Abstract 1230: The CCAAT/Enhancer Binding Protein C/EBP Beta and NFAT Cooperate to Control MCIP1.4 Expression
Background: CCAAT/Enhancer binding proteins (C/EBPs) are a family of basic leucine zipper (bZIP) transcription factors that play important roles in the regulation of cell growth and differentiation. In the heart, C/EBP beta DNA binding activity increases during ischemia/ reperfusion. The cardioprotective protein MCIP1 (Modulatory Calcineurin-Interacting Protein 1) binds to and inhibits calcineurin, an important transducer of intracellular calcium signals. We have previously shown that levels of the MCIP1.4 isoform increase in cardiomyocytes in response to multiple forms of stress including hypoxia. Futhermore, MCIP1.4 expression is controlled by calcineurin through a cluster of NFAT binding sites. Because C/EBP beta responds to hypoxia and is known to cooperate with NFAT to control the expression of several other genes, we hypothesized that C/EBP beta and NFAT might also work together to control expression of MCIP1.4.
Methods and Results: Upon examination of the proximal MCIP1.4 promoter we detected 16 putative C/EBP binding sites, some of which overlap or are adjacent to NFAT binding sites. The C/EBP beta gene is transcribed as a single mRNA, which can be further translated into three distinct isoforms, including two activators (LAP* and LAP) and a repressor (LIP). Using transient transfections of C2C12 myoblasts we tested the effect of LAP*, LAP, and LIP on expression of an MCIP1.4-luciferase reporter construct. Transfection with LAP caused a robust increase in MCIP1.4-luciferase expression, whereas neither LAP* nor LIP had an effect. LAP acted synergistically with calcineurin to increase MCIP1.4 expression. In contrast both LAP* and LIP repressed calcineurin activation of the reporter. To test the role of C/EBP beta in a physiologically relevant model of heart failure, we subjected 10-week old male mice to severe pressure overload. We compared C/EBP beta levels in control and failing hearts by western blot analysis. There was no difference in the level of either the LAP* or LAP isoform, whereas, LIP protein was significantly elevated in failing hearts.
Conclusion: These data demonstrate that NFAT and C/EBP beta cooperate to control MCIP1.4 transcription and suggest that differential processing of C/EBP beta mRNA occurs in the failing heart.