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Role of the Channel-Kinase TRPM in Cell Adhesion and Cell Migration

Li-Ting Su
MS, 1998
National Yang-Ming University
Taipei, Taiwan

Thesis Advisor: Loren W. Runnels, PhD
Graduate Program: Cellular & Molecular Pharmacology

Monday, April 28, 2008
1:30 pm


Cell migration is an integrated process, requiring the controlled interaction of numerous molecules and specific signaling pathways. Here we report a role for TRPM7, a bifunctional protein with ion channel and kinase activities, in cell adhesion and cell migration. The channel-kinase colocalized to peripheral adhesion complexes with m-calpain in HEK-293 cells, where TRPM7 regulates cell adhesion by controlling the activity of the protease. Our research revealed that overexpression of TRPM7 in HEK-293 cells caused cell rounding with a concomitant loss of cell adhesion that is dependent upon the proteinís channel but not its kinase activities. Knockdown of m-calpain blocked TRPM7-induced cell rounding and cell detachment. Silencing of TRPM7 by RNA interference, however, strengthened cell adhesion and increased the number of peripheral adhesion complexes in HEK-293 cells. Together, our results suggest a model in which TRPM7 regulates cell adhesion through m-calpain by mediating local influx of calcium into peripheral adhesion complexes. To verify TRPM7ís impact on cell adhesion and cell migration we created a Swiss 3T3 fibroblast cell line in which TRPM7 levels have been knocked-down by RNA interference. TRPM7-knockdown fibroblasts showed a spindled-constricted cell morphology. Compared to wildtype and non-silencing control cells, immunocytochemical staining revealed that TRPM7-knockdown fibroblasts had severely reduced actomyosin fibers and focal contacts. Reducing TRPM7 levels also markedly impaired the ability of fibroblasts to spread on fibronectin and largely abolished cell polarity in a cellular wound assay. Re-expression of TRPM7 rescued cell morphology and the ability of fibroblasts to spread on fibronectin. Biochemical analysis revealed that during cell spreading, Rho/Rac activities were misregulated and turnover of m-calpain substrates were reduced in TRPM7-knockdown fibroblasts compared to wildtype and shRNA control cells. Recent studies have demonstrated that during cell spreading, Rho activity is regulated by FAK and m-calpain is activated by ERK1/2, whose activity is also regulated by FAK. Surprisingly, we found levels of the fibronectin receptor, integrin alpha5beta1, were strongly reduced in TRPM7-knockdown cells. The above data supports a model in which TRPM7 regulates cell adhesion and migration through FAK by controlling protein levels of integrin alpha5beta1.

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