Hepatic Stellate Cell Derived MMP9 in Liver Fibrogenesis

描述（由申请人提供）：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在美国是一个主要的健康问题。在肝纤维化过程中，肝星状细胞（HSC）经历一种被称为活化的表型变化，通过这种变化，储存维生素a的细胞被分化为肌成纤维细胞样细胞。在HSC激活后，正常的基底膜样ECM在窦周间隙被胶原纤维所取代。有可信的证据表明IL-1在肝、肾和肺的纤维形成中起作用。尽管已知正常ECM环境对维持HSC处于“静止”状态的重要性，但HSC如何在不同三维（3D） ECM中对纤维化刺激作出反应仍然未知。为此，我们获得了一个新的发现，3D I型胶原对il -1 α诱导的原mmp - 9和MMP-13的表达和活化，通过HSC， ECM的蛋白水解降解和HSC活化发挥了最强大的协同作用。为了支持MMP-9在HSC激活和纤维化中的作用，在MMP-9缺失的小鼠中，胆管结合法导致肝纤维化减轻，尽管肝细胞损伤相当。一旦完全激活，HSC失去对产生MMP-9和MMP-13的双重信号的反应性，我们认为这一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定位于纤维发生前沿的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mmp9和MMP-13来激发和延续早期HSC的激活，而在完全激活的HSC中，这种MMP诱导性的丧失是进行性纤维化的基础。针对这一假设，我们将解决以下四个具体目标：1)确定来自3D 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激活物；3)阐明为什么完全激活的HSC会关闭对双重信号的响应性；4)通过动物模型检测IL-1和MMP-9对肝纤维化的贡献。对于目标1和目标3，将使用功能的损失或增益方法来测试关键信号分子的要求。为了目的2，我们将测试最近在人类皮肤中发现的一种类似凝乳胰蛋白酶的proMMP-9激活因子是否在双信号刺激下的HSC中表达，同时对新的激活因子进行系统的分子搜索。最后，我们将使用敲除和敲入的方法来测试IL-1信号和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.