INTRODUCTION

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Retinoids, the natural and synthetic derivatives of vitamin A, are known to regulate a broad range of biological processes, including vertebrate development, growth, and differentiation (24, 40, 56). The common denominator for these various effects is the ability of retinoids to trigger regulatory switches, modifying the repertoire of genes expressed by a given cell (24). The effects of retinoids are mediated by two families of ligand-responsive regulators, i.e., retinoic acid (RA) receptors (RARs) and retinoid X receptors (RXRs), which are members of the nuclear receptor superfamily (8, 22, 23, 30, 31, 42-45, 52). RARs bind and are activated by all-trans-RA and 9-cis-RA, whereas RXRs bind and are activated only by 9-cis-RA (26, 38). Both types of receptors are coded for by three genes (, , and ) from which multiple isoforms can be generated by the use of alternative promoters or differential splicing (29, 37, 71). Differential tissue distribution of the various isoforms suggests that they may possess functional specificity, and given the pleiotropic effects of RA, it has been suggested that a fine tuning of retinoid receptor expression levels might be an essential requirement for correct development (9, 14, 58). RARs and RXRs modulate the expression of their target genes by binding to RA response elements (RAREs) (46, 67). One such RARE is located in the RAR₂ promoter region, and it has been shown that this response element mediates RAR₂ induction in response to RA in several cell lines and tissues (13, 27, 62). This autoregulation of the RAR gene is thought to play an important role in amplifying the RA response, thereby enhancing the final biological response. This is supported by recent studies which have shown that altered receptor expression can be associated with tumor development: for example, RAR is not expressed in certain malignant tumors, including lung carcinomas and breast carcinomas (21, 63, 72, 73). A subclone of the RA-responsive murine P19 embryonal carcinoma cells carrying an RAR mutation was found to be RA resistant (55). Similarly, an RA-resistant subclone of HL60 (human myeloid leukemia) cells was found to have a dominant negative RAR (11), while F9 murine teratocarcinoma cells with disrupted RAR genes exhibit an altered differentiation response (7). Other studies, however, suggest that individual retinoid receptors may not perform unique functions, since mice carrying null mutations for RAR₁ or RAR₂ showed no obvious abnormalities (31, 39).

Retinoids have also been implicated in many aspects of neuronal differentiation. Depending on the time of RA administration, teratogenic doses of RA cause defects in neural tube closure (3), and one of the most sensitive teratogenic targets is the neural crest (50).

Neuroblastoma (NB) is the most common extracranial malignant solid tumor of childhood; it arises from neural crest cell derivatives, and retinoids can induce its differentiation in vitro (5, 12, 66), generating a neuronal phenotype and causing a marked reduction in cell proliferation (1, 2, 60, 61). In contrast to the differentiation-promoting activity of RA, the synthetic analog N-(4-hydroxyphenyl)retinamide dramatically suppresses NB cell growth by inducing programmed cell death (54). NB cells therefore appear to represent a suitable model to investigate the mechanisms of neuronal cell death apoptosis and its relation to differentiation. RAR and RAR have been found to be constitutively expressed in NB cells, while RAR upregulation depends on the presence of RA (19, 69). RAR, which could be involved in the ontogenesis of the nervous system (47, 58), is one of the genes known to be induced by RA in cells that have the neuronal phenotype (10). The effects of RA on the differentiation of NB cells do not depend entirely on the induction of RAR, since in the NB cell line LAN-5, RAR₂ is constitutively expressed but differentiation takes place only in the presence of high concentration of RA, while the LAN-1 cell line can differentiate in the presence of RA even in the absence of RAR₂ (19, 69).

The present study was undertaken to identify the roles that the different RAR subtypes play in the RA response and differentiation pathway. The SK-N-BE2(c) cell line, selected for these studies, expresses RAR and RXR subtypes, and RAR₂ induction has been reported as an early marker of the RA response (19). We show here that RAR₁ controls the expression of the RAR₂ gene and, most interestingly, that the levels of RAR₁, independently of RA addition, are critical for the expression of different cell phenotypes with modified growth rates. In addition, we observed a correlation between the levels of RAR₁ and the induction of differentiation or apoptosis.

	MATERIALS AND METHODS

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Cell cultures. The human NB SK-N-BE2(c) cells used in this study were kindly provided by G. Tonini (G. Gaslini Children's Hospital, Genova, Italy) and grown in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 1% glutamine, and 1% nonessential amino acids.

RT-PCR. Reverse transcription-PCR (RT-PCR) was performed under previously described conditions (18, 20) with, as primers, specific oligonucleotides that allow the unequivocal distinction between receptor subtypes and isoforms (20). For each analysis the quantity and quality of RNA were normalized by the coanalysis of -actin messenger (20).

Plasmids. Plasmids pECE-RAR (, , and ) and pECE-RXR have been previously described (35, 53). For stable RAR₁ transfection, the BamHI insert from pSG5 hRAR₁, kindly provided by P. Chambon, was inserted into the BamHI site of the eukaryotic expression vector pH Apr-1-neo (25), and the correct sense orientation was determined by restriction analysis. To obtain the RAR₁-specific antisense expression vector, the BalI/BstXI fragment from pSG5 hRAR₁ was made blunt and cloned into the EcoRV site of pBluescript SK. The EcoRI/HindIII insert was subsequently cloned into the EcoRI/HindIII sites of pH Apr-1-neo and analyzed for the correct orientation.

Stable transfections. The recombinant constructs were stably transfected into SK-N-BE2(c) cells by the DOTAB method (Boehringer Mannheim) and screened with 400 µg of G418 (Gibco BRL) per ml. Clones were obtained through serial dilutions. To allow for cell growth, total transfectants and clones were cultured in the presence of the specific antagonist and routinely frozen within 1 week of culture. Experiments utilizing transfected cells were conducted on freshly thawed cells cultured in regular medium. Antagonists were added when needed. The expression of exogenous RAR₁ sense and antisense cDNAs was evaluated by RT-PCR or Northern blotting.

Transient transfection and CAT assay. Transient transfections were carried out by using a modified calcium phosphate precipitation procedure, as described previously (53), with green monkey kidney cells (CV-1) grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS. To measure the transcriptional activation, TREpal-tk linked with the chloramphenicol acetyltransferase (CAT) gene was used as a reporter gene. Briefly, 100 ng of reporter gene, 200 ng of -galactosidase expression vector (pCH110; Pharmacia), and 50 ng of receptor expression vector were mixed with carrier DNA (pBluescript; Stratagene) to give a total of 1,000 ng of total DNA per well. After the cells were grown in the presence of the various retinoids for 24 h, CAT and -galactosidase activities were assayed as previously described (53). CAT activity was normalized for transfection efficiency by the corresponding -galactosidase activity.

Western blotting and immunostaining analyses. Ten micrograms of DNA-binding proteins (4) was resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis on 10% gels and electroblotted onto a polyvinylidene difluoride (PVDF) membrane. The membrane was reacted with specific anti-RAR and anti-RXR antibodies (Santa Cruz), and protein bands were visualized after addition of enhanced chemiluminescence detection reagent (Amersham) by following the manufacturer's protocol.

Evaluation of apoptosis and DNA fragmentation detection. Cells were plated at 15,000/ml on chamber slides and grown as described above. After fixation with cold 2% formaldehyde in phosphate-buffered saline (PBS), the cells were washed with cold PBS and the nuclei were stained with a solution containing 50 µg of propidium iodide per ml, 0.1% Triton X-100, 0.1% Na citrate, and 20 µg of RNase A per ml in PBS for 15 to 20 min at room temperature. Apoptotic nuclei were identified by fluorescence microscopy. DNA fragmentation was measured on floating and adherent cells; 2 × 10⁶ cells for each experiment were lysed and treated as described by Bissonette et al. (6).

