HGF and Signaling Pathways in Hepatic Tissue Assembly

描述（由申请人提供）：这是一份修订后的续期申请，目前已进入第五年，旨在了解HGF和信号通路在肝组织组装中的作用。我们最近的研究表明，HGF及其受体Met，以及EGF受体和EGFR相关配体)构成了仅有的两种受体酪氨酸激酶，能够向肝细胞传递有丝分裂信号（在无血清培养基中，当给药于整个动物，大鼠或小鼠时）。我们还发现HGF/Met和EGFR是仅有的两种能够促进培养的肝细胞向胆道上皮细胞转分化的信号系统。HGF/Met和EGFR的这些独特作用是非常显著的。许多其他受体酪氨酸激酶在肝细胞中表达，它们的配体能够激活同源受体，但有丝分裂信号仅限于HGF/Met和EGFR。我们最近通过ShRNA对HGF/Met或EGFR进行了短期敲低（KD），并证明两者都对肝脏再生有抑制作用，一个信号不能完全补偿另一个信号，并且影响的范围和复杂性不同。EGFR的KD随后是ErbB家族其他成员的代偿性增加和MET的上调。在每种情况下，促凋亡通路都有显著但非致死的激活。我们在大鼠中进一步扩展了这些研究，并在部分肝切除术（PHx）后进行了HGF/Met和EGFR的双KD。结果是完全的肝功能衰竭，大多数肝细胞大量凋亡和坏死，肝实质塌陷。在没有PHx的情况下进行双KD时，没有看到这种效果。当EGFR和MET的有丝分裂信号都无法发挥作用时，参与肝再生的许多细胞因子（如TNF和TGFb1）也可以作为肝细胞死亡和肝衰竭的因子，本研究旨在确定导致肝衰竭的信号。除实验研究外，我们还将分析上述受体以及能够诱导肝细胞死亡和肝衰竭的细胞因子在暴发性肝坏死患者肝材料中的表达和激活状态。最后，我们将评估EGFR KD后出现的代偿机制的重要性，并确定取消这些机制是否也可能导致肝衰竭。这些研究的结合可能使我们了解导致肝再生早期信号异常失调导致大面积肝坏死的机制，并允许基于合理的机制方案设计预防大面积肝坏死的治疗干预措施。

DESCRIPTION (provided by applicant): This is a revised renewal application for a grant, now in its 5th year, aiming to understand the role of HGF and signaling pathways in hepatic tissue assembly. Our recent studies have demonstrated that HGF and its receptor Met, as well as EGF receptor and the EGFR associated ligands) constitute the only two receptor tyrosine kinases capable of delivering a mitogenic signal to hepatocytes (in serum-free media and when administered to the whole animal, rat or mouse). We have also showed that HGF/Met and EGFR are the only two signaling systems capable of promoting transdifferentiation of hepatocytes to biliary epithelial cells in culture. These unique effects of HGF/Met and EGFR are highly significant. Many other receptor tyrosine kinases are expressed in hepatocytes and their ligands are capable of activating the cognate receptor, yet mitogenic signals are limited to HGF/Met and EGFR. We have recently applied short term knock down (KD) of either HGF/Met or EGFR by ShRNA and demonstrated that both have inhibitory effects on liver regeneration, that the one signal cannot compensate totally for the other, and that the effects are different in scope and complexity. KD of EGFR was followed by compensatory increases in other members of the ErbB family and upregulation of MET. In each instance, there was significant but not lethal activation of pro-apoptotic pathways. We further extend these studies in rats and performed a double KD of both HGF/Met and EGFR followed by partial hepatectomy (PHx). The result was complete liver failure, with massive apoptosis and necrosis of most hepatocytes and collapse of the hepatic parenchyma. This effect was not seen when double KD was performed without PHx. The proposed study will aim to determine the signals that lead to liver failure under the hypothesis that many cytokines involved in liver regeneration (e.g. TNF and TGFb1) can also function as agents that bring hepatocyte death and liver failure when the mitogenic signals of both EGFR and MET fail to function. In addition to the experimental studies, we will also analyze expression and status of activation of the above receptors as well cytokines capable of inducing hepatocyte death and liver failure in human liver material obtained from cases of fulminant hepatic necrosis. Finally we will assess the importance of the compensatory mechanisms emerging after KD of EGFR and determine whether abrogation of these mechanisms is capable to also lead to liver failure by itself. The combination of these studies may allow us to understand the mechanisms that lead to massive hepatic necrosis as an aberrant dis-coordination of the early signals involved in liver regeneration and allow design of therapeutic interventions for prevention of massive hepatic necrosis based on rational mechanistic schemes. PUBLIC HEALTH RELEVANCE: The proposed studies aim to bring to bear the knowledge gained from liver regeneration to the elucidation of the pathways leading to liver failure. Fulminant hepatic failure (FHF) remains as one of the most lethal and costly human diseases curable only by liver transplantation. The failure of the liver to regenerate and the potential failure of the two mitogenic receptors (EGFR and MET) in combination with altered pathways involved in regeneration that can also induce hepatocyte apoptosis (e.g. TNF) are very likely to provide rational mechanistic understanding and therapeutic options in management of this serious clinical problem.