The human preproendothelin-1 gene: possible regulation by endothelial phosphoinositide turnover signaling.

Preproendothelin-1 (ppET-1) mRNA has previously been demonstrated to be markedly induced in cultured endothelial cells (EC) by the addition to the medium of thrombin, an agent known to stimulate phosphoinositide turnover in EC. In this study, the mechanism of regulation of ppET-1 mRNA expression was investigated in cultured human umbilical vein EC by RNA blot analysis with cloned ppET-1 gene as a probe. The mRNA for ppET-1 was rapidly upregulated by O-tetradecanoylphorbol-13-acetate (TPA) (0.5 microM) and by ionomycin (5 microM) within 10 min of addition to the medium, but not by forskolin (50 microM). The rapid induction of ppET-1 mRNA by TPA or ionomycin occurred even in the presence of cycloheximide, indicating that the mRNA induction does not require de novo protein synthesis. The ppET-1 mRNA was an extremely unstable species of mRNA with an apparent half-life of about 15 min. However, the half-life of ppET-1 mRNA was not appreciably affected by TPA or ionomycin, suggesting that the mRNA induction by these agents is mostly due to an activation of the transcription of the mRNA. These observations indicate that the production of ET-1 in human EC can be controlled by a transcriptional gene regulation directly coupled to the intracellular signals from the phosphoinositide-turnover pathway, i.e., activation of protein kinase C and increase in intracellular Ca2+. These mechanisms are discussed in relation to information on the primary structure of cloned ppET-1 gene.