title: Mapping of the pressure response element of the c-fos gene by direct DNA injection into beating hearts.
abstract: 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.
authors.affiliation: Molecular Medicine Division, Beth Israel Hospital, Boston, Massachusetts 02215.
authors.forename: T
authors.initials: T
authors.lastname: Aoyagi
authors.forename: S
authors.initials: S
authors.lastname: Izumo
mesh_terms: 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
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D026362:Serum Response Factor; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase