Regulation of calcineurin through transcriptional induction of the calcineurin A beta promoter in vitro and in vivo.
The calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway has been shown to be of critical importance in regulating the growth response of cardiac myocytes. We have previously demonstrated that calcineurin A(beta) (CnA(beta)) mRNA and protein are increased in response to growth stimulation, although the precise regulatory mechanism underlying CnA(beta) upregulation is not clear. Here, we isolated the mouse CnA(beta) promoter and characterized its responsiveness to growth stimuli in vitro and in vivo. A 2.3-kb promoter fragment was strongly activated by phenylephrine and endothelin-1 stimulation and by cotransfection with constitutively active CnA, NFATc4, and GATA4. Using chromatin immunoprecipitation, sequence regions were identified within the 2.3-kb promoter that associated with NFAT and GATA4, as well as with acetylated histone H3, following agonist stimulation. Consistent with the chromatin immunoprecipitation experiments, deletion of the distal half of the CnA(beta) promoter severely reduced NFAT, GATA4, and hypertrophic agonist-mediated activation. To investigate in vivo activity, we generated beta-galactosidase (LacZ) containing transgenic mice under the control of the CnA(beta) 2.3-kb promoter. CnA(beta)-LacZ mice showed expression in the heart that was cyclosporine sensitive, as well as expression in the central nervous system and skeletal muscle from early embryonic stages through adulthood. CnA(beta)-LacZ mice were subjected to cardiac pressure overload stimulation and crossbreeding with mice containing cardiac-specific transgenes for activated calcineurin and NFATc4, which revealed inducible expression in the heart. These results indicate that the CnA(beta) 2.3-kb promoter is specifically activated by hypertrophic stimuli through a positive feedback mechanism involving NFAT and GATA4 transcription factors, suggesting transcriptional induction of CnA(beta) expression as an additional means of regulating calcineurin activity in the heart.
Oka
Toru
T
Cincinnati Children's Hospital Medical Center, Division of Molecular Cardiovascular Biology, 3333 Burnet Ave., MLC7020, Cincinnati OH 45229-3039, USA.
Dai
Yan-Shan
YS
Molkentin
Jeffery D
JD
D000818:Animals; D019703:Calcineurin / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D006332:Cardiomegaly / Q000201:enzymology / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D050991:GATA4 Transcription Factor; D005786:Gene Expression Regulation / Q000502:physiology*; D017930:Genes, Reporter; D051379:Mice; D008822:Mice, Transgenic; D009206:Myocardium / Q000201:enzymology; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*
D004268:DNA-Binding Proteins; D050991:GATA4 Transcription Factor; C484171:Gata4 protein, mouse; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D014157:Transcription Factors; D019703:Calcineurin