Structural and functional alterations of cell-cell junction in ischemic renal tubular cells

我们用体外模型研究了缺血性肾小管细胞中细胞-细胞连接的结构和功能改变;培养的肾小管细胞和体内模型中的ATP耗竭; Goldblatt肾。E-cadherin是建立和维持上皮细胞极性的关键分子，在缺血的离体和体内肾小管细胞中，E-cadherin被降解为80 kDa和35 kDa的片段。这可能代表不可避免的细胞死亡;来自应激的细胞凋亡。另一方面，紧密连接（TJ）膜蛋白claudin-1，沿细胞膜沿着重新分布，而不interemalization，虽然occludin，另一种膜TJ蛋白，在缺血后的内化和降解晚于E-钙粘蛋白，这表明TJ可以在肾缺血后以不同的方式从E-钙粘蛋白-连环蛋白复合物中分解。我们还研究了再灌注后重新建立细胞-细胞连接的分子机制。由于Src酪氨酸激酶在再灌注后被激活，我们以温度敏感的v-src MDCK细胞为模型，研究了激活的Src激酶对细胞-细胞连接的影响。v-Src在允许的温度下磷酸化细胞内大多数酪氨酸残基上的连接蛋白。这种反应被蛋白酶体抑制剂MG 132显著减弱。此外，具有泛素连接酶活性的c-Cbl在允许温度下显著降低，这被MG 132抑制。这表明细胞间连接的组装和再组装可能受蛋白水解途径，特别是泛素-蛋白酶体途径的控制。因此，我们将阐明蛋白酶体通过Src酪氨酸激酶活性调节肾小管细胞功能性细胞-细胞连接的分子机制。

We examined the structural and functional alterations of cell-cell junction in ischemic renal tubular cells with an in vitro model ; ATP depletion in cultured renal tubular cells and an in vivo model; Goldblatt kidney. E-cadherin, a key molecule that playing an important role to establish and to maintain epithelial cell polarity, was degraded to 80 kDa and 35 kDa fragments, which were detected by two different kinds of antibodies, in ischemic in vitro and in vivo renal tubular cells. This may represent non-escapable cell death; apoptosis from the stress. On the other hand, claudin-1, a tight junction (TJ) membrane protein, redistributed along the plasma membrane without intemalization, although occludin, another membrane TJ protein, was internalized and degraded later than that of E-cadherin after ischemia, indicating that the TJ could be disassembled in a different manner from that of E-cadherin-catenin complexes after renal ischemia. We also examined the molecular mechanism to re-establish cell-cell junction after reperfusion. Since Src tyrosine kinase has been activated after reperfusion, we examined the effect of activated Src kinase on cell-cell junction with temperaturesensitive v-src MDCK cells as a model. v-Src phosphorylated most ofjunctional proteins on the tyrosine residue(s) in the cells in permissive temperature. This reaction was markedly attenuated by a proteasome inhibitor, MG 132. In addition, c-Cbl, possessing ubiquitin ligase activity, significantly decreased in permissive temperature, which was inhibited by MG 132. This suggests that the assembly and reassembly of cell-cell junction might be controlled by a proteolytic pathway, especially by the ubiquitin-proteasome pathway. Thus, we are going to clarify the molecular mechanism(s) of the proteasome to regulate functional cell-cell junction through Src tyrosine kinase activity in renal tubular cells.