Rho GTpase in the Lens - Role in Growth and Development

描述(申请人提供)：眼晶状体的发育包括赤道上皮细胞的受控增殖和逐渐分化为终末分化的晶状体纤维。调节这些细胞过程的细胞内信号机制及其在白内障发生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，在晶状体生长发育和维持晶状体结构完整性方面发挥关键作用。这一假说的有力证据来自我们最近的研究，该研究基于不同模型系统中Rho GTPase功能的药理和遗传调节，包括晶状体器官和上皮细胞培养，以及C3外酶转基因小鼠模型。Rho GTP酶在细胞培养和体内失活导致晶状体上皮细胞和纤维细胞形态、肌动蛋白细胞骨架组织化和细胞黏附特性的改变。此外，Rho GTPase功能敲除的小鼠发生了白内障，并表现出细胞外基质和基底膜的编码蛋白以及应激信号和凋亡途径的蛋白的差异表达。此外，几种生长因子激活了晶状体上皮细胞中的Rho GTP酶(Rho和Rac)。这些观察结果支持Rho GTPase(Rho和Rac)介导的信号机制在晶状体发育、生长和完整性中的关键作用。为了阐明RhoGTP酶调控晶状体生长发育的机制，我们拟研究1.RhoA和RhoB GTP酶及其下游效应因子Rho激酶在RhoB基因敲除小鼠晶状体上皮细胞增殖和细胞命运中的作用，以及用腺病毒载体在人晶状体上皮细胞系中过表达RhoA或Rho激酶的显性突变。2.Rac GTPase和ROS激活的应激信号通路(C-Jun-N末端激酶-JNK1和p38 MAPK)在生长因子诱导的人晶状体上皮细胞(Akt和NFkappaB)增殖和存活(Akt和NFkappaB)中的作用。3.在晶状体生长和上皮细胞增殖的背景下，RAC/NADPH氧化酶对晶状体蛋白酪氨酸磷酸酶活性的调节。这些研究的完成将揭示Rho/Rho激酶和Rac GTPase介导的信号转导的意义，以及应激激活信号通路在晶状体上皮细胞增殖、存活和凋亡中的作用。更好地了解调节晶状体生长和维持晶状体透明度的信号转导通路，可以为深入了解白内障形成的病因和开发某些类型白内障的药物治疗提供新的途径。

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.