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 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.
Highlights
The addition of phorbol esters to the culture medium inhibits the growth of endothelial cells [1]
Previous studies have addressed this paradox and have shown that the PKC activator PDBu exerts a bidirectional effect on growth of human vascular endothelial cells; short-term application of phorbol esters efficiently recruits quiescent endothelial cells into the G1-phase of the cell cycle, whereas persistent activation of protein kinase C subsequently slows the progression though G1 and thereby delays entry of the cells into S-phase [6, 7]; the net effect of continuous treatment with phorbol esters is a suppression of endothelial cell proliferation [1, 6]
If -PDBu is applied continuously, the subsequent onset of [3H]thymidine incorporation into DNA, i.e. S-phase entry, is delayed by ϳ10 h compared with cells that have been treated with physiological stimuli such as bFGF or fetal calf serum (FCS) (Fig. 1A)
Summary
The growth of endothelial cells [1] This observation is difficult to reconcile with the fact that stimulation of the cellular targets of phorbol esters, i.e. the isoforms of protein kinase C, leads to sustained activation of several signaling pathways that are required for the recruitment of quiescent cells into the cell cycle. The ideal antiangiogenic regimen ought to block the action of all endothelial mitogens in a reversible fashion In theory, this may be achieved by inhibiting a signaling pathway onto which all receptor-generated intracellular stimuli converge to drive the cells through the cell cycle, i.e. the components of the cell cycle machinery that are required for progression through G1. This effect is likely to account for the delayed S-phase entry and the resulting growth inhibition
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