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RECEPTORS AND SIGNAL TRANSDUCTION

Requirement of Ca²⁺ influx- and phosphatidylinositol 3-kinase-mediated m-calpain activity for shear stress-induced endothelial cell polarity

Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo, Japan

Submitted 24 February 2007 ; accepted in final form 22 June 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 Ca²⁺-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 µ-calpain, suppressed the activity. Either removal of extracellular Ca²⁺ or application of an intracellular Ca²⁺ chelator (BAPTA/AM) or nonselective cation channel blocker (Gd³⁺) 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 [GenBank] ). 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 Ca²⁺ 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.

mechanotransduction; morphological change; focal adhesion reorganization; fluid flow

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