Kurlandsky S B, Xiao J H, Duell E A, Voorhees J J, Fisher G J
Department of Dermatology, University of Michigan, Ann Arbor 48109-0528.
J Biol Chem. 1994 Dec 30;269(52):32821-7.
The biological activity of all-trans retinol, in human keratinocytes, was investigated through metabolic and functional analyses that assessed the capacity for retinol uptake and metabolism and the mechanism of retinol-induced activation of gene transcription. Human keratinocytes converted all-trans retinol predominantly to retinyl esters, which accounted for 60 and 90% of cell-associated radiolabel after a 90-min pulse and a 48-h chase, respectively. Human keratinocytes also metabolized all-trans retinol to low levels of all-trans retinoic acid (11.47-131.3 ng/mg of protein) in a dose-dependent manner, between 0.3 and 10 microM added retinol. Small amounts of 13-cis retinoic acid (5.47-8.62 ng/mg of protein) were detected, but 9-cis retinoic acid was detected only when keratinocytes were incubated with radiolabeled retinol. There was no accumulation of the oxidized catabolic metabolites 4-hydroxy- or 4-oxoretinoic acid; however, 5,6-epoxy retinoic acid was detected at pharmacological levels (10 and 30 microM) of added retinol. Biological activity of retinol was assessed through analysis of two known retinoic acid-mediated responses: 1) reduction of type I epidermal transglutaminase and 2) activation of a retinoic acid receptor-dependent reporter gene, beta RARE3-tk-CAT. Both all-trans retinol and all-trans retinoic acid reduced type I epidermal transglutaminase in a dose-dependent manner; however, the ED50 for all-trans retinol (10 nM) was 10 times greater than for all-trans retinoic acid (1 nM). All-trans retinol also stimulated beta RARE3-tk-CAT reporter gene activity in a dose-dependent manner. Half-maximal induction was observed at 30 nM retinol, which was again 10-fold greater than observed with all-trans retinoic acid. Cotransfection of human keratinocytes with expression vectors for dominant negative mutant retinoic acid and retinoid X receptors reduced retinol-induced beta RARE3-tk-CAT reporter gene activation by 80%. Inhibition of conversion of all-trans retinol or all-trans retinaldehyde to all-trans retinoic acid by citral reduced beta RARE3-tk-CAT activity 98 and 86%, respectively. These data demonstrate that retinol-induced responses in human keratinocytes are mediated by its tightly regulated conversion to retinoic acid, which functions as a ligand to activate nuclear retinoic acid receptors.
通过代谢和功能分析，研究了全反式视黄醇在人角质形成细胞中的生物活性，这些分析评估了视黄醇摄取和代谢的能力以及视黄醇诱导基因转录激活的机制。人角质形成细胞将全反式视黄醇主要转化为视黄酯，在90分钟脉冲和48小时追踪后，视黄酯分别占细胞相关放射性标记的60%和90%。人角质形成细胞还以剂量依赖的方式将全反式视黄醇代谢为低水平的全反式维甲酸（11.47 - 131.3 ng/mg蛋白质），添加的视黄醇浓度在0.3至10 μM之间。检测到少量的13 - 顺式维甲酸（5.47 - 8.62 ng/mg蛋白质），但仅在角质形成细胞与放射性标记的视黄醇孵育时才检测到9 - 顺式维甲酸。氧化分解代谢产物4 - 羟基或4 - 氧代视黄酸没有积累；然而，在添加视黄醇的药理水平（10和30 μM）下检测到了5,6 - 环氧视黄酸。通过分析两种已知的维甲酸介导的反应来评估视黄醇的生物活性：1）I型表皮转谷氨酰胺酶的减少和2）维甲酸受体依赖性报告基因βRARE3 - tk - CAT的激活。全反式视黄醇和全反式维甲酸均以剂量依赖的方式降低I型表皮转谷氨酰胺酶；然而，全反式视黄醇的ED50（10 nM）比全反式维甲酸（1 nM）大10倍。全反式视黄醇还以剂量依赖的方式刺激βRARE3 - tk - CAT报告基因的活性。在30 nM视黄醇时观察到半数最大诱导，这再次比全反式维甲酸观察到的值大10倍。用人维甲酸和类视黄醇X受体的显性负突变体表达载体共转染人角质形成细胞，使视黄醇诱导的βRARE3 - tk - CAT报告基因激活降低了80%。柠檬醛抑制全反式视黄醇或全反式视黄醛向全反式维甲酸的转化，分别使βRARE3 - tk - CAT活性降低98%和86%。这些数据表明，视黄醇在人角质形成细胞中诱导的反应是由其严格调控的向维甲酸的转化介导的，维甲酸作为配体激活核维甲酸受体。
