Marikar Y, Wang Z, Duell E A, Petkovich M, Voorhees J J, Fisher G J
Department of Dermatology, University of Michigan, Ann Arbor 48109-0609, USA.
J Invest Dermatol. 1998 Sep;111(3):434-9. doi: 10.1046/j.1523-1747.1998.00297.x.
Tissue levels of all-trans retinoic acid (RA) are maintained through coordinated regulation of biosynthesis and breakdown. The major pathway for all-trans RA inactivation is initiated by 4-hydroxylation. A novel cytochrome P-450 (CYP26) that catalyzes 4-hydroxylation of all-trans 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-trans 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-trans 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-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 activation of RA receptors by all-trans RA. In summary, RA 4-hydroxylase is a unique, highly specific cytochrome P-450 isoenzyme, whose expression is regulated by its natural substrate, all-trans RA, through activation of RA receptors. RA 4-hydroxylase functions to limit the levels, and thereby the biologic activity of all-trans RA in HaCaT cells.
全反式维甲酸（RA）的组织水平通过生物合成与分解的协同调节得以维持。全反式RA失活的主要途径由4-羟基化起始。一种催化全反式RA 4-羟基化的新型细胞色素P-450（CYP26）最近已被克隆。我们研究了永生化人角质形成细胞HaCaT中RA 4-羟化酶的调节及特性。在未添加类视黄醇的情况下，RA 4-羟化酶（CYP26）的mRNA和蛋白检测量极低。添加全反式RA可迅速诱导RA 4-羟化酶mRNA（2小时内）及活性（6小时内）。mRNA和活性的诱导均是短暂的，48小时内恢复至基线水平，且完全依赖mRNA合成（即被放线菌素D阻断）。特异性激活核RA受体的合成类视黄醇CD367也可迅速诱导RA 4-羟化酶活性。然而，与全反式RA不同，这种诱导作用持续时间长（>48小时）。这种差异归因于HaCaT细胞中CD367缺乏代谢失活。抑制RA受体依赖性基因转录的CD2665可阻断类视黄醇对RA 4-羟化酶的诱导，表明其由RA受体介导。添加针对10个不同哺乳动物P-450亚家族的过量未标记底物，不会与全反式RA竞争RA 4-羟化酶活性。RA 4-羟化酶不会使9-顺式RA或13-顺式RA羟基化。酮康唑对RA 4-羟化酶活性的抑制增强了全反式RA对RA受体的激活作用。总之，RA 4-羟化酶是一种独特的、高度特异性的细胞色素P-450同工酶，其表达受天然底物全反式RA通过激活RA受体来调节。RA 4-羟化酶的作用是限制HaCaT细胞中全反式RA的水平，从而限制其生物活性。
