Multiple Molecular Mechanisms for Adiponectin Therapy in Hepatic Fibrogenesis

描述（由申请人提供）：最近的研究提供了关于肝纤维化发展的分子和细胞基础的重要信息，肝纤维化可导致肝硬化，需要肝移植。我们在前一个资助期的工作集中在两个关键的脂肪细胞因子瘦素和脂联素的作用，我们已经令人信服地证明，瘦素是一个明确的促纤维化细胞因子，而脂联素阻止纤维化。 我们的初步数据和目前的建议与本实验室的长期目标是一致的：了解与慢性肝创伤愈合相关的基本机制。脂联素促进肝星状细胞（HSC）中的分子环境-例如减少金属蛋白酶I组织抑制剂[TIMPI]表达，其有利于致密细胞外基质的溶解，这是肝硬化发病机制的基础。在体外，脂联素处理的HSC阻断瘦素介导的信号转导，导致促纤维化反应和所需的脂联素受体1，但似乎并不完全依赖于腺苷一磷酸激酶（AMPK）。由于有证据表明晚期肝纤维化是可逆的，因此目前建议的总体目标是确定脂联素促进肝纤维化消退的潜在机制。该建议的中心假设是脂联素通过抑制粘着斑激酶（FAK）的活化，防止粘着斑（FA）的形成，从而抑制活化的HSC的关键纤维化特性，从而促进肝纤维化的消退。中心假设将通过以下三个假设驱动的具体目的进行检验：具体目的1：确定脂联素抑制FAK活化，从而防止活化HSC中FA的形成并抑制与肝纤维化相关的基因。初步数据显示，脂联素阻止FAK的磷酸化的蛋白酪氨酸磷酸酶，SHP-2和脂联素阻止FA组装在体外和体内。具体目标2：证实脂联素激活激活蛋白1（AP-1）是脂联素转录激活基质金属蛋白酶（MMP）MMP-1/13，并通过灭活FAK抑制α（I）胶原基因转录的机制。初步数据表明，脂联素增加MMP-1/13 mRNA和各自的启动子活性;但是，尽管在体外存在球状脂联素（gAd），针对FAK的小发夹RNA（shFAK）阻断α2（I）胶原基因表达。具体目标3：通过体内和离体方法阐明脂联素逆转HSC活化、促进HSC衰老并最终导致纤维化消退的机制。初步数据表明，虽然CCl 4处理的脂联素敲除小鼠产生FA，但通过免疫荧光法评估，脂联素的腺病毒递送抑制FA组装;并且，离体，脂联素显著抑制HSC-α平滑肌肌动蛋白（α-SMA）表达。

DESCRIPTION (provided by applicant): Recent research has provided significant information regarding the molecular and cellular basis for the development of hepatic fibrosis that can lead to cirrhosis, and need for liver transplantation. Our work in the prior funding period focused on the role of two key adipocytokines-leptin and adiponectin-in which we have convincingly demonstrated that leptin is clearly a pro-fibrogenic cytokine while adiponectin prohibits fibrosis. Our preliminary data and the current proposal are consistent with the long-term objectives of this laboratory: to understand basic mechanisms associated with chronic liver wound-healing. Adiponectin promotes a molecular environment in hepatic stellate cells (HSCs)-e.g. reduced tissue inhibitor of metalloproteinase I [TIMPI] expression that favors dissolution of dense extracellular matrix, the basis for the pathogenesis of cirrhosis. In vitro, adiponectin treated HSCs blocked leptin-mediated signal transduction that leads to pro-fibrogenic responses and required adiponectin receptor 1, but does not appear to depend entirely on adenosine monophosphate kinase (AMPK). The overall goal of the current proposal is to determine the potential mechanisms whereby adiponectin can promote resolution of hepatic fibrosis since there is evidence advanced fibrosis is reversible. The central hypothesis of this proposal is that adiponectin promotes the resolution of hepatic fibrosis by inhibiting activation of focal adhesion kinase (FAK), preventing formation of focal adhesions (FAs), which results in the suppression of key fibrogenic properties of the activated HSC. The central hypothesis will be tested by the following three hypothesis- driven specific aims: SPECIFIC AIM 1: To determine that adiponectin suppresses FAK activation thereby preventing formation of FAs in activated HSCs and inhibits genes associated with hepatic fibrosis. Preliminary data reveal adiponectin prevents phosphorylation of FAK by the protein tyrosine phosphatase, SHP-2; and adiponectin prevents FA assembly both in vitro and in vivo. SPECIFIC AIM 2: To demonstrate that adiponectin activation of activator protein 1 (AP-1) is the mechanism responsible for adiponectin's transcriptional activation of matrix metalloproteinases (MMPs) MMP-1/13; and, for inhibiting α(I) collagen gene transcription through deactivation of FAK. Preliminary data demonstrate that adiponectin increases MMP-1/13 mRNA and respective promoter activity; but, small hairpin RNA against FAK (shFAK) blocks α2(I) collagen gene expression in spite of the presence of globular adiponectin (gAd) in vitro. SPECIFIC AIM 3: To elucidate, by in vivo and ex vivo approaches, the mechanisms whereby adiponectin reverses HSC activation, promotes HSC senescence, and ultimately leads to resolution of fibrosis. Preliminary data indicate that while CCl4 treated adiponectin knock-out mice develop FAs as assessed by immunofluorescence, adenoviral delivery of adiponectin suppressed FA assembly; and, ex vivo, adiponectin markedly suppresses HSC- alpha smooth muscle actin (α-SMA) expression.