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.     

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.     

Analysis of the Factors Involved in the Retinoic Acid-Induced Differentiation of a Retinoid-Hypersensitive Embryonal Carcinoma Cell Mutant

Retinoic acid (RA) has been known to play an important role in cellular growth and differentiation as well as in vertebrate development. Many in vitro cell cultures also respond to RA by differentiating. Perhaps the most widely studied of these cultures are embryonal carcinoma (EC) cells. We have used an RA-hypersensitive EC cell mutant, created by retroviral insertion, to analyze the activity of the identifiable components in the RA response pathway. We have analyzed the mRNA expression patterns of the retinoic acid receptors (RARs) α, β, and γ, the retinoid X receptors (RXRs) α, β, and γ, and the cellular retinoic acid binding proteins (CRABPs) I and II. Our results indicate that CRABP I, RAR β, and RAR γ mRNAs are expressed differentially between parent and RA-hypersensitive mutant cells. All three messages are present at higher basal levels and at earlier times after RA addition in the mutant relative to parental cells. All other elements examined are equivalently expressed. Therefore analyses of the expression patterns of CRABPs, RARs, and RXRs in these RA-hypersensitive cells point to the probable importance of CRABP I, RAR β, and RAR γ in the RA induction pathway and also indicate that CRABP II and RXR γ are not likely to be critical elements in the early differentiative response of cells to RA.     

Retinoids Differentially Regulate the Proliferation of Colon Cancer Cell Lines

In this study, the proliferative effects of retinoids were examined in the MC-26 and LoVo colon adenocarcinoma cell lines. The proliferation of the LoVo cell line was not altered in the presence of the retinoidsall trans-retinoic acid (atRA) and 9-cis-retinoic acid (9-cis-RA). Both retinoids, however, stimulated the growth, as measured by cell proliferation, of MC-26 cells.atRA and 9-cis-RA were equipotent in increasing MC-26 cell proliferation, suggesting that the growth stimulation is mediated by one or more retinoic acid receptors (RARs). To determine the RAR which might be responsible for this growth stimulatory effect, we characterized the RAR subtypes which were present in both cell lines. mRNA for the RARα, RARβ, and RARγ were detected in the MC-26 cell. Of the RARs present in MC-26 cells, the RARα does not mediate the growth stimulatory effects of retinoids, for a selective RARα antagonist was unable to prevent the retinoid-induced increase in MC-26 cell growth. RARα, RARβ, and RARγ mRNA are also expressed in the LoVo cell line; the lack of growth-stimulation by retinoids in LoVo cells, therefore, does not seem to be due to the absence of RARs. The results obtained in these experiments demonstrate that the growth response elicited by retinoids can vary between colon cancer cells and that the differences in response may not be solely determined by the RAR subtypes which are expressed in a colon cancer cell line.     

RAR/RXR binding dynamics distinguish pluripotency from differentiation associated cis-regulatory elements

In mouse embryonic cells, ligand-activated retinoic acid receptors (RARs) play a key role in inhibiting pluripotency-maintaining genes and activating some major actors of cell differentiation.To investigate the mechanism underlying this dual regulation, we performed joint RAR/RXR ChIP-seq and mRNA-seq time series during the first 48 h of the RA-induced Primitive Endoderm (PrE) differentiation process in F9 embryonal carcinoma (EC) cells. We show here that this dual regulation is associated with RAR/RXR genomic redistribution during the differentiation process. In-depth analysis of RAR/RXR binding sites occupancy dynamics and composition show that in undifferentiated cells, RAR/RXR interact with genomic regions characterized by binding of pluripotency-associated factors and high prevalence of the non-canonical DR0-containing RA response element. By contrast, in differentiated cells, RAR/RXR bound regions are enriched in functional Sox17 binding sites and are characterized with a higher frequency of the canonical DR5 motif. Our data offer an unprecedentedly detailed view on the action of RA in triggering pluripotent cell differentiation and demonstrate that RAR/RXR action is mediated via two different sets of regulatory regions tightly associated with cell differentiation status.