title: NOX4 mediates hypoxia-induced proliferation of human pulmonary artery smooth muscle cells: the role of autocrine production of transforming growth factor-{beta}1 and insulin-like growth factor binding protein-3.
abstract: Persistent hypoxia can cause pulmonary arterial hypertension that may be associated with significant remodeling of the pulmonary arteries, including smooth muscle cell proliferation and hypertrophy. We previously demonstrated that the NADPH oxidase homolog NOX4 mediates human pulmonary artery smooth muscle cell (HPASMC) proliferation by transforming growth factor-beta1 (TGF-beta1). We now show that hypoxia increases HPASMC proliferation in vitro, accompanied by increased reactive oxygen species generation and NOX4 gene expression, and is inhibited by antioxidants, the flavoenzyme inhibitor diphenyleneiodonium (DPI), and NOX4 gene silencing. HPASMC proliferation and NOX4 expression are also observed when media from hypoxic HPASMC are added to HPASMC grown in normoxic conditions, suggesting autocrine stimulation. TGF-beta1 and insulin-like growth factor binding protein-3 (IGFBP-3) are both increased in the media of hypoxic HPASMC, and increased IGFBP-3 gene expression is noted in hypoxic HPASMC. Treatment with anti-TGF-beta1 antibody attenuates NOX4 and IGFBP-3 gene expression, accumulation of IGFBP-3 protein in media, and proliferation. Inhibition of IGFBP-3 expression with small interfering RNA (siRNA) decreases NOX4 gene expression and hypoxic proliferation. Conversely, NOX4 silencing does not decrease hypoxic IGFBP-3 gene expression or secreted protein. Smad inhibition does not but the phosphatidylinositol 3-kinase (PI3K) signaling pathway inhibitor LY-294002 does inhibit NOX4 and IGFBP-3 gene expression, IGFBP-3 secretion, and cellular proliferation resulting from hypoxia. Immunoblots from hypoxic HPASMC reveal increased TGF-beta1-mediated phosphorylation of the serine/threonine kinase (Akt), consistent with hypoxia-induced activation of PI3K/Akt signaling pathways to promote proliferation. We conclude that hypoxic HPASMC produce TGF-beta1 that acts in an autocrine fashion to induce IGFBP-3 through PI3K/Akt. IGFBP-3 increases NOX4 gene expression, resulting in HPASMC proliferation. These observations add to our understanding hypoxic pulmonary vascular remodeling.
authors.affiliation: Division of Respiratory, Critical Care, and Occupational Pulmonary Medicine, University of Utah Health Sciences Centerand VA Medical Center, Salt Lake City, Utah 84132, USA.
authors.forename: Saleh
authors.initials: S
authors.lastname: Ismail
authors.forename: Anne
authors.initials: A
authors.lastname: Sturrock
authors.forename: Ping
authors.initials: P
authors.lastname: Wu
authors.forename: Barbara
authors.initials: B
authors.lastname: Cahill
authors.forename: Kimberly
authors.initials: K
authors.lastname: Norman
authors.forename: Thomas
authors.initials: T
authors.lastname: Huecksteadt
authors.forename: Karl
authors.initials: K
authors.lastname: Sanders
authors.forename: Thomas
authors.initials: T
authors.lastname: Kennedy
authors.forename: John
authors.initials: J
authors.lastname: Hoidal
mesh_terms: D019898:Autocrine Communication; D015687:Cell Hypoxia / Q000235:genetics / Q000502:physiology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D015870:Gene Expression; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3; D018969:Insulin-Like Growth Factor Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D008954:Models, Biological; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism*; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D053773:Transforming Growth Factor beta1 / Q000096:biosynthesis*
chemical_list: C515497:IGFBP3 protein, human; D018972:Insulin-Like Growth Factor Binding Protein 3; D018969:Insulin-Like Growth Factor Binding Proteins; D017382:Reactive Oxygen Species; D053773:Transforming Growth Factor beta1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt