Sequential activation of protein kinase C (PKC)-α and PKC-ε contributes to sustained Raf/ERK1/2 activation in endothelial cells under mechanical strain

Endothelial cells (ECs) are constantly subjected to hemodynamic forces including cyclic pressure-induced strain. The role of protein kinase C (PKC) in cyclic strain-treated ECs was studied. PKC activities were induced as cyclic strain was initiated. Cyclic strain to ECs caused activation of PKC-alpha and -epsilon. The translocation of PKC-a and -e but not PKC-beta from the cytosolic to membrane fraction was observed. An early transient activation of PKC-a versus a late but sustained activation of PKC-E was shown after the onset of cyclic strain. Consistently, a sequential association of PKC-alpha and -epsilon with the signaling molecule Raf-1 was shown. ECs treated with a PKC inhibitor (calphostin C) abolished the cyclic strain-induced Raf-1 activation. ECs under cyclic strain induced a sustained activation of extracellular signal-regulated protein kinases (ERK1/2), which was inhibited by treating ECs with calphostin C. ECs treated with a specific Ca2+-dependent PKC inhibitor (Go 6976) showed an inhibition in the early phase of ERK1/2 activation but not in the late and sustained phase. ECs transfected with the antisense to PKC-a, the antisense to PKC-epsilon, or the inhibition peptide to PKC-c reduced strain-induced ERK1/2 phosphorylation in a temporal manner. PKC-a mediated mainly the early ERK1/2 activation, whereas PKC-e was involved in the sustained ERK1/2 activation. Strained ECs increased transcriptional activity of Elk1 (an ERK1/2 substrate). ECs transfected with the antisense to each PKC isoform reduced Elk1 and monocyte chemotactic protein-1 promotor activity. Our findings conclude that a sequential activation of PKC isoforin (alpha and epsilon contribute to Raf/ERK1/2 activation, and PKC-epsilon appears to play a key role in endothelial adaptation to hemodynamic environment.