1: J Virol. 2005 Oct;79(19):12554-65. Attenuated rabies virus activates, while pathogenic rabies virus evades, the host innate immune responses in the central nervous system. Wang ZW, Sarmento L, Wang Y, Li XQ, Dhingra V, Tseggai T, Jiang B, Fu ZF. Department of Pathology, College of Veterinary Medicine, University of Georgia, 501 D.W. Brooks Drive, Athens, GA 30602, USA. Rabies virus (RV) induces encephalomyelitis in humans and animals. However, the pathogenic mechanism of rabies is not fully understood. To investigate the host responses to RV infection, we examined and compared the pathology, particularly the inflammatory responses, and the gene expression profiles in the brains of mice infected with wild-type (wt) virus silver-haired bat RV (SHBRV) or laboratory-adapted virus B2C, using a mouse genomic array (Affymetrix). Extensive inflammatory responses were observed in animals infected with the attenuated RV, but little or no inflammatory responses were found in mice infected with wt RV. Furthermore, attenuated RV induced the expression of the genes involved in the innate immune and antiviral responses, especially those related to the alpha/beta interferon (IFN-alpha/beta) signaling pathways and inflammatory chemokines. For the IFN-alpha/beta signaling pathways, many of the interferon regulatory genes, such as the signal transduction activation transducers and interferon regulatory factors, as well as the effector genes, for example, 2'-5'-oligoadenylate synthetase and myxovirus proteins, are highly induced in mice infected with attenuated RV. However, many of these genes were not up-regulated in mice infected with wt SHBRV. The data obtained by microarray analysis were confirmed by real-time PCR. Together, these data suggest that attenuated RV activates, while pathogenic RV evades, the host innate immune and antiviral responses. PMID: 16160183 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: J Rheumatol. 2005 Sep;32(9):1650-3. The Jak-STAT pathway in rheumatoid arthritis. Walker JG, Smith MD. Publication Types: Editorial PMID: 16142855 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Gut. 2005 Aug 24; [Epub ahead of print] Altered expression and activation of STATs (signal transduction and activator of transcription) in HCV infection: in vivo and in vitro studies. Larrea E, Aldabe R, Molano E, Fernandez-Rodriguez CM, Ametzazurra A, Civeira MP, Prieto J. CIMA/Clinica Universitaria, Spain. BACKGROUND: STATs play a critical role in antiviral defense. STAT3 is also important in cell protection against inflammatory damage. STAT proteins are activated by interferons and by hepatoprotective cytokines of IL6-superfamily, including cardiotrophin- 1. METHODS: We analyzed the status of STATs in HCV- infected livers and the relationship between the expression and activation of STATs and HCV replication in Huh7 cells transfected with HCV genomic replicon. RESULTS: STAT3alpha expression was reduced in HCV-infected livers showing inverse correlation with serum ALT. In patients with HCV infection nuclear staining for phosphorylated-STAT3 was faint in parenchymal cells (although conspicuous in infiltrating leukocytes), in contrast with the strong nuclear staining in hepatocytes from control livers. Expression and activation of STAT1 (a factor activated by both IFN& [alpha] and IFNgamma) were increased in HCV-infected livers particularly in those with high inflammatory activity. Conversely, phosphorylated-STAT2 (a factor selectively activated by IFNalpha) was undetectable in livers with HCV infection, a finding that was associated with marked down-regulation of the two functional subunits of IFNalpha receptor. HCV replication in Huh7 cells caused STAT3alpha down-regulation and blocked STAT3 phosphorylation by either IFNalpha or cardiotrophin-1. HCV replication in Huh7 cells also inhibited STAT1 and STAT2 activation by IFNalpha while there was no impairment of STAT1 phosphorylation by the pro-inflammatory cytokine IFNgamma. CONCLUSIONS: STAT3 is down-regulated in HCV- infected livers and in Huh7 cells bearing the full-length HCV replicon. HCV replication is associated with impaired Jak-STAT signaling by antiviral and cytoprotective cytokines. These effects may favor viral replication while facilitating the progression of liver disease. PMID: 16120756 [PubMed - as supplied by publisher] --------------------------------------------------------------- 4: J Biol Chem. 2005 Oct 7;280(40):34306-15. Epub 2005 Aug 17. Modification of the Stat1 SH2 domain broadly improves interferon efficacy in proportion to p300/CREB-binding protein coactivator recruitment. Zhang Y, Takami K, Lo MS, Huang G, Yu Q, Roswit WT, Holtzman MJ. Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA. A normal level of interferon (IFN) responsiveness via the Stat1 transcription factor is critical to the host, since decreased Stat1 signaling causes immune compromise and increased signaling is associated with inflammatory and neoplastic disease. Here we report how this balance may be influenced by novel alterations in the efficiency of Stat1 signaling. To enable disulfide-dependent and spontaneous formation of active Stat1 homodimer (as was done previously for Stat3), we engineered Stat1-CC with double-cysteine substitutions in the Src homology 2 (SH2)-homodimerization domain (at Ala-656 and Asn-658). In this case, however, mutant and wild-type Stat1 exhibited no difference inspontaneousdimerization. Moreover, Stat1-CC still required ligand-dependent Tyr-701 phosphorylation for function and exhibited hyperresponsiveness to IFN-beta (that depends on Stat1/Stat2 heterodimerization) as well as IFN-gamma (that depends on Stat1/Stat1 homodimerization). Hyperresponsivenss of Stat1-CC was accompanied by increased capacities for Tyr-701 phosphorylation and DNA binding, but these features were also found in a similarly substituted serine mutant (Stat1-SS) that showed no hyperresponsiveness to IFN-gamma. This finding raised the possibility that SH2 domain mutations also influence downstream transcriptional efficiency. Indeed, each of these mutations also enhanced recruitment of the normally rate-limiting p300/CREB-binding Protein (CBP) coactivator to the transcriptional complex in proportion to the level of IFN-driven transactivation and gene expression. Additional modifications indicated that the mutant residues in the SH2 domain appeared to cooperate with Ser-727 in the C-terminal domain to regulate p300/CBP interaction with Stat1. The profile of IFN responsiveness translated into the same progressive increase in the level of viral clearance from Stat1- to Stat1-SS- to Stat1-CC-expressing cells. Thus, SH2 domain determinants may be modified to direct better Stat1 phosphorylation, DNA binding, and coactivator recruitment to fully improve IFN efficacy. PMID: 16107341 [PubMed - in process] --------------------------------------------------------------- 5: J Virol. 2005 Aug;79(16):10180-9. Composition and assembly of STAT-targeting ubiquitin ligase complexes: paramyxovirus V protein carboxyl terminus is an oligomerization domain. Ulane CM, Kentsis A, Cruz CD, Parisien JP, Schneider KL, Horvath CM. Mount Sinai School of Medicine, New York, New York 10029, USA. Transcription regulators STAT1 and STAT2 are key components of the interferon signaling system leading to innate antiviral immunity. The related STAT3 protein is a regulator of interleukin-6-type cytokine signals and can contribute to both cell growth and death important for cancer gene regulation and tumor survival. These three STAT proteins are targeted for proteasome-mediated degradation by RNA viruses in the Rubulavirus genus of the Paramyxoviridae. A single viral protein, the V protein, assembles STAT-specific ubiquitin ligase complexes from cellular components. Simian virus 5 (SV5) targets STAT1, human parainfluenza virus 2 targets STAT2, and mumps virus targets both STAT1 and STAT3. Analysis of the V-dependent degradation complex (VDC) composition and assembly revealed several features contributing to targeting specificity. SV5 and mumps V proteins require STAT2 to recruit the STAT1 target, yet mumps V protein binds STAT3 independent of STAT1 and STAT2. All Rubulavirus V proteins tested require cellular DDB1 to target STATs for degradation but differ in the use of Roc1, which is essential for mumps V STAT3 targeting. Protein interaction analysis reveals that paramyxovirus V proteins can homo- and heterooligomerize and that the conserved cysteine-rich zinc-binding C-terminal domain is necessary and sufficient for oligomerization. Purified SV5 V protein spontaneously assembles into spherical macromolecular particles, and similar particles constitute SV5 and mumps VDC preparations. PMID: 16051811 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Drug News Perspect. 2005 May;18(4):243-9. JAK/STAT-dependent gene regulation by cytokines. Hebenstreit D, Horejs-Hoeck J, Duschl A. Department of Molecular Biology, University of Salzburg, Austria. daniel.hebenstreit@sbg.ac.at The Janus kinase-signal transducer and activator of transcription (Jak-STAT) pathway is essential for the signal transduction of many cytokines. Dysregulation of Jak-STAT signaling is associated with various human diseases. Recent studies have helped to shed some light on regulatory mechanisms that modify quantity and quality of the signaling response. Here, we summarize our current knowledge on Jak-STAT signaling. 2005 Prous Science. All rights reserved Publication Types: Review Review, Tutorial PMID: 16034480 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: J Immunol. 2005 Aug 1;175(3):1686-93. Augmentation of effector CD8+ T cell generation with enhanced granzyme B expression by IL-27. Morishima N, Owaki T, Asakawa M, Kamiya S, Mizuguchi J, Yoshimoto T. Intractable Immune System Disease Research Center, Tokyo Medical University, Tokyo, Japan. IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation. We have recently demonstrated that IL-27 has a potent antitumor activity, which is mainly mediated through CD8+ T cells, and also has an adjuvant activity to induce epitope-specific CTL in vivo. In this study, we further investigated the in vitro effect of IL-27 on CD8+ T cells of mouse spleen cells. In a manner similar to CD4+ T cells, IL-27 activated STAT1, -2, -3, -4, and -5, and augmented the expression of T-bet, IL-12Rbeta2, and granzyme B, and slightly that of perforin in naive CD8+ T cells stimulated with anti-CD3. IL-27 induced synergistic IFN-gamma production with IL-12 and proliferation of naive CD8+ T cells. Moreover, IL-27 enhanced proliferation of CD4+ T cell-depleted spleen cells stimulated by allogeneic spleen cells and augmented the generation of CTL. In STAT1-deficient naive CD8+ T cells, IL-27-induced proliferation was not reduced, but synergistic IFN-gamma production with IL-12 was diminished with decreased expression of T-bet, IL-12Rbeta2, granzyme B, and perforin. In T-bet-deficient naive CD8+ T cells, IL-27-induced proliferation was hardly reduced, but synergistic IFN-gamma production with IL-12 was diminished with decreased expression of IL-12Rbeta2, granzyme B, and perforin. However, IL-27 still augmented the generation of CTL from T-bet-deficient CD4+ T cell-depleted spleen cells stimulated by allogeneic spleen cells with increased granzyme B expression. These results suggest that IL-27 directly acts on naive CD8+ T cells in T-bet-dependent and -independent manners and augments generation of CTL with enhanced granzyme B expression. PMID: 16034109 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Mol Cell Biol. 2005 Jul;25(13):5456-65. Stat1 and Stat2 but not Stat3 arbitrate contradictory growth signals elicited by alpha/beta interferon in T lymphocytes. Gimeno R, Lee CK, Schindler C, Levy DE. Department of Pathology and Microbiology, 550 First Ave. MSB548, New York University School of Medicine, New York, New York 10016, USA. Alpha/beta interferon (IFN-alpha/beta) triggers antiviral and antiproliferative responses in target cells through modulation of gene expression. The JAK-STAT pathway is the major mediator of these biological effects through the activation of the transcription factors STAT1 and STAT2, and gene ablation studies have demonstrated that both STAT1 and STAT2 are required for most antiviral responses induced by IFN-alpha/beta. However, additional signaling pathways are also activated by IFN. Here, we show that these additional pathways provoke a proliferative response in activated T lymphocytes. While activation of IFN-stimulated gene factor 3 produces a dominant inhibitory signal capable of overriding the mitogenic response, absence of either STAT1 or STAT2 leads to a proliferative response to IFN. Growth stimulation by IFN-alpha/beta is independent of other STAT proteins, particularly of STAT3, since T lymphocytes from STAT1-STAT3 double-knockout mice are growth stimulated by IFN-alpha/beta treatment. IFN-alpha/beta can cooperate with numerous T-cell mitogens, including interleukin-2 (IL-2), IL-4, IL-7, and IL-12, and can contribute to the rapid restoration of the thymus following glucocorticoid-mediated ablation. These results underscore the complexity of the cellular response to IFN and suggest that the ultimate outcome of IFN action results from a balance between growth-inhibitory and -stimulatory effects. PMID: 15964802 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: J Biol Chem. 2005 Jul 8;280(27):25849-53. Epub 2005 May 9. Interferon alpha activates NF-kappaB in JAK1-deficient cells through a TYK2-dependent pathway. Yang CH, Murti A, Valentine WJ, Du Z, Pfeffer LM. Department of Pathology and Laboratory Medicine, University of Tennessee Health Science Center and the University of Tennessee Cancer Institute, Memphis, Tennessee 38163, USA. In addition to activating members of the STAT transcription factor family, interferon alpha/beta (IFNalpha/beta) activates the NF-kappaB transcription factor. To determine the role of the Janus tyrosine kinase (JAK)-STAT pathway in NF-kappaB activation by IFN, we examined NF-kappaB activation in JAK1-deficient mutant human fibrosarcoma cells. In wild-type fibrosarcoma cells (2fTGH), IFN activates STAT1, STAT2, and STAT3, as well as NF-kappaB complexes comprised of p50 and p65. In contrast, in JAK1-deficient cells, IFN induces NF-kappaB activation and NF-kappaB dependent gene transcription but does not activate these STAT proteins and has no effect on STAT-dependent gene transcription. Expression of a catalytically inactive TYK2 tyrosine kinase in JAK1-deficient cells, as well as in the highly IFN-sensitive Daudi lymphoblastoid cell line, abrogates NF-kappaB activation by IFN. Moreover, IFN does not promote NF-kappaB activation in TYK2-deficient mutant fibrosarcoma cells. Our results demonstrate a dichotomy between the classical JAK-STAT pathway and the NF-kappaB signaling pathway. In the IFN signaling pathway leading to STAT activation, both JAK1 and TYK2 are essential, whereas NF-kappaB activation requires only TYK2. PMID: 15883164 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: Am J Gastroenterol. 2005 Jan;100(1):64-72. Comment in: Am J Gastroenterol. 2005 Jan;100(1):73-4. Activation pattern of signal transducers and activators of transcription (STAT) factors in inflammatory bowel diseases. Mudter J, Weigmann B, Bartsch B, Kiesslich R, Strand D, Galle PR, Lehr HA, Schmidt J, Neurath MF. Department of 1st Medical Clinic, Department of Pathology, Johannes-Gutenberg-University of Mainz, Langenbeckstrasse 1, 55101 Mainz, Germany. OBJECTIVES: Cytokine signaling pathways involving transcription factors of the signal transducers and activators of transcription (STAT) family play a key role in the pathogenesis of inflammatory bowel diseases (IBD). STAT proteins are latent cytoplasmic transcription factors that induce transcription upon phosphorylation, dimerization, and nuclear translocation. However, their activation pattern in IBD is poorly understood. The aim of our study was to characterize STAT-expression in IBD. METHODS: Mononuclear cells were isolated from 36 colonic specimens of Crohn's disease, ulcerative colitis, or from control patients. Cells were stimulated overnight with antibodies against human CD2 and CD28 and mononuclear cells were analyzed by flow cytometry. Alternatively, CD4(+) T cells were immunomagnetically separated and then assessed by flow cytometry. Intracellular stainings of the following transcription factors were performed: STAT-1, STAT-2, STAT-3, STAT-4, and STAT-6. In addition, immunofluorescence staining on cryosections for phosphorylated STAT-1 and STAT-3 was performed. RESULTS: Average expression of the IFN-gamma inducible transcription factor STAT-1 was increased in Crohn's disease as compared to patients with ulcerative colitis and control patients. However, levels of phospho-STAT-1 were surprisingly not markedly upregulated in IBD as compared to controls. In contrast, STAT-3 and phospho-STAT-3 levels were significantly increased in IBD patients as compared to controls (p < 0.01). No differences could be detected in STAT-6 levels. Finally, average expression of STAT-2, which is involved in type I interferon signalling, was downregulated in IBD as compared to control patients. CONCLUSIONS: The analysis of STAT activation patterns could serve as a helpful tool to characterize intestinal inflammation. Furthermore, the IL-6/STAT-3 rather than the IFN-gamma/STAT-1 signaling pathway emerges as a key target for the development of future therapeutic concepts in IBD. PMID: 15654782 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: J Cell Physiol. 2005 Apr;203(1):209-16. JAK and STAT proteins are expressed and activated by IFN-gamma in rat pancreatic acinar cells. Gallmeier E, Schafer C, Moubarak P, Tietz A, Plossl I, Huss R, Goke B, Wagner AC. Department of Internal Medicine II, Klinikum Grosshadern, Ludwig-Maximilians-University, Munich, Germany. egallme1@jhmi.edu The development of acute pancreatitis (AP) is triggered by acinar events, but the subsequent extra-acinar events, particularly a distinct immune response, appear to determine its severity. Cytokines modulate this immune response and are derived not only from immunocytes but also from pancreatic acinar cells. We studied whether pancreatic acinar cells were also capable of responding to cytokines. The JAK/STAT-pathway represents the main effector for many cytokines. Therefore, expression and regulation of JAK and STAT proteins were investigated in rat pancreatic acinar cells. Western blotting showed expression of JAK1, JAK2, Tyk2, and STAT1, STAT2, STAT3, STAT5, STAT6. In addition, STAT1 was reversibly tyrosine-phosphorylated upon the procedure of acinar cell isolation. In contrast, STAT3-phosphorylation occurred spontaneously after pancreas removal and was not reversible within 8 h. STAT1 phosphorylation was also observed upon treatment with IFN-gamma but not upon EGF, TNF-alpha or IL-6, and inhibited by the JAK2-inhibitor AG-490. Immunohistochemistry revealed cytoplasmic expression of unphosphorylated STAT1 in untreated acinar cells and nuclear translocation of phosphorylated STAT1 following IFN-gamma-treatment. Interestingly, although CCK leads to the activation of multiple stress pathways in pancreatic acinar cells, we found no influence of CCK on phosphorylation of STAT1, STAT3, or STAT5 in the pancreas. In conclusion, our data provide further evidence that pancreatic acinar cells are able to interact with immune cells. Besides stimulating immune cells via cytokine secretion, acinar cells are in turn capable of responding to IFN-gamma via JAK2 and STAT1 which may have an impact on the development of AP. 2004 Wiley-Liss, Inc. PMID: 15493010 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: Biochem Biophys Res Commun. 2004 Nov 12;324(2):518-28. STAT3 induces anti-hepatitis C viral activity in liver cells. Zhu H, Shang X, Terada N, Liu C. Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA. Hepatitis C virus (HCV) infection is a leading cause a of chronic liver disease worldwide. The main therapeutic regimen is the combination of interferon alpha (IFN) and the nucleoside analog, Ribavirin. IFN initiates an intracellular antiviral state by the JAK-STAT signaling pathway, including a presumed role for STAT1 and STAT2. We have previously shown that the STAT3 activation occurs during IFN treatment of human hepatoma cells, suggesting that the STAT3-mediated pathway is relevant to IFN-induced antiviral activity. In this study, we investigate the role of activated STAT3 in the induction of anti-HCV activity in human hepatoma cells. We demonstrate that the STAT3 activation is involved in efficient IFN-induced anti-HCV activity. Using an inducible, cytokine-independent, STAT3 activation system, in which the entire coding region of STAT3 is fused with the ligand-binding domain of the estrogen receptor, we demonstrate that: activated STAT3 is tightly regulated in a stably transfected cell line by an estrogen analog, 4-HT; activated STAT3 initiates efficient anti-HCV activity in a HCV subgenomic replicon cell line; and activation of STAT3 is associated with the induction of a potential antiviral gene, 1-8U. In addition, we show that the cytokine IL-6, a potent STAT3 activator, inhibits HCV subgenomic RNA replication through STAT3 activation and ERK pathway. These results strongly suggest that STAT3 activation is capable of initiating intracellular antiviral pathways. PMID: 15474458 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Expert Opin Ther Targets. 2004 Oct;8(5):409-22. STAT proteins as novel targets for cancer drug discovery. Turkson J. Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, SRB 22214, Tampa, FL 33612, USA. turksonj@moffitt.usf.edu Signal transducer and activator of transcription (STAT) proteins are latent cytoplasmic transcription factors that were discovered in the context of cytokine and growth factor signalling. Normal STAT signalling is tightly controlled with finite kinetics, which is in keeping with standard cellular responses. However, persistent STAT activation has also been observed and is frequently associated with malignant transformation. Constitutive activation of STAT proteins, notably of Stat3 and Stat5, is detected in many human tumour cells and cells transformed by oncoproteins that activate tyrosine kinase signalling pathways. It is well-established that constitutively active Stat3 is one of the molecular abnormalities that has a causal role in oncogenesis. Aberrant Stat3 promotes uncontrolled growth and survival through dysregulation of gene expression, including cyclin D1, c-Myc, Bcl-xL, Mcl-1 and survivin genes, and thereby contributes to oncogenesis. Moreover, recent studies reveal that persistently active Stat3 induces tumour angiogenesis by upregulation of vascular endothelial growth factor induction, and modulates immune functions in favour of tumour immune evasion. Overall, studies have validated Stat3 as a novel target for cancer therapy, and hence provided the rationale for developing small-molecule Stat3 inhibitors. This review will discuss current evidence for the critical role of aberrant STAT signalling in malignant transformation, and examine the validity as well as the therapeutic potential of Stat3 as a cancer target. An update on the efforts to develop novel Stat3 inhibitors for therapeutic application will also be provided. Publication Types: Review PMID: 15469392 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Curr Pharm Des. 2004;10(23):2839-50. The role of STATs in inflammation and inflammatory diseases. Pfitzner E, Kliem S, Baus D, Litterst CM. Institute for Biomedical Research, Georg-Speyer-Haus, Paul-Ehrlich-Str. 42-44, 60596 Frankfurt am Main, Germany. e.pfitzner@em.uni-frankfurt.de The immune response is regulated by the concerted action of pro- and anti-inflammatory cytokines. The deregulation of this process causes immunological disorders like allergic and autoimmune diseases. The Janus Kinase (JAK)--Signal transducer and activator of transcription (STAT) pathway is one major signaling pathway converting the cytokine signal into gene expression programs regulating the proliferation and differentiation of the immune cells. Several members of the STAT protein family in particular STAT1, STAT2, STAT3, STAT4 and STAT6 act as transcription factors in modulating pro- and anti-inflammatory responses. Here we review the evidence for the involvement of the different STAT proteins in inflammation, autoimmune and allergic diseases. We discuss novel approaches to interfere with the function of these signaling transcription factors for therapeutic purpose. Publication Types: Review PMID: 15379672 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Nippon Rinsho. 2004 Jul;62 Suppl 7(Pt 1):448-58. [Human interferon: molecular mechanism of its antiviral activities] [Article in Japanese] Yoshida I. Department of Microbiology and Immunology, Asahikawa Medical College. Publication Types: Review Review, Tutorial PMID: 15359840 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: J Immunol. 2004 Sep 15;173(6):3871-7. An indispensable role for STAT1 in IL-27-induced T-bet expression but not proliferation of naive CD4+ T cells. Kamiya S, Owaki T, Morishima N, Fukai F, Mizuguchi J, Yoshimoto T. Intractable Immune System Disease Research Center, Tokyo Medical University, Japan. IL-27 is a novel IL-12 family member that plays a role in the early regulation of Th1 initiation, induces proliferation of naive CD4+ T cells, and synergizes with IL-12 in IFN-gamma production. It has been recently reported that IL-27 induces T-bet and IL-12Rbeta2 expression through JAK1/STAT1 activation. In the present study, we further investigated the JAK/STAT signaling molecules activated by IL-27 and also the role of STAT1 in IL-27-mediated responses using STAT1-deficient mice. In addition to JAK1 and STAT1, IL-27-activated JAK2, tyrosine kinase-2, and STAT2, -3, and -5 in naive CD4+ T cells. The activation of STAT2 and STAT5, but not of STAT3, was greatly diminished in STAT1-deficient naive CD4+ T cells. Comparable proliferative response to IL-27 was observed between STAT1-deficient and wild-type naive CD4+ T cells. In contrast, IL-27 hardly induced T-bet and subsequent IL-12Rbeta2 expression, and synergistic IFN-gamma production by IL-27 and IL-12 was impaired in STAT1-deficient naive CD4+ T cells. Moreover, IL-27 augmented the expression of MHC class I on naive CD4+ T cells in a STAT1-dependent manner. These results suggest that IL-27 activates JAK1 and -2, tyrosine kinase-2, STAT1, -2, -3, and -5 in naive CD4+ T cells and that STAT1 plays an indispensable role in IL-27-induced T-bet and subsequent IL-12Rbeta2 expression and MHC class I expression as well but not proliferation, while STAT3 presumably plays an important role in IL-27-induced proliferation. Copyright 2004 The American Association of Immunologists, Inc. PMID: 15356135 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: Mol Endocrinol. 2004 Dec;18(12):2997-3010. Epub 2004 Aug 19. Synergistic signaling by corticotropin-releasing hormone and leukemia inhibitory factor bridged by phosphorylated 3',5'-cyclic adenosine monophosphate response element binding protein at the Nur response element (NurRE)-signal transducers and activators of transcription (STAT) element of the proopiomelanocortin promoter. Mynard V, Latchoumanin O, Guignat L, Devin-Leclerc J, Bertagna X, Barre B, Fagart J, Coqueret O, Catelli MG. Institut Cochin, Departement d'Endo-crinologie, 24 rue du Faubourg Saint Jacques, 75014 Paris, France. Leukemia inhibitory factor (LIF) cooperates with CRH at the pituitary level to induce POMC gene transcription, resulting in activation of the pituitary-adrenal axis. However, the underlying molecular mechanisms remain elusive. Here, we show that the NurRE-signal transducers and activators of transcription (STAT) composite element of the POMC promoter was the predominant target of the LIF-CRH synergy. Whereas NurRE or STAT sites alone conferred synergy, the maximal response was found with the NurRE-STAT reporter, suggesting that direct DNA binding of both transcription factors is required for an optimal synergy. During LIF-CRH stimulation, Nur77 and activated STAT1-3 were bound to the composite element, and the binding of each factor was abolished by appropriate mutations. CREB was also detected in this complex in a stimulation-dependent and DNA binding-independent manner. Nur77 and STAT1-3 bound to the NurRE-STAT site were each sufficient for CREB recruitment. Recombinant CREB directly interacted with recombinant Nur77 or STAT1-3. Moreover, CREB-Nur77 interaction was increased by CREB phosphorylation at Ser-133 and the dominant-negative mutant CREB-M1 efficiently inhibited the synergistic LIF-CRH response. This synergism was also inhibited after transfection of CREB-small interfering RNA. We conclude that both CREB phosphorylation at Ser-133 and level of CREB expression are crucial in LIF-CRH synergism where CREB, without direct DNA binding, could improve the stability of Nur77 and STAT1-3 binding to POMC promoter and facilitate the recruitment of coactivators. This novel intrapituitary signaling mechanism may have more general implications in cross talks between cAMP-protein kinase A and Janus kinase-STAT pathways. PMID: 15319449 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: J Virol. 2004 Sep;78(17):9285-94. Blocking of the alpha interferon-induced Jak-Stat signaling pathway by Japanese encephalitis virus infection. Lin RJ, Liao CL, Lin E, Lin YL. Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China. The induction of alpha/beta interferon (IFN-alpha/beta) is a powerful host defense mechanism against viral infection, and many viruses have evolved strategies to overcome the antiviral effects of IFN. In this study, we found that IFN-alpha had only some degree of antiviral activity against Japanese encephalitis virus (JEV) infection, in contrast to another flavivirus, dengue virus serotype 2, which was highly sensitive to IFN-alpha in the cultured cell system. JEV infection appeared to render cells resistant to IFN-alpha since the IFN-alpha-induced luciferase reporter activity driven by the IFN-stimulated response element (ISRE) was gradually reduced as the JEV infection progressed. Since the biological activities of IFNs are triggered by the Janus kinase (Jak) signal transducer and activation of transcription (Stat) signaling cascade, we then studied the activation of Jak-Stat pathway in the virus-infected cells. The IFN-alpha-stimulated tyrosine phosphorylation of Stat1, Stat2, and Stat3 was suppressed by JEV in a virus replication and de novo protein synthesis-dependent manner. Furthermore, JEV infection blocked the tyrosine phosphorylation of IFN receptor-associated Jak kinase, Tyk2, without affecting the expression of IFN-alpha/beta receptor on the cell surface. Consequently, expression of several IFN-stimulated genes in response to IFN-alpha stimulation was also reduced in the JEV-infected cells. Overall, our findings suggest that JEV counteracts the effect of IFN-alpha/beta by blocking Tyk2 activation, thereby resulting in inhibition of Jak-Stat signaling pathway. PMID: 15308723 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: Biol Reprod. 2004 Oct;71(4):1330-9. Epub 2004 Jun 9. Presence of permanently activated signal transducers and activators of transcription in nuclear interchromatin granules of unstimulated mouse oocytes and preimplantation embryos. Truchet S, Chebrout M, Djediat C, Wietzerbin J, Debey P. USM 503 MNHN, UMR 8646 CNRS-MNHN, U565 INSERM, Departement Regulation, Developpement et Diversite Moleculaire, Museum National d'Histoire Naturelle, 75005 Paris, France. sandrine.truchet@ibpc.fr We previously described that mouse oocytes and preimplantation embryos express the two subunits of interferon-gamma receptor. We now report that, despite the presence of STAT1 (signal transducer and activator of transcription 1) at both the mRNA and protein levels, interferon gamma (IFNgamma) as well as IFNalpha are unable to trigger massive nuclear translocation of STAT1 in these cells, even at high cytokine concentrations. Conversely, nuclear accumulation of STAT1 was readily observed in murine L929 somatic cells under the same conditions. However, in the absence of any stimulation, both tyrosine (Y701p) and serine (S727p) phosphorylated forms of STAT1 were already detected in the nuclei of oocytes and early embryos. Phosphorylated STAT1 appeared concentrated in large nuclear dots, which were identified by indirect immunofluorescence and electron microscopy as clusters of interchromatin granules (IGCs or speckles). A similar distribution was also observed for the serine (S727p) phosphorylated form of STAT3 as well as for tyrosine (Y689p) phosphorylated STAT2. Western blot analysis confirmed that STAT factors present in mouse oocytes are predominantly phosphorylated. In parallel, we showed that the transcription of two IFNgamma-target genes, namely interferon regulatory factor-1 (IRF-1) and suppressor of cytokine signaling-1 (SOCS-1) is indeed increased in two-cell embryos in response to IFNgamma. Altogether, our results suggest that, despite the lack of massive nuclear accumulation of STAT1 in response to exogenous IFNs and the permanent presence of phosphorylated STATs in the nucleus, JAK/ STAT pathways are functional during early development. PMID: 15189833 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: J Leukoc Biol. 2004 Aug;76(2):416-22. Epub 2004 Jun 3. IFN-alpha regulates IL-21 and IL-21R expression in human NK and T cells. Strengell M, Julkunen I, Matikainen S. Department of Microbiology, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finland. mari.strengell@ktl.fi Interleukin (IL)-21 is a T cell-derived cytokine that regulates innate and adaptive immune responses. IL-21 receptor (IL-21R), which is expressed in natural killer (NK) and T cells, is structurally homologous to IL-2Rbeta and IL-15Ralpha. These receptors also share a common cytokine receptor gamma-chain with IL-4, IL-7, and IL-9. Macrophage- or dendritic cell-derived interferon (IFN)-alpha/beta is a key cytokine in regulation of NK and T cell functions. We demonstrate here that in addition to activating IFN-gamma gene expression, IFN-alpha/beta and IL-12 enhance the mRNA expression of IL-21 in activated human T cells. In addition, IFN-alpha/beta enhanced T cell receptor stimulation-induced IL-21 and IFN-gamma gene expression in resting T cells. The promoter analysis of IL-21 gene revealed a putative IFN-gamma activation site element, which was found to bind signal transducer and activator of transcription 1 (STAT1), STAT2, STAT3, and STAT4 proteins in IFN-alpha/beta-stimulated NK or T cell extracts. In contrast to IL-21 expression, IFN-alpha/beta down-regulated IL-21R mRNA expression in NK and T cells. IFN-alpha/beta-induced down-regulation of IL-21R expression resulted in reduced STAT3 phosphorylation and DNA binding after IL-21 stimulation. In conclusion, our results suggest a novel role for IFN-alpha/beta in the regulation of IL-21 response. PMID: 15178704 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: J Biol Chem. 2004 Jul 30;279(31):32269-74. Epub 2004 May 27. Role of the interleukin (IL)-28 receptor tyrosine residues for antiviral and antiproliferative activity of IL-29/interferon-lambda 1: similarities with type I interferon signaling. Dumoutier L, Tounsi A, Michiels T, Sommereyns C, Kotenko SV, Renauld JC. Ludwig Institute for Cancer Research, Brussels Branch and the Experimental Medicine Unit, Universite de Louvain, B-1200 Brussels, Belgium. Interferon (IFN)-lambda 1, -lambda 2, and -lambda 3 are the latest members of the class II cytokine family and were shown to have antiviral activity. Their receptor is composed of two chains, interleukin-28R/likely interleukin or cytokine or receptor 2 (IL-28R/LICR2) and IL-10R beta, and mediates the tyrosine phosphorylation of STAT1, STAT2, STAT3, and STAT5. Here, we show that activation of this receptor by IFN-lambda 1 can also inhibit cell proliferation and induce STAT4 phosphorylation, further extending functional similarities with type I IFNs. We used IL-28R/LICR2-mutated receptors to identify the tyrosines required for STAT activation, as well as antiproliferative and antiviral activities. We found that IFN-lambda 1-induced STAT2 tyrosine phosphorylation is mediated through tyrosines 343 and 517 of the receptor, which showed some similarities with tyrosines from type I IFN receptors involved in STAT2 activation. These two tyrosines were also responsible for antiviral and antiproliferative activities of IFN-lambda 1. By contrast, STAT4 phosphorylation (and to some extent STAT3 activation) was independent from IL-28R/LICR2 tyrosine residues. Taken together, these observations extend the functional similarities between IFN-lambdas and type I IFNs and shed some new light on the mechanisms of activation of STAT2 and STAT4 by these cytokines. PMID: 15166220 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: Virology. 2004 May 20;323(1):141-52. Multiple signal transducers and activators of transcription are induced by EBV LMP-1. Zhang L, Hong K, Zhang J, Pagano JS. Nebraska Center for Virology, School of Biological Sciences, University of Nebraska, Lincoln, NE 68588, USA. lzhang2@unlnotes.unl.edu Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) is required for EBV immortalization of primary B cells in vitro. Signal transducers and activators of transcription (STATs) play a pivotal role in the initiation and maintenance of certain cancers. STAT proteins, especially STAT-1, -3, and -5, are persistently tyrosine phosphorylated or activated in many cancers. We show here that EBV-infected type III latency cells, in which the EBV oncoprotein, LMP-1 is expressed, express high levels of four STATs (STAT-1, -2, -3, and -5A) and that LMP-1 is responsible for the induction of three (STAT-1, -2, and -3). In addition, the C-terminal activator region 1 (CTAR-1) and CTAR-2 of LMP-1 cooperatively induced the expression of STAT-1. The cooperativity was evident when CTAR-1 and CTAR-2 were present in cis, but not in trans. Furthermore, NF-kappaB is an essential factor involved in the induction of STAT-1. Most of the induced STATs were not phosphorylated at the critical tyrosine residue activated by many cytokines. However, the induced STATs, at least STAT-1, were functional because it could be activated by interferon (IFN) and could upregulate an IFN-inducible gene. Finally, expression of STAT-1, but not STAT-2 and -3, is associated with EBV transformation. The association of the expression of STAT-1, -2, -3, and -5A with EBV type III latency and the expression of STAT-1 in the EBV transformation process may be part of the viral programming that regulates viral latency and cellular transformation. PMID: 15165826 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: Allergy. 2004 May;59(5):505-14. Differential expression of IL-10 receptor by epithelial cells and alveolar macrophages. Lim S, Caramori G, Tomita K, Jazrawi E, Oates T, Chung KF, Barnes PJ, Adcock IM. Department of Thoracic Medicine, Imperial College School of Medicine, National Heart and Lung Institute, Dovehouse Street, London, UK. BACKGROUND: Interleukin (IL)-10 is a pleiotropic cytokine with a broad spectrum of immunosuppressive and anti-inflammatory effects. IL-10 secretion from alveolar macrophages is defective in patients with asthma and lower concentrations of IL-10 are found in bronchoalveolar lavage (BAL) from asthmatic patients than in normal control subjects. Reduced IL-10 may result in exaggerated and more prolonged inflammatory responses in asthmatic airways. IL-10 acting through the IL-10 receptor (IL-10R) stimulates the transcription factors STAT1 and STAT3. METHODS: We investigated IL-10 and IL-10R expression in normal and asthmatic bronchial epithelium and BAL macrophages using reverse transcription-polymerase chain reaction, immunohistochemistry and Western blotting. The functional effect of IL-10 was examined using granulocyte-macrophage-colony stimulating factor, enzyme-linked immunosorbent assay and Western blotting for phosphorylated STAT1 and STAT3. RESULTS: IL-10 was not expressed in epithelial cells; furthermore these cells did not express the IL-10R and had no functional response to exogenous IL-10. Bronchial epithelial cells expressed variable levels of phosphorylated STAT1 and STAT3 with no change in expression between normal subjects and asthmatics. IL-10 protein and IL-10R expression was detected in alveolar macrophages from all subjects. CONCLUSION: Our study suggests that the bronchial epithelium is not a source of IL-10 and cannot respond to exogenous IL-10 because of a lack of IL-10R expression. PMID: 15080831 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: Int J Immunopathol Pharmacol. 2004 Jan-Apr;17(1):5-14. Novel shift of Jak/Stat signalling characterizes the protective effect of aurintricarboxylic acid (ATA) from tumor necrosis factor-alpha toxicity in human B lymphocytes. Marchisio M, Grimley PM, Di Baldassarre A, Santavenere E, Miscia S. Cell Signaling Unit, Department of Biomorphology, University of Chieti, Chieti, Italy. Previous results demonstrated that the occurrence of death in human peripheral B lymphocytes by TNF-alpha was paralleled by the activation of the cytoplasmic Jak1 and Tyk2 protein kinases, along with the recruitment of transcription factors Stat3 and Stat5b. In this study we demonstrate that the balance of survival signals in the presence of TNF-alpha was altered by the addition of a salicylate compound, the endonuclease inhibitor aurintricarboxylic acid (ATA). Apoptosis effected by TNF-alpha alone was suppressed by ATA and this event was paralleled by phosphorylation and nuclear translocation of Jak2, Stat2, Stat4 and NF-kB, along with inhibition of caspase activation. These results confirm that among the different cellular responses evoked by TNF-alpha in human B cells, recruitment of Jak/Stat proteins and possible related gene modulation represent contributing factors and address the issue of the development of potential therapeutic strategies aimed at the control of systemic or local effects produced by TNF-alpha. PMID: 15000861 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 25: J Am Soc Nephrol. 2004 Feb;15(2):276-85. Comment in: J Am Soc Nephrol. 2004 Feb;15(2):504-5. Activation of the signal transducer and activator of transcription signaling pathway in renal proximal tubular cells by albumin. Nakajima H, Takenaka M, Kaimori JY, Hamano T, Iwatani H, Sugaya T, Ito T, Hori M, Imai E. Department of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, Osaka, Japan. Renal proximal tubular cells activated by reabsorption of protein are thought to play significant roles in the progression of kidney diseases. It was hypothesized that the signal transducer and activator of transcription (STAT) proteins may be activated by proteinuria in proximal tubular cells. To test this hypothesis, murine proximal tubular cells were treated with albumin (30 mg/ml medium) for various lengths of time. The results showed that albumin could activate Stat1 and Stat5 within 15 min in proximal tubular cells. The activation of STATs was mediated mostly by Jak2 and required no protein synthesis. In addition, activation of Stat1 occurred even after neutralization of IFN-gamma. The activation of STATs was inhibited by N-acetyl-L-cysteine, a precursor of glutathione and a reactive oxygen species (ROS) scavenger, and fluorescence-activated cell sorter analysis showed upregulation of intracellular ROS after albumin overloading, suggesting that albumin per se could generate ROS in proximal tubular cells. The activation of STATs occurred by way of the ROS generating system, and especially through the membrane-bound NADPH oxidase system. Reduced activities of glutathione peroxidase and catalase could also be responsible for the accumulation of intracellular ROS. Hence, not only the ROS generating system, but also the ROS scavenging system may contribute to the induction of ROS by albumin. These findings support the hypothesis that proximal tubular cells are activated and generate ROS by reabsorption of abundant urinary proteins filtered through the glomerular capillaries, and as a consequence, various IFN-gamma-inducible proteins are synthesized through IFN-gamma-independent activation of STAT signaling. PMID: 14747374 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: J Biol Chem. 2004 Mar 26;279(13):12379-85. Epub 2004 Jan 13. Identification of a novel conserved motif in the STAT family that is required for tyrosine phosphorylation. Gamero AM, Sakamoto S, Montenegro J, Larner AC. Department of Immunology, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA. The rapid transcriptional activation of cellular genes by either type 1 interferons (IFNalpha/beta) or type 2 interferon (IFNgamma) is responsible for many of the pleiotropic effects of these cytokines, including their antiviral, antigrowth, and immunomodulatory activities. Interferon-stimulated gene expression is mediated by transcription factors termed Stats, which upon being tyrosine-phosphorylated, translocate to the nucleus and bind enhancers of interferon-activated genes. We have recently characterized a new Jurkat cell variant, named H123, where IFNalpha stimulates programmed cell death. H123 clones that are resistant to the apoptotic actions of IFNalpha have been selected. One of these clones (Clone 8) is defective in its responses to IFNalpha with regard to activation of genes that require tyrosine phosphorylation of Stat2. Stimulation of Clone 8 cells with IFNalpha induces normal tyrosine phosphorylation of Stat1 and Stat3. Sequencing of Stat2 RNA reveals a substitution of proline 630 located within the Src homology 2 domain of Stat2 to leucine (P630L). Pro-630 and its adjacent amino acids are conserved in all Stat family members but are absent in other proteins that contain Src homology 2 domains. Expression of Stat2 P630L in cells inhibits IFNalpha-stimulated gene expression. These results not only define a critical motif in Stat2 required for its transcriptional activity, but they also provide evidence that resistance to type one IFNs can be mediated by mutations in Stat2 as well as those previously described for Stat1. PMID: 14722125 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: Int J Oncol. 2004 Feb;24(2):365-72. Effectiveness of Type I interferons in the treatment of multidrug resistant osteosarcoma cells. Manara MC, Serra M, Benini S, Picci P, Scotlandi K. Laboratorio di Ricerca Oncologica, Istituti Ortopedici Rizzoli, I-40136 Bologna, Italy. P-glycoprotein overexpression is an important adverse prognostic marker for osteosarcoma (OS) patients, which is associated with higher risk for developing metastases as a consequence of the limited responsiveness to standard treatments of P-glycoprotein overexpressing OS cells. The use of cytokines has been advocated as a possible therapeutic approach to overcome multidrug resistance (MDR), being active on cell lines that are resistant to conventional drugs. In this study, we evaluated in vitro effects of interferons (IFNs) on MDR P-glycoprotein overexpressing OS cells. Type I IFNs, but not IFNgamma, showed tangible inhibitory effects on OS cell growth, which were higher in MDR cell lines compared to parental cells. The higher sensitivity of P-glycoprotein overexpressing cells to Type I IFNs correlates with higher expression of the activator of the transcription (STAT)-2 and (STAT)-3, two intracellular mediators of the IFNalpha and IFNbeta signaling pathways, whereas no differences were observed with respect to the expression or activation of the Type I IFN receptor and STAT-1. Exposure of OS MDR cells to Type I IFN decreased the expression of P-glycoprotein. This effect resulted in a significantly increased chemosensitivity of MDR cells to doxorubicin. Therefore, our data support the use of IFNalpha or IFNbeta in the treatment of osteosarcoma patients who overexpress P-glycoprotein in their primary tumors, and respond insufficiently to current therapeutic regimens. PMID: 14719113 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: Biochem J. 2004 Apr 1;379(Pt 1):199-208. Interferon-gamma inhibits interferon-alpha signalling in hepatic cells: evidence for the involvement of STAT1 induction and hyperexpression of STAT1 in chronic hepatitis C. Radaeva S, Jaruga B, Kim WH, Heller T, Liang TJ, Gao B. Section on Liver Biology, Laboratory of Physiologic Studies, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 12420 Parklawn Drive, MSC 8115, Bethesda, MD 20892, USA. IFN-gamma (interferon-gamma) modulates IFN-alpha therapy in chronic hepatitis C infection; however, the underlying mechanism remains unclear. Here we demonstrate that long-term (3-6 days) but not short-term (up to 1 day) IFN-gamma treatment of human hepatoma Hep3B cells attenuates IFN-alpha activation of STAT1 (signal transducers and activators of transcription factor 1), STAT2 and STAT3, but enhances IFN-gamma and interleukin 6 activation of STATs. Prolonged exposure to IFN-gamma also significantly induces STAT1 protein expression without affecting STAT2, STAT3 and ERK (extracellular-signal-regulated kinase) 1/2 protein expression. To determine the role of STAT1 protein overexpression in regulation of IFN-alpha signalling, Hep3B cells were stably transfected with wild-type STAT1. Overexpression of STAT1 via stable transfection enhances IFN-gamma activation of STAT1, but surprisingly attenuates IFN-alpha activation of STAT1, STAT2 and STAT3 without affecting Janus kinase activation. This STAT1-mediated inhibition does not require STAT1 tyrosine phosphorylation because overexpression of dominant-negative STAT1 with a mutation on tyrosine residue 701 also blocks IFN-alpha activation of STAT1, STAT2 and STAT3. Moreover, overexpression of STAT1 blocks IFN-alpha-activated STAT2 translocation from IFN-alpha receptor 2 to IFN-alpha receptor 1, a critical step in IFN-alpha signalling activation. Finally, significantly higher levels of STAT1 protein expression, which is probably induced by IFN-gamma, are detected in the majority of hepatitis C virus-infected livers compared with healthy controls. In conclusion, long-term IFN-gamma treatment inhibits IFN-alpha-activated signals most probably, at least in part, through the induction of STAT1 protein expression, which could partly contribute to IFN-alpha treatment failure in hepatitis C patients. PMID: 14690454 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: Mol Vis. 2003 Dec 16;9:715-22. Expression of cytokine signal transduction components in the postnatal mouse retina. Rhee KD, Yang XJ. Department of Ophthalmology, Jules Stein Eye Institute, David Geffen School of Medicine, Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA. PURPOSE: Members of the ciliary neurotrophic factor (CNTF) family of cytokines have been shown to influence neuronal differentiation during retinal development and enhance cell survival in various retinal degeneration models. However, the cellular mechanism of CNTF signaling and the target cell types for CNTF in the developing retina remain unidentified. The purpose of this study is to characterize expression patterns of proteins involved in cytokine signal transduction in the mouse retina, thus to assess the potential responsiveness of different retinal cell types to CNTF-like cytokine signals. METHODS: The expression profiles of various cytokine signal transduction components, including receptor subunits CNTF receptor alpha (CNTFRa) and gp130, intracellular protein kinases, Jak2 and Tyk2, as well as latent transcription factors, STAT1 and STAT3, were determined by immunohistochemical staining of mouse retinal sections derived from different postnatal stages. In addition, the distribution of ERK was studied by immunofluorescent staining. RESULTS: In the neonatal retina, intense staining signals for gp130, CNTFRalpha, Jak2, Tyk2, STAT1, and STAT3 were present in the differentiated ganglion cell layer and the developing inner plexiform layer of the mouse retina. Detectable staining signals were also observed in the ventricular zone of the early postnatal mouse retina. From P5 to P10, cytokine signaling molecules also accumulated in the developing outer plexiform layer. In the adult retina, cytokine signaling components examined were localized to the ganglion cell layer, the inner nuclear layer, and the two plexiform layers. In addition, regions corresponding to the inner and/or outer segments of the photoreceptor cells showed positive staining for cytokine signaling components. In contrast, the ERK2 protein kinase was found throughout the neonatal retina. In the mature retina, ERK2 was concentrated in the ganglion cells and the inner plexiform layer, while a lesser expression of ERK2 was detected in the inner nuclear layer, the outer plexiform layers, and the outer nuclear layer. CONCLUSIONS: In the neonatal mouse retina, signaling components of the Jak-STAT pathway and ERK2 are differentially expressed. All cytokine signaling components included in this study are expressed in the differentiated inner retina as well as in cells occupying the ventricular zone, suggesting that both postmitotic neurons and proliferative progenitors may directly respond to CNTF-like cytokines during postnatal development. The distribution of cytokine signaling pathway components in the adult mouse retina is consistent with previous findings that ganglion cells and Muller glia are the primary target cell types for CNTF. PMID: 14685141 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: Tanpakushitsu Kakusan Koso. 2003 Dec;48(16):2241-6. [STAT: transcriptional regulator of cytokine signaling] [Article in Japanese] Sekine K, Kim KW, Miyajima A. ksekine@iam.u-tokyo.ac.jp Publication Types: Review Review, Tutorial PMID: 14661581 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 31: J Biol Chem. 2004 Jan 23;279(4):2461-9. Epub 2003 Oct 24. Reactive oxygen species mediate virus-induced STAT activation: role of tyrosine phosphatases. Liu T, Castro S, Brasier AR, Jamaluddin M, Garofalo RP, Casola A. Department of Pediatrics, University of Texas Medical Branch, Galveston, Texas 77555-0366, USA. Respiratory syncytial virus (RSV) is the leading cause of epidemic respiratory tract illness in children in the United States and worldwide. RSV infection of airway epithelial cells induces formation of reactive oxygen species (ROS), whose production mediates the expression of cytokines and chemokines involved the immune/inflammatory responses of the lung. In this study, we have investigated the role of ROS in RSV-induced signal transducers and activators of transcription (STAT) activation and interferon regulatory factor (IRF) gene expression in human airway epithelial cells. Our results indicate that RSV replication induces IRF-1 and -7 gene transcription, a response abrogated by antioxidants. RSV infection induces binding of STAT to the IRF-1 gamma-interferon-activated sequence (GAS) and IRF-7 interferon-stimulated responsive element (ISRE). STAT1 and STAT3 bind IRF-1 GAS, whereas STAT1, STAT2, IRF-1, and IRF-9 bind IRF-7 ISRE. Antioxidant treatment blocks RSV-induced STAT binding to both the IRF-1 GAS and IRF-7 ISRE by inhibition of inducible STAT1 and STAT3 tyrosine phosphorylation, suggesting that RSV-induced ROS formation is required for STAT activation and IRF gene expression. Although protein tyrosine phosphorylation is necessary for RSV-induced STAT activation, Janus kinase and Src kinase activation do not mediate this effect. Instead, RSV infection inhibits intracellular tyrosine phosphatase activity, which is restored by antioxidant treatment. Pharmacological inhibition of tyrosine phosphatases induces STAT activation. Together, these results suggest that modulation of phosphatases could be an important mechanism of virus-induced STAT activation. Treatment of alveolar epithelial cells with the NAD(P)H oxidase inhibitor diphenylene iodonium abolishes RSV-induced STAT activation, indicating that NAD(P)H oxidase-produced ROS are required for downstream activation of the transcription factors IRF and STAT in virus-infected airway epithelial cells. PMID: 14578356 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 32: J Virol. 2003 Nov;77(21):11842-5. Erratum in: J Virol. 2003 Dec;77(24):13457. Hendra virus V protein inhibits interferon signaling by preventing STAT1 and STAT2 nuclear accumulation. Rodriguez JJ, Wang LF, Horvath CM. Immunobiology Center, Mount Sinai School of Medicine, New York, New York 10029, USA. The V protein of the recently emerged paramyxovirus, Nipah virus, has been shown to inhibit interferon (IFN) signal transduction through cytoplasmic sequestration of cellular STAT1 and STAT2 in high-molecular-weight complexes. Here we demonstrate that the closely related Hendra virus V protein also inhibits cellular responses to IFN through binding and cytoplasmic sequestration of both STAT1 and STAT2, but not STAT3. These findings demonstrate a V protein-mediated IFN signal evasion mechanism that is a general property of the known Henipavirus species. PMID: 14557668 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 33: J Cell Physiol. 2003 Nov;197(2):157-68. STAT proteins: from normal control of cellular events to tumorigenesis. Calo V, Migliavacca M, Bazan V, Macaluso M, Buscemi M, Gebbia N, Russo A. Section of Molecular Oncology, Department of Oncology, Regional Reference Center for the Biomolecular Characterization of Neoplasms and Genetic Screening of Hereditary Tumors, University of Palermo, Palermo, Italy. Signal transducers and activators of transcription (STAT) proteins comprise a family of transcription factors latent in the cytoplasm that participate in normal cellular events, such as differentiation, proliferation, cell survival, apoptosis, and angiogenesis following cytokine, growth factor, and hormone signaling. STATs are activated by tyrosine phosphorylation, which is normally a transient and tightly regulates process. Nevertheless, several constitutively activated STATs have been observed in a wide number of human cancer cell lines and primary tumors, including blood malignancies and solid neoplasias. STATs can be divided into two groups according to their specific functions. One is made up of STAT2, STAT4, and STAT6, which are activated by a small number of cytokines and play a distinct role in the development of T-cells and in IFNgamma signaling. The other group includes STAT1, STAT3, and STAT5, activated in different tissues by means of a series of ligands and involved in IFN signaling, development of the mammary gland, response to GH, and embriogenesis. This latter group of STATS plays an important role in controlling cell-cycle progression and apoptosis and thus contributes to oncogenesis. Although an increased expression of STAT1 has been observed in many human neoplasias, this molecule can be considered a potential tumor suppressor, since it plays an important role in growth arrest and in promoting apoptosis. On the other hand, STAT3 and 5 are considered as oncogenes, since they bring about the activation of cyclin D1, c-Myc, and bcl-xl expression, and are involved in promoting cell-cycle progression, cellular transformation, and in preventing apoptosis. Publication Types: Review Review, Tutorial PMID: 14502555 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 34: Brain Res Mol Brain Res. 2003 Aug 19;116(1-2):135-46. Initiation and maintenance of CNTF-Jak/STAT signaling in neurons is blocked by protein tyrosine phosphatase inhibitors. Jiao J, Kaur N, Lu B, Reeves SA, Halvorsen SW. Department of Pharmacology and Toxicology, School of Medicine and Biomedical Sciences, 102 Farber Hall, The State University of New York at Buffalo, Buffalo, NY 14214-3000, USA. Cytokines, including interferon-gamma and ciliary neurotrophic factor (CNTF), act in common through tyrosine kinase-based Jak/STAT signaling pathways. We found that activation of the Jak/STAT pathway by both interferon-gamma and CNTF in nerve cells was rapidly terminated by tyrosine phosphatase inhibitors. Exposure of human neuroblastoma cells, BE(2)-C, first to tyrosine phosphatase inhibitors (either phenylarsine oxide or PTP inhibitor-2) prevented Jak1, STAT1 and STAT3 activation elicited subsequently by either CNTF or interferon-gamma. In contrast, exposure of these cells to phosphatase inhibitors after initial stimulation by CNTF or interferon-gamma prevented the normal time-dependent decrease of total cellular phosphotyrosine-STAT levels as expected, while excluding already formed phosphotyrosine-STAT from the nucleus. Thus, treatment of nerve cells with a tyrosine phosphatase inhibitor blocked nuclear signal transduction. A similar inhibition of CNTF-Jak/STAT signaling was observed following tyrosine phosphatase inhibition in SH-SY5Y human neuroblastoma cells, HMN-1 mouse motor neuron-neuroblastoma hybrid cells, HepG2 human hepatoma cells and embryonic chick ciliary ganglion and retinal neurons. Expression of dominant-negative forms of the tyrosine phosphatases, SHP-1 and/or SHP-2, in BE(2)-C cells had no effect on CNTF activation of STAT or on the ability of phosphatase inhibitors to block signaling. Further, results from H-35 cells expressing gp130 receptor subunits lacking functional SHP-2 binding sites revealed normal cytokine activation of Jak and STAT that was inhibited by phosphatase inhibitors. These findings suggest a critical control for regulating the initiation of Jak/STAT signaling requiring tyrosine phosphatase activity. PMID: 12941469 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 35: J Clin Invest. 2003 Aug;112(4):535-43. Dysregulated Sonic hedgehog signaling and medulloblastoma consequent to IFN-alpha-stimulated STAT2-independent production of IFN-gamma in the brain. Wang J, Pham-Mitchell N, Schindler C, Campbell IL. The Scripps Research Institute, SP315, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA. The type I IFNs (IFN-alpha and IFN-beta), which are crucial in antiviral defense and immune regulation, signal via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway with activation of STAT1 and STAT2. Here, the function of STAT2 was studied in transgenic mice (termed GIFN/STAT2-/-) with CNS production of IFN-alpha. Surprisingly, GIFN/STAT2-/-, but not GIFN/STAT1-null, transgenic mice, with CNS production of IFN-alpha, died prematurely with medulloblastoma. An immune response also induced in the brain of the GIFN/STAT2-/- mice was associated with IFN-gamma gene expression by CD3+ T cells and the activation of the STAT1, STAT3, STAT4, and STAT5 molecules. Expression of the Sonic hedgehog (Shh) and the downstream transcriptional factor Gli-1 genes, implicated in the pathogenesis of medulloblastoma, was found to be significantly increased and cotranscribed in cerebellar granule neurons of the GIFN/STAT2-/- mice. IFN-gamma, but not IFN-alpha, induced STAT1-dependent expression of the Shh gene in cultured cerebellar granule neurons. Thus, there is an unexpected and extraordinarily adverse biological potency of IFN-alpha in the CNS when the primary signal transduction molecule STAT2 is absent. Moreover, a hitherto unknown role is indicated for the immune system in the pathogenesis of developmental disorders and tumorigenesis of the CNS via dysregulated Shh signaling mediated by IFN-gamma. PMID: 12925694 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 36: J Virol. 2003 Jul;77(13):7635-44. STAT protein interference and suppression of cytokine signal transduction by measles virus V protein. Palosaari H, Parisien JP, Rodriguez JJ, Ulane CM, Horvath CM. Immunobiology Center, Mount Sinai School of Medicine, One Gustave L. Levy Pl., Box 1630, New York, NY 10029, USA. Measles virus, a paramyxovirus of the Morbillivirus genus, is responsible for an acute childhood illness that infects over 40 million people and leads to the deaths of more than 1 million people annually (C. J. Murray and A. D. Lopez, Lancet 349:1269-1276, 1997). Measles virus infection is characterized by virus-induced immune suppression that creates susceptibility to opportunistic infections. Here we demonstrate that measles virus can inhibit cytokine responses by direct interference with host STAT protein-dependent signaling systems. Expression of the measles V protein prevents alpha, beta, and gamma interferon-induced transcriptional responses. Furthermore, it can interfere with signaling by interleukin-6 and the non-receptor tyrosine kinase, v-Src. Affinity purification demonstrates that the measles V protein associates with cellular STAT1, STAT2, STAT3, and IRF9, as well as several unidentified partners. Mechanistic studies indicate that while the measles V protein does not interfere with STAT1 or STAT2 tyrosine phosphorylation, it causes a defect in IFN-induced STAT nuclear accumulation. The defective STAT nuclear redistribution is also observed in measles virus-infected cells, where some of the STAT protein is detected in cytoplasmic bodies that contain viral nucleocapsid protein and nucleic acids. Interference with STAT-inducible transcription may provide a novel intracellular mechanism for measles virus-induced cytokine inhibition that links innate immune evasion to adaptive immune suppression. PMID: 12805463 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 37: Melanoma Res. 2003 Jun;13(3):219-29. The JAK/STAT pathway is not sufficient to sustain the antiproliferative response in an interferon-resistant human melanoma cell line. Jackson DP, Watling D, Rogers NC, Banks RE, Kerr IM, Selby PJ, Patel PM. Cancer Research UK Clinical Centre, St James's University Hospital, Leeds, UK. d.p.jackson@cancermed.leeds.ac.uk The mechanism of resistance of malignant melanoma to treatment with interferon-alpha is unknown, and currently there is no reliable method of predicting response. Signalling via the JAK/STAT pathway is known to mediate many interferon-regulated events and has been implicated in mediating the antiproliferative response. The objective of this study was to determine whether defects in JAK/STAT signalling may be responsible for interferon resistance. The in vitro response to interferon was determined in a panel of established melanoma cell lines, and the components and functioning of the JAK/STAT pathway were examined in sensitive and resistant cell lines. Two melanoma cell lines, characterized as sensitive (MM418) and resistant (MeWo) to the antiproliferative effect of interferon, were both shown by Western blotting to possess all the protein components of the JAK/STAT pathway, and were shown to be capable of producing functional transcription factors using an electrophoretic mobility shift assay and a ribonuclease protection assay of known interferon-induced genes. In addition, both cell lines had intact antiviral and HLA upregulation responses. These data suggest that there is no defect in the JAK/STAT pathway per se in the MeWo cell line, and that the substantial resistance to interferon must be mediated through components either downstream or additional to this signalling pathway. Others have shown JAK/STAT defects to be responsible for interferon resistance in some melanoma cell lines. However, our results highlight the likely heterogeneity in the mechanisms leading to interferon resistance both in cell lines and tumours, and suggest that a clinical assay based on analysis of components of the JAK/STAT pathway may have only limited use as a predictor of interferon response. PMID: 12777975 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 38: J Immunol. 2003 Jun 1;170(11):5373-81. Stimulation of primary human endothelial cell proliferation by IFN. Gomez D, Reich NC. Department of Pathology, Stony Brook University, Stony Brook, NY 11794, USA. The IFN family of cytokines has pleiotropic roles in immunity and development. In this study, we provide evidence that IFN can stimulate the proliferation of primary human endothelial cells. This is in contrast to the growth-suppressive effects of IFN observed on transformed human cells, thereby underscoring the distinctive responses of primary human cells. The growth-stimulatory effect of IFN was determined by an increase in DNA synthesis assessed with [(3)H]thymidine incorporation, an increase in G(2) and M cell cycle phases assessed with flow cytometric analysis, and an increase in cell number. Distinct cell types, including primary human fibroblast and smooth muscle cells, were also growth stimulated by IFN. Neutralizing Abs to IFN were used to demonstrate the growth response was mediated specifically by the IFN cytokine. The signaling pathway of type I IFNs activates STAT1 and STAT2. In primary endothelial cells, we demonstrate that STAT3 and STAT5 are also activated, and these STATs may contribute to cellular proliferation. To evaluate possible effectors of positive growth, DNA microarray analyses were performed to assess gene induction in response to IFN. These results reveal changes in the RNA levels of genes in endothelial cells that encode proteins involved in cellular proliferation. PMID: 12759411 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 39: J Virol. 2003 Jun;77(11):6385-93. STAT3 ubiquitylation and degradation by mumps virus suppress cytokine and oncogene signaling. Ulane CM, Rodriguez JJ, Parisien JP, Horvath CM. Immunobiology Center, Mount Sinai School of Medicine, New York, New York 10029, USA. Mumps virus is a common infectious agent of humans, causing parotitis, meningitis, encephalitis, and orchitis. Like other paramyxoviruses in the genus Rubulavirus, mumps virus catalyzes the proteasomal degradation of cellular STAT1 protein, a means for escaping antiviral responses initiated by alpha/beta and gamma interferons. We demonstrate that mumps virus also eliminates cellular STAT3, a protein that mediates transcriptional responses to cytokines, growth factors, nonreceptor tyrosine kinases, and a variety of oncogenic stimuli. STAT1 and STAT3 are independently targeted by a single mumps virus protein, called V, that assembles STAT-directed ubiquitylation complexes from cellular components, including STAT1, STAT2, STAT3, DDB1, and Cullin4A. Consequently, mumps virus V protein prevents responses to interleukin-6 and v-Src signals and can induce apoptosis in STAT3-dependent multiple myeloma cells and transformed murine fibroblasts. These findings demonstrate a unique cytokine and oncogene evasion property of mumps virus that provides a molecular basis for its observed oncolytic properties. PMID: 12743296 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 40: Gastroenterology. 2003 May;124(5):1465-75. Expression of hepatitis c virus proteins inhibits interferon alpha signaling in the liver of transgenic mice. Blindenbacher A, Duong FH, Hunziker L, Stutvoet ST, Wang X, Terracciano L, Moradpour D, Blum HE, Alonzi T, Tripodi M, La Monica N, Heim MH. Department of Research, University Hospital Basel, Switzerland. BACKGROUND & AIMS Hepatitis C virus (HCV) is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma worldwide. The majority of patients treated with interferon alpha do not have a sustained response with clearance of the virus. The molecular mechanisms underlying interferon resistance are poorly understood. Interferon-induced activation of the Jak-STAT (signal transducer and activator of transcription) signal transduction pathway is essential for the induction of an antiviral state. Interference of viral proteins with the Jak-STAT pathway could be responsible for interferon resistance in patients with chronic HCV. METHODS: We have analyzed interferon-induced signal transduction through the Jak-STAT pathway in transgenic mice that express HCV proteins in their liver cells. STAT activation was investigated with Western blots, immunofluorescence, and electrophoretic mobility shift assays. Virus challenge experiments with lymphocytic choriomeningitis virus were used to demonstrate the functional importance of Jak-STAT inhibition. RESULTS: STAT signaling was found to be strongly inhibited in liver cells of HCV transgenic mice. The inhibition occurred in the nucleus and blocked binding of STAT transcription factors to the promoters of interferon-stimulated genes. Tyrosine phosphorylation of STAT proteins by Janus kinases at the interferon receptor was not inhibited. This lack in interferon response resulted in an enhanced susceptibility of the transgenic mice to infection with a hepatotropic strain of lymphocytic choriomeningitis virus. CONCLUSIONS: Interferon-induced intracellular signaling is impaired in HCV transgenic mice. Interference of HCV proteins with interferon-induced intracellular signaling could be an important mechanism of viral persistence and treatment resistance. PMID: 12730885 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 41: J Biol Chem. 2003 Jun 13;278(24):21869-77. Epub 2003 Apr 2. A novel function for a glucose analog of blood group H antigen as a mediator of leukocyte-endothelial adhesion via intracellular adhesion molecule 1. Zhu K, Amin MA, Kim MJ, Katschke KJ Jr, Park CC, Koch AE. Department of Medicine, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Avenue, Chicago, IL 60611, USA. The 4A11 antigen is a unique cytokine-inducible antigen up-regulated on rheumatoid arthritis synovial endothelium compared with normal endothelium. In soluble form, this antigen, Lewisy-6/H-5-2 (Ley/H), or its glucose analog, 2-fucosyllactose (H-2g), mediates angiogenesis. The Ley/H antigen is structurally related to the soluble E-selectin ligand, sialyl Lewisx, and is selectively expressed in skin, lymphoid organs, thymus, and synovium, suggesting that it may be important in leukocyte homing or adhesion. In the present study, we used H-2g as a functional substitute to demonstrate a novel property for Ley/H antigen in inducing leukocyte-endothelial adhesion. H-2g significantly enhanced the expression of human dermal microvascular endothelial cells (HMVECs) intercellular adhesion molecule-1 (ICAM-1), but not vascular cell adhesion molecule-1, E-selectin, and P-selectin. Immunoprecipitation and Western blotting showed glycolipids Ley-6, H-5-2, or the glucose analog H-2g quickly activated human microvascular endothelial cell line-1 (HMEC-1) Janus kinase 2 (JAK2) and that the JAK2 inhibitor, AG-490, completely inhibited HMVEC ICAM-1 expression and HL-60 adhesion to HMEC-1s. Use of a JAK/signal transducer and activator of transcription (STAT) profiling system confirmed that H-2g selectively activated STAT3 but not STAT1 and STAT2. AG-490 inhibited H-2g-induced Erk1/2 and PI3K-Akt activation, suggesting that JAK2 is upstream of the Erk1/2 and PI3K-Akt pathways. Furthermore, the JAK2 inhibitor AG-490, the Erk1/2 inhibitor PD98059, or the phosphatidylinositol 3-kinase inhibitor LY294002 or antisense oligodeoxynucleotides directed against JAK2, Erk1/2, or phosphatidylinositol 3-kinase blocked H-2g-induced HMVEC ICAM-1 expression and HL-60 adhesion to HMEC-1s. Hence, H-2g signals through JAK2 and its downstream signal transducers STAT3, Erk1/2, and phosphatidylinositol 3-kinase result in ICAM-1 expression and cell adhesion. Potential treatment strategies through the inhibition of JAK-dependent pathways to target H-2g signals may provide a useful approach in inflammation-driven diseases like rheumatoid arthritis. PMID: 12672794 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 42: Mol Cell Biol. 2003 Feb;23(4):1316-33. Rho family GTPases are required for activation of Jak/STAT signaling by G protein-coupled receptors. Pelletier S, Duhamel F, Coulombe P, Popoff MR, Meloche S. Institut de recherches cliniques de Montreal and Department of Pharmacology, Universite de Montreal, Montreal, Quebec, Canada. As do cytokine receptors and receptor tyrosine kinases, G protein-coupled receptors (GPCRs) signal to Janus kinases (Jaks) and signal transducers and activators of transcription (STATs). However, the early biochemical events linking GPCRs to this signaling pathway have been unclear. Here we show that GPCR-stimulated Rac activity and the subsequent generation of reactive oxygen species are necessary for activating tyrosine phosphorylation of Jaks and STAT-dependent transcription. The requirement for Rac activity can be overcome by addition of hydrogen peroxide. Expression of activated mutants of Rac1 is sufficient to activate Jak2 and STAT-dependent transcription, and the activation of Jak2 correlates with the ability of Rac1 to bind to NADPH oxidase subunit p67(phox). We further show that GPCR agonists stimulate tyrosine phosphorylation of STAT1 and STAT3 proteins in a Rac-dependent manner. The tyrosine phosphorylation of STAT3 is biphasic; the first peak of phosphorylation is weak and correlates with rapid activation of Jaks by GPCRs, whereas the second peak is stronger and requires the synthesis of an autocrine factor. Rho also plays an essential role in the induction of STAT transcriptional activity. Our results highlight a novel role for Rho GTPases in mediating the regulatory effects of GPCRs on STAT-dependent gene expression. PMID: 12556491 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 43: Biochem J. 2003 Mar 1;370(Pt 2):391-6. Cloning of a new type II cytokine receptor activating signal transducer and activator of transcription (STAT)1, STAT2 and STAT3. Dumoutier L, Lejeune D, Hor S, Fickenscher H, Renauld JC. The Ludwig Institute for Cancer Research, Brussels Branch, and the Experimental Medicine Unit, Christian de Duve Institute of Cellular Pathology, Universite Catholique de Louvain, avenue Hippocrate 74, B-1200-Brussels, Belgium. In the present paper, we report the identification of a new gene encoding a transmembrane protein of 520 amino acids, showing 22% amino acid identity with the extracellular domain of the interleukin (IL)-20 receptor. This gene, termed likely interleukin or cytokine receptor-2 ( LICR2 ), is located on chromosome 1, at 25 kb from the IL22R (IL-22 receptor) gene, and is constitutively expressed in most tissues. A chimaeric receptor, consisting of the extracellular domain of the IL-10 receptor alpha chain and the intracellular domain of LICR2, activated signal transducer and activator of transcription (STAT)1, STAT2, STAT3 and STAT5 upon IL-10 stimulation, in a Janus kinase 1-dependent manner. In contrast, none of the IL-10-related cytokines described so far could activate LICR2-transfected cells, suggesting that LICR2 is a signalling receptor for a new cytokine of the IL-10 family. PMID: 12521379 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 44: J Immunol. 2003 Jan 15;170(2):749-56. Down-modulation of responses to type I IFN upon T cell activation. Dondi E, Rogge L, Lutfalla G, Uze G, Pellegrini S. Unite de Signalisation des Cytokines and Laboratoire de Immunoregulation, Institut Pasteur, Paris, France. The immunomodulatory role of type I IFNs (IFN-alpha/beta) in shaping T cell responses has been demonstrated, but the direct effects of IFN on T cells are still poorly characterized. Particularly, because IFN exert an antiproliferative activity, it remains elusive how the clonal expansion of effector T cells can paradoxically occur in the event of an infection when large amounts of IFN are produced. To address this issue, we have studied the effects of type I IFN in an in vitro differentiation model of human primary CD4(+) T cells. We found that IFN-alpha treatment of resting naive T cells delayed their entry into the cell cycle after TCR triggering. Conversely, the ongoing expansion of effector T cells was not inhibited by the presence of IFN. Moreover, activated T cells showed a significantly reduced induction of IFN-sensitive genes, as compared with naive precursors, and this decline occurred independently of subset-specific polarization. The residual type I IFN response measured in activated T cells was found sufficient to inhibit replication of the vesicular stomatitis virus. Our data suggest that the activation of T lymphocytes includes regulatory processes that restrain the transcriptional response to IFN and allow the proliferation of effector cells in the presence of this cytokine. PMID: 12517937 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 45: Transplantation. 