Peroxisome proliferator-activated receptor-α-mediated transcription of miR-301a and miR-454 and their host gene SKA2 regulates endothelin-1 and PAI-1 expression in sickle cell disease.

Caryn S Gonsalves,Vijay K Kalra,Stanley M Tahara,Chen Li,Punam Malik

doi:10.1042/bsr20150190

Abstract

Endothelin-1 (ET-1) and plasminogen activator inhibitor-1 (PAI-1) play important roles in pulmonary hypertension (PH) in sickle cell disease (SCD). Our previous studies show higher levels of placenta growth factor (PlGF) in SCD correlate with increased plasma levels of ET-1, PAI-1, and other physiological markers of PH. PlGF-mediated ET-1 and PAI-1 expression occurs via activation of hypoxia-inducible factor-1α (HIF-1α). However, relatively little is understood regarding post-transcriptional regulation of PlGF-mediated expression of ET-1 and PAI-1. Herein, we show PlGF treatment of endothelial cells reduced levels of miR-301a and miR-454 from basal levels. In addition, both miRNAs targeted the 3'-UTRs of ET-1 and PAI-1 mRNAs. These results were corroborated in the mouse model of SCD [Berkeley sickle mice (BK-SS)] and in SCD subjects. Plasma levels of miR-454 in SCD subjects were significantly lower compared with unaffected controls, which correlated with higher plasma levels of both ET-1 and PAI-1. Moreover, lung tissues from BK-SS mice showed significantly reduced levels of pre-miR-301a and concomitantly higher levels of ET-1 and PAI-1. Furthermore, we show that miR-301a/miR-454 located in the spindle and kinetochore-associated protein-2 (SKA2) transcription unit was co-transcriptionally regulated by both HIF-1α and peroxisome proliferator-activated receptor-α (PPAR-α) as demonstrated by SKA2 promoter mutational analysis and ChIP. Finally we show that fenofibrate, a PPAR-α agonist, increased the expression of miR-301a/miR-454 and SKA2in human microvascular endothelial cell line (HMEC) cells; the former were responsible for reduced expression of ET-1 and PAI-1. Our studies provide a potential therapeutic approach whereby fenofibrate-induced miR-301a/miR-454 expression can ameliorate PH and lung fibrosis by reduction in ET-1 and PAI-1 levels in SCD.

Highlights

Pulmonary disease, both acute and chronic, is the second most common cause of hospitalization and a leading cause of both morbidity and mortality in adults with sickle cell disease (SCD)[1,2]
Our previous studies show higher circulatory levels of placenta growth factor (PlGF) in the Berkeley sickle mice (BK-SS) model and SCD patients are associated with increased levels of plasma ET-1 and other markers of pulmonary hypertension (PH), including plasminogen activator inhibitor-1 (PAI-1) [6]
We previously showed that expression of ET-1 and PAI-1 was transcriptionally regulated by hypoxia-inducible factor-1α (HIF-1α), independent of hypoxia [8,23]

Summary

INTRODUCTION

Both acute and chronic, is the second most common cause of hospitalization and a leading cause of both morbidity and mortality in adults with sickle cell disease (SCD). Other studies show that SCD patients have elevated steady state plasma levels of circulating tissue factor [14] and plasminogen activator inhibitor-1 (PAI-1), both of which increase further during sickle vaso-occlusive crises [15]. Our studies showed that miR-301a and miR-454, target the 3 -UTRs of ET-1 and PAI-1 mRNAs. A physiological relationship between miR-301a and miR-454 was demonstrated in vitro, as both miRNAs attenuated PlGF-mediated secretion, in human microvascular endothelial cell line (HMEC-1), of both ET-1 and PAI-1. A physiological relationship between miR-301a and miR-454 was demonstrated in vitro, as both miRNAs attenuated PlGF-mediated secretion, in human microvascular endothelial cell line (HMEC-1), of both ET-1 and PAI-1 These miRNAs co-located in an intron of the spindle and kinetochore-associated protein-2 (SKA2) gene were co-transcriptionally regulated by peroxisome proliferatoractivated receptor-α (PPAR-α) and HIF-1α. The present study, to the best of our knowledge, is the first demonstration that PPAR-α co-regulates the transcription of SKA2, miR-301a and miR-454; the ET-1 mRNA has complementary sites in the 3 -UTR for the seed sequences of these miRNAs

MATERIALS AND METHODS

Method Forward primer

Findings

DISCUSSION