Kooistra T, Bosma P J, Toet K, Cohen L H, Griffioen M, van den Berg E, le Clercq L, van Hinsbergh V W
Gaubius Laboratory IVVO-TNO, Leiden, The Netherlands.
Arterioscler Thromb. 1991 Jul-Aug;11(4):1042-52. doi: 10.1161/01.atv.11.4.1042.
Activation of protein kinase C leads to a strong induction of tissue-type plasminogen activator (t-PA) expression in endothelial cells. Using endothelial cells from human umbilical vein (HUVECs) and human aorta (HAECs), we have studied this regulation of t-PA and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), at the mRNA level and have compared their induction with the expression of platelet-derived growth factors A and B (PDGF-A and PDGF-B) and the proto-oncogenes c-jun and c-fos. Treatment of HUVECs with exogenous bacterial phospholipase C or the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol led to a threefold and a twofold increase, respectively, in t-PA concentrations in 24-hour-conditioned medium. Similarly, the more stable protein kinase C activator 4 beta-phorbol-12-myristate-13-acetate (PMA) caused about a 10-fold increase in t-PA antigen levels. This effect of PMA is maximal between 8 and 16 hours at a concentration of 10 nM and is fully accounted for by parallel increases in t-PA mRNA levels. An increase in intracellular cyclic adenosine monophosphate levels by forskolin (10 microM) slightly diminished t-PA expression but further enhanced the PMA-induced increases in t-PA synthesis and mRNA levels by at least twofold. PMA also enhanced the mRNA levels of two other important endothelium-expressed genes, PDGF-A and PDGF-B, with a time profile similar to that of t-PA, with peak values about fivefold higher than control values. Forskolin did not further stimulate this PMA-induced PDGF expression in HUVECs, which suggests a regulatory mechanism different from that of t-PA. Qualitatively very similar induction patterns of t-PA, PDGF-A, and PDGF-B were seen with HAECs. In contrast to t-PA and PDGF, PAI-1 mRNA and antigen levels increased only slightly after PMA treatment of HUVECs or HAECs; forskolin alone or in combination with PMA diminished the expression of PAI-1. The induction of t-PA mRNA by PMA was dependent on protein synthesis and was preceded by a strong transient increase in c-jun and c-fos mRNA levels; the induction of c-fos but not of c-jun was potentiated by forskolin. Because the products of these two proto-oncogenes form dimeric complexes for which specific binding sites are present in the t-PA promoter region, they may mediate the protein kinase C-dependent increase in t-PA gene expression, including the stimulating action of cyclic adenosine monophosphate.
蛋白激酶C的激活导致内皮细胞中组织型纤溶酶原激活物（t-PA）表达的强烈诱导。利用人脐静脉内皮细胞（HUVECs）和人主动脉内皮细胞（HAECs），我们在mRNA水平研究了t-PA及其抑制剂纤溶酶原激活物抑制剂-1（PAI-1）的这种调节，并将它们的诱导情况与血小板衍生生长因子A和B（PDGF-A和PDGF-B）以及原癌基因c-jun和c-fos的表达进行了比较。用外源性细菌磷脂酶C或合成二酰基甘油1-油酰基-2-乙酰甘油处理HUVECs，分别导致24小时条件培养基中t-PA浓度增加三倍和两倍。同样，更稳定的蛋白激酶C激活剂4β-佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）使t-PA抗原水平增加约10倍。PMA的这种作用在浓度为10 nM时，在8至16小时之间达到最大值，并且完全由t-PA mRNA水平的平行增加所解释。福斯高林（10 microM）使细胞内环状单磷酸腺苷水平升高，略微降低了t-PA的表达，但进一步增强了PMA诱导的t-PA合成增加和mRNA水平，至少增加了两倍。PMA还增强了另外两个在内皮细胞中重要表达基因PDGF-A和PDGF-B的mRNA水平，其时间模式与t-PA相似，峰值比对照值高约五倍。福斯高林没有进一步刺激HUVECs中PMA诱导的PDGF表达，这表明其调节机制与t-PA不同。HAECs中t-PA、PDGF-A和PDGF-B的诱导模式在质量上非常相似。与t-PA和PDGF相反，PMA处理HUVECs或HAECs后，PAI-1 mRNA和抗原水平仅略有增加；单独使用福斯高林或与PMA联合使用会降低PAI-1的表达。PMA对t-PA mRNA的诱导依赖于蛋白质合成，并且在c-jun和c-fos mRNA水平强烈短暂增加之前；福斯高林增强了c-fos但没有增强c-jun的诱导。因为这两个原癌基因的产物形成二聚体复合物，其在t-PA启动子区域存在特异性结合位点，所以它们可能介导蛋白激酶C依赖性的t-PA基因表达增加，包括环状单磷酸腺苷的刺激作用。
