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.     

Retinoic acid resistance of the variant embryonal carcinoma cell line RAC65 is caused by expression of a truncated RARα

P19 embryonal carcinoma (EC) cells differentiate when treated with retinoic acid (RA). The P19 EC-derived mutant cell line RAC65 is resistant to the differentiation-inducing activity of RA. We show that these cells express a truncated retinoic acid receptor alpha(mRAR alpha-RAC65), probably due to the integration of a transposon-like element in the RAR alpha gene. This receptor lacks 71 C-terminal amino acids and terminates in the ligand-binding domain. In CAT assays in RAC65 cells, mRAR alpha-RAC65 fails to trans-activate the RAR beta promoter, which contains a RA-response element. In wild-type P19 EC cells mRAR alpha-RAC65 functions as a dominant-negative repressor of RA-induced RAR beta activation. Gel retardation assays demonstrate that mRAR alpha-RAC65 is still able to bind to the RA-response element of the RAR beta promoter, indicating that competition with functional RARs for the same binding site leads to the observed dominant-negative effect. In addition, in two RAC65 clones in which wild-type hRAR alpha was stably transfected RA-sensitivity was restored and in one RAR beta expression could be induced by RA. Taken together, these data show that the primary cause of RA-resistance of RAC65 cells is the expression of a defective RAR alpha, which prevents the trans-activation of RA-responsive genes and results in a loss of the ability to differentiate.     

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.     

Retinoic acid receptor beta mediates the growth-inhibitory effect of retinoic acid by promoting apoptosis in human breast cancer cells.

Retinoids are known to inhibit the growth of hormone-dependent but not that of hormone-independent breast cancer cells. We investigated the involvement of retinoic acid (RA) receptors (RARs) in the differential growth-inhibitory effects of retinoids and the underlying mechanism. Our data demonstrate that induction of RAR beta by RA correlates with the growth-inhibitory effect of retinoids. The hormone-independent cells acquired RA sensitivity when the RAR beta expression vector was introduced and expressed in the cells. In addition, RA sensitivity of hormone-dependent cells was inhibited by a RAR beta-selective antagonist and the expression of RAR beta antisense RNA. Introduction of RAR alpha also restored RA sensitivity in hormone-independent cells, but this restoration was accomplished by the induction of endogenous RAR beta expression. Furthermore, we show that induction of apoptosis contributes to the growth-inhibitory effect of RAR beta. Thus, RAR beta can mediate retinoid action in breast cancer cells by promoting apoptosis. Loss of RAR beta, therefore, may contribute to the tumorigenicity of human mammary epithelial cells.