Sequential Activation of Protein Kinase C (PKC)-α and PKC-ε Contributes to Sustained Raf/ERK1/2 Activation in Endothelial Cells under Mechanical Strain

Abstract

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-α and -ε. The translocation of PKC-α and -ε but not PKC-β from the cytosolic to membrane fraction was observed. An early transient activation of PKC-α versus a late but sustained activation of PKC-ε was shown after the onset of cyclic strain. Consistently, a sequential association of PKC-α and -ε 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-α, the antisense to PKC-ε, or the inhibition peptide to PKC-ε reduced strain-induced ERK1/2 phosphorylation in a temporal manner. PKC-α mediated mainly the early ERK1/2 activation, whereas PKC-ε 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 (α and ε) contribute to Raf/ERK1/2 activation, and PKC-ε appears to play a key role in endothelial adaptation to hemodynamic environment. 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-α and -ε. The translocation of PKC-α and -ε but not PKC-β from the cytosolic to membrane fraction was observed. An early transient activation of PKC-α versus a late but sustained activation of PKC-ε was shown after the onset of cyclic strain. Consistently, a sequential association of PKC-α and -ε 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-α, the antisense to PKC-ε, or the inhibition peptide to PKC-ε reduced strain-induced ERK1/2 phosphorylation in a temporal manner. PKC-α mediated mainly the early ERK1/2 activation, whereas PKC-ε 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 (α and ε) contribute to Raf/ERK1/2 activation, and PKC-ε appears to play a key role in endothelial adaptation to hemodynamic environment. endothelial cell protein kinase C extracellular signal-regulated protein kinase phosphorylated ERK monocyte chemotactic protein-1 diacylglycerol phosphatidylinositide phosphatidylcholine mitogen-activated protein kinase endothelin-1 early growth response-1 intercellular adhesion molecule polyacrylamide gel electrophoresis mitogen-activated protein kinase/extracellular signal-regulated kinase kinase Vascular endothelial cells (ECs)1 are constantly under the influence of hemodynamic forces including flow-induced shear stress and pressure-generated cyclic strain. These hemodynamic forces play an essential role in maintaining vascular integrity by inducing the release of vasoactive substances and modulating gene expression (1Chien S. Li S. Shyy Y.J. Hypertension. 1998; 31: 162-169Crossref PubMed Google Scholar, 2Sadoshima J. Izumo S. EBMO J. 1993; 12: 1681-1692Crossref PubMed Scopus (567) Google Scholar). Studies have examined how intracellular signals are involved in transmitting mechanical forces into second messengers and subsequently gene expression (3Takahashi M. Berk B.C. J. Clin. Invest. 1996; 98: 2623-2631Crossref PubMed Scopus (189) Google Scholar, 4Zou Y. Hu Y. Metzler B. Xu Q. Int. J. Mol. Med. 1998; 1: 827-834PubMed Google Scholar). Shear flow stimulates the signals involved in the ERK1/2 and JNK pathways. These signals may result in the induction of various genes' expression including platelet-derived growth factor (5Bao X. Lu C. Frangos J.A. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 996-1003Crossref PubMed Scopus (209) Google Scholar), Egr-1 (6Schwatchtgen J.L. Houston P. Campbell C. Sukhatme V. Braddock M. J. Clin. Invest. 1998; 101: 2540-2549Crossref PubMed Scopus (175) Google Scholar), c-fos (7Jalali S. Li Y.S. Sotoudeh M. Yuan S. Li S. Chien S. Shyy Y.J. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 227-234Crossref PubMed Scopus (218) Google Scholar), monocyte chemotactic protein-1 (MCP-1) (8Ito W.D. Arras M. Winkler B. Scholz D. Schaper J. Schaper W. Circ. Res. 1997; 80: 829-837Crossref PubMed Scopus (413) Google Scholar), and intercellular adhesion molecule-1 (ICAM-1) (9Chiu J.J. Wung B.S. Shyy Y.J. Hsieh S.J. Wang D.L. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3570-3577Crossref PubMed Scopus (166) Google Scholar). Because rhythmic distension of the vessel wall is a component of pulsatile flow, cyclic strain on vessel walls plays an important role in modulating gene expression. Earlier studies from our laboratory showed that ECs under cyclic strain increase their expression of MCP-1 (10Wang D.L. Wung B.S. Shyy Y.J. Lin C.F. Chao Y.J. Usami S. Chien S. Circ. Res. 1995; 77: 294-302Crossref PubMed Google Scholar, 11Wung B.S. Cheng J.J. Chao Y.J. Lin J. Shyy Y.J. Wang D.L. Am. J. Physiol. 1996; 270: H1462-H1468PubMed Google Scholar, 12Wung B.S. Cheng J.J. Hsieh H.J. Shyy Y.J. Wang D.L. Circ. Res. 1997; 81: 1-7Crossref PubMed Scopus (224) Google Scholar), ICAM-1 (13Cheng J.J. Wung B.S. Chao Y.J. Wang D.L. Hypertension. 1998; 31: 125-130Crossref PubMed Scopus (90) Google Scholar, 14Cheng J.J. Wung B.S. Chao Y.J. Wang D.L. Hypertension. 1996; 28: 386-391Crossref PubMed Scopus (100) Google Scholar), and early growth response-1 (Egr-1) (15Wung B.S. Cheng J.J. Wang D.L. Circ. Res. 1999; 84: 804-812Crossref PubMed Scopus (112) Google Scholar). Signaling pathways involving ERK1/2 and c-Jun N-terminal kinase participate in mechanical force-induced gene expression (3Takahashi M. Berk B.C. J. Clin. Invest. 1996; 98: 2623-2631Crossref PubMed Scopus (189) Google Scholar, 12Wung B.S. Cheng J.J. Hsieh H.J. Shyy Y.J. Wang D.L. Circ. Res. 1997; 81: 1-7Crossref PubMed Scopus (224) Google Scholar, 16Liang F. Lu S. Gardner D.G. Hypertension. 2000; 35: 188-192Crossref PubMed Google Scholar). However, the initial events and the following networks of signaling pathways are still poorly understood. Cyclic strain to ECs activates intracellular second messengers. Activation of protein kinase C (PKC) is associated with an increase of phosphatidyl inositol turnover and intracellular calcium (17Evans L. Frenkel L. Brophy C.M. Rosales O. Sai Sudhaker C.B. Li G. Du W. Sumpio B.E. J. Physiol. 1997; 272: C650-C656Google Scholar). PKC is activated by diacylglycerol (DAG), which is derived either from phosphatidylinositide (PI) or phosphatidylcholine (PC). PKC isoforms in human ECs have been identified that cover PKC-α, PKC-δ, PKC-ε, and PKC-ξ (18Haller Z. Ziegler W. Lindschau C. Luft F.C. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 678-686Crossref PubMed Scopus (51) Google Scholar). PKC-α belongs to a Ca2+-dependent group, and the isoforms PKC-ε and PKC-ξ belong to a Ca2+-independent group. Studies have indicated that PKC is involved in shear stress- and cyclic strain-induced gene expression of platelet-derived growth factor and Et-1 in ECs (19Morita T. Kurihara H. Maemura K. Yoshizumi M. Nagai R. Yazaki Y. Circ. Res. 1994; 75: 630-636Crossref PubMed Google Scholar, 20Morawietz H. Talanow R. Szibor M. Rueckschloss U. Schubert A. Bartling B. Darmer D. Holtz J. J. Physiol. 2000; 525: 761-770Crossref PubMed Scopus (141) Google Scholar). Indeed, PKC-ε is required for fluid shear stress-mediated activation of ERK1/2 in ECs (21Traub O. Monia B.P. Dean N.M. Berk B.C. J. Biol. Chem. 1997; 272: 31251-31257Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In smooth muscle cells, stretching promotes DNA synthesis via activation of PKC (22Mills I. Cohen C.R. Kamal K. Li G. Shin T. Du W. Sumpio B.E. J. Cell. Physiol. 1997; 170: 228-234Crossref PubMed Scopus (99) Google Scholar). Our previous studies demonstrated that cyclic strain to ECs increases gene expression of MCP-1, which is regulated by PKC (10Wang D.L. Wung B.S. Shyy Y.J. Lin C.F. Chao Y.J. Usami S. Chien S. Circ. Res. 1995; 77: 294-302Crossref PubMed Google Scholar). Further studies indicated that cyclic strain induces the Ras/Raf-1/ERK1/2 signaling pathway and results in an increase of gene expression of MCP-1 and Egr-1 (11Wung B.S. Cheng J.J. Chao Y.J. Lin J. Shyy Y.J. Wang D.L. Am. J. Physiol. 1996; 270: H1462-H1468PubMed Google Scholar, 15Wung B.S. Cheng J.J. Wang D.L. Circ. Res. 1999; 84: 804-812Crossref PubMed Scopus (112) Google Scholar). The upstream signaling pathway and/or signaling network that lead to activation of Ras/Raf-1/ERK1/2 by cyclic strain remain unclear. Among the likely signaling networks, different PKC isoforms have been shown to modulate the ERK1/2 signaling pathway under different stimuli (23Liao D.F. Monia B. Dean N. Berk B.C. J. Biol. Chem. 1997; 272: 6146-6150Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 24Soh, J. W., Lee, E. H., and I. B. Scholar). However, of of the PKC isoforms involved in the signaling pathway endothelial to cyclic strain not been In the that ECs subjected to cyclic strain increase PKC activities and that PKC-α and PKC-ε are activated for Raf/ERK1/2 activation. PKC-α and PKC-ε contribute to the early and late phase of ERK1/2 activation, in cyclic strain-treated The of activated by cyclic strain to adaptation including gene induction in Our results of PKC in signaling in ECs under a hemodynamic environment. The strain of a to a by a J.A. J. 1994; PubMed Scopus Google Scholar). ECs on a membrane were by a that an strain of a of ECs were in and cell was and PKC activity was on an that a peptide and a that the phosphorylated of the peptide ECs were into and and the and were as cytosolic and membrane to the phosphorylation in cell was were in and subjected to The PKC isoforms were with PKC phosphorylation in to or was were and an of phosphorylated ERK1/2 was the activation specific to the phosphorylated activation of was with was as second and results were a An Elk1 was from that and An MCP-1 the gene Y.J. Lin Lu Y. M. Chien S. U. S. A. 1995; PubMed Scopus Google was was the and the was to the to PKC-α or PKC-ε were by The of and antisense to PKC-α were and The of and antisense to PKC-ε were and The of the PKC-ε peptide was These and the as (21Traub O. Monia B.P. Dean N.M. Berk B.C. J. Biol. Chem. 1997; 272: 31251-31257Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, P. H. H. J. Clin. Invest. 1996; 98: PubMed Scopus Google Scholar, A. C. U. Lindschau C. R. Luft F.C. H. Circ. Res. 1997; 81: PubMed Scopus Google Scholar, M. J.A. D. E. E. R. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), were transfected into ECs the ECs were with and a inhibitor were by a the of protein from each was with The was with protein for The was and in the ECs were and with The activity in the was in and of The was with The were and the gel was by were are as was as Our previous studies demonstrated that PKC is involved in cyclic strain-induced gene expression of Et-1 and MCP-1 in ECs (10Wang D.L. Wung B.S. Shyy Y.J. Lin C.F. Chao Y.J. Usami S. Chien S. Circ. Res. 1995; 77: 294-302Crossref PubMed Google Scholar, D.L. Wung B.S. Wang J.J. J. Cell. Physiol. 1995; PubMed Scopus Google Scholar). the role of PKC in cyclic strain-induced endothelial PKC activity and PKC isoforms in ECs were ECs under cyclic strain increased their PKC These increased PKC activities as the cyclic strain to PKC activation phosphorylation of that were activated after cyclic phosphorylation of PKC-α and PKC-ε as an of PKC activation was shown in ECs under cyclic strain induced phosphorylation of on PKC-α and that an activated for to In the phosphorylation of on PKC-ε showed activation after of strain and that to following of ECs after strain the cytosolic and membrane were PKC isoforms from each fraction were PKC isoforms PKC-α, and were identified with after cyclic strain for induced their PKC-α activity as shown by the PKC-α from the cytosolic to the membrane fraction These PKC-α to the cytosolic The of PKC-α to the cytosolic fraction after cyclic strain. the PKC-ε was not the early phase but activation and in an activated after cyclic strain In to the of PKC-α and PKC-ε from the cytosolic to the membrane the PKC-β isoform was not activated and in the cytosolic fraction the cyclic strain These are with the sequential of phosphorylation on of shown in B. Our results a sequential of PKC-α and PKC-ε to the membrane fraction in ECs after the onset of cyclic strain. The phosphorylation of Raf-1 by PKC isoforms been as an activation of PKC on the signaling pathway W. G. G. R. H. H. G. D. 1993; PubMed Scopus Google Scholar, G. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus (98) Google Scholar). demonstrated that cyclic strain activates the Ras/Raf-1/ERK1/2 pathway J.J. Wung B.S. Chao Y.J. Wang D.L. Hypertension. 1996; 28: 386-391Crossref PubMed Scopus (100) Google Scholar). that PKC is involved in Raf-1 activation, ECs were with a PKC calphostin with cyclic strain shown in the cyclic inducing an increased phosphorylation of Raf-1 in activation was by treating ECs with calphostin C. the role of PKC isoforms in the signaling the association of each PKC isoform with Raf-1 in ECs was by Raf-1 with from of ECs and by with to PKC-α or an of Raf-1 was shown in the PKC isoform association with Raf-1 in a temporal In ECs under PKC-ε was associated with but PKC-α was However, ECs subjected to cyclic strain for in a increased association of PKC-α with association of PKC-α with Raf-1 after of cyclic strain. In PKC-ε was associated with Raf-1 These findings of Raf-1 phosphorylation via PKC and sequential association of PKC isoforms with Raf-1 are with the of temporal of PKC-α and PKC-ε in ECs after cyclic strain demonstrated that cyclic strain to ECs induces Egr-1 gene which is mediated via the Ras/Raf-1/ERK1/2 signaling pathway (15Wung B.S. Cheng J.J. Wang D.L. Circ. Res. 1999; 84: 804-812Crossref PubMed Scopus (112) Google Scholar). that the ERK1/2 signaling pathway is ECs after cyclic strain for various were and phosphorylated ERK1/2 was with ERK1/2 Cyclic strain to to ECs after induced ERK1/2 activity ERK1/2 activity in a phosphorylated as cyclic strain and late activation of to ECs and of the cyclic were inhibited after treating ECs with calphostin C Consistently, cyclic strain-induced ERK1/2 kinase as phosphorylation of was inhibited in ECs with calphostin C In to the activation of PKC-ε, PKC-α activation is which PKC isoforms contribute to the early phase versus the late but sustained phase of ERK1/2 ECs were with a specific Ca2+-dependent PKC and subjected to cyclic strain. shown in the early phase of cyclic strain-induced ERK1/2 activity was inhibited after of In strain-induced ERK1/2 activity in the late but sustained phase was not by inhibitor These results that a sequential activation of PKC-α and PKC-ε is involved in cyclic strain-induced ERK1/2 activation in the role of each PKC isoform in strain-induced ERK1/2 ECs were with antisense to PKC-α or ECs transfected with an antisense to a PKC isoform reduced the protein expression of that PKC isoform in ECs Consistently, antisense to PKC-α and PKC-ε inhibited PKC activity in ECs after cyclic strain for and ECs were subjected to cyclic strain for ECs transfected with antisense to PKC-α showed an inhibition of ERK1/2 phosphorylation In ECs transfected with the not ERK1/2 activity. that PKC-α is required for early ERK1/2 activity. However, PKC-α not play a role the late phase of ERK1/2 activation, antisense ECs not ERK1/2 phosphorylation after cyclic strain In ECs transfected with an antisense to PKC-ε abolished the strain-induced ERK1/2 activity phase. PKC-ε involved in late phase of ERK1/2 was by the inhibition of ERK1/2 activity in ECs transfected with the peptide to PKC-ε of that PKC-α is required for the early PKC-ε mainly to the late and sustained phase of cyclic strain-induced ERK1/2 activation in and PKC -ε cyclic strain-induced ERK1/2 ECs were transfected with either or to PKC-α or PKC-ε for after ECs were subjected to cyclic strain for ECs were transfected with or antisense to PKC-α or PKC-ε or inhibition peptide to PKC-ε for after ECs were subjected to strain for cell was for an to of protein to each are shown by the ERK for each are of the Ras/Raf-1/ERK1/2 signaling pathway is to the activation of transcriptional including protein-1 and PKC isoforms contribute to strain-induced ERK1/2 activity and ERK1/2 activation increases the transcriptional activity of Elk1 by PKC isoforms the transcriptional activity of Elk1 which the protein of the to the activation of was with a of of the and the into These ECs were subjected to cyclic strain. ECs were transfected with the antisense to PKC-α or PKC-ε, strain-induced Elk1 transcriptional activities were reduced to to that of ECs In ECs transfected with not the Elk1 induction by cyclic strain. ECs transfected with an antisense to PKC-α or PKC-ε MCP-1 promotor activity These results demonstrated that cyclic strain to ECs increases ERK1/2 which is subsequently by an increase of transcriptional activity of These results the of PKC isoforms and in modulating the signaling pathway in ECs under cyclic strain. our results that PKC isoforms are involved in endothelial to cyclic strain. The