Specific Activation of Retinoic Acid Receptors (RARs) and Retinoid X Receptors Reveals a Unique Role for RARγ in Induction of Differentiation and Apoptosis of S91 Melanoma Cells

Remco A Spanjaard,Timothy J Eberlein,Bruno Charpentier,William W Chin,Patricia J Lee,Masato Ikeda

doi:10.1074/jbc.272.30.18990

Abstract

Retinoic acid (RA) and 9-cis-RA induce growth arrest and differentiation of S91 melanoma cells. RA activates retinoic acid receptors (RARs), whereas 9-cis-RA activates both RARs and retinoid X receptors (RXRs). Both classes of receptors function as ligand-dependent transcription factors. S91 melanoma cells contain mRNA for RXRalpha, RXRbeta, RARalpha, RARgamma, and RARbeta in low levels. Among these, only RARbeta gene transcription is induced by retinoids. However, at present the individual role(s) for each RXR and RAR isoform in these processes is unclear. We assessed the function of all isoforms in the S91 melanoma model by using RXR and RAR isoform-specific retinoids to study their effects on cell growth, RARbeta expression, and differentiation. Activation of each of the endogenous RXR or RAR isoforms induces RARbeta gene expression, and blocks cellular proliferation. However, only the RARgamma-ligands cause additional differentiation toward a melanocytic phenotype, which coincides with substantial apoptosis well before morphological changes are apparent. Apoptosis is completely dependent on de novo protein synthesis but cannot be induced by changes in activities of AP-1, protein kinase C, and protein kinase A, nor can it be blocked by the presence of the antioxidant glutathione. These results argue against a specific role for RARbeta, but suggest that RARgamma has a critical role in a genetic switch between melanocytes and melanoma, and induction of ligand-dependent apoptosis.

Highlights

Retinoid receptors, which include retinoic acid (RA)1 receptors (RARs) and retinoid X receptors (RXRs), are members of a large group of ligand-dependent transcription factors [1, 2]
Apoptosis is completely dependent on de novo protein synthesis but cannot be induced by changes in activities of AP-1, protein kinase C, and protein kinase A, nor can it be blocked by the presence of the antioxidant glutathione. These results argue against a specific role for RARb, but suggest that RARg has a critical role in a genetic switch between melanocytes and melanoma, and induction of ligand-dependent apoptosis
Ligands Which Bind and Activate RARa, RARb, RARg, or RXR Cause Growth Arrest—For the studies described here, we selected the following synthetic retinoids, and determined their Kd values for each retinoic acid receptors (RARs) isoform isoforms used in these studies

Summary

Introduction

Retinoid receptors, which include retinoic acid (RA) receptors (RARs) and retinoid X receptors (RXRs), are members of a large group of ligand-dependent transcription factors [1, 2]. Ligand-bound receptors can modulate the expression of genes containing appropriate response elements (RA response element, RARE). Upon treatment with RA or 9-cis-RA, a reversible conversion of malignant melanoma into a benign, melanocytic phenotype takes place suggesting that a specific genetic program is induced and maintained by RARs and/or RXRs [23, 25,26,27]. Administration of either of these two retinoids results in rapid up-regulation of RARb expression, followed by cessation of cell division, and morphological differentiation [23,24,25]. We set out to answer these questions by using a number of RXR and RAR isoform-specific agonists to evaluate their effects on cell growth, RARb expression, and morphological differentiation. Our results suggest that all receptors can induce growth arrest and transcriptional activation of the RARb gene. Only the RARg ligands cause morphological differentiation which, interestingly, occurs concomitantly with sub-

Methods

Results

Conclusion