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: 7825296
• 负责人: Judith A West-Mays
• 金额: $ 20.76万
• 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 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7825296
• 关键词: Anterior; Cataract; Conditioned Culture Media; Crystalline Lens; Data; Development; Disease; E-Cadherin; Epithelial; Epithelial Cells; Exhibits; Family member; Fibrosis; Figs - dietary; Gelatinase A; Gelatinase B; Genes; Genetic; Goals; In Vitro; Knockout Mice; MAP Kinase Gene; MAP Kinase Signaling Pathways; MAPK14 gene; MMPI; Malignant Neoplasms; Matrix Metalloproteinases; Mediating; Mesenchymal; Mitogen-Activated Protein Kinases; Modeling; Mus; Mutant Strains Mice; Myofibroblast; Pathology; Pathway interactions; Phenotype; Phosphorylation; Play; Rattus; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Smooth Muscle Actin Staining Method; System; Testing; Time; Western Blotting; Wound Healing; cytokine; in vitro Model; in vivo; kinase inhibitor; laser capture microdissection; lens; morphogens; mouse model; prevent; programs; repaired

The cytokine TGFP 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 TGFP 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 TGFp-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 TGFP induces ASC we have shown that treatment with agents that inhibit enzymatic activity of Matrix Metalloproteinase (MMP) family members (MMPIs) suppresses the TGFp-induced cataractous changes. Further preliminary findings suggest the testable hypothesis that MMPIs act to inhibit TGFP- induced EMT and ASC in the lens by suppressing MMP-mediated E-cadherin degradation by shedding. The RSmad, SmadS, is a common effector of TGFP 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) TGFpl can induce EMT in the lens, demonstrating the involvement of SmadS-independent signaling in ASC formation. Additional findings suggest that the hypothesis that TGFp-induced ASC involves signaling MAP kinase pathways, specifically p38,which act independently of Smad3. To test these hypotheses we will utilize the in vitro TGFp-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 pSSMAPKin 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 TGFp-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.

细胞因子TGF β是一种多效性形态原，其调节组织修复表型，并且还发挥调节细胞凋亡的作用。 在纤维化修复病理学的发展中起重要作用。在眼睛的透镜中，纤维化病理 由TGF β介导的白内障的发病机制包括前囊下白内障（ASC）和后囊膜混浊（PCO）。 ASC和PCO纤维化之前的细胞变化包括透镜增殖增加 上皮细胞（LEC），其经历上皮-间充质转化（EMT）， 肌成纤维细胞，涉及E-钙粘蛋白的损失和诱导的α-平滑肌肌动蛋白（aSMA）表达。的 该项目的长期目标是确定TGF β介导的信号，这些信号改变了遗传组成， ASC期间透镜上皮细胞的表型。使用先前开发的大鼠透镜培养模型，其中 外源性TGF β诱导ASC，我们已经表明，用抑制ASC的酶活性的试剂处理， 基质金属蛋白酶（MMP）家族成员（MMPIs）抑制TGF β诱导的白内障 变化进一步的初步研究结果表明，MMPIs抑制TGF-β 1表达的假说是可检验的。 通过抑制MMP介导的E-钙粘蛋白降解诱导透镜中的EMT和ASC， 脱落RSmad，SmadS，是TGF β信号传导的常见效应物，其已经在免疫组织化学中鉴定。 透镜。然而，我们已经发现使用两种不同的小鼠模型，在不存在SmadS的情况下（在Smad 3 KO中）， TGF β 1可以诱导透镜中的EMT，证明了SmadS非依赖性信号转导参与了晶状体上皮细胞的EMT。 ASC形成。另外的发现表明，TGF β诱导的ASC涉及信号转导的假设， MAP激酶途径，特别是p38，其作用独立于Smad 3。为了验证这些假设，我们 将利用体外TGF β诱导的ASC大鼠透镜模型，以及遗传修饰小鼠，包括 MMP-2、MMP-9和SmadS KO小鼠。MMPIs对E-cadherin表达和脱落的影响将被进一步研究。 使用RT-QPCR结合激光捕获显微切割、蛋白质印迹和免疫印迹进一步检查， 免疫定位在ASC形成中对激活的pSSMAPK的需求将在两种情况下进行研究。 使用特异性Pp 38激酶抑制剂的体内和体外模型。这些数据将有助于确定 TGF β介导的途径控制ASC中的EMT和纤维化。此外，由于基因和信号传导 ASC形成的机制与其他纤维化疾病和癌症中发生的机制相似， 从这些透镜研究中获得的信息也可能与这些疾病实体相关。