Egr-1 negatively regulates expression of the sodium-calcium exchanger-1 in cardiomyocytes in vitro and in vivo.
OBJECTIVE
Increased expression of the transcription factor early growth response gene-1 (Egr-1) accompanies catecholamine infusion. Catecholamine-treated, Egr-1-deficient (-/-) mice show exacerbated cardiac damage when compared to similarly treated wild-type (+/+) mice, suggesting that Egr-1 reduces heart damage. We sought to identify Egr-1-mediated cardiac sparing genes.


METHODS
Microarray analyses identified increased sodium calcium exchanger-1 (NCX1) expression in catecholamine-treated -/- mice. Immunoblots assessed NCX1 expression in +/+, -/-, and transgenic mice overexpressing Egr-1 in heart and cardiac differentiated H9c2 cells harboring wild-type Egr-1 (wtEgr-1) or NAB-binding ablating mutations. Chromatin immunoprecipitation (ChIP) used anti-Egr-1 antibody coupled to amplification of purified Egr-1/associated DNA.


RESULTS
Immunoblots revealed a two- to threefold increase in NCX1 in catecholamine-stimulated and naive -/- versus +/+ mice. In contrast, transgenic mice overexpressing Egr-1 in heart had 30% of normal NCX1 protein. Thus, the in vivo data indicate that Egr-1 negatively controls NCX1 expression. In vitro cardiac differentiated H9c2 cells overexpressing wtEgr-1 also showed 30% NCX1 expression. However, cells overexpressing NAB-ablating Egr-1 mutations showed four- to fivefold increased NCX1 expression. NCX1 promoter DNA was specifically amplified from Egr-1/associated DNA. Thus, the in vitro results indicate that Egr-1/NAB interactions are critical for NCX1 repression at the NCX1 promoter.


CONCLUSIONS
NCX1 is responsible for calcium exit from cardiomyocytes, and continued overexpression is thought to be detrimental. We propose that one way Egr-1 action is cardiac sparing is by promoting a reduction in NCX1 expression.
Wang
Chunlei
C
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, 3755 chemin Côte Ste Catherine, Montréal, Québec, Canada H3T 1E2.
Dostanic
Stevan
S
Servant
Nicolas
N
Chalifour
Lorraine E
LE
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004280:Dobutamine / Q000494:pharmacology; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D015918:Infusion Pumps, Implantable; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D009921:Metaproterenol / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D010656:Phenylephrine / Q000494:pharmacology; D011433:Propranolol / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*; D013268:Stimulation, Chemical; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D017874:Immediate-Early Proteins; D019831:Sodium-Calcium Exchanger; D014157:Transcription Factors; C110374:sodium-calcium exchanger 1; D010656:Phenylephrine; D004280:Dobutamine; D009921:Metaproterenol; D012964:Sodium; D011433:Propranolol; D007545:Isoproterenol; D002118:Calcium