Common variation in EDN1 regulatory regions highlights the role of PPARγ as a key regulator of Endothelin in vitro

Abstract

Pulmonary Arterial Hypertension (PAH) is a rare disease caused by the obliteration of the pulmonary arterioles, increasing pulmonary vascular resistance and eventually causing right heart failure. Endothelin-1 is a vasoconstrictor peptide whose levels are indicators of disease progression and its pathway is one of the commonest targeted by current treatments. We sequenced the EDN1 untranslated regions of a small subset of PAH patients, predicted the effect in silico, and used a luciferase assay with the different genotypes to analyze its influence on gene expression. Finally, we used siRNAs against the major transcription factors predicted for these regions (PPAR{gamma}, KLF4, and VDR) to assess Endothelin-1 expression in cell culture and validate the binding sites. First, we detected a SNP in the 5UTR (rs397751713) and another in the 3regulatory region (rs2859338) that increased luciferase activity in vitro depending on their genotype. We determined in silico that KLF4/PPAR{gamma} could be binding in the rs397751713 and VDR in rs2859338. By using siRNAs and luciferase, we determined that PPAR{gamma} binds differentially in rs397751713. PPAR{gamma} and VDR Knock-Down, increased EDN1 mRNA levels and Endothelin-1 production in PAECs, while PPAR{gamma} and KLF4 Knock-Down increased the Endothelin-1 production in HeLa. In conclusion, common variants in EDN1 regulatory regions could alter Endothelin-1 levels. We were able to validate that PPAR{gamma} binds in rs397751713 and is key to regulate Endothelin-1. Also, KLF4 and VDR regulate Endothelin-1 production in a cell-dependent manner, but for VDR this interaction does not happen by binding directly to the regions we studied.