Regulated expression of endothelin converting enzymes in glomerular endothelial cells

Endothelin converting enzyme (ECE) constitutes a potential regulatory site for the production of active mature endothelins. Two cDNAs (ECE-1 and -2) encoding ECE have recently been cloned, but the regulation of the expression of these ECE has not been clarified. In the study presented here, an attempt was made to determine whether or not glomerular endothelial cells (GEN) express ECE-1 and -2, and to learn how the expression of ECE-1 and -2 is regulated by kinase-mediated signaling systems. Ribonuclease protection assay revealed the expression of ECE-1 and -2 in cultured GEN, and the expression was increased approximately 2.5- and approximately 1.8-fold, respectively, by treatment with 10(-7) M 12-O-tetradecanocyl-phorbol-13-acetate (TPA) for 4 hours. These increases in ECE-1 and -2 expression with TPA were inhibited by cotreatment with calphostin C (10(-7) M). In contrast, 24-h treatment with 10(-7) M TPA significantly decreased the expression of ECE-1 and -2, indicating that the expression was tightly regulated by protein kinase C (PKC)-dependent mechanism(s). Actinomycin D (1 microgram/mL) abolished the TPA-induced increase of ECE-1 and -2 mRNA, whereas TPA treatment did not affect the mRNA stability of ECE-1 and -2, thus suggesting that TPA-induced increases of ECE-1 and -2 mRNA resulted from the transcriptional activation of ECE-1 and -2, gene, rather than from the increase of mRNA stability. In addition to the regulation by PKC, the effects of protein kinase A and G on ECE-1 and -2 expression were also examined. Treatment with chlorophenyl-thio cyclic AMP (200 microM) for 24 h decreased ECE-1 and -2 expression to approximately 50% and approximately 40% of the control value, respectively. 8-bromo-3', 5'-cyclic GMP also decreased ECE-1 and -2 expression to approximately 80% and approximately 25% of the control value, respectively. These results demonstrate that the expression of ECE-1 and -2 is regulated by kinase-mediated signaling systems, with the most prominent regulatory effect shown by protein kinase C.