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

Open AccessPublished:November 11, 2014DOI:https://doi.org/10.1074/jbc.M114.600775
      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.Elevated plasma levels of PlGF are associated with increased endothelin-1 and pulmonary hypertension (PH) in SCD.

      Results

      miR-199a2, which targets HIF-1α mRNA, located in host gene DNM3os is co-transcriptionally regulated by PPARα.

      Conclusion

      PPARα agonist induction of miR-199a2 reduced ET-1 levels.

      Significance

      PPARα agonist reduction of ET-1 levels via induced miR-199a2 provides an alternative strategy to ameliorate PH.

      Introduction

      Endothelin and nitric oxide are two opposing vasoactive factors that regulate vascular tone. In blood vessels, the endothelin system plays an important role in basal vasoconstriction and participates in the pathology of diseases such as hypertension, cardiovascular disease, and atherosclerosis (
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      The abbreviations used are: PH
      pulmonary hypertension
      DNM3os
      Dynamin 3 opposite strand
      ET-1
      endothelin-1
      HIF-1α
      hypoxia inducible factor-1 α
      HMEC-1
      human microvascular cell line
      HLMVEC
      human lung microvascular endothelial cells
      PlGF
      placenta growth factor
      SCD
      sickle cell disease
      miR
      microRNA
      MRE
      microRNA response element
      PPARα
      peroxisome proliferator-activated receptor-α
      BK-SS
      Berkeley sickle
      qRT-PCR
      quantitative real time PCR
      SFM
      serum-free medium
      sc
      scrambled.
      PH occurs in 10–30% of patients with sickle cell disease (SCD) and is associated with higher incidence of mortality in adults (
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      ). Reduced bioavailability of nitric oxide caused by quenching of NO by extracellular hemoglobin (
      • Hsu L.L.
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      Hemolysis in sickle cell mice causes pulmonary hypertension due to global impairment in nitric oxide bioavailability.
      ,
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      • Gladwin M.T.
      The relationship between the severity of hemolysis, clinical manifestations and risk of death in 415 patients with sickle cell anemia in the US and Europe.
      ) and increased levels of endothelin-1 (ET-1) have been implicated in PH in SCD (
      • Rybicki A.C.
      • Benjamin L.J.
      Increased levels of endothelin-1 in plasma of sickle cell anemia patients.
      ,
      • Sundaram N.
      • Tailor A.
      • Mendelsohn L.
      • Wansapura J.
      • Wang X.
      • Higashimoto T.
      • Pauciulo M.W.
      • Gottliebson W.
      • Kalra V.K.
      • Nichols W.C.
      • Kato G.J.
      • Malik P.
      High levels of placenta growth factor in sickle cell disease promote pulmonary hypertension.
      ).
      The endothelins are a family of small peptides that include ET-1, ET-2, and ET-3 and are produced primarily by vascular endothelial cells and to a lesser extent by vascular smooth muscle cells, airway epithelial cells, macrophages, and fibroblast cells (
      • Shao D.
      • Park J.E.
      • Wort S.J.
      The role of endothelin-1 in the pathogenesis of pulmonary arterial hypertension.
      ). ET-1 expression is induced by hypoxia subsequent to HIF-1α activation (
      • Yamashita K.
      • Discher D.J.
      • Hu J.
      • Bishopric N.H.
      • Webster K.A.
      Molecular regulation of the endothelin-1 gene by hypoxia: contributions of hypoxia-inducible factor-1, activator protein-1, GATA-2, AND p300/CBP.
      ). We showed that placenta growth factor (PlGF) induces the expression of ET-1 in cultured human pulmonary microvascular endothelial cells via activation of hypoxia inducible factor (HIF-1α), independently of hypoxia (
      • Patel N.
      • Gonsalves C.S.
      • Malik P.
      • Kalra V.K.
      Placenta growth factor augments endothelin-1 and endothelin-B receptor expression via hypoxia-inducible factor-1α.
      ). Placenta growth factor is elaborated from bone marrow erythroid cells, and its levels are significantly increased in patients with chronic hemolytic anemia, including SCD, because of a compensatory erythroid hyperplastic response (
      • Perelman N.
      • Selvaraj S.K.
      • Batra S.
      • Luck L.R.
      • Erdreich-Epstein A.
      • Coates T.D.
      • Kalra V.K.
      • Malik P.
      Placenta growth factor activates monocytes and correlates with sickle cell disease severity.
      ). Moreover, our studies show elevated PlGF levels in Berkeley sickle (BK-SS) mice and SCD patients are associated with increased levels of ET-1 and clinical markers of pulmonary hypertension (
      • Sundaram N.
      • Tailor A.
      • Mendelsohn L.
      • Wansapura J.
      • Wang X.
      • Higashimoto T.
      • Pauciulo M.W.
      • Gottliebson W.
      • Kalra V.K.
      • Nichols W.C.
      • Kato G.J.
      • Malik P.
      High levels of placenta growth factor in sickle cell disease promote pulmonary hypertension.
      ). These studies clearly indicate the importance of PlGF in regulation of ET-1 and its role in PH in SCD.
      We previously showed reduced expression of miR-30c and miR-301a in human pulmonary microvascular endothelial cells, in response to PlGF (
      • Patel N.
      • Tahara S.M.
      • Malik P.
      • Kalra V.K.
      Involvement of miR-30c and miR-301a in immediate induction of plasminogen activator inhibitor-1by placenta growth factor in human pulmopnary endothelial cells.
      ). These miRNAs target the 3′-UTR of PAI-1 mRNA and are implicated in regulating the steady state level of this mRNA under basal conditions (
      • Patel N.
      • Tahara S.M.
      • Malik P.
      • Kalra V.K.
      Involvement of miR-30c and miR-301a in immediate induction of plasminogen activator inhibitor-1by placenta growth factor in human pulmopnary endothelial cells.
      ). Others have shown participation of miR-199a as a post-transcriptional regulator of HIF-1α under hypoxia for cell proliferation of non-small cell lung cancer cells (
      • Ding G.
      • Huang G.
      • Liu H.-D.
      • Liang H.-X.
      • Ni Y.-F.
      • Ding Z.-H.
      • Ni G.-Y.
      • Hua H.W.
      MiR-199a suppresses the hypoxia-induced proliferation of non-small cell lung cancer cells through targeting HIF1α.
      ). However, the regulation of miR-199a availability in the context of SCD has not been previously studied, especially in the absence of hypoxia (
      • Patel N.
      • Gonsalves C.S.
      • Malik P.
      • Kalra V.K.
      Placenta growth factor augments endothelin-1 and endothelin-B receptor expression via hypoxia-inducible factor-1α.
      ,
      • Ding G.
      • Huang G.
      • Liu H.-D.
      • Liang H.-X.
      • Ni Y.-F.
      • Ding Z.-H.
      • Ni G.-Y.
      • Hua H.W.
      MiR-199a suppresses the hypoxia-induced proliferation of non-small cell lung cancer cells through targeting HIF1α.
      ). This type of post-transcriptional regulation would be expected to directly affect downstream HIF-1α-dependent gene expression events.
      In the present study, we show that PlGF reduced miR-199a2 levels in cultured human endothelial cells and that miR-199a2 targets the HIF-1α 3′-UTR for turnover. The miR-199a2 precursor is transcribed as a Dynamin-3 opposite strand (DNM3os) RNA polymerase II transcript as previously shown for the mouse DNM3 locus (
      • Loebel D.A.
      • Tsoi B.
      • Wong N.
      • Tam P.P.
      A conserved noncoding intronic transcript at the mouse Dnm3 locus.
      ). The human DNM3os transcript is also predicted to include miR-214 in the same transcription unit (
      • el Azzouzi H.
      • Leptidis S.
      • Dirkx E.
      • Hoeks J.
      • van Bree B.
      • Brand K.
      • McClellan E.A.
      • Poels E.
      • Sluimer J.C.
      • van den Hoogenhof M.M.
      • Armand A.-S.
      • Yin X.
      • Langley S.
      • Bourajjaj M.
      • Olieslagers S.
      • Krishnan J.
      • Vooijs M.
      • Kurihara H.
      • Stubbs A.
      • Pinto Y.M.
      • Krek W.
      • Mayr M.
      • da Costa Martins P.A.
      • Schrauwen P.
      • De Windt L.J.
      The hypoxia-inducible microRNA cluster miR-199a∼214 targets myocardial PPARδ and impairs mitochondrial fatty acid oxidation.
      ). Our studies for the first time show that DNM3os and premiR-199a2 transcription was co-regulated by PPARα. Moreover, PPARα agonist augmented the expression of premiR-199a2 and concomitantly attenuated the activation of HIF-1α, thus reducing the expression of ET-1 in cultured endothelial cells. It is pertinent to note that Staels and co-workers (
      • Glineur C.
      • Gross B.
      • Neve B.
      • Rommens C.
      • Chew G.T.
      • Martin-Nizard F.
      • Rodríguez-Pascual F.
      • Lamas S.
      • Watts G.F.
      • Staels B.
      Fenofibrate inhibits endothelin-1 expression by peroxisome proliferator-activated receptor α-dependent and independent mechanisms in human endothelial cells.
      ) showed that the PPARα agonist fenofibrate attenuated ET-1 expression in endothelial cells by increasing expression of transcriptional repressor Kruppel-like factor 11 (KLF11) and its binding to the ET-1 gene promoter. In our studies, fenofibrate acted to increase transcription from the DNM3os locus leading to higher miR-199a2 expression with a resultant decline of HIF-1α activity and subsequent reduction of ET-1 transcription.
      These studies were corroborated in SCD patients, wherein we observed significantly reduced plasma levels of miR-199a-5p, indicating that decreased levels of miR-199a-5p contributed to increased levels of HIF-1α and its regulated gene, i.e. ET-1. We also 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 miR-199a2 to reduction of ET-1, thus providing a rationale for a therapeutic approach to attenuate ET-1 levels and ameliorate pulmonary hypertension in SCD.

      DISCUSSION

      We have previously shown that plasma levels of PlGF, an angiogenic growth factor, produced by erythroid cells, are high in SCD patients. This correlates with an increase in plasma levels of ET-1 and tricuspid regurgitant jet velocity; the latter is a marker of PH in SCD (
      • Sundaram N.
      • Tailor A.
      • Mendelsohn L.
      • Wansapura J.
      • Wang X.
      • Higashimoto T.
      • Pauciulo M.W.
      • Gottliebson W.
      • Kalra V.K.
      • Nichols W.C.
      • Kato G.J.
      • Malik P.
      High levels of placenta growth factor in sickle cell disease promote pulmonary hypertension.
      ). Similar to patients with SCD, Berkeley sickle mice exhibit elevated plasma levels of PlGF and ET-1 and right ventricular hypertrophy (
      • Sundaram N.
      • Tailor A.
      • Mendelsohn L.
      • Wansapura J.
      • Wang X.
      • Higashimoto T.
      • Pauciulo M.W.
      • Gottliebson W.
      • Kalra V.K.
      • Nichols W.C.
      • Kato G.J.
      • Malik P.
      High levels of placenta growth factor in sickle cell disease promote pulmonary hypertension.
      ). Moreover, stimulated erythroid expression of PlGF in normal mice, up to the levels seen in sickle mice, results in increased ET-1 production and associated right ventricular hypertrophy with pulmonary changes within a time period of 8 weeks (
      • Sundaram N.
      • Tailor A.
      • Mendelsohn L.
      • Wansapura J.
      • Wang X.
      • Higashimoto T.
      • Pauciulo M.W.
      • Gottliebson W.
      • Kalra V.K.
      • Nichols W.C.
      • Kato G.J.
      • Malik P.
      High levels of placenta growth factor in sickle cell disease promote pulmonary hypertension.