Flow cytometric analysis. Adherent and floating cells were fixed in 70% ethanol, washed twice in PBS, and resuspended in DNA staining solution containing 30 µg of propidium iodide per ml and 0.5 mg of RNase A per ml. DNA flow cytometric measurements were performed on an EPICS Elite instrument (Coulter Corporation, Miami, Fla.), and the Muticycle program (Phoenix Flow Systems, San Diego, Calif.) was used for the analysis of the cell cycle distribution as well as for the evaluation of apoptotic cells.

Cell proliferation assay. To study anchorage-dependent cell growth, mock-transfected SK-N-BE2(c) cells and sense or antisense transgene cells were seeded at 1,000 to 3,000 cells per well (depending on the time in culture) in 96-well plates and grown in regular medium or treated with various concentrations of retinoid antagonists. Media were changed every 48 h. The number of viable cells was measured by the capacity of cells to reduce nitroblue tetrazolium with a colorimetric cell proliferation kit (MTT assay; Promega) (51).

	RESULTS

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Induction of RAR₂ correlates with a transient decrease of RAR₁ in SK-N-BE2(c) cells. We used an RT-PCR protocol that allows for semiquantitative analysis of RAR and RXR subtype and isoform expression (20). We observed that SK-N-BE2(c) cells constitutively express RAR₁, RAR₁, RAR₂, RXR, and RXR (Fig. 1A). Consistent with previous observations (19), exposure to physiological concentrations of RA (10 nM) led to the induction of RAR₂ mRNA, whereas the mRNA levels for the other retinoid receptors were not affected (Fig. 1B). Figure 2A shows that SK-N-BE2(c) cells constitutively express high levels of RAR₁ mRNA; RAR₂ expression is weak, but upon stimulation by RA, it increases strongly in a time-dependent manner. RAR₂ induction correlated with a decrease in RAR₁ mRNA, suggesting that expression of both RAR₁ and RAR₂ is controlled by RA in NB cells. RAR₁, RAR₂, and RXR and - expression did not change after exposure to RA (data not shown). To assess whether the RAR₁ mRNA decrease was associated with a decrease of its protein, nuclear extracts were analyzed. Western blot analysis of nuclear extracts from control cells and cells exposed to 10 nM RA revealed a reduction in RAR₁ protein at 2 h after RA addition (Fig. 2B). When the same blot was reprobed with anti-RAR and anti-RXR antibodies, no differences between RA-treated and control cells could be detected. Antibodies to RAR did not recognize specific bands in these analyses.

View larger version (59K): [in this window] [in a new window]   FIG. 1.   Analysis of RAR and RXR expression in the NB cell line SK-N-BE2(c). One microgram of total cellular RNA was analyzed by RT-PCR with a nested reaction protocol for RAR or RXR subtypes and isoforms as described in Materials and Methods. (A) Control cell cells; (B) cells treated for 24 h with 10 nM RA. The left lane in each panel contains molecular size markers (X174 RFDNA/HaeIII fragments [GIBCO]).

View larger version (26K): [in this window] [in a new window]   FIG. 2.   Time course of RA-regulated expression of RAR2 and RAR1. (A) SK-N-BE2(c) cells were plated at 106 cells per 25-cm2 tissue culture flask, and after an overnight incubation at 37°C, RA was added (time zero) to a final concentration of 10 nM. At various times after RA addition, total RNA was isolated and 1 µg was analyzed by RT-PCR for RAR or RXR expression as described in the text. Values for RAR and RXR mRNAs were normalized to that for -actin mRNA used as internal standard for each RNA sample. The degree of amplification was quantitated by scanning densitometry and plotted as a ratio of RAR to -actin or RXR to -actin. Only data relative to RAR2 and RAR1 are reported, since no modulations were observed for the remaining RARs and RXRs. Five independent experiments with very similar results were conducted. OD, optical density. (B) Ten micrograms of DNA-binding proteins obtained from control cells and cells exposed to 10 nM RA was electrophoresed on SDS-polyacrylamide gels, transferred to PVDF membranes, and probed with antibodies against RAR, RAR, or RXR. Lanes 1, control cells; lanes 2, cells exposed to RA for 90 min; lanes 3 to lane 9, treated cells collected every 30 min. Prestained molecular size standards were used to identify bands of the correct molecular weight.

