Characterization of the Murine A1 Adenosine Receptor Promoter, Potent Regulation by GATA-4 and Nkx2.5

Abstract

Adenosine acts via A1 adenosine receptors (A1ARs) in the heart and brain to potently influence mammalian physiology. A1ARs are expressed very early in embryonic development, and A1ARs are among the earliest expressed G protein coupled receptors in the heart and brain. To understand the biologic basis of A1AR expression, a genomic fragment containing the murine A1AR promoter was cloned. Reporter assay studies using DDT1 MF2 cells that express A1ARs revealed that 500 base pairs of the proximal A1AR promoter contained essential elements for A1AR gene expression. Transgenic mice with A1AR proximal promoter coupled with the ␤-galactosidase reporter gene had heavy labeling of the brain and atria, consistent with normal patterns of A1AR expression. Within the proximal A1AR promoter, putative binding sites for cardiac transcription factors GATA and Nkx2.5 were identified. Co-expression studies revealed that GATA-4 and Nkx2.5 could individually drive A1AR promoter activity and act synergistically to activate A1AR expression. These observations suggest that embryonic A1AR expression involves activation of the A1AR promoter by GATA-4 and Nkx2.5.

GATA-4 Gene Organization and Analysis of Its Promoter

“…For the reporter gene assay, 1ϫ10 5 (TM3 and TM4 cells) or 2ϫ10 5 (P19.CL6 cells) cells were seeded into 2 ml medium containing 7% (v/v) FBS in each well of a 6-well culture plate, and then cultured for 24 h. 53) Lipofectamine · (Invitrogen) was used for transfection (duplicate) of plasmid DNA [reporter plasmid (0.5 mg) and pSV-b-Gal (0.5 mg) per well], according to the manual supplied by the manufacturer, except that the volume was quadrupled. Lysis buffer (90 ml) (Promega) was added to the phosphate-buffered saline [10 mM sodium phosphate buffer (pH 7.2), 137 mM NaCl, 3 mM KCl] (PBS)-washed plate 48 h after the start of transfection, and a cell lysate was prepared (12000ϫg, 10 min at 4°C).…”

“…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.…”

Roles of Cardiac Transcription Factors in Cardiac Hypertrophy

“…117,118 The expression of several cardiac genes in the heart of Csx/Nkx2-5-deficient embryos (including MLC2v, ANP, BNP, CARP, MEF2-C, eHAND/HAND1, N-myc, Iroquois homeobox gene 4, and HOP) was reduced. [117][118][119][120][121][122] In addition, direct downstream targets for Csx/ Nkx2-5 (such as ANP, 52,123 cardiac ␣-actin, 124 A 1 adenosine receptor, 125 calreticulin, 126 connexin40, 127 and NCX1) have been identified. 128 These results indicate a functional role of Csx/Nkx2-5 in the transcriptional regulation of a cardiac gene program.…”

A1 adenosine receptors play an essential role in protecting the embryo against hypoxia

“…Embryonic cardiac A 1 AR expression appears to be driven by the transcription factors GATA4 and Nkx2.5, which are expressed in the heart during early embryogenesis (12). As soon as the heart begins to beat, A 1 ARs regulate embryonic heart rate, and adenosine is the dominant prenatal humoral regulator of the fetal heart rate (13).…”