Inhibition of tumor necrosis factor alpha-induced prostaglandin E2 production by the antiinflammatory cytokines interleukin-4, interleukin-10, and interleukin-13 in osteoarthritic synovial fibroblasts: distinct targeting in the signaling pathways.
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OBJECTIVE: To investigate the effects of the antiinflammatory cytokines interleukin-4 (IL-4), IL-10, and IL-13 on tumor necrosis factor alpha (TNFalpha)-induced prostaglandin E2 (PGE2) release in the cellular signaling cascade on human osteoarthritis (OA) synovial fibroblasts. METHODS: Human OA synovial fibroblasts were cultured to explore the impact of IL-4, IL-10, and IL-13 on TNFalpha binding to TNF receptors (TNFR), soluble TNFR (sTNFR), cytoplasmic phospholipase A2 (cPLA2), and cyclooxygenase-2 (COX-2) production, and on the binding activity of the transcription factors nuclear factor kappaB (NF-kappaB), CCAAT-enhancer binding protein (C/EBP), activator protein 2 (AP-2), and cyclic AMP response element-binding protein (CREB). RESULTS: IL-4, IL-10, and IL-13 at 5 ng/ml dramatically reduced TNFalpha-induced PGE2 release by approximately 90% (P < 0.0001). IL-4 up-regulated the level of TNFalpha-induced TNFR by 47% (P < 0.06), while IL-10 down-regulated it by 71% (P < 0.02); IL-13 had no effect. Although statistical significance was not reached, all 3 cytokines up-regulated the basal level of sTNFR-55. IL-4 and IL-10, while not altering the basal level of sTNFR-75, significantly increased the TNFalpha-stimulated release of sTNFR-75. IL-4, IL-10, and IL-13 reduced the TNFalpha-induced COX-2 level, and IL-4 and IL-10 reduced the cPLA2 level. IL-4 had no effect on TNFalpha up-regulation of NF-kappaB, and a slight decrease was noted with IL-10 and IL-13 at the highest concentration used (5 ng/ml). IL-4 and IL-13 decreased the TNFa-induced C/EBP accumulation in a dose-dependent manner, while IL-10 up-regulated its basal level. AP-2 and CREB were not induced by TNFalpha. CONCLUSION: The results indicate that these antiinflammatory cytokines reversed the TNFalpha-induced release of PGE2 by OA synovial fibroblasts, by acting at various levels of the TNFa-dependent signaling cascade. These data shed new light on the mechanisms by which these cytokines reduce inflammatory processes. (+info)
Mitogen-activated protein kinase phosphatase-1 (MKP-1) expression is induced by low oxygen conditions found in solid tumor microenvironments. A candidate MKP for the inactivation of hypoxia-inducible stress-activated protein kinase/c-Jun N-terminal protein kinase activity.
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Pathophysiological hypoxia is an important modulator of gene expression in solid tumors and other pathologic conditions. We observed that transcriptional activation of the c-jun proto-oncogene in hypoxic tumor cells correlates with phosphorylation of the ATF2 transcription factor. This finding suggested that hypoxic signals transmitted to c-jun involve protein kinases that target AP-1 complexes (c-Jun and ATF2) that bind to its promoter region. Stress-inducible protein kinases capable of activating c-jun expression include stress-activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK) and p38 members of the mitogen-activated protein kinase (MAPK) superfamily of signaling molecules. To investigate the potential role of MAPKs in the regulation of c-jun by tumor hypoxia, we focused on the activation SAPK/JNKs in SiHa human squamous carcinoma cells. Here, we describe the transient activation of SAPK/JNKs by tumor-like hypoxia, and the concurrent transcriptional activation of MKP-1, a stress-inducible member of the MAPK phosphatase (MKP) family of dual specificity protein-tyrosine phosphatases. MKP-1 antagonizes SAPK/JNK activation in response to diverse environmental stresses. Together, these findings identify MKP-1 as a hypoxia-responsive gene and suggest a critical role in the regulation of SAPK/JNK activity in the tumor microenvironment. (+info)
Transcription factors in neuroendocrine regulation: rhythmic changes in pCREB and ICER levels frame melatonin synthesis.
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Neurotransmitter-driven activation of transcription factors is important for control of neuronal and neuroendocrine functions. We show with an in vivo approach that the norepinephrine cAMP-dependent rhythmic hormone production in rat pineal gland is accompanied by a temporally regulated switch in the ratio of a transcriptional activator, phosphorylated cAMP-responsive element-binding protein (pCREB), and a transcriptional inhibitor, inducible cAMP early repressor (ICER). pCREB accumulates endogenously at the beginning of the dark period and declines during the second half of the night. Concomitant with this decline, the amount of ICER rises. The changing ratio between pCREB and ICER shapes the in vivo dynamics in mRNA and, thus, protein levels of arylalkylamine-N-acetyltransferase, the rate-limiting enzyme of melatonin synthesis. Consequently, a silenced ICER expression in pinealocytes leads to a disinhibited arylalkylamine-N-acetyltransferase transcription and a primarily enhanced melatonin synthesis. (+info)
Identification of an oxygen-responsive element in the 5'-flanking sequence of the rat cytosolic phosphoenolpyruvate carboxykinase-1 gene, modulating its glucagon-dependent activation.
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The glucagon-stimulated transcription of the cytosolic phosphoenolpyruvate carboxykinase-1 (PCK1) gene is mediated by cAMP and positively modulated by oxygen in primary hepatocytes. Rat hepatocytes were transfected with constructs containing the first 2500, 493 or 281 bp of the PCK1 5'-flanking region in front of the chloramphenicol acetyltransferase (CAT) reporter gene. With all three constructs glucagon induced CAT activity with decreasing efficiency maximally under arterial pO2 and to about 65% under venous pO2. Rat hepatocytes were then transfected with constructs containing the first 493 bp of the PCK1 5'-flanking region in front of the luciferase (LUC) reporter gene, which were block-mutated at the CRE1 (cAMP-response element-1; -93/-86), putative CRE2 (-146/-139), promoter element (P) 1 (-118/-104), P2 (-193/-181) or P4 (-291/-273) sites. Glucagon induced LUC activity strongly when the P1 and P2 sites were mutated and weakly when the P4 site was mutated; induction of the P1, P2 and P4 mutants was positively modulated by the pO2. Glucagon also induced LUC activity strongly when the putative CRE2 site was altered; however, induction of the CRE2 mutant was not modulated by the pO2. Glucagon did not induce LUC activity when the CRE1 site was modified. These experiments suggested that the CRE1 but not the putative CRE2 was an essential site necessary for the cAMP-mediated PCK1 gene activation by glucagon and that the putative CRE2 site was involved in the oxygen-dependent modulation of PCK1 gene activation. To confirm these conclusions rat hepatocytes were transfected with simian virus 40 (SV40)-promoter-driven LUC-gene constructs containing three CRE1 sequences (-95/-84), three CRE2 sequences (-148/-137) or three CRE1 sequences plus two CRE2 sequences of the PCK1 gene in front of the SV40 promoter. Glucagon induced LUC activity markedly when the CRE1, but not when the CRE2, sites were in front of the SV40-LUC gene; however, induction of the (CRE1)3SV40-LUC constructs was not modulated by the pO2. Glucagon also induced LUC activity very strongly when the CRE1 and CRE2 sites were combined; induction of the (CRE1)3(CRE2)2SV40-LUC constructs was positively modulated by the pO2. These findings corroborated that sequences of the putative CRE2 site were responsible for the modulation by oxygen of the CRE1-dependent induction by glucagon of PCK1 gene transcription. (+info)
Mice homozygous for a truncated form of CREB-binding protein exhibit defects in hematopoiesis and vasculo-angiogenesis.
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CREB-binding protein (CBP) and the closely related adenovirus E1A-associated 300-kD protein (p300) function as coactivators of transcription factors such as CREB, c-Fos, c-Jun, c-Myb, and several nuclear receptors. To study the roles of CBP in embryonic development, we generated CBP homozygous mutant mouse embryos that expressed a truncated form of CBP protein (1-1084 out of 2441 residues). The embryos died between embryonic days 9.5 (E9.5) and E10.5 and exhibited a defect in neural tube closure. They appeared pale and showed decreases in erythroid cells and colony-forming cells (CFCs) in the yolk sac, suggesting defects in primitive hematopoiesis. Immunohistochemistry with an anti-PECAM antibody showed a lack of vascular network formation. Organ culture of para-aortic splanchnopleural mesoderm (P-Sp) with stromal cells (OP9) showed an autonomous abnormality of putative endothelial precursors, which may induce the microenvironmental defect in hematopoiesis. In addition, these defects were partially rescued by the addition of VEGF to this culture. Our analyses demonstrate that CBP plays an essential role in hematopoiesis and vasculo-angiogenesis. (+info)
Negative cooperativity between juxtaposed E-box and cAMP/TPA responsive elements in the cholecystokinin gene promoter.
