Requirement of Ca influx- and phosphatidylinositol 3-kinase-mediated m-calpain activity for shear stress-induced endothelial cell polarity
نویسندگان
چکیده
Miyazaki T, Honda K, Ohata H. Requirement of Ca influxand phosphatidylinositol 3-kinase-mediated m-calpain activity for shear stress-induced endothelial cell polarity. Am J Physiol Cell Physiol 293: C1216–C1225, 2007. First published June 27, 2007; doi:10.1152/ajpcell.00083.2007.—Proteolytic activity in sheared human umbilical vein endothelial cells (HUVECs) was measured using a fluorogenic substrate and laser scanning confocal microscopy to clarify the key role of an intracellular Ca -sensitive protease, calpain, in these cells in response to shear stress. Within physiological shear range, activity in the cells was enhanced in shear-dependent fashion. Short interfering RNA-induced silencing of m-calpain, but not of -calpain, suppressed the activity. Either removal of extracellular Ca or application of an intracellular Ca chelator (BAPTA/AM) or nonselective cation channel blocker (Gd ) reduced proteolytic activity. Furthermore, activity was suppressed by phosphatidylinositol bisphosphate (PIP2) chelator (neomycin) or phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002); in contrast, activity, which was partially inhibited by ERK kinase inhibitor (U0126, PD98059), was unaffected by PLC inhibitor (U73122). Moreover, Akt phosphorylation downstream of PI3K, which was elicited by shear, was attenuated by neomycin but not by calpain inhibitor (calpeptin). Following assessment of shear stress-induced focal adhesion (FA) and cytoskeletal dynamics using interference reflection/green fluorescence protein-actin microscopy, we found that either calpain or PI3K inhibition impaired shear stress-induced polarization of FAs via stabilization of FA structures. Additionally, HUVEC alignment and cytoskeletal remodeling, which was accompanied by calpain-mediated cleavage of vinculin and talin, were also elicited by prolonged application of shear and impaired by m-calpain knockdown. Thus, these results revealed that physiological shear stress elicits Ca influx-sensitive activation of m-calpain in HUVECs. This activity is facilitated primarily through the PI3K pathway; furthermore, it is essential for subsequent FA reorganization and cell alignment under shear conditions.
منابع مشابه
Requirement of Ca2+ influx- and phosphatidylinositol 3-kinase-mediated m-calpain activity for shear stress-induced endothelial cell polarity.
Proteolytic activity in sheared human umbilical vein endothelial cells (HUVECs) was measured using a fluorogenic substrate and laser scanning confocal microscopy to clarify the key role of an intracellular Ca(2+)-sensitive protease, calpain, in these cells in response to shear stress. Within physiological shear range, activity in the cells was enhanced in shear-dependent fashion. Short interfer...
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