Rho GTpase in the Lens - Role in Growth and Development

描述（由申请人提供）：眼透镜的发育涉及赤道上皮细胞受控增殖和渐进分化为终末分化的透镜纤维。调节这些细胞过程的细胞内信号传导机制及其在白内障形成中的可能作用尚未被表征。我们推测Rho GTP酶（Rho和Rac）通过调节细胞骨架组织和生长因子刺激的应激反应信号通路在透镜的生长和发育以及维持透镜结构完整性中起关键作用。这一假说的有力证据来自我们最近的研究，这些研究基于不同模型系统中Rho GTdR功能的药理学和遗传学调节，包括透镜器官和上皮细胞培养物以及C3-胞外酶转基因小鼠模型。Rho GT3在细胞培养和体内的失活导致透镜上皮和纤维细胞形态、肌动蛋白细胞骨架结构和细胞粘附特性的改变。此外，Rho GT3功能敲除小鼠发生白内障，并表现出编码细胞外基质和基底膜蛋白以及应激信号传导和凋亡途径蛋白的基因的差异表达。此外，几种生长因子激活了透镜上皮细胞中的Rho GTP酶（Rho和Rac）。这些观察结果支持Rho GT3（Rho和Rac）介导的信号传导机制在透镜发育、生长和完整性中的关键作用。为了阐明Rho GTP酶调节透镜生长和发育的机制基础，我们建议研究1。RhoA和RhoB GTP酶及其下游效应物Rho激酶在透镜上皮细胞增殖和细胞命运中的作用，使用RhoB敲除小鼠，以及通过使用腺病毒载体在人透镜上皮细胞系中过表达RhoA或Rho激酶的显性负突变体。2. Rac GT3和活性氧（ROS）激活的应激信号通路（C-jun-N-末端激酶-JNK 1和p38 MAPK）在生长因子诱导的人透镜上皮细胞增殖和细胞存活（Akt和NF κ B）中的作用，使用NADPH氧化酶抑制剂和通过Rac GT3的显性负性和组成性活性突变体的过表达。3.在透镜生长和上皮细胞增殖的背景下，Rac/NADPH氧化酶对器官培养晶状体和透镜上皮细胞中透镜蛋白酪氨酸磷酸酶活性的调节这些研究的完成将阐明Rho/Rho激酶和Rac GTP酶介导的信号传导的意义以及应激激活的信号传导途径在透镜上皮细胞增殖、存活和凋亡中的作用。更好地了解调节透镜生长和保持透镜透明度的信号转导途径可以深入了解白内障形成的病因学，并为开发某些类型白内障的医学治疗提供新的方法。

DESCRIPTION (provided by applicant): The development of the ocular lens involves controlled proliferation and progressive differentiation of equatorial epithelial cells into terminally differentiated lens fibers. Intracellular signaling mechanisms that regulate these cellular processes and their possible role in cataractoqenesis have not been characterized. We hypothesize that Rho GTPases (Rho and Rac) play a critical role in lens growth and development and in maintenance of lens structural integrity by regulating cytoskeletal organization and growth factor stimulated stress-response signaling pathways. Strong evidence for this hypothesis derives from our recent studies based on pharmacological and genetic modulation of Rho GTPase function in different model systems including lens organ and epithelial cell culture, and a C3-exoenzyme transgenic mouse model. Inactivation of Rho GTPase in cell culture and in vivo resulted in alterations of lens epithelial and fiber cell morphology, actin cytoskeletal orqanization and cell adhesive characteristics. Additionally, the Rho GTPase functional knock-out mouse developed cataract and exhibited differential expression of genes encodinq proteins of the extracellular matrix and basement membrane, as well as proteins of stress signaling and apoptotic pathways. Further, several growth factors activated Rho GTPases (both Rho and Rac) in lens epithelial cells. These observations support a critical role for Rho GTPase (Rho and Rac)-mediated signaling mechanisms in lens development, growth and integrity. To elucidate the mechanistic basis (es) by which Rho GTPases regulate lens growth and development, we propose to investigate 1. The roles of RhoA and RhoB GTPases and their down stream effector-Rho kinase, in lens epithelial cell proliferation and cell fate using a RhoB knockout mouse, and by overexpressing dominant neqative mutants of RhoA or Rho kinase in a human lens epithelial cell line using adenoviral vectors. 2. The role of Rac GTPase and reactive oxygen species (ROS)-activated stress signaling pathways (C-jun-N-terminal kinase-JNK1 and p38 MAPK) in growth factor-induced proliferation and cell survival (Akt and NFkappaB) of human lens epithelial cells using an NADPH oxidase inhibitor and by overexpression of dominant negative and constitutively active mutants of Rac GTPase. 3. Regulation of lens protein tyrosine phosphatase activity by Rac/NADPH oxidase in organ-cultured lenses and in lens epithelial cells, in the context of lens growth and epithelial cell proliferation. The completion of these studies should unravel the significance of Rho/Rho kinase and Rac GTPase-mediated signaling and the role of stress -activated signaling pathways in proliferation, survival and apoptosis of lens epithelial cells. Better understanding of signal transduction pathways regulating lens growth and maintenance of lens transparency could provide insights into the etiology of cataract formation and into novel approaches towards developing medical treatments for certain types of cataract.