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-␣ and -⑀. The translocation of PKC-␣ and -⑀ but not PKC-␤ from the cytosolic to membrane fraction was observed. An early transient activation of PKC-␣ versus a late but sustained activation of PKC-⑀ was shown after the onset of cyclic strain. Consistently, a sequential association of PKC-␣ and -⑀ 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 signalregulated 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-␣, the antisense to PKC-⑀, or the inhibition peptide to PKC-⑀ reduced strain-induced ERK1/2 phosphorylation in a temporal manner. PKC-␣ mediated mainly the early ERK1/2 activation, whereas PKC-⑀ 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 (␣ and ⑀) contribute to Raf/ERK1/2 activation, and PKC-⑀ appears to play a key role in endothelial adaptation to hemodynamic environment.

Increased megakaryocytopoiesis in Lyn-deficient mice

“…Furthermore, a recent report demonstrates that Erk phosphorylation is normal in raf-1 À/À (knockout) mice (Kamata et al, 2004). Lastly, in some systems, Erk1/2 has been shown to be activated directly by protein kinase C (PKC) (Ezumi et al, 1998;Cheng et al, 2001). It is not completely understood how TPO stimulation leads to the activation of PI3K, but recent studies have begun to investigate novel signaling mechanisms in which TPO-induced MAPK activation is mediated by PI3K Rojnuckarin et al, 2001).…”

Calcium/Calmodulin-dependent Protein Kinase II Binds to Raf-1 and Modulates Integrin-stimulated ERK Activation

“…Raf-1 is a substrate of several kinases (Src, protein kinase C, protein kinase A, and Akt) that, together with Ras, provide co-stimulatory signals that lead to Erk activation in response to several receptor signals (35). Activation of PKC contributes to Raf/Erk-1/2 activation in response to cyclic pressure-induced strain in endothelial cells (36). Ras-mediated activation of Erk-1/2, either by insulin, insulin like-growth factor I, interleukin-8 and -2, platelet-derived growth factor, vasopressin, T cell receptor, and adrenergic receptors is increased by Raf-1 co-activation by PI3-K (37)(38)(39)(40)(41)(42)(43).…”

Cdc42- and Rac1-mediated endothelial lumen formation requires Pak2, Pak4 and Par3, and PKC-dependent signaling

“…To identify more precisely the relevant PKC isoforms in this pathway, we utilized an RNAi-based approach in which we suppressed the conventional isoform PKCα, novel isoforms PKCδ and PKCε and atypical isoform PKCζ. PKCα, PKCδ and PKCε have been shown to be involved in angiogenesis, EC permeability, tumor metastasis and inflammation, whereas PKCζ is a key member of the Cdc42-mediated polarity complex (Cheng et al, 2001;Griner and Kazanietz, 2007;Macara, 2004b;Orr et al, 2006;Saha et al, 2003;Taylor et al, 2006). RNAi-mediated suppression of PKCα did not have any effect on EC lumen and tube formation, as assessed by the number and area of EC lumens compared with the luciferase control (Fig.…”