      ). These studies showed that PlGF-induced ET-1 expression in vivo plays a significant role in development of PH in SCD.
      Our previous studies in cultured endothelial cells show that PlGF induces expression of ET-1 via activation of HIF-1α, independently of hypoxia. This involves binding of HIF-1α to hypoxia response elements in the promoter of ET-1 (
      • Patel N.
      • Gonsalves C.S.
      • Malik P.
      • Kalra V.K.
      Placenta growth factor augments endothelin-1 and endothelin-B receptor expression via hypoxia-inducible factor-1α.
      ).
      In the present study, we examined the post-transcriptional mechanism of PlGF-mediated ET-1 expression. We showed that PlGF treatment of endothelial cells (HMEC) attenuated both premiR-199a2/premiR-214 and mature miR-199a2/miR-214 levels. Based on several public data sets, both miR-199a2 and miR-214 are predicted to target multiple genes. Previous studies showed that miR-199a2 targets the 3′-UTR of HIF-1α mRNA in mouse cardiac myocytes (
      • Rane S.
      • He M.
      • Sayed D.
      • Vashistha H.
      • Malhotra A.
      • Sadoshima J.
      • Vatner D.E.
      • Vatner S.F.
      • Abdellatif M.
      Downregulation of miR-199a derepresses hypoxia-inducible factor-1α and sirtuin 1 and recapitulates hypoxia preconditioning in cardiac myocytes.
      ). Consequently we focused our studies on the human HIF-1α 3′-UTR as it harbors a complementary sequence to the miR-199a2 seed sequence (positions +16 to +38 of the 3′-UTR); HIF-1α mRNA has no complementary sites for miR-214 binding. The specificity of miR-199a2 binding to the HIF-1α mRNA miR-199a MRE was established using a luciferase reporter gene containing the HIF-1α 3′-UTR. This translation reporter responded to synthetic miR-199a2 mimic, which attenuated basal and PlGF-induced luciferase reporter activity. Moreover, the functionality of the MRE sequence was established by mutation of the seed region complement for miR-199a2 in the 3′-UTR of the luciferase reporter, thus confirming the presence of the miR-199a response element in the 3′-UTR of HIF-1α mRNA. These results also strongly implicated miR-199a2 involvement in post-transcriptional regulation of HIF-1α expression in response to PlGF. Indeed, transfection of HMEC cells with miR-199a mimic attenuated endogenous HIF-1α mRNA and protein and concomitantly attenuated basal and PlGF-induced ET-1 expression.
      Previous studies have shown miR-199a is down-regulated in cardiac myocytes in response to low oxygen tension, which is required for the up-regulation of target HIF-1α (
      • Rane S.
      • He M.
      • Sayed D.
      • Vashistha H.
      • Malhotra A.
      • Sadoshima J.
      • Vatner D.E.
      • Vatner S.F.
      • Abdellatif M.
      Downregulation of miR-199a derepresses hypoxia-inducible factor-1α and sirtuin 1 and recapitulates hypoxia preconditioning in cardiac myocytes.
      ). Hypoxia was also shown to regulate expression of several other miRNAs that post-transcriptionally modulate levels of a variety of mRNAs, including HIF-1α (
      • Nallamshetty S.
      • Chan S.Y.
      • Loscalzo J.
      Hypoxia: a master regulator of microRNA biogenesis and activity.
      ). Indeed, our studies demonstrated that hypoxia reduces miR-199a2 levels in endothelial cells, which is manifested by increased HIF-1α expression as previously shown (
      • Gonsalves C.S.
      • Kalra V.K.
      Hypoxia-mediated expression of 5-lipoxygenase-activating protein involves HIF-1α and NF-κB and microRNAs 135a and 199a-5p.
      ).
      miR-199a2 is located within the DNM3os transcription unit. This long noncoding RNA transcript is responsible for synthesis of the miRNA cluster containing miR-199a2 and miR-214 and is involved in skeletal development (
      • Watanabe T.
      • Sato T.
      • Amano T.
      • Kawamura Y.
      • Kawamura N.
