Rho GTpase in the Lens - Role in Growth and Development

晶状体中的 Rho GTpase - 在生长和发育中的作用

• 批准号: 7012175
• 负责人: P VASANTHA Rao
• 金额: $ 26.32万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7012175
• 关键词: DNA replication; JUN kinase; NAD(P)H dehydrogenase; apoptosis; biological signal transduction; cell differentiation; cell growth regulation; cell line; cell proliferation; clinical research; developmental neurobiology; enzyme activity; free radical oxygen; genetically modified animals; growth factor; guanine nucleotide binding protein; laboratory mouse; lens; mitogen activated protein kinase; nuclear factor kappa beta; organ culture; oxidative stress; protein tyrosine phosphatase; terminal nick end labeling; transfection /expression vector

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.

描述（由申请人提供）：眼晶状体的发育涉及赤道上皮细胞的受控增殖和逐步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传导机制及其在白内障发生中可能的作用尚未得到表征。我们假设 Rho GTPases（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，在晶状体生长和发育以及维持晶状体结构完整性中发挥关键作用。这一假设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 GTPase 功能的药理学和遗传调节，包括晶状体器官和上皮细胞培养物以及 C3-外切酶转基因小鼠模型。 Rho GTPase 在细胞培养物和体内的失活导致晶状体上皮和纤维细胞形态、肌动蛋白细胞骨架组织和细胞粘附特性的改变。此外，Rho GTPase功能性敲除小鼠出现白内障，并表现出编码细胞外基质和基底膜蛋白以及应激信号和凋亡途径蛋白的基因差异表达。此外，几种生长因子激活晶状体上皮细胞中的 Rho GTPases（Rho 和 Rac）。这些观察结果支持 Rho GTPase（Rho 和 Rac）介导的信号传导机制在晶状体发育、生长和完整性中的关键作用。为了阐明 Rho GTPases 调节晶状体生长和发育的机制基础，我们建议使用以下方法研究 1. RhoA 和 RhoB GTPases 及其下游效应器 Rho 激酶在晶状体上皮细胞增殖和细胞命运中的作用RhoB 敲除小鼠，并使用腺病毒载体在人晶状体上皮细胞系中过表达 RhoA 或 Rho 激酶的显性失活突变体。 2. Rac GTPase 和活性氧 (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增殖和细胞存活 (Akt 和 NFkappaB) 中的作用使用 NADPH 氧化酶抑制剂并通过 Rac GTPase 的显性失活和组成型活性突变体的过表达来对晶状体上皮细胞进行研究。 3.在晶状体生长和上皮细胞增殖的背景下，器官培养晶状体和晶状体上皮细胞中Rac/NADPH氧化酶对晶状体蛋白酪氨酸磷酸酶活性的调节。这些研究的完成应该揭示 Rho/Rho 激酶和 Rac GTPase 介导的信号传导的重要性以及应激激活的信号传导途径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途径可以深入了解白内障形成的病因学以及开发针对某些类型白内障的医疗方法。