Decoy oligonucleotide characterization of GATA-4 transcription factor in hypertrophic agonist induced responses of cardiac myocytes

Abstract

GATA-4 transcription factor is required for normal cardiac development. However, it is unknown whether GATA-4 is an essential mediator of hypertrophic responses in the heart. Rat B-type natriuretic peptide (BNP) gene promoter contains a region of two adjacent GATA binding sites (between -68 and -97) with high affinity for GATA-4. In order to block GATA-4 dependent signaling in cultured neonatal rat ventricular myocytes we administered a synthetic 30-bp phosphorothioated double-stranded DNA complementary to the rat BNP promoter region (between -68 and -97) as a "decoy" cis-element to bind GATA-4. GATA decoy oligodeoxynucleotide treatment of cardiomyocytes blocked GATA-4 DNA binding activity in electrophoretic mobility shift analysis and decreased baseline expression of cardiac natriuretic peptides and GATA-dependent promoter activity. In contrast, blocked GATA-4 DNA binding did not prevent endothelin-1 or phenylephrine induced expression of cardiac natriuretic peptides. Mutation of GATA binding sites at -80 and -91 rat BNP promoter downregulated baseline but did not affect endothelin-1 or angiotensin II induced promoter activity. Additively, GATA decoy oligodeoxynucleotide treatment was insufficient to block endothelin-1 induced activation of protein synthesis or sarcomeric protein assembly. In conclusion, a targeted disruption of GATA-4 DNA binding activity is insufficient to prevent hypertrophic agonist induced responses of ventricular myocytes.

Endothelin-1-specific Activation of B-type Natriuretic Peptide Gene via p38 Mitogen-activated Protein Kinase and Nuclear ETS Factors

“…Plasmid for Rous sarcoma virus-promoter linked to ␤-galactosidase gene (RSV-␤gal) and pGL3-Basic plasmid-expressing luciferase gene were purchased from Clontech (Palo Alto, CA). Mutations (QuikChange site-directed mutagenesis, Stratagene, La Jolla, CA) to the Ϫ534 bp 5Ј-flanking region of rBNP promoter-driven pGL3-Basic plasmid (Ϫ534BNP-luc) (20) were introduced using oligonucleotides (coding strand, mutated nucleotides are in boldface) of 5Ј-GCTACCAG-AGTGCCCAGCCTCCGTGCAGCCCGGCCC-3Ј, 5Ј-CTGGAAGTGTTT-TTGACAGTTCACCCCATAAAGCCCC-3Ј and 5Ј-GGCAGGAATGTGT-CTTGCAAATCAGATGCAACCCCACCCCTAC-3Ј, for ETS binding sequence at Ϫ498 bp, AP-1 binding sequence at Ϫ373 bp and tandem GATA binding sequence at Ϫ90/Ϫ81 bp of rBNP promoter (GenBank TM accession number M60266), respectively. All mutations were confirmed by nucleotide sequencing (ABI-Prism 310, PerkinElmer Life Sciences, Foster City, CA).…”

“…Cell Culture and Transfection-Neonatal rat ventricular myocytes were prepared from 2-to 4-day-old Sprague-Dawley rats and transfected with liposome-mediated transfection (20). The Animal Use and Care Committee of the University of Oulu approved the experimental design.…”

“…Northern blots were hybridized with specific cDNA probes for rat BNP, rat ANP, and rat ribosomal 18 S (20) labeled with [ 32 P]dCTP (Amersham Biosciences) with a T7 Quick Prime kit (Amersham Biosciences). To measure the secretion of BNP, samples of incubation medium were subjected to radioimmunoassay of BNP (20).…”

“…Sarcomeric Organization-Cardiac myocytes were grown on collagen-coated glass coverslips and exposed to ET-1 (100 nM) for 24 h. ␣-Actin filaments of fixed myocytes were labeled with Alexa Fluor TM 488 Phalloidin (Molecular Probes Inc., Eugene, OR) (20). Representative images were taken with laser confocal microscope (LSM 510, Zeiss, Jena, Germany).…”

“…Physical and functional interactions of GATA-4 with its cofactors, nuclear factor of activated T lymphocytes (NFAT) and serum response factor (SRF), are induced in ET-1-treated cardiac myocytes (17,18). However, inhibitory strategies targeted against either GATA-4 protein synthesis or DNA binding affinity have produced different results in ET-1-induced hypertrophic growth of cardiac myocytes, suggesting an increasing complexity of ET-1-dependent pathways (19,20). To elucidate the nuclear mechanisms of ET-1-activated cell signaling, we studied rat BNP (rBNP) as a cardiac myocyte-specific target gene for ET-1 and identified a novel signaling mechanism initiating from cell membrane ET A receptor and leading to p38 MAPK-and ETS factor-dependent induction of the rBNP gene.…”