title: Myocardin-related Transcription Factor Regulates Nox4 Protein Expression: LINKING CYTOSKELETAL ORGANIZATION TO REDOX STATE.
abstract: TGFβ-induced expression of the NADPH oxidase Nox4 is essential for fibroblast-myofibroblast transition. Rho has been implicated in Nox4 regulation, but the underlying mechanisms are largely unknown. Myocardin-related transcription factor (MRTF), a Rho/actin polymerization-controlled coactivator of serum response factor, drives myofibroblast transition from various precursors. We have shown that TGFβ is necessary but insufficient for epithelial-myofibroblast transition in intact epithelia; the other prerequisite is the uncoupling of intercellular contacts, which induces Rho-dependent nuclear translocation of MRTF. Because the Nox4 promoter harbors a serum response factor/MRTF cis-element (CC(A/T)6GG box), we asked if MRTF (and thus cytoskeleton organization) could regulate Nox4 expression. We show that Nox4 protein is robustly induced in kidney tubular cells exclusively by combined application of contact uncoupling and TGFβ. Nox4 knockdown abrogates epithelial-myofibroblast transition-associated reactive oxygen species production. Laser capture microdissection reveals increased Nox4 expression in the tubular epithelium also during obstructive nephropathy. MRTF down-regulation/inhibition suppresses TGFβ/contact disruption-provoked Nox4 protein and mRNA expression, Nox4 promoter activation, and reactive oxygen species production. Mutation of the CC(A/T)6GG box eliminates the synergistic activation of the Nox4 promoter. Jasplakinolide-induced actin polymerization synergizes with TGFβ to facilitate MRTF-dependent Nox4 mRNA expression/promoter activation. Moreover, MRTF inhibition prevents Nox4 expression during TGFβ-induced fibroblast-myofibroblast transition as well. Although necessary, MRTF is insufficient; Nox4 expression also requires TGFβ-activated Smad3 and TAZ/YAP, two contact- and cytoskeleton-regulated Smad3-interacting coactivators. Down-regulation/inhibition of TAZ/YAP mitigates injury-induced epithelial Nox4 expression in vitro and in vivo. These findings uncover new MRTF- and TAZ/YAP-dependent mechanisms, which link cytoskeleton remodeling and redox state and impact epithelial plasticity and myofibroblast transition.
authors.affiliation: From the Keenan Research Centre for Biomedical Science of the St. Michael's Hospital.
authors.forename: Matthew
authors.initials: M
authors.lastname: Rozycki
authors.affiliation: From the Keenan Research Centre for Biomedical Science of the St. Michael's Hospital, the Department of Biology, Section for Cell Biology and Physiology, University of Copenhagen, Copenhagen DK-2100, Denmark.
authors.forename: Janne Folke
authors.initials: JF
authors.lastname: Bialik
authors.affiliation: From the Keenan Research Centre for Biomedical Science of the St. Michael's Hospital.
authors.forename: Pam
authors.initials: P
authors.lastname: Speight
authors.affiliation: From the Keenan Research Centre for Biomedical Science of the St. Michael's Hospital.
authors.forename: Qinghong
authors.initials: Q
authors.lastname: Dan
authors.affiliation: the Department of Biology, Section for Cell Biology and Physiology, University of Copenhagen, Copenhagen DK-2100, Denmark.
authors.forename: Teresa E T
authors.initials: TE
authors.lastname: Knudsen
authors.affiliation: From the Keenan Research Centre for Biomedical Science of the St. Michael's Hospital.
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Szeto
authors.affiliation: From the Keenan Research Centre for Biomedical Science of the St. Michael's Hospital.
authors.forename: Darren A
authors.initials: DA
authors.lastname: Yuen
authors.affiliation: From the Keenan Research Centre for Biomedical Science of the St. Michael's Hospital, Departments of Surgery and.
authors.forename: Katalin
authors.initials: K
authors.lastname: Szászi
authors.affiliation: the Department of Biology, Section for Cell Biology and Physiology, University of Copenhagen, Copenhagen DK-2100, Denmark.
authors.forename: Stine F
authors.initials: SF
authors.lastname: Pedersen
authors.affiliation: From the Keenan Research Centre for Biomedical Science of the St. Michael's Hospital, Departments of Surgery and Biochemistry, University of Toronto, Toronto, Ontario M5B 1T8, Canada and kapusa@smh.ca.
authors.forename: András
authors.initials: A
authors.lastname: Kapus
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D003599:Cytoskeleton / Q000378:metabolism*; D004848:Epithelium / Q000473:pathology; D005355:Fibrosis; D015971:Gene Expression Regulation, Enzymologic; D007684:Kidney Tubules / Q000378:metabolism / Q000473:pathology; D018374:LLC-PK1 Cells; D008297:Male; D008648:Mesoderm / Q000378:metabolism / Q000473:pathology; D008810:Mice, Inbred C57BL; D024510:Muscle Development; D058628:Myofibroblasts / Q000378:metabolism / Q000473:pathology; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism; D010084:Oxidation-Reduction; D058105:Polymerization; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D013552:Swine; D014157:Transcription Factors / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D000199:Actins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D014157:Transcription Factors; D019255:NADPH Oxidases
keywords: NADPH oxidase; Yes-associated protein (YAP); cytoskeleton; epithelium; kidney; myocardin; myofibroblast; promoter; transforming growth factor β (TGF-β)