THE CYCLIN-DEPENDENT KINASE INHIBITOR P21(CIP1) MEDIATES THE GROWTH-INHIBITORY EFFECT OF PHORBOL ESTERS IN HUMAN VENOUS ENDOTHELIAL-CELLS

Long-term application of the phorbol ester phorbol 12,18-dibutyrate (P DBu) inhibits the proliferation of human venous endothelial cells, The cyclin-dependent kinase inhibitor p21(cip1) is a potential candidate mediating the PDBu-induced delayed entry of the cells into S-phase (by similar to 10 h when compared with cells stimulated with basic fibrob last growth factor (bFGF)), Levels of p21(cip1) (protein and mRNA) rap idly rise (within similar to 2 h) in endothelial cells treated with th e active isomer beta-PDBu, but not with alpha-PDBu; this effect is blo cked by the mitogen-activated protein kinase kinase-l (Mek1) inhibitor PD098059 and by the protein kinase C (PRC) antagonists GF109203X and rottlerin (selective for PKC-delta), but not Go 6976 (selective for Ca 2+-dependent PRC isoforms), Rapamycin blocks the PDBu-induced accumula tion of p21(cip1) (but not of the cognate mRNA), indicating an action of PKC on p21(cip1) mRNA translation, If endothelial cells are recruit ed into the cell cycle by bFGF, p21(cip1) mRNA and protein levels rise initially (within 2 h) and decline subsequently such that p21(cip1) d rops to a minimum prior to the initiation of DNA synthesis (i.e. after similar to 12 h). In bFGF-stimulated cells, changes in p21(cip1) mRNA and protein are strictly linked, In contrast, the levels of p21(cip1) mRNA decline substantially (>10 h) before the protein decreases in PD Bu-stimulated cells, Thus, PKC (presumably PKC-delta) regulates the am ounts of p21(cip1) in endothelial cells at the level of mRNA accumulat ion and translation, leading to a rapid and robust induction; followin g persistent PKC activation, p21(cip1) remains elevated despite reduce d mRNA levels, indicating an enhanced stability of the protein, The bF GF-mediated increase in p21(cip1) is blocked by the Mek1 inhibitor, bu t not by GF109203X; hence, in endothelial cells, induction of p21(cip1 ) by PKC- and growth factor-dependent signaling is achieved by distinc t pathways that converge and require activation of the mitogen-activat ed protein kinase cascade, The beta-PDBu-induced delayed S-phase entry and drop in p21(cip1) are reversed if GF109203X is added 4 h after be ta-PDBu to prevent persistent PKC activation, These observations indic ate a cause and effect relation between sustained p21(cip1) elevations and the delay in S-phase entry induced by beta-PDBu.