Hypoxia mediates mutual repression between microRNA-27a and PPARγ in the pulmonary vasculature.

Pulmonary hypertension (PH) is a serious disorder that causes significant morbidity and mortality. The pathogenesis of PH involves complex derangements in multiple pathways including reductions in peroxisome proliferator-activated receptor gamma (PPARγ). Hypoxia, a common PH stimulus, reduces PPARγ in experimental models. In contrast, activating PPARγ attenuates hypoxia-induced PH and endothelin 1 (ET-1) expression. To further explore mechanisms of hypoxia-induced PH and reductions in PPARγ, we examined the effects of hypoxia on selected microRNA (miRNA or miR) levels that might reduce PPARγ expression leading to increased ET-1 expression and PH. Our results demonstrate that exposure to hypoxia (10% O2) for 3-weeks increased levels of miR-27a and ET-1 in the lungs of C57BL/6 mice and reduced PPARγ levels. Hypoxia-induced increases in miR-27a were attenuated in mice treated with the PPARγ ligand, rosiglitazone (RSG, 10 mg/kg/d) by gavage for the final 10 d of exposure. In parallel studies, human pulmonary artery endothelial cells (HPAECs) were exposed to control (21% O2) or hypoxic (1% O2) conditions for 72 h. Hypoxia increased HPAEC proliferation, miR-27a and ET-1 expression, and reduced PPARγ expression. These alterations were attenuated by treatment with RSG (10 µM) during the last 24 h of hypoxia exposure. Overexpression of miR-27a or PPARγ knockdown increased HPAEC proliferation and ET-1 expression and decreased PPARγ levels, whereas these effects were reversed by miR-27a inhibition. Further, compared to lungs from littermate control mice, miR-27a levels were upregulated in lungs from endothelial-targeted PPARγ knockout (ePPARγ KO) mice. Knockdown of either SP1 or EGR1 was sufficient to significantly attenuate miR-27a expression in HPAECs. Collectively, these studies provide novel evidence that miR-27a and PPARγ mediate mutually repressive actions in hypoxic pulmonary vasculature and that targeting PPARγ may represent a novel therapeutic approach in PH to attenuate proliferative mediators that stimulate proliferation of pulmonary vascular cells

microRNA levels in hypoxia-exposed C57BL/6 mice and HPAECs.

<p><b>A.</b> C57BL/6 mice were exposed to normoxia (NOR, 21% O₂) or hypoxia (HYP, 10% O₂) for 3-wks. qRT-PCR was performed on lung tissue for selected miRNAs predicted to regulate PPARγ expression. miRNA levels are expressed relatives to RNU6B and normalized to control values. *p<0.05 vs. NOR, n = 6–7. <b>B.</b> HPAEC were exposed to NOR or HYP (1% O₂) in vitro for 72 h. n = 3, * p<0.05 vs. NOR. C. HPAECs were also examined following exposure to hypoxia for 0, 24, 48, or 72 h. HPAEC miRNA was isolated and subjected to qRT-PCR analysis along with RNU6B. Each bar represents the mean ± SE miR-27a relative to RNU6B expressed as fold change vs. SCR. n = 3, *p<0.05 vs. NOR. ⁺p<0.05 vs. 24 h.</p

Putative pathways by which PPARγ and miR-27a regulate ET-1 in PH.

<p>The current findings indicate that hypoxia increases lungs miR-27a expression and that increases in miR-27a reduce PPARγ levels which stimulates increased ET-1 levels and pulmonary vascular cell proliferation. Conversely, activating PPARγ with rosiglitazone attenuated hypoxic upregulation of miR-27a and ET-1. These findings suggest that PPARγ ligands attenuate alterations in miR-27a and ET-1 levels to reduce PH.</p

PPARγ is a target gene of miR-27a.

<p><b>A.</b> Schematic illustration of the human PPARγ 3′UTR which contains a putative binding site (open arrowhead) for miR-27a. The miR-27a seed sequence is shown in bold font. <b>B.</b> HPAECs were incubated in a 24-well plate (1×10⁴ cells/well) for 24 h, washed with PBS, and then fresh growth medium was added before transfection with PPARγ-3′UTR luciferase reporter constructs. Wild-type vector (psiCHECK2) or psiCHECK2-PPARγ construct, or mut-3′UTR construct with or without 30 nM of miR-27a mimic or scrambled miRNA (SCR) were transiently co-transfected into HPAECs. After 72 h, HPAEC lysates were harvested and analyzed for <i>Renilla</i> and firefly luciferase activities. Each bar represents <i>Renilla</i> luciferase activity in relative light units normalized to firefly luciferase activity and expressed relative to HPAECs treated with SCR constructs. n = 3, * p<0.05 vs. SCR-PPARγ-3′UTR constructs.</p

Knockdown of transcription factors, SP1 and EGR1, attenuates miR-27a expression.

<p>HPAEC were treated with 10(SCR) or siRNA duplexes to SP1 or EGR1 for 72 hours. HPAEC mRNA or proteins were then isolated. qRT-PCR was performed demonstrating decreases in SP1 (<b>A</b>) or EGR1 (<b>C</b>) mRNA levels following treatment with siRNA. Each bar represents the mean ± SE SP1 or EGR1 relative to 9S in the same sample expressed as fold-change vs. cells treated with scrambled siRNA (SCR). n = 3, *p<0.05 vs. SCR. Western blotting was employed to detect SP1 (<b>A</b>) or EGR1 (<b>C</b>) protein levels. Representative blots depicting SP1 or EGR1 protein depletion in siRNA-treated HPAEC is shown. n = 3, *p<0.05 vs. SCR. HPAECs were treated with SCR or siRNA duplexes to SP1 or EGR1, and miRNA was isolated and subjected to qRT-PCR analysis along with RNU6B. Each bar represents the mean ± SE miR-27a relative to RNU6B expressed as fold change vs. SCR. n = 3, *p<0.05 vs. SCR. miR-27a levels were downregulated following SP1 (<b>B</b>) or EGR1 (<b>D</b>) depletion in HPAECs.</p

siRNA-mediated reductions in PPARγ increased HPAEC ET-1, proliferation, and miR-27a levels.

<p>HPAEC were treated with scrambled (SCR) or siRNA (100 nM) to PPARγ for 72 hours. HPAEC mRNA or proteins were then isolated. In selected studies, HPAEC were subjected to MTT assays of proliferation. <b>A.</b> qRT-PCR was performed demonstrating decreases in PPARγ mRNA levels following treatment with siRNA. Each bar represents the mean ± SE PPARγ or ET-1 relative to 9S in the same sample expressed as fold-change vs. cells treated with scrambled siRNA (SCR). n = 4–9, *p<0.05 vs. SCR. <b>B.</b> HPAECs were treated with 100 nM SCR or PPARγ siRNA, and proteins were subjected to Western blotting for PPARγ, ET-1, or β-actin (ACTB). As illustrated by these representative Western blots, PPARγ depletion reduced HPAEC PPARγ protein levels, and increased levels of ET-1. <b>C.</b> HPAEC were treated with 100 nM SCR or siPPARγ and subjected to MTT proliferation assays. n = 6. *p<0.05 vs. SCR. <b>D.</b> HPAECs were treated with SCR or siPPARγ, and miRNA was isolated and subjected to qRT-PCR analysis along with RNU6B. Each bar represents the mean ± SE miR-27a relative to RNU6B expressed as fold changed vs. SCR. n = 4, *p<0.05 vs. SCR. <b>E.</b> miR-27a levels were upregulated in lungs from endothelial-targeted PPARγ knockout (ePPARγ KO) mice. miR-27a levels in lungs from endothelial-targeted PPARγ KO mice were determined with qRT-PCR. Each bar represents the mean ± SE level of miR-27a relative to RNU6B expressed as fold-change vs. littermate controls (LitCon), n = 3–6. *p<0.05 vs. LitCon.</p

A PPARγ ligand attenuates hypoxic increases in miR-27a levels in mouse lung or in HPAECs.

<p><b>A.</b> Whole lung homogenates were collected from mice exposed to normoxia (NOR, 21% O₂) or hypoxia (HYP, 10% O₂) for 3-weeks. During the last 10-days of this exposure, selected animals were also treated ± rosiglitazone (RSG, 10 mg/kg/d by gavage) as we have reported. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079503#pone.0079503-Nisbet1" target="_blank">[12]</a> Lung miR-27a levels were measured with qRT-PCR and are expressed relative to lung RNU6B. *p<0.05 vs. NOR, ⁺p<0.05 vs. HYP, n = 3–4. <b>B.</b> HPAECs were exposed to NOR (21% O₂) or HYP (1% O₂) for 72 h<b>.</b> Selected cells were treated during the final 24 h of exposure with RSG (10 µM). Each bar represents the mean miR-27a level relative to RNU6B ± SE. *p<0.05 vs. NOR, ⁺p<0.05 vs. HYP, n = 3–6.</p

Anti-miR-27a reduced HPAEC miR-27a levels, HPAEC proliferation, and ET-1 levels and increased PPARγ expression.

<p>HPAECs were treated with either scrambled (SCR) or 25–50 nM anti-miR-27a for 72 hours. HPAEC were then collected, and RNA was isolated. qRT-PCR was performed for miR-27a, PPARγ, and ET-1 levels. Western blotting was employed to detect PPARγ and ET-1 protein levels. HPAEC proliferation was determined using MTT assays. Each bar represents the mean ± SE miR-27a/RNU6B (<b>A</b>), proliferation (<b>B</b>), PPARγ or ET-1 mRNA relative to ribosomal S9 (9S RNA) (<b>C</b>), or PPARγ or ET-1 protein relative to CDK4 protein (<b>D</b>) expressed as fold-change vs. SCR. *p<0.05 vs. scrambled (SCR) miRNA, n = 3–5.</p