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（由适用提供）：我们将Rho激酶Rock1确定为人类失败心脏中的caspase-3目标。裂解导致组成性活跃的Rho激酶，岩石1。我们还证明了ROCK1的遗传缺失抑制了应激诱导的心脏纤维化。但是，岩石1和将岩石1与心脏纤维化联系起来的分子信号传导的功能仍然晦涩。该研究的目的是确定岩石的纤维化作用？1并研究岩石1-介导的心脏纤维化的分子机制。为了解决这些问题，我们产生了表达岩石的转基因小鼠？观察到明显的心脏纤维化，在转基因小鼠中明显上调TGF。自从Rho激酶的激活增加了SRF活性以来，我们评估了转基因心脏，发现SRF活性的增加。我们的初步结果表明SRF是TGF¿1的潜在调节剂。我们还发现小鼠NF-的表达和活性的强劲增加。因此，中心假设是，通过分别通过激活SRF和NF-？B的激活，通过上调TGF信号传导和其他促纤维化细胞因子，岩石1在心肌细胞中的组成型激活足以导致心脏纤维化。三个目标将完成。目的是确定岩石1在完整心脏中的促纤维效应。转基因小鼠将在基本和压力挑战条件下进行研究。纤维化表型将在岩石高和低表达水平的两种小鼠线上确定。将进行Rho激酶抑制剂的救援实验。 AIM II是阐明岩石1-介导的心脏纤维化的分子机制。已经提出了与Rho激酶和纤维化反应之间关联的信号通路，并考虑了大量初步数据。提出的机制包括TGF¿1和NF- b介导的细胞因子的上调。该假设将在心肌细胞和转基因小鼠的体内进行体外检验。为了研究SRF定向的TGF¿1调节，将通过广泛的实验（包括1）荧光素酶，EMSA和CHIP分析来验证TGF¿1启动子/增强子区域中确定的CIS元素； 2）通过表达lacz的转基因小鼠，由野生或突变的顺式元素驱动； 3）通过分析tgf¿1在srf null小鼠心脏中的表达，tgf？1水平的下降。 AIM III是确定内源性Rock1抑制剂RND3是否丢失了岩石？1介导的纤维化心肌病。纤维化表型，Rho激酶活性，TGF？在正常和应力挑战条件下，将评估NF- b信号传导。该提案的结果将是在纤维化心脏重塑中的Rho激酶，TGF¿1和NF-？B信号传导之间建立联系。该提案的创新包括1）岩石的纤维化作用的演示； 2）阐明岩石1-> srf-> tgf？1->纤维化和岩石1-> nf-？b-> cytokines->纤维化信号传导途径； 3）RND3在心脏重塑中的作用； 4）操纵Rho激酶活性和胱天蛋白酶清洁的影响是候选治疗靶标。

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