GRK2/Epac1调控肝星状细胞表型转化在肝纤维化形成中的作用及机制

Liver fibrosis is a common end-result of a wide variety of chronic liver diseases following various types of injuries. It is often associated with severe morbidity and significant mortality. In liver, the activation of resident hepatic stellate cells (HSC) into myofibroblasts through the acquisition of a contractile, proliferative, inflammatory, and fibrogenic phenotype is a critical event in fibrogenesis. Activated stellate cells/myofibroblasts are the primary source of extracellular matrix (ECM), whose accumulation leads to the progressive replacement of the liver parenchyma by scar. Currently, clinical reports suggested that advanced liver fibrosis is potentially reversible. Thus, it is essential to develop therapeutic strategies to counteract liver fibrosis.. Exchange protein directly activated by cAMP 1 (Epac1) is a more recently discovered effector of cAMP signaling. Epac1 is a guanine nucleotide exchange factor catalyzing the exchange of GDP for GTP for the Ras-like GTPases Rap1 and Rap2, resulting in their activation. Subcellular localization of Epac1 is dynamic and spatiotemporally regulated. Published work showed that the serine-threonine kinase G protein-coupled receptor kinase 2 (GRK2) inhibits Epac1-mediated pain signaling. Specifically, we showed that hepatic fibrosis is associated with a decrease in GRK2 levels and increase in Epac1 levels in fibrotic liver of rats. However, the role of GRK2/Epac1 in modulation of HSC phenotype transformation, and the mechanism via which GRK2 controls Epac1-mediated signaling in hepatic fibrogenesis remained to be identified.. This study was designed to investigate whether GRK2/Epac1 modulates HSC phenotype transformation in hepatic fibrogenesis and its possible mechanism. To that end, we will choose human samples, which are biopsies from chronic hepatitis patients showing different degrees of fibrosis or control liver samples showing normal histology or minimal change, to determine the possible relationship between GRK2, Epac1 expression and the degree of fibrosis. In vitro studies, the molecular mechanism for which GRK2-mediated controlling Epac1 activity in HSC phenotype transformation will be identified. Meanwhile, mice with a stellate cell-specific knockout of GRK2 or Epac1 were used to compare the severity of hepatic fibrosis with wide type mice in vivo. Furthermore, effect of targeting GRK2/Epac1 on liver fibrosis animal model was assessed to explore the underlying possibilities of therapeutics. Altogether, the present studies will be helpful to discover some new mechanisms underlying the liver fibrosis, and develop a novel therapy target for treatment of hepatic fibrosis.

肝星状细胞（HSC）的表型转化是肝纤维化形成的关键事件。课题组前期研究发现，在肝纤维化大鼠模型中，肝脏G蛋白偶联受体激酶2(GRK2)表达降低，Epac1表达升高。然而，GRK2/Epac1在HSC表型转化中的作用及机制尚不清楚。 我们推测GRK2通过磷酸化或其他机制调节Epac1活性，参与了HSC表型转化及纤维化的形成。本项目采用肝纤维化临床标本，分析GRK2、Epac1与纤维化严重程度的关系。体外研究GRK2如何通过磷酸化调节Epac1活性，影响HSC表型转化；体内应用HSC特异性GRK2或Epac1敲除小鼠，比较其与野生型小鼠肝纤维化形成差异。在此基础上，以GRK2/Epac1为靶点，给予激动剂或抑制剂，考察是否可以抑制肝纤维化的发生。本课题的实施将阐明HSC上GRK2/Epac1在肝纤维化形成中的作用机制，为肝纤维化的病理分子机制提供新的依据，也为肝纤维化治疗的新靶点提供实验基础。

肝星状细胞（HSC）的表型转化是肝纤维化形成的关键事件，是肝脏胶原等细胞外基质的主要来源。课题组的前期研究发现，在肝纤维化大鼠模型中，肝脏G蛋白偶联受体激酶2（GRK2）表达降低，Epac1表达升高。然而，GRK2/Epac1在HSC表型转化中的作用及机制尚不清楚。根据前期研究结果，我们推测HSC上Epac1参与了肝纤维化HSC的表型转化，GRK2可通过磷酸化或其他机制调节Epac1活性，参与了HSC表型转化及肝纤维化的形成，试图阐明HSC中GRK2/Epac1在肝纤维化形成中的作用机制。首先，激光共聚焦法检测肝纤维化患者肝组织中Epac1、GRK2表达差异，发现肝纤维化患者中α-SMA、Epac1表达增强，而GRK2表达减弱。其次，建立了CCl4诱导的肝纤维化小鼠模型，结果显示Epac1表达随造模时间的延长逐渐增加，而GRK2表达降低。体外选用HSC，检测Epac1对HSC功能及下游相关信号通路的影响，结果表明选择性激动Epac1增强HSC的增殖、迁移、活化及胶原合成能力，升高Rap1-GTP水平及p-Akt、p-ERK表达；干扰Epac1可抑制HSC中Rap1的活化，并抑制其下游Akt、ERK信号的激活，从而降低HSC的活化及胶原合成能力。免疫荧光双染结果显示Epac1与GRK2在HSC中存在共定位，干扰GRK2促进HSC中Epac1/Rap1及下游Akt、ERK的激活，促进HSC的表型转化，且Akt、ERK通路抑制剂可部分逆转由低表达GRK2引起的HSC活化及胶原合成；过表达GRK2可降低Rap1的活化及其下游信号的激活，抑制HSC表型转化。进一步探讨HSC中GRK2对Epac1的调节机制，使用缺失激酶活性的GRK2突变体质粒转染HSC，结果发现缺失激酶活性的GRK2对HSC中Epac1/Rap1活化及HSC表型转化无明显抑制作用；免疫共沉淀检测结果表明，与过表达GRK2组相比，转染GRK2突变体质粒后，明显减弱GRK2与Epac1之间增强的相互作用。以上结果提示GRK2可能通过磷酸化Epac1使其失活，抑制Rap1及下游Akt、ERK信号通路的激活，从而抑制HSC表型转化。为以GRK2/Epac1为可能作用靶点研发治疗肝纤维化新策略提供了重要的理论依据和实验基础，具有潜在的应用价值。