Common Variation in EDN1 Regulatory Regions Highlights the Role of PPARγ as a Key Regulator of Endothelin in vitro

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 (EDN1) is a vasoconstrictor peptide whose levels are indicators of disease progression and its pathway is one of the most common targeted by current treatments. We sequenced the EDN1 untranslated regions of a small subset of patients with PAH, 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 (TFs) predicted for these regions [peroxisome proliferator-activated receptor gamma (PPAR gamma), Kruppel-Like Factor 4 (KLF4), and vitamin D receptor (VDR)] to assess EDN1 expression in cell culture and validate the binding sites. First, we detected a single nucleotide polymorphism (SNP) in the 5' untranslated region (UTR; rs397751713) and another in the 3'regulatory region (rs2859338) that altered luciferase activity in vitro depending on their genotype. We determined in silico that KLF4/PPAR gamma could bind to the rs397751713 and VDR to rs2859338. By using siRNAs and luciferase assays, we determined that PPAR gamma binds differentially to rs397751713. PPAR gamma and VDR Knock-Down (KD) increased the EDN1 mRNA levels and EDN1 production in porcine aortic endothelial cells (PAECs), while PPAR gamma and KLF4 KD increased the EDN1 production in HeLa. In conclusion, common variants in EDN1 regulatory regions could alter EDN1 levels. We were able to validate that PPAR gamma binds in rs397751713 and is a key regulator of EDN1. In addition, KLF4 and VDR regulate EDN1 production in a cell-dependent manner, but VDR does not bind directly to the regions we studied.

Automated summary

Pulmonary Arterial Hypertension (PAH) is a rare disease caused by the obliteration of the pulmonary arterioles, leading to right heart failure. Endothelin-1 (EDN1) is a peptide that constricts blood vessels and its levels are associated with disease progression in PAH. In this study, we sequenced the untranslated regions of EDN1 in PAH patients and identified two single nucleotide polymorphisms (SNPs) that can affect gene expression. We found that the transcription factors PPAR gamma, KLF4, and VDR can bind to these SNPs and regulate EDN1 production in different cell types.