PKC-EPSILON IS REQUIRED FOR MECANOSENSITIVE ACTIVATION OF ERK1 2 IN ENDOTHELIAL-CELLS/

Mechano-sensitive regulation of endothelial cells (EC) function by she ar stress is critical for flow-induced vasodilation and gene expressio n. Previous studies by our laboratory demonstrated that shear stress a ctivates the 44- and 42-kDa extracellular signal-regulated kinases (ER K1/2) in EC in a time-and force-dependent manner. ERK1/2 activation wa s inhibited by protein kinase C (PKC) down-regulation with phorbol 12, 13-dibutyrate (1 mu M for 24 h) but not by calcium chelation with BAPT A-AM (acetoxymethyl ester of BAPTA) (75 mu M for 30 min), suggesting t hat a novel PKC isoform (delta, epsilon, eta, theta) mediates shear st ress-induced ERK1/2 activation. Western blotting with PKC isoform-spec ific antibodies demonstrated expression of PKC-alpha, -epsilon, and -z eta isoforms in EC. PKC-epsilon was specifically inhibited by transfec tion with antisense PKC-epsilon phosphorothioate oligonucleotides (1,0 00 nM for 6 h). Antisense treatment decreased PKC-epsilon protein leve ls by 80 +/- 13% after 72 h and completely inhibited shear stress-stim ulated ERK1/2 activation. Scrambled PKC-epsilon oligonucleotides and a ntisense 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 inhibi t ERK1/2 activation by EGF or bradykinin but did inhibit ERK1/2 activa tion upon EC adhesion to fibronectin. These results define a pathway f or shear stress-mediated ERK1/2 activation and establish a new functio n for PKC-epsilon as part of a mechano-sensitive signal transduction p athway in EC.