Differential glucocorticoid responses of CYP3A23 and CYP3A2 are mediated by selective binding of orphan nuclear receptors.
CYP3A2 and CYP3A23 are two cytochrome P450 genes in rat that are differentially regulated in both their constitutive activities and their responsiveness to glucocorticoids, the prototypic CYP3A inducers. CYP3A2 displays 20-25% of the response to glucocorticoids as CYP3A23 despite extensive sequence homology in their 5'-regulatory regions. Promoter deletion analyses revealed that the CYP3A2 -57 to -168 region, homologous to the CYP3A23 dexamethasone-responsive region, mediated its low level activation. When this region was analyzed by DNase I footprinting, three binding sites were shown to correspond to the functional elements described for CYP3A23: DexRE-1, DexRE-2, and Site A (J. M. Huss and C. B. Kasper (1998) J. Biol. Chem. 273: 16155-16162). The CYP3A2 DexRE-2 and Site A elements bear two mismatches each from the CYP3A23 elements but displayed similar binding patterns in footprinting and gel-shift analyses as their CYP3A23 counterparts. The region containing 3A2DexRE-1 has six mismatches and displayed unique footprinting and gel-shift patterns compared to 3A23DexRE-1. Functional assays revealed that four mismatches within the DexRE-1 and DexRE-2 elements accounted for the differential inducibility of the two isoforms. We propose that the reduced responsiveness of CYP3A2 is the result of preferential binding of COUP-TF at the CYP3A2 DexRE-1 site. In contrast, CYP3A23 DexRE-1 associates with an accessory factor(s) that acts in concert with downstream sites to mediate the strong glucocorticoid induction response observed for CYP3A23. Site A mismatches did not influence induction magnitude but were responsible for basal activity differences. Higher CYP3A23 basal activity appears to be due to an E-box in 3A23SiteA that interacts with USF1, a ubiquitous bHLH/leucine zipper transcription factor. This site is disrupted in the corresponding 3A2SiteA. Hence, 4 nucleotide mismatches within two elements account for the difference in glucocorticoid induction, and a single mismatch is responsible for the fivefold difference in the basal activities of CYP3A2 and CYP3A23.
Huss
J M
JM
Department of Oncology, Environmental Toxicology Program, McArdle Laboratory for Cancer Research, Madison, Wisconsin, 53706, USA.
Wang
S I
SI
Kasper
C B
CB
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D020137:Base Pair Mismatch / Q000235:genetics; D001483:Base Sequence; D001667:Binding, Competitive; D051838:COUP Transcription Factor I; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology*; D004790:Enzyme Induction / Q000187:drug effects; D051557:Hepatocyte Nuclear Factor 4; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000276:immunology / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013250:Steroid Hydroxylases / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000276:immunology / Q000378:metabolism; D014407:Tumor Cells, Cultured; D051720:Upstream Stimulatory Factors
D051838:COUP Transcription Factor I; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C497591:Nr2f1 protein, rat; D010750:Phosphoproteins; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C497182:Usf1 protein, rat; D003907:Dexamethasone; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C465138:Cyp3a23-3a1 protein, rat; D051544:Cytochrome P-450 CYP3A; C022657:steroid hormone 6-beta-hydroxylase