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

• 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
• 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

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ROCK 1鉴定为人类衰竭心脏中的半胱天冬酶-3靶标。切割导致在组成型活性Rho激酶，岩石？1.我们还证明了ROCK 1基因缺失抑制应激诱导的心脏纤维化。但是，ROCK的功能呢？1和连接ROCK 1与心脏纤维化的分子信号传导仍然不清楚。该研究的目的是确定ROCK？1，探讨ROCK？1-介导的心脏纤维化。为了解决这些问题，我们产生了转基因小鼠表达ROCK？1在心脏模仿人类心脏病。在转基因小鼠中观察到明显的心脏纤维化，TGF？1显著上调。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。我们的初步结果表明SRF作为TGF β 1的潜在调节剂。我们还发现NF-？小鼠中的B表达和活性。因此，中心假设是心肌细胞中ROCK 1的组成性激活足以通过SRF和NF-κ B的激活上调TGF β信号和其他促纤维化细胞因子导致心脏纤维化。分别为B。将完成三个目标。 目的I是确定ROCK的促纤维化作用？1个在完整的心脏中。将在基础和应激挑战条件下研究转基因小鼠。将在两个小鼠品系中测定ROCK？1.将进行Rho激酶抑制剂的拯救实验。目的二是阐明ROCK？1-介导的心脏纤维化。Rho激酶和纤维化反应之间的信号通路已经提出了大量的初步数据。提出的机制包括TGF-β 1和NF-？B介导的细胞因子。该假设将在体外心肌细胞和转基因小鼠体内进行测试。为了研究SRF介导的TGF β 1调控，将通过广泛的实验来验证TGF β 1启动子/增强子区域中鉴定的顺式元件，包括1）荧光素酶、EMSA和CHIP测定; 2）通过表达由野生或突变顺式元件驱动的lacZ的转基因小鼠; 3）通过分析TGF β 1在SRF敲除小鼠心脏中的表达，其中TGF β 1的减少是由SRF介导的。预计为1级。目的III是确定内源性ROCK 1抑制剂Rnd 3的丢失是否重演ROCK？1-介导的纤维化心肌病。纤维化表型、Rho激酶活性、TGF？而NF-？将在正常和应激挑战条件下评估B信号传导。 该提案的结果将是建立Rho激酶，TGF-β 1和NF-？纤维化心脏重构中的B信号。该提案的创新包括：1）证明ROCK的纤维化作用？1; 2）阐明ROCK 1->SRF->TGF？1->纤维化和ROCK 1->NF-？B->细胞因子->纤维化信号传导途径; 3）揭示Rnd 3在心脏重塑中的作用;和4）操纵Rho激酶活性和半胱天冬酶切割作为候选治疗靶点的意义。

项目成果

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
• 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.

• DOI: 10.1016/j.cophys.2017.07.005
• 发表时间: 2018-03
• 期刊: Current opinion in physiology
• 影响因子: 2.5
• 作者: Dai Y;Luo W;Chang J
• 通讯作者: Chang J