1: Oncogene. 2005 Oct 6;24(44):6689-98. Nitric oxide upregulates the cyclooxygenase-2 expression through the cAMP-response element in its promoter in several cancer cell lines. Park SW, Sung MW, Heo DS, Inoue H, Shim SH, Kim KH. Department of Tumor Biology, College of Medicine, Seoul National University, Chongno-gu, Korea. We previously showed that nitric oxide (NO) induces overexpression of cyclooxygenase-2 (COX-2) and production of prostaglandin E(2) in cancer cells. Here, we investigated the mechanisms by which NO induces COX-2 expression in cancer cells. We found that the cAMP-response element (CRE) is a critical factor in NO-induced COX-2 expression in all cells tested. We found that in cancer cells, three transcription factors (TFs) - cAMP response element-binding protein (CREB), activating transcription factor-2 (ATF-2) and c-jun, bound the CRE in the COX-2 promoter, and their activities were increased by addition of the NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP). NO-induced activation of soluble guanylate cyclase (sGC), p38 and c-Jun NH(2)-terminal kinase (JNK) upregulated the three TFs, leading to COX-2 overexpression. Addition of dibutyryl-cGMP (db-cGMP) induced COX-2 expression in a manner similar to SNAP; this induction was blocked by a p38 inhibitor (SB202190), but not by a JNK inhibitor (SP600125). NO-induced cGMP was found to activate CREB and ATF-2 in a p38, but not c-jun-dependent manner, while NO induced JNK in a cGMP-independent manner, leading to subsequent activation of c-jun and ATF-2. These results suggest that the low concentrations of endogenous NO present in cancer cell may induce the expression of many genes, including COX-2, which promotes the growth and survival of tumor cells. PMID: 16007171 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: J Neurochem. 2005 Sep;94(5):1457-70. Epub 2005 Jun 30. Integration of G protein signals by extracellular signal-regulated protein kinases in SK-N-MC neuroepithelioma cells. Chan AS, Yeung WW, Wong YH. Department of Biochemistry, the Molecular Neuroscience Center, and the Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong. Mammalian cells often receive multiple extracellular stimuli under physiological conditions, and the various signaling inputs have to be integrated for the processing of complex biological responses. G protein-coupled receptors (GPCRs) are critical players in converting extracellular stimuli into intracellular signals. In this report, we examined the integration of different GPCR signals by mitogen-activated protein kinases (MAPKs) using the SK-N-MC human brain neuroepithelioma cells as a neuronal model. Stimulation of the Gi-coupled neuropeptide Y1 and Gq-coupled muscarinic M1 acetylcholine receptors, but not the Gs-coupled dopamine D1 receptor, led to the activation of extracellular signal-regulated kinase (ERK). All three receptors were also capable of stimulating c-Jun NH2-terminal kinases (JNK) and p38 MAPK. The Gi-mediated ERK activation was completely suppressed upon inhibition of Src tyrosine kinases by PP1, while the Gq-induced response was suppressed by both PP1 and the Ca2+ chelator, BAPTA-AM. In contrast, activations of JNK and p38 by Gs-, Gi-, and Gq-coupled receptors were sensitive to PP1 and BAPTA-AM pretreatments. Simultaneous stimulation of Gi- and Gq-coupled receptors resulted in the synergistic activation of ERK, but not JNK or p38 MAPK. The Gi/Gq-induced synergistic ERK activation was PTX-sensitive, and appeared to be a co-operative effect between Ca2+ and Src family tyrosine kinases. Enhanced ERK activation was associated with an increase in CREB phosphorylation, while the JNK and p38-responsive transcription factor ATF-2 was weakly enhanced upon Gi/Gq-induction. This report provides evidence that G protein signals can be integrated at the level of MAPK, resulting in differential effects on ERK, JNK and p38 MAPK in SK-N-MC cells. PMID: 15992362 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: J Immunol. 