Decoy oligonucleotide characterization of GATA-4 transcription factor in hypertrophic agonist induced responses of cardiac myocytes.
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.
Pikkarainen
Sampsa
S
Department of Pharmacology and Toxicology, Biocenter Oulu, Faculty of Medicine, University of Oulu, P.O. Box 5000, 90014 Oulu, Finland.
Kerkelä
Risto
R
Pöntinen
Juhani
J
Majalahti-Palviainen
Theresa
T
Tokola
Heikki
H
Eskelinen
Sinikka
S
Vuolteenaho
Olli
O
Ruskoaho
Heikki
H
D000804:Angiotensin II; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D006332:Cardiomegaly / Q000209:etiology / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019332:Endothelin-1 / Q000378:metabolism; D050991:GATA4 Transcription Factor; D005786:Gene Expression Regulation; D009206:Myocardium / Q000378:metabolism*; D020097:Natriuretic Peptide, Brain / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D010656:Phenylephrine / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D019332:Endothelin-1; D050991:GATA4 Transcription Factor; D009838:Oligodeoxyribonucleotides; D014157:Transcription Factors; D000804:Angiotensin II; D020097:Natriuretic Peptide, Brain; D010656:Phenylephrine; D009320:Atrial Natriuretic Factor