Mechanisms of Antifibrotic Actions of Relaxin in the Liver

描述(由申请人提供)： 肝纤维化是由酗酒、肝炎等原因引起的慢性肝损伤，进展为肝硬变和肝功能障碍。最近的研究集中在肝星状细胞(HSC)在肝纤维化和肝硬变中的作用，HSC是纤维状胶原的主要来源。正常情况下，HSC表现为静止的表型，功能是以维甲酸的形式储存脂质。随着肝损伤，HSC失去其维甲酸储备，并采用激活的表型，其特征是胶原分泌增加。促纤维化细胞因子转化生长因子-2(TGF2)是HSC活化的主要因素。第二个主要因素是过氧化物酶体增殖物激活受体3(PPAR3)的表达缺失。PPAR3的存在对于维持HSC静止的成脂表型至关重要。因此，靶向抑制TGF2信号转导，激活PPAR3，是治疗纤维化肝病的有效途径。我们的工作主要集中在激素松弛素及其在肝脏中的抗肝纤维化作用。松弛素能抑制HSC的纤维化表型，减少体内肝纤维化模型中胶原的沉积。在肝外成纤维细胞中，松弛素通过抑制TGF2信号通路发挥抗纤维化作用，但目前对松弛素抑制肝星状细胞纤维化表型的机制知之甚少。初步数据表明，松弛素通过一种涉及TGF2信号的下游介导物Smad2和Smad3的机制来抑制HSC中的TGF2信号。此外，我们还发现松弛素能激活PPAR3，但这种作用的信号机制尚不清楚。与这些发现一致的是，松弛素治疗增加了体内已建立的肝纤维化治疗中对PPAR3激动剂的反应。本研究的目的是通过验证松弛素拮抗TGF2信号和上调PPAR3活性的假说，来确定松弛素抗肝纤维化作用的机制。为了验证这一假设，提出了三个特定的目标：特定的目标#1.确定TGF2信号在肝星状细胞中松弛拮抗的机制。工作假设是，松弛素腺苷酸环化酶的激活和下游通路的激活导致Smad2和Smad3的磷酸化和核移位的扰动。特定目的#2.确定松弛素激活肝星状细胞PPAR3的机制。根据初步数据，工作假设是松弛蛋白信号以一种不依赖于配体的方式激活PPAR3。具体目标#3：确定松弛素合用PPAR3激动剂治疗广泛性纤维化和肝硬变的疗效。工作假设是，在已建立的肝纤维化和肝硬变的小鼠模型中，松弛素会增加对PPAR3配体的反应。为了实现目标1，将利用肝星状细胞来确定松弛素抑制TGF2信号转导的机制。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，报告构建结合共沉淀和信号通路操作将被用来确定松弛素激活PPAR3的机制。最后，对于Aim 3，将使用已建立的纤维化和从肝硬变中恢复的小鼠模型来确定松弛素和PPAR3配体吡格列酮联合治疗的效果。这项拟议的研究意义重大，因为了解松弛蛋白在TGF2和PPAR3通路上的机制将有助于开发新的方法来操纵这些信号通路。这项申请中提出的研究可能会导致有针对性的治疗策略来治疗肝病，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.