Egr-1 negatively regulates calsequestrin expression and calcium dynamics in ventricular cells.
OBJECTIVE
The transcription factor early growth response-1 (Egr-1) is increased in models of cardiac pathology; however, it is unclear how Egr-1 impacts the heart. We sought to identify how Egr-1 regulates expression of proteins involved in cardiomyocyte calcium homeostasis.


METHODS
Protein expression was measured by immunoblotting in control cardiac differentiated H9c2 cells or in H9c2 cells overexpressing wild-type Egr-1 (Egr-1) or an Egr-1 (I293F) mutant. Microspectrofluorimetry of fura-2-loaded cells was used to study calcium dynamics. Chromatin immunoprecipitation with anti-Egr-1 antibody was used to identify Egr-1-associated DNA.


RESULTS
Calsequestrin (CSQ) expression was reduced in Egr-1- and profoundly reduced in I293F-expressing cells. Calreticulin, triadin, sarcoendoplasmic reticulum ATPase 2a, phospholamban, and phosphoserine 16-phospholamban expression was unaffected. Calcium release from CSQ-dependent ryanodine-sensitive stores was reduced in Egr-1 and absent in I293F-expressing cells. In contrast, calcium release from calreticulin-dependent inositol 1,4,5-trisphosphate stores was unaffected. In vivo and in vitro chromatin immunoprecipitation demonstrated Egr-1 binding to the CSQ2 promoter. The Egr-1-binding region contains overlapping Egr-1, SP1, and nuclear factor of activated T-cells (NFAT) sites and a CpG island. Reciprocal immunoprecipitation coupled to immunoblots indicated Egr-1:NFAT3 binding was present in all cells lines. Treatment with cyclosporin A, inhibition of DNA methylation using 5-azadeoxycytidine, or inhibition of protein acetylation using sodium butyrate reduced CSQ expression.


CONCLUSIONS
Our data suggest that Egr-1:DNA binding at the promoter, DNA methylation, and protein acetylation are important in CSQ repression. Moreover, we demonstrate that a reduction in CSQ protein is associated with abnormal calcium dynamics. We conclude that Egr-1 acts as a transcriptional repressor at the CSQ promoter, resulting in downregulation of CSQ, the major calcium storage protein that links excitation-contraction coupling in the cardiac sarcoendoplasmic reticulum.
Kasneci
Amanda
A
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, 3755 chemin Côte Ste Catherine, Montréal, Québec, Canada H3T 1E2.
Kemeny-Suss
Naomi M
NM
Komarova
Svetlana V
SV
Chalifour
Lorraine E
LE
D000107:Acetylation; D000818:Animals; D001665:Binding Sites; D020013:Calcium Signaling; D002135:Calcium-Binding Proteins; D002155:Calsequestrin / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D019175:DNA Methylation; D015536:Down-Regulation; D051766:Early Growth Response Protein 1 / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006352:Heart Ventricles / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009154:Mutation; D032383:Myocytes, Cardiac / Q000378:metabolism*; D050778:NFATC Transcription Factors / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D051381:Rats; D013997:Time Factors; D014162:Transfection
D002135:Calcium-Binding Proteins; D002155:Calsequestrin; D002352:Carrier Proteins; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D050778:NFATC Transcription Factors; C514388:casq2 protein, mouse; C057137:Casq1 protein, rat