Farrell C R, Boado R J, Pardridge W M
Department of Medicine, UCLA School of Medicine 90024-1682.
Brain Res Mol Brain Res. 1992 Oct;15(3-4):221-6. doi: 10.1016/0169-328x(92)90112-o.
The in vitro angiogenesis of endothelium obtained from peripheral tissues is stimulated by phorbol esters. The present studies examine the effects of phorbol esters or serum factors on GLUT1 glucose transporter, cytoplasmic actin, and beta-tubulin messenger RNA levels and gene transcription rates in bovine brain capillary endothelial cells grown in tissue culture. Messenger RNA levels were measured by Northern blot analysis and transcription rates were quantified by nuclear run-on assays. Although cytoplasmic actin mRNA levels in cultured brain endothelium were comparable to levels found in isolated capillaries isolated in vivo, there was a profound down-regulation of the GLUT1 glucose transporter mRNA in the cultured endothelium. The GLUT1 mRNA level was increased by exposure to 12-O-tetra-decanoyl-phorbol 13-acetate (TPA). Both serum and TPA enhanced cytoplasmic actin and beta-tubulin mRNA levels in cultured cells; the serum effect on cytoskeletal mRNA persisted through at least 24 h of exposure whereas the TPA stimulation was maximal by 2 h of exposure and lost following 8 h. Both serum and TPA increased cytoplasmic actin mRNA levels approximately 2- to 3-fold greater than the increase in beta-tubulin mRNA levels. GLUT1 and actin transcription rates were measured with the nuclear run-on assay, but no stimulation was observed following 3 h exposure to 200 nM TPA. In conclusion, these studies show that GLUT1 glucose transporter, cytoplasmic actin, and beta-tubulin mRNA levels in bovine brain capillary endothelial cells are regulated by both serum factors and phorbol ester, which activates the protein kinase C pathway, and that the mechanism of the phorbol ester effect is post-transcriptional.
佛波酯可刺激从外周组织获取的内皮细胞进行体外血管生成。本研究检测了佛波酯或血清因子对组织培养的牛脑微血管内皮细胞中葡萄糖转运蛋白1（GLUT1）、细胞质肌动蛋白和β-微管蛋白信使核糖核酸（mRNA）水平以及基因转录速率的影响。通过Northern印迹分析测量mRNA水平，通过核转录分析对转录速率进行定量。虽然培养的脑内皮细胞中的细胞质肌动蛋白mRNA水平与体内分离的毛细血管中的水平相当，但培养的内皮细胞中GLUT1葡萄糖转运蛋白mRNA却有显著下调。暴露于12-O-十四烷酰佛波醇13-乙酸酯（TPA）可使GLUT1 mRNA水平升高。血清和TPA均可提高培养细胞中细胞质肌动蛋白和β-微管蛋白的mRNA水平；血清对细胞骨架mRNA的影响在至少24小时的暴露后仍持续存在，而TPA刺激在暴露2小时时达到最大值，并在8小时后消失。血清和TPA使细胞质肌动蛋白mRNA水平的增加幅度均比β-微管蛋白mRNA水平的增加幅度大约高2至3倍。用核转录分析测量了GLUT1和肌动蛋白的转录速率，但在暴露于200 nM TPA 3小时后未观察到刺激作用。总之，这些研究表明，牛脑微血管内皮细胞中的GLUT1葡萄糖转运蛋白、细胞质肌动蛋白和β-微管蛋白mRNA水平受血清因子和激活蛋白激酶C途径的佛波酯的调控，且佛波酯作用的机制是转录后水平的。
