HGF/HGFR Axis and Fatty Liver Disease

 描述(由申请人提供)：脂肪肝(FLD)在美国和其他地方是一个主要的公共卫生问题。肝脏实质细胞(肝细胞)在调节血糖和脂肪的动态平衡中起着重要作用，这被认为是通过胰岛素-胰岛素受体(I-IR)轴的适当作用而实现的。在2型糖尿病(T2D)中，这一途径的失调可导致非酒精性FLD(NAFLD)--一种表现出一系列病理变化的疾病，包括肝细胞脂肪堆积(脂肪变性)、脂肪变性肝细胞的炎症和坏死(NASH)、纤维化、肝硬变和肝癌，这些在分子水平上尚不清楚。肝脏胰岛素抵抗被认为是这一过程的驱动因素，其原因是肝细胞缺乏最佳的胰岛素受体(IR)功能。最近，我们发现肝细胞生长因子(HGF)-MET信号轴控制肝脏的葡萄糖和脂肪代谢，并且对于最佳的肝脏胰岛素反应是必不可少的。鉴于HGF-MET信号轴不仅控制新陈代谢，还调节细胞生长和存活(即抑制细胞死亡)，我们认为肝脏中HGF-MET信号减弱(即“HGF抵抗”)就像一把双刃剑，导致肝细胞代谢紊乱的恶性循环，以及无法克服(或生存)由脂毒性等侮辱引起的肝损伤。RIPK1(受体相互作用蛋白激酶1)已经成为控制炎症和坏死的主开关。基于我们突破性的初步数据研究了肝细胞生长因子-MET轴对RIPK1在肝细胞中的影响，我们假设，在正常肝细胞条件下，HGF-MET轴通过直接酪氨酸磷酸化RIPK1促进RIPK1蛋白小体的降解来负性调节RIPK1的酶活性，从而保护肝细胞免受RIPK1‘S促炎和促坏死作用的影响；在肝细胞脂毒性和非酒精性脂肪肝的背景下，HGF-MET信号的减少和缺陷导致RIPK1免于HGF-MET介导的负性调控，从而释放RIPK1依赖的肝细胞炎症和坏死细胞死亡。我们认为，HGF-MET信号的恢复将抵消这一效应。我们概述了检验这一新假设的三个具体目标。目的1-我们将利用定点突变和体外肝细胞培养系统研究MET对RIPK1酪氨酸磷酸化的功能影响，以确定这种修饰会抑制RIPK1的酶活性并促进其蛋白酶体的降解，从而防止RIPK1介导的炎症和坏死。我们还将测试MET信号中断导致RIPK1活性失控，从而释放RIPK1介导的肝细胞死亡的假设。调控RIPK1的分子机制还不是很清楚。我们已经发现MET直接使Tyr384上的RIPK1磷酸化，下调RIPK1的活性，并标志着它的蛋白酶体降解。因此，在这一目标下的研究将破译酪氨酸激酶生长因子受体MET对RIPK1的分子调控。目的2-我们将使用功能丧失和功能获得的小鼠模型直接验证我们的假设，即HGF-MET介导的RIPK1调节在脂肪肝疾病背景下对肝脏动态平衡具有生物学和病理学意义。我们认为，脂毒性引起的HGF-MET不足会加剧RIPK1诱导的肝脏炎症和坏死，而HGF-MET信号的恢复将抵消这一作用。我们发现在人和小鼠脂肪肝中，RIPK1表达上调，HGF/MET表达下调。推动脂肪性肝病进展的分子机制尚不清楚。我们认为，脂肪肝细胞中HGF/MET信号的减少导致RIPK1逃避MET介导的负调控，从而导致RIPK1诱导的肝细胞坏死。我们将诱发脂肪肝(通过喂食高脂饮食)，在肝脏特异性丢失- 功能(使用LMETKO和LRIPK1 KO)和功能获得(白蛋白-HGF转基因)小鼠模型。目的3-我们将通过脂毒性来确定RIPK1在肝细胞中上调的分子基础。我们假设，脂毒性引起的HGF/MET信号的任何缺陷都会促进RIPK1的积聚。脂肪毒性也可能通过直接抑制蛋白酶体来稳定RIPK1。我们将使用肝细胞培养系统和体内模型进行一系列全面的实验，以测试这两条途径中的一条或两条是否活跃，以确定脂肪肝病中RIPK1失调的分子基础。这项拟议的研究将建立一种新的范式，通过下调RIPK1的促炎和促死亡活性，HGF-Met等酪氨酸激酶生长因子受体系统可以预防肝脏炎症和坏死。我们的研究将为在NASH或酒精性肝炎等炎症性肝病的临床环境中操纵这些关键介质(HGF-MET和RIPK1)提供理论基础。

 DESCRIPTION (provided by applicant): Fatty Liver Disease (FLD) is a leading public health problem in the U.S. and elsewhere. Liver parenchymal cells (hepatocytes) play an essential role in regulating glucose and fat homeostasis which is believed to be achieved by the proper action of the Insulin-Insulin Receptor (I-IR) axis. Dysregulation of this pathway in Type 2 Diabetes (T2D) can result in non-alcoholic FLD (NAFLD) - a disorder showing a spectrum of pathologies from hepatocyte fat accumulation (steatosis), inflammation and necrosis of steatotic hepatocytes (NASH), fibrosis, cirrhosis and liver cancer that are not well understood at the molecular level. Hepatic insulin resistance is believed to be a driver of this process and results from lack of optimum insulin receptor (IR) function in hepatocytes. Recently, we established that the Hepatocyte Growth Factor (HGF)-MET signaling axis controls hepatic glucose and fat metabolism and is essential for optimum hepatic insulin response. Given the fact that the HGF-MET signaling axis not only controls metabolism but also modulates cell growth and survival (i.e. suppression of cell death), we propose that diminished HGF-MET signalling (that is, "HGF Resistance") in the liver acts as a double-edged sword causing a vicious cycle of hepatocyte metabolic derangement coupled with an inability to overcome (or survive) liver damage provoked by insults such as lipotoxicity. RIPK1 (Receptor Interacting Protein Kinase 1) has emerged as the master switch that dictates inflammation and necrosis. Based on our groundbreaking preliminary data examining the impact of the HGF- MET axis on RIPK1 in hepatocytes, we hypothesize that, under normal conditions in hepatocytes, the HGF-MET axis negatively regulates RIPK1 enzymatic activity through directly tyrosine phosphorylating RIPK1 promoting RIPK1 proteosomal degradation in order to protect hepatocytes from RIPK1's pro- inflammatory and pro-necrotic actions, and that, in the setting of hepatocyte lipotoxicity and NAFLD, reduced and defective HGF-MET signaling results in escape of RIPK1 from HGF-MET-mediated negative regulation unleashing RIPK1-dependent hepatocyte inflammation and necrotic cell death. We propose that restoration of HGF-MET signaling will negate this effect. We outline three specific aims to test this novel hypothesis. Aim 1 - we will investigate the functional consequences of tyrosine phoshorylation of RIPK1 by MET using site directed mutagenesis and in vitro hepatocytic cell cuture systems to establish that this modification results in inhibition of RIPK1 enzymatic activity and promotes its proteosomal degradation - hence, RIPK1 mediated inflammation and necrosis are prevented. We will also test the hypothesis that disruption of MET signaling results in uncontrolled RIPK1 activity unleashing RIPK1- mediated hepatocyte death. The molecular mechanisms that regulate RIPK1 are not well understood. We have discovered that MET directly tyrosine phosphorylates RIPK1 on Tyr384 downregulating RIPK1 kinase activity and marking it for proteosomal degradation. Thus, studies under this aim will decipher the molecular regulation of RIPK1 by the tyrosine kinase growth factor receptor MET. Aim 2 - we will directly test our hypothesis that HGF-MET mediated regulation of RIPK1 has biological and pathological implications to liver homeostasis in the setting of fatty liver disease using loss- and gain-of-function mouse models. We believe that HGF-MET insufficiency caused by lipotoxicity exacerbates RIPK1-induced liver inflammation and necrosis and that restoration of HGF-MET signaling will negate this effect. We have discovered that RIPK1 is upregulated and HGF/MET are downregulated in human and mouse fatty liver. The molecular mechanisms that drive progression of fatty liver disease are not well understood. We propose that reduced HGF/MET signaling in fatty hepatocytes causes RIPK1 to escape MET-mediated negative regulation driving RIPK1-induced hepatocyte necrosis. We will induce fatty liver disease (by feeding a high fat diet) in liver specific loss-of function (using LMETKO and LRIPK1 KO) and gain- of-function (Albumin-HGF Transgenic) mouse models. Aim 3 - we will determine the molecular basis of RIPK1 upregulation in hepatocytes by lipotoxicity. We hypothesize that any defect in HGF/MET signaling provoked by lipotoxicity will promote RIPK1 accumulation. Liptoxicity may also stabilizes RIPK1 via direct inhibition of the proteasome. We will perform a series of comprehensive experiments using a hepatocyte culture system and in vivo models to test if one or both of these pathways are active to define the molecular bases of RIPK1 dysregulation in fatty liver disease. The proposed studies will establish a new paradigm by which tyrosine kinase growth factor receptor systems like HGF-Met prevent hepatic inflammation and necrosis by downmodulating the pro-inflammatory and pro- death activity of RIPK1. Our studies will provide rationale for manipulating these key mediators (HGF-MET and RIPK1) in the clinical setting of inflammatory hepatic conditions such as NASH or alcoholic hepatitis.