Retinoic Acid Receptor-γ in Human Epidermis Preferentially Traps All-Trans Retinoic Acid as its Ligand Rather Than 9-cis Retinoic Acid

Abstract

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.