The cyclin-dependent kinase inhibitor p21cip1 mediates the growth inhibitory effect of phorbol esters in human venous endothelial cells.

Long-term application of the phorbol ester phorbol 12,13-dibutyrate (PDBu) inhibits the proliferation of human venous endothelial cells. The cyclin-dependent kinase inhibitor p21cip1 is a potential candidate mediating the PDBu-induced delayed entry of the cells into S-phase (by approximately 10 h when compared with cells stimulated with basic fibroblast growth factor (bFGF)). Levels of p21cip1 (protein and mRNA) rapidly rise (within approximately 2 h) in endothelial cells treated with the active isomer beta-PDBu, but not with alpha-PDBu; this effect is blocked by the mitogen-activated protein kinase kinase-1 (Mek1) inhibitor PD098059 and by the protein kinase C (PKC) antagonists GF109203X and rottlerin (selective for PKC-delta), but not Gö 6976 (selective for Ca2+-dependent PKC isoforms). Rapamycin blocks the PDBu-induced accumulation of p21cip1 (but not of the cognate mRNA), indicating an action of PKC on p21(cip1) mRNA translation. If endothelial cells are recruited into the cell cycle by bFGF, p21cip1 mRNA and protein levels rise initially (within 2 h) and decline subsequently such that p21cip1 drops to a minimum prior to the initiation of DNA synthesis (i.e. after approximately 12 h). In bFGF-stimulated cells, changes in p21cip1 mRNA and protein are strictly linked. In contrast, the levels of p21cip1 mRNA decline substantially (>10 h) before the protein decreases in PDBu-stimulated cells. Thus, PKC (presumably PKC-delta) regulates the amounts of p21cip1 in endothelial cells at the level of mRNA accumulation and translation, leading to a rapid and robust induction; following persistent PKC activation, p21(cip1) remains elevated despite reduced mRNA levels, indicating an enhanced stability of the protein. The bFGF-mediated increase in p21cip1 is blocked by the Mek1 inhibitor, but not by GF109203X; hence, in endothelial cells, induction of p21cip1 by PKC- and growth factor-dependent signaling is achieved by distinct pathways that converge and require activation of the mitogen-activated protein kinase cascade. The beta-PDBu-induced delayed S-phase entry and drop in p21cip1 are reversed if GF109203X is added 4 h after beta-PDBu to prevent persistent PKC activation. These observations indicate a cause and effect relation between sustained p21cip1 elevations and the delay in S-phase entry induced by beta-PDBu.