ucn-1028-c and Inflammation

Inducible nitric-oxide synthase (iNOS) plays a central role in the regulation of vascular function and response to injury. A central mediator controlling iNOS expression is transforming growth factor-beta (TGF-beta), which represses its expression through a mechanism that is poorly understood. We have identified a binding site in the iNOS promoter that interacts with the nuclear heterodimer TCF11/MafG using chromatin immunoprecipitation and mutation analyses. We demonstrate that binding at this site acts to repress the induction of iNOS gene expression by cytokines. We show that this repressor is induced by TGF-beta1 and by Smad6-short, which enhances TGF-beta signaling. In contrast, the up-regulation of TCF11/MafG binding could be suppressed by overexpression of the TGF-beta inhibitor Smad7, and a small interfering RNA to TCF11 blocked the suppression of iNOS by TGF-beta. The binding of TCF11/MafG to the iNOS promoter could be enhanced by phorbol 12-myristate 13-acetate and suppressed by the protein kinase C inhibitor staurosporine. Moreover, the induction of TCF11/MafG binding by TGF-beta and Smad6-short could be blocked by staurosporine, and the effect of TGF-beta was blocked by the selective protein kinase C inhibitor calphostin C. Consistent with the in vitro data, we found suppression of TCF11 coincident with iNOS up-regulation in a rat model of endotoxemia, and we observed a highly significant negative correlation between TCF11 and nitric oxide production. Furthermore, treatment with activated protein C, a serine protease effective in septic shock, blocked the down-regulation of TCF11 and suppressed endotoxin-induced iNOS. Overall, our results demonstrate a novel mechanism by which iNOS expression is regulated in the context of inflammatory activation.

Topics: Animals; Carcinogens; Cells, Cultured; Dimerization; Disease Models, Animal; Endotoxemia; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Hepatocyte Nuclear Factor 1-beta; Humans; Inflammation; MafG Transcription Factor; Male; Mutation; Naphthalenes; Nitric Oxide Synthase Type II; Protein Kinase C; Rats; Rats, Sprague-Dawley; Repressor Proteins; Response Elements; RNA, Small Interfering; Signal Transduction; Smad6 Protein; Smad7 Protein; Staurosporine; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta1

1. Roles of histamine in the production of vascular endothelial growth factor (VEGF) in the carrageenin-induced granulation tissue in rats were analysed in vitro and in vivo. 2. Incubation of the minced granulation tissue in the presence of histamine (1 and 10 microM) increased the content of VEGF protein in the conditioned medium in a time- and concentration-dependent manner. The levels of VEGF mRNA in the minced granulation tissue were also increased by histamine in a concentration-dependent manner. 3. The increase in the content of VEGF protein in the conditioned medium by histamine (10 microM) was suppressed by the H(2) receptor antagonist cimetidine (IC(50) 0.37 microM), but not by the H(1) receptor antagonist pyrilamine maleate, the H(3) receptor antagonist thioperamide or the cyclo-oxygenase inhibitor indomethacin. 4. The histamine-induced increase in the content of VEGF protein in the conditioned medium was inhibited by the cyclic AMP antagonist Rp-cAMP (IC(50) 6.8 microM), and the protein kinase A inhibitor H-89 (IC(50) 12.5 microM), but not by the protein kinase C inhibitors Ro 31-8425 and calphostin C or the tyrosine kinase inhibitor genistein. 5. Simultaneous injection of cimetidine (400 microg) and indomethacin (100 microg) into the air pouch of rats additively reduced the carrageenin-induced increase in VEGF protein levels and angiogenesis in the granulation tissue as assessed by using carmine dye. 6. These findings indicate that histamine has an activity to induce VEGF production in the granulation tissue via the H(2) receptor-cyclic AMP-protein kinase A pathway and augments angiogenesis in the granulation tissue.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Carrageenan; Cells, Cultured; Cimetidine; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Endothelial Growth Factors; Enzyme Inhibitors; Fibroblasts; Gene Expression Regulation; Granulation Tissue; Histamine; Histamine Antagonists; Immunohistochemistry; Indoles; Indomethacin; Inflammation; Isoquinolines; Lymphokines; Macrophages, Peritoneal; Male; Maleimides; Naphthalenes; Neovascularization, Pathologic; Piperidines; Protein Kinase C; Pyrilamine; Rats; Rats, Sprague-Dawley; Receptors, Histamine H2; RNA, Messenger; Specific Pathogen-Free Organisms; Sulfonamides; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Activation of several protein kinases contributes to the development of hyperalgesia evoked by injuries. The present study was designed to investigate the role of protein kinase C in the spinal cord in thermal hyperalgesia evoked by sciatic nerve ligation or by intraplantar injection of complete Freund's adjuvant. The paw withdrawal latency on the ipsilateral side, but not on the contralateral side, was markedly decreased after sciatic nerve ligation. Intraplantar injection of complete Freund's adjuvant also caused markedly decreases of the paw withdrawal latency. Intrathecal pretreatment with protein kinase C inhibitor calphostin C (100 and 250 ng) attenuated the decrease of the paw withdrawal latency evoked by sciatic nerve ligation. In contrast, the decrease of the paw withdrawal latency evoked by inflammation was only slightly attenuated by intrathecal pretreatment with calphostin C. The results indicate that protein kinase C in the spinal cord is involved in the development of the thermal hyperalgesia evoked by nerve ligation and is much less involved in the thermal hyperalgesia by complete Freund's adjuvant's-induced inflammation.

