Design,synthesis and evaluation of retinoids with novel bulky hydrophobic partial structures

Many synthetic retinoids contain an aromatic structure with a bulky hydrophobic fragment. In order to obtain retinoids with therapeutic potential that do not bind to or activate retinoic acid X receptors (RXRs), we focused on the introduction of novel hydrophobic moieties, that is, metacyclophane, phenalene and benzoheptalene derivatives. The designed compounds were synthesized and their agonistic activities towards RARs and RXRs were evaluated. Most of the active compounds showed selectivity for RARα and RARβ over RARγ, and higher RARβ transactivating activity seemed to correlate with higher cell differentiation-inducing activity towards promyelocytic leukemia cell line HL-60. These compounds showed no agonistic activity towards RXRs.     

Novel retinoid X receptor (RXR) antagonists having a dicarba-closo-dodecaborane as a hydrophobic moiety

We designed and synthesized novel retinoid X receptor (RXR)-selective antagonists bearing a carborane moiety. Compounds 8a-d or 9a-d themselves have no differentiation-inducing activity toward HL-60 cells and no inhibitory activity towards a retinoic acid receptor (RAR) agonist. However, they inhibit the synergistic activity of an RXR agonist, PA024, in the presence of Am80 on the cell differentiation of HL-60. Transactivation assay using RARs and RXRs suggested that the inhibitory activity of 9b resulted from the selective antagonism at the RXR site of RXR-RAR heterodimers.     

F9 embryocarcinoma cells: a cell autonomous model to study the functional selectivity of RARs and RXRs in retinoid signaling

Mouse F9 embryocarcinoma (EC) cells constitute a well established cell-autonomous model system for investigating retinoid signaling in vitro as, depending on culture conditions, retinoic acid (RA) can induce their differentiation into either primitive, parietal or visceral extraembryonic endoderm-like cells. These RA-induced differentiations are accompanied by decreases in proliferation rates, modifications of expression of subsets of RA-target genes, and induction of apoptosis. To elucidate the roles played by the multiple retinoid receptors (RARs and RXRs) in response to RA treatments, F9 EC cells lacking one or several RARs or RXRs were engineered through homologous recombination. Mutated RARs and/or RXRs were then reexpressed in given RAR or RXR null backgrounds. WT and mutant cells were also treated with different combinations of ligands selective for RXRs and/or for each of the three RAR isotypes. These studies lead to the conclusion that most RA-induced events (e.g. primitive and visceral differentiation, growth arrest, apoptosis and activation of expression of a number of genes) are transduced by RARgamma/RXRalpha heterodimers, whereas some other events (e.g. parietal differentiation) are mediated by RARalpha/RXRalpha. heterodimers. They also demonstrate that both AF-1 and AF-2 activation functions of RARs and RXRs, as well as their phosphorylation, are differentially required in these RA-induced events. In RARgamma/RXRalpha heterodimers, the phosphorylation of RARgamma is necessary for triggering primitive differentiation, while that of RXRalpha is required for growth arrest. On the other hand, phosphorylation of RARalpha is necessary for parietal differentiation. Thus, retinoid receptors are sophisticated signal integrators that transduce not only the effects of their cognate ligands, but also those of ligands that bind to membrane receptors.     

Retinoic acids regulate apoptosis of T lymphocytes through an interplay between RAR and RXR receptors

Vitamin A deficiency has been known for a long time to be accompanied with immune deficiency and susceptibility to a wide range of infectious diseases. Increasing evidence suggests that retinoic acids derived from vitamin A are involved in the functional regulation of the immune system. Of the two groups of retinoid receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs) all-trans and 9-cis retinoic acids are high affinity ligands for RARs and 9-cis retinoic acid additionally binds to RXRs. In cells, at high concentrations, all-trans retinoic acid can be converted to 9-cis retinoic acid by unknown mechanisms. Apoptosis plays a major role in shaping the T cell repertoire and one way in which retinoids may affect immune functions is to influence the various apoptosis pathways. Indeed, it has been shown that retinoic acids can induce apoptosis, increase the rate of dexamethasone-induced death and inhibit activation-induced death of thymocytes and T lymphocytes. Therefore, retinoids together with glucocorticoids may be involved in regulating positive and negative selection of T lymphocytes. Here we demonstrate that retinoids can induce apoptosis of T cells through the stimulation of RARgamma. Specific stimulation of RARalpha, on the other hand, prevents both RARgamma-dependent and TCR-mediated cell death. In all these functions 9-cis retinoic acid proved to be more effective than all-trans retinoic acid suggesting the involvement of RXRs. Based on these results a possible mechanism through which costimulation of RARs and RXRs might affect spontaneous and activation-induced death of T lymphocytes is proposed.     

Two RAREs and an overlapping CRE are involved in the hepatic transcriptional regulation of the Q10 MHC class I gene

The Q10 gene is a member of the major histocompatibility complex of the mouse that is expressed in the liver and kidney of the adult. Using transient expression assays, we found that the Q10 promoter was activated by retinoic acid (RA) and exogenous RARs and/or RXRs in a cell type-dependent manner. In addition, the basal activity of the Q10 promoter in HepG2 cells is lowered by expressing a dominant negative form of RARalpha. Incidentally, we have identified two cis-elements which consist of sequences related to retinoic acid response elements (RAREs) and a putative cAMP responsive element (CRE) the sequence of which overlaps one of the RAREs. RAR, RXR, CREB-ATF, and COUP-TF factors bind these elements and/or affect their activity. We also demonstrate that the CRE mediates part of the stimulation induced by activation of the cAMP pathway on the Q10 promoter, the residual activation being mediated by RARs. Our results suggest that Q10 expression in liver depends upon RA and the interaction between nuclear receptors that are expressed in this organ. The overlapping of the CRE with one of the RAREs together with the results of PKA activation also suggest that RA and cAMP signalling pathways are linked.     

Targeted disruption of retinoic acid receptor alpha (RAR alpha) and RAR gamma results in receptor-specific alterations in retinoic acid-mediated differentiation and retinoic acid metabolism.

F9 embryonic teratocarcinoma stem cells differentiate into an epithelial cell type called extraembryonic endoderm when treated with retinoic acid (RA), a derivative of retinol (vitamin A). This differentiation is presumably mediated through the actions of retinoid receptors, the RARs and RXRs. To delineate the functions of each of the different retinoid receptors in this model system, we have generated F9 cell lines in which both copies of either the RAR alpha gene or the RAR gamma gene are disrupted by homologous recombination. The absence of RAR alpha is associated with a reduction in the RA-induced expression of both the CRABP-II and Hoxb-1 (formerly 2.9) genes. The absence of RAR gamma is associated with a loss of the RA-inducible expression of the Hoxa-1 (formerly Hox-1.6), Hoxa-3 (formerly Hox-1.5), laminin B1, collagen IV (alpha 1), GATA-4, and BMP-2 genes. Furthermore, the loss of RAR gamma is associated with a reduction in the metabolism of all-trans-RA to more polar derivatives, while the loss of RAR alpha is associated with an increase in metabolism of RA relative to wild-type F9 cells. Thus, each of these RARs exhibits some specificity with respect to the regulation of differentiation-specific gene expression. These results provide an explanation for the expression of multiple RAR types within one cell type and suggest that each RAR has specific functions.     

