Fibrogenic Role of ROCK delta1 and Mechanism in Cardiac Remodeling

描述（由申请人提供）：我们发现Rho激酶ROCK1是人类衰竭心脏的caspase-3靶点。裂解产生了一个具有组成活性的Rho激酶ROCK?1。我们还证明了ROCK1基因缺失抑制应激诱导的心脏纤维化。然而，ROCK的功能？1和将ROCK1与心脏纤维化联系起来的分子信号仍然不清楚。该研究的目的是确定ROCK？研究ROCK？1介导的心脏纤维化。为了解决这些问题，我们产生了表达ROCK？模拟人类心脏疾病。在转基因小鼠中观察到明显的心脏纤维化，TGF¿1明显上调。由于Rho激酶的激活增加了SRF活性，我们对转基因心脏进行了评估，发现SRF活性明显增加。我们的初步结果表明SRF是TGF¿1的潜在调节因子。我们还发现NF-？B在小鼠中的表达和活性。因此，中心假设是心肌细胞中ROCK1的组成性激活足以通过激活SRF和NF-?来上调TGF¿信号和其他促纤维化细胞因子，从而导致心脏纤维化。B,分别。将完成三个目标。目的一是确定ROCK的促纤维化作用。我在完好无损的心里。转基因小鼠将在基础条件和应激条件下进行研究。我们将在ROCK?1高表达和低表达的两种小鼠系中测定其纤维化表型。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激酶与纤维化反应之间的信号通路已被提出，并有大量的初步数据。提出的机制包括TGF - 1和NF- 1的上调。B-mediated细胞因子。该假设将在体外心肌细胞和体内转基因小鼠中进行验证。为了研究srf对TGF¿1的调控，TGF¿1启动子/增强子区域已确定的顺式元件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驱动表达lacZ的转基因小鼠；3)通过分析TGF¿1在SRF无效小鼠心脏中的表达，TGF？1级。目的III是确定内源性ROCK1抑制剂Rnd3的缺失是否会重现ROCK？1介导的纤维化心肌病。纤维化表型、Rho激酶活性、TGF？和NF - ?B信号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¿1和NF-？B信号在纤维化心脏重构中的作用。该方案的创新点包括：1)论证了ROCK?1的纤维化作用；2) ROCK1->SRF->TGF？1->纤维化与ROCK1->NF-？B->细胞因子->纤维化信号通路；3)揭示Rnd3在心脏重构中的作用；4)操纵Rho激酶活性和caspase裂解作为候选治疗靶点的意义。

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. PUBLIC HEALTH RELEVANCE: The study is to determine the fibrogenic role of the constitutively active ROCK?1 in intact heart, and to elucidate the molecular mechanism involved in Rho kinase-mediated cardiac fibrosis. The genetic-manipulated murine mice are applied.