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MicroRNA-124 controls the proliferative, migratory, and inflammatory phenotype of pulmonary vascular fibroblasts.

ABSTRACT: Pulmonary hypertensive remodeling is characterized by excessive proliferation, migration, and proinflammatory activation of adventitial fibroblasts. In culture, fibroblasts maintain a similar activated phenotype. The mechanisms responsible for generation/maintenance of this phenotype remain unknown.We hypothesized that aberrant expression of microRNA-124 (miR-124) regulates this activated fibroblast phenotype and sought to determine the signaling pathways through which miR-124 exerts effects.We detected significant decreases in miR-124 expression in fibroblasts isolated from calves and humans with severe pulmonary hypertension. Overexpression of miR-124 by mimic transfection significantly attenuated proliferation, migration, and monocyte chemotactic protein-1 expression of hypertensive fibroblasts, whereas anti-miR-124 treatment of control fibroblasts resulted in their increased proliferation, migration, and monocyte chemotactic protein-1 expression. Furthermore, the alternative splicing factor, polypyrimidine tract-binding protein 1, was shown to be a direct target of miR-124 and to be upregulated both in vivo and in vitro in bovine and human pulmonary hypertensive fibroblasts. The effects of miR-124 on fibroblast proliferation were mediated via direct binding to the 3' untranslated region of polypyrimidine tract-binding protein 1 and subsequent regulation of Notch1/phosphatase and tensin homolog/FOXO3/p21Cip1 and p27Kip1 signaling. We showed that miR-124 directly regulates monocyte chemotactic protein-1 expression in pulmonary hypertension/idiopathic pulmonary arterial hypertension fibroblasts. Furthermore, we demonstrated that miR-124 expression is suppressed by histone deacetylases and that treatment of hypertensive fibroblasts with histone deacetylase inhibitors increased miR-124 expression and decreased proliferation and monocyte chemotactic protein-1 production.Stable decreases in miR-124 expression contribute to an epigenetically reprogrammed, highly proliferative, migratory, and inflammatory phenotype of hypertensive pulmonary adventitial fibroblasts. Thus, therapies directed at restoring miR-124 function, including histone deacetylase inhibitors, should be investigated.

SUBMITTER: Wang D

PROVIDER: S-EPMC4117588 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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MicroRNA-124 controls the proliferative, migratory, and inflammatory phenotype of pulmonary vascular fibroblasts.

Wang Daren D Zhang Hui H Li Min M Frid Maria G MG Flockton Amanda R AR McKeon B Alexandre BA Yeager Michael E ME Fini Mehdi A MA Morrell Nicholas W NW Pullamsetti Soni S SS Velegala Sivareddy S Seeger Werner W McKinsey Timothy A TA Sucharov Carmen C CC Stenmark Kurt R KR

Circulation research 20131011 1

<h4>Rationale</h4>Pulmonary hypertensive remodeling is characterized by excessive proliferation, migration, and proinflammatory activation of adventitial fibroblasts. In culture, fibroblasts maintain a similar activated phenotype. The mechanisms responsible for generation/maintenance of this phenotype remain unknown.<h4>Objective</h4>We hypothesized that aberrant expression of microRNA-124 (miR-124) regulates this activated fibroblast phenotype and sought to determine the signaling pathways thro ...[more]

PMID: 24122720

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Project description:BackgroundAn emerging metabolic theory of pulmonary hypertension (PH) suggests that cellular and mitochondrial metabolic dysfunction underlies the pathology of this disease. We and others have previously demonstrated the existence of hyperproliferative, apoptosis-resistant, proinflammatory adventitial fibroblasts from human and bovine hypertensive pulmonary arterial walls (PH-Fibs) that exhibit constitutive reprogramming of glycolytic and mitochondrial metabolism, accompanied by an increased ratio of glucose catabolism through glycolysis versus the tricarboxylic acid cycle. However, the mechanisms responsible for these metabolic alterations in PH-Fibs remain unknown. We hypothesized that in PH-Fibs microRNA-124 (miR-124) regulates PTBP1 (polypyrimidine tract binding protein 1) expression to control alternative splicing of pyruvate kinase muscle (PKM) isoforms 1 and 2, resulting in an increased PKM2/PKM1 ratio, which promotes glycolysis and proliferation even in aerobic environments.MethodsPulmonary adventitial fibroblasts were isolated from calves and humans with severe PH (PH-Fibs) and from normal subjects. PTBP1 gene knockdown was achieved via PTBP1-siRNA; restoration of miR-124 was performed with miR-124 mimic. TEPP-46 and shikonin were used to manipulate PKM2 glycolytic function. Histone deacetylase inhibitors were used to treat cells. Metabolic products were determined by mass spectrometry-based metabolomics analyses, and mitochondrial function was analyzed by confocal microscopy and spectrofluorometry.ResultsWe detected an increased PKM2/PKM1 ratio in PH-Fibs compared with normal subjects. PKM2 inhibition reversed the glycolytic status of PH-Fibs, decreased their cell proliferation, and attenuated macrophage interleukin-1β expression. Furthermore, normalizing the PKM2/PKM1 ratio in PH-Fibs by miR-124 overexpression or PTBP1 knockdown reversed the glycolytic phenotype (decreased the production of glycolytic intermediates and byproducts, ie, lactate), rescued mitochondrial reprogramming, and decreased cell proliferation. Pharmacological manipulation of PKM2 activity with TEPP-46 and shikonin or treatment with histone deacetylase inhibitors produced similar results.ConclusionsIn PH, miR-124, through the alternative splicing factor PTBP1, regulates the PKM2/PKM1 ratio, the overall metabolic, proliferative, and inflammatory state of cells. This PH phenotype can be rescued with interventions at various levels of the metabolic cascade. These findings suggest a more integrated view of vascular cell metabolism, which may open unique therapeutic prospects in targeting the dynamic glycolytic and mitochondrial interactions and between mesenchymal inflammatory cells in PH.