PKC-ε appears to play an important role for sustained Raf/ERK1/2 activation in ECs constantly under hemodynamic The activation of PKC isoform and the signaling pathway by gene are for adaptation to a hemodynamic environment. Earlier studies indicated that PKC activities are increased in ECs under shear or cyclic strain T. O. M. P. I. Sumpio B.E. J. Cell. 1997; PubMed Scopus Google Scholar). Our previous studies showed that PKC is involved in cyclic strain-induced Et-1 and MCP-1 gene expression (10Wang D.L. Wung B.S. Shyy Y.J. Lin C.F. Chao Y.J. Usami S. Chien S. Circ. Res. 1995; 77: 294-302Crossref PubMed Google Scholar, D.L. Wung B.S. Wang J.J. J. Cell. Physiol. 1995; PubMed Scopus Google Scholar). Cyclic strain to ECs results in a increase in (17Evans L. Frenkel L. Brophy C.M. Rosales O. Sai Sudhaker C.B. Li G. Du W. Sumpio B.E. J. Physiol. 1997; 272: C650-C656Google that to early transient PKC activity by sustained PKC activity T. O. M. P. I. Sumpio B.E. J. Cell. 1997; PubMed Scopus Google Scholar). PKC in mechanical force-induced endothelial been T. O. M. P. I. Sumpio B.E. J. Cell. 1997; PubMed Scopus Google Scholar), studies have indicated that specific isoforms PKC-ε and PKC-β are involved in endothelial (21Traub O. Monia B.P. Dean N.M. Berk B.C. J. Biol. Chem. 1997; 272: 31251-31257Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, Chien S. J. 1997; PubMed Scopus Google Scholar). The as to and how PKC isoforms are involved in to mechanical forces remain unclear. The that PKC-α and PKC-ε are sequential activated and are involved in cyclic strain-induced Raf/ERK1/2 activation. of the of PKC-α and PKC-ε from the cytosolic to the membrane fraction was a sequential PKC-α was involved in an early activation, whereas PKC-ε was activated a late but sustained ECs treated with a PKC inhibitor abolished the Raf-1 phosphorylation by cyclic strain. a sequential association of PKC-α and PKC-ε with the signaling molecule Raf-1 was shown. a PKC inhibitor inhibited strain-induced ERK1/2 activation, a role of PKC in the Raf/ERK1/2 signaling ECs treated with a Ca2+-dependent PKC inhibitor (Go 6976) showed an inhibition in the early phase but not the late sustained phase of ERK1/2 activation. ECs transfected with the antisense to PKC-α inhibited early and not late ERK1/2 activation, whereas the antisense or peptide to PKC-ε sustained ERK1/2 activation. Consistently, an antisense to PKC-α not late ERK1/2 activation. of that PKC-α and PKC-ε are activated and are required for Raf/ERK1/2 activity. ECs treated with an antisense to each PKC isoform reduced the transcriptional activity of a of a result of ERK1/2 antisense to each PKC isoform inhibited MCP-1 transcriptional activity. of results that the sequential activation of PKC-α and PKC-ε is essential for Raf/ERK1/2 activation in cyclic strain-treated The activation of ERK1/2 and activation may result in gene PKC-α belongs to the of protein kinases that are whereas PKC-ε is a protein is that ECs are subjected to hemodynamic plays an essential role in endothelial H. Talanow R. Szibor M. Rueckschloss U. Schubert A. Bartling B. Darmer D. Holtz J. J. Physiol. 2000; 525: 761-770Crossref PubMed Scopus (141) Google Scholar, B.C. H. 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Monia B.P. Dean N.M. Berk B.C. J. Biol. Chem. 1997; 272: 31251-31257Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), PKC-ε been to involved in ERK1/2 activity (21Traub O. Monia B.P. Dean N.M. Berk B.C. J. Biol. Chem. 1997; 272: 31251-31257Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In the cyclic strain PKC-α in early ERK1/2 activity. signaling events by which each PKC isoform activates ERK1/2 remain to PKC in Raf-1 activation and sequential association of PKC isoforms with Raf-1 that PKC-α and PKC-ε contribute to activation. In the that the antisense to each PKC isoform ERK1/2 activity that PKC isoforms contribute to Raf-1 activation. PKC-α been to and Raf-1 W. G. G. R. H. H. G. D. 1993; PubMed Scopus Google Scholar, G. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus (98) Google Scholar, R. Y. C. H. 1998; PubMed Scopus Google Scholar). PKC-α by treating cells with However, not Raf-1 activation by growth factor H. R. A. A. J. Mol. Cell. Biol. PubMed Scopus Google Scholar). PKC-ε activated by J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). Activation of Raf-1 via and PKC-ε phosphorylation was indicated to play a role in maintaining sustained activity of pathway H. U. V. I. J. U. Mol. Cell. Biol. 1997; 17: PubMed Scopus Google Scholar). PKC-ε was shown to a sustained phosphorylation of in growth cells C. K. P. G. 1999; Google Scholar). that PKC-α and PKC-ε the signaling pathway which on the J. W., Lee, E. H., and I. B. Scholar). Our previous demonstrated that the in the of Egr-1 is involved in cyclic strain-induced Egr-1 expression (15Wung B.S. Cheng J.J. Wang D.L. Circ. Res. 1999; 84: 804-812Crossref PubMed Scopus (112) Google Scholar). on previous and our is that PKC-α and PKC-ε were involved in Raf-1 activation and to the ERK1/2 activation. specific PKC isoforms are important in networks of signaling pathways that modulate gene expression in ECs under hemodynamic The of the is that sequential activation of PKC-α and PKC-ε is of a signaling pathway that to activation of ERK1/2 and gene In to sequential activation of PKC-α and PKC-ε and their temporal association with our antisense studies showed that is sequential activation of PKC isoforms in cyclic strain-treated The of to PKC isoforms an the of PKC in of M. J. A. H. W. 1997; Scopus Google Scholar, N.M. R. U. S. A. 1994; PubMed Scopus Google Scholar). Our findings the activation of PKC-α and a late activation of PKC-ε in ECs under cyclic strain. These results are with an that PKC-ε is required for ERK1/2 activity (21Traub O. Monia B.P. Dean N.M. Berk B.C. J. Biol. Chem. 1997; 272: 31251-31257Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). However, ERK1/2 activity is transient and to by after shear (21Traub O. Monia B.P. Dean N.M. Berk B.C. J. Biol. Chem. 1997; 272: 31251-31257Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The for and to the of the forces studied. flow a shear stress to ECs and not whereas our strain of to which ECs may but sustained PKC activation in cyclic strain-treated ECs was T. O. M. P. I. Sumpio B.E. J. Cell. 1997; PubMed Scopus Google Scholar). The that specific PKC isoforms were the that PKC isoforms may contribute to ERK1/2 activation, our results that PKC-α and PKC-ε are activated and contribute to ERK1/2 activation in ECs under cyclic strain. studies that PKC activation by intracellular of cells PKC activation, and phosphorylation of PKC isoforms have been demonstrated H. Y. Y. U. Y. U. S. A. 1997; PubMed Scopus Google Scholar). of the activation of PKC isoforms that intracellular PKC activation J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). demonstrated that including as second messengers that contribute to hemodynamic force-induced Ras/Raf-1/ERK1/2 activation and gene expression B.S. Cheng J.J. Hsieh H.J. Shyy Y.J. Wang D.L. Circ. Res. 1997; 81: 1-7Crossref PubMed Scopus (224) Google Scholar, J.J. Wung B.S. Chao Y.J. Wang D.L. Hypertension. 1998; 31: 125-130Crossref PubMed Scopus (90) Google Scholar, 15Wung B.S. Cheng J.J. Wang D.L. Circ. Res. 1999; 84: 804-812Crossref PubMed Scopus (112) Google Scholar). may modulate PKC activation in our cyclic strain-treated our strain of on the the of PKC as the of cyclic strain not as a second in growth cells M. Winkler T. S. G. R. 1999; PubMed Google and is to play a role vascular B.C. Thromb. 1999; PubMed Scopus Google Scholar). PKC activation may a in ECs subjected to growth factor and hemodynamic Our of PKC-ε in strain-induced ERK1/2 is with a previous of PKC-ε in ERK1/2 activation (21Traub O. Monia B.P. Dean N.M. Berk B.C. J. Biol. Chem. 1997; 272: 31251-31257Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The that PKC are essential signaling for of cyclic strain. PKC-α and PKC-ε as Raf-1 that lead to a on the signaling pathway and gene studies that of fluid shear flow in ECs O. Berk B.C. Arterioscler. Thromb. Vasc. Biol. 1998; 18: PubMed Scopus Google Scholar). The activation of PKC by cyclic strain not as a signaling but is important for growth and the signaling mediated via PKC in ECs hemodynamic is key for of endothelial and vascular