Anderson Susan C, Stone Cynthia, Tkach Lisa, SundarRaj Nirmala
Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Invest Ophthalmol Vis Sci. 2002 Apr;43(4):978-86.
To examine whether Rho and its downstream target, a Rho-associated kinase (ROCK), are involved in the regulation of the assembly of cadherin-mediated cell adhesion and connexin 43 (Cx43) gap junctions in corneal epithelium.
Rho and ROCK activities in rabbit corneal epithelial cells in culture were inhibited by microinjection of a Clostridium botulinum ADP-ribosyltransferase (C3) and treatment with a ROCK specific inhibitor (Y-27632), respectively. Immunocytochemical and Western blot techniques were used to study the distribution and relative concentrations of E-cadherin and Cx43. Intercellular communication via gap junctions was measured by a dye transfer assay.
Inhibition of Rho activity in the primary cultures of rabbit corneal epithelial cells by microinjecting them with C3 resulted in an inhibition of the assembly of E-cadherin-based cell-cell adhesion and Cx43 gap junctions. However, inhibition of the ROCK activity by treatment with Y-27632 inhibited the assembly of E-cadherin-based cell-cell adhesions but not Cx43 gap junctions. In fact, inhibition of ROCK resulted in an increase in the number of Cx43 gap junctions and in cell-cell communication. Culturing corneal epithelial cells in a low calcium medium prevented the formation of E-cadherin adherens junctions but not the Cx43 gap junctions.
E-cadherin adherens junctions are not a prerequisite for the assembly of Cx43 gap junctions in corneal epithelial cells. Different Rho signaling pathways are involved in the regulation of the assembly of E-cadherin mediated cell-cell adhesion and Cx43 gap junctions. Although a Rho/ROCK signaling pathway influences the assembly of E-cadherin adherens junctions, its downregulation influences Cx43 gap junction assembly.
研究Rho及其下游靶点Rho相关激酶（ROCK）是否参与角膜上皮中钙黏蛋白介导的细胞黏附及连接蛋白43（Cx43）间隙连接组装的调控。
分别通过显微注射肉毒杆菌ADP核糖基转移酶（C3）和用ROCK特异性抑制剂（Y-27632）处理，抑制培养的兔角膜上皮细胞中的Rho和ROCK活性。采用免疫细胞化学和蛋白质印迹技术研究E-钙黏蛋白和Cx43的分布及相对浓度。通过染料转移试验测量间隙连接介导的细胞间通讯。
向兔角膜上皮细胞原代培养物中显微注射C3抑制Rho活性，导致基于E-钙黏蛋白的细胞间黏附及Cx43间隙连接组装受到抑制。然而，用Y-27632处理抑制ROCK活性，抑制了基于E-钙黏蛋白的细胞间黏附组装，但未抑制Cx43间隙连接组装。事实上，抑制ROCK导致Cx43间隙连接数量增加及细胞间通讯增强。在低钙培养基中培养角膜上皮细胞可阻止E-钙黏蛋白黏附连接的形成，但不影响Cx43间隙连接的形成。
E-钙黏蛋白黏附连接并非角膜上皮细胞中Cx43间隙连接组装的先决条件。不同的Rho信号通路参与E-钙黏蛋白介导的细胞间黏附及Cx43间隙连接组装的调控。虽然Rho/ROCK信号通路影响E-钙黏蛋白黏附连接的组装，但其下调影响Cx43间隙连接的组装。
