Role of Matrix Metalloproteinases in Subcapsular Cataract Formation

描述：细胞因子转化生长因子β是一种多效性的形态原，调节组织修复表型，在纤维化修复病理学的发展中也起着重要作用。在眼的晶状体中，由转化生长因子介导的纤维化病理包括前囊下白内障(ASC)和后囊混浊(PCO)。ASC和PCO纤维化前的细胞变化包括晶状体上皮细胞(LECs)增殖增加，继而发生上皮-间充质转化(EMT)为肌成纤维细胞，涉及E-钙粘附素的丢失和诱导α-平滑肌肌动蛋白(ASMA)的表达。该项目的长期目标是确定在ASC过程中改变晶状体上皮细胞遗传组成和表型的转化生长因子介导的信号。使用以前建立的外源性转化生长因子诱导ASC的大鼠晶状体培养模型，我们已经证明，用抑制基质金属蛋白酶家族成员(MMPIs)酶活性的药物治疗可以抑制转化生长因子β诱导的白内障改变。进一步的初步研究结果表明，可检验的假说是，MMPIs通过抑制基质金属蛋白酶介导的E-钙粘附素的脱落降解，从而抑制转化生长因子β诱导的晶状体内EMT和ASC。RSmad，SMAD3，是一种常见的转化生长因子信号效应器，已在晶状体中发现。然而，我们使用两种不同的小鼠模型发现，在没有Smads的情况下(在Smad3 KO小鼠中)，转化生长因子1可以诱导晶状体中的EMT，表明Smad3非依赖的信号参与了ASC的形成。其他发现表明，转化生长因子β诱导的ASC涉及信号MAP激酶通路，特别是p38，该通路独立于Smad3发挥作用。为了验证这些假设，我们将利用体外转化生长因子诱导的ASC大鼠晶状体模型，以及转基因小鼠，包括MMP2、MMP9和Smads KO小鼠。MMPIs对E-钙粘蛋白表达和脱落的影响将通过RT-QPCR结合激光捕获显微切割、免疫印迹和免疫定位来进一步检测。在体内和体外模型中，将使用特定的Pp38激酶抑制剂来研究ASC形成过程中对激活的P38MAPK的需求。这些数据将有助于确定ASC中由转化生长因子介导的控制EMT和纤维化的途径。此外，由于ASC形成中的基因和信号机制与其他纤维化疾病和癌症中发生的相似，从这些晶状体研究中获得的信息也可能与这些疾病实体相关。

DESCRIPTION: The cytokine TGF¿ is a pleotropic morphogen that modulates the tissue repair phenotype and also plays an important role in the development of fibrotic repair pathologies. In the lens of the eye, fibrotic pathologies mediated by TGF¿ include anterior subcapsular cataracts (ASC) and posterior capsular opacification (PCO). The cellular changes that precede fibrosis in ASC and PCO include an increased proliferation of lens epithelial cells (LECs), which under go an epithelial-mesenchymal transformation (EMT) into myofibroblasts, involving loss of E-cadherin and induced a-smooth muscle actin (aSMA) expression. The long-term goal of this project is to determine the TGF¿-mediated signals, which alter the genetic makeup and phenotype of lens epithelial cells during ASC. Using a previously developed rat lens culture model in which exogenous TGF¿ induces ASC we have shown that treatment with agents that inhibit enzymatic activity of Matrix Metalloproteinase (MMP) family members (MMPIs) suppresses the TGF¿-induced cataractous changes. Further preliminary findings suggest the testable hypothesis that MMPIs act to inhibit TGF¿-induced EMT and ASC in the lens by suppressing MMP-mediated E-cadherin degradation by shedding. The RSmad, Smad3, is a common effector of TGF¿ signaling, which has been identified in the lens. However, we have found using two different mouse models that in the absence of SmadS (in Smad3 KO mice) TGF¿1 can induce EMT in the lens, demonstrating the involvement of Smad3-independent signaling in ASC formation. Additional findings suggest that the hypothesis that TGF¿-induced ASC involves signaling MAP kinase pathways, specifically p38, which act independently of Smad3. To test these hypotheses we will utilize the in vitro TGF¿-induced rat lens model of ASC, as well as genetically modified mice, including MMP-2, MMP-9 and SmadS KO mice. The effect of MMPIs on E-cadherin expression and shedding will be further examined using RT-QPCR in combination with laser capture microdissection, western blotting and immunolocalization. The requirement for activated p38MAPK in ASC formation will be investigated in both the in vivo and in vitro models using specific Pp38 kinase inhibitors. These data will aid in defining the TGF¿-mediated pathways controlling EMT and fibrosis in ASC. Furthermore, since the genes and signaling mechanisms in ASC formation are similar to those which occur in other fibrotic diseases and cancer, the information gained from these studies in the lens may also have relevance to these disease entities.