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The promoter of the cholecystokinin (CCK) gene possesses evolutionary conserved juxtaposed E-box and cAMP/TPA responsive elements (CRE/TRE). We have examined the functional interaction of these two sites. As previously noted, c-Jun/c-Fos heterodimers greatly increase promoter activity through association with the CRE/TRE. Mutation of the E-box enhanced the activation by c-Jun/c-Fos, as well as stimulation by forskolin and bFGF, that acts through the CRE/TRE site. Moreover, c-Jun/c-Fos stimulation was inhibited by co-expression of c-Myc and Max. The results indicate that factors associating with the E-box exhibit a negative cooperative effect on the activation via the CRE/TRE element. We propose that this mechanism plays a significant role in CCK gene transcription and other genes with juxtaposed E-box and CRE/TRE. (+info)
The Drosophila dCREB2 gene affects the circadian clock.
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We report the role of dCREB2, the Drosophila homolog of CREB/CREM, in circadian rhythms. dCREB2 activity cycles with a 24 hr rhythm in flies, both in a light:dark cycle and in constant darkness. A mutation in dCREB2 shortens circadian locomotor rhythm in flies and dampens the oscillation of period, a known clock gene. Cycling dCREB2 activity is abolished in a period mutant, indicating that dCREB2 and Period affect each other and suggesting that the two genes participate in the same regulatory feedback loop. We propose that dCREB2 supports cycling of the Period/Timeless oscillator. These findings support CREB's role in mediating adaptive behavioral responses to a variey of environmental stimuli (stress, growth factors, drug addiction, circadian rhythms, and memory formation) in mammals and long-term memory formation and circadian rhythms in Drosophila. (+info)
Molecular mechanism(s) of action of isoproterenol on the expression of the angiotensinogen gene in opossum kidney proximal tubular cells.
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BACKGROUND: beta-adrenoceptors are present in the renal proximal tubules. We have previously reported that isoproterenol stimulates the accumulation of intracellular cAMP and the expression of the angiotensinogen (ANG) gene in opossum kidney (OK) proximal tubular cells via the beta 1-adrenoceptor. We hypothesized that the molecular mechanism(s) of action of isoproterenol on the expression of the ANG gene is mediated via the interaction of the phosphorylated cAMP-responsive element binding protein (CREB) and the cAMP-responsive element (CRE; that is, ANG N-806/-779) in the 5'-flanking region of the rat ANG gene. METHODS: The fusion genes containing the putative ANG-CRE of the rat ANG gene inserted upstream of the rat ANG basal promoter (ANG N-53/+18) fused to a human growth hormone (hGH) gene as reporter were stably cotransfected, with or without the plasmid containing the cDNA for 43 kDa CREB, into the OK cells. The effect of various agonists and antagonists of adrenoceptors on the expression of the fusion genes was evaluated by the amount of immunoreactive hGH secreted into the culture medium. The interactions of OK cellular nuclear protein(s) with the ANG N-806/779 were determined by gel mobility shift assays and by Southwestern and Western blot analysis. RESULTS: The addition of isoproterenol, forskolin, or 8-Bromo-cAMP (8-Br-cAMP) stimulated the expression of pOGH (ANG N-806/-779/-53/+18) by 135, 150, and 160%, respectively, but not mutants of the ANG N-806/-779. The stimulatory effect of isoproterenol was blocked in the presence of propranolol, Rp-cAMP, and atenolol, but not by the presence of stauro-sporine, U73122, and ICI 118,551. Transient transfection of the plasmid containing the cDNA for the catalytic subunit of protein kinase A further enhanced the stimulatory effect of 43 kDa CREB on the expression of the fusion gene. The gel mobility shift assays revealed the the nuclear protein(s) of OK cells binds to the radioactive-labeled ANG N-806/-779. The binding of the labeled ANG N-806/-779 to the OK cell nuclear protein(s) was displaced by unlabeled ANG N-806/-779, but not by the CRE of the somatostatin gene, the CRE of the tyrosine amino-transferase gene, or the mutants of the ANG N-806/-779. Southwestern blot analysis revealed that the labeled ANG N-806/-779 binds to two nuclear species of 43 and 35 kDa proteins. Western blot analysis, however, revealed that rabbit polyclonal antibodies against the 43 kDa CREB interacted with only the 43 kDa molecular species but not with the 35 kDa species. CONCLUSION: These studies demonstrate that the stimulatory effect of isoproterenol on the expression of the ANG gene may be mediated, at least in part, via the interaction of the phosphorylated CREB and the CRE in the 5'-flanking region of the rat ANG gene. The novel 35 kDa nuclear protein that is immunologically different from the 43 kDa CREB may also play a role in the expression of the ANG gene. (+info)