Aryl hydrocarbon receptor regulation of cytochrome P4501B1 in rat mammary fibroblasts: evidence for transcriptional repression by glucocorticoids.
Cytochrome P450 1B1 (CYP1B1), which actively metabolizes polycyclic aromatic hydrocarbons, is regulated by the aryl hydrocarbon receptor (AhR) in primary cultures of rat mammary fibroblasts (RMF) and rat embryo fibroblasts (REF). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induced the 5.2-kilobase CYP1B1 mRNA in RMF (12-fold) and REF (14-fold) after a 6-hr treatment, with comparable increases in the microsomal protein. The synthetic glucocorticoid dexamethasone (DEX) suppresses TCDD-induced expression of CYP1B1 in RMF and REF. Suppression of CYP1B1 mRNA in RMF (maximal suppression, 70%) was observed when DEX was added 2 hr before TCDD, but was not observed with co-administration. The concentration dependence (EC50 approximately 10 nM) and reversal by the antagonist, RU486, implicates the glucocorticoid receptor. DEX inhibition of TCDD-induced CYP1B1 protein needed more extensive preincubation (>6 hr). TCDD induction of CYP1B1-luciferase constructs in RMF was mediated by a 265-base-pair upstream region (-810 to -1075), which was similarly suppressed (50-70%) by a 2-hr preincubation with 10(-7) M DEX via this enhancer region. Expression of the AhR is suppressed by DEX (70% after 12 hr), but not after the 2-hr period that was sufficient for suppression of transcription. The AhR nuclear translocator is not affected by this treatment. We conclude that glucocorticoid receptor rapidly suppresses activity of the AhR/AhR nuclear translocator complex in the CYP1B1 enhancer region, even though lacking glucocorticoid responsive element(s). DEX inhibits proliferation of RMF in this same concentration range (35%, EC50 approximately 5 nM), indicating additional effects on intracellular activity that may link to this suppression.
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Environmental Toxicology Center, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
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D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D065633:Cytochrome P-450 CYP1B1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004268:DNA-Binding Proteins; D003907:Dexamethasone / Q000494:pharmacology*; D004742:Enhancer Elements, Genetic / Q000187:drug effects / Q000502:physiology; D005260:Female; D005347:Fibroblasts / Q000187:drug effects* / Q000201:enzymology*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D008321:Mammary Glands, Animal / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D011247:Pregnancy; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics / Q000502:physiology*; D014157:Transcription Factors / Q000096:biosynthesis; D014158:Transcription, Genetic / Q000187:drug effects*
C497398:ARNT protein, rat; D004268:DNA-Binding Proteins; D000072317:Polychlorinated Dibenzodioxins; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D008156:Luciferases; D001189:Aryl Hydrocarbon Hydroxylases; C585499:Cyp1b1 protein, rat; D065633:Cytochrome P-450 CYP1B1