Modulation by retinoids of mRNA levels for nuclear retinoic acid receptors in murine melanoma cells

Retinoids inhibit the growth and enhance the differentiation of murine S91-C2 melanoma cells. Specific alterations in gene expression are a plausible mechanism for these effects. Since nuclear retinoic acid receptors (RAR) are likely mediators of retinoid-induced changes in gene expression, we used Northern blotting to analyze the expression of RAR alpha, RAR beta, and RAR gamma in S91-C2 cells. mRNA for both RAR alpha and RAR gamma was detected in these cells, but no RAR beta mRNA could be found. Treatment with 10(-7) and 10(-6) M beta-all-trans-retinoic acid (RA) for 24 h caused a 1.5- to 2-fold increase in RAR alpha and RAR gamma mRNA, whereas lower concentrations of RA were ineffective. RAR beta mRNA, which was undetectable in untreated cells, was detected after 24 h of treatment with a RA concentration as low as 10(-9) M, and its level increased with up to 10(-6) M RA. At the latter dose, RAR beta mRNA induction occurred by 4 h and increased progressively, reaching a plateau after 24 h of treatment. RAR beta mRNA induction at 4 h was not inhibited by cycloheximide at a concentration that suppressed protein synthesis by more than 90%. Several retinoids and related synthetic compounds, including 13-cis RA, TTNPB, Ch55, Am80, and the trifluoromethyl nonyloxyphenyl analog of RA, also induced RAR beta mRNA, whereas a 24-h treatment with 10(-6) M retinol, TTNP (a decarboxylated analog of TTNPB), or the phenyl analog of RA failed to induce RAR beta mRNA. With the exception of retinol and the trifluoromethyl nonyloxyphenyl analog of RA, the ability of the retinoids to induce RAR beta mRNA and their growth inhibitory effect were correlated. However, S91-C154, a RA-resistant mutant subclone derived from S91-C2 cells, showed mRNA levels of RAR alpha and RAR gamma and induction of RAR beta by RA similar to those detected in the sensitive S91-C2 cells. Like the S91 melanoma cells, two other mouse melanoma cell lines, K-1735P and B16-F1, constitutively expressed RAR alpha and RAR gamma mRNAs. The level of RAR beta mRNA was increased by RA only in B16-F1 cells, although the growth of both was inhibited by RA. These results demonstrate that RA can, directly and rapidly, induce the expression of mRNA for a high affinity nuclear receptor in some murine melanoma cells and that this induction is not sufficient to inhibit growth.     

Cyclic AMP analogs and retinoic acid influence the expression of retinoic acid receptor alpha, beta, and gamma mRNAs in F9 teratocarcinoma cells.

Retinoic acid (RA) receptor alpha (RAR alpha) and RAR gamma steady-state mRNA levels remained relatively constant over time after the addition of RA to F9 teratocarcinoma stem cells. In contrast, the steady-state RAR beta mRNA level started to increase within 12 h after the addition of RA and reached a 20-fold-higher level by 48 h. This RA-associated RAR beta mRNA increase was not prevented by protein synthesis inhibitors but was prevented by the addition of cyclic AMP analogs. In the presence of RA, cyclic AMP analogs also greatly reduced the RAR alpha and RAR gamma mRNA levels, even though cyclic AMP analogs alone did not alter these mRNA levels. The addition of either RA or RA plus cyclic AMP analogs did not result in changes in the three RAR mRNA half-lives. These results suggest that agents which elevate the internal cyclic AMP concentration may also affect the cellular response to RA by altering the expression of the RARs.     

Retinoic acid receptor isoform RAR gamma 1: an antagonist of the transactivation of the RAR beta RARE in epithelial cell lines and normal human keratinocytes.

The diversity of isoforms of retinoic acid (RA) receptors (RARs) and of DNA sequences of retinoic acid-responsive elements (RAREs) suggests the existence of selectivities in the RAR/RARE recognition or in the subsequent gene modulation. Such selectivities might be particularly important for RAREs involved in positive feedback, eg. the RAR beta RARE. In the present work we found that in several epithelial cell lines, reporter constructs containing the RAR beta RARE linked to the HSV-tk promoter were transactivated in the presence of RA by endogenous RARs and co-transfected RAR alpha 1 and RAR beta 2 isoforms, but not by RAR gamam 1. On the contrary, this latter isoform behaved towards the RAR beta RARE as an inhibitor of the transactivation produced by endogenous RARs and by cotransfected RAR alpha 1 and RAR beta 2. RAR gamma 1 also behaved as an antagonist of the transactivation produced by cotransfected RXR alpha. The natural RAR beta gene promoter or RAR beta RARE tk constructs were not activated by the endogenous receptors of normal human keratinocytes (NHK), which are known to contain predominantly RAR gamma 1. It was, however, possible to activate to a certain extent RAR beta RARE-reporter constructs in NHK by co-transfecting RAR alpha 1, RAR beta 2 or RXR alpha. The antagonist behavior of RAR gamma 1 towards the RAR beta RARE may explain why in certain cell types such as keratinocytes, RAR beta is neither expressed nor induced by RA.     

