PKC-epsilon is required for mechano-sensitive activation of ERK1/2 in endothelial cells.
Mechano-sensitive regulation of endothelial cells (EC) function by shear stress is critical for flow-induced vasodilation and gene expression. Previous studies by our laboratory demonstrated that shear stress activates the 44- and 42-kDa extracellular signal-regulated kinases (ERK1/2) in EC in a time- and force-dependent manner. ERK1/2 activation was inhibited by protein kinase C (PKC) down-regulation with phorbol 12,13-dibutyrate (1 microM for 24 h) but not by calcium chelation with BAPTA-AM (acetoxymethyl ester of BAPTA) (75 microM for 30 min), suggesting that a novel PKC isoform (delta, epsilon, eta, theta) mediates shear stress-induced ERK1/2 activation. Western blotting with PKC isoform-specific antibodies demonstrated expression of PKC-alpha, -epsilon, and -zeta isoforms in EC. PKC-epsilon was specifically inhibited by transfection with antisense PKC-epsilon phosphorothioate oligonucleotides (1,000 nM for 6 h). Antisense treatment decreased PKC-epsilon protein levels by 80 +/- 13% after 72 h and completely inhibited shear stress-stimulated ERK1/2 activation. Scrambled PKC-epsilon oligonucleotides and antisense PKC-alpha and PKC-zeta oligonucleotides had no effect on ERK1/2 activity. PKC-epsilon appeared specific for mechano-sensitive ERK1/2 activation, as antisense PKC-epsilon oligonucleotides did not inhibit ERK1/2 activation by EGF or bradykinin but did inhibit ERK1/2 activation upon EC adhesion to fibronectin. These results define a pathway for shear stress-mediated ERK1/2 activation and establish a new function for PKC-epsilon as part of a mechano-sensitive signal transduction pathway in EC.
Traub
O
O
Department of Pathology, University of Washington, Seattle, Washington 98195, USA.
Monia
B P
BP
Dean
N M
NM
Berk
B C
BC
D000818:Animals; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000201:enzymology*; D004789:Enzyme Activation; D005353:Fibronectins / Q000378:metabolism; D018056:Hemorheology; D006657:Histones / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D013314:Stress, Mechanical
D005353:Fibronectins; D006657:Histones; D007527:Isoenzymes; D016376:Oligonucleotides, Antisense; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D002118:Calcium