Zezula J, Sexl V, Hutter C, Karel A, Schütz W, Freissmuth M
Institute of Pharmacology, Vienna University, Währinger Strasse 13a, A-1090 Vienna, Austria.
J Biol Chem. 1997 Nov 21;272(47):29967-74. doi: 10.1074/jbc.272.47.29967.
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.
佛波酯佛波醇12,13 - 二丁酸酯（PDBu）的长期应用可抑制人静脉内皮细胞的增殖。细胞周期蛋白依赖性激酶抑制剂p21cip1是介导PDBu诱导细胞延迟进入S期（与用碱性成纤维细胞生长因子（bFGF）刺激的细胞相比，延迟约10小时）的潜在候选分子。在用活性异构体β - PDBu处理的内皮细胞中，p21cip1（蛋白质和mRNA）水平迅速升高（约2小时内），而用α - PDBu处理的细胞则不然；这种效应被丝裂原活化蛋白激酶激酶 - 1（Mek1）抑制剂PD098059以及蛋白激酶C（PKC）拮抗剂GF109203X和罗特列素（对PKC - δ有选择性）阻断，但不被Gö 6976（对Ca2 +依赖性PKC同工型有选择性）阻断。雷帕霉素阻断PDBu诱导的p21cip1积累（但不阻断同源mRNA的积累），表明PKC对p21（cip1）mRNA翻译有作用。如果内皮细胞被bFGF募集进入细胞周期，p21cip1 mRNA和蛋白质水平最初升高（2小时内），随后下降，使得p21cip1在DNA合成开始前（即约12小时后）降至最低。在bFGF刺激的细胞中，p21cip1 mRNA和蛋白质的变化严格相关。相反，在PDBu刺激的细胞中，p21cip1 mRNA水平在蛋白质下降之前大幅下降（> 10小时）。因此，PKC（可能是PKC - δ）在mRNA积累和翻译水平调节内皮细胞中p21cip1的量，导致快速而强烈的诱导；在持续的PKC激活后，尽管mRNA水平降低，但p21（cip1）仍保持升高，表明蛋白质稳定性增强。Mek1抑制剂可阻断bFGF介导的p21cip1增加，但GF109203X不能；因此，在内皮细胞中，PKC依赖性和生长因子依赖性信号传导对p21cip1的诱导是通过不同的途径实现的，这些途径汇聚并需要丝裂原活化蛋白激酶级联的激活。如果在β - PDBu加入4小时后添加GF109203X以防止PKC持续激活，则β - PDBu诱导的延迟S期进入和p21cip1下降可被逆转。这些观察结果表明持续的p21cip1升高与β - PDBu诱导的S期进入延迟之间存在因果关系。