RAR₁ limits RA-dependent transactivation of the RAR₂ gene. It has been observed that RAR₁ does not act as an RA-dependent activator of the RAR₂ promoter but acts as a transcriptional repressor (27, 28). We investigated whether overexpression of RAR₁ could reduce the RA-induced transcription of RAR₂ in SK-N-BE2(c) cells. The coding region of human RAR₁ was placed under the control of the human -actin promoter, and stable transfectants were selected. In Fig. 3A, expression levels of endogenous and transfected RAR₁ in three different clones are compared to those for control cells (transfected with the empty vector). To verify the data, RNA levels were also analyzed by Northern blotting, and two bands corresponding to 3.3 and 1.5 kb, as expected, were seen (Fig. 3B). Clones overexpressing RAR₁ were investigated in detail to assess their ability to respond to RA as measured by the activation of the RAR₂ gene. Control clones that contained empty vector and three RAR₁-overexpressing clones were grown in the presence of increasing concentrations of RA for 24 h to achieve maximal induction of RAR₂. An RT-PCR analysis of RNA samples is shown in Fig. 3C. A clear correlation between expression levels of RAR₁ and inducibility of RAR₂ was observed: 10 nM RA was sufficient to induce RAR₂ mRNA in control cells (empty vector clones), while in the RAR₁-transfected clones, increasing the levels of exogenous RAR₁ antagonized the effects of RA. Interestingly, relatively small increases in RAR₁ levels clearly affected RAR₂ expression (the relative amounts of endogenous versus transfected RAR₁ were estimated by scanning densitometry of Fig. 3A). For instance clone 1, where RAR₁ mRNA is augmented 0.5-fold, is resistant to 10 nM RA but still responds to higher concentrations of RA. When the RAR₁ levels are doubled (clone 2), 100 nM RA no longer induces the RAR₂ gene, while the most striking effect is observed in clone 3 (with the highest levels of transfected RAR₁), where the cells have become resistant to even 1 µM RA. Thus, we observed an RAR₁-dependent inhibition of the RAR₂ response to RA in the NB cells, consistent with the previously observed repression of the RAR₂ promoter by RAR₁ (27, 28) in transient-transfection experiments.

View larger version (35K): [in this window] [in a new window]   FIG. 3.   RAR1 represses RAR2 gene induction in a dose-dependent manner. (A) RT-PCR determination of RNA transcripts of endogenous (lanes E) and transfected (lanes T) RAR1 in three selected clones compared to mock-transfected SK-N-BE2(c) cells (lanes C). The levels of transfected RAR1 RNA expressed relative to the amount of endogenous RNA, which was taken as 1, were 0.5, 1, and 2 in clones 1, 2, and 3, respectively. (B) Expression of endogenous and transfected RAR1 determined by Northern blot analysis with total RNA (20 µg) to evaluate their correct sizes. (C) Cells from clones 1, 2, and 3 were treated for 24 h in the presence of increasing RA concentrations or solvent alone. RNA was extracted, and RT-PCR was used to estimate the relative amounts of RAR2 gene transcripts. RNA transcripts of the -actin gene were used to normalize the RT-PCR assays. Densitometric scanning of the gel clearly shows that a correlation exists between total RAR1 levels and the cell response to RA, evaluated as RAR2 gene induction. OD, optical density.

View larger version (41K): [in this window] [in a new window]   FIG. 4.   Analysis of RAR transcripts in RAR1 antisense transgene-transfected cells. (Left panel) Total RNAs (20 µg) from control (lane 1) and antisense transgene-transfected (lane 2) cells were analyzed by Northern blot hybridization to the BamHI insert of RAR1 cDNA. Two bands of the correct size (3.3 and 0.167 kb, respectively) can be visualized in transfected cells. (Right panel) RT-PCR for RAR expression in transfected cells grown in regular medium (C) or in the presence of 10 nM RA for 24 h. From left to right are RAR1, -2 -1, and -2. Note that RAR2 mRNA is present independent of RA addition. The left lane contains molecular size markers (X174 RFDNA/HaeIII fragments [GIBCO]).

View larger version (166K): [in this window] [in a new window]   FIG. 5.   Morphological evaluation of transfected SK-N-BE2(c) cells compared to mock-transfected cells. (a) control cells; (b) cells cultured for 4 days in the presence of 10 µM RA; (c) RAR1 sense transgene-transfected cells; (d) RAR1 antisense transgene-transfected cells.

View larger version (25K): [in this window] [in a new window]   FIG. 6.   Inhibition of cell growth in stable transfected SK-N-BE2(c) cells. (A) Ten micrograms of DNA-binding proteins obtained from mock-transfected SK-N-BE2(c) cells (lane 1), RAR1 sense transgene-transfected cells (lane 2), and RAR1 antisense transgene-transfected cells (lane 3) was electrophoresed on an SDS-polyacrylamide gel, transferred to a PVDF membrane, and probed with anti-RAR antibodies. Numbers on the left are molecular weights in thousands. (B) Recently thawed cells were kept in regular FCS-containing medium for 3 days and then seeded at 1,000 cells per well. Cell growth was evaluated every 48 h. The results were expressed as the A550 of MTT-derived formazan developed by sense and antisense RAR1 cDNA-transfected cells compared to cells transfected with the empty vector. All data shown are representative of three independent experiments conducted in triplicate. Error bars indicate standard deviations.

RAR₁ levels allow for a switch between neuronal maturation and cell death. Morphological changes and cytoskeletal protein expression are typical hallmarks of neuronal maturation in NB cell lines, and microscopic inspection of our cell lines showed a similarity between sense transgene-transfected cells (Fig. 5c) and cells exposed for 4 days to 10 µM RA (Fig. 5b). Neurofilaments, specific markers of neurons, were assessed by immunostaining in control, RA-treated, and transgene-containing cells by utilizing the 2H3 monoclonal antibody, which is specific for the 165-kDa neurofilaments. RA treatment (Fig. 7C) caused a shift in the localization of the staining from a diffuse somatic pattern (characteristic of control cells [Fig. 7B]) to an intense perinuclear and neuritic pattern (Fig. 7C), which became more apparent in RAR₁-overexpressing cells (Fig. 7D).

View larger version (98K): [in this window] [in a new window]   FIG. 7.   Morphological differentiation of sense transgene-transfected SK-N-BE2(c) cells. Effects of RA (10 µM) and RAR1 overexpression on cytoskeletal proteins were assessed by immunostaining analysis with the 2H3 monoclonal antibody against 165-kDa neurofilaments. (B) Control cells; (C) RA-treated cells; (D) RAR1-overexpressing cells. As a negative control, RAR1-overexpressing cells were reacted with anti-CD4 antibodies (A).

View larger version (79K): [in this window] [in a new window]   FIG. 8.   Apoptosis in RAR1 antisense transgene-transfected SK-N-BE2(c) cells. (A) Morphological analysis of propidium iodide-stained nuclei from control cells (a) compared to RAR1-overexpressing cells (b) and RAR1 antisense transgene-transfected cells (c). Nuclei with typical morphological features of apoptosis are indicated (arrows). (B) Agarose gel electrophoresis of DNA from mock-transfected SK-N-BE2(c) cells (lane 1), RAR1-overexpressing cells (lane 2), and RAR1 antisense transgene-transfected cells (lane 3). Identical numbers of cells from each sample were lysed. DNA was isolated and electrophoresed on a 1.2% agarose gel. The left lane contains molecular size markers (X174 RFDNA/HaeIII fragments [GIBCO]).

RAR₂- and RAR₁-specific antagonists selectively counteract RA effects. Receptor-selective antagonists can be used as alternative tools to evaluate the roles of individual receptors. We therefore used RAR- and RAR-selective antagonists to further evaluate the roles of RAR and RAR in the regulation and induction of specific programs in the NB cells. From several compounds reported to possess antagonist activity (34), we selected CD2331 and CD2366. When RAR, -, and - and RXR expression vectors were cotransfected with a TREpal-tk-CAT reporter gene into CV-1 cells, both CD2331 and CD2366 were unable to activate the reporter gene (Fig. 9). Conversely, in the presence of RA the antagonists caused a dose-dependent reduction of transactivation by RAR₂ and RAR₁, respectively (Fig. 9). At the highest nontoxic antagonist concentration (1 µM), CD2331 could completely suppress the transactivation induced by RAR₂ in the presence of 10 nM RA, while CD2366 inhibited more than 80% of RAR₁-mediated transactivation under the same conditions. The two compounds were ineffective when tested in the presence of RAR (Fig. 9) or 9-cis-RA-activated RXR (data not shown). These results show that CD2331 antagonizes selectively the transactivation of RAR₂ by RA, while CD2366 antagonizes selectively the transactivation of RAR₁. In both cases a 100-fold excess of the antagonist over RA was required for complete inhibition. If RAR₁ is directly involved in the transduction of the RA signal in SK-N-BE2(c) cells, CD2366 should be able to antagonize this action, and we could expect that the addition of the antagonist to cells exposed to RA prevents RAR gene induction. In Table 1 the inhibitory activity of CD2366 on RAR induction by RA is shown. When used at 1 µM, CD2366 completely inhibits induction of RAR mRNA by 10 nM RA and partially inhibits induction by 100 nM RA. CD2331 was also tested under the same conditions, and no inhibitory effects on RAR mRNA synthesis were observed (data not shown).

View larger version (20K): [in this window] [in a new window]   FIG. 9.   Antagonistic effects of the synthetic retinoids CD2331 and CD2366 on RA-induced activation of TREpal-tk-CAT and inhibition of specific receptor subtypes. CV-1 cells were transiently transfected with 100 ng of TREpal-tk-CAT reporter together with RAR and RXR expression plasmids (top panel), RAR2 and RXR (middle panel), or RAR1 and RXR (bottom panel). Transfected cells were treated with 10 nM RA, with the indicated concentrations of CD2366 and CD2331, or with the combination of RA and antagonists. CAT activity was assayed after 24 h as described in Materials and Methods. The activation obtained in the presence of 10 nM RA alone represents the maximum value. The data shown represent the means from two experiments carried out in duplicate, and the error bars represent standard deviations. The standard errors of the mean values were between 0.02 and 0.5.

View this table: [in this window] [in a new window]   TABLE 1.   CD2366 inhibits RAR2 mRNA induction by RA

CD2331 and CD2366 partially counteract inhibition of cell proliferation in transfected SK-N-BE2(c) cells. We also assessed the ability of the antagonists to counteract retinoid-induced growth inhibition. To avoid clonal effects due to position-insertion, total transfectant populations were studied. The transfected cultures showed an appreciable level of transgene expression (data not shown), and Western blot immunostaining revealed an increase of RAR₁ protein in sense transgene-transfected cells (Fig. 10A) and a decrease of the molecule in antisense transgene-transfected cells (Fig. 10B). Cells were seeded in the presence of increasing concentration of antagonists to evaluate their effects on cell proliferation. On the basis of the antagonist properties, cells overexpressing RAR₁ were cultured in the presence of CD2366, while CD2331 was utilized to antagonize RAR₂ being constitutively expressed in RAR₁ antisense transgene-transfected cells. Cell proliferation was evaluated by the MTT assay. Growth curves obtained in the presence of 1 µM antagonists are shown in Fig. 10. Lower antagonist concentrations were ineffective in this assay. CD2366 allowed sense transgene-transfected cells (Fig. 10A) to grow faster, with a maximal effect observed at day 8, at which point the growth rate was comparable to that of control cells that lacked the sense transgene. The growth rate of the antisense transgene-transfected cells (Fig. 10B) was partially restored by the addition of CD2331, suggesting that RAR₂ does contribute to cell growth arrest. Indeed, flow cytometric analysis of antisense transgene-transfected cells revealed an apoptotic peak (about 68%) and a decrease in the fraction of S- and G₂-plus M-phase cells (Fig. 11B), while antagonist treatment (4 days at 1 µM) decreased the apoptotic peak to 16% and normalized the cell cycle distribution (Fig. 11C).

View larger version (21K): [in this window] [in a new window]   FIG. 10.   Effect of CD2331 and CD2366 antagonists on SK-N-BE2(c) cell proliferation when transfected with RAR1 sense and antisense transgenes. Recently thawed cells were kept for 3 days in FCS-containing regular medium and then seeded at 1,000 cells/well in the presence of 1 µM antagonists. Cell growth was evaluated every 48 h by the MTT assay. Three independent experiments were conducted, with very similar results. The data shown represent the means of 10 points from a single experiment. Error bars represent standard deviations. Note that CD2366 can antagonize only RAR1, while CD2331 is specific for RAR2. Panels on the right show the relative amount of RAR1 in transfected cells. Ten micrograms of DNA-binding proteins was electrophoresed on SDS-polyacrylamide gels, transferred to PVDF membranes, and probed with antibodies against RAR1. Lanes 1, empty vector-transfected cells; lanes 2, RAR1 sense (A) and RAR1 antisense (B) transgene-transfected cells. Numbers on the left are molecular weights in thousands.

View larger version (15K): [in this window] [in a new window]   FIG. 11.   Effect of CD2331 on RAR1 antisense transgene-transfected cell cycle. Floating and adherent mock-transfected SK-N-BE2(c) cells (A), antisense transgene-transfected cells (B), and antisense transgene-transfected cells cultured for 4 days in the presence of 1 µM CD2331 (C) were analyzed by flow cytometry. Arrowheads point to apoptotic cells.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

In this study we demonstrate a ligand-sensitive transcriptional cross talk between RAR₁ and RAR₂ in SK-N-BE2(c) cells. Our findings support the existence of a regulatory interplay between members of the retinoid receptor superfamily, consistent with data reported by other groups.

Several studies have suggested that RAR₁ inhibits the activation of the RARE by other RARs (28); this function is not due to a general lack of transcriptional enhancer activity of the receptor, since other response elements are efficiently activated (28, 33). A similar conclusion was reached also by Taneja et al. (65), who studied the contribution of RARs and RXRs to the activation of RA target genes by using RAR subtype (, , or )-specific synthetic retinoids. They observed that even though all three RARs can functionally substitute for each other as activators of RA target genes, one RAR subtype can cell specifically override the activity of the other RAR subtypes, and RAR can suppress RAR₂ expression in wild-type F9 cells by a mechanism that involves the inhibition of RAR-dependent induction of RAR₂ (65). This inhibitory effect of RAR₁ is likely to be of biological significance for the containment of RA-mediated responses via activation of the RARE. It is tempting to speculate that the reciprocal tissue expression patterns of RAR and RAR might in part be due to such a mechanism. Indeed, unlike RAR, RAR and RAR show restricted and mutually exclusive spatio-temporal patterns of expression during embryonic development (15, 58). Ruberte et al. (58) have shown the presence of RAR transcripts in the closed neural tube, while RAR transcripts become undetectable at the time of neural tube closure and are absent from the central and peripheral nervous systems throughout development (59). A similar nonoverlapping distribution of RAR and RAR transcripts was also seen in the developing limb and in the inner ear region; for the latter region, RAR transcripts are present only in the otic capsule, whereas RAR transcripts are found in the mesenchyme surrounding the inner ear epithelium (59). The direct involvement of RAR in the transduction of the RA signal was also shown in vivo, as RAR null mutant mice display some of the abnormalities present in animals fed a vitamin A-deficient diet (39) and do not display some of the teratogenic effects caused by maternal RA administration (39).

