Vol. 60, Issue 6, 1332-1342, December 2001

Transcriptional Mechanism of Protein Kinase C-Induced Isoform-Specific Expression of the Gene for Endothelin-Converting Enzyme-1in Human Endothelial Cells

Hans-Dieter Orzechowski, Astrid Günther,¹ Stefan Menzel,² Andreas Zimmermann, Heiko Funke-Kaiser, Robert Real,³ Thomas Subkowski, Frank S. Zollmann, and Martin Paul

Institute of Clinical Pharmacology and Toxicology, Benjamin Franklin Medical Center, Freie Universität, Berlin, (H.-D.O., A.G., S.M., A.Z., H.F.-K., R.R., F.S.Z., M.P.), and Main Laboratory, BASF AG, Ludwigshafen, Germany (T.S.)

Isoform-specific expression of endothelin-converting enzyme (ECE)-1, the major big endothelin-processing enzyme, is controlled by alternative promoters. Signaling pathways and transcriptional mechanisms of ECE-1 mRNA expression are largely unknown. To investigate ECE-1 isoform expression after protein kinase C (PKC) activation, we used phorbol 12-myristate 13-acetate (PMA) to stimulate primary cultured human umbilical vein endothelial cells and the related EA.hy926 cell line. ECE-1a mRNA was up-regulated (approximately 3-fold), whereas mRNA of alternative isoforms (b, c, and d) was unchanged, which was confirmed on the protein level. PMA effects on mRNA expression were suppressed by the PKC inhibitors H-7 and Calphostin C. Because increased ECE-1a expression was preceded by induction of the transcription factor Ets-1, we performed gel shift assays and demonstrated specific DNA/protein interactions involving the ETS binding motif GGAA. Luciferase reporter assays showed that PMA induced ECE-1a promoter activity about 2.5-fold in EA.hy926 cells. Similarly, coexpression of Ets-1 protein resulted in a dose-dependent increase in ECE-1a promoter activity (more than 8-fold). Using gel shift assays and mutation analysis, we identified two tandemly arranged Ets-1 binding sites (EBS) at 638 and 658, respectively, that are involved in transcriptional activation of the ECE-1a promoter by PMA or Ets-1. Moreover, we also found evidence for binding of a transcriptional repressor to EBS 638. The inhibitor of mitogen-activated protein kinase kinase, PD98059, inhibited PMA effects on ECE-1a mRNA expression and promoter activity, respectively. Our results provide the first detailed analysis of signaling pathways and transcriptional mechanisms involved in isoform-specific ECE-1 gene expression.