Peroxisome Proliferator-activated Receptor-α-mediated Transcription of miR-199a2 Attenuates Endothelin-1 Expression via Hypoxia-inducible Factor-1α

Chen Li,Marthe-Sandrine Eiymo Mwa Mpollo,Caryn S Gonsalves,Stanley M Tahara,Punam Malik,Vijay K Kalra

doi:10.1074/jbc.m114.600775

Abstract

Endothelin-1, a potent vasoconstrictor, plays an important role in pulmonary hypertension (PH) in sickle cell disease (SCD). Our previous studies show that higher levels of placenta growth factor (PlGF), secreted by erythroid precursor cells, correlate with increased plasma levels of endothelin-1 (ET-1) and other functional markers of PH in SCD. PlGF-mediated ET-1 expression occurs via activation of hypoxia-inducible factor-1α (HIF-1α). However, relatively less is understood regarding how PlGF-mediated expression of HIF-1α and its downstream effector ET-1 are post-transcriptionally regulated. Herein, we show that PlGF treatment of endothelial cells resulted in reduced levels of miR-199a2, which targeted the 3'-UTR of HIF-1α mRNA and concomitantly led to augmented ET-1 expression. Plasma levels of miR-199a2 in SCD subjects were significantly lower with reciprocally high levels of plasma ET-1, unlike unaffected controls. This observation provided a molecular link between miR-199a2 and high levels of ET-1 in SCD. Furthermore, we show that miR-199a2 located in the DNM3os transcription unit was co-transcriptionally regulated by peroxisome proliferator-activated receptor α (PPARα). Binding of the latter to PPARα cis-elements in the promoter of DNM3os was demonstrated by promoter mutational analysis and ChIP. Additionally, we show that fenofibrate, a PPARα agonist, increased the expression of miR-199a2 and DNM3os; the former was responsible for reduced expression of HIF-1α and ET-1. In vivo studies of fenofibrate-fed Berkeley sickle mice resulted in increased levels of miR-199a2 and reduced levels of ET-1 in lung tissues. Our studies provide a potential therapeutic approach whereby fenofibrate-induced miR-199a2 expression can ameliorate PH by reduction of ET-1 levels.

Highlights

Elevated plasma levels of placenta growth factor (PlGF) are associated with increased endothelin-1 and pulmonary hypertension (PH) in sickle cell disease (SCD)
A predicted miRNA recognition element (MRE) for miR-199a-5p is located within nucleotides ϩ16 to ϩ38 of the 3Ј-UTR of HIF-1␣ mRNA as shown in the schematic (Fig. 1A)
PPAR␣ Agonists Mediate Expression of miR-199a2, in Turn Regulating Expression of HIF-1␣ and Its Target Gene ET-1— Because we showed that PPAR␣ agonist increased levels of miR-199a2, which targets HIF-1␣ mRNA, we anticipated that fenofibrate treatment would reduce overall HIF-1␣ activity and subsequently down-regulate expression of a key downstream target gene ET-1

Summary

Background

Elevated plasma levels of PlGF are associated with increased endothelin-1 and pulmonary hypertension (PH) in SCD. Fenofibrate acted to increase transcription from the DNM3os locus leading to higher miR-199a2 expression with a resultant decline of HIF-1␣ activity and subsequent reduction of ET-1 transcription These studies were corroborated in SCD patients, wherein we observed significantly reduced plasma levels of miR-199a5p, indicating that decreased levels of miR-199a-5p contributed to increased levels of HIF-1␣ and its regulated gene, i.e. ET-1. We showed that feeding fenofibrate diet to wild type C57BL/6NJ mice and Berkeley sickle mice augmented expression of miR-199a2, which resulted in reduced levels of ET-1 in lungs of both wild type and sickle mice These studies elucidated a novel molecular pathway linking up-regulation of miR199a2 to reduction of ET-1, providing a rationale for a therapeutic approach to attenuate ET-1 levels and ameliorate pulmonary hypertension in SCD

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION