Hepatic Stellate Cell Derived MMP9 in Liver Fibrogenesis

描述（由申请人提供）：在美国，由病毒感染、酒精滥用、肝毒性或遗传代谢紊乱引起的肝硬化是一个主要的健康问题。在肝纤维化中，肝星状细胞（HSC）经历称为活化的表型变化，通过活化，维生素A储存细胞转分化成肌纤维母细胞样细胞。HSC活化后，窦周隙中的正常基底膜样ECM被胶原纤维取代。存在IL-1在肝、肾和肺纤维化中的作用的合理证据。尽管已知正常ECM环境在维持HSC处于“静止”状态中的重要性，但HSC如何在不同的三维（3D）ECM中响应纤维化刺激仍然是未知的。为此，我们获得了一个新的发现，即3D I型胶原蛋白对IL-1 α诱导的HSC表达和激活MMP- 9和MMP-13原、ECM蛋白水解降解和HSC激活发挥最强的协同作用。为了支持MMP-9在HSC活化和纤维形成中的作用，MMP-9缺失小鼠中的胆管结扎导致减弱的肝纤维化，尽管肝细胞损伤相当。一旦完全激活，HSC失去对MMP-9和MMP-13产生的双重信号的反应性，我们认为这种缺陷反映了已知伴随晚期肝纤维化的纤维溶解活性受到抑制。在人类纤维化肝脏中，MMP-9定位于纤维化前沿的α-平滑肌肌动蛋白阳性细胞亚群中。基于这些发现，我们提出了一个假设，即I型胶原和IL-1 α作为有效的双重信号，通过诱导和激活MMP-9和MMP-13的pro-MMP-9和MMP-13，而在完全激活的HSC中，这种MMP诱导因子的丧失是进行性纤维化的基础，从而引发和维持早期HSC活化。为此，我们将从以下四个方面进行研究：1）确定来自I型胶原和IL-1 α的双重信号诱导proMMP-9的机制; 2）鉴定由IL-1 α和I型胶原诱导的proMMP-9激活剂; 3）阐明为什么完全激活的HSC关闭对双重信号的反应性; 4）检测IL-1和MMP-9在肝纤维化动物模型中的作用。对于目标1和3，将使用功能丧失或获得方法来测试关键信号分子的要求。对于目标2，我们将测试最近在人类皮肤中发现的胰凝乳蛋白酶样proMMP-9激活剂是否在双重信号刺激的HSC中表达，同时进行系统的分子搜索以寻找新的激活剂。最后，敲除和敲入方法将用于测试IL-1信号传导和MMP-9表达在体内早期和晚期肝纤维化中的作用。

DESCRIPTION (provided by applicant): Cirrhosis resulting from viral infection, alcohol abuse, hepatotoxicity, or genetic metabolic disorders is a major health problem in the United Sates. In liver fibrogenesis, hepatic stellate cells (HSC) undergo phenotypic changes called activation, by which the vitamin-A storing cells are trans-differentiated into myofibroblast-like cells. Upon HSC activation, the normal basement membrane-like ECM in the perisinusoidal space is replaced by collagenous fibers. Plausible evidence exists for the role of IL-1 in fibrogenesis in liver, kidney, and lung. Despite the known importance of the normal ECM milieu in maintaining HSC in the "quiescent" state, how HSC respond to fibrogenic stimulation in different three-dimensional (3D) ECM still remains unknown. To this end, we have obtained a novel finding that 3D type I collagen exerts the most powerful synergistic effect on IL-1alpha-induced expression and activation of pro-MMP- 9 and MMP-13 by HSC, proteolytic degradation of ECM, and HSC activation. In support the role of MMP-9 in HSC activation and fibrogenesis, bile duct ligation in MMP-9 null mice results in attenuated liver fibrosis despite comparable hepatocellular damage. Once fully activated, HSC lose the responsiveness to the dual signals for production of MMP-9 and MMP-13, the defect that we believe mirrors the suppressed fibrolytic activity known to accompany advanced liver fibrosis. In human fibrotic livers, MMP-9 is localized in a subpopulation of alpha-smooth muscle actin-positive cells in the leading edge of fibrogenesis. Based on these findings, we propose a hypothesis that type I collagen and IL-1alpha serve as potent dual signals to provoke and perpetuate early HSC activation via induction and activation of pro-MMP9 and MMP-13 while the loss of this MMP inducibility in fully-activated HSC underlie progressive fibrogenesis. Toward this hypothesis, we will address the following four specific aims: 1) To determine the mechanism by which dual signals from 3D type I collagen and IL-1alpha induce proMMP-9; 2) To identify a proMMP-9 activator(s) that is induced by IL-1alpha and type I collagen; 3) To elucidate why fully-activated HSC switch off the responsiveness to the dual signals; 4) To test the contribution of IL-1 and MMP-9 to liver fibrosis in animal models. For the aim 1 and 3, the loss or gain of function approach will be used to test the requirements of key signaling molecules. For the aim 2, we will test whether a chymotrypsin-like proMMP-9 activator recently identified in human skin, is expressed in HSC stimulated by the dual signals while pursuing a systematic molecular search for new activators. Lastly, knock-out and knock-in methods will be used to test the roles of IL-1 signaling and MMP-9 expression in early and late liver fibrogenesis in vivo.