      • Kawaguchi H.
      • Yamashita N.
      • Kurihara H.
      • Nakaoka T.
      Dnm3os, a non-coding RNA, is required for normal growth and skeletal development in mice.
      ,
      • Desvignes T.
      • Contreras A.
      • Postlethwait J.H.
      Evolution of the miR199–214 cluster and vertebrate skeletal development.
      ). Accordingly it was necessary to determine whether PlGF regulated synthesis of the long DNM3os transcript, as previously observed, or instead led to independent transcription of premir-199a2. Our studies showed that PlGF coordinately attenuated RNA levels of DNM3os, premiR-199a2, and premiR-214 in cultured human endothelial cells. As a consequence of reduced miR-199a2 in PlGF-treated conditions, we expected increased levels of HIF-1α and ET-1 expression as was observed.
      We observed significant reduction of miR-199a in plasma from patients with SCD compared with unaffected sibling controls. Reduced levels of miR-199a in SCD would be expected to decrease the turnover of HIF-1α mRNA leading to increased HIF-1α protein and subsequent increased levels of ET-1, as was observed (
      • Sundaram N.
      • Tailor A.
      • Mendelsohn L.
      • Wansapura J.
      • Wang X.
      • Higashimoto T.
      • Pauciulo M.W.
      • Gottliebson W.
      • Kalra V.K.
      • Nichols W.C.
      • Kato G.J.
      • Malik P.
      High levels of placenta growth factor in sickle cell disease promote pulmonary hypertension.
      ). Thus, both in vitro and in vivo results support this model (Fig. 7G) whereby reduced expression of miR-199a2 leads to increased expression of HIF-1α in SCD and is reflected by increased HIF-1α-dependent gene expression.
      Recent studies by others show that myocardial hypoxia in mice, induced by transverse aortic constriction, led to increased expression of DNM3os and concomitant miR-199a/miR214 expression (
      • el Azzouzi H.
      • Leptidis S.
      • Dirkx E.
      • Hoeks J.
      • van Bree B.
      • Brand K.
      • McClellan E.A.
      • Poels E.
      • Sluimer J.C.
      • van den Hoogenhof M.M.
      • Armand A.-S.
      • Yin X.
      • Langley S.
      • Bourajjaj M.
      • Olieslagers S.
      • Krishnan J.
      • Vooijs M.
      • Kurihara H.
      • Stubbs A.
      • Pinto Y.M.
      • Krek W.
      • Mayr M.
      • da Costa Martins P.A.
      • Schrauwen P.
      • De Windt L.J.
      The hypoxia-inducible microRNA cluster miR-199a∼214 targets myocardial PPARδ and impairs mitochondrial fatty acid oxidation.
      ). Herein, it was shown that miR-199a/miR-214 target PPARδ mRNA for turnover, thus resulting in decreased PPARδ activity leading to defective mitochondrial respiration, as a consequence of flawed fatty acid substrate metabolism. It was suggested in their model, that miR-199a could also target HIF-1α mRNA for turnover. Reduction of HIF-1α levels would lead to decreased DNM3os transcription and miRNA synthesis because the promoter for the latter has HRE and Twist binding sites (
      • el Azzouzi H.
      • Leptidis S.
      • Dirkx E.
      • Hoeks J.
      • van Bree B.
      • Brand K.
      • McClellan E.A.
      • Poels E.
      • Sluimer J.C.
      • van den Hoogenhof M.M.
      • Armand A.-S.
      • Yin X.
      • Langley S.
      • Bourajjaj M.
      • Olieslagers S.
      • Krishnan J.
      • Vooijs M.
      • Kurihara H.
      • Stubbs A.
      • Pinto Y.M.
      • Krek W.
      • Mayr M.
      • da Costa Martins P.A.
      • Schrauwen P.
      • De Windt L.J.
      The hypoxia-inducible microRNA cluster miR-199a∼214 targets myocardial PPARδ and impairs mitochondrial fatty acid oxidation.
