Document Detail


Requirement of Ca2+ influx- and phosphatidylinositol 3-kinase-mediated m-calpain activity for shear stress-induced endothelial cell polarity.
MedLine Citation:
PMID:  17596297     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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 interfering RNA-induced silencing of m-calpain, but not of micro-calpain, suppressed the activity. Either removal of extracellular Ca(2+) or application of an intracellular Ca(2+) chelator (BAPTA/AM) or nonselective cation channel blocker (Gd(3+)) reduced proteolytic activity. Furthermore, activity was suppressed by phosphatidylinositol bisphosphate (PIP(2)) 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(2+) 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.
Authors:
Takuro Miyazaki; Kazuo Honda; Hisayuki Ohata
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2007-06-27
Journal Detail:
Title:  American journal of physiology. Cell physiology     Volume:  293     ISSN:  0363-6143     ISO Abbreviation:  Am. J. Physiol., Cell Physiol.     Publication Date:  2007 Oct 
Date Detail:
Created Date:  2007-10-09     Completed Date:  2007-12-13     Revised Date:  2013-06-03    
Medline Journal Info:
Nlm Unique ID:  100901225     Medline TA:  Am J Physiol Cell Physiol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  C1216-25     Citation Subset:  IM    
Affiliation:
Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan. taku@pharm.showa-u.ac.jp
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MeSH Terms
Descriptor/Qualifier:
Actins / metabolism
Calcium / metabolism*
Calpain / antagonists & inhibitors,  genetics,  metabolism*
Cell Polarity / physiology*
Cells, Cultured
Chromones / pharmacology
Cytoskeleton / drug effects,  metabolism
Dipeptides / pharmacology
Endothelial Cells / cytology,  drug effects,  metabolism*
Flavonoids / pharmacology
Focal Adhesions / drug effects,  metabolism
Gadolinium / pharmacology
Humans
Microscopy, Confocal
Morpholines / pharmacology
Neomycin / pharmacology
Phosphatidylinositol 3-Kinases / antagonists & inhibitors,  metabolism*
Phosphorylation / drug effects
Protein Kinase Inhibitors / pharmacology
Proto-Oncogene Proteins c-akt / metabolism
Pseudopodia / drug effects,  metabolism
RNA, Small Interfering / genetics
Stress, Mechanical
Talin / metabolism
Vinculin / metabolism
Chemical
Reg. No./Substance:
0/2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; 0/Actins; 0/Chromones; 0/Dipeptides; 0/Flavonoids; 0/Morpholines; 0/Protein Kinase Inhibitors; 0/RNA, Small Interfering; 0/Talin; 117591-20-5/calpeptin; 125361-02-6/Vinculin; 1404-04-2/Neomycin; 154447-36-6/2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; 7440-54-2/Gadolinium; 7440-70-2/Calcium; EC 2.7.1.-/Phosphatidylinositol 3-Kinases; EC 2.7.11.1/Proto-Oncogene Proteins c-akt; EC 3.4.22.-/Calpain; EC 3.4.22.-/m-calpain

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