Dissociation of vasoconstrictor-stimulated basic fibroblast growth factor expression from hypertrophic growth in cultured vascular smooth muscle cells. Relevant roles of protein kinase C.
Thromboxane A2 (TXA2) and angiotensin II (Ang II) stimulate vascular smooth muscle hypertrophy by upregulating endogenous synthesis of basic fibroblast growth factor (bFGF). Because mitogenic phorbol esters can also stimulate bFGF formation, we investigated the role of protein kinase C (PKC) in vascular smooth muscle cell (VSMC) bFGF formation and hypertrophy. Preliminary characterization of PKC isoform expression in VSMC by use of polymerase chain reaction identified PKC alpha, delta, epsilon, and zeta. Western analysis confirmed the presence of these isoforms in cultured VSMC lines and demonstrated downregulation of PKC alpha, delta, and epsilon by phorbol 12-myristate 13-acetate (PMA) but not TXA2 or Ang II. PKC activation with 100 nmol/L PMA stimulated VSMC mitogenesis measured as incorporation of [3H]leucine and [3H]thymidine and increased cell number. Like TXA2 and Ang II, PMA increased endogenous VSMC bFGF in a time-dependent manner, whereas an inactive phorbol ester had no such effect. Addition of an antisense oligodeoxynucleotide against bFGF prevented PMA-stimulated bFGF expression and inhibited PMA-stimulated growth, suggesting that bFGF synthesis is necessary for VSMC growth stimulated by PMA. To clarify the role of PKC in vasoconstrictor-stimulated VSMC production of bFGF and hypertrophy, PKC was down-regulated by prolonged exposure to PMA or was inhibited with calphostin C or staurosporine before the addition of TXA2 or Ang II. PKC inhibition prevented TXA2-stimulated and Ang II--stimulated VSMC hypertrophy without attenuating the observed increase in bFGF expression. Furthermore, PKC inhibition with calphostin C inhibited VSMC mitogenesis stimulated by exogenous bFGF.(ABSTRACT TRUNCATED AT 250 WORDS)
Ali
S
S
Department of Internal Medicine, University of Cincinnati, OH 45267-0542.
Becker
M W
MW
Davis
M G
MG
Dorn
G W
GW
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D005346:Fibroblast Growth Factors / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation; D015151:Immunoblotting; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D010703:Phorbol Esters / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000235:genetics / Q000502:physiology*; D051381:Rats; D013928:Thromboxane A2 / Q000494:pharmacology; D014158:Transcription, Genetic
D010703:Phorbol Esters; D000804:Angiotensin II; D013928:Thromboxane A2; D005346:Fibroblast Growth Factors; D011493:Protein Kinase C