ABSTRACT
Retinoids are important regulators of epithelial differentiation. AGN 193109 is a high-affinity antagonist and inverse agonist for the nuclear retinoic acid receptors (RARs). Paradoxically, both AGN 193109 and retinoid agonists inhibit the expression of the differentiation marker MRP-8 in normal human keratinocytes (NHKs). TTNPB, an RAR agonist, and AGN 193109 mutually antagonize MRP-8 inhibition at both mRNA and protein levels. We find that this antagonism, which is greatest at an AGN 193109:TTNPB ratio of about 10:1, is absent when either compound is in significant excess. The potent RARalpha-specific agonist, AGN 193836, has no effect on MRP-8 regulation. These data indicate that inverse agonists and agonists suppress MRP-8 in NHKs through RARgamma using distinct and mutually inhibitory mechanisms. The activity of AGN 193109 on MRP-8 is cell type specific. In differentiating ECE16-1 cervical cells, TTNPB inhibits while AGN 193109 induces MRP-8 mRNA levels. The effect of AGN 193109 on genes inhibited by retinoid agonists in NHKs is also selective; expression of the differentiation markers transglutaminase 1 and keratin 6 is not down-regulated by AGN 193109 whereas stromelysin-1 expression is suppressed. These results show a complex gene and cell context-specific interplay between agonist and inverse agonist for the regulation of gene expression.
Subject(s)
Keratinocytes/drug effects , Naphthalenes/metabolism , Naphthalenes/pharmacology , Receptors, Retinoic Acid/metabolism , Calcium-Binding Proteins/metabolism , Calgranulin A , Cells, Cultured , Dose-Response Relationship, Drug , Gene Expression Regulation , Humans , Keratins/pharmacology , Matrix Metalloproteinase 3/pharmacology , Retinoids/agonists , Reverse Transcriptase Polymerase Chain Reaction , Transglutaminases/pharmacology , Retinoic Acid Receptor gammaABSTRACT
Retinoids down-regulate the expression of metalloproteinases, cytokines, and other genes involved in cell proliferation and inflammation. Tazarotene (AGN 190168), a retinoic acid receptor (RAR)-specific retinoid, is effective in the treatment of psoriasis, a hyperproliferative and inflammatory skin disease. Because negative regulation of genes appears to be important in the antiproliferative and antiinflammatory action of retinoids, we studied the down-regulation of genes in skin raft cultures by this antipsoriatic retinoid. By subtraction hybridization, we found that migration inhibitory factor-related protein (MRP-8) and skin-derived anti-leukoproteinase (SKALP) are down-regulated by AGN 190168. MRP-8 and SKALP are overexpressed in psoriatic lesions as compared to the normal epidermis, and they are markers of hyperproliferative keratinocyte differentiation. We also show that MRP-8 expression is retinoid inhibitable in cultured keratinocytes induced to differentiate with 10% serum or IFN-gamma, and that MRP-8 is inhibited by RAR but not by retinoid X receptor-specific retinoids in a dose-dependent manner. Finally, MRP-8, SKALP, and the previously characterized differentiation marker, transglutaminase I, are all down-regulated in vivo in psoriatic lesions after treatment with AGN 190168 in comparison to placebo. Taken together, these data suggest that these markers may be down-regulated by tazarotene in psoriasis through direct action on keratinocyte gene expression rather than by an overall tazarotene effect on lesional therapeutic status.
Subject(s)
Keratinocytes/cytology , Psoriasis/pathology , Receptors, Retinoic Acid/genetics , Antigens, Differentiation/genetics , Antineoplastic Agents/pharmacology , Biomarkers , Calcium-Binding Proteins/genetics , Calgranulin A , Cell Differentiation/physiology , Cells, Cultured/chemistry , Cells, Cultured/drug effects , Cells, Cultured/enzymology , DNA, Complementary/genetics , Dose-Response Relationship, Drug , Down-Regulation/physiology , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/physiology , Glycogen Debranching Enzyme System/genetics , Humans , Interferon-gamma/pharmacology , Keratinocytes/chemistry , Keratinocytes/enzymology , Male , Nicotinic Acids/pharmacology , Proteinase Inhibitory Proteins, Secretory , Proteins/genetics , Psoriasis/genetics , Psoriasis/metabolism , RNA, Messenger/metabolism , Retinoids/pharmacology , Sensitivity and Specificity , Serine Proteinase Inhibitors/genetics , Skin/cytology , Teratogens/pharmacologyABSTRACT
Inverse agonists are ligands that are capable of repressing basal receptor activity in the absence of an agonist. We have designed a series of C-1-substituted acetylenic retinoids that exhibit potent antagonism of retinoic acid receptor (RAR)-mediated transactivation. Comparison of these related retinoid antagonists for their ability to repress basal RAR transcriptional activity demonstrates that the identity of the C-1 substituent differentiates these ligands into two groups: RAR inverse agonists and neutral antagonists. We show that treatment of cultured human keratinocytes with a RAR inverse agonist, but not a RAR neutral antagonist, leads to the repression of the serum-induced differentiation marker MRP-8. While RAR-selective agonists also repress expression of MRP-8, cotreatment with a RAR inverse agonist and a RAR agonist results in a mutual repression of their individual inhibitory activities, indicating the distinct modes of action of these two disparate retinoids in modulating MRP-8 expression. Our data indicate that RARs, like beta2-adrenoreceptors, are sensitive to inverse agonists and that this new class of retinoids will provide insight into the molecular mechanisms of RAR function in skin and other responsive tissues.