Role of Matrix Metalloproteinases in Subcapsular Cataract Formation

基质金属蛋白酶在囊下白内障形成中的作用

• 批准号: 7024380
• 负责人: Judith A West-Mays
• 金额: $ 21.6万
• 依托单位: MCMASTER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7024380
• 关键词: cadherins; cataract; lens; model; role

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.

描述：细胞因子 TGF 是一种多效性形态发生素，可调节组织修复表型，并且在纤维化修复病理学的发展中发挥重要作用。在眼睛的晶状体中，TGF 介导的纤维化病理包括前囊膜下白内障 (ASC) 和后囊膜混浊 (PCO)。 ASC 和 PCO 纤维化之前的细胞变化包括晶状体上皮细胞 (LEC) 增殖增加，其经历上皮间质转化 (EMT) 转化为肌成纤维细胞，涉及 E-钙粘蛋白的丧失和诱导的 a-平滑肌肌动蛋白 (aSMA) 表达。该项目的长期目标是确定 TGFβ 介导的信号，这些信号会改变 ASC 过程中晶状体上皮细胞的基因组成和表型。使用先前开发的大鼠晶状体培养模型，其中外源性TGFβ诱导ASC，我们已经证明用抑制基质金属蛋白酶（MMP）家族成员（MMPI）酶活性的药物治疗可以抑制TGFβ诱导的白内障变化。进一步的初步研究结果提出了可检验的假设，即 MMPI 通过抑制 MMP 介导的 E-钙粘蛋白脱落来抑制晶状体中 TGFβ 诱导的 EMT 和 ASC。 RSmad（Smad3）是 TGFβ 信号传导的常见效应器，已在晶状体中得到鉴定。然而，我们发现使用两种不同的小鼠模型，在没有 SmadS 的情况下（在 Smad3 KO 小鼠中），TGF¿1 可以诱导晶状体中的 EMT，证明不依赖于 Smad3 的信号传导参与 ASC 的形成。其他研究结果表明，TGFβ 诱导的 ASC 涉及 MAP 激酶信号通路，特别是 p38，其作用独立于 Smad3。为了测试这些假设，我们将利用体外 TGFβ 诱导的 ASC 大鼠晶状体模型，以及转基因小鼠，包括 MMP-2、MMP-9 和 SmadS KO 小鼠。将使用 RT-QPCR 结合激光捕获显微切割、蛋白质印迹和免疫定位进一步检查 MMPI 对 E-钙粘蛋白表达和脱落的影响。将使用特定的 Pp38 激酶抑制剂在体内和体外模型中研究 ASC 形成中激活的 p38MAPK 的需求。这些数据将有助于确定 TGFβ 介导的控制 ASC 中 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基因和信号机制相似，因此从晶状体中的这些研究中获得的信息也可能与这些疾病实体相关。