title: SMAD3 augments FoxO3-induced MuRF-1 promoter activity in a DNA-binding-dependent manner.
abstract: Muscle-specific RING finger-1 (MuRF-1), a ubiquitin ligase and key regulator of proteasome-dependent protein degradation, is highly expressed during skeletal muscle atrophy. The transcription factor forkhead box O3 (FoxO3) induces MuRF-1 expression, but the direct role of other major atrophy-related transcription factors, such as SMAD3, is largely unknown. The goal of this study was to determine whether SMAD3 individually regulates, or with FoxO3 coordinately regulates, MuRF-1 expression. In cultured myotubes or human embryonic kidney cells, MuRF-1 mRNA content and promoter activity were increased by FoxO3 but not by SMAD3 overexpression. However, FoxO3 and SMAD3 coexpression synergistically increased MuRF-1 mRNA and promoter activity. Mutation of the SMAD-binding element (SBE) in the proximal MuRF-1 promoter or overexpression of a SMAD3 DNA-binding mutant attenuated FoxO3-dependent MuRF-1 promoter activation, showing that SMAD binding to DNA is required for optimal activation of FoxO3-induced transcription of MuRF-1. Using chromatin immunoprecipitation, SMAD3 DNA binding increased FoxO3 abundance and SBE mutation reduced FoxO3 abundance on the MuRF-1 promoter. Furthermore, SMAD3 overexpression dose-dependently increased FoxO3 protein content, and coexpression of FoxO3 and SMAD3 synergistically increased FoxO-dependent gene transcription [assessed with a FoxO response element (FRE)-driven reporter]. Collectively, these results show that SMAD3 regulates transcription of MuRF-1 by increasing FoxO3 binding at a conserved FRE-SBE motif within the proximal promoter region, and by increasing FoxO3 protein content and transcriptional activity. These data are the first to indicate that two major transcription factors regulating protein degradation, FoxO3 and SMAD3, converge to coordinately and directly regulate transcription of MuRF-1.
authors.affiliation: Department of Kinesiology, Human Performance Laboratory, College of Health and Human Performance, East Carolina University, Greenville, North Carolina; Department of Biochemistry and Molecular Biology, Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina; and East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, North Carolina.
authors.forename: Lance M
authors.initials: LM
authors.lastname: Bollinger
authors.affiliation: Department of Kinesiology, Human Performance Laboratory, College of Health and Human Performance, East Carolina University, Greenville, North Carolina; Department of Biochemistry and Molecular Biology, Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina; and East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, North Carolina.
authors.forename: Carol A
authors.initials: CA
authors.lastname: Witczak
authors.affiliation: Department of Kinesiology, Human Performance Laboratory, College of Health and Human Performance, East Carolina University, Greenville, North Carolina; East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, North Carolina.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Houmard
authors.affiliation: Department of Kinesiology, Human Performance Laboratory, College of Health and Human Performance, East Carolina University, Greenville, North Carolina; Department of Biochemistry and Molecular Biology, Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina; and East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, North Carolina braultj@ecu.edu.
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Brault
mesh_terms: D000328:Adult; D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D005260:Female; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism; D009124:Muscle Proteins / Q000096:biosynthesis / Q000235:genetics*; D009133:Muscular Atrophy / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis; D020218:Response Elements; D044843:SKP Cullin F-Box Protein Ligases / Q000096:biosynthesis; D051900:Smad3 Protein / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D000070584:Tripartite Motif Proteins; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; C499517:FOXO3 protein, human; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D009124:Muscle Proteins; D012333:RNA, Messenger; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D000070584:Tripartite Motif Proteins; D004247:DNA; C486691:Fbxo32 protein, mouse; D044843:SKP Cullin F-Box Protein Ligases; C440908:TRIM63 protein, human; D044767:Ubiquitin-Protein Ligases
keywords: cell culture; protein degradation; protein-DNA interaction; transcription factors; ubiquitin ligase