Endogenous retinoic acid receptor (RAR)-retinoid X receptor (RXR) heterodimers are the major functional forms regulating retinoid-responsive elements in adult human keratinocytes. Binding of ligands to RAR only is sufficient for RAR-RXR heterodimers to confer ligand-dependent activation of hRAR beta 2/RARE (DR5).

We have examined how retinoic acid receptors (RARs) and retinoid X receptors (RXRs) at physiological concentrations regulate distinct retinoid-responsive elements, hRAR beta 2/beta RARE (DR5) and rCRBPII/RXRE (DR1), in keratinocytes from human skin, a major retinoid target. In vitro, endogenous RAR gamma and RXRs bound to these elements as heterodimers (RAR.RXR) but not homodimers (RAR.RAR or RXR.RXR). In cultured keratinocytes, all-trans retinoic acid, 9-cis retinoic acid, and CD367 activated beta RARE but not RXRE via endogenous RAR.RXR (ED50 = 2.3, 3.8, and 0.3 nM, respectively) whereas SR11237 showed no significant effect. All-trans retinoic acid, 9-cis retinoic acid, and SR11237 activated RXRE via overexpressed RXR.RXR (ED50 = 110, 120, and 11 nM, respectively), indicating interconversion between retinoic acid isomers, whereas co-overexpression of RAR alpha or RAR gamma suppressed this activation. Unlike 9cRA, CD367 neither induced formation of nor activated RXR.RXR. Overexpression of RAR or RXR mutated in transactivation domain AF-2 suppressed endogenous receptor activity over beta RARE. Our data suggest that 1) in keratinocytes, RAR.RXR-mediated pathway dominates over that mediated by RXR.RXR; 2) RAR-selective CD367 and RXR-selective SR11237 can be used to identify these two distinct pathways, respectively; 3) beta RARE is mainly regulated by RAR.RXR, in which RAR alone confers ligand inducibility whereas AF-2 of unliganded RXR is required for transactivation by liganded RAR AF-2; 4) lack of RXRE activity in keratinocytes is due to low endogenous levels of RXR.RXR and inhibition by RAR.RXR; and 5) interaction among RXRs is much lower than that between RAR and RXR.