title: Primary involvement of NADPH oxidase 4 in hypoxia-induced generation of reactive oxygen species in adipose-derived stem cells.
abstract: We have previously demonstrated that hypoxia stimulates adipose-derived stem cells (ASCs) through the generation of reactive oxygen species (ROS). However, the precise mechanism involved in the ROS generation by ASCs is not well understood. We sought to investigate in this work: (1) which subtype of NADPH oxidase (Nox) is primarily expressed in ASCs; (2) where Nox4 is localized in ASCs; and (3) whether silencing of Nox4 attenuates hypoxia-enhanced function of ASC. We used 2',7'-dichlorofluorescin diacetate (DCF-DA) as an indicator of ROS generation and found that the fluorescence intensity of DCF-DA was significantly increased after hypoxia exposure (2% oxygen). In addition, hypoxia enhanced the proliferation and migration of ASCs and upregulated the mRNA expression of Oct4 and Rex1. Quantitative analysis of mRNA expression of Nox family in ASCs demonstrated that Nox4 is primarily expressed in ASCs, while immunofluorescence assay showed that Nox4 is mainly localized in the perinuclear region and overlaps with Mitotracker, a mitochondria marker. Silencing of Nox4 by siRNA treatment downregulated the RNA and protein expression of Nox4, which significantly reduced the ROS generation under hypoxia. In addition, Nox4 silencing significantly reduced the proliferation and migration of ASCs and downregulated the mRNA expression of Oct4 and Rex1. Phosphorylation of platelet-derived growth factor receptor-β, AKT, and ERK1/2 also diminished following Nox4 silencing. In a nutshell, these results suggest that Nox4 is primarily expressed in ASCs and plays a pivotal role in the hypoxia-enhanced stimulation of ASCs.
authors.affiliation: Department of Applied Bioscience, CHA University, Seoul, Korea.
authors.forename: Ji Hye
authors.initials: JH
authors.lastname: Kim
authors.forename: Seung-Yong
authors.initials: SY
authors.lastname: Song
authors.forename: Sang Gyu
authors.initials: SG
authors.lastname: Park
authors.forename: Sun U
authors.initials: SU
authors.lastname: Song
authors.forename: Ying
authors.initials: Y
authors.lastname: Xia
authors.forename: Jong-Hyuk
authors.initials: JH
authors.lastname: Sung
mesh_terms: D053687:Adult Stem Cells / Q000201:enzymology* / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D015687:Cell Hypoxia; D002465:Cell Movement; D002467:Cell Nucleus / Q000201:enzymology; D049109:Cell Proliferation; D002478:Cells, Cultured; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D006801:Humans; D008928:Mitochondria / Q000201:enzymology; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction; D050151:Subcutaneous Fat / Q000166:cytology*
chemical_list: D002352:Carrier Proteins; D009687:Nuclear Proteins; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; C477883:REXO1 protein, human; D017382:Reactive Oxygen Species; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human