Fibrogenic Role of ROCK delta1 and Mechanism in Cardiac Remodeling

ROCK delta1 的纤维化作用及其在心脏重构中的机制

基本信息

批准号：
8666794
负责人：
Jiang Chang
金额：
$ 35.53万
依托单位：
TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-01 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8666794
关键词：
Actins Address Adenoviruses Angiotensin II Animals Attenuated Binding Bioinformatics Biological Assay Cardiac Cardiac Myocytes Cardiomyopathies Caspase Cell Culture Techniques Collagen Culture Media Data Depressed mood Doppler Echocardiography EMSA Elements Endothelin-1 Enhancers Enzyme-Linked Immunosorbent Assay Fibrosis Gel Genetic Goals Heart Heart Diseases Histology Human Indium Interleukin-1 Interleukin-6 Knockout Mice LacZ Genes Link Luciferases Measurement Mediating Molecular Morphology Mus Mutant Strains Mice Nuclear Outcome Phenotype Phosphorylation Platelet Factor 4 Proteins ROCK1 gene Regulation Regulatory Element Rho-associated kinase Role Serum Serum Response Factor Signal Pathway Signal Transduction Smad Proteins Smooth Muscle Actin Staining Method Stimulus Stress TNF gene Testing Tissues Transforming Growth Factors Transgenic Mice Transgenic Organisms Up-Regulation caspase-3 chromatin immunoprecipitation connective tissue growth factor constriction cytokine experimental analysis fasudil in vitro testing in vivo inhibitor/antagonist innovation kinase inhibitor mutant novel osteopontin pressure promoter public health relevance research study response rho therapeutic target transcription factor

项目摘要

DESCRIPTION (provided by applicant): We identified Rho kinase ROCK1 as caspase-3 target in human failing hearts. The cleavage resulted in a constitutively active Rho kinase, ROCK?1. We also demonstrated that genetic deletion of ROCK1 inhibited stress-induced cardiac fibrosis. However, the function of ROCK?1 and molecular signaling linking ROCK1 to cardiac fibrosis remain obscure. The goal of the study is to determine the fibrogenic role of ROCK?1 and investigate molecular mechanism of ROCK?1-mediated cardiac fibrosis. To address these questions, we generated transgenic mice expressing ROCK?1 in heart to mimic human heart disease. Overt cardiac fibrosis was observed with marked upregulation of TGF¿1 in the transgenic mice. Since activation of Rho kinase increased SRF activity, we assessed the transgenic heart and found manifest increase in SRF activity. Our preliminary results suggested SRF as a potential regulator of TGF¿1. We also found robust increases in NF-?B expression and activity in the mice. Therefore, the central hypothesis is that constitutive activation of ROCK1 in cardiomyocytes is sufficient to result in cardiac fibrosis by upregulating TGF¿ signaling and other pro-fibrotic cytokines through activation of SRF and NF-?B, respectively. Three aims will be completed. Aim I is to determine the pro-fibrotic effect of ROCK?1 in intact heart. The transgenic mice will be studied under basal and stress challenging conditions. The fibrogenic phenotype will be determined in two mouse lines with high and low expression level of ROCK?1. A rescue experiment by Rho kinase inhibitor will be conducted. Aim II is to elucidate the molecular mechanisms of ROCK?1-mediated cardiac fibrosis. The signaling pathway linked between Rho kinase and fibrotic response has been proposed with a considerable amount of preliminary data. The proposed mechanism includes the upregulation of TGF¿1 and NF-?B-mediated cytokines. The hypothesis will be tested in vitro in cardiomyocytes and in vivo in the transgenic mice. To investigate SRF-directed TGF¿1 regulation, the identified cis elements in TGF¿1 promoter/enhancer region will be verified by extensive experiments including 1) luciferase, EMSA and CHIP assay; 2) through transgenic mice expressing lacZ driven by either the wild or mutant cis elements; 3) by analyzing expression of TGF¿1 in SRF null mouse heart, where the decrease in TGF?1 level is expected. Aim III is to determine if the loss of endogenous ROCK1 inhibitor, Rnd3, recapitulates ROCK?1-mediated fibrotic cardiomyopathy. The fibrotic phenotype, Rho kinase activity, TGF? and NF-?B signaling will be assessed under normal and stress challenging conditions. The outcome of the proposal will be to establish links between the activation of Rho kinase, TGF¿1 and NF-?B signaling in fibrotic cardiac remodeling. The innovation of the proposal includes 1) demonstration of fibrogenic role of ROCK?1; 2) elucidation of the ROCK1->SRF->TGF?1->fibrosis and the ROCK1->NF-?B->cytokines->fibrosis signaling pathways; 3) revelation the role of Rnd3 in cardiac remodeling; and 4) implication of manipulating Rho kinase activity and caspase cleavage as candidate therapeutic targets.

描述（由申请人提供）：我们将 Rho 激酶 ROCK1 鉴定为人类衰竭心脏中的 caspase-3 靶点。裂解产生了持续活跃的 Rho 激酶 ROCK?1。我们还证明了 ROCK1 的基因缺失可抑制应激诱导的心脏纤维化。然而，ROCK?1 的功能以及将 ROCK1 与心脏纤维化联系起来的分子信号传导仍然不清楚。该研究的目的是确定 ROCK1 与心脏纤维化之间的关系。 ROCK?1 的纤维形成作用并研究 ROCK?1 介导的心脏纤维化的分子机制为了解决这些问题，我们培育了在心脏中表达 ROCK?1 的转基因小鼠，以模拟人类心脏疾病，观察到 TGF 显着上调。 ¿ 1 在转基因小鼠中，由于 Rho 激酶的激活增加了 SRF 活性，我们评估了转基因心脏，发现 SRF 活性明显增加。 1. 我们还发现小鼠体内 NF-κB 表达和活性显着增加，因此，核心假设是心肌细胞中 ROCK1 的组成性激活足以通过上调 TGF 导致心脏纤维化。分别通过激活 SRF 和 NF-κB 信号传导和其他促纤维化细胞因子将完成三个目标，即确定 ROCK?1 在完整心脏中的促纤维化作用。将在 ROCK?1 高表达水平和低表达水平的两个小鼠系中确定纤维形成表型，目的是阐明 Rho 激酶抑制剂的分子机制。 ROCK?1 介导的心脏纤维化已通过大量初步数据提出，其中包括 TGF¿ 1 和 NF-κB 介导的细胞因子将在体外心肌细胞和转基因小鼠体内进行测试以研究 SRF 介导的 TGF¿ 1 调控，TGF¿ 中已鉴定的顺式元件1 启动子/增强子区域将通过大量实验进行验证，包括 1) 荧光素酶、EMSA 和 CHIP 测定；2) 通过野生或突变顺式元件表达 lacZ 的转基因小鼠；3) 通过分析 TGF¿ SRF 缺失小鼠心脏中的 TGF?1 水平下降是预期的，目标 III 是确定内源性 ROCK1 抑制剂 Rnd3 的丧失是否再现了 ROCK?1 介导的纤维化心肌病的纤维化表型、Rho 激酶活性。 TGF? 和 NF-?B 信号传导将在正常和应激条件下进行评估。该提案的结果将是建立 Rho 激酶、TGF¿ 激活之间的联系。该提案的创新点包括：1）ROCK1-1的纤维化作用的论证；2）ROCK1->SRF->TGF-1->纤维化和ROCK1-的阐明。 >NF-κB->细胞因子->纤维化信号通路；3）揭示Rnd3在心脏重塑中的作用；4）调控的意义； Rho 激酶活性和 caspase 裂解作为候选治疗靶点。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

RND3 promotes Snail 1 protein degradation and inhibits glioblastoma cell migration and invasion.

RND3促进Snail 1蛋白降解并抑制胶质母细胞瘤细胞迁移和侵袭

DOI：
10.18632/oncotarget.12396
发表时间：
2016-12-13
期刊：
Oncotarget
影响因子：
0
作者：
Liu B;Dong H;Lin X;Yang X;Yue X;Yang J;Li Y;Wu L;Zhu X;Zhang S;Tian D;Wang J;Cai Q;Mao S;Chen Q;Chang J
通讯作者：
Chang J

Rho kinase signaling and cardiac physiology.