Effects of retinoids on the production of tumour necrosis factor-alpha and nitric oxide by lipopolysaccharide-stimulated rat Kupffer cells in vitro: evidence for participation of retinoid X receptor signalling pathway

Kupffer cells play important roles in the development of liver injury by producing cytokines and free radicals. In consequence inhibition of these inflammatory mediators will be one of the targets for treating liver diseases. Retinoids modulate a wide variety of functions of monocytes/macrophages. Cellular effects of retinoids are mediated by two families of nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We examined the effects of several kinds of natural and synthetic retinoids on the production of tumour necrosis factor-α (TNF-α) and nitric oxide (NO) by LPS-stimulated rat Kupffer cells in vitro. Of the various retinoids tested, 9-cis-retinoic acid (9-cis-RA) and Ro 13-6307, which are agonists of both RARs and RXRs, suppressed the production of TNF-α and NO in a concentration-dependent fashion, whereas three types of RAR-selective agonists, Ro 13-7410, Ro 40-6055 and Ro 19-0645 did not show any effect. Furthermore, the RARα antagonist, Ro 41-5253, did not prevent the effects induced by 9-cis-RA. The results suggest that these effects of 9-cis RA and Ro 13-6307 were induced by the RXRs-dependent signalling pathway. © 1997 John Wiley & Sons, Ltd.     

Retinoic acid receptors and retinoid X receptors in the rat testis during fetal and postnatal development: immunolocalization and implication in the control of the number of gonocytes

Retinoids have pleiotropic effects on embryonic development and are essential for spermatogenesis in the adult, where they act via nuclear retinoid receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We used immunohistochemistry to examine the cellular localization of RARs and RXRs in the rat testis from Day 13.5 postconception (13.5 dpc) until Day 8 postpartum (8 dpp), and these findings were compared with those for immature and adult testes. RARalpha and RARbeta were detected in the interstitial tissue from 14.5 dpc, with intense staining in the gonocytes from 20. 5 dpc to 8 dpp. The nuclei of all cell types stained faintly for RARgamma from 8 dpp. Immunoreactivity for RXRalpha was intense in the gonocytes from 13.5 dpc and in the Leydig cells from 16.5 dpc, and persisted throughout the period studied. RXRbeta was always detected in the Leydig cells and during a short neonatal period in the gonocytes. RXRgamma gave a faint reaction in the nuclei of all cell types from 20.5 dpc. Unexpectedly, immunostaining for all the receptors tested, except RARgamma and RXRgamma, was detected in the cytoplasmic compartment of the cells of fetal and neonatal testes, while it was found in the nuclei in immature and adult testes. In cultures of dispersed testicular cells from 3 dpp pups, retinoic acid had a dose-dependent deleterious effect on the survival of the gonocytes and, to a lesser extent, of the somatic cells. These results suggest that retinoids act on the testicular development, especially on germ cells, via RARs and/or RXRs.     

Terminal differentiation in keratinocytes involves positive as well as negative regulation by retinoic acid receptors and retinoid X receptors at retinoid response elements.

Terminal differentiation of epidermal keratinocytes is inhibited by 1 microM retinoic acid, a concentration which induces differentiation in a number of cell types, including F9 teratocarcinoma cells. The molecular basis for these opposing retinoid responses is unknown, although retinoic acid receptors (RARs) and retinoid X receptors (RXRs) have been detected in both cell types. When F9 cells are stably transfected with a truncated RAR alpha lacking the E/F domain necessary for ligand binding and RAR/RXR dimerization, action at retinoid response elements is suppressed and cells produce a retinoic acid-resistant phenotype; i.e., they are blocked in differentiation (A. S. Espeseth, S. P. Murphy, and E. Linney, Genes Dev. 3:1647-1656, 1989). If retinoid receptors influence epidermal differentiation only in a negative fashion, then suppression of transactivation at retinoid response elements would be expected to enhance, rather than block, keratinocyte differentiation. In this study, we show that surprisingly, even though constitutive expression of an analogous truncated RAR gamma in keratinocytes specifically suppressed transactivation at retinoid response elements, keratinocytes were blocked, rather than enhanced, in their ability to undergo morphological and biochemical features of differentiation. These findings demonstrate a direct and hitherto unrecognized role for RARs and RXRs in positively as well as negatively regulating epidermal differentiation. Additionally, our studies extend those of Espeseth et al. (Genes Dev. 3:1647-1656, 1989), indicating a novel RAR function independent of the E/F domain.