Retinoid receptors involved in the effects of retinoic acid on rat testis development

We have previously shown that retinoic acid (RA) is able to act on the development of Leydig, Sertoli, and germ cells in the testis in culture (Livera et al., Biol Reprod 2000; 62:1303-1314). To identify which receptors mediate these effects, we have now added selective agonists and antagonists of retinoic acid receptors (RARs) or retinoid X receptors (RXRs) in the same organotypic culture system. The RAR alpha agonist mimicked most of the effects of RA on the cultured fetal or neonatal testis, whereas the RAR beta, gamma, and pan RXR agonists did not. The RAR alpha agonist decreased the testosterone production, the number of gonocytes, and the cAMP response to FSH of fetal testis explanted at 14.5 days postconception (dpc). The RAR alpha agonist disorganized the cords of the 14.5-dpc cultured testis and increased the cord diameter in cultured 3-days-postpartum (dpp) testis in the same way as RA. All these RA effects could be reversed by an RAR alpha antagonist and were unchanged by an RAR beta/gamma antagonist. The RAR beta agonist, however, increased Sertoli cell proliferation in the 3-dpp testis in the same way as RA, and this effect was blocked by an RAR beta antagonist. The RAR gamma and the pan RXR agonists had no selective effect. These results suggest that all the effects of RA on development of the fetal and neonatal testis are mediated via RAR alpha, except for its effect on Sertoli cell proliferation, which involves RAR beta.     

Variable expression of retinoie acid receptor (RARβ) mRNA in human oral and epidermal keratinocytes; relation to keratin 19 expression and keratinization potential

Previous studies have revealed that the cells that form the different regions of the oral and epidermal stratified squamous epithelia represent a number of intrinsically distinct keratinocyte subtypes, each of which is developmentally programmed to preferentially express a particular pattern of keratins and type of suprabasal histology. Retinoic acid (RA) is known to modulate stratified squamous epithelial differentiation, including expression of the basal cell keratin K19 and the suprabasal keratins K1/K10 and K4/K13. We have found that all keratinocyte subtypes are similar in their steady state levels of RAR alpha and RAR gamma mRNAs in culture and that these levels are only minimally affected by RA. In contrast, RAR beta mRNA expression varies greatly among keratinocyte subtypes and, in eight of ten cell strains examined, directly correlated with their levels of K19 mRNA. Exposure to 10(-6) M RA increases the levels of RAR beta and K19 mRNA; conversely, complete removal of RA from the medium results in reduced levels of these messages. RA does not coordinately induce RAR beta and K19 messages in nonkeratinocyte cell types: fibroblasts cultured in the presence of 10(-6) M RA express very high levels of RAR beta mRNA but do not express detectable K19, and mesothelial cells decrease their levels of RAR beta and K19 mRNA in response to 10(-6) M RA. The correlation between RAR beta and K19 mRNA levels in most keratinocyte subtypes suggests a role for RAR beta in specifying patterns of keratin expression and suprabasal differentiation in stratified squamous epithelia.     

Analysis of the ligand-binding domain of human retinoic acid receptor alpha by site-directed mutagenesis.

Three subtypes of retinoic acid receptors (RAR), termed RAR alpha, RAR beta, and RAR gamma, have been described. They are composed of different structural domains, including distinct domains for DNA and ligand binding. RARs specifically bind all-trans-retinoic acid (RA), 9-cis-RA, and retinoid analogs. In this study, we examined the functional role of cysteine and arginine residues in the ligand-binding domain of hRAR alpha (hRAR alpha-LBD, amino acids 154 to 462). All conserved cysteine and arginine residues in this domain were mutated by site-directed mutagenesis, and the mutant proteins were characterized by blocking reactions, ligand-binding experiments, transactivation assays, and protease mapping. Changes of any cysteine residue of the hRAR alpha-LBD had no significant influence on the binding of all-trans RA or 9-cis RA. Interestingly, residue C-235 is specifically important in antagonist binding. With respect to arginine residues, only the two single mutations of R-276 and R-394 to alanine showed a dramatic decrease of agonist and antagonist binding whereas the R272A mutation showed only a slight effect. For all other arginine mutations, no differences in affinity were detectable. The two mutations R217A and R294A caused an increased binding efficiency for antagonists but no change in agonist binding. From these results, we can conclude that electrostatic interactions of retinoids with the RAR alpha-LBD play a significant role in ligand binding. In addition, antagonists show distinctly different requirements for efficient binding, which may contribute to their interference in the ligand-inducible transactivation function of RAR alpha.     

Aggregation and cell cycle dependent retinoic acid receptor mRNA expression in P19 embryonal carcinoma cells.

Differentiation of P19 EC cells along different pathways into derivatives resembling cells of the three embryonic germ layers is accompanied by characteristic differences in modulation of expression of each of the three retinoic acid receptor genes, RAR alpha, -beta and -gamma. Differentiation induced by addition of RA to P19 EC cells cultured in monolayer is accompanied by a rapid increase in expression of both RAR alpha and -beta. Induction of RAR beta occurs in a characteristic biphasic manner, suggesting that multiple factors and/or different mechanisms are involved in controlling its expression. RAR beta mRNA is induced to a far higher level during early aggregation in the presence of RA than during early differentiation in monolayer, suggesting that the direction of differentiation depends on the number and/or ratio of alpha and beta type of RA receptors. Aggregation of P19 EC cells in the presence of RA, but not DMSO, is accompanied by repression of RAR gamma, suggesting that the expression of RAR beta and RAR gamma during neuroectodermal differentiation is mutually exclusive. The effects of RA on RAR expression are significantly greater in G1 than in S-phase of the cell cycle. These results extend previous observations that commitment to differentiation is cell cycle dependent and indicates that critical target gene regulation in response to RA has to take place in G1 for differentiation to occur.