The Cyclin-dependent Kinase Inhibitor p21^cip1Mediates the Growth Inhibitory Effect of Phorbol Esters in Human Venous Endothelial Cells*

Abstract

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 p21^cip1 is a potential candidate mediating the PDBu-induced delayed entry of the cells into S-phase (by ∼10 h when compared with cells stimulated with basic fibroblast growth factor (bFGF)). Levels of p21^cip1 (protein and mRNA) rapidly rise (within ∼2 h) in endothelial cells treated with the active isomer β-PDBu, but not with α-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-δ), but not Gö 6976 (selective for Ca²⁺-dependent PKC isoforms). Rapamycin blocks the PDBu-induced accumulation of p21^cip1 (but not of the cognate mRNA), indicating an action of PKC on p21^cip1mRNA translation. If endothelial cells are recruited into the cell cycle by bFGF, p21^cip1 mRNA and protein levels rise initially (within 2 h) and decline subsequently such that p21^cip1 drops to a minimum prior to the initiation of DNA synthesis (i.e. after ∼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 PDBu-stimulated cells. Thus, PKC (presumably PKC-δ) regulates the amounts of p21^cip1 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 p21^cip1 is blocked by the Mek1 inhibitor, but not by GF109203X; hence, in endothelial cells, induction of p21^cip1 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 β-PDBu-induced delayed S-phase entry and drop in p21^cip1 are reversed if GF109203X is added 4 h after β-PDBu to prevent persistent PKC activation. These observations indicate a cause and effect relation between sustained p21^cip1elevations and the delay in S-phase entry induced by β-PDBu.