title: Activation of Wnt/β-catenin signaling by hydrogen peroxide transcriptionally inhibits NaV1.5 expression.
abstract: Oxidants and canonical Wnt/β-catenin signaling have been shown to decrease cardiac Na(+) channel activity by suppressing NaV1.5 expression. Our aims are to determine if hydrogen peroxide (H2O2), one oxidant of reactive oxygen species (ROS), activates Wnt/β-catenin signaling and promotes β-catenin nuclear activity, leading to suppression of NaV1.5 expression and if this suppression requires the interaction of β-catenin with its nuclear partner, TCF4 (also called TCF7L2) to decrease SCN5a promoter activity. The results demonstrated that H2O2 increased β-catenin, but not TCF4 nuclear localization determined by immunofluorescence without affecting total β-catenin protein level. Furthermore, H2O2 exerted a dose- and time-dependent suppressive effect on NaV1.5 expression. RT-PCR and/or Western blot analyses revealed that overexpressing active form of β-catenin or stabilizing β-catenin by GSK-3β inhibitors, LiCl and Bio, suppressed NaV1.5 expression in HL-1 cells. In contrast, destabilization of β-catenin by a constitutively active GSK-3β mutant (S9A) upregulated NaV1.5 expression. Whole-cell recording showed that LiCl significantly inhibited Na(+) channel activity in these cells. Using immunoprecipitation (IP), we showed that β-catenin interacted with TCF4 indicating that β-catenin as a co-transfactor, regulates NaV1.5 expression through TCF4. Analyses of the SCN5a promoter sequences among different species by using VISTA tools indicated that SCN5a promoter harbors TCF4 binding sites. Chromatin IP assays demonstrated that both β-catenin and TCF4 were recruited in the SCN5a promoter, and regulated its activity. Luciferase promoter assays exhibited that β-catenin inhibited the SCN5a promoter activity at a dose-dependent manner and this inhibition required TCF4. Small interfering (Si) RNA targeting β-catenin significantly increased SCN5a promoter activity, leading to enhanced NaV1.5 expression. As expected, β-catenin SiRNA prevents H2O2 suppressive effects on both SCN5a promoter activity and NaV1.5 expression. Our findings indicate that H2O2 inhibits NaV1.5 expression by activating the Wnt/β-catenin signaling and β-catenin interacts with TCF4 to transcriptionally suppress cardiac NaV1.5 expression.
authors.affiliation: Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, UCLA, 10833 Le Conte Ave., CHS 13-145E, Los Angeles, CA 90095-1732, USA.
authors.forename: Ning
authors.initials: N
authors.lastname: Wang
authors.affiliation: Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, UCLA, 10833 Le Conte Ave., CHS 13-145E, Los Angeles, CA 90095-1732, USA.
authors.forename: Rong
authors.initials: R
authors.lastname: Huo
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA.
authors.forename: Benzhi
authors.initials: B
authors.lastname: Cai
authors.affiliation: Division of Cardiology, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA.
authors.forename: Bo
authors.initials: B
authors.lastname: Ye
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA.
authors.forename: Xiang
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA.
authors.forename: Faqian
authors.initials: F
authors.lastname: Li
authors.affiliation: Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, UCLA, 10833 Le Conte Ave., CHS 13-145E, Los Angeles, CA 90095-1732, USA; Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA. Electronic address: HaodongXu@mednet.ucla.edu.
authors.forename: Haodong
authors.initials: H
authors.lastname: Xu
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D005786:Gene Expression Regulation / Q000187:drug effects; D000071679:Glycogen Synthase Kinase 3 beta / Q000037:antagonists & inhibitors / Q000235:genetics; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D018021:Lithium Chloride / Q000494:pharmacology; D051379:Mice; D009154:Mutation; D032383:Myocytes, Cardiac / Q000378:metabolism; D062554:NAV1.5 Voltage-Gated Sodium Channel / Q000235:genetics* / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011401:Promoter Regions, Genetic / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D000073940:Transcription Factor 4 / Q000235:genetics* / Q000378:metabolism; D060449:Wnt Signaling Pathway / Q000187:drug effects; D051176:beta Catenin / Q000235:genetics* / Q000378:metabolism
chemical_list: C495267:CTNNB1 protein, mouse; D062554:NAV1.5 Voltage-Gated Sodium Channel; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C568321:Scn5a protein, mouse; C090743:Tcf4 protein, mouse; D000073940:Transcription Factor 4; D051176:beta Catenin; D006861:Hydrogen Peroxide; D000071679:Glycogen Synthase Kinase 3 beta; D018021:Lithium Chloride
keywords: Hydrogen peroxide; Na(V)1.5; Oxidant; SCN5a promoter; TCF4; β-Catenin