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 differenti ally regulated in both their constitutive activities and their responsivene ss 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 reveale d that the CYP3A2 -57 to -168 region, homologous to the CYP3A23 dexamethaso ne-responsive region, mediated its low level activation. When this region w as analyzed by DNase I footprinting, three binding sites were shown to corr espond 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: 16155-16 162). 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 containin g 3A2DexRE-1 has six mismatches and displayed unique footprinting and gel-s hift patterns compared to 3A23DexRE-1. Functional assays revealed that four mismatches within the DexRE-1 and DexRE-2 elements accounted for the diffe rential inducibility of the two isoforms. We propose that the reduced respo nsiveness 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 acces sory factor(s) that acts in concert with downstream sites to mediate the st rong glucocorticoid induction response observed for CYP3A23. Site A mismatc hes did not influence induction magnitude but were responsible for basal ac tivity differences. Higher CYP3A23 basal activity appears to be due to an E -box in 3A23SiteA that interacts with USF1, a ubiquitous bHLH/leucine zippe r transcription factor. This site is disrupted in the corresponding 3A2Site A Hence, 4 nucleotide mismatches within two elements account for the differ ence in glucocorticoid induction, and a single mismatch is responsible for the fivefold difference in the basal activities of CYP3A2 and CYP3A23. (C) 1999 Academic Press.