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

Members of the CYP2C subfamily, highly represented in rat liver, are constitutively expressed genes that encode enzymes that metabolize endogenous substrates such as steroids. Expression of P4502C11 and P4502C12 in rat liver is regulated in a sex-specific manner that ultimately depends on the sexually differentiated secretory pattern of GH release. Additional members of this family, also expressed in rat liver, had been cloned and the role of GH in regulating P4502C6 and P4502C7 was investigated.

P4502C6 was minimally affected by hypophysectomy or GH treatment whereas 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 induction seen in vivo. In contrast, retinoids and especially all-trans retinoic acid (RA) had a profound effect which was demonstrated to be at the transcriptional level. The importance of vitamin A for CYP2C7 expression in vivo was confirmed by the low abundance of P4502C7 mRNA levels in vitamin A deficient rats.

In vitro footprinting analysis of 200 bp upstream of the transcription start site in the CYP2C7 gene revealed binding of proteins to sequences with apparent homology to orphan nuclear receptor binding sites. Similar sequences were found in CYP2C11, 2C12 and 2C13. The functional role of these sites was investigated, however, the results indicated that the orphan receptor binding elements are redundant in the rat CYP2C genes. It cannot be excluded that they have a more significant function in the complete promoter context.

Next we addressed the question if retinoids and/or GH could restore the low expression seen in vitamin A deficient animals and also why micromolar concentrations of retinoids were required for maximal induction in vitro. Both RA and GH partially restored the P4502C7 mRNA levels in vitamin A deficient rats; with a combination of the treatments a complete restoration was achieved. The P450 inhibitors ketoconazole and SKF-525A reduced the RA induction of P4502C7 mRNA in primary hepatocytes by 75% whereas the induction of RARB2 mRNA was not affected. In attempts to identify possible metabolites of RA that could be involved in the induction of CYP2C7 gene, cellular extracts were analyzed by HPLC. Two retinoid metabolites of intermediate polarity to RA and 4-oxo-RA were found but their identities have not yet been solved. Furthermore, the RA induction of P4502C7 was sensitive to a block of protein synthesis whereas that of RARB2 was not. These data indicate that the mechanism of retinoid regulation of CYP2C7 is different from the classical regulation of RARB2 and other retinoid responsive genes. An mutual dependency of GH and vitamin A at different levels could also be inferred from these studies.