Langer D J, Kuo A, Kariko K, Ahuja M, Klugherz B D, Ivanics K M, Hoxie J A, Williams W V, Liang B T, Cines D B
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104.
Circ Res. 1993 Feb;72(2):330-40. doi: 10.1161/01.res.72.2.330.
Binding of urokinase-type plasminogen activator (u-PA) to specific receptors (u-PAR) on the surface of endothelial cells contributes to the regulation of plasmin-dependent processes such as fibrinolysis and angiogenesis. We studied the effect of raising intracellular levels of cyclic AMP (cAMP) and/or activating protein kinase C on the expression of u-PAR in cultured human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with forskolin stimulated a time- and concentration-dependent increase in the expression of u-PAR, measured both by an increase in the specific binding of radiolabeled single-chain u-PA (scu-PA) and by increased binding of anti-u-PAR antibodies. Maximal increase in u-PAR expression (81 +/- 11% above control, n = 11) was not associated with a change in receptor affinity for scu-PA when HUVEC were incubated for 20 hours at 37 degrees C with 50 microM forskolin. Receptor induction by forskolin was inhibited when HUVEC were preincubated with deoxyadenosine monophosphate (DAM), an inhibitor of adenylyl cyclase. A similar increase in receptor expression (128 +/- 27% above control, n = 3) was induced by the cAMP analogue 8-bromoadenosine 3':5'-cyclic monophosphate (50 mM). Forskolin induced an approximately twofold increase in the expression of a single approximately 1.4-kb u-PAR messenger RNA (mRNA) transcript within 2 hours. Phorbol myristate acetate (PMA) also stimulated a time- and concentration-dependent increase in specific scu-PA binding. The maximal increase in u-PAR expression (254 +/- 27% above control, n = 11) was observed when HUVEC were preincubated with 10 nM PMA for 20 hours. Induction of u-PAR by PMA was inhibited when HUVEC were preincubated with either cycloheximide or H7 but was unaffected by DAM. u-PAR induced by PMA showed a reduced affinity for scu-PA (Kd, 14 +/- 2 nM versus 3.6 +/- 0.6 nM, p < 0.001; n = 8). PMA stimulation for 20 hours resulted in a sixfold increase in a single approximately 1.4-kb u-PAR mRNA transcript, with increased levels detectable within 30 minutes. Coincubation of HUVEC with optimal concentrations of forskolin and PMA for 20 hours produced a fully additive increase in u-PAR expression at both the mRNA and protein levels. These data suggest that both cAMP-dependent and protein kinase C-dependent protein kinase pathways may independently regulate u-PAR expression in human endothelial cells.
尿激酶型纤溶酶原激活剂（u-PA）与内皮细胞表面的特异性受体（u-PAR）结合，有助于调节纤溶酶依赖性过程，如纤维蛋白溶解和血管生成。我们研究了提高细胞内环磷酸腺苷（cAMP）水平和/或激活蛋白激酶C对培养的人脐静脉内皮细胞（HUVEC）中u-PAR表达的影响。用福斯高林孵育HUVEC可刺激u-PAR表达呈时间和浓度依赖性增加，这通过放射性标记的单链u-PA（scu-PA）特异性结合的增加以及抗u-PAR抗体结合的增加来测定。当HUVEC在37℃用50μM福斯高林孵育20小时时，u-PAR表达的最大增加（比对照高81±11%，n = 11）与受体对scu-PA的亲和力变化无关。当HUVEC用腺苷酸环化酶抑制剂脱氧单磷酸腺苷（DAM）预孵育时，福斯高林对受体的诱导作用受到抑制。cAMP类似物8-溴腺苷3':5'-环一磷酸（50 mM）诱导了类似的受体表达增加（比对照高128±27%，n = 3）。福斯高林在2小时内使单一约1.4-kb的u-PAR信使核糖核酸（mRNA）转录物的表达增加约两倍。佛波酯肉豆蔻酸酯（PMA）也刺激了scu-PA特异性结合的时间和浓度依赖性增加。当HUVEC用10 nM PMA预孵育20小时时，观察到u-PAR表达的最大增加（比对照高254±27%，n = 11）。当HUVEC用放线菌酮或H7预孵育时，PMA对u-PAR的诱导作用受到抑制，但不受DAM影响。PMA诱导的u-PAR对scu-PA的亲和力降低（解离常数Kd，14±2 nM对3.6±0.6 nM，p < 0.001；n = 8）。PMA刺激20小时导致单一约1.4-kb的u-PAR mRNA转录物增加六倍，30分钟内即可检测到水平升高。将HUVEC与最佳浓度的福斯高林和PMA共同孵育20小时，在mRNA和蛋白质水平上u-PAR表达均产生完全相加的增加。这些数据表明，cAMP依赖性和蛋白激酶C依赖性蛋白激酶途径可能独立调节人内皮细胞中u-PAR的表达。