Topics: Animals; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hindlimb; Hyperalgesia; Inflammation; Injections, Spinal; Ligation; Male; Mice; Mice, Inbred ICR; Naphthalenes; Pain Measurement; Protein Kinase C; Sciatic Nerve

Previously, we reported that TGF-beta2 regulates the C3 gene expression in a dose- and time-dependent manner in monocytes. To extend these studies, we examined the role of PKC in the TGF-beta2-mediated induction of C3 expression by the human monocyte cell line, U937. Treatment of U937 cells with the PKC inhibitors, H7 and calphostin C, suppressed TGF-beta2-mediated induction of C3 protein levels, but not mRNA levels, in a dose-dependent manner. At the highest concentrations of H7 and calphostin C, C3 protein levels were inhibited 50% and 93%, respectively, compared to control levels. Treatment of U937 cells with HA1004, a weak PKC inhibitor used as a control for H7, did not inhibit induction of C3 protein levels. Down-modulating PKC with a prolonged exposure of U937 cells to PMA also suppressed TGF-beta2-mediated C3 protein induction by as much as 82%. Incubating cell extracts isolated from TGF-beta2-treated U937 cells with the PKC substrate, MIBP(4-14), resulted in increased substrate phosphorylation compared to cell extracts isolated from untreated cells. Addition of calphostin C suppressed the increased substrate phosphorylation by TGF-beta2. Furthermore, biosynthetic labeling of U937 cells treated with TGF-beta2 and calphostin C demonstrated an accumulation of C3 protein within cell lysates compared to controls. Collectively, these studies suggest a role for PKC in the secretion of C3 protein during TGF-beta2-mediated regulation of C3 expression in U937 cells.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Complement C3; Drug Interactions; Humans; Inflammation; Isoenzymes; Isoquinolines; Monocytes; Naphthalenes; Protein Kinase C; Signal Transduction; Sulfonamides; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta

Experiments from our and other laboratories have shown that specific inhibitors of protein kinase C (PKC) inhibited the secretion of nitric oxide, TNF alpha, and IL-1 beta from lipopolysaccharide (LPS)-stimulated macrophages, suggesting an important role for PKC in the inflammatory response. The present study was designed to investigate the mechanism whereby LPS stimulates PKC activity in inflammatory macrophages. Mouse macrophages were stimulated with 0-1 microgram/ml LPS for 0-18 hours, and PKC activity was detected in cell lysates. PKC isoform specificity was determined by blocking PKC activity with isoform-specific antibodies. Treatment of macrophages with 1 microgram/ml LPS induced a two-fold increase in PKC activity within 15 minutes and an additional more significant peak of PKC activity appeared 3 hours post-LPS stimulation. A lower dose of LPS (10 ng/ml) induced the later peak only. The enhancement in PKC activity induced by LPS occurred in both the cytosol and membrane fractions, but the enhancement in the membrane fraction was significantly greater than in the cytosol. The increase in PKC activity in both peaks was abolished only by the addition of anti-PKC-epsilon antibody. The present experiments suggest that PKC activation is an important pathway in the LPS-induced secretory response of macrophages and that PKC-epsilon is the major isoform involved.

Topics: Animals; Antibodies; Enzyme Activation; Enzyme Inhibitors; Female; Inflammation; Isoenzymes; Lipopolysaccharides; Macrophage Activation; Macrophages; Mice; Mice, Inbred Strains; Naphthalenes; Nitric Oxide; Porphyromonas gingivalis; Precipitin Tests; Protein Kinase C; Protein Kinase C-epsilon