Regulation of Calcineurin through Transcriptional Induction of the calcineurin Aβ Promoter In Vitro and In Vivo

Calcineurin (PP2B) is a calcium-calmodulin activated serine-threonine protein phosphatase that is conserved from yeast to humans in its overall structure and biochemistry. Calcineurin exists as a heterotrimer between a 57- to 61-kDa catalytic A subunit (CnA) and two smaller, 16- to 19-kDa EF-hand-containing calcium binding proteins referred to as calcineurin B (CnB) and calmodulin (27). In vertebrates, three unlinked loci encode the catalytic subunit (CnAα, CnAβ, and CnAγ), while two loci encode the regulatory subunit (CnB1 and CnB2) (44). The CnAα, CnAβ, and CnB1 genes are each expressed in a ubiquitous pattern throughout the body, while CnAγ and CnB2 expression is more restricted to the brain and testis (5, 26, 39, 47). Once activated, calcineurin directly dephosphorylates a family of transcription factors referred to as nuclear factor of activated T cells (NFAT) within the cytoplasm, exposing a nuclear localization sequence and promoting translocation into the nucleus where they participate in the transcriptional induction of diverse genes in a wide array of cell types (25). There are four calcineurin-regulated NFAT transcription factors, NFATc1 to -c4, each of which is expressed in the heart (52). Calcineurin-NFAT signaling plays diverse regulatory roles in multiple vertebrate cell types. In the heart, calcineurin has been implicated as a pivotal regulator of the myocyte hypertrophic growth response (38). Calcineurin-NFAT was originally implicated as a cardiac effector through a search for factors that interacted with the cardiac tissue-enriched zinc finger containing transcription factor GATA4, which identified NFATc4 as a critical cofactor (38). NFATc4 and GATA4 synergistically activated the brain natriuretic peptide (BNP) promoter, which is normally upregulated by stimuli that elicit a hypertrophic growth response. Expression of constitutively active forms of CnA or NFATc4 in the mouse heart by transgenesis produced profound cardiac hypertrophy, demonstrating the sufficiency of this pathway in regulating the hypertrophic growth response (38). The necessity of this pathway in mediating cardiac growth responses has been confirmed through the use of the calcineurin inhibitory agents cyclosporine A (CsA) and FK506, as well as by gene targeting of the CnAβ gene and by transgene-mediated expression of calcineurin inhibitory peptides in the heart (4, 36, 37). While calcineurin-NFAT signaling regulates the cardiac hypertrophic growth response, the mechanism whereby calcium activates this pathway in a cell type experiencing cyclical release of calcium is uncertain. Previous studies have established the requirement for sustained increases in intracellular calcium concentration to mediate calcineurin activation and the nuclear localization of NFATs (13-15, 48, 49). In addition to changes in calcium concentration, calcineurin phosphatase activity can be altered through changes in the activity or concentration of calcineurin modulatory factors or by changes in coregulatory signaling pathways. We have previously observed an increase in CnAβ mRNA and protein expression in hypertrophied neonatal cardiac myocytes and hypertrophied adult hearts, with no coincident change in CnAα levels (20, 46), supporting the existence of yet another way of upregulating calcineurin in the heart. However, the underlying mechanism whereby CnAβ expression is augmented in cardiac myocytes undergoing hypertrophy has not been evaluated.