2004 Aug 15;173(4):2552-61. Transcriptional regulation of the human TLR9 gene. Takeshita F, Suzuki K, Sasaki S, Ishii N, Klinman DM, Ishii KJ. Section of Retroviral Immunology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA. takesita@yokohama-cu.ac.jp To clarify the molecular basis of human TLR9 (hTLR9) gene expression, the activity of the hTLR9 gene promoter was characterized using the human myeloma cell line RPMI 8226. Reporter gene analysis and EMSA demonstrated that hTLR9 gene transcription was regulated via four cis-acting elements, cAMP response element, 5'-PU box, 3'-PU box, and a C/EBP site, that interacted with the CREB1, Ets2, Elf1, Elk1, and C/EBPalpha transcription factors. Other members of the C/EBP family, such as C/EBPbeta, C/EBPdelta, and C/EBPepsilon, were also important for TLR9 gene transcription. CpG DNA-mediated suppression of TLR9 gene transcription led to decreased binding of the trans-acting factors to their corresponding cis-acting elements. It appeared that suppression was mediated via c-Jun and NF-kappaB p65 and that cooperation among CREB1, Ets2, Elf1, Elk1, and C/EBPalpha culminated in maximal transcription of the TLR9 gene. These findings will help to elucidate the mechanism of TLR9 gene regulation and to provide insight into the process by which TLR9 evolved in the mammalian immune system. PMID: 15294971 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: J Cell Biochem. 2004 Apr 1;91(5):915-25. SPARC regulates TGF-beta1-dependent signaling in primary glomerular mesangial cells. Francki A, McClure TD, Brekken RA, Motamed K, Murri C, Wang T, Sage EH. Department of Vascular Biology, The Hope Heart Institute, Seattle, Washington 98104, USA. Secreted protein acidic and rich in cysteine (SPARC), a member of the family of matricellular proteins, regulates the interaction of cells with pleiotropic factors and proteins of the extracellular matrix (ECM). Although it has been appreciated that transforming growth factor beta 1 (TGF-beta1) induces SPARC and collagen type I, we have recently shown that SPARC regulates the expression of TGF-beta1 and collagen type I in renal mesangial cells via a TGF-beta1-dependent pathway, and have proposed a reciprocal, autocrine regulatory feedback loop between SPARC and TGF-beta1. Herein, we sought to determine how SPARC regulates TGF-beta1-dependent signal transduction. Our data indicate that SPARC modulates the TGF-beta1-dependent phosphorylation of Smad-2 in primary mesangial cells derived from wild-type and SPARC-null mice. We also show that SPARC regulates the levels and activation of the stress-activated c-jun-N-terminal kinase (JNK) in mesangial cells by augmentation of the stimulatory effects of TGF-beta1. Furthermore, we found that SPARC increases the levels and the activity of the transcription factor c-jun. These effects of SPARC on the TGF-beta1 signaling pathway appear to be mediated through an interaction with the TGF-beta1-receptor complex, but only in the presence of TGF-beta1 bound to its cognate type II receptor. That SPARC is directly involved in the regulation of the TGF-beta1 signaling cascade is consistent with the paradigm that matricellular proteins modulate interactions among cells, growth factors, and their respective receptors. Copyright 2004 Wiley-Liss, Inc. PMID: 15034927 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Atherosclerosis. 2003 Aug;169(2):259-67. Oxidised LDL decreases VEGFR-1 expression in human monocyte-derived macrophages. Salomonsson L, Svensson L, Pettersson S, Wiklund O, Ohlsson BG. Wallenberg Laboratory for Cardiovascular Research, The Cardiovascular Institute, Sahlgrenska Academy, Goteborg University, SE-413 45 Goteborg, Sweden. linda.salomonsson@wlab.gu.se Monocyte infiltration followed by differentiation into macrophages and accumulation of oxidised LDL (oxLDL) comprise early stages of atherosclerosis. Vascular endothelial growth factor (VEGF), which is upregulated by oxLDL, may contribute to atherogenesis through monocyte recruitment, increased vascular permeability and promotion of intraplaque vessels. The VEGF receptor-1 (VEGFR-1/Flt-1) mediates monocyte migration towards VEGF and regulates the levels of available VEGF through ligand-entrapment. In this study we investigated the effect of oxLDL on VEGFR-1 expression in human monocyte-derived macrophages. mRNA expression was estimated using RT-PCR, protein secretion was measured with ELISA and the amount of membrane-bound VEGFR-1 was analysed using flow cytometry analysis. Binding of transcription factors to the promoter was studied with EMSA. Incubation with oxLDL decreased VEGFR-1 mRNA expression in a time- and dose-dependent manner, followed by a subsequent decrease in protein secretion of VEGFR-1 and a reduction of the amount of receptor expressed on the cell surface. Furthermore, the PPARgamma agonists 9-hydroxy-(S)-10,12-octadecadienoic acid (9-HODE) and darglitazone also decreased VEGFR-1 mRNA expression. Incubation of macrophages with oxLDL or 9-HODE decreased binding of the transcription factor AP-1 (c-jun/ATF-2) to the VEGFR-1 promoter. Together, these data suggest that oxLDL decreases VEGFR-1 expression in macrophages, probably through a PPARgamma-dependent reduction in the AP-1 transcriptional activity. This implies that oxLDL has effects on the biological availability of VEGF, besides its direct effect on VEGF expression. PMID: 12921977 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: J Mol Endocrinol. 2003 Aug;31(1):105-21. Genomic structure and transcriptional regulation of the human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase gene. Nandy A, Jenatschke S, Hartung B, Milde-Langosch K, Bamberger AM, Gellersen B. IHF Institute for Hormone and Fertility Research, University of Hamburg, Falkenried 88, 20251 Hamburg, Germany. The NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) is a catabolic enzyme that controls the biological activities of prostaglandins by converting them into inactive keto-metabolites. Here we report the genomic organisation of the complete human PGDH gene and characterise its transcriptional regulation. The PGDH gene spans about 31 kb on chromosome 4 and contains 7 exons. Within 2.4 kb of the 5'-flanking sequence we identified two regions with clustered putative transcription factor binding sites. The distal promoter element PGDH-DE (positions-2152/-1944 relative to the start codon) contains binding sites for Ets and activating protein-1 (AP-1) flanked by two cAMP-responsive element-binding protein binding sites (CREB1, CREB2), whereas the proximal element PGDH-PE (-235/-153) includes an Ets and an AP-1 binding sequence. By electrophoretic mobility shift assay, no high affinity binding of Ets or AP-1 factors was observed with PGDH-PE, whereas we confirmed interaction of members of the Ets, AP-1 and CREB families of transcription factors with PGDH-DE. Transcriptional control of the PGDH promoter was assessed by transiently transfecting JEG-3 choriocarcinoma cells. A luciferase reporter gene construct containing the PGDH-PE was not induced by c-jun/c-fos in the absence or presence of co-expressed Ets-1. A construct carrying the PGDH-DE in front of the minimal homologous promoter was activated by co-transfection of expression vectors for AP-1 proteins. Mutation of the AP-1 or CREB2 site reduced the response to c-jun/c-fos, whereas mutation of the Ets site of the distal element reduced basal promoter activity. CREB activated the PGDH-DE construct through the CREB1 site. These results defined the distal element as an integrator of transcriptional regulation by AP-1, Ets and CREB proteins. PMID: 12914529 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Proc Natl Acad Sci U S A. 2003 Feb 4;100(3):1420-5. Epub 2003 Jan 13. Erratum in: Proc Natl Acad Sci U S A. 2004 Dec 28;101(52):18262. Comment in: Proc Natl Acad Sci U S A. 2003 Feb 4;100(3):768-70. Beneficial effects of antioxidants and L-arginine on oxidation-sensitive gene expression and endothelial NO synthase activity at sites of disturbed shear stress. de Nigris F, Lerman LO, Ignarro SW, Sica G, Lerman A, Palinski W, Ignarro LJ, Napoli C. Department of Medicine-0682, University of California, San Diego, CA 92093, USA. Atherogenesis is enhanced in arterial segments exposed to disturbed blood flow, indicating the active participation of the hemodynamic environment in lesion formation. Turbulent shear stress selectively regulates responsive genes in the endothelium and increases the damage induced by free radicals. The purpose of the present study was to evaluate the effects of intervention with antioxidants and l-arginine on endothelial NO synthase (eNOS) and oxidation-sensitive gene perturbation induced by disturbed flow in vitro and in vivo. Both human endothelial cells exposed to shear stress and high atherosclerosis-prone areas of hypercholesterolemic low-density lipoprotein receptor knockout (LDLR(-/-)) mice showed increased activities of redox-transcription factors (ELK-1, p-Jun, and p-CREB) and decreased expression of eNOS. Intervention with antioxidants and l-arginine reduced the activation of redox-transcription factors and increased eNOS expression in cells and in vivo. These results demonstrate that atherogenic effects induced by turbulent shear stress can be prevented by cotreatment with antioxidants and l-arginine. The therapeutic possibility to modulate shear stress-response genes may have important implications for the prevention of atherosclerosis and its clinical manifestations. PMID: 12525696 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: J Biol Chem. 1999 Feb 5;274(6):3887-96. Regulation of the transglutaminase I gene. Identification of DNA elements involved in its transcriptional control in tracheobronchial epithelial cells. Medvedev A, Saunders NA, Matsuura H, Chistokhina A, Jetten AM. Cell Biology Section, Laboratory of Pulmonary Pathobiology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA. The transglutaminase I (TGase I) gene encodes an enzyme that catalyzes the cross-linking of structural proteins involved in the formation of the cornified envelope during squamous cell differentiation. To identify DNA elements important for the transcriptional control of the TGase I gene, we analyzed the ability of a 2.9-kilobase pair (kb) upstream regulatory region to control the expression of a reporter gene in vivo and in vitro. Transgenic mice bearing the pTG(-2.9kb)CAT construct exhibited the same pattern of tissue-specific expression of CAT as reported for TGase I. Deletion analysis in transiently transfected rabbit tracheal epithelial cells indicated that two sequences from bp -490 to -470 and from -54 to -37 are involved in the activation of TGase I transcription. Point mutation analysis and mobility shift assays showed that the sequence located between -54 and -37 is a functional Sp1-like transcription element. Sp1 and Sp3, but not Sp2, are part of nuclear protein complexes from differentiated RbTE cells binding to this site. The element TGATGTCA between bp -490 and -470 is contained in a larger 22-bp palindrome and resembles the consensus cAMP response element-binding protein (CREB)/AP-1 element recognized by dimeric complexes of members of the CREB, ATF, Fos, and Jun families. Mutations in this sequence greatly reduced promoter activity. Supershift analysis identified CREB1, JunB, c-Fos, Fra-1, and c-Jun in protein complexes isolated from differentiated rabbit tracheal epithelial cells binding to this site. Our study shows that the Sp1- and CREB/AP-1-like sites act in concert to stimulate transcription of the TGase I gene. The 2.9-kb promoter region could guide expression of specific genes in the granular layer of the epidermis and could be useful in gene therapy. PMID: 9920944 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: J Immunol. 1997 Nov 15;159(10):4676-85. Transcriptional regulation of the junB gene in B lymphocytes: role of protein kinase A and a membrane Ig-regulated protein phosphatase. Amato SF, Nakajima K, Hirano T, Chiles TC. Department of Biology, Boston College, Chestnut Hill, MA 02167, USA. We have examined herein whether membrane Ig (mIg) stimulates junB transcription through a protein kinase A (PKA)-dependent or PKA-independent pathway. PKA phosphotransferase activity was not increased following mIg cross-linking of Bal17 B cells. However, junB transcriptional activation was dependent upon PKA activity, as evidenced by inhibition of goat anti-mouse IgM-stimulated junB promoter-chloramphenicol acetyltransferase reporter gene activity in transfected Bal17 B cells treated with the PKA inhibitor H-89. mIg-stimulated junB promoter-chloramphenicol acetyltransferase activity was also blocked in B cells expressing a specific PKA inhibitor peptide, whereas in vivo expression of an inactive PKA inhibitor peptide variant was not inhibitory. Expression of a mutant cAMP response element binding protein (CREB) containing an inactivated kinase A phosphoacceptor site at Ser133 reduced mIg-stimulated junB transcription. Okadaic acid increased CREB1 phosphorylation at Ser133 and junB transcriptional activation, suggesting the action of protein phosphatase-1 (PP-1) or -2A (PP-2A). Extracts from unstimulated B cells exhibited phosphatase activity against an in vitro PKA-phosphorylated peptide containing the Ser133 phosphoacceptor site. The involvement of a phosphatase activity in regulating mIg-stimulated junB transcription is supported by our finding that extracts from goat anti-mouse IgM-stimulated B cells exhibited a significantly reduced level of Ser133 phosphatase activity. Hence, the level of CREB1 phosphorylation is governed by the balance between PKA and phosphatase activities. junB transcriptional activation results in part from mIg signals that negatively regulate a CREB1-targeted PP-1 or PP-2A activity. PMID: 9366390 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: J Virol. 1995 Jun;69(6):3420-32. Protein domains involved in both in vivo and in vitro interactions between human T-cell leukemia virus type I tax and CREB. Yin MJ, Paulssen EJ, Seeler JS, Gaynor RB. Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-8594, USA. Gene expression from the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) is mediated by three cis-acting regulatory elements known as 21-bp repeats and the transactivator protein Tax. The 21-bp repeats can be subdivided into three motifs known as A, B, and C, each of which is important for maximal gene expression in response to Tax. The B motif contains nucleotide sequences known as a cyclic AMP response element (CRE) or tax-response element which binds members of the ATF/CREB family of transcription factors. Though mutations of this element in the HTLV-I LTR eliminate tax activation, Tax will not activate most other promoters containing these CRE sites. In this study, we investigated the mechanism by which Tax activates gene expression in conjunction with members of the ATF/CREB family. We found that Tax enhanced the binding of one member of the ATF/CREB family, CREB 1, to each of the three HTLV-I LTR 21-bp repeats but not another member designated CRE-BP1 or CREB2. Tax enhanced the binding of CREB1 to nonpalindromic CRE binding sites such as those found in the HTLV-I LTR, but Tax did not enhance the binding of CREB1 to palindromic CRE binding sites such as found in the somatostatin promoter. This finding may help explain the failure of Tax to activate promoters containing consensus CRE sites. These studies were extended by use of the mammalian two-hybrid system. Tax was demonstrated to interact directly with CREB1 but not with other bZIP proteins, including CREB2 and Jun. Site-directed mutagenesis of both Tax and CREB1 demonstrated that the amino terminus of Tax and both the basic and the leucine zipper regions of CREB1 were required for direct interactions between these proteins both in vivo and in vitro. This interaction occurred in vivo and thus did not require the presence of the HTLV-I 21-bp repeats, as previously suggested. These results define the domains required for interaction between Tax and CREB that are likely critical for the activation of HTLV-I gene expression. PMID: 7745688 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Nucleic Acids Res. 1994 Apr 25;22(8):1463-9. Different binding specificities and transactivation of variant CRE's by CREB complexes. Benbrook DM, Jones NC. Department of Obstetrics and Gynecology, University of Oklahoma Health Sciences Center, Oklahoma City 73190. The DNA binding specificities of CREB1 and CREB2 homodimers and the CREB2/cJun heterodimer were analyzed with a CASTing technique. All but one of the selected sequences