We have demonstrated that in our system RAR₁ can repress RAR₂ induction and RAR₂ levels determine the inhibition of cell proliferation and induction of apoptosis. A correlation between high levels of RAR expression and apoptosis has also been observed in vivo in cells in the interdigital regions of the developing limb, in the fusion region of the neural tube, and in the palate (32, 48). Repression of RAR gene transcription by RAR₁ most likely involves competitive binding between RAR and RAR-RXR heterodimers to the RARE. Both RAR and RAR have been shown to bind to this RARE, but while RAR is an effective activator of this response element, RAR is not (28). This might explain why even relatively small increases in RAR can have substantial effects on RAR induction even in the presence of RA and why high RA concentrations can still induce RAR₂ through RAR upregulation (69). Not unexpectedly, RAR₁ may also be able to substitute for some (but not all) RAR₂ functions (Fig. 5), since overexpression of RAR₁ leads to a phenotype similar to that observed in normal cells in the presence of 10 µM RA. It is well known that RAR₁ has high constitutive activity (28) that may allow it to substitute for the RAR₁ and RAR₂ activity observed in wild-type cells at 10 µM RA. The apparent discrepancy between results obtained when expressing RAR antisense or inhibiting RAR by a specific antagonist are most easily explained by the very different modes of action of these two agents. RAR₁ antagonists allow continuous blocking of the RARE by RAR-RXR heterodimers. In fact, it is likely that the antagonist represses the activation of RARE-containing genes by constitutively inhibiting RAR activity (28). Thus, RAR antagonists can still allow repression of RAR expression and thereby avoid apoptosis but allow cell proliferation. In contrast, RAR antisense expression eliminates or reduces RAR expression, thereby allowing binding of RAR-RXR or RAR-RXR heterodimers to the RARE and induction of RAR (by low concentrations of RA present under the growth conditions) and thus progression towards apoptosis. The orphan receptor nur77 might also function as an activator of the RARE under those conditions and add to RAR induction (68). The observation that RAR antagonists strongly inhibit this pathway indicates that induction of other RAR-responsive genes is part of this signaling cascade.

Although it has been shown that certain RAR-selective compounds with retinoid-like activities can induce apoptosis (16, 41), it is generally believed that RAR can induce cell differentiation. In fact, only RAR can mediate the RA-induced differentiation of wild-type F9 cells (65), and the overexpression of RAR directly induces terminal differentiation of human embryonal carcinoma NT2/D1 cells into a neuronal phenotype (49). Conversely, the overexpression of RAR and - and RXR does not produce maturation or growth-inhibitory effects (49). In agreement with these findings, we detect a differentiated phenotype in SK-N-BE2(c) cells overexpressing RAR₁; the direct involvement of RAR in cell growth arrest and differentiation is further demonstrated by the effects of an RAR-selective antagonist capable of restoring the normal cell growth rate.

A functional redundancy between RARs in vivo and in vitro has been described (31, 57, 64), and the upregulation of the remaining RARs in F9 cells, in which a single RAR has been disrupted, may be sufficient for the maintenance of several functions (64). This is not the case for our antisense transgene-transfected cells, where the loss of RAR₁ and its repressor role cannot be replaced by RAR₂, suggesting that RAR₁ has a particular role in the regulation of genes that control cell growth and differentiation. Thus, our data suggest that both RAR₁ and RAR₂ can control cell growth but that they play distinct roles in determining cell differentiation or apoptosis.

Our study also shows that changes in RAR isoform expression levels can lead to dramatically different effects on the fate of a cell population. Thus, a high level of complexity appears to govern nuclear receptor function in vivo.