Glucocorticoid regulation of the rat cytochrome P450c (P450IA1) gene: receptor binding within intron I.
The actions of polycyclic aromatic hydrocarbons and glucocorticoids to regulate the expression of cytochrome P450c were investigated using cultured fetal rat hepatocytes. Cytochrome P450c mRNA content, determined by Northern blot analysis, was induced in cells treated with 1,2-benzanthracene from levels undetectable in untreated cells. When dexamethasone was included in the culture medium together with 1,2-benzanthracene there was a further 2-fold increase in the induction of cytochrome P450c mRNA. The concentration of dexamethasone required for a half-maximal increase in cytochrome P450c mRNA content was approximately 10(-9) M. By nuclear run-on transcription assays, treatment with 1,2-benzanthracene induced cytochrome P450c transcription 5.3-fold over untreated cells. In the presence of dexamethasone and 1,2-benzanthracene, there was a further 2-fold increase in cytochrome P450c transcription. Southwestern blotting and exonuclease footprinting methods have identified binding interactions of a purified glucocorticoid receptor fraction with portions of the cytochrome P450c gene within the first intron. Using a chimeric plasmid containing the first intron, the first exon, and 824 bp of 5'-flanking region of the cytochrome P450c gene, chloramphenicol acetyltransferase activity was induced in transfected HepG2 hepatoma cells by the addition of 1,2-benzanthracene. The addition of dexamethasone induced a further 2.2-fold increase in activity. Deletion of the first intron within the chimeric plasmid abolished responsiveness to dexamethasone. It is concluded that glucocorticoids act together with polycyclic aromatic hydrocarbons to increase the levels of cytochrome P450c expressed in the fetal rat hepatocyte, and that this action is mediated by the glucocorticoid receptor. A glucocorticoid responsive element, which binds the glucocorticoid receptor, has been identified within the first intron of the cytochrome P450c gene. These results suggest that glucocorticoids play a significant role in the response of the hepatic cytochrome P450c gene to xenobiotics.
Mathis
J M
JM
Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas 75235.
Houser
W H
WH
Bresnick
E
E
Cidlowski
J A
JA
Hines
R N
RN
Prough
R A
RA
Simpson
E R
ER
D000199:Actins / Q000235:genetics; D000818:Animals; D001551:Benz(a)Anthracenes / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D003907:Dexamethasone / Q000494:pharmacology*; D004357:Drug Synergism; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007438:Introns / Q000187:drug effects*; D007700:Kinetics; D009710:Nucleotide Mapping; D012333:RNA, Messenger / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D014158:Transcription, Genetic / Q000187:drug effects
D000199:Actins; D001551:Benz(a)Anthracenes; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; C028697:dexamethasone receptor; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; C030935:benz(a)anthracene