Title: Retinoic Acid Receptors Regulate Expression of Retinoic Acid 4-Hydroxylase that Specifically Inactivates All-Trans Retinoic Acid in Human Keratinocyte HaCaT Cells
Abstract: Tissue levels of all-<i>trans</i> retinoic acid (RA) are maintained through coordinated regulation of biosynthesis and breakdown. The major pathway for all-<i>trans</i> RA inactivation is initiated by 4-hydroxylation. A novel cytochrome P-450 (CYP26) that catalyzes 4-hydroxylation of all-<i>trans</i> RA has recently been cloned. We have investigated regulation and properties of RA 4-hydroxylase in immortalized human keratinocyte HaCaT cells. In the absence of added retinoid, RA 4-hydroxylase (CYP26) mRNA and protein were minimally detected. Addition of all-<i>trans</i> RA rapidly induced RA 4-hydroxylase mRNA (within 2 h) and activity (within 6 h). Induction of both mRNA and activity was transient, returning to baseline within 48 h, and completely dependent on mRNA synthesis (i.e., blocked by actinomycin D). The synthetic retinoid CD367, which specifically activates nuclear RA receptors, also rapidly induced RA 4-hydroxylase activity. This induction, however, unlike that of all-<i>trans</i> RA, was long-lived (>48 h). This difference was attributable to lack of metabolic inactivation of CD367 in HaCaT cells. CD2665, which inhibits RA receptor-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-450 subfamilies did not compete with all-<i>trans</i> RA for RA 4-hydroxylase activity. RA 4-hydroxylase did not hydroxylate 9-<i>cis</i> RA or 13-<i>cis</i> RA. Inhibition of RA 4-hydroxylase activity by ketoconazole potentiated activation of RA receptors by all-<i>trans</i> RA. In summary, RA 4-hydroxylase is a unique, highly specific cytochrome P-450 isoenzyme, whose expression is regulated by its natural substrate, all-<i>trans</i> RA, through activation of RA receptors. RA 4-hydroxylase functions to limit the levels, and thereby the biologic activity of all-<i>trans</i> RA in HaCaT cells.