Retinoid Signaling Is Attenuated by Proteasome-Mediated Degradation of Retinoid Receptors in Human Keratinocyte HaCaT Cells

Abstract

The biological actions of retinoids are mediated by nuclear retinoid receptors, RAR and RXR, which are ligand-activated transcription factors. We investigated the mechanism of attenuation of retinoid receptor activity in human keratinocyte HaCaT cells. Treatment of HaCaT cells with all-trans-retinoic acid or 9-cis-retinoic acid reduced RARγ and RXRα protein levels by one-half within 24 h. In contrast, retinoid treatment did not alter RARγ or RXRα mRNA levels, suggesting that retinoids stimulate breakdown of their receptors. Pulse-chase studies revealed that retinoid treatment of HaCaT cells reduced RARγ and RXRα half-lives by 50%, indicating that retinoids accelerate breakdown of their receptors. The proteasome inhibitor MG132 prevented retinoid-induced receptor loss. Furthermore, MG132 potentiated retinoid-induced receptor activity, as assessed by expression of the retinoid-regulated CRABP-II gene in HaCaT cells. These data demonstrate that retinoids attenuate retinoid receptor function by enhancing proteasome-mediated retinoid receptor breakdown in HaCaT cells. Proteasome-mediated degradation of RARγ or RXRα in vitro was significantly reduced by the corepressor SMRT, which binds unliganded retinoid receptors. This protection from degradation was markedly diminished by ligand, which causes SMRT to dissociate from receptors. Ligand failed to relieve protection from degradation by SMRT of a mutant form of RXRα that binds SMRT in the presence and absence of ligand. Addition of coactivators TIF1, TIF2, and RIP140 had no effect on degradation of RARγ or RXRα. In summary, ligand binding to retinoid receptors promotes proteasome-mediated receptor degradation via dissociation of SMRT. Ligand-stimulated receptor degradation results in attenuation of retinoid signaling.