The Role of GATA, CArG, E-box, and a Novel Element in the Regulation of Cardiac Expression of the Na+-Ca2+ Exchanger Gene

Abstract

The cardiac Na ؉ -Ca 2؉ exchanger (NCX1) is the principal Ca 2؉ efflux mechanism in cardiocytes. The exchanger is up-regulated in both cardiac hypertrophy and failure. In this report, we identify the cis-acting elements that control cardiac expression and ␣-adrenergic up-regulation of the exchanger gene. Deletion analysis revealed that a minimal cardiac promoter fragment from ؊184 to ؉172 is sufficient for cardiac expression and ␣-adrenergic stimulation. Mutational analysis revealed that both the CArG element at ؊80 and the GATA element at ؊50 were required for cardiac expression. Gel mobility shift assay supershift analysis demonstrated that the serum response factor binds to the CArG element and GATA-4 binds to the GATA element. Point mutations in the ؊172 E-box demonstrated that it was required for ␣-adrenergic induction. In addition, deletion analysis revealed one or more enhancer elements in the first intron (؉103 to ؉134) that are essential for phenylephrine up-regulation but bear no homology to any known transcription element. Therefore, this work demonstrates that SRF and GATA-4 are critical for NCX1 expression in neonatal cardiomyocytes and that the ؊172 E-box in addition to a novel enhancer element(s) are required for phenylephrine up-regulation of NCX1 and may mediate its hypertrophic up-regulation.The Na ϩ -Ca 2ϩ exchanger (NCX1) 1 catalyzes the electrogenic exchange of one intracellular calcium ion for three extracellular sodium ions across the plasma membrane in many mammalian cells. Transport is reversible and can facilitate calcium entry, which in the heart is capable of triggering calcium release from the sarcoplasmic reticulum (1). The exchanger is most abundant in the heart, where it regulates Ca 2ϩ fluxes across the sarcolemma and serves a critical role in the maintenance of the cellular calcium balance for excitation-contraction coupling. Na ϩ -Ca 2ϩ exchanger activity in cardiomyocytes is regulated by several factors. It is activated by cytosolic Ca 2ϩ and MgATP (2) and inhibited by cytosolic sodium (3) and ATP depletion (4). A high affinity Ca 2ϩ -binding domain has been identified in the large cytoplasmic loop (residues 371-508) that is believed to be responsible for calcium regulation (5). It is also inhibited by the exchanger inhibitory peptide, which corresponds to a 20-amino acid segment at the N terminus of the large cytoplasmic loop (6). A recent study has demonstrated that the exchanger is phosphorylated via a protein kinase C-dependent pathway and that NCX1 phosphorylation appears to coincide with up-regulation of exchanger activity (7).In addition, the exchanger is regulated at the transcriptional level in cardiac hypertrophy, ischemia, and failure. In the feline model of acute right ventricular hypertrophy, NCX1 message levels are rapidly up-regulated following pressure overload (8,9). An increase in NCX1 mRNA expression is also observed in cultured cardiac myocytes following ␣-adrenergic stimulation by phenylephrine or exposure to veratridine. Importantly, the exchanger is also...

Chronic Administration of KB-R7943 Induces Up-regulation of Cardiac NCX1

“…We utilized an adenoviral construct of the full-length Ncx1 promoter that contains 1831 bp of the promoter and the entire first exon fused to the luciferase reporter gene. The 1831Ncx1 construct has been previously shown to be sufficient for cardiac restricted expression that is up-regulated in response to ␣-adrenergic stimulation in both adult and neonatal cardiomyocytes (32)(33)(34). Adult cardiomyocytes infected with the 1831Ncx1 adenovirus were treated with 10 M KB-R7943 for 48 h. Inhibition of reverse mode NCX1 activity with KB-R7943 treatment resulted in a 3-fold up-regulation of the 1831Ncx1 promoter-driven luciferase expression when compared with control ( Fig.…”

GATA-4 Gene Organization and Analysis of Its Promoter

“…Since knockout mice exhibited specification of the cardiac cell lineage, 3,4) and ES cells with homozygous GATA-4(Ϫ/Ϫ) contributed to the heart tissue in chimeric embryos, 3) GATA-4 in procardiomyocytes or visceral endoderm cells likely regulate the expression of a morphogenic signaling molecule(s) that directs the migration of procardiomyocytes. Although GATA-4 knockout mice express cardiac contractile proteins, 3,4) a number of studies have suggested that both structural and regulatory genes in the cardiac muscle cells are targets of GATA-4, such as those of A1 adenosine receptor, 5) angiotensin II type Ia receptor, 6) atrial natriuretic peptide, 7) brain natriuretic peptide, [7][8][9] cardiac a actin, 10) cardiac troponin C, 11) cardiac troponin I, 9) cardiac-restricted ankyrin repeat protein, 12) amyosin heavy-chain, 9,13) b-myosin heavy-chain, 9) myosin light chain 1/3, 14) Na ϩ /Ca 2ϩ exchanger 1, 15,16) Nkx-2.5, 17,18) slow myosin heavy chain, 19) and corin. 20) It is also evident that GATA-4 functions in the regulation of both epithelial cell differentiation and gene expression in the gut 21,22) and gonads [23][24][25][26][27] in addition to the heart.…”

The Transcription Factors GATA4 and dHAND Physically Interact to Synergistically Activate Cardiac Gene Expression through a p300-dependent Mechanism

“…In cardiac myocytes, GATA4 is thought to play a particularly important role in regulating expression of most cardiac-expressed genes, including ␣-myosin heavy chain (␣-MHC), 1 cardiac troponin-C, atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), cardiac troponin-I, sodium/ calcium exchanger, cardiac-restricted ankyrin repeat protein, A1 adenosine receptor, m2 muscarinic receptor, and myosin light chain 1/3 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). In addition to directly controlling cardiac structural and regulatory gene expression, cardiac-expressed GATA factors indirectly support tissue-specific gene expression by regulating expression of other transcription factors.…”