Project description:BackgroundChanges in metabolism have been suggested to contribute to the aberrant phenotype of vascular wall cells, including fibroblasts, in pulmonary hypertension (PH). Here, we test the hypothesis that metabolic reprogramming to aerobic glycolysis is a critical adaptation of fibroblasts in the hypertensive vessel wall that drives proliferative and proinflammatory activation through a mechanism involving increased activity of the NADH-sensitive transcriptional corepressor C-terminal binding protein 1 (CtBP1).MethodsRNA sequencing, quantitative polymerase chain reaction,13C-nuclear magnetic resonance, fluorescence-lifetime imaging, mass spectrometry-based metabolomics, and tracing experiments with U-13C-glucose were used to assess glycolytic reprogramming and to measure the NADH/NAD+ ratio in bovine and human adventitial fibroblasts and mouse lung tissues. Immunohistochemistry was used to assess CtBP1 expression in the whole-lung tissues. CtBP1 siRNA and the pharmacological inhibitor 4-methylthio-2-oxobutyric acid (MTOB) were used to abrogate CtBP1 activity in cells and hypoxic mice.ResultsWe found that adventitial fibroblasts from calves with severe hypoxia-induced PH and humans with idiopathic pulmonary arterial hypertension (PH-Fibs) displayed aerobic glycolysis when cultured under normoxia, accompanied by increased free NADH and NADH/NAD+ ratios. Expression of the NADH sensor CtBP1 was increased in vivo and in vitro in fibroblasts within the pulmonary adventitia of humans with idiopathic pulmonary arterial hypertension and animals with PH and cultured PH-Fibs, respectively. Decreasing NADH pharmacologically with MTOB or genetically blocking CtBP1 with siRNA upregulated the cyclin-dependent genes (p15 and p21) and proapoptotic regulators (NOXA and PERP), attenuated proliferation, corrected the glycolytic reprogramming phenotype of PH-Fibs, and augmented transcription of the anti-inflammatory gene HMOX1. Chromatin immunoprecipitation analysis demonstrated that CtBP1 directly binds the HMOX1 promoter. Treatment of hypoxic mice with MTOB decreased glycolysis and expression of inflammatory genes, attenuated proliferation, and suppressed macrophage numbers and remodeling in the distal pulmonary vasculature.ConclusionsCtBP1 is a critical factor linking changes in cell metabolism to cell phenotype in hypoxic and other forms of PH and a therapeutic target.

Project description:Quiescent cells are considered to be dormant. However, recent studies suggest that quiescent fibroblasts possess active metabolic profile and certain functional characteristics. We previously observed that serum-starved quiescent fibroblasts respond to proinflammatory stimuli by robust expression of cyclooxygenase-2 (COX-2), which declines after the quiescent fibroblasts are driven to proliferation. In this study, we elucidated the underlying signaling and transcriptional mechanism and identified by microarray genes with similar differential expression. By using pharmacological inhibitors coupled with gene silencing, we uncovered the key role of protein kinase C ? (PKC?) and extracellular signal regulated protein kinase 1/2 (ERK1/2) signaling in mediating COX-2 expression in quiescent cells. Surprisingly, COX-2 expression in proliferative cells was not blocked by PKC? or ERK1/2 inhibitors due to intrinsic inhibition of PKC? and ERK1/2 in proliferative cells. Restrained COX-2 transcription in proliferative cells was attributable to reduced NF-?B binding. Microarray analysis identified 35 genes whose expressions were more robust in quiescent than in proliferative cells. A majority of those genes belong to proinflammatory cytokines, chemokines, adhesive molecules and metalloproteinases, which require NF-?B for transcription. Quiescent fibroblasts had a higher migratory activity than proliferative fibroblasts as determined by the transwell assay. Selective COX-2 inhibition reduced migration which was restored by prostaglandin E(2). As COX-2 and inflammatory mediators induce DNA oxidation, we measured 8-hydroxydeoxyguanosine (8-OHdG) in quiescent vs. proliferative fibroblasts. PMA-induced 8-OHdG accumulation was significantly higher in quiescent than in proliferative fibroblasts. These findings indicate that quiescent fibroblasts (and probably other quiescent cells) are at the forefront in mounting inflammatory responses through expression of an array of proinflammatory genes via the PKC?/ERK1/2 signaling pathway.

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MicroRNA-124 controls the proliferative, migratory, and inflammatory phenotype of pulmonary vascular fibroblasts.

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MicroRNA-124 controls the proliferative, migratory, and inflammatory phenotype of pulmonary vascular fibroblasts.

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