Protein kinase C and the stimulation of tissue plasminogen activator release from human endothelial cells. Dependence on the elevation of messenger RNA.
Tumor-promoting phorbol esters stimulate tissue plasminogen activator (tPA) release from human endothelial cells, and simultaneous elevation of cyclic AMP potentiates this response 5-fold (Santell, L., and Levin, E. G. (1988) J. Biol. Chem. 263, 16802-16808). A similar effect on tPA mRNA was observed, with phorbol myristate acetate inducing a 3.5-fold increase in steady state tPA mRNA levels and forskolin enhancing that increase to 25-fold. Peak levels occurred at 8 h after agonist addition and returned to baseline levels by 16 h. As was found with tPA antigen secretion, delayed addition of forskolin reduced the level of potentiation, and, at 6 h after phorbol 12-myristate 13-acetate (PMA), forskolin was no longer effective. The protein synthesis inhibitor cycloheximide did not inhibit the rise in tPA mRNA levels in response to PMA/forskolin nor the decline in mRNA levels between 8 and 12 h. However, peak levels (8 h) were approximately 1.5-fold higher than in cultures not treated with cycloheximide. The effect of two inhibitors of protein kinases, H-7 and staurosporine, on PMA-induced tPA antigen secretion and tPA mRNA levels were examined. H-7 and staurosporine inhibited PMA, and PMA/forskolin induced tPA secretion in a dose-dependent manner. This effect was time-dependent; the inhibitory effect was reduced with delayed H-7 addition, and, by 6 h after PMA treatment, no inhibition was observed. H-7 and staurosporine also inhibited the PMA/forskolin-induced increase in tPA mRNA levels and were less effective the later they were added. The same time-dependent effect on the potentiation of PMA-induced tPA mRNA levels by forskolin was observed. Again, delayed addition reduced the effect, and, by 6 h, potentiation was absent. The results of this study indicate that changes in mRNA levels in response to PMA and PMA/forskolin precede and determine those that occur to tPA antigen secretion. In addition, the maximal response is dependent upon the prolonged activation of an H-7- and cAMP-sensitive pathway.
Levin
E G
EG
Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, California 92037.
Marotti
K R
KR
Santell
L
L
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D015152:Blotting, Northern; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics*; D019311:Staurosporine; D013449:Sulfonamides; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D000470:Alkaloids; D002121:Calcium Channel Blockers; D007546:Isoquinolines; D010879:Piperazines; D012333:RNA, Messenger; D013449:Sulfonamides; D005576:Colforsin; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D004247:DNA; C043204:N-(2-guanidinoethyl)-5-isoquinolinesulfonamide; D003513:Cycloheximide; D011493:Protein Kinase C; D010959:Tissue Plasminogen Activator; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate