ABSTRACT
Most studies have reported an up-regulation of retinoic acid receptor (RAR) mRNA expression by all-trans retinoic acid (RA). We aimed to study the effect of RA on RAR protein levels in MCF-7 human breast cancer cells. Incubation of these cells with 10(-6) M RA induced a rapid breakdown of both RARalpha and RARgamma in spite of the accumulation of their mRNAs. Proteasome specific inhibitors blocked the RA-induced breakdown of RARs. Furthermore, RA enhanced the formation of the complex between RARalpha and ubiquitin in a concentration- and time-dependent manner, suggesting the involvement of ubiquitin and proteasome in this reaction. Retinoid X receptor alpha (RXRalpha) was also decreased, albeit to a lesser extent, in RA-treated cells. Use of synthetic receptor agonists and antagonists clearly showed that the effect of the retinoid on the breakdown of the retinoid receptors is receptor-ligand agonist-dependent and blunted by the antagonist. An electrophoretic mobility shift assay, using nuclear extracts from RA-treated cells, showed that a reduction in complex formation with hormone response elements correlated with the reduction of RAR and RXR protein. These data suggest that RA induces the breakdown of RARs through a process involving ubiquitination and that this phenomenon causes a reduction in the formation of DNA-receptor complexes.
Subject(s)
Antineoplastic Agents/pharmacology , Cysteine Endopeptidases/metabolism , Multienzyme Complexes/metabolism , Receptors, Retinoic Acid/metabolism , Tretinoin/pharmacology , Breast Neoplasms , Cysteine Proteinase Inhibitors/pharmacology , DNA/drug effects , DNA/metabolism , Humans , Leupeptins/pharmacology , Nuclear Proteins/metabolism , Proteasome Endopeptidase Complex , Response Elements/drug effects , Retinoic Acid Receptor alpha , Tumor Cells, Cultured , Ubiquitins/metabolism , Retinoic Acid Receptor gammaABSTRACT
4-Hydroxyphenylretinamide (4-HPR) is a synthetic retinoid with minimal toxicity and favorable pharmacokinetics during long-term administration to patients in clinical trials. Since 4-HPR binds poorly to the retinoic acid receptors, the issue of whether 4-HPR exerts its biological actions via classical retinoid receptor pathways remains to be resolved. We have previously reported that stable expression of a truncated retinoic acid receptor alpha, RARalpha403, transduced in NIH 3T3 cells by a retroviral vector, rendered the cells resistant to retinoic acid for growth inhibition and induction of tissue transglutaminase (TGase II). Here, we report that stable expression of the dominant negative construct RARalpha403 fails to blunt growth inhibition and TGase II induction by 4-HPR, a potent chemopreventive retinoid, in the same cells. These data show that retinoic acid receptors do not mediate either growth inhibition or induction of TGase II activity by 4-HPR in mouse fibroblast cells.