      ). By contrast, our present studies showed that hypoxia and PlGF in cultured endothelial cells reduced expression of DNM3os, premiR-199a2, and premiR-214, resulting in higher expressed levels of HIF-1α and increased expression of ET-1, with the latter a key indicator of HIF-1α-dependent transcription. These results are consistent with previous studies that showed hypoxia up-regulated ET-1 expression via activation of HIF-1α (
      • Yamashita K.
      • Discher D.J.
      • Hu J.
      • Bishopric N.H.
      • Webster K.A.
      Molecular regulation of the endothelin-1 gene by hypoxia: contributions of hypoxia-inducible factor-1, activator protein-1, GATA-2, AND p300/CBP.
      ,
      • Kourembanas S.
      • Marsden P.A.
      • McQuillan L.P.
      • Faller D.V.
      Hypoxia induces endothelin gene expression and secretion in cultured human endothelium.
      ). Further work is warranted to delineate the mechanism used during hypoxia to regulate DNM3os transcription.
      We examined the transcriptional regulation of miR-199a2 and its host gene DNM3os in cultured endothelial cells. Utilizing PPARα shRNA and PPARα agonist, fenofibrate, we showed PPARα up-regulated the expression of DNM3os, pre-miR199a2, and miR-199a2 RNAs. The effect was specific for PPARα because an antagonist to PPARα, GW6471, antagonized the stimulatory effect of fenofibrate. In the promoter of DNM3os, there are three predicted PPARα cis-binding elements proximal to the transcription start site. These PPARα binding elements were involved in DNM3os transcription as demonstrated by DNM3os-promoter-luciferase reporter assays and response to PPARα agonist, fenofibrate. We refined our analysis of the PPARα cis-binding sites to establish whether one or more of these PPARα elements were required for DNM3os transcription in response to fenofibrate. Single PPARα site mutations were constructed for this purpose whereupon it was found that two of the three PPARα sites were required and functional for PPARα-dependent transcription of DNM3os, as determined by response to fenofibrate. These results were further extended by analysis of physiological interactions of PPARα with the DNM3os promoter in intact HMEC-1 cells as detected by ChIP assay. Thus, the transcription activity of the DNM3os locus under native conditions paralleled the transcription activity observed in reporter assays.
      The present study demonstrated for the first time to the best of our knowledge that transcription factor PPARα co-regulated the transcription of DNM3os and miR-199a2. This relationship between PPARα, HIF-1α, and a downstream target gene, ET-1, is summarized in our working model (Fig. 7G).
      Previous studies show that the PPARα agonist fenofibrate attenuates ET-1 expression in a PPARα-dependent mechanism via activation of a Kruppel-like factor (KLF-11) (
      • Glineur C.
      • Gross B.
      • Neve B.
      • Rommens C.
      • Chew G.T.
      • Martin-Nizard F.
      • Rodríguez-Pascual F.
      • Lamas S.
      • Watts G.F.
      • Staels B.
      Fenofibrate inhibits endothelin-1 expression by peroxisome proliferator-activated receptor α-dependent and independent mechanisms in human endothelial cells.
      ) and by inhibition of AP-1 signaling pathways (
      • Delerive P.
      • Martin-Nizard F.
      • Chinetti G.
      • Trottein F.
      • Fruchart J.-C.
      • Najib J.
      • Duriez P.
      • Staels B.
      Peroxisome proliferator-activated receptor activators inhibit thrombin-induced endothelin-1 production in human vascular endothelial cells by inhibiting the activator protein-1 signaling pathway.
      ). As previously reported, fibrates suppress HIF-1α activation in cancer cells in response to hypoxia (
      • Zhou J.
      • Zhang S.
      • Xue J.
      • Avery J.
      • Wu J.
      • Lind S.E.
      • Ding W.Q.
      Activation of peroxisome proliferator-activated receptor α (PPARα) suppresses hypoxia-inducible factor-1α (HIF-1α) signaling in cancer cells.
