Regulation of cytochrome P4502C7 : Effects of growth hormone and vitamin A

Sammanfattning: Members of the CYP2C subfamily, highly represented in rat liver, are consti-tutively expressed genes that encode enzymes that metabolize endogenous substratessuch as steroids. Expression of P4502C11 and P4502C12 in rat liver is regulated ina sex-specific manner that ultimately depends on the sexually differentiated secre-tory pattern of GH release. Additional members of this family, also expressed in ratliver, had been cloned and the role of GH in regulating P4502C6 and P4502C7 wasinvestigated. P4502C6 was minimally affected by hypophysectomy or GH treatmentwhereas P4502C7 was reduced by hypophysectomy and restored by GH treatment.To further investigate the regulation of P4502C7, primary rat hepatocytes were used.The GH induction of 2C7 was low in this cell-system compared to the inductionseen in vivo. In contrast, retinoids and especially all-trans retinoic acid (RA) had aprofound effect which was demonstrated to be at the transcriptional level. The im-portance of vitamin A for CYP2C7 expression in vivo was confirmed by the lowabundance of P4502C7 mRNA levels in vitamin A deficient rats. In vitro footprinting analysis of 200 bp upstream of the transcription start sitein the CYP2C7 gene revealed binding of proteins to sequences with apparent ho-mology to orphan nuclear receptor binding sites. Similar sequences were found inCYP2C11,2C12 and 2C13. The functional role of these sites was investigated, how-ever, the results indicated that the orphan receptor binding elements are redundantin the rat CYP2C genes. It cannot be excluded that they have a more significantfunction in the complete promoter context. Next we addressed the question if retinoids and/or GH could restore the lowexpression seen in vitamin A deficient animals and also why micromolar concentra-tions of retinoids were required for maximal induction in vitro. Both RA and GHpartially restored the P4502C7 mRNA levels in vitamin A deficient rats; with acombination of the treatments a complete restoration was achieved. The P450 in-hibitors ketoconazole and SKF-525A reduced the RA induction of P4502C7 mRNAin primary hepatocytes by 75% whereas the induction of RARB2 mRNA was notaffected. In attempts to identify possible metabolites of RA that could be involvedin the induction of CYP2C7 gene, cellular extracts were analyzed by HPLC. Tworetinoid metabolites of intermediate polarity to RA and 4-oxo-RA were found buttheir identities have not yet been solved. Furthermore, the RA induction of P4502C7was sensitive to a block of protein synthesis whereas that of RARB2 was not. Thesedata indicate that the mechanism of retinoid regulation of CYP2C7 is different fromthe classical regulation of RARB2 and other retinoid responsive genes. An mutualdependency of GH and vitamin A at different levels could also be inferred fromthese studies.Keywords: P450, growth hormone, vitamin A, gene regulation, orphan receptorsISBN 91-628-1986-0