Dexamethasone-mediated up-regulation of human CYP2A6 involves the glucocorticoid receptor and increased binding of hepatic nuclear factor 4 alpha to the proximal promoter.
Human cytochrome P450 2A6 (CYP2A6) metabolizes various clinically relevant compounds, including nicotine- and tobacco-specific procarcinogens; however, transcriptional regulation of this gene is poorly understood. We investigated the role of the glucocorticoid receptor (GR) in transcriptional regulation of CYP2A6. Dexamethasone (DEX) increased CYP2A6 mRNA and protein levels in human hepatocytes in primary culture. This effect was attenuated by the GR receptor antagonist mifepristone (RU486; 17beta-hydroxy-11beta-[4-dimethylamino phenyl]-17alpha-[1-propynyl]estra-4,9-dien-3-one), suggesting that induction of CYP2A6 by DEX was mediated by the GR. In gene reporter assays, DEX caused dose-dependent increases in luciferase activity that was also prevented by RU486 and progressive truncations of the CYP2A6 promoter delineated DEX-responsiveness to a -95 to +12 region containing an hepatic nuclear factor 4 (HNF4) alpha response element (HNF4-RE). Mutation of the HNF4-RE abrogated HNF4alpha- and DEX-mediated transactivation of CYP2A6. In addition, overexpression of HNF4alpha increased CYP2A6 transcriptional activity by 3-fold. DEX increased HNF4alpha mRNA levels by 4-fold; however, the amount of HNF4alpha nuclear protein was unaltered. Electrophoretic mobility shift, chromatin immunoprecipitation (ChIP), and streptavidin DNA binding assays revealed that DEX increased binding of HNF4alpha to the HNF4-RE and that an interaction of GR and HNF4alpha occurred at this site. Moreover, ChIP assays indicated that histone H4 acetylation of the CYP2A6 proximal promoter chromatin was increased by DEX that may allow for increased binding of HNF4alpha to the HNF4-RE in human hepatocytes. These findings indicate that increased expression of CYP2A6 by DEX is mediated by the GR via a nonconventional transcriptional mechanism involving interaction of HNF4alpha with an HNF4-RE rather than a glucocorticoid response element.
Onica
Tania
T
Department of Biomedical Sciences, University of Guelph, Guelph, ON, Canada. gkirby@uoguelph.ca
Nichols
Kathleen
K
Larin
Meghan
M
Ng
Lorraine
L
Maslen
Ann
A
Dvorak
Zdenek
Z
Pascussi
Jean-Marc
JM
Vilarem
Marie-Josée
MJ
Maurel
Patrick
P
Kirby
Gordon M
GM
D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis* / Q000235:genetics; D002478:Cells, Cultured; D065637:Cytochrome P-450 CYP2A6; D003907:Dexamethasone / Q000494:pharmacology*; D004790:Enzyme Induction / Q000187:drug effects / Q000502:physiology; D006367:HeLa Cells; D051557:Hepatocyte Nuclear Factor 4 / Q000235:genetics / Q000378:metabolism*; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology*
D051557:Hepatocyte Nuclear Factor 4; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6