varied from the consensus CRE (TGACGTCA) by three nucleotides or less. The profile of variations selected and the binding affinity for these sequences were unique for each CREB complex. The affinities were not effected by the palindromic nature of the sequences, but were strongly effected by flanking sequences. The strength of DNA binding in vitro correlated with the degree of transactivation observed in JEG-3 cells transfected with reporter plasmids harboring CRE variants, when hybrid CREB proteins fused to the VP16 activation domain were expressed. When native CREB proteins were expressed, the correlation was attenuated by the nature of the variant sequence. A CRE variant (TGACATCA) found in several natural promoters, exhibited the lowest basal transcription rate of the variants and a lower level of induction than expected when compared with the in vitro binding data. These results indicate that transactivation of DNA sequence elements is strongly effected by the strength of transcription factor binding, and that individual sequences can attenuate the level of induction. PMID: 8190638 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: Genomics. 1993 Jan;15(1):197-9. Localization of the murine activating transcription factor 4 gene to mouse chromosome 15. Mielnicki LM, Elliott RW, Pruitt SC. Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263. Restriction fragment length variant analysis employing a mouse cDNA probe was used to localize the gene encoding murine activating transcription factor 4 (ATF-4) to mouse chromosome 15 in close proximity to Sis (the cellular homolog of the simian sarcoma virus oncoprotein). Previous studies suggest that conserved linkage relationships exist between this region of mouse chromosome 15 and human chromosome 22q. The chromosomal locations of genes encoding most members of the ATF and cyclic AMP response element binding protein (CREB) subfamily of b-zip proteins have not been determined. This study demonstrates that the location of the gene for murine ATF-4 is not linked to the genes for JUN family members, CREB1 and CREB2. Further mapping of individual ATF/CREB subfamily members in the mouse will provide insight into the evolution of this multigene family. PMID: 8432535 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: EMBO J. 1990 Aug;9(8):2537-42. Multiple cDNA clones encoding nuclear proteins that bind to the tax-dependent enhancer of HTLV-1: all contain a leucine zipper structure and basic amino acid domain. Yoshimura T, Fujisawa J, Yoshida M. Department of Viral Oncology, Cancer Institute, Tokyo, Japan. A trans-activator protein, p40tax, of human T cell leukemia virus type 1 (HTLV-1) activates its own promoter and cellular promoters of IL-2, IL-2 receptor alpha and GM-CSF genes. We isolated three cDNA clones encoding cellular proteins that bind to the p40tax-dependent enhancer of HTLV-1 by screening a lambda gt11 cDNA library of an HTLV-1 infected cell line. All three proteins, TREB5, TREB7 and TREB36, contained a leucine zipper structure and basic amino acid domain, which are conserved in FOS, JUN and CREB, and also had multiple potential phosphorylation sites. The proteins expressed in Escherichia coli bound to the p40tax-dependent enhancer of the 21 bp sequence, but not to an inactive mutant carrying a mutation in the CRE region. In DNase I footprint analysis, all three proteins protected the 21 bp sequences in the LTR; however, the patterns were not identical to each other. TREB7 and TREB36 protected all three repeats of the 21 bp, but TREB5 protected only the second repeat. TREB7 and TREB36 protected the 5' and middle portions of the 21 bp which are essential for p40tax-mediated trans-activation, whereas TREB5 and CREB1 protected a narrower part of the middle region of the second 21 bp repeat containing the CRE consensus sequence. These structural features and DNA binding properties suggest that TREB proteins are members of a CREB protein family and that some of them (i.e., TREB7 and TREB36) may be involved in p40tax-mediated trans-activation. PMID: 2196176 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------