ENHANCED SYNTHESIS OF PROTEOGLYCANS BY VASCULAR ENDOTHELIAL-CELLS TREATED WITH PHORBOL ESTER

We investigated the biosynthesis of proteoglycans (PG) in endothelial cells following their treatment with phorbol 12-myristate 13-acetate ( PMA). Confluent cultures of bovine aortic endothelial cells were incub ated in the presence and absence of PMA (100 ng/ml) and then pulsed wi th [S-35]sulfate, [H-3]glucosamine, or [S-35]sulfate plus [H-3]leucine for varying times in the absence of PMA. Alternatively, confluent end othelial cells were simultaneously incubated with PMA and [(35)]sulfat e for varying times. The metabolically labeled PG in the cell layer an d medium were analyzed. Both short-term and prolonged exposure of endo thelial cells to PMA significantly stimulated PG synthesis, regardless of the experimental conditions. [S-35]sulfate incorporation into newl y synthesized PG in PMA-treated cells also increased by 1.7-fold and 3 .6-fold over control cells, following a 15-min and 30-min pulse, respe ctively. Cycloheximide markedly inhibited the increased synthesis of P G in PMA-treated cells, while actinomycin D produced a moderate inhibi tion. PG secretion was increased in PMA-treated cells compared with co ntrol cells, while there was no significant difference in PG degradati on between the two cultures. PG from control and PMA-treated endotheli al cell cultures did not differ in composition or hydrodynamic sizes. The incorporation of [H-3]leucine into total cellular proteins decreas ed significantly following exposure of endothelial cells to PMA. Endot helial cells exposed to PMA for 3 h had significantly more protein kin ase C (PKC) activity than did control cells. Inhibition of PKC by calp hostin C abolished the PMA-mediated stimulation of PG synthesis in end othelial cells. The results indicate that PMA stimulates PG synthesis in endothelial cells either directly or indirectly through a PKC depen dent mechanism.