The CCAAT/Enhancer Binding Protein β (C/EBPβ) Cooperates with NFAT to Control Expression of the Calcineurin Regulatory Protein RCAN1-4

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/EBP beta) as an NFAT binding partner that cooperates with NFAT to regulate RCAN1-4 expression. Numerous C/EBP beta binding sites are conserved in the RCAN1-4 proximal promoter. Overexpression of C/EBP beta increased activity of both the endogenous mouse Rcan1-4 gene and a human RCAN1-4 luciferase reporter. Binding of C/EBP beta to multiple sites in the promoter was verified using electrophoretic mobility shift assays and chromatin immunoprecipitation. Adirect interaction between C/EBP beta and NFAT was demonstrated by co-immunoprecipitation of proteins and complex formation at NFAT-C/EBP beta composite sites. Depletion of endogenous C/EBP beta decreased maximal activation of RCAN1-4 expression by calcineurin, whereas inhibition of calcineurin did not alter the ability of C/EBP beta to activate RCAN1-4 expression. Together, these findings suggest that calcineurin/NFAT activation of RCAN1-4 expression is in part dependent upon C/EBP beta, whereas activation by C/EBP beta is not dependent on calcineurin and may provide a calcineurin-independent pathway for regulating RCAN1-4 expression. Importantly, nuclear localization, C/EBP beta 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.