Zheng Shizhong, Chen Anping
Department of Pathology, School of Medicine, St. Louis University, St. Louis, MO 63104, USA.
Am J Physiol Gastrointest Liver Physiol. 2007 Jan;292(1):G113-23. doi: 10.1152/ajpgi.00200.2006. Epub 2006 Sep 7.
Activation of hepatic stellate cells (HSC), the major effectors of hepatic fibrogenesis, is coupled with sequential alterations in gene expression, including an increase in receptors for transforming growth factor-beta (TGF-beta) and a dramatic reduction in the peroxisome proliferator-activated receptor-gamma (PPAR-gamma). The relationship between them remains obscure. We previously demonstrated that curcumin induced gene expression of PPAR-gamma in activated HSC, leading to reducing cell proliferation, inducing apoptosis and suppressing expression of extracellular matrix genes. The underlying molecular mechanisms are largely unknown. We recently observed that stimulation of PPAR-gamma activation suppressed gene expression of TGF-beta receptors in activated HSC, leading to the interruption of TGF-beta signaling. This observation supported our assumption of an antagonistic relationship between PPAR-gamma activation and TGF-beta signaling in HSC. In this study, we further hypothesize that TGF-beta signaling might negatively regulate gene expression of PPAR-gamma in activated HSC. The present report demonstrates that exogenous TGF-beta1 inhibits gene expression of PPAR-gamma in activated HSC, which is eliminated by the pretreatment with curcumin likely by interrupting TGF-beta signaling. Transfection assays further indicate that blocking TGF-beta signaling by dominant negative type II TGF-beta receptor increases the promoter activity of PPAR-gamma gene. Promoter deletion assays, site-directed mutageneses, and gel shift assays localize two Smad binding elements (SBEs) in the PPAR-gamma gene promoter, acting as curcumin response elements and negatively regulating the promoter activity in passaged HSC. The Smad3/4 protein complex specifically binds to the SBEs. Overexpression of Smad4 dose dependently eliminates the inhibitory effects of curcumin on the PPAR-gamma gene promoter and TGF-beta signaling. Taken together, these results demonstrate that the interruption of TGF-beta signaling by curcumin induces gene expression of PPAR-gamma in activated HSC in vitro. Our studies provide novel insights into the molecular mechanisms of curcumin in the induction of PPAR-gamma gene expression and in the inhibition of HSC activation.
肝星状细胞（HSC）是肝纤维化形成的主要效应细胞，其激活与基因表达的一系列改变相关，包括转化生长因子-β（TGF-β）受体增加以及过氧化物酶体增殖物激活受体-γ（PPAR-γ）显著减少。它们之间的关系仍不清楚。我们之前证明姜黄素可诱导活化的HSC中PPAR-γ的基因表达，从而减少细胞增殖、诱导凋亡并抑制细胞外基质基因的表达。其潜在的分子机制 largely 未知。我们最近观察到刺激PPAR-γ激活可抑制活化的HSC中TGF-β受体的基因表达，导致TGF-β信号传导中断。这一观察结果支持了我们关于HSC中PPAR-γ激活与TGF-β信号传导之间存在拮抗关系的假设。在本研究中，我们进一步假设TGF-β信号传导可能负向调节活化的HSC中PPAR-γ的基因表达。本报告表明外源性TGF-β1抑制活化的HSC中PPAR-γ的基因表达，姜黄素预处理可能通过中断TGF-β信号传导消除这种抑制作用。转染实验进一步表明，通过显性负性II型TGF-β受体阻断TGF-β信号传导可增加PPAR-γ基因的启动子活性。启动子缺失实验、定点诱变实验和凝胶迁移实验确定了PPAR-γ基因启动子中的两个Smad结合元件（SBE），它们作为姜黄素反应元件并负向调节传代HSC中的启动子活性。Smad3/4蛋白复合物特异性结合到SBE上。Smad4的过表达剂量依赖性地消除姜黄素对PPAR-γ基因启动子和TGF-β信号传导的抑制作用。综上所述，这些结果表明姜黄素中断TGF-β信号传导可在体外诱导活化的HSC中PPAR-γ的基因表达。我们的研究为姜黄素诱导PPAR-γ基因表达及抑制HSC激活的分子机制提供了新的见解。
