in IL-6 signal transduction leading to target gene induction. PMID: 7533107 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 110: J Immunol. 1994 Nov 15;153(10):4573-82. E1A repression of IL-6-induced gene activation by blocking the assembly of IL-6 response element binding complexes. Takeda T, Nakajima K, Kojima H, Hirano T. Division of Molecular Oncology, Osaka University Medical School, Japan. Some viral products interfere with host antiviral defense mechanisms. Adenovirus E1A represses IFN signal transduction pathways which induces gene activation and an antiviral state. Both IFN and IL-6 activate Jak/Tyk protein tyrosine kinases and the STAT (signal transducer and activator of transcription) family proteins. We showed that 12S E1A repressed IL-6 signals activating the junB promoter and the two IL-6 response elements (REs), JRE-IL6 and type II IL-6 RE (also called acute phase response element), required for IL-6-induced activation of the junB promoter and the type II acute phase reactant genes, respectively, in hepatocytes. Conserved region 1 of the 12S E1A was responsible for the repression. Target molecules of the repression by E1A appeared to be IL-6-inducible DNA-binding proteins acting on the IL-6 REs. In a rat 3Y1 cell line stably expressing E1A, the levels of IL-6-induced IL-6 RE binding proteins were severely reduced compared with those in a parental 3Y1 cell line. Moreover, we found that the levels of the STAT family proteins including Stat1-alpha (p91), Stat1-beta (p84), Stat2 (p113), and Stat3 were decreased by the stable expression of adenovirus E1A. The E1A-induced reduction in the amount of DNA-binding proteins seemed to be partly responsible for the decreased transcriptional activity of the IL-6 RE-driven gene expression in response to IL-6. This repression mechanism may be applicable to the E1A repression of IFN-gamma-induced gene activation. PMID: 7963529 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 111: Biochem Biophys Res Commun. 1994 Jul 29;202(2):1181-7. Transcriptional activation of the IL-6 response element in the junB promoter is mediated by multiple Stat family proteins. Fujitani Y, Nakajima K, Kojima H, Nakae K, Takeda T, Hirano T. Division of Molecular Oncology, Osaka University Medical School, Japan. IL-6 signals activate an IL-6 response element in the junB promoter, JRE-IL6, in a Ras-independent manner. IL-6 rapidly induced a DNA-binding activity to the Ets binding site of JRE-IL6 (JEBS), one of necessary DNA motifs of the IL-6 response element. The IL-6-induced JEBS-binding activity was indistinguishable from those to acute phase response element (APRE) in both kinetics of induction and its DNA-binding specificity. Purified APRE binding factors (APRFs) from IL-6-stimulated rat liver were found to be composed of multiple Stat3-related proteins and Stat1. Moreover the purified APRFs specifically made a complex with JRE-IL6 with the same mobility as that observed in the crude extracts. These results indicate that an immediate early signal of IL-6 leading to activation of JRE-IL6 is mediated by STAT family transcription factors. PMID: 7519422 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------