Embryonic stem cell differentiation into smooth muscle cells is mediated by Nox4-produced H2O2.
NADPH oxidase (Nox4) produces reactive oxygen species (ROS) that are important for vascular smooth muscle cell (SMC) behavior, but the potential impact of Nox4 in stem cell differentiation is unknown. When mouse embryonic stem (ES) cells were plated on collagen IV-coated dishes/flasks, a panel of SMC-specific genes was significantly and consistently upregulated. Nox4 expression was markedly correlated with such a gene induction as confirmed by real-time PCR, immunofluorescence, and Western blot analysis. Overexpression of Nox4 specifically resulted in increased SMC marker production, whereas knockdown of Nox4 induced a decrease. Furthermore, SMC-specific transcription factors, including serum response factor (SRF) and myocardin were activated by Nox4 gene expression. Moreover, Nox4 was demonstrated to drive SMC differentiation through generation of H(2)O(2). Confocal microscopy analysis indicates that SRF was translocated into the nucleus during SMC differentiation in which SRF was phosphorylated. Additionally, autosecreted transforming growth factor (TGF)-beta(1) activated Nox4 and promoted SMC differentiation. Interestingly, cell lines generated from stem cells by Nox4 transfection and G418 selection displayed a characteristic of mature SMCs, including expression of SMC markers and cells with contractile function. Thus we demonstrate for the first time that Nox4 is crucial for SMC differentiation from ES cells, and enforced Nox4 expression can maintain differentiation status and functional features of stem cell-derived SMCs, highlighting its impact on vessel formation in vivo and vascular tissue engineering in the future.
Xiao
Qingzhong
Q
Cardiovascular Div., King's College London, The James Black Centre, 125 Coldharbour Lane, London SE5 9NU.
Luo
Zhenling
Z
Pepe
Anna Elena
AE
Margariti
Andriani
A
Zeng
Lingfang
L
Xu
Qingbo
Q
D000818:Animals; D019898:Autocrine Communication; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D024141:Collagen Type IV / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000201:enzymology*; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D006861:Hydrogen Peroxide / Q000378:metabolism*; D051379:Mice; D032389:Myocytes, Smooth Muscle / Q000201:enzymology*; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D026362:Serum Response Factor / Q000378:metabolism; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000378:metabolism; D015854:Up-Regulation; D000099251:Myocardin
D024141:Collagen Type IV; D006861:Hydrogen Peroxide; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta; D026362:Serum Response Factor; D015534:Trans-Activators; D053773:Transforming Growth Factor beta1; D000099251:Myocardin; C422943:Nox4 protein, mouse