2002 Aug 15;74(3):396-402. Constitutive activation of Jak/STAT proteins in Epstein-Barr virus-infected B-cell lines from patients with posttransplant lymphoproliferative disorder. Nepomuceno RR, Snow AL, Robert Beatty P, Krams SM, Martinez OM. Department of Surgery, Division of Liver Transplantation, Stanford University School of Medicine, CA 94305-5492, USA. BACKGROUND: Posttransplant lymphoproliferative disease (PTLD) is a major complication after bone marrow and solid organ transplantation. The disease encompasses a spectrum of abnormal, Epstein-Barr virus (EBV)-associated B-cell proliferations. We have previously shown that EBV-infected, spontaneous lymphoblastoid cell lines (SLCL) derived from PTLD patients require autocrine interleukin (IL)-10 to proliferate. To determine if cytokine signal transduction is involved in the autonomous growth of the SLCL, the activation states of the Jak/STAT signaling pathway proteins were analyzed in three different SLCL, termed JB7, MF4, and VB4. METHODS: The tyrosine phosphorylation (P-tyr) states of the Janus kinases (Jaks) and signal transducers and activators of transcription (STAT) proteins were examined by immunoprecipitation and immunoblot. Activated STAT dimer formation was determined by electromobility shift assays. RESULTS: All three SLCL, but not the Daudi Burkitt's lymphoma B-cell line, expressed the four known Jak kinases constitutively tyrosine phosphorylated, with particularly high levels of P-tyr Jak1 in the JB7 line. STAT1 and STAT3, but not STAT2 or STAT5, are also constitutively activated in all SLCL. The ability of the activated STAT proteins to form DNA-binding dimers was confirmed by electromobility shift assay. The SLCL, but not the Daudi line, express activated STAT complexes composed of STAT1 and STAT3. Another EBV-infected B-cell line, isolated from a lymph node biopsy after kidney transplantation, is phenotypically similar to the other SLCL in both surface antigen and activated STAT1 and STAT3 expression. CONCLUSION: These data support the presence of a constitutively active autocrine signaling pathway consistent with IL-10 in the SLCL. PMID: 12177620 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 46: J Biol Chem. 2002 Sep 20;277(38):35635-41. Epub 2002 Jun 24. STAT3 activation by type I interferons is dependent on specific tyrosines located in the cytoplasmic domain of interferon receptor chain 2c. Activation of multiple STATS proceeds through the redundant usage of two tyrosine residues. Velichko S, Wagner TC, Turkson J, Jove R, Croze E. Department of Immunology, Berlex Biosciences Inc., Richmond, California 94804 and the Molecular Oncology and Drug Discovery Programs, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612. Human type I interferons (IFNs) play an important role in the regulation of antiviral defense mechanisms, immunomodulatory activities, and growth control. Recent efforts have demonstrated the importance of IFNs in the activation of signal transducers and activators of transcription (STATs). The role of STAT1 and STAT2 in IFN-dependent JAK-STAT signaling is well established; however, the role of STAT3 and its activation by IFNs remains unclear. Understanding the IFN-dependent regulation of STAT3 is of increasing interest because recent studies have demonstrated that STAT3 may play a role in cancer. Studies have revealed that STAT3 is constitutively active in a number of cancer cell lines and that overexpression of an active form of STAT3 transforms normal fibroblasts. Therefore, STAT3 exhibits properties indicative of known oncogenes. In this report, we define the role of the type I IFN receptor in STAT3 activation and identify for the first time tyrosine residues present in the cytoplasmic domain of IFNAR2c that are critical for STAT3 activation. The regulation of STAT3 activation by IFNs was measured in a human lung fibrosarcoma cell line lacking IFNAR2c but stably expressing various IFNAR2c tyrosine mutants. We show here that in addition to IFN-dependent tyrosine phosphorylation of STAT3, activation using a STAT3-dependent electrophoretic mobility shift assay and a STAT3-specific reporter can also be demonstrated. Furthermore, we demonstrate that type I IFN-dependent activation of STAT3 proceeds through a novel mechanism that is dependent on two tyrosines, Tyr(337) and Tyr(512), present in IFNAR2c and contained within a conserved six-amino acid residue motif, GxGYxM. Surprisingly, both tyrosines were previously shown to be required for type I IFN-dependent STAT1 and STAT2 activation. Our results reveal that type I IFNs activate multiple STATs via the overlapping usage of two tyrosine residues located in the cytoplasmic domain of IFNAR2c. PMID: 12105218 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 47: Mol Cell Endocrinol. 2002 Mar 28;189(1-2):213-9. Genes induced by growth hormone in a model of adipogenic differentiation. Shang CA, Thompson BJ, Teasdale R, Brown RJ, Waters MJ. Department of Physiology and Pharmacology, Institute for Molecular Biology, University of Queensland, Qld 4072, Brisbane, Australia. A substantial number of GH regulated genes have been reported in mature hepatocytes, but genes involved in GH-initiated cell differentiation have not yet been identified. Here we have studied a well-characterised model of GH-dependent differentiation, adipogenesis of 3T3-F442A preadipocytes, to identify genes rapidly induced by GH. Using the suppression subtractive hybridisation technique, we have identified eight genes induced within 60 min of GH treatment, and verified these by northern analysis. Six were identifiable as Stat 2, Stat 3, thrombospondin-1, oncostatin M receptor beta chain, a DEAD box RNA helicase, and muscleblind, a developmental transcription factor. Bioinformatic approaches assigned one of the two remaining unknown genes as a novel 436 residue serine/threonine kinase. As each of the identified genes have important developmental roles, they may be important in initiating GH-induced adipogenesis. PMID: 12039079 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 48: J Urol. 2002 Apr;167(4):1859-62. Selective activation of members of the signal transducers and activators of transcription family in prostate carcinoma. Ni Z, Lou W, Lee SO, Dhir R, DeMiguel F, Grandis JR, Gao AC. Department of Urology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA. PURPOSE: Cytokines, hormones and growth factors use signal transducers and activators of transcription (STAT) signaling pathways to control various biological responses, including development, differentiation, cell proliferation and survival. Constitutive activation of STATs has been found in a wide variety of human tumors. In this study we examined the activity of STATs in primary human prostate tissues. MATERIALS AND METHODS: STAT activity was determined in 104 human primary prostate tissues, including 42 tumors, 42 matched normal prostates adjacent to tumors and 20 normal prostates from donors without cancer by electromobility shift assay. RESULTS: Significant levels of activated Stat4 and Stat6 were detected in primary prostate tissues. However, little or no expression of active Stat1, Stat2 or Stat5 was detected in primary prostate tissues. Significantly higher levels of constitutive Stat6 activity were found in prostate carcinomas compared with levels in normal tissue adjacent to tumors and normal prostates from donors without prostate cancer. There was no significant difference in Stat6 activity in normal prostate tissues adjacent to tumors and normal prostates from donors without prostate cancer. The levels of Stat4 activity varied but failed to yield statistically significant differences among tumors, matched normal prostates adjacent to tumors and normal prostates from donors without cancer. CONCLUSIONS: We have previously shown that Stat3 is activated in prostate cancer. The results of the current study demonstrate that in addition to Stat3, Stat6 is selectively activated in prostate cancer. PMID: 11912448 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 49: Gastroenterology. 2002 Apr;122(4):1020-34. Interferon-alpha activates multiple STAT signals and down-regulates c-Met in primary human hepatocytes. Radaeva S, Jaruga B, Hong F, Kim WH, Fan S, Cai H, Strom S, Liu Y, El-Assal O, Gao B. Section on Liver Biology, Laboratory of Physiologic Studies, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland 20892, USA. BACKGROUND & AIMS: Interferon (IFN)-alpha therapy is currently the primary choice for viral hepatitis and a promising treatment for hepatocellular carcinoma (HCC). Primary mouse and rat hepatocytes respond poorly to IFN-alpha stimulation. Thus, it is very important to examine the IFN-alpha signal pathway in primary human hepatocytes. METHODS: The IFN-alpha-activated signals and genes in primary human hepatocytes and hepatoma cells were examined by Western blotting and microarray analyses. RESULTS: Primary human hepatocytes respond very well to IFN-alpha stimulation as shown by activation of multiple signal transducer and activator of transcription factor (STAT) 1, 2, 3, 5, and multiple genes. The differential response to IFN-alpha stimulation in primary human and mouse hepatocytes may be caused by expression of predominant functional IFN-alpha receptor 2c (IFNAR2c) in primary human hepatocytes vs. expression of predominant inhibitory IFNAR2a in mouse hepatocytes. Microarray analyses of primary human hepatocytes show that IFN-alpha up-regulates about 44 genes by over 2-fold and down-regulates about 9 genes by 50%. The up-regulated genes include a variety of antiviral and tumor suppressors/proapoptotic genes. The down-regulated genes include c-myc and c-Met, the hepatocyte growth factor (HGF) receptor. Down-regulation of c-Met is caused by IFN-alpha suppression of the c-Met promoter through down-regulation of Sp1 binding and results in attenuation of HGF-induced signals and cell proliferation. CONCLUSIONS: IFN-alpha directly targets human hepatocytes, followed by activation of multiple STATs and regulation of a wide variety of genes, which may contribute to the antiviral and antitumor activities of IFN-alpha in human liver. PMID: 11910354 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 50: Am J Pathol. 2002 Jan;160(1):271-88. Regulation of signal transducer and activator of transcription and suppressor of cytokine-signaling gene expression in the brain of mice with astrocyte-targeted production of interleukin-12 or experimental autoimmune encephalomyelitis. Maier J, Kincaid C, Pagenstecher A, Campbell IL. Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California 92037, USA. Interleukin (IL)-12 and interferon (IFN)-gamma are implicated in the pathogenesis of immune disorders of the central nervous system (CNS). To define the basis for the actions of these cytokines in the CNS, we examined the temporal and spatial regulation of key signal transducers and activators of transcription (STATs) and suppressors of cytokine signaling (SOCS) in the brain of transgenic mice with astrocyte production of IL-12 or in mice with experimental autoimmune encephalomyelitis (EAE). In healthy mice, with the exception of STAT4 and STAT6, the expression of a number of STAT and SOCS genes was detectable. However, in symptomatic transgenic mice and in EAE significant up-regulation of STAT1, STAT2, STAT3, STAT4, IRF9, and SOCS1 and SOCS3 RNA transcripts was observed. Although the increased expression of STAT1 RNA was widely distributed and included neurons, astrocytes, and microglia, STAT4 and STAT3 and SOCS1 and SOCS3 RNA was primarily restricted to the infiltrating mononuclear cell population. The level and location of the STAT1, STAT3, and STAT4 proteins overlapped with their corresponding RNA and additionally showed nuclear localization indicative of activation of these molecules. Thus, in both the glial fibrillary acidic protein-IL-12 mice and in EAE the CNS expression of key STAT and SOCS genes that regulate IL-12 (STAT4) and IFN-gamma (STAT1, SOCS1, and SOCS3) receptor signaling is highly regulated and compartmentalized. We conclude the interaction between these positive and negative signaling circuits and their distinct cellular locations likely play a defining role in coordinating the actions of IL-12 and IFN-gamma during the pathogenesis of type 1 immune responses in the CNS. PMID: 11786421 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 51: Oncogene. 2001 Nov 1;20(50):7326-33. Regulation of STAT protein synthesis by c-Cbl. Blesofsky WA, Mowen K, Arduini RM, Baker DP, Murphy MA, Bowtell DD, David M. Department of Biology and UCSD Cancer Center, University of California San Diego, La Jolla, California 92093-0322, USA. Many cytokines and growth factors induce transcription of immediate early response genes by activating members of the Signal Transducers and Activators of Transcription (STAT) family. Although significant progress has been made in understanding the events that lead to the activation of STAT proteins, less is known about the regulation of their expression. Here we report that murine embryonic fibroblasts derived from c-Cbl-deficient mice display significantly increased levels of STAT1 and STAT5 protein. In contrast, STAT2 and STAT3 expression, as well as the levels of the tyrosine kinases Jak1 and Tyk2, appear to be regulated independently of c-Cbl. Interestingly, the half-life of STAT1 was unaffected by the presence of c-Cbl, indicating that c-Cbl acts independently of STAT1 degradation. Further analysis revealed similar levels of STAT1 mRNA, however, a dramatically increased rate of STAT1 protein synthesis was observed in c-Cbl-deficient cells. Thus, our findings demonstrate an additional control mechanism over STAT1 function, and also provide a novel biological effect of the Cbl protein family. PMID: 11704862 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 52: J Biol Chem. 2001 Dec 14;276(50):47004-12. Epub 2001 Oct 15. Down-modulation of type 1 interferon responses by receptor cross-competition for a shared Jak kinase. Dondi E, Pattyn E, Lutfalla G, Van Ostade X, Uze G, Pellegrini S, Tavernier J. Laboratoire de Signalisation des Cytokines, Institut Pasteur, 25, rue du Dr. Roux, 75724 Paris, cedex 15 France. In contrast to the large number of class I and II cytokine receptors, only four Janus kinase (Jak) proteins are expressed in mammalian cells, implying the shared use of these kinases by many different receptor complexes. Consequently, if receptor numbers exceed the amount of available Jak, cross-interference patterns can be expected. We have engineered two model cellular systems expressing two different exogenous Tyk2-interacting receptors. A receptor chimera was generated wherein the extracellular part of the interferon type 1 receptor (Ifnar1) component of the interferon-alpha/beta receptor is replaced by the equivalent domain of the erythropoietin receptor. Despite Tyk2 activation, erythropoietin treatment of cells expressing this erythropoietin receptor/Ifnar1 chimera did not evoke any detectable IFN-type response. However, a dose-dependent interference with signal transduction via the endogenous Ifnar complex was found for STAT1, STAT2, STAT3, Tyk2, and Jak1 activation, for gene induction, and for antiviral activity. In a similar approach, cells expressing the beta1 chain of the interleukin-12 receptor showed a reduced transcriptional response to IFN-alpha as well as reduced STAT and kinase activation. In both model systems, titration of the Tyk2 kinase away from the Ifnar1 receptor chain accounts for the observed cross-interference. PMID: 11602573 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 53: J Invest Dermatol. 2001 Sep;117(3):583-9. Constitutive and interleukin-7- and interleukin-15-stimulated DNA binding of STAT and novel factors in cutaneous T cell lymphoma cells. Qin JZ, Kamarashev J, Zhang CL, Dummer R, Burg G, Dobbeling U. Department of Dermatology, University Hospital of Zurich, Zurich, Switzerland. On testing cutaneous T cell lymphoma cell lines and skin lesions, we found that the transcription factors STAT2, STAT3, STAT5, and STAT6 (STAT, signal transducer and activator of transcription) were present in the nuclei of these cells and that the binding to their specific DNA binding sites was stimulated by interleukin-7 and interleukin-15. DNA binding studies also revealed the presence of three additional DNA factors in cutaneous T cell lymphoma cells that bound to the same sequences and could also be stimulated by interleukin-7 and interleukin-15. One of these novel factors was also present in the adult T cell leukemia cell line Jurkat and malignant T cells from the blood of Sezary syndrome patients, but not in normal peripheral blood lymphocytes. It may therefore be a marker of T cell leukemia. It seems to interfere with the binding of STAT1 to the sis inducible element, suggesting that the DNA binding activity of STAT1 in cutaneous T cell lymphoma cells is disturbed. PMID: 11564163 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 54: Proc Natl Acad Sci U S A. 2001 Jul 31;98(16):9050-5. An essential role of the JAK-STAT pathway in ischemic preconditioning. Xuan YT, Guo Y, Han H, Zhu Y, Bolli R. Experimental Research Laboratory, Division of Cardiology, University of Louisville and the Jewish Hospital Heart and Lung Institute, Louisville, KY 40292, USA. The goal of this study was to determine the role of the Janus tyrosine kinase (JAK)-signal transducers and activators of transcription (STAT) pathway in the late phase of ischemic preconditioning (PC). A total of 230 mice were used. At 5 min after ischemic PC (induced with six cycles of 4-min coronary occlusion/4-min reperfusion), immunoprecipitation with anti-phosphotyrosine (anti-pTyr) antibodies followed by immunoblotting with anti-JAK antibodies revealed increased tyrosine phosphorylation of JAK1 (+257 +/- 53%) and JAK2 (+238 +/- 35%), indicating rapid activation of these two kinases. Similar results were obtained by immunoblotting with anti-pTyr-JAK1 and anti-pTyr-JAK2 antibodies. Western analysis with anti-pTyr-STAT antibodies demonstrated a marked increase in nuclear pTyr-STAT1 (+301 +/- 61%) and pTyr-STAT3 (+253 +/- 60%) 30 min after ischemic PC, which was associated with redistribution of STAT1 and STAT3 from the cytosolic to the nuclear fraction and with an increase in STAT1 and STAT3 gamma-IFN activation site DNA-binding activity (+606 +/- 64%), indicating activation of STAT1 and STAT3. No nuclear translocation or tyrosine phosphorylation of STAT2, STAT4, STAT5A, STAT5B, or STAT6 was observed. Pretreatment with the JAK inhibitor AG-490 20 min before the six occlusion/reperfusion cycles blocked the enhanced tyrosine phosphorylation of JAK1 and JAK2 and the increased tyrosine phosphorylation, nuclear translocation, and enhanced DNA-binding activity of STAT1 and STAT3. The same dose of AG-490 abrogated the protection against myocardial infarction and the concomitant up-regulation of inducible NO synthase (iNOS) protein and activity observed 24 h after ischemic PC. Taken together, these results demonstrate that ischemic PC induces isoform-selective activation of JAK1, JAK2, STAT1, and STAT3, and that ablation of this response impedes the up-regulation of iNOS and the concurrent acquisition of ischemic tolerance. This study demonstrates that the JAK-STAT pathway plays an essential role in the development of late PC. The results reveal a signaling mechanism that underlies the transcriptional up-regulation of the cardiac iNOS gene and the adaptation of the heart to ischemic stress. PMID: 11481471 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 55: J Interferon Cytokine Res. 2001 Jun;21(6):445-50. Signal transducers and activators of transcription (Stat) are detectable in mouse trigeminal ganglion neurons. Kriesel JD, Jones BB, Hwang IP, Dahms KM, Spruance SL. Department of Ophthalmology, The John A. Moran Eye Center, University of Utah School of Medicine, Salt Lake City, UT 84132, USA. jkriesel@med.utah.edu We studied signal transducers and activators of transcription (Stat) expression in mouse trigeminal ganglia (TG) to gain an understanding of herpes simplex virus (HSV) infection and reactivation. Mouse TG were harvested and were either frozen for Western blot analysis or preserved in 4% paraformaldehyde for subsequent immunohistochemistry study. The thawed specimens were homogenized, and nuclear/cytoplasmic extractions were performed for Western blots and immunoprecipitation. Immunohistochemistry showed that Stat1, Stat3, Stat4, Stat5b, and phosphotyrosine Stat3 localized to TG neurons, not surrounding satellite cells. Western blot of TG nuclear and cytoplasmic extracts confirmed the presence of these Stat at the appropriate molecular weights. Stat2 was undetectable in TG by these methods. Immunoprecipitation of TG nuclear extracts did not confirm the presence of Stat-Stat dimers in these specimens. These studies show that several Stat, including phosphotyrosine Stat3, are present in TG neurons, the site of HSV latency, where they could act upon latent viral DNA to effect reactivation. PMID: 11440643 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 56: J Interferon Cytokine Res. 2001 Jun;21(6):379-88. The interferon-inducible Stat2:Stat1 heterodimer preferentially binds in vitro to a consensus element found in the promoters of a subset of interferon-stimulated genes. Ghislain JJ, Wong T, Nguyen M, Fish EN. Biologie Moleculaire du Developpement, Ecole Normale Superieure, France. Regulated expression of type I interferon (IFN)-stimulated genes (ISG) requires the binding of the signal transducer and activator of transcription (Stat) complexes to enhancer elements located in the ISG promoters. These enhancer elements include the IFN-stimulated response element (ISRE) and the palindromic IFN-gamma activation site (GAS) element. Regulated expression of ISRE containing ISG depends on IFN-stimulated gene factor 3 (ISGF3), a heterodimer involving Stat1 and Stat2 in association with a DNA-binding adapter protein, p48/IFN regulatory factor-9 (IRF-9). Several GAS binding Stat complexes involving Stat1, Stat3, Stat4, and Stat5 have been described, but their contribution to GAS-dependent ISG expression remains to be established. We and others previously identified an IFN-alpha-inducible Stat2:1 heterodimer that exhibits binding to the GAS element of the IRF-1 gene. These previous studies raise the possibility that Stat2:1 may participate in the transcriptional activation of the subset of ISG containing GAS elements. To address this question, we performed a PCR-assisted binding site selection procedure to define the Stat2:1 recognition sequence. The data reveal that Stat2:1 preferentially binds to a palindromic sequence similar to the consensus GAS element found in the promoter of several ISG. Our results establish that in addition to the classic complex formation involving Stat2, Stat1, and p48 associations, Stat2:1 heterodimers are formed in response to IFN treatment that may play an important role in the transcriptional regulation of certain ISG. PMID: 11440635 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 57: J Anat. 2001 May;198(Pt 5):581-9. Immunolocalisation of the janus kinases (JAK)--signal transducers and activators of transcription (STAT) pathway in human epidermis. Nishio H, Matsui K, Tsuji H, Tamura A, Suzuki K. Osaka Medical College, Department of Legal Medicine, Takatsuki, Japan. The janus kinases (JAK) and signal transducers and activators of the transcription (STAT) pathway have been shown to be activated by a number of cytokines or growth factors and to play significant roles in the differentiation of various cell types. In the present study, we investigated the distribution of the JAK-STAT pathway using immunohistochemistry in the human epidermis. Each element of the pathway showed abundant and differential expression in the epidermis. The differential distribution of the elements was most strikingly observed in the horny keratinised cell and granular layers of the epidermis. JAK2, JAK3, STAT1 and STAT5 were expressed in high amounts, and JAK1, TYK2, STAT2, STAT3, STAT4 and STAT6 to a much lesser extent in the horny cell layer. JAK3, TYK2, STAT2, STAT3, STAT4 and STAT6 were more abundantly expressed in the granular layer than the lower layers of the epidermis. JAK1, STAT1 and STAT5 were expressed at almost the same levels in the various layers of the epidermis. These results show that elements of the JAK-STAT pathway are abundantly and differentially expressed in the epidermis. It is suggested that each element of the pathway may play a role at a distinct stage of keratinocyte differentiation. PMID: 11430697 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 58: Methods Enzymol. 2001;333:138-51. STAT proteins: signal tranducers and activators of transcription. Bromberg J, Chen X. Memorial Sloan Kettering Cancer Center, New York, New York 10021, USA. Publication Types: Review Review, Tutorial PMID: 11400331 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 59: Int J Hematol. 2001 Apr;73(3):271-7. Role of Jak kinases and STATs in cytokine signal transduction. Leonard WJ. Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1674, USA. The Janus family tyrosine kinase-signal transducer and activator of transcription (Jak-STAT) signaling pathway is broadly used by interferons and type I cytokines. These cytokines and interferons activate Janus family tyrosine kinases (Jak kinases), which in turn phosphorylate and thereby activate STAT proteins. Before activation, STAT proteins are cytosolic proteins; after activation, however, they are translocated to the nucleus where they function as transcription factors. This review summarizes salient features of the Jak-STAT pathway and focuses on the functional role of the different Jak kinases and STATs in vivo. Publication Types: Review Review, Tutorial PMID: 11345192 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 60: Endocrinology. 2001 May;142(5):1786-94. Interferon-tau (IFNtau) regulation of IFN-stimulated gene expression in cell lines lacking specific IFN-signaling components. Stewart MD, Johnson GA, Bazer FW, Spencer TE. Center for Animal Biotechnology and Genomics, Department of Animal Science, Texas A&M University, College Station, Texas 77843-2471, USA. Interferon-tau (IFNtau) is a unique type I IFN secreted by the ruminant conceptus that acts in a paracrine manner on the endometrial epithelium to signal pregnancy recognition. In the ovine endometrium, IFNtau suppresses estrogen receptor alpha and oxytocin receptor gene expression, but increases or induces expression of IFN-simulated genes (ISGs), including signal transducer and activator of transcription-1 (STAT1), STAT2, ISG factor-3gamma (ISGF3gamma)/p48/IFN regulatory factor-9, and 2',5'-oligoadenylate synthetase (OAS). Human fibroblast cell lines lacking specific IFN signaling components were employed to determine the roles of STAT1, STAT2, and ISGF3gamma in the effects of IFNtau on ISG protein expression. Results indicated that STAT1alpha or STAT1beta is required for IFNtau effects on STAT2, ISGF3gamma, and OAS (40/46, 69/71, and 100 kDa). STAT2 is required for effects on STAT1, ISGF3gamma, and all OAS forms. ISGF3gamma is required for effects of IFNtau on STAT2 and 40/46- and 69/71-kDa OAS and plays a role in the effects of IFNtau on 100-kDa OAS and STAT1. Mutation of Tyr(701), but not Ser(727), of STAT1 abolished the effects of IFNtau on ISG expression. Mutation of the SH2 domain of STAT1 abolished the effects of IFNtau on all ISGs and reduced increases in 100-kDa OAS. These data illustrate the importance of transcription factors composed of STAT1, STAT2, and ISGF3gamma in the signaling pathway mediating the effects of IFNtau on ISG expression. PMID: 11316742 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 61: J Biol Chem. 2001 Jun 29;276(26):24113-21. Epub 2001 Apr 17. G-protein-independent activation of Tyk2 by the platelet-activating factor receptor. Lukashova V, Asselin C, Krolewski JJ, Rola-Pleszczynski M, Stankova J. Immunology Division, Department of Pediatrics and the Department of Anatomy and Cell Biology, Universite de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada. Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid with multiple physiological and pathological effects. PAF exerts its activity through a specific heptohelical G-protein coupled receptor, expressed on a variety of cell types, including leukocytes. In this study, we showed that PAF induced a rapid tyrosine phosphorylation of the Tyk2 kinase in the monocytic cell lines U937 and MonoMac-1. PAF-initiated Tyk2 phosphorylation was also observed in COS-7 cells transiently transfected with the human PAF receptor (PAFR) and Tyk2 cDNAs. In addition, we found that Tyk2 co-immunoprecipitated and co-localized with PAFR, independently of ligand binding. Deletion mutants of Tyk2 indicated that the N terminus of the kinase was important for the binding to PAFR. Activation of Tyk2 was followed by a time-dependent 2-4-fold increase in the level of tyrosine phosphorylation of signal transducers and activators of transcription 1 (STAT1), STAT2, and STAT3 and a sustained 2.5-fold increase in STAT5 tyrosine phosphorylation. In MonoMac-1 cells, STAT1 and STAT3 translocated to the nucleus following PAF stimulation, and their translocation in transiently transfected COS-7 cells was shown to be dependent on the presence of Tyk2. In addition, when COS-7 cells were transfected with PAFR and constructs containing PAFR promoter 1, coupled to the luciferase reporter gene, PAF induced a 3.6-fold increase in promoter activation in the presence of Tyk2. Finally, PAFR mutants that could not couple to G-proteins were found to effectively mediate Tyk2 activation and signaling. Taken together, these findings suggest an important role for the Janus kinase/STAT pathway in PAFR signaling, independent of G-proteins, and in the regulation of PAF receptor expression by its ligand. PMID: 11309383 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 62: Curr Opin Cell Biol. 2001 Apr;13(2):211-7. The Stat family in cytokine signaling. Ihle JN. Department of Biochemistry, Howard Hughes Medical Institute, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, Tennessee 38105, USA. james.ihle@stjude.org During the past few years studies from several laboratories have utilized gene disruption approaches to define the function of members of the Stat family of transcription factors. The results have demonstrated that each family member has unique, critical, non-redundant functions in signal transduction through members of the cytokine receptor superfamily. Many of the family members mediate functions associated with innate or acquired immunity. With the availability of mice deficient in one or more of the Stats, critical experiments are possible to evaluate the roles of Stat signal transduction pathways in cellular transformation as well as evaluating their specific roles in a range of cellular responses to cytokines. Publication Types: Review Review, Tutorial PMID: 11248555 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 63: Biochem Biophys Res Commun. 2001 Mar 9;281(4):907-12. Differential expression of signal transducers and activators of transcription during human adipogenesis. Harp JB, Franklin D, Vanderpuije AA, Gimble JM. Department of Nutrition, University of North Carolina at Chapel Hill, North Carolina 27599, USA. jharp@sph.unc.edu Signal Transducers and Activators of Transcription (STATs) display unique expression patterns upon induction of differentiation of murine 3T3-L1 preadipocytes into adipocytes. During differentiation, expression of STAT1 and STAT5 increase, while STAT3 and STAT6 remain relatively unchanged. Here, we determined whether human subcutaneous preadipocytes expressed STATs and if the pattern of expression changed during adipogenesis. We found by Western blot analysis that freshly isolated preadipocytes expressed STAT1, STAT3, STAT5, and STAT6, but not STAT2 and STAT4. Induction of preadipocyte differentiation with 1-methyl-3-isobutylxanthine, dexamethasone, insulin, and BRL49653 decreased expression of STAT1, and increased expression of STAT3 and STAT5. STAT6 expression did not change during adipogenesis. Changes in expression of CCAAT/enhancer binding protein beta (C/EBPbeta), C/EBPdelta, C/EBPalpha, and peroxisome proliferator-activated receptor gamma were similar to murine cell lines. These results suggest that unlike the traditional adipogenic transcription factors, unique differences exist in STAT expression patterns between murine and human adipose cells. Copyright 2001 Academic Press. PMID: 11237746 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 64: Zh Evol Biokhim Fiziol. 2000 Nov-Dec;36(6):504-8. [STAT pathway of the intracellular signaling] [Article in Russian] Nikol'skii NN, Vasilenko KP. Publication Types: Review Review, Tutorial PMID: 11212528 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 65: Bioessays. 2001 Feb;23(2):161-9. Activation of STAT proteins and growth control. Bromberg JF. Department of Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, NY 10021, USA. bromberj@mskcc.org This review will discuss how STAT (Signal Transducers and Activators of Transcription) proteins, a group of transcription factors that transmit signals from the extracellular surface of cells to the nucleus, are involved in growth control. I will discuss the anatomy of a STAT protein, how it works as a transcription factor, the molecules that regulate its "activity", the phenotypes of mice that lack individual STAT proteins and their involvement in growth, differentiation, apoptosis, and transformation. Finally, a number of examples will be presented of how dysregulated STAT signaling may be involved in the pathogenesis of cancer. Publication Types: Review Review, Tutorial PMID: 11169589 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 66: Endocrinology. 2001 Jan;142(1):98-107. Interferon-tau activates multiple signal transducer and activator of transcription proteins and has complex effects on interferon-responsive gene transcription in ovine endometrial epithelial cells. Stewart DM, Johnson GA, Vyhlidal CA, Burghardt RC, Safe SH, Yu-Lee LY, Bazer FW, Spencer TE. Center for Animal Biotechnology and Genomics, Department of Animal Science, Texas A&M University, University College of Veterinary Medicine, College Station, Texas 77843, USA. Interferon-tau (IFNtau), a type I IFN produced by sheep conceptus trophectoderm, is the signal for maternal recognition of pregnancy. Although it is clear that IFNtau suppresses transcription of the estrogen receptor alpha and oxytocin receptor genes and induces expression of various IFN-stimulated genes within the endometrial epithelium, little is known of the signal transduction pathway activated by the hormone. This study determined the effects of IFNtau on signal transducer and activator of transcription (STAT) activation, expression, DNA binding, and transcriptional activation using an ovine endometrial epithelial cell line. IFNtau induced persistent tyrosine phosphorylation and nuclear translocation of STAT1 and -2 (10 min to 48 h), but transient phosphorylation and nuclear translocation of STAT3, -5a/b, and -6 (10 to <60 min). IFNtau increased expression of STAT1 and -2, but not STAT3, -5a/b, and -6. IFN-stimulated gene factor-3 and STAT1 homodimers formed and bound an IFN-stimulated response element (ISRE) and gamma-activated sequence (GAS) element, respectively. IFNtau increased transcription of GAS-driven promoters at 3 h, but suppressed their activity at 24 h. In contrast, the activity of an ISRE-driven promoter was increased at 3 and 24 h. These results indicate that IFNtau activates multiple STATs and has differential effects on ISRE- and GAS-driven gene transcription. PMID: 11145571 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 67: J Interferon Cytokine Res. 2000 Nov;20(11):971-82. The neutralization of type I IFN biologic actions by anti-IFNAR-2 monoclonal antibodies is not entirely due to inhibition of Jak-Stat tyrosine phosphorylation. Novick D, Nabioullin RR, Ragsdale W, McKenna S, Weiser W, Garone L, Burkins C, Kim SH, Rubinstein M, Tepper MA, Arulanandam AR. Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel. A panel of monoclonal antibodies (mAb) derived against human interferon-alpha/beta receptor-2 (IFNAR-2) was evaluated for their ability to antagonize the biologic effects of type 1 interferons (IFN-alpha1, IFN-alpha2a, and IFN-beta). Anti-IFNAR-2 mAb 117.7, 35.9, 53.2, and 51.44 neutralized type I IFN-mediated antiviral, antiproliferative, and major histocompatibility complex (MHC) class I upregulation functions. However, only mAb 51.44 neutralized IFN-alpha2a and IFN-beta-mediated natural killer (NK) cell cytotoxicity. In BIAcore and cell binding studies, only mAb 51.44 and 234.28 inhibited IFN-alpha2a and IFN-beta binding to its receptor. The receptor blockade by mAb 51.44 and 234.28 resulted in the inhibition of IFN-alpha2a and IFN-beta-induced tyrosine phosphorylation of Jak1, Tyk2, Stat1/2/3, and IFNAR-1/2 and inhibition of IFN-stimulated gene factor 3 (ISGF3) formation. mAb 117.7, 35.9, and 53.2, although antagonists of IFN's biologic activities, did not block the binding of IFN-alpha/beta to its receptor. The 117.7 mAb, representative of this class of receptor nonblocking mAb, induced hyper-tyrosine phosphorylation of IFNAR-2 in the presence of IFN-alpha/beta but did not inhibit IFN-alpha/beta-induced Jak-Stat tyrosine phosphorylation and ISGF3 complex formation. These results show that the neutralization of type I IFN biologic actions by anti-IFNAR-2 mAb cannot be entirely explained by inhibition of Jak-Stat tyrosine phosphorylation. PMID: 11096454 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 68: Trends Biochem Sci. 2000 Oct;25(10):496-502. STAT proteins and transcriptional responses to extracellular signals. Horvath CM. Immunobiology Center, Box 1630, East Building Room 12-20D, The Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA. curt.horvath@mssm.edu Signal transducer and activator of transcription (STAT) transcription factors are implicated in programming gene expression in biological events as diverse as embryonic development, programmed cell death, organogenesis, innate immunity, adaptive immunity and cell growth regulation in organisms ranging from slime molds to insects to man. Rapid progress has unearthed much about the activation of STATs by Janus kinases (JAKs) and other tyrosine kinases and their ability to interface with other signaling systems. Once inside the nucleus, the STATs bind to promoters and join other transcriptional activators in the regulation of gene expression. Publication Types: Review Review, Tutorial PMID: 11050435 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 69: Sheng Li Ke Xue Jin Zhan. 1997 Apr;28(2):145-7. [A novel family of transcription factors: signal transducers and activators of transcription] [Article in Chinese] Huang SH, Qin CH. Publication Types: Review Review, Tutorial PMID: 11038710 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 70: Br J Haematol. 2000 Jun;109(4):823-8. JAK2 tyrosine kinase inhibitor tyrphostin AG490 downregulates the mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription (STAT) pathways and induces apoptosis in myeloma cells. De Vos J, Jourdan M, Tarte K, Jasmin C, Klein B. INSERM U 475, Immunopathologie des Maladies Tumorales et Autoimmunes, Montpellier, France. Cytokines of the interleukin 6 (IL-6) family, which activates the signal transducer gp130, are major survival and growth factors for human multiple myeloma (MM) cells. The signal transduction of gp130 involves the Janus tyrosine kinases (JAK) JAK1, JAK2 and Tyk2 and then the downstream effectors comprising the signal transducer and activator of transcription 3 (STAT3) and mitogen-activated protein kinase (MAPK) pathways. We evaluated the effects of the JAK2 inhibitor tyrphostin AG490 on MM cells. We found that AG490 suppressed cell proliferation and induced apoptosis in IL-6-dependent MM cell lines. JAK2 kinase activity, ERK2 and STAT3 phosphorylation were inhibited. These results suggest that the chemical blocking of the gp130 signalling pathway at the JAK level could be a relevant therapeutic approach to MM. PMID: 10929036 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 71: J Biol Chem. 2000 Sep 15;275(37):28902-10. FER kinase activation of Stat3 is determined by the N-terminal sequence. Priel-Halachmi S, Ben-Dor I, Shpungin S, Tennenbaum T, Molavani H, Bachrach M, Salzberg S, Nir U. Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel. p94(fer) and p51(ferT) are two tyrosine kinases that share identical SH2 and kinase domains but differ in their N-terminal regions. To further explore the cellular functions of these two highly related tyrosine kinases, their subcellular distribution profiles and in vivo phosphorylation activity were followed using double immunofluorescence assay. When combined with immunoprecipitation analysis, this assay showed that p94(fer) can lead to the tyrosine phosphorylation and activation of Stat3 but not of Stat1 or Stat2. Native p94(fer) exerted this activity when residing in the cytoplasm. However, modified forms of p94(fer), which are constitutively nuclear, could also lead to the phosphorylation of Stat3. Endogenous Stat3 and p94(fer) co-immunoprecipitated with each other, thus proving the interaction of these two proteins in vivo. Unlike p94(fer), p51(ferT) did not induce the phosphorylation of Stat3 but led to the phosphorylation of other nuclear proteins. Replacing the unique 43-amino acid-long N-terminal tail of p51(ferT) with a parallel segment from the N-terminal tail of p94(fer) did not change the subcellular localization of p51(ferT) but enabled it to activate Stat3. Thus the different N-terminal sequences of p94(fer) and p51(ferT) can affect their ability to induce phosphorylation of Stat3 and most probably direct their different cellular functions. PMID: 10878010 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 72: J Biol Chem. 2000 Apr 28;275(17):12661-6. Distinct mechanisms of STAT phosphorylation via the interferon-alpha/beta receptor. Selective inhibition of STAT3 and STAT5 by piceatannol. Su L, David M. Department of Biology and UCSD Cancer Center, University of California San Diego, La Jolla, California 92093-0322, USA. Interferon-alpha (IFNalpha) can activate several members of the signal transducers and activator of transcription (STAT) transcription factor family, a process that requires the tyrosine kinases Jak1 and Tyk2. Here we provide evidence that IFNalpha-mediated activation of various STAT proteins is regulated by distinct mechanisms. Piceatannol, previously reported as a Syk/ZAP70-specific kinase inhibitor, selectively inhibits the tyrosine phosphorylation of STAT3 and STAT5, but not of STAT1 and STAT2. This inhibition is paralleled by the loss of Jak1 and IFNAR1 tyrosine phosphorylation in response to IFNalpha, whereas Tyk2 and IFNAR2 tyrosine phosphorylation is unaffected. Last, the IFNalpha-induced serine phosphorylation of STAT1 and STAT3 is not inhibited by piceatannol but is sensitive to the Src kinase-specific inhibitor PP2. Thus, our results not only demonstrate that the IFNalpha/beta receptor utilizes distinct mechanisms to trigger the tyrosine phosphorylation of specific STAT proteins, but they also indicate a diverging pathway that leads to the serine phosphorylation of STAT1 and STAT3. PMID: 10777558 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 73: Med Clin (Barc). 2000 Feb 19;114(6):227-34. [The JAK-STAT signaling pathway and its role in oncogenesis, immunomodulation and development] [Article in Spanish] Duarte RF, Frank DA. Harvard Medical School, Boston, Massachusetts, USA. duarte@rfhsm.ac.uk Publication Types: Review Review, Tutorial PMID: 10757107 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 74: Int J Cancer. 2000 Mar 1;85(5):720-5. Defective Jak-STAT signal transduction pathway in melanoma cells resistant to growth inhibition by interferon-alpha. Pansky A, Hildebrand P, Fasler-Kan E, Baselgia L, Ketterer S, Beglinger C, Heim MH. Department of Research, University Hospital Basel, Basel, Switzerland. Advanced malignant melanoma is an aggressive malignancy with poor prognosis. Current therapeutic strategies have a modest success rate. The most promising treatment consists of a combination of chemotherapy with interferon-alpha, but complete response rates remain less than 15%. Interferon-alpha is also effective in adjuvant therapy for non-advanced melanoma treated surgically. The molecular mechanisms leading to loss of growth restraints and gain of growth-promoting functions during carcinogenesis of malignant melanoma are not understood in detail. Here, we studied 9 human melanoma cell lines with regard to growth inhibition by interferon-alpha and defects in intracellular signal transduction through the Jak-STAT pathway. In 3 cell lines, we found a complete loss of growth restraint by interferon-alpha. In all of them, different components of the Jak-STAT pathway were defective. Since signal transduction through the Jak-STAT pathway is necessary for antiviral and antiproliferative effects of interferons, we conclude that defects in this pathway may be one of the mechanisms that lead to cancer progression through loss of growth-restraining functions. Moreover, our results indicate that a subgroup of melanomas could be completely resistant to interferon-alpha and should therefore not be treated with this cytokine. Copyright 2000 Wiley-Liss, Inc. PMID: 10699955 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 75: EMBO J. 2000 Feb 1;19(3):392-9. A small amphipathic alpha-helical region is required for transcriptional activities and proteasome-dependent turnover of the tyrosine-phosphorylated Stat5. Wang D, Moriggl R, Stravopodis D, Carpino N, Marine JC, Teglund S, Feng J, Ihle JN. Department of Biochemistry, St Jude Children's Research Hospital, Memphis, TN 38105, USA. Cytokines induce the tyrosine phosphorylation and associated activation of signal transducers and activators of transcription (Stat). The mechanisms by which this response is terminated are largely unknown. Among a variety of inhibitors examined, the proteasome inhibitors MG132 and lactacystin affected Stat4, Stat5 and Stat6 turnover by significantly stabilizing the tyrosine-phosphorylated form. However, these proteasome inhibitors did not affect downregulation of the tyrosine-phosphorylated Stat1, Stat2 and Stat3. With Stat5 isoforms, we have observed that tyrosine-phosphorylated carboxyl-truncated forms of Stat5 proteins were considerably more stable than phosphorylated wild-type forms of the protein. Also, the C-terminal region of Stat5 could confer proteasome-dependent downregulation to Stat1. With a series of C-terminal deletion mutants, we have defined a relatively small, potentially amphipathic alpha-helical region that is required for the rapid turnover of the phosphorylated Stat5 proteins. The region is also required for transcriptional activation, suggesting that the functions are linked. The results are consistent with a model in which the transcriptional activation domain of activated Stat5 is required for its transcriptional activity and downregulation through a proteasome-dependent pathway. PMID: 10654938 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 76: FEBS Lett. 1999 Dec 17;463(3):360-4. Cell swelling activates STAT1 and STAT3 proteins in cultured rat hepatocytes. Meisse D, Dusanter-Fourt I, Husson A, Lavoinne A. Groupe de Biochimie et Physiopathologie Digestive et Nutritionnelle (GBPDN), Institut Federatif de Recherche Multidisciplinaire sur les Peptides no. 23 (IFRMP), UFR Medecine-Pharmacie de Rouen, 22 boulevard Gambetta, 76183, Rouen, France. In this paper, we demonstrated that in cultured rat hepatocytes cell swelling induced the activation of STAT1 and STAT3 proteins without any effect on STAT4, STAT5 and STAT6 proteins. Cell swelling induced an activation of STAT proteins through an increase in the phosphorylation of the tyrosine residue also phosphorylated by interleukin-6, but without any activation of JAK kinases. The signaling pathway by which cell swelling activated STAT1 and STAT3 is discussed. PMID: 10606754 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 77: Adv Pharmacol. 2000;47:113-74. Cytokines and STAT signaling. Schindler C, Strehlow I. Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA. Publication Types: Review PMID: 10582086 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 78: Proc Natl Acad Sci U S A. 1999 Nov 23;96(24):14007-12. Inhibition of DNA methyltransferase stimulates the expression of signal transducer and activator of transcription 1, 2, and 3 genes in colon tumor cells. Karpf AR, Peterson PW, Rawlins JT, Dalley BK, Yang Q, Albertsen H, Jones DA. The Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA. Inhibitors of DNA methyltransferase, typified by 5-aza-2'-deoxycytidine (5-Aza-CdR), induce the expression of genes transcriptionally down-regulated by de novo methylation in tumor cells. We utilized gene expression microarrays to examine the effects of 5-Aza-CdR treatment in HT29 colon adenocarcinoma cells. This analysis revealed the induction of a set of genes that implicated IFN signaling in the HT29 cellular response to 5-Aza-CdR. Subsequent investigations revealed that the induction of this gene set correlates with the induction of signal transducer and activator of transcription (STAT) 1, 2, and 3 genes and their activation by endogenous IFN-alpha. These observations implicate the induction of the IFN-response pathway as a major cellular response to 5-Aza-CdR and suggests that the expression of STATs 1, 2, and 3 can be regulated by DNA methylation. Consistent with STAT's limiting cell responsiveness to IFN, we found that 5-Aza-CdR treatment sensitized HT29 cells to growth inhibition by exogenous IFN-alpha2a, indicating that 5-Aza-CdR should be investigated as a potentiator of IFN responsiveness in certain IFN-resistant tumors. PMID: 10570189 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 79: J Biol Chem. 1999 Nov 5;274(45):32507-11. Catalytically active TYK2 is essential for interferon-beta-mediated phosphorylation of STAT3 and interferon-alpha receptor-1 (IFNAR-1) but not for activation of phosphoinositol 3-kinase. Rani MR, Leaman DW, Han Y, Leung S, Croze E, Fish EN, Wolfman A, Ransohoff RM. Department of Neurosciences, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA. TYK2, a Janus kinase, plays both structural and catalytic roles in type I interferon (IFN) signaling. We recently reported (Rani, M. R. S., Gauzzi, C., Pellegrini, S., Fish, E., Wei, T., and Ransohoff, R. M. (1999) J. Biol. Chem. 274, 1891-1897) that catalytically active TYK2 was necessary for IFN-beta to induce the beta-R1 gene. We now report IFN-beta-mediated activation of STATs and other components in U1 (TYK2-null) cell lines that were complemented with kinase-negative (U1.KR930) or wild-type TYK2 (U1.wt). We found that IFN-beta induced phosphorylation on tyrosine of STAT3 in U1.wt cells but not in U1.KR930 cells, whereas STAT1 and STAT2 were activated in both cell lines. Additionally, IFN-beta-mediated phosphorylation of interferon-alpha receptor-1 (IFNAR-1) was defective in IFN-beta treated U1.KR930 cells, but evident in U1.wt cells. In U1A-derived cells, the p85/p110 phosphoinositol 3-kinase isoform was associated with IFNAR-1 but not STAT3, and the association was ligand-independent. Further, IFN-beta treatment stimulated IFNAR-1-associated phosphoinositol kinase activity equally in either U1.wt or U1.KR930 cells. Our results indicate that catalytically active TYK2 is required for IFN-beta-mediated tyrosine phosphorylation of STAT3 and IFNAR-1 in intact cells. PMID: 10542297 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 80: Eur J Biochem. 1999 Oct 1;265(1):251-7. Different protein turnover of interleukin-6-type cytokine signalling components. Siewert E, Muller-Esterl W, Starr R, Heinrich PC, Schaper F. Institut fur Biochemie der RWTh Aachen, Germany. Interleukin (IL)-6 and IL-6-type cytokines signal through the gp130/Jak/STAT signal transduction pathway. The key components involved are the signal transducing receptor subunit gp130, the Janus kinases Jak1, Jak2 and Tyk2, STAT1 and STAT3 of the family of signal transducers and activators of transcription, the protein tyrosine phosphatase SHP2 and the suppressors of cytokine signalling SOCS1, SOCS2 and SOCS3. Whereas considerable information has been accumulated concerning the time-course of activation for the individual signalling molecules, data on the availability of the proteins involved in IL-6-type cytokine signal transduction are scarce. Nevertheless, availability of these molecules, determined by the balance of protein synthesis and degradation, also influences IL-6-type cytokine signal transduction. Here, we present a comprehensive set of data on the half-lives of the key molecules involved in the IL-6 signal transduction pathway. The turnover rates for the various proteins differ substantially. Three groups of signalling proteins can be discriminated: whereas the feedback inhibitors SOCS1, SOCS2 and SOCS3 are very short-lived, STAT1, STAT3 and SHP2 have an extremely slow turnover rate. Interestingly, the half-life of STAT3beta, a splice variant of STAT3alpha, is reduced to almost 50% of the half-life of STAT3alpha. The Janus kinases Jak1, Jak2, Tyk2 and gp130 show intermediate half-lives. Our data imply that signalling components activated by post-translational modifications are long-lived whereas the activity of very short-lived proteins is regulated mainly at the transcriptional level. PMID: 10491180 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 81: Am J Hum Genet. 1999 Oct;65(4):959-65. STAT5 signaling in sexually dimorphic gene expression and growth patterns. Davey HW, Wilkins RJ, Waxman DJ. AgResearch, Ruakura Research Centre, Hamilton, New Zealand. daveyh@agresearch.cri.nz The past 10 years have seen enormous advances in our understanding of how cytokine signals are mediated intracellularly. Of particular significance was the discovery of a family of seven Signal Transducer and Activators of Transcription (STAT) proteins. Each of these has now been studied in detail, and appropriate gene-disrupted mouse models are available for all except STAT2 (Leonard and O'Shea 1998). Fetal lethality is observed in Stat3-deficient mice, and various immunodeficiencies characterize mice with disrupted Stat1, Stat4, and Stat6 genes, which is consistent with impaired signaling from the specific cytokines that activate each of these proteins. The recent characterization of Stat5-deficient mice has led to several unanticipated findings that point to diverse biological functions for the two STAT5 forms, STAT5a and STAT5b. These include roles for one or both STAT5 forms in the immune system, hematopoiesis, sexually dimorphic growth, mammary development, hair growth, deposition of adipose tissue, and pregnancy. Here we review the hormone- and cytokine-activated signaling pathways in which STAT5 participates and the extensive evidence, from laboratory animals, that these factors are required for sex-specific aspects of development, including control of body size. Finally, we consider human growth disorders that may involve defects in STAT5-dependent signal transduction. Publication Types: Review Review, Tutorial PMID: 10486314 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 82: J Biol Chem. 1999 Sep 3;274(36):25343-9. Stat protein transactivation domains recruit p300/CBP through widely divergent sequences. Paulson M, Pisharody S, Pan L, Guadagno S, Mui AL, Levy DE. Department of Pathology and Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, New York 10016, USA. The signal transduction and activator of transcription (Stat) gene family has been highly conserved throughout evolution. Gene duplication and divergence has produced 7 mammalian Stat genes, each of which mediates a distinct process. While some Stat proteins are activated by multiple cytokines, Stat2 is highly specific for responses to type I interferon. We have cloned mouse Stat2 and found that while its sequence was more divergent from its human homologue than any other mouse-human Stat pairs, it was fully functional even in human cells. Overall sequence identity was only 69%, compared with 85-99% similarity for other Stat genes, and several individual domains that still served similar or identical functions in both species were even less well conserved. The coiled-coil domain responsible for interaction with IRF9 was only 65% identical and yet mouse Stat2 interacted with either human or mouse IRF9; the carboxyl terminus was only 30% identical and yet both regions functioned as equal transactivation domains. Both mouse and human transactivation domains recruited the p300/CBP coactivator and were equally sensitive to inhibition by adenovirus E1A protein. Interestingly, the Stat3 carboxyl terminus also functioned as a transactivator capable of recruiting p300/CBP, as does the Stat1 protein, although with widely differing potencies. Yet these proteins share no sequence similarity with Stat2. These data demonstrate that highly diverged primary sequences can serve similar or identical functions, and that the minimal regions of similarity between human and mouse Stat2 may define the critical determinants for function. PMID: 10464260 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 83: J Interferon Cytokine Res. 1999 Jul;19(7):797-801. Activation of the Jak-Stat pathway in cells that exhibit selective sensitivity to the antiviral effects of IFN-beta compared with IFN-alpha. Grumbach IM, Fish EN, Uddin S, Majchrzak B, Colamonici OR, Figulla HR, Heim A, Platanias LC. Section of Hematology-Oncology, University of Illinois at Chicago and West Side VA Hospital, 60607-7173, USA. We determined whether selective activation of components of the Jak-Stat pathway by different type I interferons (IFN) occurs in human myocardial fibroblasts that exhibit much higher sensitivity to the antiviral effects of IFN-beta than of IFN-alpha. Similar levels of activation of the Tyk2 kinase and the Stat3 transcription factor were induced in response to either IFN-beta or IFN-alpha treatment. However, activation of the Jak1 tyrosine kinase was detectable only in IFN-beta-treated but not IFN-alpha-treated cells. Consistent with this, tyrosine phosphorylation of Stat1 and Stat2 and formation of the IFN-stimulated gene factor 3 (ISGF3) complex occurred to a much higher degree in response to IFN-beta stimulation. These findings demonstrate that differential activation of distinct components of the Jak-Stat pathway by different type I IFN can occur. Furthermore, they strongly suggest that such selective activation accounts for the occurrence of differences in the antiviral properties of distinct type I IFN in certain cell types. PMID: 10454351 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 84: Biochem Biophys Res Commun. 1999 Aug 19;262(1):14-9. Evidence for the involvement of JAK/STAT pathway in the signaling mechanism of interleukin-17. Subramaniam SV, Cooper RS, Adunyah SE. Department of Biochemistry, Meharry Medical College, Nashville, Tennessee 37208, USA. Interleukin-17 is a T-cell-derived pro-inflammatory cytokine, exhibiting multiple biological activities in a variety of cells and believed to fine tune all general phases of hematopoietic response. However, the signaling mechanism of this novel cytokine remains unknown. Here, we report for the first time that the early signaling events triggered by interleukin-17 involve tyrosine phosphorylation of several members of the JAK and STAT proteins in human U937 monocytic leukemia cells. Immunoprecipitation with specific antibodies followed by Western blot analysis with antiphosphotyrosine antibody has shown that in U937 cells, interleukin-17 induces time-dependent stimulation of tyrosine phosphorylation of JAK 1, 2 and 3, Tyk 2 and STAT 1, 2, 3 and 4 within 0.5 to 30 min. Interleukin-17-mediated tyrosine phosphorylation of these proteins strongly suggests that the JAK/STAT signaling pathway may play a major role in transducing signals from interleukin-17 receptors to the nucleus. Copyright 1999 Academic Press. PMID: 10448060 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 85: J Biol Chem. 1999 Aug 20;274(34):24059-65. Urokinase induces activation and formation of Stat4 and Stat1-Stat2 complexes in human vascular smooth muscle cells. Dumler I, Kopmann A, Wagner K, Mayboroda OA, Jerke U, Dietz R, Haller H, Gulba DC. Franz Volhard Clinic and Max-Delbruck Center for Molecular Medicine, Virchow Klinikum-Charite, Humboldt University of Berlin, D-13125 Berlin, Germany. dumler@fvk-berlin.de Urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR) act in concert to stimulate cytoplasmic signaling machinery and transcription factors responsible for cell migration and proliferation. Recently we demonstrated that uPA activates the Janus kinase/signal transducers and activators of transcription (Stat1) signaling in human vascular smooth muscle and endothelial cells. However, the important question whether other transcription factors of the Stat family, in addition to Stat1, are involved in the uPAR-related signaling has not been addressed. In this study, we demonstrate that Stat4 and Stat2, but not Stat3, Stat5, or Stat6, are rapidly activated in response to uPA. We demonstrate further that Stat4 and Stat2 rapidly and transiently translocate to the cell nucleus where they bind specifically to the regulatory DNA elements. Analysis of Stat complexes formed in response to uPA revealed a Stat2-Stat1 heterodimer, which lacks p48, a DNA-binding protein known to combine with Stat1-Stat2. This new uPA-induced Stat2-Stat1 heterodimer binds to GAS (the interferon-gamma activation site) distinct from the interferon-stimulated response element to which the p48 protein containing complexes generally bind. We conclude that uPA activates a specific and unusual subset of latent cytoplasmic transcription factors in human vascular smooth muscle cells that suggests a critical role of uPA in these cells. PMID: 10446176 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 86: Circ Res. 1999 Apr 30;84(8):876-82. Stimulation of different subtypes of angiotensin II receptors, AT1 and AT2 receptors, regulates STAT activation by negative crosstalk. Horiuchi M, Hayashida W, Akishita M, Tamura K, Daviet L, Lehtonen JY, Dzau VJ. Cardiovascular Research, Department of Medicine, Harvard Medical School, Brigham and Women's Hospital, Boston, Mass., USA. horiuchi@m.chime-u.ac.jp Angiotensin II type 2 (AT2) receptor exerts an inhibitory action on cell growth. In the present study, we report that the stimulation of AT2 receptor in AT2 receptor cDNA-transfected rat adult vascular smooth muscle cells (VSMCs) inhibited angiotensin II type 1 (AT1) receptor-mediated tyrosine phosphorylation of STAT (signal transducers and activators of transcription) 1alpha/beta, STAT2, and STAT3 without influence on Janus kinase. AT2 receptor activation also inhibited the tyrosine phosphorylation of STAT1alpha/beta induced by interferon-gamma, epidermal growth factor, and platelet-derived growth factor. Similar effects of AT2 receptor were observed in R3T3 fibroblast and mouse fetal VSMCs, which express endogenous AT2 receptor. Moreover, AT2 receptor inhibited serine phosphorylation of STAT1alpha and STAT3 via the inhibition of extracellular signal-regulated kinase (ERK) activation. Stimulation of AT2 receptor inhibited the binding of STATs with sis-inducing element in c-fos promoter, resulting in decreased c-fos expression. Taken together, our results suggest that AT2 receptor can crosstalk negatively with multiple families of growth receptors by inhibiting ERK and STAT activation. PMID: 10222333 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 87: Proc Natl Acad Sci U S A. 1999 Apr 27;96(9):5007-12. The intracellular domain of interferon-alpha receptor 2c (IFN-alphaR2c) chain is responsible for Stat activation. Kotenko SV, Izotova LS, Mirochnitchenko OV, Lee C, Pestka S. Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854-5635, USA. Type I IFNs activate the Jak-Stat signal transduction pathway. The IFN-alpha receptor 1 (IFN-alphaR1) subunit and two splice variants of the IFN-alphaR2 subunit, IFN-alphaR2c and IFN-alphaR2b, are involved in ligand binding. All these receptors have been implicated in cytokine signaling and, specifically, in Stat recruitment. To evaluate the specific contribution of each receptor subunit to Stat recruitment we employed chimeric receptors with the extracellular domain of either IFN-gammaR2 or IFN-gammaR1 fused to the intracellular domains of IFN-alphaR1, IFN-alphaR2b, and IFN-alphaR2c. These chimeric receptors were expressed in hamster cells. Because human IFN-gamma exhibits no activity on hamster cells, the use of the human IFN-gamma receptor extracellular domains allowed us to avoid the variable cross-species activity of the type I IFNs and eliminate the possibility of contributions of endogenous type I IFN receptors into the Stat recruitment process. We demonstrate that Stat recruitment is solely a function of the IFN-alphaR2c intracellular domain. When chimeric receptors with the human IFN-gammaR1 extracellular domain and various human IFN-alpha receptor intracellular domains were expressed in hamster cells carrying the human IFN-gammaR2 subunit, only the IFN-alphaR2c subunit was capable of supporting IFN-gamma signaling as measured by MHC class I induction, antiviral protection, and Stat activation. Neither the IFN-alphaR2b nor the IFN-alphaR1 intracellular domain was able to recruit Stats or support IFN-gamma-induced biological activities. Thus, the IFN-alphaR2c intracellular domain is necessary and sufficient to activate Stat1, Stat2, and Stat3 proteins. PMID: 10220409 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 88: Cytokine. 1999 Mar;11(3):192-9. Ciliary neurotrophic factor and phorbol ester each decrease selected STAT3 pools in neuroblastoma cells by proteasome-dependent mechanisms. Malek RL, Halvorsen SW. Department of Biochemical Pharmacology, School of Pharmacy, State University of New York at Buffalo, Buffalo, NY 14260-1200, USA. Many cytokines and growth factors activate common signal transduction pathways and yet are able to elicit distinct cell-specific responses. We are defining mechanisms regulating signalling molecules in order to understand how cytokines can produce unique responses. It was found that individual members of the signal transducer and activator of transcription (STAT) family are regulated by ciliary neurotrophic factor (CNTF) and by protein kinase C. Treatment of SH-SY5Y human neuroblastoma cells with the phorbol ester, 12- O -tetradecanoylphorbol 13-acetate (TPA), for 4-5 h caused a 60% decline in both STAT2 and STAT3 levels and no decline in levels of STATs 1, 5 or 6, or in Jaks 1 or 2. The decline in STAT3 was inhibited by treatment with MG132, an inhibitor of proteasome-dependent protein degradation. Treatment of cells with CNTF induced a rapid tyrosine phosphorylation of STAT3 followed by a time-dependent decay of this signal. Loss of tyrosine phosphorylated STAT3 was inhibited by MG132 but did not require protein kinase C activity. These results suggest that STAT3 availability can be controlled by proteasome-dependent pathways activated either by protein kinase C or by cytokines. Copyright 1999 Academic Press. PMID: 10209066 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 89: J Biol Chem. 1999 Feb 12;274(7):4045-52. The proximal tyrosines of the cytoplasmic domain of the beta chain of the type I interferon receptor are essential for signal transducer and activator of transcription (Stat) 2 activation. Evidence that two Stat2 sites are required to reach a threshold of interferon alpha-induced Stat2 tyrosine phosphorylation that allows normal formation of interferon-stimulated gene factor 3. Nadeau OW, Domanski P, Usacheva A, Uddin S, Platanias LC, Pitha P, Raz R, Levy D, Majchrzak B, Fish E, Colamonici OR. Department of Pathology, University of Tennessee, Memphis, Tennessee 38163, USA. The precise role of the different subunits (alpha/IFNAR1 and betaL/IFNAR2) of the type I interferon receptor (IFN-R) in the activation of signal transducer and activator of transcription (Stat) 1, Stat2, and Stat3 has not yet been established. In this report we demonstrate that there are functionally redundant phosphotyrosine-dependent and -independent binding sites for Stat2 in the alpha and beta subunits of the type I IFN-R. Expression of a type I IFN-R containing only the constitutive Stat2 site or the proximal tyrosines of betaL, but not the docking site on the alpha chain (Tyr466 and Tyr481), supported low levels of Stat2 activation. However, the presence of only one intact Stat2 site did not lead to induction of interferon-stimulated gene factor 3 (ISGF3) or an antiviral state. Normal levels of Stat2 tyrosine phosphorylation, induction of ISGF3, and an antiviral effect always required the proximal tyrosines of betaL and at least one of the other Stat2 sites (Tyralpha466, 481 or betaL404-462). These data suggest that a threshold of Stat2 tyrosine phosphorylation is required for complete activation of ISGF3. Interestingly, a receptor in which all tyrosines were mutated to phenylalanine shows normal Stat3 phosphorylation and low levels of activation of Stat1. PMID: 9933596 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 90: Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10626-31. Inhibition of Stat1-mediated gene activation by PIAS1. Liu B, Liao J, Rao X, Kushner SA, Chung CD, Chang DD, Shuai K. Division of Hematology-Oncology, Department of Medicine, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA. STAT (signal transducer and activator of transcription) proteins are latent cytoplasmic transcription factors that become activated by tyrosine phosphorylation in response to cytokine stimulation. Tyrosine phosphorylated STATs dimerize and translocate into the nucleus to activate specific genes. Different members of the STAT protein family have distinct functions in cytokine signaling. Biochemical and genetic analysis has demonstrated that Stat1 is essential for gene activation in response to interferon stimulation. Although progress has been made toward understanding STAT activation, little is known about how STAT signals are down-regulated. We report here the isolation of a family of PIAS (protein inhibitor of activated STAT) proteins. PIAS1, but not other PIAS proteins, blocked the DNA binding activity of Stat1 and inhibited Stat1-mediated gene activation in response to interferon. Coimmunoprecipitation analysis showed that PIAS1 was associated with Stat1 but not Stat2 or Stat3 after ligand stimulation. The in vivo PIAS1-Stat1 interaction requires phosphorylation of Stat1 on Tyr-701. These results identify PIAS1 as a specific inhibitor of Stat1-mediated gene activation and suggest that there may exist a specific PIAS inhibitor in every STAT signaling pathway. PMID: 9724754 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 91: J Gen Virol. 1998 Aug;79 ( Pt 8):2007-12. Biological efficacy and signal transduction through STAT proteins of recombinant duck interferon in duck hepatitis B virus infection. Heuss LT, Heim MH, Schultz U, Wissmann D, Offensperger WB, Staeheli P, Blum HE. Department of Medicine, University Hospital Freiburg, Germany. The aim of this study was to characterize the interferon induced intracellular signals in duck hepatocytes and to investigate the effects of duck interferon on virus replication in duck hepatitis B virus (DHBV) infected ducks. Interestingly, duck interferon was found to activate intracellular signal transduction pathways similar to those of its mammalian counterparts. An interferon stimulated gel shift activity like that of gene factor 3 is induced, as well as serum inducible element binding factors homologous to serum inducible factor A (SIF-A), SIF-B and SIF-C. Duck interferon induced signal transducer and activator of transcription activation is not inhibited by DHBV infection of hepatocytes. DHBV infected ducks treated for 10 days with recombinant duck interferon show a decrease in viral DNA in hepatocytes, and in many cases disappearance of viraemia. These findings confirm the usefulness of the DHBV infection model for the study of human hepatitis B virus infection. PMID: 9714251 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 92: Eur J Biochem. 1998 Jun 15;254(3):514-9. Interferon-alpha activates signal transducers and activators of transcription 5 and 6 in Daudi cells. Fasler-Kan E, Pansky A, Wiederkehr M, Battegay M, Heim MH. Department of Research, University Hospital Basel, Switzerland. In most cells studied so far, interferon-alpha (IFN-alpha) activates signal transducer and activator of transcription (Stat1), Stat2 and Stat3, whereas interferon-gamma (IFN-gamma) induces Stat1 only. In general, each of the several dozens of cytokines, growth factors and hormones that signal through the Janus kinases-signal transducers and activators of transcription (Jak-STAT) pathway activates a distinct subset of STATs, and this selectivity is thought to be essential for the specificity of the cellular responses toward these ligands. Here, we have studied the pattern of STAT activation in the human lymphoblastoid cell line Daudi in response to IFN-alpha. In addition to Stat1, Stat2 and Stat3 activation, IFN-alpha was found to directly induce activation of Stat5 and Stat6. Cell-type-specific activation of additional STATs could be responsible for cell-type-specific responses to IFN-alpha. PMID: 9688261 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 93: J Biol Chem. 1998 Jul 24;273(30):18701-4. Activation of Raf-1 by interferon gamma and oncostatin M requires expression of the Stat1 transcription factor. Stancato LF, Yu CR, Petricoin EF 3rd, Larner AC. Laboratory of Cellular and Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA. A primary signaling cascade responsible for the expression of cytokine-stimulated immediate early genes involves the activation of the Jak/Stat pathway. In addition to being tyrosine-phosphorylated, several signal transducers and activators of transcription (Stats), including Stat1alpha, Stat3, and Stat4, are phosphorylated on a conserved serine residue, which is a consensus phosphorylation site for mitogen-activated protein kinases (MAPKs). Serine phosphorylation of Stat1alpha is required for maximal transcriptional activation of early response genes by interferon gamma (IFNgamma) as well as the antiviral and antigrowth actions of this cytokine. Incubation of cells with either IFNgamma or oncostatin M (OSM) activates Raf-1, a serine/threonine kinase responsible for the ultimate activation of p42 MAPK. To examine whether any of the signaling components that are required for activation of the Jak/Stat pathway are also necessary for activation of Raf-1 by IFNs and OSM, we examined activation of Raf-1 in cell lines that are deficient in either Stat1alpha or Stat2. Unexpectedly, incubation of Stat1-deficient, but not Stat2-deficient cells with IFNgamma or OSM for 5 min displayed no increase in Raf-1 activity. In peripheral blood lymphocytes Raf-1 was associated with Stat1, and this interaction was disrupted after incubation of cells with IFNgamma. Stat1-negative cells reconstituted with either Stat1alpha or Stat1alpha with a point mutation in the site where it is serine-phosphorylated displayed normal activation of Raf-1 by IFNgamma and OSM. However, activation of Raf-1 was not observed in lines that expressed Stat1alpha containing a mutation in its tyrosine phosphorylation site or in its SH2 domain. These results provide the first example of a novel role of Stat1alpha not as a transcription factor, but as a protein which may function to scaffold signaling components required for activation of the distinct Raf/MEK/MAPK signaling cascade. PMID: 9668040 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 94: Eur J Clin Invest. 1998 May;28(5):398-406. Growth effects of alpha-interferon but not of bombesin or angiotensin II are mediated by activation of STAT proteins. Pansky A, Hildebrand P, Heim MH, Eberhard M, Kissel T, Beglinger C. Department of Research, University Hospital, Basle, Switzerland. BACKGROUND: The recently discovered Jak/STAT signal transduction pathway is associated with cytokine or growth factor receptors; whether members of the G protein-coupled receptor superfamily also activate this pathway is not yet clear. As a first member, the angiotensin (AT)1A receptor has been demonstrated to phosphorylate Jak and STAT proteins. Bombesin, a neurotransmitter and growth factor in many cells and tissues, activates its G protein-coupled receptor and in addition phosphorylates proteins that might be members of the Jak/STAT family. This study investigated whether bombesin- or angiotensin-mediated growth effects are associated with STAT protein activation. METHODS: Functional receptors were characterized using ligand-binding studies, second-messenger activation and determination of ligand-mediated growth effects. STAT protein activation was analysed by electrophoretic mobility shift assay (EMSA) using labelled DNA response elements recognizing all known STAT proteins. RESULTS: Functional bombesin receptors mediating mitogenic effects were demonstrated on Swiss 3T3 fibroblasts, human melanoma cells (A375-6) and primary human lung fibroblasts; however, bombesin-related STAT protein activation was not observed by EMSA. Interferon-alpha typically activated a STAT1-STAT2-p48 heterotrimer, as well as STAT1-3 hetero- and homodimers in human melanoma cells and significantly inhibited growth of this cell line in vitro. Functional AT1A receptors on primary rat cardiac fibroblasts mediated angiotensin-stimulated growth effects but, in contrast to recently published data, did not activate any known STAT protein. CONCLUSION: Interferon alpha-stimulated growth inhibition is mediated by activation of the Jak/STAT pathway, whereas bombesin or AT1A receptor-mediated effects on cellular proliferation do not involve phosphorylation of STAT proteins. PMID: 9650014 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 95: Proc Natl Acad Sci U S A. 1998 May 12;95(10):5568-72. STAT3 complements defects in an interferon-resistant cell line: evidence for an essential role for STAT3 in interferon signaling and biological activities. Yang CH, Murti A, Pfeffer LM. University of Tennessee Health Science Center, Department of Pathology, 899 Madison Avenue, Memphis, TN 38163, USA. STAT proteins play critical roles in the signal transduction pathways for various cytokines. The type I interferons (IFNalpha/beta) promote the DNA-binding activity of the transcription factors STAT1, STAT2, and STAT3. Although the requirement for STAT1 and STAT2 in IFNalpha/beta signaling and action is well documented, the biological importance of STAT3 to IFN action has not yet been addressed. We found that STAT3 plays a critical role in signal transduction by IFNalpha/beta. A human cell line that is resistant to the antiviral and antiproliferative activities of IFN but is still IFN-responsive by virtue of STAT1 and STAT2 activation was found to be defective in STAT3 activation and in induction of NF-kappaB DNA-binding activity. Expression of STAT3 in these resistant cells complemented these signaling defects and also markedly increased cellular sensitivity to the antiviral and antiproliferative effects of IFN. Because STAT3 is involved in the induction of NF-kappaB DNA-binding activity and in the induction of antiviral and antiproliferative activity, our results place STAT3 as an important upstream element in type I IFN signal transduction and in the induction of biological activities. Therefore, our results indicate that STAT1 and STAT2 are not the only STATs required for the expression of the key biological activities of IFNalpha/beta. PMID: 9576923 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 96: Cancer Res. 1998 May 1;58(9):1914-9. Prolactin activates Stat1 but does not antagonize Stat1 activation and growth inhibition by type I interferons in human breast cancer cells. Schaber JD, Fang H, Xu J, Grimley PM, Rui H. Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA. Type I interferons (IFN alpha and IFN beta) are presently used in the adjuvant treatment of several human cancers. However, these cytokines have demonstrated only modest success in breast cancer therapy, and research efforts have focused on improving their efficacy. Recent progress in understanding the molecular mechanisms underlying the antiproliferative effects of IFNs has identified the cytoplasmic transcription factor Stat1 as a critical mediator. It is, therefore, possible that IFN-induced growth inhibition of mammary epithelial cells is counteracted by other cytokines that also use Stat1. One such candidate IFN-antagonist with particular relevance to breast cancer is the mammotropic hormone prolactin (PRL). The main goal of this study was to examine whether PRL would interfere with type I IFN (IFN alpha/beta) signal transduction by competing for limited cytoplasmic Stat factors. A second aim was to test whether pretreatment of mammary tumor cell lines with IFN gamma could enhance the effect of IFN alpha/beta. By analyzing the effect of PRL on IFN alpha/beta-induced tyrosine phosphorylation of Stat proteins and their binding to IFN-regulated genes, we now report that costimulation of PRL receptors did not interfere with IFN alpha/beta signals in several human breast cancer cell lines, including T47D, MCF-7, and BT-20. Specifically, PRL did not affect IFN alpha/beta-induced tyrosine phosphorylation or heterodimerization of Stat1 and Stat2 in any cell line. Instead, IFN alpha/beta- and PRL-induced tyrosine phosphorylation of Stat1 was additive and occurred without evidence of competition for limited concentrations of cytoplasmic Stat1. A similar additive relationship was observed on IFN alpha/beta- and PRL-induced Stat3 tyrosine phosphorylation. Furthermore, electrophoretic mobility shift assays showed that type I IFNs induced predominantly Stat1-Stat2 or Stat1-Stat3 heteromeric complexes with various IFN-response elements of IFN-stimulated genes, whereas PRL induced Stat1 homodimers. Despite significant mutual use of Stats by IFNs and PRL, these results indicated a high degree of signaling specificity in the two receptor systems, and that cytoplasmic levels of Stat proteins were not limiting. Similarly, PRL did not interfere with the growth-inhibitory effect of IFN beta. On the other hand, the study indicated that pretreatment of human breast cancer cell lines with IFN gamma enhanced the growth-inhibitory action of type I IFNs, suggesting a possible avenue for improving the effect of type I IFNs in the treatment of breast cancer patients. PMID: 9581833 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 97: J Immunol. 1998 Mar 15;160(6):2742-50. Induction of Jak/STAT signaling by activation of the type 1 TNF receptor. Guo D, Dunbar JD, Yang CH, Pfeffer LM, Donner DB. Department of Microbiology and Immunology and the Walther Oncology Center, Indiana University School of Medicine, Indianapolis 46202, USA. Cellular responses to TNF are initiated by either of two cell surface receptors, the type 1 TNF receptor (TNFR1) and the type 2 TNF receptor (TNFR2). Although neither receptor contains an intrinsic protein tyrosine kinase, such activity has been implicated in TNF action. In this study, we show that murine TNF induces the tyrosine phosphorylation and activation of the intracellular Janus tyrosine kinases Jak1, Jak2, and Tyk2 in murine 3T3-L1 adipocytes. Activation of Jak kinases by TNF was associated with tyrosine phosphorylation of STAT1, STAT3, STAT5, and STAT6, but not STAT2 or STAT4, showing that TNF acts on a specific subset of these latent cytoplasmic transcription factors in 3T3-L1 adipocytes. Agonist antiserum to TNFR1 induced Jak kinase and STAT protein phosphorylation. Phosphorylation of Jak proteins was also induced by human TNF, which selectively binds to TNFR1 on murine cells. 35S-labeled Jak kinases were precipitated from a cell-free system and from lysates of 3T3-L1 adipocytes by a glutathione S-transferase fusion protein containing the cytoplasmic domain of TNFR1. These results suggest that the cytoplasmic domain of TNFR1 can directly interact with and form signaling complexes with Jak kinases. Jak2 was precipitated from HeLa cells by antiserum to TNFR1, directly demonstrating their association in vivo. Thus, TNF activates a Jak/STAT signal-transduction cascade by acting through TNFR1. PMID: 9510175 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 98: Oncogene. 1998 Jan 29;16(4):505-15. Activation of Stat5 by platelet-derived growth factor (PDGF) is dependent on phosphorylation sites in PDGF beta-receptor juxtamembrane and kinase insert domains. Valgeirsdottir S, Paukku K, Silvennoinen O, Heldin CH, Claesson-Welsh L. Ludwig Institute for Cancer Research, Biomedical Center, Uppsala, Sweden. Signal transducers and activators of transcription (Stats) are known to transduce signals from the cell surface to the nucleus in cytokine receptor signaling. We examined the capacity of platelet-derived growth factor (PDGF) receptor to interact with and activate Stat molecules. Activation of the PDGF beta-receptor led to tyrosine phosphorylation of Stat1, Stat3 and Stat5, which was accompanied by specific DNA-binding activities. These events were only weakly stimulated by the activated PDGF alpha-receptor. In cells expressing PDGF beta-receptors mutated at Tyr579, Tyr581 or Tyr775, tyrosine phosphorylation as well as DNA-binding activity of Stat5 was reduced. Immobilized peptides containing phosphorylated Tyr579, Tyr581 or Tyr775 bound Stat5, suggesting direct binding of Stat5 to these tyrosine residues of the PDGF beta-receptor. Members of the Janus kinase family were also shown to interact with the PDGF beta-receptor, and to a lesser extent with the alpha-receptor, but their importance for PDGF-induced Stat activation remains to be determined. PMID: 9484840 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 99: Circ Res. 1998 Feb 9;82(2):244-50. Biphasic activation of the JAK/STAT pathway by angiotensin II in rat cardiomyocytes. Kodama H, Fukuda K, Pan J, Makino S, Sano M, Takahashi T, Hori S, Ogawa S. Cardiopulmonary Division, Keio University, Tokyo, Japan. This study was designed to demonstrate the characteristic pattern of angiotensin II-induced JAK/STAT (indicating just another kinase/signal transducer and activator of transcription) activation in cultured rat cardiomyocytes by comparing it with leukemia inhibitory factor (LIF)-induced activation. Angiotensin II (10(-7) mol/L) induced rapid phosphorylation of JAK2 and Tyk2, but not JAK1, and phosphorylated STAT1 and STAT2, but not STAT3, in the early stage up to 30 minutes. The time course of JAK/STAT activation by angiotensin II was apparently slower than that by LIF. Interestingly, angiotensin II phosphorylated STAT3 and rephosphorylated STAT1 in the late stage at 120 minutes. We also found that angiotensin II induced the formation of interferon-stimulating gene factor (ISGF) complexes biphasically, in the early stage at 15 to 30 minutes and in the late stage at 120 minutes, and that angiotensin II induced delayed activation of the sis-inducing factor (SIF) complex at 120 minutes. Formation of ISGF and SIF complexes in response to angiotensin II paralleled the phosphorylation pattern of STAT1 and STAT3 and was quite different from those obtained in response to LIF. The phosphorylation of STAT1 was suppressed by pretreatment with the angiotensin II type-1 (AT1) receptor antagonist CV11974, but the delayed addition of CV11974 failed to suppress phosphorylation of STAT3 at 120 minutes. In conclusion, angiotensin II-induced JAK/STAT activation in rat cardiomyocytes is biphasic and entirely different from LIF-induced activation. PMID: 9468195 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 100: J Biol Chem. 1998 Jan 9;273(2):1058-63. Granulocyte-macrophage colony-stimulating factor-activated signaling pathways in human neutrophils. Selective activation of Jak2, Stat3, and Stat5b. Al-Shami A, Mahanna W, Naccache PH. Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, and Department of Medicine, Faculty of Medicine, Laval University, Ste-Foy, Quebec G1V 4G2, Canada. Granulocyte-macrophage colony stimulating factor (GM-CSF) regulates many of the biological functions of human neutrophils. This includes the stimulation of protein synthesis and the tyrosine phosphorylation of various proteins among which is JAK2. The present study was aimed at characterizing in detail the pattern of activation by GM-CSF of the JAK/STAT pathway in human neutrophils. The results obtained show that the stimulation of human neutrophils by GM-CSF specifically led to tyrosine phosphorylation of JAK2 and had no effect on JAK1, JAK3, or TYK2. Furthermore, GM-CSF induced the tyrosine phosphorylation of STAT3 and STAT5 but not of STAT1, STAT2, STAT4, or STAT6. Tyrosine phosphorylation of STAT3 was transient reaching its maximum at 15 min. STAT5 presented a different pattern of tyrosine phosphorylation. The anti-STAT5 antibodies identified two proteins at 94 and 92 kDa. The 94-kDa STAT5 was constitutively tyrosine phosphorylated and showed no change upon GM-CSF stimulation. On the other hand, the 92-kDa STAT5 was tyrosine phosphorylated within 1 min of GM-CSF treatment and this was maintained for at least 30 min. By the use of specific antibodies, it was determined that only STAT5B, and not STAT5A, was tyrosine phosphorylated in GM-CSF-treated neutrophils. Furthermore, GM-CSF treatment induced an increase in the ability of STAT3 and STAT5B, but not STAT5A, to bind DNA probes. The specificity of the pattern of activation of the JAK/STAT pathway suggests that it may be directly linked to the modulation of the functions of mature nondividing, human neutrophils by GM-CSF. PMID: 9422769 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 101: Blood. 1998 Jan 15;91(2):570-6. Interferon-alpha resistance in a cutaneous T-cell lymphoma cell line is associated with lack of STAT1 expression. Sun WH, Pabon C, Alsayed Y, Huang PP, Jandeska S, Uddin S, Platanias LC, Rosen ST. Department of Pediatrics, Northwestern University Medical School, Chicago, IL, USA. Interferon-alpha (IFN alpha) mediates its biological effects through activation of the JAK-STAT signaling pathway and it has been shown to be one of most effective therapeutic agents for a number of hematological malignancies, including cutaneous T-cell lymphoma (CTCL). Nevertheless, its efficacy is limited by the development of clinical resistance but the reasons for resistance in CTCL are unknown. Here, we report the development of an IFN alpha-resistant CTCL cell line (HUT78R), characterized by its ability to proliferate in high concentration of recombinant IFN alpha, which can be used as a model system to study IFN resistance. The levels of IFN receptor expression and binding affinity were found to be comparable between the parental sensitive (HUT78S) and resistant (HUT78R) cells. However, IFN alpha stimulation failed to induce interferon-stimulated gene factor 3 (ISGF3) complex formation in HUT78R cells. In addition, the expression of the IFN-inducible 2-5 OAS gene was significantly reduced in HUT78R cells, suggesting the presence of a defect in the Jak-STAT signaling pathway. Our results showed that the IFN alpha-activated form of a latent transcriptional factor STAT1 was not found in HUT78R cells, whereas activated STAT2 and STAT3 were clearly detectable. By Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses, we found that HUT78R cells do not express any STAT1 protein or mRNA, suggesting the possibility of a null mutation in the STAT1 gene. Resistance to the growth inhibitory effect of IFN alpha in CTCL cells may result from lack of STAT1 expression. PMID: 9427711 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 102: Eur J Clin Invest. 1997 Nov;27(11):948-55. Specific activation of AP-1 but not Stat3 in regenerating liver in mice. Heim MH, Gamboni G, Beglinger C, Gyr K. Department of Research, University Hospital, Basle, Switzerland. Liver regeneration following partial hepatectomy is regulated by hepatotrophic factors whose precise roles are still elusive. In cell culture studies, some of them have been shown to activate members of the family of the signal transducers and activators of transcription (Stat). To test this contention in vivo, nuclear extracts were isolated from livers of partially hepatectomized and sham-operated mice killed at 30 different time points between zero and 108 h after surgery. Stat3 DNA binding is rapidly induced after surgery in both partially hepatectomized and sham-operated mice. Maximum activation of Stat3 is achieved 4-6 h after resection, and elevated Stat3 activation is detected as late as 60 h after surgery in both groups. Activated Stat5 is found sporadically in both sham-operated and resected mice but appears to be absent in the first 12 h after partial hepatectomy. Neither Stat1, Stat2, Stat4 nor Stat6 is induced during the time of observation. In contrast, AP-1 DNA binding activity is specifically induced in regenerating mouse liver. PMID: 9395792 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 103: Circ Res. 1997 Nov;81(5):656-63. Leukemia inhibitory factor, a potent cardiac hypertrophic cytokine, activates the JAK/STAT pathway in rat cardiomyocytes. Kodama H, Fukuda K, Pan J, Makino S, Baba A, Hori S, Ogawa S. Department of Internal Medicine, Keio University, Tokyo, Japan. Leukemia inhibitory factor (LIF) is a member of the interleukin-6 family of cytokines, which induces a wide range of responses in a variety of cells. The aim of this study was to investigate whether LIF induces cardiomyocyte hypertrophy and transmits signals through the JAK/STAT (indicating just another kinase/signal transducer and activator of transcription) pathway in primary cultured neonatal rat cardiomyocytes. LIF increased protein content and [3H]phenylalanine uptake in cardiomyocytes in a dose-dependent manner. LIF (10(3) U/mL) induced rapid tyrosine phosphorylation of gp130, JAK1, JAK2, STAT1, and STAT3 but not Tyk2 or STAT2. LIF also induced autokinase activity of JAK1 in a time-dependent manner. Gel shift assays for interferon gamma activation site/interferon-stimulated responsive element and sis-inducible element (SIE) revealed that LIF induced dimerization of STAT1 and STAT3 and formation of sis-inducing factor complexes, which subsequently interacted with SIE in the promoter. Preincubation with anti-STAT1 and anti-STAT3 antibodies inhibited the binding of SIF complexes. In conclusion, LIF induces cardiac hypertrophy and directly stimulates the JAK/STAT pathway in cardiomyocytes. PMID: 9351438 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 104: Blood. 1997 Oct 1;90(7):2574-82. The IRS-pathway operates distinctively from the Stat-pathway in hematopoietic cells and transduces common and distinct signals during engagement of the insulin or interferon-alpha receptors. Uddin S, Fish EN, Sher D, Gardziola C, Colamonici OR, Kellum M, Pitha PM, White MF, Platanias LC. Department of Medicine, University of Illinois at Chicago and West Side Veterans Affairs Hospital, 60607-7173, USA. Binding of interferon-alpha (IFN-alpha) to its receptor on hematopoietic cells activates the signal transducers and activators of transcription (Stat)- and insulin receptor substrate (IRS)-pathways, and regulates expression of antiproliferative and antiviral activities. However, it remains unknown whether these two pathways cooperate in the generation of IFN-alpha responses or function independently, and whether IRS-proteins transduce distinct downstream signals in response to IFNs or insulin/insulin-like growth factor (IGF)-1-mediated activation. Our data show that in response to IFN-alpha treatment, IRS-1 functions selectively as a docking protein for the SH2 domains of the p85 subunit of the PI 3'-kinase, but not the SH2 domain of Grb-2 which is engaged during insulin/IGF-1 signaling. In studies with THP-1 human myelomonocytic cells and 32D mouse myeloid cells, which are IRS-defective, we found that the IFN-alpha-regulated activation of Stat-1, Stat-2, and Stat-3 does not require the function of the IRS-system. Furthermore, THP-1 cells are responsive to the protective effect of IFN-alpha against vesicular stomatitis virus. Both 32D and THP-1 cells were resistant to the growth inhibitory effect of IFN-alpha, but this effect was not reversible by expression of IRS-1 or IRS-2 alone in 32D cells. Taken altogether these data show that: (1) The IRS-system transduces common and distinct signals in response to IFN-alpha or insulin/lGF-1 stimulation of hematopoietic cells. (2) The IRS-pathway operates separately from the Stat-pathway, and its function is not essential for the generation of the antiviral effect of IFN-alpha. (3) Neither the IRS- nor the Stat-pathways alone are sufficient to mediate the antiproliferative effects of IFN-alpha in hematopoietic cells, and additional signaling elements are required. PMID: 9326223 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 105: Circ Res. 1997 Oct;81(4):611-7. Role of angiotensin II in activation of the JAK/STAT pathway induced by acute pressure overload in the rat heart. Pan J, Fukuda K, Kodama H, Makino S, Takahashi T, Sano M, Hori S, Ogawa S. Cardiopulmonary Division, Keio University, Tokyo, Japan. This study was designed to determine whether the JAK/STAT (indicating just another kinase/signal transducer and activator of transcription) pathway is activated in cardiac hypertrophy induced in vivo by pressure overload in rats and to demonstrate whether angiotensin II is involved in the activation of the JAK/STAT pathway. Acute pressure overload was produced by constricting the abdominal aorta of Wistar rats. Immunoprecipitation-Western blot analysis revealed that pressure overload activated JAK1, JAK2, and Tyk2 as early as 5 minutes and that STAT1, STAT2, and STAT3 were tyrosine-phosphorylated rapidly after exposure to the pressure overload. Phosphorylation of STAT1 and STAT2 peaked in the early stage at 5 to 15 minutes, whereas that of STAT3 peaked in the late stage at 60 minutes. Gel mobility shift of the interferon gamma activation site/interferon alpha-stimulating response element was observed immediately after the aortic banding, whereas the band of sis-inducing element was shifted in the late stage at 60 minutes. Both cilazapril (angiotensin II-converting enzyme inhibitor) and E4177 (angiotensin II type 1 [AT1] receptor antagonist) significantly suppressed the phosphorylation of Tyk2 and partially inhibited the phosphorylation of JAK2, but neither affected JAK1. Coimmunoprecipitation of the AT1 receptor with JAK2 or Tyk2 was clearly observed at 5 minutes and peaked at 15 minutes (20-fold the control value). These results indicate that the JAK/STAT pathway is activated by acute pressure overload in rats and that angiotensin II is involved in activating Tyk2, and partially activating JAK2, via the AT1 receptor. Both angiotensin II-dependent and -independent pathways take part in activating the JAK/STAT pathway in the pressure-overloaded rat heart. PMID: 9314843 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 106: J Immunol. 1997 Sep 1;159(5):2212-21. IL-6 triggers cell growth via the Ras-dependent mitogen-activated protein kinase cascade. Ogata A, Chauhan D, Teoh G, Treon SP, Urashima M, Schlossman RL, Anderson KC. Dana-Farber Cancer Institute, and Department of Medicine, Harvard Medical School, Boston, MA 02215, USA. IL-6 mediates growth of some human multiple myeloma (MM) cells and IL-6-dependent cell lines. Although three IL-6 signaling pathways (STAT1, STAT3, and Ras-dependent MAPK cascade) have been reported, cascades mediating IL-6-triggered growth of MM cells and cell lines are not defined. In this study, we therefore characterized IL-6 signaling cascades in MM cell lines, MM patient cells, and IL-6-dependent B9 cells to determine which pathway mediates IL-6-dependent growth. IL-6 induced phosphorylation of JAK kinases and gp130, regardless of the proliferative response of MM cells to this growth factor. Accordingly, we next examined downstream IL-6 signaling via the STAT3, STAT1, and Ras-dependent mitogen-activated protein kinase (MAPK) cascades. IL-6 triggered phosphorylation of STAT1 and/or STAT3 in MM cells independent of their proliferative response to IL-6. In contrast, IL-6 induced phosphorylation of Shc and its association with Sos1, as well as phosphorylation of MAPK, only in MM cells and B9 cells that proliferated in response to IL-6. Moreover, MAPK antisense, but not sense, oligonucleotide inhibited IL-6-induced proliferation of these cells. These data suggest that STAT1 and/or STAT3 activation may occur independently of the proliferative response to IL-6, and that activation of the MAPK cascade is an important distal pathway for IL-6-mediated growth. PMID: 9278309 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 107: Mol Cell Biol. 1997 Jul;17(7):3833-40. Beta interferon and oncostatin M activate Raf-1 and mitogen-activated protein kinase through a JAK1-dependent pathway. Stancato LF, Sakatsume M, David M, Dent P, Dong F, Petricoin EF, Krolewski JJ, Silvennoinen O, Saharinen P, Pierce J, Marshall CJ, Sturgill T, Finbloom DS, Larner AC. Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892, USA. Activation of early response genes by interferons (IFNs) and other cytokines requires tyrosine phosphorylation of a family of transcription factors termed signal transducers and activators of transcription (Stats). The Janus family of tyrosine kinases (Jak1, Jak2, Jak3, and Tyk2) is required for cytokine-induced tyrosine phosphorylation and dimerization of the Stat proteins. In order for IFNs to stimulate maximal expression of Stat1alpha-regulated genes, phosphorylation of a serine residue in the carboxy terminus by mitogen-activated protein kinase (MAPK) is also required. In HeLa cells, both IFN-beta and oncostatin M (OSM) stimulated MAPK and Raf-1 enzyme activity, in addition to Stat1 and Stat3 tyrosine phosphorylation. OSM stimulation of Raf-1 correlated with GTP loading of Ras, whereas IFN-beta activation of Raf-1 was Ras independent. IFN-beta- and OSM-induced Raf-1 activity could be coimmunoprecipitated with either Jak1 or Tyk2. Furthermore, HeLa cells lacking Jak1 displayed no activation of STAT1alpha, STAT3, and Raf-1 by IFN-beta or OSM and also demonstrated no increase in the relative level of GTP-bound p21ras in response to OSM. The requirement for Jak1 for IFN-beta- and OSM-induced activation of Raf-1 was also seen in Jak1-deficient U4A fibrosarcoma cells. Interestingly, basal MAPK, but not Raf-1, activity was constitutively enhanced in Jak1-deficient HeLa cells. Transient expression of Jak1 in both Jak-deficient HeLa cells and U4A cells reconstituted the ability of IFN-beta and OSM to activate Raf-1 and decreased the basal activity of MAPK, while expression of a kinase-inactive form of the protein showed no effect. Moreover, U4A cells selected for stable expression of Jak1, or COS cells transiently expressing Jak1 or Tyk2 but not Jak3, exhibited enhanced Raf-1 activity. Therefore, it appears that Jak1 is required for Raf-1 activation by both IFN-beta and OSM. These results provide evidence for a link between the Jaks and the Raf/MAPK signaling pathways. PMID: 9199317 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 108: Proc Natl Acad Sci U S A. 1997 Jun 24;94(13):6764-9. Constitutive activation of a slowly migrating isoform of Stat3 in mycosis fungoides: tyrphostin AG490 inhibits Stat3 activation and growth of mycosis fungoides tumor cell lines. Nielsen M, Kaltoft K, Nordahl M, Ropke C, Geisler C, Mustelin T, Dobson P, Svejgaard A, Odum N. Institute of Medical Microbiology and Immunology, Section A, University of Copenhagen, 2200 N Copenhagen, Denmark. M.Nielsen@SB.IMMI.KU.DK Mycosis fungoides (MF) is a low-grade cutaneous T cell lymphoma of unknown etiology. In this report, the Jak/Stat (Janus kinase/signal transducer and activator of transcription) signaling pathway was investigated in tumor cell lines established from skin biopsy specimens from a patient with MF. Jaks link cytokine receptors to Stats, and abnormal Jak/Stat signaling has been observed in some hemopoietic cancers. In MF tumor cells, a slowly migrating isoform of Stat3, Stat3(sm), was found to be constitutively activated, i.e., (i) Stat3(sm) was constitutively phosphorylated on tyrosine residues, and tyrosine phosphorylation was not enhanced by growth factor stimulation; (ii) band shift assays and immunoprecipitations of DNA/Stat complexes showed constitutive DNA-binding properties of Stat3(sm); and (iii) Stat3(sm) was constitutively associated with Jak3. The abnormal activation of Stat3(sm) was highly specific. Thus, neither the fast migrating isoform of Stat3 (Stat3(fm)) nor other Stats (Stat1, Stat2, and Stat4 through Stat6) were constitutively activated. The Jak kinase inhibitor, tyrphostin AG490, blocked the constitutive activation of Stat3(sm) and inhibited spontaneous as well as interleukin 2-induced growth of MF tumor cells. In conclusion, we have provided evidence for an abnormal Jak/Stat signaling and growth regulation in tumor cells obtained from affected skin of an MF patient. PMID: 9192639 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 109: Cell Growth Differ. 1997 Jun;8(6):687-98. Retinoic acid induces signal transducer and activator of transcription (STAT) 1, STAT2, and p48 expression in myeloid leukemia cells and enhances their responsiveness to interferons. Matikainen S, Ronni T, Lehtonen A, Sareneva T, Melen K, Nordling S, Levy DE, Julkunen I. National Public Health Institute, Helsinki, Finland. IFNs are antiproliferative cytokines that have growth-inhibitory effects on various normal and malignant cells. Therefore, they have been used in the treatment of certain forms of cancer, such as chronic myelogenous leukemia and hairy cell leukemia. However, there is little evidence that IFNs would be effective in the treatment of acute myelogenous leukemia, and molecular mechanisms underlying IFN unresponsiveness have not been clarified. Here we have studied the activation and induction of IFN-specific transcription factors signal transducer and activator of transcription (STAT) 1, STAT2, and p48 in all-trans-retinoic acid (ATRA)-differentiated myeloid leukemia cells using promyelocytic NB4, myeloblastic HL-60, and monoblastic U937 cells as model systems. These cells respond to ATRA by growth inhibition and differentiation. We show that in undifferentiated NB4 cells, 2',5'-oligoadenylate synthetase and MxB gene expression is not activated by IFN-alpha, possibly due to a relative lack of signaling molecules, especially p48 protein. However, during ATRA-induced differentiation, steady-state STAT1, STAT2, and especially p48 mRNA and corresponding protein levels were elevated both in NB4 and U937 cells, apparently correlating to an enhanced responsiveness of these cells to IFNs. ATRA treatment of NB4 cells sensitized them to IFN action as seen by increased IFN-gamma activation site DNA-binding activity or by efficient formation of IFN-alpha-specific ISGF3 complex and subsequent oligoadenylate synthetase and MxB gene expression. Lack of p48 expression could be one of the mechanisms of promyelocytic leukemia cell escape from growth-inhibitory effects of IFN-alpha. PMID: 9186002 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 110: J Biol Chem. 1997 Apr 4;272(14):9550-5. The amino acid residues immediately carboxyl-terminal to the tyrosine phosphorylation site contribute to interleukin 6-specific activation of signal transducer and activator of transcription 3. Inoue M, Minami M, Matsumoto M, Kishimoto T, Akira S. Institute for Molecular and Cellular Biology, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565, Japan. Signal transducers and activators of transcription (Stat) proteins play an important role in signaling through a variety of cytokine and growth factor receptors. Each of the Stat proteins is activated in a ligand-specific manner. Only the Src homology 2 (SH2) domains of Stat1 and Stat2 are critical for the ligand-specific activation of interferon signaling. In this study we determined the domains in Stat3 protein that contribute to interleukin 6 (IL-6)-specific phosphorylation. Based on evidence that Stat3, but not Stat1, is activated in the presence of low levels of IL-6 and soluble IL-6 receptor, we constructed various swap mutants between Stat3 and Stat1 and examined their response to IL-6 after their transient expression into COS7 cells. The region upstream of the SH2 domain was exchangeable between Stat1 and Stat3, whereas the region carboxyl-terminal to the SH2 domain of Stat3 was critical to phosphorylation by IL-6. However, unlike Stat1 and Stat2 in interferon signaling, the swap mutant in which 5 amino acid residues just carboxyl-terminal to the tyrosine phosphorylation site (Tyr705) in Stat3 was replaced by the corresponding region derived from Stat1 was not phosphorylated in response to IL-6. Substituting 1 amino acid (Lys709) at position +4 relative to Tyr705 abolished the tyrosine phosporylation of Stat3 in response to IL-6. Co-immunoprecipitation experiments demonstrated that these mutants were associated with gp130 at an extent similar to wild-type Stat3. Taken together, these results show that the amino acid residues immediately carboxyl-terminal to the tyrosine phosphorylation site are involved in IL-6-specific activation of Stat3. PMID: 9083098 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 111: J Clin Invest. 1997 Feb 1;99(3):447-56. Differential human multiple myeloma cell line responsiveness to interferon-alpha. Analysis of transcription factor activation and interleukin 6 receptor expression. Jelinek DF, Aagaard-Tillery KM, Arendt BK, Arora T, Tschumper RC, Westendorf JJ. Department of Immunology, Mayo Clinic/Foundation, Rochester, Minnesota 55905, USA. jelinek.diane@mayo.edu Although IFN-alpha is commonly used as maintenance treatment for multiple myeloma patients, its effectiveness is varied. In this study, we have used a panel of IL-6 responsive myeloma cell lines that vary remarkably in responsiveness to IFN-alpha. Three cell lines were growth arrested by IFN-alpha; however, IFN-alpha significantly stimulated growth of the fourth cell line, KAS-6/1. Our studies have focused on elucidating the mechanism of differential IFN-alpha responsiveness. First, we have shown that IFN-alpha-stimulated growth of the KAS-6/1 cells did not result from induction of autocrine IL-6 expression. Second, analysis of Stats 1, 2, and 3 and IFN regulatory factor-1 (IRF-1) and IRF-2 activation failed to reveal differences between the IFN-alpha growth-arrested or growth-stimulated cells. Third, although IFN-alpha treatment of the IFN-alpha growth-inhibited cell lines reduced IL-6 receptor (IL-6R) expression, IFN-alpha also reduced KAS-6/1 IL-6R expression. Finally, although IFN-alpha treatment reduced IL-6R numbers on each cell line, analysis of Stat protein activation revealed that the receptors were still functional. We conclude that myeloma cell responsiveness to IFN-alpha is heterogeneous and that mechanisms of IFN-alpha-mediated growth inhibition other than IL-6R downregulation must exist in myeloma. Identification of these mechanisms may allow development of agents that are more universally effective than IFN-alpha. PMID: 9022078 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 112: Clin Exp Immunol. 1996 Oct;106(1):179-86. IL-2 signalling in T and natural killer (NK) cells associated with their class I-non-restricted killing activity. Yoneda K, Osaki T, Yamamoto T. Department of Oral Surgery, Kochi Medical School Kohasu, Japan. The signal transduction of IL-2 in NK cells and T cells was compared. On 5 min incubation of these cells with IL-2, we observed tyrosine phosphorylation of 105-kD and 110-kD proteins in NK cells and of 95-kD and 110-kD proteins in T cells. The phosphorylation reached maximal levels in 15 min in both NK and T cells, but the levels were higher in NK cells, which showed superior killing against Daudi cells. With this phosphorylation, p52rhc was also tyrosine-phosphorylated and p21ras was activated by the short term (10 min) treatment of NK and T cells with IL-2. These signals were completely suppressed by anti-IL-2R beta MoAb, but only slightly suppressed by anti-IL-2R alpha MoAb, correlated with the suppression of the class-I-non-restricted cytotoxic activity of NK and T cells by these MoAbs. When tyrosine phosphorylation was inhibited by herbimycin A and genistein, the cytotoxic activities of NK and T cells were nearly completely suppressed. In addition, the tyrosine phosphorylation of JAK3 by IL-2 was more prominent in NK cells than in T cells, but JAK1, JAK2, STAT1 alpha, STAT2 and STAT3 were not phosphorylated. These results indicate that the IL-2 signal flows downstream via both ras-dependent and ras-independent pathways and that the superior killing activity of NK cells depends on their high susceptibility to protein tyrosine phosphorylation by IL-2. PMID: 8870717 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 113: Int Immunol. 1996 Aug;8(8):1205-11. The genomic structure and chromosomal localization of the mouse STAT3 gene. Shi W, Inoue M, Minami M, Takeda K, Matsumoto M, Matsuda Y, Kishimoto T, Akira S. Division of Immunology, Osaka University, Japan. A variety of cytokines induce the tyrosine phosphorylation of signal transducers and activators of transcription (STATs). Activation of the same STAT proteins by distinct cytokines and activation of different STAT proteins by each cytokine are thought to contribute to redundancy and pleiotropy of cytokine actions respectively. STAT3 is rapidly tyrosine phosphorylated in response to IL-6, ciliary neurotrophic factor, oncostatin M, leukemia inhibitory factor, IL-11, granulocyte colony stimulation factor and epidermal growth factor. In this report we have isolated and characterized the mouse genomic structure of STAT3. The mouse STAT3 gene consisted of 24 exons which spanned > 37 kb. The structure of the mouse STAT3 gene was almost identical to that of the human STAT2 gene, including the number and size of exons, indicating that the exon-intron organization had already been accomplished before these two genes duplicated, and then these genes evolved to respond to different ligands. By molecular linkage analysis with interspecific backcross mice the STAT3 gene mapped at 1.4 cM proximal to D11Mit59 on mouse chromosome 11. The promoter region contained potential regulatory elements such as GATA, NF-IL-6, PEBP2, Sp-1, AP-2 binding sites, cAMP response element, CAAT box and E-box. Transient expression of constructs harboring the 5' flanking region of the STAT3 gene fused to the luciferase gene showed that a 160 bp sequence upstream of the transcription start site conferred a basal and an IL-6-inducible promoter activity. PMID: 8918689 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 114: J Biol Chem. 1996 May 24;271(21):12408-13. Application of genomic DNA affinity chromatography identifies multiple interferon-alpha-regulated Stat2 complexes. Ghislain JJ, Fish EN. Department of Microbiology, University of Toronto, Ontario, Canada. Interferon-alpha (IFN-alpha)-induced signal transduction is mediated by the phosphorylation-activation of the signal transducer and activator of transcription (STAT) proteins Stat1, Stat2, and Stat3. Previous studies have shown that these activated STATs dimerize to form four distinct STAT complexes which translocate to the nucleus and activates transcription by binding to specific promoter elements. The interferon-stimulated gene factor-3 (ISGF3) consists of Stat2 and Stat1 heterodimers in association with a DNA-binding protein, p48, that binds to the interferon stimulated response element. Homo-and heterodimers of Stat1 and Stat3 bind to the palindromic interferon response element (pIRE). In this report we demonstrate the utility of a biochemical procedure that we have developed, based on genomic DNA affinity chromatography, for the identification of IFN-alpha-induced STAT complexes. Using this approach, we identified ISGF3-independent Stat2-containing STAT complexes. Results from the analysis of Stat2 complexes in the electrophoretic mobility shift assay were consistent with genomic DNA affinity chromatography results and identified a Stat2:1 complex that binds with low affinity to the pIRE of the interferon regulatory factor-1 gene. Immunoprecipitation studies of Stat2 revealed an IFN-alpha dependent co-precipitation of both Stat1 and Stat3. Taken together, our results suggest that IFN-alpha activates, in addition to ISGF3, other Stat2-containing STAT complexes, one of which binds to an element related to the interferon regulatory factor-1 pIRE. PMID: 8647845 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 115: Mol Cell Biol. 1996 Apr;16(4):1595-603. Activation and association of Stat3 with Src in v-Src-transformed cell lines. Cao X, Tay A, Guy GR, Tan YH. Signal Transduction Laboratory, Institute of Molecular and Cell Biology, National University of Singapore. STAT proteins are a group of latent cytoplasmic transcription factors which function as signal transducers and activators of transcription. Stat1 and -2 were originally identified to function in interferon signaling, and Stat1 was also found to be activated by epidermal growth factor (EGF) and other cytokines. New members of the STAT gene family are identified. Among them, Stat3 has 52.5% amino acid sequence homology with Stat1 and is activated by platelet-derived growth factor (PDGF), colony-stimulating factor 1 (CSF-1), EGF, interleukin-6, and other cytokines. Treatment of cells with EGF activates Stat1 and Stat3, which become phosphorylated on tyrosine residues to form homo - or heterodimers and translocate into the nucleus, binding to the sis-inducible element (SIE) in the c-fos promoter. Somatic cell genetic analyses demonstrated that Jaks, a family of nontransmembrane protein tyrosine kinases, are required for the activation of Stat1 and Stat2 in interferon-treated cells. However, little is known about the activation of Stat3 by growth factors. Here we report that in all v-Src-transformed cell lines examined, Stat3 is constitutively activated to bind to DNA and the phosphorylation of tyrosine on Stat3 is enhanced by the induction of v-Src expression. We also report that Src is shown to be associated with Stat3 in vivo, as well as in vitro, and phosphorylates Stat3 in vitro. Stat3 is also activated by CSF-1, possibly through CSF-1 receptor-c Src association in NIH 3T3 cells overexpressing CSF-1 receptors. Together, the data suggest that Src is involved in activation of Stat3 in growth factor signal transduction. PMID: 8657134 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 116: Mol Cell Biol. 1996 Apr;16(4):1419-24. Inhibition of alpha interferon but not gamma interferon signal transduction by phorbol esters is mediated by a tyrosine phosphatase. Petricoin E 3rd, David M, Igarashi K, Benjamin C, Ling L, Goelz S, Finbloom DS, Larner AC. Division of Cytokine Biology, Center for Biologics Evaluation and Research, Bethesda, Maryland 20892, USA. Previous studies have indicated that the expression of viral oncoproteins, cell transformation, or phorbol ester treatment of cells can inhibit alpha/beta interferon (IFN-alpha/beta)-induced gene expression. The mechanisms by which these promoters of cell growth exert their inhibitory effects vary, but in most instances they involve a disruption of the IFN-alpha/beta-induced transcription complex ISGF3 such that the DNA-binding component of this complex (the 48-kDa ISGF3gamma protein) does not bind to the interferon-stimulated response element (ISRE). In this report, we demonstrated that phorbol ester treatment of human peripheral blood monocytes dramatically inhibits activation of IFN-alpha/B-stimulated early response genes but by a mechanism which does not involve abrogation of the ISRE binding of ISGF3gamma. Phorbol ester treatment of monocytes inhibited IFN alpha-stimulated tyrosine phosphorylation of the transcription factors Stat1alpha, Stat2, and Stat3 and of the tyrosine kinase Tyk2 but had no effect on IFN-gamma activation of Stat1alpha. IFNalpha-stimulated tyrosine phosphorylation of Jak1 and the alpha subunit of the IFN-alpha receptor were unaffected by phorbol 12-myristate 13-acetate (PMA). Moreover, PMA caused the dephosphorylation of Tyk2 but not of Jak1, which was activated by IFN. Pretreatment of cells with vanadate prevented the effects of PMA with regard to PMA-induced Tyk2 dephosphorylation. These observations suggest that PMA exerts its inhibitory effects by activation of a tyrosine phosphatase which selectively regulates Tyk2 but not Jak1 activity. PMID: 8657115 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 117: J Biol Chem. 1996 Feb 23;271(8):4134-7. Preassociation of STAT1 with STAT2 and STAT3 in separate signalling complexes prior to cytokine stimulation. Stancato LF, David M, Carter-Su C, Larner AC, Pratt WB. Department of Pharmacology, University of Michigan Medical School, Ann Arbor, 48109, USA. A variety of cytokines and growth factors act through an induction of gene expression mediated by a family of latent transcription factors called STAT (signal transducers and activators of transcription) proteins. Ligand-induced tyrosine phosphorylation of the STATs promotes their homodimer and heterodimer formation and subsequent nuclear translocation. We demonstrate here that STAT protein heterocomplexes exist prior to cytokine treatment. When unstimulated HeLa cells are ruptured in hypotonic buffer without salt or detergent, immunoadsorption of either STAT1 or STAT2 from the resulting cytosol yields coimmunoadsorption of the other STAT protein. Similarly, STAT1-STAT3 heterocomplexes are coimmunoadsorbed from hypotonic cytosol. STAT1 and STAT2 or STAT1 and STAT3 translated in reticulocyte lysate spontaneously form heterocomplexes when the translation lysates are mixed at 0 degrees C. Our data suggest that interferon-alpha /beta-induced tyrosine phosphorylation increases the stability of a preexisting, latent, STAT1-STAT2 signaling complex. Newly translated STAT1 binds in equilibrium fashion to STAT2 and STAT3, but we show that STAT2 and STAT3 exist in separate heterocomplexes with STAT1, consistent with a model in which STAT1 contains a common binding site for other STAT proteins. PMID: 8626752 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 118: Blood. 1996 Jan 15;87(2):439-46. Thrombopoietin induces tyrosine phosphorylation of Stat3 and Stat5 in human blood platelets. Miyakawa Y, Oda A, Druker BJ, Miyazaki H, Handa M, Ohashi H, Ikeda Y. Department of Internal Medicine, Keio University, Tokyo, Japan. Thrombopoietin is known to be essential for megakaryocytopoiesis and thrombopoiesis. Recently, we and others have shown that thrombopoietin induces rapid tyrosine phosphorylation of Jak2 and other proteins in human platelets and BaF3 cells, genetically engineered to express c-Mpl, a receptor for thrombopoietin. The Jak family of tyrosine kinases are known to mediate some of the effects of cytokines or hematopoietic growth factors by recruitment and tyrosine phosphorylation of a variety of Stat (signal transducers and activators of transcription) proteins. Hence, we have investigated whether Stat proteins are present in platelets and, if so, whether they become tyrosine phosphorylated in response to thrombopoietin. We immunologically identified Stat1, Stat2, Stat3, and Stat5 in human platelet lysates. Thrombopoietin induced tyrosine phosphorylation of Stat3 and Stat5 in these cells. Thrombopoietin also induced tyrosine phosphorylation of Stat3 and Stat5 in FDCP-2 cells genetically engineered to constitutively express human c-Mpl. Thus, our data indicate that Stat3 and Stat5 may be involved in signal transduction after ligand binding to c-Mpl and that this event may have a role in megakaryopoiesis/thrombopoiesis or possibly a mature platelet function such as aggregation. Publication Types: Review Review, Tutorial PMID: 8555464 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 119: Adv Immunol. 1996;61:147-99. Interleukin-2 receptor signaling mechanisms. Karnitz LM, Abraham RT. Department of Immunology, Mayo Clinic, Rochester, Minnesota 55905, USA. Publication Types: Review PMID: 8834496 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 120: Cell Signal. 1995 Nov;7(8):739-45. Regulation of the Jak/STAT signalling pathway. Finbloom DS, Larner AC. Food and Drug Administration, Center for Biologics Evaluation and Research, Division of Cytokine Biology, Bethesda, MD 20892-4555, USA. Publication Types: Review Review, Tutorial PMID: 8593242 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 121: J Biol Chem. 1995 Sep 15;270(37):21785-92. Configuration of the interferon-alpha/beta receptor complex determines the context of the biological response. Ghislain J, Sussman G, Goelz S, Ling LE, Fish EN. Department of Microbiology, University of Toronto, Ontario, Canada. Constituents of the Type 1 interferon (IFN) receptor (IFNABR) identified to date include the alpha and beta transmembrane subunits and the associated intracellular kinases, Jak 1 and Tyk 2. In this report, we demonstrate that a human cell type that expresses both subunits of IFNABR, together with Jak 1 and Tyk 2, exhibits a limited binding capacity for and is only partially sensitive to the effects of IFN-alpha/beta, despite adequate levels of the cytoplasmic transcription factors Stat1, Stat2, and Stat3. Specifically, a low affinity interaction between IFN-alpha/beta and cell surface receptors results in ISGF3 (Stat1:2) activation and an antiviral response, yet no IFN-inducible growth inhibition. Using a panel of murine cells that are variably configured with respect to the human IFNABR-alpha/beta subunits, we provide evidence that an additional component(s) encoded on human chromosome 21 is required to confer high affinity binding and IFN-inducible growth inhibition to cells that express the alpha and beta subunits of the IFNABR. The data indicate that transcriptional activation that leads to an antiviral response is mediated by IFN-alpha/beta activation of IFNABR-alpha and IFNABR-beta in the context of a low affinity interaction, yet a high affinity interaction is necessary for signal transducing events that mediate growth inhibition. We provide evidence that the extent of ISGF3 activation correlates directly with the magnitude of an antiviral but not a growth inhibitory response. PMID: 7665599 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 122: Blood. 1995 Jul 15;86(2):557-71. Signal transduction by the receptors for thrombopoietin (c-mpL) and interleukin-3 in hematopoietic and nonhematopoietic cells. Morella KK, Bruno E, Kumaki S, Lai CF, Fu J, Wang HM, Murray L, Hoffman R, Timour M, Benit L, et al. Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA. Antisense oligonucleotide to the translation initiation sequence of human c-mpI reduced the proliferation of human CD34+ bone marrow cells in response to interleukin-3 (IL-3) alone or to the combination of IL-3 and thrombopoietin (TPO). To investigate the molecular basis for these cytokine interactions, we analyzed the relationship between the receptor subunits for IL-3 and TPO and determined whether both receptors activate identical signal transduction pathways. The function of the receptor subunits was characterized in transiently transfected hepatoma cells and fibroblasts by the activation of gene expression via specific regulatory elements and by the stimulation of DNA-binding activity of STAT proteins. Although c-mpl and IL-3 receptor (IL-3R) reconstituted a qualitatively comparable gene regulatory response, there was no detectable functional interaction between their respective receptor subunits. By comparing the receptor action in different cell lines, we observed that in human hepatoma cells the signaling of c-mpI was 100-fold less sensitive to TPO than in rat hepatoma cells. However, IL-3R signaling was comparable between the two cell types, suggesting that c-mpI and IL-3R do not use identical signal transducing mechanisms. The cytoplasmic domains necessary for c-mpI signaling were determined by testing deletion mutants. The membrane-proximal box 1 sequence motif was critical for gene regulation and for STAT protein activation that seemed to involve the Janus kinase 2 (JAK2). Because IL-3R was less dependent on JAK2 than c-mpI, different levels of JAK2 expression may account, in part, for the quantitative difference in IL-3 and TPO response among various cell lines. PMID: 7605989 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 123: EMBO J. 1995 Jun 15;14(12):2847-56. Thrombopoietin activates a STAT5-like factor in hematopoietic cells. Pallard C, Gouilleux F, Benit L, Cocault L, Souyri M, Levy D, Groner B, Gisselbrecht S, Dusanter-Fourt I. U363 INSERM, ICGM, Hopital Cochin, Paris, France. Thrombopoietin (TPO) is a newly cloned cytokine which is the major regulator of circulating platelet levels, acting on both proliferation and differentiation of megakaryocytes. We have investigated the ability of TPO to activate the JAK/STAT pathway in megakaryocytic cell lines. We used either the granulocyte-macrophage colony-stimulating factor (GM-CSF)- and/or erythropoietin (EPO)-dependent UT7 cell line in which the murine TPO receptor (mumpl) had been transfected (mumpl-UT7 transfectants) or the MO7E and DAMI cells which express endogenous human TPO receptors. We demonstrated that TPO activates the kinase JAK2 and a STAT5-like transcriptional factor but not STAT1, STAT2, STAT3 or STAT4, in a very rapid and transient manner. In order to better ascertain the specificity of the activation of STAT5-related factor by TPO, we investigated the effect of other cytokines/growth factors. Both GM-CSF and EPO activated the STAT5-like factor. In contrast, neither interferon (IFN)-gamma nor the mitogenic stem cell factor (SCF) activated STAT5, although IFN-gamma did activate STAT1 in those cells. The hematopoietic DNA binding activity related to STAT5 was identified as a p97 tyrosine-phosphorylated protein band which exhibited identical gel mobility to the mammary STAT5. Because v-mpl, a truncated form of the TPO receptor c-mpl, was shown to be oncogenic, we tested the activity of v-mpl on STAT5 and found STAT5 constitutively activated in two different v-mpl-expressing cells, the transiently transfected Cos7 cells and the stable v-mpl-UT7 transfectants. Overall, our data indicate that STAT5 is widely expressed in hematopoietic cells and activated by a number of cytokines, including TPO, GM-CSF and EPO, but not by IFN-gamma or SCF. PMID: 7796811 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 124: Annu Rev Biochem. 1995;64:621-51. Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. Schindler C, Darnell JE Jr. Department of Medicine, Columbia University Medical Center, New York, N.Y., USA. Cytokines and growth factors regulate multiple aspects of cell growth through their interactions with specific receptors. These receptors initiate signals directed at both the cytoplasmic and the nuclear compartments. Many of the nuclear signals culminate in the induction of new genes. Characterization of the ability of IFN-alpha to rapidly induce new genes has led to the identification of a new signaling paradigm, the JAK-STAT (Signal Transducers and Activators of Transcription) pathway. In the IFN-alpha pathway, two receptor associated tyrosine kinases from the JAK family, Jak1 and Tyk2, mediate the activation of two latent cytoplasmic transcription factors, Stat1 and Stat2. More recent studies have not only determined that this pathway is used extensively, but have led to the identification of additional components (e.g., Jak2, Jak3, Stat3, Stat4, Stat5, and Stat6). This review will examine how these components mediate the transduction of signal directly from receptor to nucleus. Publication Types: Review PMID: 7574495 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 125: EMBO J. 1994 Dec 1;13(23):5605-15. Activation of JAK kinases and STAT proteins by interleukin-2 and interferon alpha, but not the T cell antigen receptor, in human T lymphocytes. Beadling C, Guschin D, Witthuhn BA, Ziemiecki A, Ihle JN, Kerr IM, Cantrell DA. Lymphocyte Activation Laboratory, London, UK. The activation of Janus protein tyrosine kinases (JAKs) and signal transducer and activator of transcription (STAT) proteins by interleukin (IL)-2, the T cell antigen receptor (TCR) and interferon (IFN) alpha was explored in human peripheral blood-derived T cells and the leukemic T cell line Kit225. An IL-2-induced increase in JAK1 and JAK3, but not JAK2 or Tyk2, tyrosine phosphorylation was observed. In contrast, no induction of tyrosine phosphorylation of JAKs was detected upon stimulation of the TCR. IFN alpha induced the tyrosine phosphorylation of JAK1 and Tyk2, but not JAK2 or JAK3. IFN alpha activated STAT1, STAT2 and STAT3 in T cells, but no detectable activation of these STATs was induced by IL-2. However, IL-2 regulates the DNA binding and tyrosine phosphorylation of two STAT-like protein complexes which do not include STAT1, STAT2 or STAT3. STAT4 is not activated by IL-2. The activation of STAT5 cannot be excluded, so the IL-2-activated complexes most probably include at least one novel STAT. No STAT activity was detected in TCR-stimulated lymphocytes, indicating that the JAK/STAT pathway defined in this study constitutes an IL-2R-mediated signaling event which is not shared by the TCR. Finally, in other cell types the correlation between JAK1 activation and the induction of STAT1 has suggested that JAK1 may activate STAT1. The observation that IL-2 and IFN alpha activate JAK1 to a comparable degree, but only IFN alpha activates STAT1, indicates that JAK1 activation is not the only determining factor for STAT1 activation. Moreover, the data show that JAK1 stimulation is also not sufficient for STAT3 activation. PMID: 7988557 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 126: 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] ---------------------------------------------------------------