Fisher G J, Datta S C, Voorhees J J
Department of Dermatology, University of Michigan Medical Center, Ann Arbor 48109-0609, USA.
J Invest Dermatol. 1998 Mar;110(3):297-300. doi: 10.1046/j.1523-1747.1998.00112.x.
The biologic activity of retinoids is mediated through nuclear retinoic acid receptors (RAR), which are ligand-activated transcription factors. RAR directly bind and are activated by two naturally occurring isomers of retinoic acid (RA), all-trans retinoic acid (t-RA) and 9-cis retinoic acid (9c-RA). Human skin predominantly expresses RAR-gamma (approximately 87%) and RAR-alpha makes up the remainder. Recombinant RAR-gamma preferentially binds t-RA over 9c-RA in cell-free assays containing mixtures of the two retinoic acid isomers. We have investigated the ligand-binding properties of RAR in human epidermis. [3H]All-trans retinol (t-ROL) added to suspensions of intact epidermal cells was metabolically converted to [3H]t-RA, which bound to RAR. No binding of [3H]9c-RA to RAR was detected. Binding of [3H]t-RA, formed from [3H]t-ROL, was abolished by adding unlabeled t-RA, but was unaffected by adding unlabeled 9c-RA. Intact epidermal cells were incubated with mixtures of [3H]9c-RA and [3H]t-RA in varying ratios, and the amount of each labeled retinoid bound to RAR was measured. At ratios of 9c-RA to t-RA of 3:1 or lower, only [3H]t-RA was bound by RAR. Incubation of cells with [3H]9c-RA alone resulted in substantial (38%) binding of [3H]t-RA to RAR, in addition to binding of [3H]9c-RA, due to isomerization of [3H]9c-RA to [3H]t-RA. RAR in nuclear extracts from epidermal cells also displayed strong preferential binding of t-RA over 9c-RA. Competition studies revealed that 9c-RA was 6-fold less effective than t-RA at displacing [3H]t-RA bound to RAR in nuclear extracts. At ratios of 9c-RA to t-RA of 4:1 or lower, RAR in nuclear extracts bound t-RA exclusively. At higher ratios, [3H]9c-RA binding increased steeply. RAR-alpha in nuclear extracts bound both 9c-RA and t-RA without preference, whereas RAR-gamma displayed strong preferential binding of t-RA over 9c-RA. The level of endogenous t-RA exceeds that of 9c-RA in human skin in vivo, and significant isomerization of topically applied 9c-RA and 13c-RA to t-RA occurs. The relative abundance of t-RA in human skin, and preferential binding of t-RA by RAR-gamma, indicate that t-RA is the primary ligand mediating RAR-dependent responses in human skin under physiologic conditions, and under pharmacologic conditions when t-RA, 9c-RA, or 13c-RA are applied to skin.
类视黄醇的生物活性是通过核视黄酸受体（RAR）介导的，RAR是配体激活的转录因子。RAR直接结合并被视黄酸（RA）的两种天然存在的异构体激活，即全反式视黄酸（t-RA）和9-顺式视黄酸（9c-RA）。人类皮肤主要表达RAR-γ（约87%），其余为RAR-α。在含有两种视黄酸异构体混合物的无细胞试验中，重组RAR-γ优先结合t-RA而非9c-RA。我们研究了人类表皮中RAR的配体结合特性。添加到完整表皮细胞悬液中的[3H]全反式视黄醇（t-ROL）经代谢转化为[3H]t-RA，后者与RAR结合。未检测到[3H]9c-RA与RAR的结合。由[3H]t-ROL形成的[3H]t-RA的结合，在加入未标记的t-RA后被消除，但加入未标记的9c-RA则无影响。完整表皮细胞与不同比例的[3H]9c-RA和[3H]t-RA混合物孵育，并测量与RAR结合的每种标记类视黄醇的量。当9c-RA与t-RA的比例为3:1或更低时，只有[3H]t-RA被RAR结合。细胞单独与[3H]9c-RA孵育，除了[3H]9c-RA的结合外，还导致大量（38%）的[3H]t-RA与RAR结合，这是由于[3H]9c-RA异构化为[3H]t-RA。表皮细胞核提取物中的RAR也表现出对t-RA而非9c-RA的强烈优先结合。竞争研究表明，在取代与核提取物中RAR结合的[3H]t-RA方面，9c-RA的效力比t-RA低6倍。当9c-RA与t-RA的比例为4:1或更低时，核提取物中的RAR仅结合t-RA。在更高比例时，[3H]9c-RA的结合急剧增加。核提取物中的RAR-α对9c-RA和t-RA的结合无偏好，而RAR-γ表现出对t-RA而非9c-RA的强烈优先结合。在体内人类皮肤中，内源性t-RA的水平超过9c-RA，并且局部应用的9c-RA和13c-RA会大量异构化为t-RA。人类皮肤中t-RA的相对丰度以及RAR-γ对t-RA的优先结合表明t-RA是在生理条件下以及在将t-RA、9c-RA或13c-RA应用于皮肤的药理条件下介导人类皮肤中RAR依赖性反应的主要配体。
