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

描述（由申请人提供）：由过度饮酒、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化及其多种并发症，并在全球范围内造成巨大的医疗保健负担。其特征在于通过生长因子受体酪氨酸激酶（RTK）和GPCR的Gi偶联趋化因子/细胞因子家族的异常信号传导，例如，CCRs），由慢性损伤/炎症引起，导致主要由活化的肝星状细胞（HSC）产生的细胞外基质的过度沉积。响应于纤维化刺激，静止的HSC转化为产生胶原的肌成纤维细胞样细胞。这种关键转化的关键促纤维化触发因素是PI 3 K-Akt途径，而关键抗纤维化刺激是cAMP。PI 3 K-Akt通路对于KC的趋化性也是至关重要的，KC在肝纤维化中发挥另一组关键作用。协调驱动不受限制的PI 3 K-Akt信号传导并同时减少这些不同类型受体下游的cAMP，最终导致肝纤维化的确切机制仍然未知。最近发现了一种由G蛋白亚基G？i和GIV，其非受体GEF已被鉴定，其抑制cAMP产生，并增强由生长因子受体受体和GPCR两者启动的PI 3 K-Akt信号。假设由GIV-Gi信号传导轴驱动的这些信号传导程序激活HSC和KC并驱动肝纤维化，并且抑制该轴可以停止和逆转纤维化。为了检验这一假设，将在小鼠模型和来自人类受试者的临床样品中的急性和慢性肝损伤过程中研究GIV的表达、翻译后修饰和G蛋白结合特性（目的1）; GIV对肝纤维化的影响以及纤维化受体下游的HSC和KC中的促纤维化和抗纤维化信号传导程序，例如，TGF β R、PDGFR和CCR以及细胞过程，通过分别在野生型和HSC/KC特异性GIV- /-小鼠以及从它们分离的HSC/KC中诱导肝损伤来研究胶原蛋白的产生、凋亡、趋化性和增殖（目的2）;如果肝纤维化需要Gi-GIV复合物，以及靶向抑制Gi-GIV界面是否可以作为抑制和/或抑制GIV的策略。或逆转纤维化将在肝硬化的鼠模型中使用界面的高度特异性小分子抑制剂来靶向破坏肝脏中的功能复合物（Aim 3）; 最后，将在患有慢性病毒性肝炎的人类受试者的历史队列中评价肝活检中GIV的丰度是否可以预测向肝硬化的进展，从而作为预后标志物，所述受试者具有向晚期纤维化和肝硬化的可变时间线（目的4）。获得的见解将阐明这种新型Gi-GIV信号复合物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，到其在人类肝硬化中的更广泛意义的水平，并帮助确定该复合物是否可以作为标记物和治疗靶点，从而为个性化医疗提供机会。

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., TGFßR, 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.