Enhanced GLUT1 glucose transporter and cytoskeleton gene expression in cultured bovine brain capillary endothelial cells after treatment with phorbol esters and serum

Abstract

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 β-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 β-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 β-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 β-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.