Mechanisms of Antifibrotic Actions of Relaxin in the Liver

肝脏松弛素的抗纤维化作用机制

• 批准号: 8244936
• 负责人: ROBERT G BENNETT
• 金额: --
• 依托单位: OMAHA VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8244936
• 关键词: Adenylate Cyclase; Adopted; Affect; Agonist; Alcohol abuse; Alcohol withdrawal syndrome; Alcoholic Liver Diseases; Antiviral Agents; Area; Binding; Biochemical; Cells; Cessation of life; Chronic; Cirrhosis; Collagen; Combined Modality Therapy; Data; Deposition; Development; Endocrine; Environment; Excision; Extracellular Matrix; Extrahepatic; Fibrillar Collagen; Fibrosis; General Population; Goals; HCV Cirrhosis; Health; Healthcare; Healthcare Systems; Hepatic Stellate Cell; Hepatitis; Hepatocyte; Hormones; Human; Injury; Investigation; Knowledge; Lead; Ligands; Lipids; Liver; Liver Cirrhosis; Liver Dysfunction; Liver Failure; Liver Fibrosis; Liver diseases; Maintenance; Mediator of activation protein; Modeling; Molecular; Molecular Biology; Nuclear Translocation; Outcome; PPAR gamma; Pathologic; Pathway interactions; Peroxisome Proliferator-Activated Receptors; Phenotype; Phosphorylation; Pioglitazone; Population; Receptor Activation; Recovery; Relaxin; Reporter; Research; Resolution; Resources; Retinoids; Role; Signal Pathway; Signal Transduction; Signaling Protein; Small Interfering RNA; Source; System; Testing; Time; Tissues; Transforming Growth Factor beta; Transforming Growth Factors; Transplantation; Treatment Efficacy; United States; Veterans; Work; base; cytokine; design; effective therapy; expectation; experience; in vivo; in vivo Model; innovation; mouse model; novel strategies; prevent; public health relevance; receptor; relaxin receptor; response; therapeutic effectiveness; therapeutic target; treatment strategy

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. PUBLIC HEALTH RELEVANCE: Potential Impact on Veterans Health Care: Cirrhosis affects 900,000 people and causes 36,000 deaths annually in the U.S. The major causes of cirrhosis (hepatitis C virus and alcohol abuse) are significantly more prevalent in Veterans than in the general population, and therefore treatment of fibrosis and cirrhosis an area of particular concern in the VA Health Care System. Treatment options are currently limited to removal of the cause of the injury, which is not always possible or effective, or transplantation. The research proposed in this application may lead to targeted therapeutic strategies for the treatment of liver disease, a major health concern in the Veteran population.

描述（由申请人提供）： 肝纤维化是由酗酒，肝炎和其他原因引起的慢性肝损伤引起的，发展为肝硬化和肝功能障碍。最近的研究集中在肝星状细胞（HSC）的作用上，肝脏胶原蛋白在肝纤维化和肝硬化中的主要来源。通常，HSC显示出静止的表型，并以类维生素的形式存储脂质的功能。由于肝损伤，HSC失去了类维生体储存，并采用活化的表型，其特征是胶原蛋白分泌增加。 HSC激活的主要因素是纤维化细胞因子转化生长因子2（TGF2）。第二个主要因素是过氧化物酶体增殖物激活受体3（PPAR3）的表达丧失。 PPAR3的存在对于维持HSC的静态脂肪生成表型至关重要。因此，靶向抑制TGF2信号传导和PPAR3的激活是治疗纤维化肝病的有前途方法。 我们的工作集中在肝脏中的激素松弛蛋白及其抗纤维化作用上。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蛋白沉积。在肝外成纤维细胞中，松弛素抗纤维化作用的机制是通过抑制TGF2信号传导的，但目前对HSC纤维化表型的抑制作用的机制知之甚少。初步数据表明，松弛素通过涉及TGF2信号传导下游介质，蛋白质SMAD2和SMAD3的机制抑制HSC中的TGF2信号传导。此外，我们已经表明，松弛素会激活PPAR3，但是该作用的信号传导机制尚不清楚。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的PPAR3激动剂的反应。该提案的目的是通过测试弛豫蛋白拮抗TGF2信号传导并上调PPAR3活性的假设来确定松弛蛋白的抗纤维化作用的机制。为了检验这一假设，提出了三个具体目标：特定目的＃1。确定肝星状细胞中TGF2信号传导的松弛素拮抗作用的机制。工作假设是，腺苷环酶的松弛激活和下游途径的激活导致SMAD2和SMAD3磷酸化和核转运的扰动。特定目标＃2。确定肝星状细胞中松弛素激活PPAR3的机理。基于初步数据，工作假设是松弛蛋白信号传导以配体独立的方式激活PPAR3。特定目标＃3。确定将松弛素与PPAR3激动剂结合使用以治疗广泛的纤维化和肝硬化的治疗功效。工作假设是，在已建立的肝纤维化和肝硬化的小鼠模型中，松弛素将增加对PPAR3配体的反应。 为了达到目标1，将使用肝星状细胞确定抑制TGF2信号的机制。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AIM 2，将使用与共沉淀和信号通路操作结合使用的记者构建体来识别松弛蛋白的PPAR3激活机理。最后，对于AIM 3，将使用已建立的纤维化和从肝硬化中恢复的小鼠模型来确定松弛蛋白和PPAR3配体吡格列酮的联合治疗的效果。拟议的研究很重要，因为了解TGF2和PPAR3途径上松弛素的机制将允许开发新方法来操纵这些信号通路。在本申请中提出的研究可能导致针对治疗肝病的有针对性的治疗策略，这是美国的主要健康问题。 公共卫生相关性： 对退伍军人卫生保健的潜在影响：肝硬化会影响90万人，每年在美国每年36,000人死亡。在退伍军人中，肝硬化的主要原因（乙型肝炎病毒和酒精滥用）比一般人群中的普遍性要大得多，因此在纤维化和纤维化治疗中，对VA Health Carey System的尤其关注。目前，治疗方案仅限于去除伤害原因，这并非总是可能或有效或移植。在本应用中提出的研究可能导致针对治疗肝病的有针对性的治疗策略，这是退伍军人人口的主要健康问题。