| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2000: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
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.
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