G Protein pathways as Novel Therapeutic and Diagnostic Targets in Liver Fibrosis

描述(申请人提供)：过量饮酒、病毒性肝炎、自身免疫性疾病和非酒精性脂肪性肝炎(NASH)引起的肝纤维化可进展为肝硬变及其多种并发症，在全球范围内构成巨大的医疗负担。它的特点是在慢性损伤/炎症的刺激下，通过生长因子受体酪氨酸激酶(RTK)和GPCRs的GI偶联趋化因子/细胞因子家族(如CCRs)发出异常信号，导致细胞外基质过度沉积，主要由活化的肝星状细胞(HSCs)产生。作为对纤维化刺激的反应，静止的HSCs转化为产生胶原的肌成纤维细胞样细胞。PI3K-Akt通路是这一关键转化的关键促纤维化触发因素，而cAMP则是关键的抗纤维化刺激因素。PI3K-Akt通路对KCs的趋化也是至关重要的，KCs在肝纤维化中起着另一组关键作用。协调驱动不受限制的PI3K-Akt信号并同时降低这些不同类别受体下游的cAMP最终导致肝纤维化的确切机制(S)尚不清楚。最近发现了一种新的信号复合体，它由G蛋白亚基G？I和GIV组成，其非受体的GfG能抑制cAMP的产生，并增强由生长因子受体和GPCRs共同启动的PI3K-Akt信号。假设由GIV-GI信号轴驱动的这些信号程序激活HSCs和KCs，并推动肝纤维化，抑制这一轴可能阻止和逆转纤维化。为了验证这一假说，将在小鼠急性和慢性肝损伤模型和人类临床样本中研究GIV的表达、翻译后修饰和G蛋白结合特性(目标1)；通过分别诱导野生型和HSC/KC特异性GIV-/-小鼠和从它们分离的HSCs/KCs的肝损伤，研究GIV对肝纤维化的影响，以及纤维化受体(如转化生长因子受体、PDGFR和CCRs)下游的HSCs和KCs中的促纤维化和抗纤维化信号程序以及对细胞过程的影响，如胶原生成、凋亡、趋化和增殖(目标2)；如果肝纤维化需要GI-GIV复合体，以及靶向抑制GI-GIV界面是否可以作为抑制和/或逆转纤维化的策略，将在肝硬变小鼠模型中使用高特异性小分子抑制剂来靶向破坏肝脏中的功能复合体(目标3)； 最后，肝脏活检组织中GIV的丰度是否可以预测进展为肝硬变，从而作为预后标志，将在一组历史上患有慢性病毒性肝炎的受试者中进行评估，这些受试者到晚期纤维化和肝硬化的时间线是可变的(目标4)。所获得的见解将阐明这种新型的GI-GIV信号复合体在肝纤维化中的作用，从分子和细胞水平，到整个动物水平，最后到其在人类肝硬变中的更广泛影响的水平，并有助于确定该复合体是否可以作为标记物和治疗靶点，从而为个性化药物提供机会。

DESCRIPTION (provided by applicant): Liver fibrosis caused by excessive alcohol consumption, viral hepatitis, autoimmune diseases and non-alcoholic steatohepatitis (NASH) can progress to cirrhosis and its multiple complications, and represents a massive health care burden worldwide. It is characterized by aberrant signaling by growth factor receptor tyrosine kinases (RTKs) and Gi-coupled chemokine/cytokine family of GPCRs, e.g., the CCRs), incited by chronic injury/inflammation, causing excessive deposition of extracellular matrix, mainly produced by activated hepatic stellate cells (HSCs). In response to fibrogenic stimuli, quiescent HSCs transform into a collagen- producing myofibroblast-like cell. A key profibrotic trigger for this critical transformation is the PI3K-Akt pathway, whereas a key antifibrotic stimulus is cAMP. The PI3K-Akt pathway is also critical for chemotaxis of KCs, which play another set of critical role in liver fibrosis. The precise mechanism(s) that coordinately drives unrestricted PI3K-Akt signaling and simultaneously reduces cAMP downstream of these diverse classes of receptors culminating into liver fibrosis remains unknown. Recently a novel signaling complex comprised of G protein subunit, G?i and GIV, its non-receptor GEF has been identified, which inhibits cAMP production, and enhances PI3K-Akt signals initiated by both growth factor receptor receptors and GPCRs. It is hypothesized that these signaling programs driven by the GIV-Gi signaling axis activates HSCs and KCs and drives liver fibrosis, and that inhibiting this axis may halt and reverse fibrosis. To test this hypothesis, the expression, posttranslational modifications and G-protein binding properties of GIV will be investigated during the course of acute and chronic liver injury in murine models and in clinical samples from human subjects (Aim 1); GIV's impact on liver fibrosis and pro- and antifibrotic signaling programs in HSCs and KCs downstream of fibrogenic receptors, e.g., TGF?R, PDGFR and CCRs and on cellular processes e.g., collagen production, apoptosis, chemotaxis, and proliferation will be interrogated by inducing liver injury in wild-type and HSC/KC-specific GIV- /- mice and HSCs/KCs isolated from them, respectively (Aim 2); if the Gi-GIV complex is required for liver fibrosis, and whether targeted inhibition of the Gi-GIV interface could serve as a strategy to inhibit and/or reverse fibrosis will be interrogated in murine models of cirrhosis using highly specific small molecule inhibitors of the interface for targeted disruption of the functional complex in the liver (Aim 3); and finally, whether the abundance of GIV in liver biopsies can predict the progression to cirrhosis and thereby, serve as a prognostic marker will be evaluated in a historic cohort of human subjects with chronic viral hepatitis who had a variable time-line to advanced fibrosis and cirrhosis (Aim 4). Insights gained will elucidate the role of this novel Gi-GIV signaling complex i liver fibrosis from a molecular and cellular level, to whole animal level, and finally, to the leve of its broader implications in a human liver cirrhosis, and help determine if the complex could serve as a marker and as a therapeutic target, thereby presenting an opportunity for personalized medicine.