P158	Isoform-specific regulation of endothelin-converting enzyme-1 during monocytic differentiation.
N.Draheim, M.Paul, H-D.Orzechowski
Charité - Campus Benjamin Franklin, Institute of Clinical Pharmacology and Toxicology, Berlin, DE.

Processing of inactive big endothelin (ET)-1 by endothelin-converting enzymes (ECEs) is essential for the synthesis of the biologically active endothelins. Previous observations of increased expression of ECE-1 protein in macrophages invading atherosclerotic lesions in human arteries have suggested a specific pathophysiological role of this metalloprotease during progression of atherosclerotic disease. The mechanisms by which transcription of the ECE1 gene in human monocytic cells is regulated are largely unknown. We, therefore, analyzed the mechanism of ECE-1 upregulation in cultured human HL-60 monocytic cells. Using PMA as diffentiation-inducing agent, we found a dose-dependent increase in ECE-1 mRNA levels which reached its maximum at 300 nM PMA. Compared to DMSO-treated control cells, ECE-1 mRNA levels were significantly increased to about 180% at 24 hours in the HL-60 subpopulation which became adherent upon PMA stimulation. In HL-60 cells which remained in the supernatant, the increase of ECE-1 mRNA was less pronounced (about 130% of controls). Pretreatment with the transcriptional inhibitor actinomycin D completely inhibited the increase in ECE-1 mRNA expression. Using RNase protection assay, we found that the PMA-induced increase in ECE-1 mRNA levels mainly based on a strong induction of ECE-1d and, though to a much lesser extent, ECE-1b isoforms, whereas expression of the alternative isoforms, ECE-1c and ECE-1a, was unchanged. Using 5'RACE, the preferred initiation sites of transcriptional induction of ECE-1d and ECE-1b were determined. Our results demonstrate a distinct association of enhanced ECE-1 expression with monocyte/ macrophage differentiation which may be of pathophysiological significance in human atherosclerosis.

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