Sequential activation of protein kinase C (PKC)-alpha and PKC-epsilon 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-alpha and -epsilon but not PKC-beta from the cytosolic to membrane fraction was observed. An early transient activation of PKC-alpha versus a late but sustained activation of PKC-epsilon 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 Ca(2+)-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-alpha, the antisense to PKC-epsilon, or the inhibition peptide to PKC-epsilon reduced strain-induced ERK1/2 phosphorylation in a temporal manner. PKC-alpha mediated mainly the early ERK1/2 activation, whereas PKC-epsilon 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 isoform (alpha and epsilon) contribute to Raf/ERK1/2 activation, and PKC-epsilon appears to play a key role in endothelial adaptation to hemodynamic environment.
Cheng
J J
JJ
Cardiovascular Division, Institute of Biomedical Sciences, Academia Sinica, 11529 Taipei, Taiwan, Republic of China.
Wung
B S
BS
Chao
Y J
YJ
Wang
D L
DL
D000818:Animals; D001324:Autoantigens / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D004268:DNA-Binding Proteins; D004730:Endothelium, Vascular / Q000201:enzymology*; D004789:Enzyme Activation; D007527:Isoenzymes / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D015221:Potassium Channels / Q000235:genetics; D011493:Protein Kinase C / Q000502:physiology*; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D011518:Proto-Oncogene Proteins; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D013314:Stress, Mechanical; D014157:Transcription Factors; D014158:Transcription, Genetic; D050807:ets-Domain Protein Elk-1
D001324:Autoantigens; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D016376:Oligonucleotides, Antisense; D015221:Potassium Channels; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D002118:Calcium