title: Hypoxia downregulates PPARγ via an ERK1/2-NF-κB-Nox4-dependent mechanism in human pulmonary artery smooth muscle cells.
abstract: The ligand-activated transcription factor peroxisome proliferator-activated receptor γ (PPARγ) regulates metabolism, cell proliferation, and inflammation. Pulmonary hypertension (PH) is associated with reduced PPARγ expression, and hypoxia exposure regimens that cause PH reduce PPARγ expression. This study examines mechanisms of hypoxia-induced PPARγ downregulation in vitro and in vivo. Hypoxia reduced PPARγ mRNA and protein levels, PPARγ activity, and the expression of PPARγ-regulated genes in human pulmonary artery smooth muscle cells (HPASMCs) exposed to 1% oxygen for 72 h. Similarly, exposure of mice to hypoxia (10% O₂) for 3 weeks reduced PPARγ mRNA and protein in mouse lung. Inhibiting ERK1/2 with PD98059 or treatment with siRNA directed against either NF-κB p65 or Nox4 attenuated hypoxic reductions in PPARγ expression and activity. Furthermore, degradation of H₂O₂ using PEG-catalase prevented hypoxia-induced ERK1/2 phosphorylation and Nox4 expression, suggesting sustained ERK1/2-mediated signaling and Nox4 expression in this response. Mammalian two-hybrid assays demonstrated that PPARγ and p65 bind directly to each other in a mutually repressive fashion. We conclude from these results that hypoxic regimens that promote PH pathogenesis and HPASMC proliferation reduce PPARγ expression and activity through ERK1/2-, p65-, and Nox4-dependent pathways. These findings provide novel insights into mechanisms by which pathophysiological stimuli such as hypoxia cause loss of PPARγ activity and pulmonary vascular cell proliferation, pulmonary vascular remodeling, and PH. These results also indicate that restoration of PPARγ activity with pharmacological ligands may provide a novel therapeutic approach in selected forms of PH.
authors.affiliation: Department of Medicine, Atlanta Veterans Affairs and Emory University Medical Centers, Atlanta, GA 30033, USA.
authors.forename: Xianghuai
authors.initials: X
authors.lastname: Lu
authors.forename: Kaiser M
authors.initials: KM
authors.lastname: Bijli
authors.forename: Allan
authors.initials: A
authors.lastname: Ramirez
authors.forename: Tamara C
authors.initials: TC
authors.lastname: Murphy
authors.forename: Jennifer
authors.initials: J
authors.lastname: Kleinhenz
authors.forename: C M
authors.initials: CM
authors.lastname: Hart
mesh_terms: D000818:Animals; D002374:Catalase / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D006976:Hypertension, Pulmonary / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000860:Hypoxia / Q000378:metabolism / Q000473:pathology; D020935:MAP Kinase Signaling System / Q000235:genetics; D051379:Mice; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D047495:PPAR gamma / Q000235:genetics / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D034741:RNA, Small Interfering; D015398:Signal Transduction
chemical_list: D016328:NF-kappa B; D047495:PPAR gamma; D034741:RNA, Small Interfering; C070295:catalase-polyethylene glycol; D011092:Polyethylene Glycols; D006861:Hydrogen Peroxide; D002374:Catalase; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human
keywords: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; C/EBPα; CCAAT/enhancer binding protein-α; CDK4; ELISA; ERK1/2; Free radicals; GAL4; GAPDH; HEK 293; HPAEC; HPASMC; Hypoxia; MAPK; MTT; NADPH oxidase 4; NF-κB; Nox4; PH; PPARγ; PPRE; PPREx3-Tk-luc; Pulmonary hypertension; Renilla luciferase reporter-containing plasmid; SDS–PAGE; Signaling mechanism; Transcription factors; Vascular wall cells; aP2; adipocyte protein-2; cyclin-dependent kinase 4; enzyme-linked immunosorbent assay; extracellular signal-regulated kinase 1/2; glyceraldehyde phosphate dehydrogenase; human embryonic kidney 293 cell; human pulmonary artery endothelial cell; human pulmonary artery smooth muscle cell; mammalian expression vector containing CMV promoter; mitogen-activated protein kinase; nuclear factor-κB; pCMV; pRL-TK; peroxisome proliferator response element; peroxisome proliferator-activated receptor-γ; pulmonary hypertension; siRNA; small interfering RNA; sodium dodecyl sulfate–polyacrylamide gel electrophoresis; thymidine kinase luciferase containing reporter plasmid with a PPARγ response element; transcription factor for galactose-inducible genes in yeast