Hepatic Stellate Cell Derived MMP9 in Liver Fibrogenesis

描述(申请人提供)：由病毒感染、酗酒、肝毒性或遗传代谢紊乱引起的肝硬变在美国是一个主要的健康问题。在肝纤维化形成过程中，肝星状细胞(HSC)经历被称为激活的表型变化，通过这种表型变化，储存维生素A的细胞被反式分化为肌成纤维细胞样细胞。当HSC被激活时，窦周隙中正常的基底膜样细胞外基质被胶原纤维取代。有可信的证据表明IL-1在肝、肾和肺的纤维化形成中起作用。尽管已知正常的细胞外基质环境对维持HSC处于“静止”状态的重要性，但在不同的三维(3D)细胞外基质中，HSC对纤维化刺激的反应仍不清楚。为此，我们获得了一个新的发现，3DI型胶原对IL-1α诱导的HSC诱导的前-MMP9和MMP13的表达和激活、ECM的蛋白降解和HSC的激活具有最强的协同作用。为了支持基质金属蛋白酶-9在肝星状细胞激活和纤维化形成中的作用，在基质金属蛋白酶-9基因缺失的小鼠中，胆管结扎导致了肝纤维化的减轻，尽管肝细胞损伤相似。一旦被完全激活，HSC就会失去对产生基质金属蛋白酶-9和基质金属蛋白酶-13的双重信号的反应性，我们认为这一缺陷反映了已知的伴随晚期肝纤维化而抑制的纤维溶解活性。在人类纤维化肝脏中，基质金属蛋白酶-9定位于纤维化形成前沿的α-平滑肌肌动蛋白阳性细胞亚群。基于这些发现，我们提出了一个假设，即I型胶原和IL-1α作为强有力的双重信号，通过诱导和激活前MMP9和MMP13来激发和维持早期HSC的激活，而在完全激活的HSC中，这种MMPs诱导能力的丧失是进行性纤维化形成的基础。针对这一假说，我们将致力于以下四个具体目标：1)确定3DI型胶原和IL-1α双重信号诱导基质金属蛋白酶-9原的机制；2)确定由IL-1α和I型胶原诱导的原基质金属蛋白酶-9激活剂(S)；3)阐明完全激活的肝星状细胞关闭对双重信号的反应性的原因；4)检测IL-1和基质金属蛋白酶-9在动物模型肝纤维化中的作用。对于目标1和目标3，将使用功能损失或获得方法来测试关键信号分子的要求。为了达到目的2，我们将测试最近在人类皮肤中发现的凝乳酶样原基质金属蛋白酶-9激活剂是否在双重信号刺激下在HSC中表达，同时进行系统的分子研究寻找新的激活剂。最后，采用敲除和敲入的方法检测IL-1信号和基质金属蛋白酶-9在体内早期和晚期肝纤维化形成中的作用。

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.