MicroRNA 648 Targets ET-1 mRNA and Is Cotranscriptionally Regulated with MICAL3 by PAX5

Abstract

c Pulmonary hypertension (PHT) is associated with high mortality in sickle cell anemia (SCA). Previously, we showed that elevated levels of placenta growth factor (PlGF) in SCA patients correlate with increased levels of the potent vasoconstrictor endothelin-1 (ET-1) and PHT. Moreover, PlGF induced the expression of ET-1 via hypoxia-inducible factor 1␣. Here, we show a novel example of ET-1 posttranscriptional regulation by PlGF via action of microRNA 648 (miR-648), which is subject to transcriptional coregulation with its host gene, MICAL3 (microtubule-associated monooxygenase, calponin, and LIM domain containing 3gene). PlGF repressed expression of miR-648 in endothelial cells. Luciferase reporter assays using wild-type and mutant ET-1 3= untranslated region (UTR) constructs, and transfection of miR-648 mimics showed that miR-648 targets the 3= UTR of ET-1 mRNA. Since miR-648 is located in a 5=-proximal intron of MICAL3, we examined which of three potential promoters was responsible for its expression. The MICAL3 distal promoter (P1) was the predominant promoter used for transcription of premiR-648, and it was under positive control by PAX5 (paired box protein 5) transcription factor, as demonstrated by the loss and gain of function of PAX5 activity, and chromatin immunoprecipitation analysis. These studies provide a novel link wherein PlGF-mediated downregulation of PAX5 attenuates miR-648 expression leading to increased ET-1 levels that are known to induce PHT in SCA. Endothelin-1 (ET-1), a 21-amino-acid peptide hormone primarily synthesized and secreted by endothelium in vivo, is involved in proliferation of smooth muscle cells and vascular tone (1-4). In the vasculature, the endothelin system, comprising endothelin ligands (ET-1, ET-2, and ET-3), endothelin receptors (ET-AR and ET-BR), and two activating peptidases, has a basal vasoconstricting role; dysfunction contributes to the development of diseases such as hypertension and other cardiovascular diseases (3). Hypoxia is a potent inducer of ET-1 gene expression in endothelial cells via activation of hypoxia-inducible factor 1␣ (HIF-1␣) (5, 6).Pulmonary hypertension (PHT), occurs in ca. 10% of adults with sickle cell anemia (SCA), and its diagnosis is associated with a 38 to 40% mortality at 2 to 6 years (7,8). Sickle mice develop PHT with increasing age, manifested as high pulmonary artery pressures and right ventricular hypertrophy (9). Physiological factors implicated in PHT in SCA include endothelial dysfunction, pulmonary vasoconstriction, and vascular remodeling, all of which are associated with chronic hemolysis, hypoxia, hemostatic activation, and inflammation (8, 10-12). ET-1 and nitric oxide (NO) are mutually opposing pulmonary vasoactive factors that regulate pulmonary vascular tone. It is postulated that hemolysis leads to quenching of NO by extracellular/cell-free hemoglobin, thereby reducing NO bioavailability (11, 13), which in turn leads to the clinical manifestations of sickle PHT (13-15). Numerous studies in SCA and other hemolytic anem...

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

“…Furthermore, PlGF was found to attenuate the expression of miR-199a-5p by 10-fold (48). 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.…”

Placenta growth factor mediated gene regulation in sickle cell disease

“…Since miR-648 post-transcriptionally modulated ET-1 expression, the plasma levels of miR-648 were examined in SCA patients and healthy matched controls. These data show miR-648 levels are ~4-times lower in SCA patients compared to unaffected controls (71). These studies in vivo support the inverse relationship between miR-648 and ET-1 observed in cultured endothelial cells in response to PlGF (71).…”

“…The role of miRNAs as biomarkers and regulators of gene expression and their role in physiological and pathological processes have been addressed in several reviews (66–70). In the context of SCA, studies show PlGF attenuates levels of miR-648, with seed sequence complementary to the 3′-UTR of ET-1, in cultured endothelial cells (71). To examine the transcription of miR-648, a bioinformatics analysis of the gene encompassing miR-648 was examined in UCSC browser.…”

“…miR-648 is located in a 5′-proximal intron of MICAL3 gene (a member of the MICAL family of flavoprotein monoxygenases) (72). Studies show both MICAL3 and pre-miR648 are co-transcribed, and PAX5 transcription factor is involved in the transcription of MICAL3 (71). Upon synthesis of MICAL3 pre-mRNA, excision of the intron containing pre-miR648 occurs and concomitantly leads to maturation of miRNA as miR-648.…”

Preeclamptic plasma stimulates the expression of miRNAs, leading to a decrease in endothelin-1 production in endothelial cells

“…Recently, it was demonstrated that microRNAs (miRNAs) are involved in the post-transcriptional regulation of the ET-1 gene (EDN1) [22][23][24][25][26]. miRNAs are small (18-24 nucleotides) endogenous noncoding single-stranded RNAs that post-transcriptionally regulate gene expression by repressing protein expression [27].…”