Negative Regulation of Inducible Nitric-oxide Synthase Expression Mediated through Transforming Growth Factor-β-dependent Modulation of Transcription Factor TCF11

Abstract

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-␤ (TGF-␤), 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-␤1 and by Smad6-short, which enhances TGF-␤ signaling. In contrast, the up-regulation of TCF11/MafG binding could be suppressed by overexpression of the TGF-␤ inhibitor Smad7, and a small interfering RNA to TCF11 blocked the suppression of iNOS by TGF-␤. 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-␤ and Smad6-short could be blocked by staurosporine, and the effect of TGF-␤ 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 upregulation 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.Nitric oxide (NO) is an important regulator and mediator of numerous and diverse processes, including vascular function, neurotransmission, tumor biology, and innate immune response (reviewed in Ref. 1). The production and balance of NO from inducible nitric-oxide synthase (iNOS) 3 play an important role in septic shock because overexpression mediates vascular collapse and cardiac dysfunction (2) and contributes to multiple organ dysfunction (reviewed in Ref.3). Selective iNOS inhibition has been shown to attenuate the hemodynamic alterations in sepsis models (4). During acute inflammatory insult, iNOS is synergistically induced by the TH-1 cytokines tumor necrosis factor-␣, interleukin-1␤, and interferon-␥ (5). Following cytokine activation and iNOS induction, vascular smooth muscle cells produces high levels of NO (6). A key factor in controlling iNOS expression is transforming growth factor-␤ (TGF-␤), which has been shown to suppress the expression of iNOS at the mRNA level in a variety of cells (6 -8), and anti-TGF antibody has been shown to block the suppression of iNOS in the vasculature (9). Moreover, in an endotoxin model of septic shock, TGF-␤1 treatment markedly reduced iNOS mRNA in several organs and blocked the lipopolysaccharide (LPS)-induced hypotension (10).Although t...

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Role of testis‐specific high‐mobility‐group protein in transcriptional regulation of inducible nitric oxide synthase expression in the liver of endotoxic shock mice

“…A large number of studies have demonstrated that elevated expression of iNOS contribute to increased production of NO; NO is harmful to many tissues and cells and plays an important role in septic shock and other diseases [4][5][6][7][8][36][37][38][39][40]. However, the molecular mechanism that regulates transcriptional expression of iNOS is incompletely defined.…”

“…It is conceivable that regulatory mechanisms that control the timing and intensity of NO production by iNOS exist so that protective effects outweigh detrimental ones. The regulation of iNOS expression and the related signal transduction pathways have been well documented in endotoxic shock animal models and various types of cells treated with LPS [4][5][6][7][8]. However, the molecular mechanism involved in regulating transcription of iNOS has not been fully clarified.…”

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