Rho and Rho-kinase (ROCK) signaling in adherens and gap junction assembly in corneal epithelium.
Rho and Rho-kinase (ROCK) signaling in adherens and gap junction assembly in corneal epithelium.
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发表时间：
2002-04
影响因子：
4.4
通讯作者：
S. Anderson;Cynthia Stone;Lisa Tkach;N. Sundarraj
中科院分区：
文献类型：
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作者：
S. Anderson;Cynthia Stone;Lisa Tkach;N. Sundarraj
目的研究Rho及其下游靶点Rho相关激酶（ROCK）是否参与调节角膜上皮细胞钙粘蛋白介导的细胞粘附和缝隙连接蛋白43（Cx43）的组装。方法用肉毒梭菌ADP核糖基转移酶（C3）和ROCK特异性抑制剂Y-27632分别抑制培养的兔角膜上皮细胞Rho和ROCK活性。采用免疫细胞化学和Western blot技术研究E-cadherin和Cx43在大鼠海马神经元中的分布和相对浓度。通过染料转移测定来测量经由间隙连接的细胞间通信。结果C3抑制原代培养的兔角膜上皮细胞Rho活性，可抑制E-cadherin的细胞间粘附和Cx43缝隙连接的组装。然而，通过用Y-27632处理抑制ROCK活性抑制了基于E-钙粘蛋白的细胞-细胞粘附的组装，但不抑制Cx43间隙连接。事实上，ROCK的抑制导致Cx43间隙连接和细胞间通讯数量的增加。在低钙培养基中培养角膜上皮细胞可阻止E-钙粘蛋白粘附连接的形成，但不能阻止Cx43间隙连接的形成。结论E-cadherin粘附连接不是角膜上皮细胞Cx43缝隙连接组装的先决条件。不同的Rho信号通路参与调节E-cadherin介导的细胞-细胞粘附和Cx43间隙连接的组装。虽然Rho/ROCK信号通路影响E-钙粘蛋白粘附连接的组装，但其下调影响Cx43间隙连接组装。
PURPOSE 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.