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

我们用体外模型检测了缺血肾小管细胞细胞连接的结构和功能改变；培养肾小管细胞ATP耗竭及体内模型的建立戈德布拉特肾脏。E-cadherin是在上皮细胞极性的建立和维持中起重要作用的关键分子，在体外和体内缺血肾小管细胞中被降解为80 kDa和35 kDa片段，并被两种不同的抗体检测到。这可能代表不可避免的细胞死亡；应激导致细胞凋亡。另一方面，紧密连接（TJ）膜蛋白claudin-1在缺血后沿着质膜重新分布，而不被内化，尽管另一种膜TJ蛋白occludin在缺血后被内化和降解的时间晚于E-cadherin，这表明肾缺血后TJ的解聚方式与E-cadherin-catenin复合物的解聚方式不同。我们还研究了再灌注后重建细胞-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，研究了活化的Src激酶对细胞-细胞连接的影响。在允许的温度下，v-Src使细胞中酪氨酸残基上的大多数连接蛋白磷酸化。这种反应被蛋白酶体抑制剂mg132明显减弱。此外，具有泛素连接酶活性的c-Cbl在允许温度下显著降低，MG 132对其有抑制作用。这表明细胞-细胞连接的组装和重组可能受到蛋白质水解途径，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酪氨酸激酶活性在肾小管细胞中调节功能性细胞-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.