title: The CCAAT/enhancer binding protein beta (C/EBPbeta) cooperates with NFAT to control expression of the calcineurin regulatory protein RCAN1-4.
abstract: Regulator of calcineurin 1 (RCAN1) inhibits the protein phosphatase calcineurin and is required for appropriate immune responses, synaptic plasticity, vascular tone, angiogenesis, and cardiac remodeling. Expression of the RCAN1-4 isoform is under the control of the calcineurin-responsive transcription factor NFAT. Typically, NFATs act in cooperation with other transcription factors to achieve maximal activation of gene expression. In this study, we identify the CCAAT/enhancer binding protein beta (C/EBPbeta) as an NFAT binding partner that cooperates with NFAT to regulate RCAN1-4 expression. Numerous C/EBPbeta binding sites are conserved in the RCAN1-4 proximal promoter. Overexpression of C/EBPbeta increased activity of both the endogenous mouse Rcan1-4 gene and a human RCAN1-4 luciferase reporter. Binding of C/EBPbeta to multiple sites in the promoter was verified using electrophoretic mobility shift assays and chromatin immunoprecipitation. A direct interaction between C/EBPbeta and NFAT was demonstrated by co-immunoprecipitation of proteins and complex formation at NFAT-C/EBPbeta composite sites. Depletion of endogenous C/EBPbeta decreased maximal activation of RCAN1-4 expression by calcineurin, whereas inhibition of calcineurin did not alter the ability of C/EBPbeta to activate RCAN1-4 expression. Together, these findings suggest that calcineurin/NFAT activation of RCAN1-4 expression is in part dependent upon C/EBPbeta, whereas activation by C/EBPbeta is not dependent on calcineurin and may provide a calcineurin-independent pathway for regulating RCAN1-4 expression. Importantly, nuclear localization, C/EBPbeta DNA binding activity and occupancy of the Rcan1-4 promoter increased in mouse models of heart failure demonstrating in vivo activation of this pathway to regulate Rcan1-4 expression and ultimately shape the dynamics of calcineurin-dependent signaling.
authors.affiliation: Department of Internal Medicine Cardiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA.
authors.forename: Misook
authors.initials: M
authors.lastname: Oh
authors.forename: Asim
authors.initials: A
authors.lastname: Dey
authors.forename: Robert D
authors.initials: RD
authors.lastname: Gerard
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Hill
authors.forename: Beverly A
authors.initials: BA
authors.lastname: Rothermel
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D019703:Calcineurin / Q000378:metabolism; D002135:Calcium-Binding Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D005786:Gene Expression Regulation; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism*; D050778:NFATC Transcription Factors / Q000378:metabolism*; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D022782:CCAAT-Enhancer-Binding Protein-beta; D002135:Calcium-Binding Proteins; C511512:DSCR1 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D009124:Muscle Proteins; D050778:NFATC Transcription Factors; D019703:Calcineurin