      ); we also observed that fibrates attenuated PlGF-induced HIF-1α mRNA levels in endothelial cells. In the present study, we showed PlGF-induced ET-1 transcription was completely attenuated by fenofibrate. A more detailed examination of the ET-1 promoter was undertaken, whereupon we observed that the 669-bp, 5′-flanking DNA contained KLF-11 and HRE cis-binding elements. The relative contributions of the KLF-11 sites and the single HRE were examined in luciferase reporter constructs by comparing a truncated promoter (176 bp) to the full-length promoter. The former completely lacked the three KLF-11 sites but still maintained responsiveness to PlGF and HIF-1α. Based on the results of this analysis combined with fenofibrate response, we estimated that HIF-1α contributed ∼60% to the overall expression of ET-1; the remaining contribution to expression was effected by derepression of the ET-1 gene by PlGF-mediated release of KLF-11 from the promoter by another regulatory pathway.
      Fibrates are approved by the Food and Drug Administration for the treatment of dyslipidemia (
      • Lalloyer F.
      • Staels B.
      Fibrates, glitazones, and peroxisome proliferator-activated receptors.
      ). These PPAR-α agonists exhibit improvement of flow-mediated dilation of bronchial arteries in type 2 diabetes mellitus patients (
      • Glineur C.
      • Gross B.
      • Neve B.
      • Rommens C.
      • Chew G.T.
      • Martin-Nizard F.
      • Rodríguez-Pascual F.
      • Lamas S.
      • Watts G.F.
      • Staels B.
      Fenofibrate inhibits endothelin-1 expression by peroxisome proliferator-activated receptor α-dependent and independent mechanisms in human endothelial cells.
      ) and diabetic retinopathy in type I diabetic animal models (
      • Chen Y.
      • Hu Y.
      • Lin M.
      • Jenkins A.J.
      • Keech A.C.
      • Mott R.
      • Lyons T.J.
      • Ma J.X.
      Therapeutic effects of PPARα agonists on diabetic retinopathy in type 1 diabetes models.
      ). Thus, we asked whether this drug could be potentially beneficial in SCD treatment, given our experimental observation that miR-199a2 synthesis was positively regulated by PPARα. As a first step, we examined the effect of feeding fenofibrate to Berkeley sickle mice. Results from these studies demonstrated that 10-week administration of fenofibrate to these mice increased miR-199a2 in the lung tissues of these mice, as well as WT control mice. There was a significant reduction of HIF-1α and ET-1 mRNAs in isolated endothelial cells from these lungs, thus demonstrating that fenofibrate was effective in vivo as a means of attenuating HIF-1α levels and decreasing ET-1 expression, consistent with the in vitro model (Fig. 7G). However, long term (6–12 months) fenofibrate feeding studies in a larger cohort of BK-SS mice coupled with physiological measurements of pulmonary functions are warranted to conclusively demonstrate a beneficial effect of fenofibrate in alleviating PH in the SCA animal model.
      In conclusion, our data showed that miR-199a2 has a significant role in overall regulation of HIF-1α activity. It is clear that in addition to post-transcriptional regulatory events regulating the basal level of this factor through regulated proteolysis, there is also a mechanism for modulating synthesis of this important transcription factor at the level of mRNA translation. In the course of our studies, we discovered that PPARα is capable of regulating the expression of miR-199a2 via transcription of its host gene DNM3os. In sickle subjects, miR-199a2 levels are significantly reduced, which in turn leads to increased levels of HIF-1α and abnormal levels of ET-1, the latter of which is correlated as causal in PH (
      • Sundaram N.
      • Tailor A.
      • Mendelsohn L.
      • Wansapura J.
      • Wang X.
      • Higashimoto T.
      • Pauciulo M.W.
      • Gottliebson W.
      • Kalra V.K.
      • Nichols W.C.
      • Kato G.J.
      • Malik P.
      High levels of placenta growth factor in sickle cell disease promote pulmonary hypertension.
      ). These studies provide a mechanistic link for the transcriptional regulation of miR-199a2 by fibrates, thus providing a therapeutic rationale for attenuating elevated ET-1 levels observed during pulmonary hypertension in SCA.

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