RETINOIC ACID RECEPTORS REGULATE EXPRESSION OF RETINOIC ACID 4-HYDROXYLASE THAT SPECIFICALLY INACTIVATES ALL-TRANS-RETINOIC ACID IN HUMAN KERATINOCYTE HACAT CELLS

Tissue levels of all-trans retinoic acid (RA) are maintained through c oordinated regulation of biosynthesis and breakdown. The major pathway for all-trans RA inactivation is initiated by 4-hydroxylation. A nove l cytochrome P-450 (CYP26) that catalyzes 4-hydroxylation of all-trans RA has recently been cloned. We have investigated regulation and prop erties of RA 4-hydroxylase in immortalized human keratinocyte HaCaT ce lls. In the absence of added retinoid, RA 4-hydroxylase (CYP26) mRNA a nd protein were minimally detected. Addition of all-trans RA rapidly i nduced RA.4-hydroxylase mRNA (within 2 h) and activity (within 6 h). I nduction of both mRNA and activity was transient, returning to baselin e within 48 h, and completely dependent on mRNA synthesis (i.e., block ed by actinomycin D). The synthetic retinoid CD367, which specifically activates nuclear RA receptors, also rapidly induced RA Q-hydroxylase activity. This induction, however, unlike that of all-trans RA, was l ong-lived (>48 h). This difference was attributable to lack of metabol ic inactivation of CD367 in HaCaT cells. CD2665, which inhibits RA rec eptor-dependent gene transcription, blocked retinoid induction of RA 4 -hydroxylase, indicating that it is mediated by RA receptors. Addition of excess unlabeled substrates specific for 10 distinct mammalian P-4 50 subfamilies did not compete with all-trans RA for RA 4-hydroxylase activity. RA 4-hydroxylase did not hydroxylate 9-cis RA or 13-cis RA. Inhibition of RA 4-hydroxylase activity by ketoconazole potentiated ac tivation of RA receptors by all-tuans RA. In summary, RA 4-hydroxylase is a unique, highly specific cytochrome P-450 isoenzyme, whose expres sion is regulated by its natural substrate, all-trans RA, through acti vation of RA receptors. RA 4-hydroxylase functions to limit the levels , and thereby the biologic activity of all-trans RA in HaCaT cells.