Mechanisms of Antifibrotic Actions of Relaxin in the Liver

描述（由申请人提供）： 肝纤维化是由酒精滥用、肝炎等原因引起的慢性肝损伤，发展为肝硬化和肝功能障碍的结果。肝星状细胞（HSC）是肝纤维化和肝硬化中纤维胶原的主要来源，近年来的研究主要集中在HSC的作用上。正常情况下，HSC表现为静止表型，并以类维生素A的形式储存脂质。随着肝损伤，HSC失去其类维生素A储存并采用活化表型，其特征在于胶原分泌增加。HSC活化的主要因素是促纤维化细胞因子转化生长因子-2（TGF 2）。第二个主要因素是过氧化物酶体增殖物激活受体3（PPAR 3）表达的丧失。PPAR 3的存在对于维持HSC的静止、成脂表型是至关重要的。因此，靶向抑制TGF 2信号传导和激活PPAR 3是治疗纤维化肝病的有希望的方法。 我们的工作集中在激素松弛素及其在肝脏中的抗纤维化作用。松弛素抑制HSC的纤维化表型，并减少体内肝纤维化模型中的胶原沉积。在肝外成纤维细胞中，松弛素的抗纤维化作用的机制是通过抑制TGF 2信号传导，但目前对松弛素抑制HSC纤维化表型的机制知之甚少。初步数据表明，松弛素通过涉及TGF 2信号传导的下游介质，蛋白质Smad 2和Smad 3的机制抑制HSC中的TGF 2信号传导。此外，我们已经表明松弛素激活PPAR 3，但这种作用的信号转导机制尚不清楚。与这些发现一致，松弛素治疗增加了对体内已建立的肝纤维化治疗中的PPAR 3激动剂的反应。该建议的目的是通过测试松弛素拮抗TGF 2信号传导和上调PPAR 3活性的假设来确定松弛素的抗纤维化作用的机制。为了验证这一假设，提出了三个具体目标：具体目标#1。确定松弛素拮抗肝星状细胞中TGF 2信号传导的机制。工作假设是松弛素激活腺苷酸环化酶和下游途径的激活导致Smad 2和Smad 3磷酸化和核转位的扰动。具体目标#2。确定松弛素激活肝星状细胞中PPAR 3的机制。基于初步数据，工作假设是松弛素信号传导以配体非依赖性方式激活PPAR 3。具体目标#3确定松弛素联合PPAR 3激动剂治疗广泛纤维化和肝硬化的疗效。工作假设是松弛素将增加建立的肝纤维化和肝硬化小鼠模型中对PPAR 3配体的反应。 为了实现目标1，将使用肝星状细胞来确定松弛素抑制TGF 2信号传导的机制。这将通过使用特定的下游激活剂和siRNA方法来操纵信号通路来实现。对于目标2，报告构建体结合共沉淀和信号传导通路操作将用于鉴定松弛素激活PPAR 3的机制。最后，对于目标3，将使用建立的纤维化和从肝硬化恢复的小鼠模型来确定松弛素和PPAR 3配体吡格列酮的组合治疗的效果。这项研究意义重大，因为了解松弛素对TGF 2和PPAR 3通路的作用机制将有助于开发操纵这些信号通路的新方法。本申请中提出的研究可能会导致针对肝脏疾病治疗的靶向治疗策略，这是美国的一个主要健康问题。

DESCRIPTION (provided by applicant): Hepatic fibrosis results from chronic liver injury induced by alcohol abuse, hepatitis and other causes, progressing to cirrhosis and liver dysfunction. Recent studies have focused on the role of hepatic stellate cells (HSC), the major source of fibrillar collagen in hepatic fibrosis and cirrhosis. Normally, HSC display a quiescent phenotype, and function to store lipids in the form of retinoids. With liver injury, HSC lose their retinoid stores and adopt an activated phenotype, characterized by increased collagen secretion. A major factor in HSC activation is the profibrotic cytokine transforming growth factor-2 (TGF2). A second major factor is the loss of expression of the peroxisome proliferator-activated receptor 3 (PPAR3). The presence of PPAR3 is critical for maintenance of the quiescent, adipogenic phenotype of HSC. Therefore, targeted inhibition of TGF2 signaling, and activation of PPAR3, are promising approaches to the treatment of fibrotic liver diseases. Our work has focused on the hormone relaxin and its antifibrotic effects in the liver. Relaxin inhibits the fibrotic phenotype of HSC, and decreases collagen deposition in models of liver fibrosis in vivo. In extrahepatic fibroblastic cells, the mechanism of relaxin's antifibrotic effects was through inhibition of TGF2 signaling, but at present little is known about the mechanism of relaxin inhibition of the fibrotic phenotype of HSC. Preliminary data suggests that relaxin inhibits TGF2 signaling in HSC through a mechanism involving the downstream mediators of TGF2 signaling, the proteins Smad2 and Smad3. Furthermore, we have shown that relaxin activates PPAR3, but the signaling mechanisms for this effect are unknown. Consistent with these findings, relaxin treatment increased the response to a PPAR3 agonist in the treatment of established hepatic fibrosis in vivo. The objective of this proposal is to determine the mechanisms of the antifibrotic actions of relaxin by testing the hypothesis that relaxin antagonizes TGF2 signaling and upregulates PPAR3 activity. To test this hypothesis, three Specific Aims are proposed: Specific Aim #1. Determine the mechanism of relaxin antagonism of TGF2 signaling in hepatic stellate cells. The working hypothesis is that relaxin activation of adenylyl cyclase and activation of downstream pathways results in perturbation of Smad2 and Smad3 phosphorylation and nuclear translocation. Specific Aim #2. Determine the mechanism of PPAR3 activation by relaxin in hepatic stellate cells. Based on preliminary data, the working hypothesis is that relaxin signaling activates PPAR3 in a ligand- independent manner. Specific Aim #3. Determine the therapeutic efficacy of using relaxin in combination with PPAR3 agonists to treat extensive fibrosis and cirrhosis. The working hypothesis is that relaxin will increase the response to PPAR3 ligands in mouse models of established hepatic fibrosis and cirrhosis. To achieve Aim 1, hepatic stellate cells will be used determine the mechanism for relaxin inhibition of TGF2 signaling. This will be achieved by using specific downstream activators and siRNA approaches to manipulate signaling pathways. For Aim 2, reporter constructs in conjunction with coprecipitation and signaling pathway manipulations will be used to identify the mechanism of PPAR3 activation by relaxin. Finally, for Aim 3, mouse models of established fibrosis and recovery from cirrhosis will be used to determine the effect of the combined treatment of relaxin and the PPAR3 ligand pioglitazone. The proposed research is significant, because understanding the mechanism for relaxin on the TGF2 and PPAR3 pathways will allow the development of new approaches to manipulate these signaling pathways. The research proposed in this application may lead to targeted therapeutic strategies for the treatment of liver disease, a major health concern in the United States.