Endothelial proliferation, migration, and differentiation are blunted by conditionally expressed protein kinase C pseudosubstrate peptides.
Peptides based on the pseudosubstrate (PS) sequence of conventional protein kinase C isoenzymes (alpha, beta, gamma) specifically inhibit PKC activity in permeabilized cells, but whether PS can be used to study the role of PKC in the proliferation or migration of intact endothelial cells (EC) and angiogenesis is unknown. Peptides based on the PKCeta pseudosubstrate (etaPS) sequence were 3.5- to 8-fold more potent in inhibiting the PKCalpha, delta, epsilon, or eta kinase activity than was the peptide based on the PKCalpha pseudosubstrate (alphaPS) sequence. Thus, etaPS was conditionally overexpressed in intact EC and compared to alphaPS. Serum-induced growth of EC expressing etaPS was significantly slower than that of control EC. etaPS EC demonstrated slower rate of serum stimulated migration than that of either control or alphaPS EC. Expression of either etaPS or alphaPS produced slower rates of PMA induced EC migration, as compared to control EC. In an in vitro three-dimensional assay in which EC organize into capillary tubules, the EC that expressed etaPS formed fewer such tubules. This study shows that pseudosubstrate inhibitors derived from PKCeta are more potent both in vitro and in vivo than one based on the conventional isoenzyme PKCalpha. These data further support a role for PKC in proliferation and migration of intact EC, and angiogenesis.
Harrington
E O
EO
Vascular Biology Unit, Harvard Medical School, Boston, Massachusetts, 02215, USA. harrington@brown.edu
Doyle
K E
KE
Brunelle
J L
JL
Ware
J A
JA
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D017209:Apoptosis; D002196:Capillaries / Q000201:enzymology; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D002465:Cell Movement; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D004338:Drug Combinations; D004730:Endothelium, Vascular / Q000166:cytology*; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D007797:Laminin / Q000378:metabolism; D020935:MAP Kinase Signaling System; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D051571:Protein Kinase C-alpha; D011509:Proteoglycans / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D013997:Time Factors
D000893:Anti-Inflammatory Agents; D004338:Drug Combinations; D007527:Isoenzymes; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D012313:RNA; D003907:Dexamethasone; D003094:Collagen; C089212:protein kinase D; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha