Endothelin-1 is differentially regulated by clofibrate and the +138D/I polymorphism

Remmler, Cornelia , Dr. Haenisch, Sierk. UK-SH Institute for Experimental and Clinical Pharmacology, Hospitalstr. 4, 24105 Kiel, Germany.

The expression of endothelin-1 (ET-1) is affected by angiotensin II (AngII) and clofibrate (Co). Studies concerning the regulation of ppET-1 have revealed putatively binding sites of GATA-2 (-136 to -131 bp) and AP1 (-108 to -102 bp) in the promoter region. The +138D/I polymorphism alters the ET-1 expression and mRNA stability. We aimed to investigate the regulation of ET-1 in relation to the D variant in an in vitro model. The mRNA expression of ppET-1 in endothelial EA.hy926 cells was determined after 2, 6, 12 and 24h using AngII or Co. Reporter gene constructs were designed consisting the ppET-1 5’-UTR and parts of the promoter, including the GATA-2 and AP1 binding site. Further constructs were generated by mutagenesis. Luciferase assays were performed with or without 1 µM AngII or 600 µM Co. Gel shift assays were performed with nuclear extract of EA.hy926 cells. The mRNA expression was only slightly affected by AngII, 6 and 60 µM Co, whereas 600 µM Co showed clear induction after 2 hours of incubation. The luciferase activity was dependent on the -136/-131 and the -108/-102 promoter region, genotype dependent differences could be observed in all constructs having at least one mutation. AngII did not affect the activity of any construct, while Co enforced the luciferase activity due to functional -136/-131 region. Gel shift assays revealed a binding protein at the 5’-UTR probe for both variants. Comparing GATA and AP1 consensus sequences with the ppET-1 promoter sequence different binding patterns were observed. The significant influence of the 5’-UTR +138 I variant on ppET1 expression could be confirmed and the results suggest that both, the -136/-131 and the -108/-102 promoter region are crucial for activity of ppET-1. The activation through Co differed related to the -136/-131 binding site. The results suggest that the ppET-1 regulation via AngII and Co is caused by other transcription factors than GATA-2 and AP1 and additional post-translational mechanisms.