Mapping of the pressure response element of the c-fos gene by direct DNA injection into beating hearts.
In vivo gene transfer into various organs of postnatal animals has recently been demonstrated. We applied this technique in order to map the promoter element of the protooncogene c-fos in the myocardium that responds to left ventricular pressure overload in vivo. Beating rat hearts were directly injected with a recombinant plasmid containing a 356-base pair upstream regulatory element of the mouse c-fos gene fused to a reporter gene. Two days after the injection, the hearts were excised and buffer-perfused. Acute pressure overload was applied for 2 h by inflating an intraventricular balloon. Pressure overload increased the myocardial reporter gene activity by 3-8-fold (p < 0.01). Deletion and point mutations in the serum response element (SRE) of the c-fos promoter resulted in loss of pressure-induced reporter gene expression, indicating that the SRE is necessary for pressure response. The SRE alone was sufficient to confer pressure responsiveness to the minimal c-fos promoter, confirming that the pressure response element coincides with the SRE. A construct containing a point mutation at the p62TCF binding site of the SRE did not respond to pressure overload or to a protein kinase C activator but retained responsiveness to a calcium ionophore. This suggests that pressure overload may activate the c-fos promoter by a protein kinase C-dependent pathway. Thus, a directly injected gene can be regulated in the host cells by a physical stimulus to the intact organ. The in vivo DNA injection technique is a useful method to study complex organ physiology at the molecular level.
Aoyagi
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Molecular Medicine Division, Beth Israel Hospital, Boston, Massachusetts 02215.
Izumo
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D000818:Animals; D001483:Base Sequence; D001794:Blood Pressure; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA; D004268:DNA-Binding Proteins; D005786:Gene Expression Regulation / Q000502:physiology*; D018014:Gene Transfer Techniques; D016762:Genes, fos; D006321:Heart / Q000502:physiology*; D006352:Heart Ventricles; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D051381:Rats; D017208:Rats, Wistar; D026362:Serum Response Factor
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D026362:Serum Response Factor; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase