Thrombin-induced expression of the KC gene in cultured aortic endothelial cells. Involvement of proteolytic activity and protein kinase C.
The KC gene, first identified in platelet-derived growth factor-stimulated BALB/c 3T3 cells, shares structural similarities with a new family of genes that code for secreted cytokines which appear to be involved in wound healing and inflammation. Thrombin is a coagulation system proteinase likely to be present in vivo at sites of tissue injury. This enzyme is known to stimulate multiple responses in cultured endothelial cells (EC), including the production of eicosanoids, the expression of growth factor genes and the adhesion of leukocytes. The present experiments were designed to examine the effect of thrombin on KC mRNA expression in EC and to explore the molecular mechanisms involved. Thrombin caused a marked concentration-dependent increase in the steady state level of KC mRNA in confluent porcine aortic EC. The level of KC mRNA reached a peak 2 h after thrombin treatment and returned to near control levels by 8 h. Thrombin that was pretreated with phenylmethylsulfonyl fluoride (PMSF) to block proteolytic activity did not stimulate KC gene expression. Trypsin (2 micrograms/ml) but not PSMF-trypsin also caused a substantial increase in the level of KC mRNA. We postulated a role for protein kinase C in thrombin-induced KC gene expression since previous work had demonstrated a similar EC response to phorbol esters. This hypothesis was further supported by the finding that thrombin-induced KC expression was suppressed by the C kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, but not by its structural analogue. The results of the present study demonstrate that thrombin augments KC mRNA expression by vascular EC in a process that requires intact proteinase activity. The activation of protein kinase C may be a necessary component of the intracellular signalling pathway involved in this response.
Department of Vascular Cell Biology and Atherosclerosis Research, Cleveland Clinic Foundation, OH 44195.
X Y
XY
Shen
T A
TA
Hamilton
P E
PE
DiCorleto
D000818:Animals; D001685:Biological Factors / Q000235:genetics*; D015152:Blotting, Northern; D016207:Cytokines; D004730:Endothelium, Vascular / Q000502:physiology*; D015870:Gene Expression / Q000187:drug effects; D006133:Growth Substances / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D011112:Polymyxin B / Q000494:pharmacology; D011493:Protein Kinase C / Q000502:physiology*; D013552:Swine; D013917:Thrombin / Q000494:pharmacology*; D013997:Time Factors; D014945:Wound Healing
D001685:Biological Factors; D016207:Cytokines; D006133:Growth Substances; D008070:Lipopolysaccharides; D011493:Protein Kinase C; D013917:Thrombin; D011112:Polymyxin B