title: Smad-independent pathway involved in transforming growth factor β1-induced Nox4 expression and proliferation of endothelial cells.
abstract: NADPH oxidase-derived reactive oxygen species are important for various cellular functions, including proliferation. Endothelial cells predominantly express the Nox4 isoform of NADPH oxidase, but it is not entirely clear how it is regulated. In this study, we investigated the signalling pathways involved in transforming growth factor-β1 (TGF-β1)-induced Nox4 expression and the proliferation of human microvascular endothelial cells (HMECs). TGF-β1 stimulated Nox4 messenger RNA and protein expression in HMECs. TGF-β1-induced Nox4 also increased hydrogen peroxide production, which was inhibited by diphenyleneiodonium and EUK134. The acute treatment of HMECs with TGF-β1 enhanced the phosphorylation of Smad2 and extracellular signal-regulated kinase (ERK) 1/2, without affecting p38MAPK, Akt, or Jun N-terminal kinase 1/2 (JNK1/2) pathways. Further, inhibition of Smad2 signalling using an inhibitor of activin receptor-linked kinase 5 SB431542 reduced TGF-β1-induced Nox4 expression, while inhibition of ERK1/2 with the inhibitor of mitogen-activated protein kinase kinase 1/2 U0126 decreased both basal and TGF-β1-induced Nox4 expression. Inhibition of ERK1/2 phosphorylation with U0126 did not affect Smad2 phosphorylation. Finally, TGF-β1 enhanced endothelial cell proliferation, which was reduced by U0126 but not by SB431542. These findings suggest that the non-canonical pathway ERK1/2 regulates Nox4 expression and may be involved in TGF-β1-induced proliferation of endothelial cells, which is vital during angiogenesis and vascular development.
authors.affiliation: Centre for Eye Research Australia, University of Melbourne, Level 1, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia.
authors.forename: Nora Y
authors.initials: NY
authors.lastname: Hakami
authors.forename: Henry
authors.initials: H
authors.lastname: Wong
authors.forename: Manisha H
authors.initials: MH
authors.lastname: Shah
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Dusting
authors.forename: Fan
authors.initials: F
authors.lastname: Jiang
authors.forename: Hitesh M
authors.initials: HM
authors.lastname: Peshavariya
mesh_terms: D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051785:Smad Proteins / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
chemical_list: D012333:RNA, Messenger; D051785:Smad Proteins; D053773:Transforming Growth Factor beta1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human