©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence that Retinoid X Receptors Mediate Retinoid-dependent Transcriptional Activation of the Retinoic Acid Receptor Gene in S91 Melanoma Cells (*)

(Received for publication, November 23, 1994; and in revised form, April 28, 1995)

Remco A. Spanjaard (§) , Akira Sugawara , Masato Ikeda , William W. Chin

From the Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Howard Hughes Medical Institute and Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

S91 melanoma cells are growth arrested and differentiate when treated with retinoids. These processes correlate with expression of the retinoic acid receptor (RAR) gene, which is induced through a retinoic acid response element (RARE). We wished to determine which endogenous retinoid receptors (RARs and retinoid X receptors, RXRs) mediate induction of the RAR gene. We show that RXR and RXR are constitutively expressed. Electrophoretic mobility shift assays with nuclear extracts show specific binding to the RARE (Complex I) in untreated cells, which can be supershifted by antibodies against RXRs but not by anti-RAR antibodies. After 48 h of treatment with retinoic acid, Complex I is replaced by a faster migrating Complex II, which can be supershifted by anti-RAR and anti-RXR antibodies. This suggests that induction of the RAR gene is largely mediated by RXRs only. Accordingly, we also find that 9-cis RA, which activates both RAR and RXR, is a more potent inducer of the RAR gene than RA, which only activates RAR. After 48 h, all RXRs appear to be titrated by the newly synthesized RAR into an RARRXR heterodimer complex. Thus, it appears that the RARE is sequentially occupied by RXR dimers and RARRXR heterodimers.


INTRODUCTION

Retinoids, a group of chemically related molecules derived from vitamin A (retinol), regulate a large number of biological processes in vertebrate development, cell growth, differentiation, and homeostasis (1, 2, 3) . The actions of retinoids are mediated by two classes of nuclear receptors: retinoic acid receptors (RARs), ()which bind all-trans-retinoic acid (RA) and 9-cis-retinoic acid (9-cis-RA) with similar affinities, and retinoid X receptors (RXRs), which bind 9-cis-RA with much higher affinity than RA(3, 4, 5, 6) . RARs and RXRs belong to an extensive gene family of ligand-dependent transcription factors that together form the steroid/thyroid hormone receptor superfamily(7, 8) . There are three types of both RARs and RXRs (, , and ), encoded by separate genes, that have different spatio/temporal expression patterns(9, 10, 11, 12, 13, 14) . In addition, they are also subject to alternative splicing and/or alternate promoter choice, thereby generating a large family of mainly N-terminally different receptor isoforms(1, 5) . Thus, it is conceivable that certain RARRXR isoforms may serve specific genetic programs.

RARs and RXRs bind specific DNA elements in the promoter region of target genes called RA response elements (RAREs). The majority of RAREs appears to consist of two direct repeats (DR), or half-sites, of the sequence AGGTCA spaced by five nucleotides (DR5)(15, 16) . Numerous in vitro experiments have shown that RARs and RXRs bind with low affinity as homodimers to DR5, but, when mixed, RARRXR heterodimers can form that bind with much higher affinity than either homodimer. These data, together with transfection experiments in mammalian cells and yeast, suggest that the heterodimer is the transcriptionally active species when both partners are present (17, 18, 19, 20, 21, 22, 23, 24, 25, 26) . However, it was recently found that an element with a spacing of one nucleotide (DR1) is also bound by RARRXR heterodimers but in a transcriptionally inactive conformation. In contrast, in the presence of 9-cis-RA, RXR homodimers specifically can bind and activate transcription from promoters with this element(27, 28) .

An interesting aspect of retinoids is their effect on differentiation of neoplastic cells, both in vivo and in vitro (1, 3 and references therein). For instance, there are over 200 retinoid-sensitive tumor cell lines that could provide a useful model system to gain insight into how retinoids affect malignant growth(29) . In this report, we have focused on the murine melanoma cell line S91. Upon treatment with RA, these cells become growth arrested and display an enhanced differentiated phenotype, exemplified by the formation of more and longer dendritic extensions and an increase in melanin synthesis(30, 31, 32) . The occurrence of malignant melanoma has been steadily on the rise(33) , and prognosis is still poor when discovered in advanced state. Unfortunately, little is known about the molecular events of malignant change in melanocytes, and S91 cells may be very useful for these studies. Previous reports have shown that RAR and RAR are constitutively expressed in S91 cells but that RAR is rapidly induced by treatment with retinoids. RAR expression is maximal after 24 h and is independent of de novo protein synthesis(32, 34) . Interestingly, retinoids that fail to induce RAR do not cause growth arrest(32) , and differentiation becomes phenotypically apparent only after RAR mRNA levels have reached their highest levels(30) . One interpretation is that a certain threshold level of RAR is required to facilitate differentiation of S91 cells. It is, therefore, important to know which proteins regulate RAR expression. Retinoid-dependent induction of the RAR2 gene (the major RAR isoform) is mediated through an RARE in the RAR2 promoter (RARE, a DR5-like element), which has been well characterized in in vitro studies(35, 36, 37) . In this report, we investigated which endogenous, intracellular RARRXR isoforms mediate the retinoid-dependent induction of the RAR gene in S91 cells. Our data provide evidence that, surprisingly, a complex containing RXRs, but not RARRXR heterodimers, largely regulates RAR gene expression.


MATERIALS AND METHODS

RNA Isolation Procedures and Northern Blotting

Poly(A) RNA was extracted from cells grown at about 60% confluency in 5-8 flasks (162 cm) for each time point, using a poly(A) RNA isolation kit (Stratagene, La Jolla, CA). Total RNA was isolated from 2 flasks for each time point according to (38) . 1 µg of poly(A) RNA or 15 µg of total RNA was subjected to electrophoresis through a denaturing formaldehyde-agarose gel (1%), transferred to a nylon membrane (Duralon-UV, Stratagene, La Jolla, CA) according to (39) , and cross-linked in a Stratagene Stratalinker. Northern blots were hybridized with the following DNA restriction fragments, which had been gel-purified and random-primed in the presence of [-P]dCTP: RAR, EcoRI-EagI fragment from pSG5RAR; RXR, AvaI fragment from pBSRXR; RXR, EcoRI-NheI fragment from pBSRXR and a cyclophilin BamHI fragment. Blots were washed in 0.1 SSC, 0.1% SDS at 55 °C and autoradiographed. Quantitation of Northern blots was performed on a Molecular Dynamics PhosphorImager (Sunnyvale, CA) using ImageQuant software (version 3.3).

Nuclear Extracts, EMSA, and Oligonucleotides

Procedures for obtaining nuclear extracts from cells (grown in 1-2 flasks (162 cm) at about 60% confluency for each time point) and EMSA conditions were essentially performed as described previously(40) ; typically, 1.5-3 µg of nuclear extract was used. Incubation with gel-purified P-end-labeled oligonucleotides was performed in the presence of 1 µg of salmon sperm DNA. The reaction mix minus probe was incubated for 15 min at room temperature; probe (50,000 cpm, 5 fmol) was added and incubated for another 30 min, and then put on ice for 10 min before electrophoresis on 4% polyacrylamide gel. For antibody supershifts, 1 µl of antiserum was added at this point and incubated at 4 °C for another 2 h before electrophoresis. The specificity and characterization of the RXR antibodies will be described elsewhere(54) . The RAR antibody does not cross-react with RAR or RAR. The RARc antibody reacts about equally well with RAR and RAR, less well with RAR in immunoprecipitations, and about equally well with all three RAR-isoforms in EMSA (data not shown).()

The following oligonucleotides were used: RARE (-61/-29), AGCTTCCGGGAAGGGTTCACCGAAAGTTCACTCGCATAAGGCCCTTCCCAAGTGGCTTTCAAGTGAGCGTATTCGA; TK minimal promoter (-46/+1), AGCGGTCCGAGGTCCACTTCGCATATTAAGGTGACGCGTGTGGCCTCGAACCAGGCTCCAGGTGAAGCGTATAATTCCACTGCGCACACCGGAGCTTCGA; CTF binding site from TK promoter (-96/-62), TCGACAGCGTCTTGTCATTGGCGAATTCGAACACGCAGATGGTCGCAGAACAGTAACCGCTTAAGCTTGTGCGTCTACAGCT; DR1, AGCTAGTTACTTATTGAGGTCAGAGGTCAAGTTACGTCAATGAATAACTCCAGTCTCCTGTTCAATGCTCGA.

Western Blot

10 µg of nuclear extract was loaded on a 10% SDS slab gel and analyzed by SDS-polyacrylamide gel electrophoresis. A 1:1000 dilution of primary antibody was used, followed by a 1:2000 dilution of a peroxidase-coupled goat-anti-rabbit secondary antibody. Visualization of bands was done by using an ECL detection kit (Amersham Corp.) Exposure time was about 30 s on x-ray film.

Cell Culture Conditions

S91 cells (ATTC CCL 53.1) were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37 °C in 5% CO in humidified air. Solutions of RA and 9-cis-RA were made fresh every 24 h in ethanol. Plates that did not receive ligand received ethanol instead. Working concentrations were 10 or 10M, and plates were kept in the dark as much as possible.

Chemical Modification Interference Assay

The oligonucleotide with the RARE was cloned in the HindIII site of pBluescript and confirmed by dideoxy sequencing. The coding strand was labeled by cutting the plasmid with XhoI followed by Klenow-fill-in in the presence of [-P]dCTP, dTTP, and dGTP. The plasmid was then cut with SpeI. Similarly, the noncoding strand was labeled by digesting the plasmid with SpeI and Klenow-fill-in in the presence of [-P]dCTP and dTTP followed by digestion with XhoI. Both probes were gel-purified and chemically modified by dimethyl sulfate or KMnO, essentially as described previously(41, 55) . EMSA was performed in 6% polyacrylamide gel with 6 µg and 3 µg of the nuclear extracts at the 0 (Complex I) and 48 (Complex II) h time points, respectively. Free and bound probe was eluted, digested as described(41, 55) , and run on a 8% DNA sequence gel. Quantitation of individual bands was performed by PhosphorImager scanning analysis, and normalized for loading differences.


RESULTS

S91 Cells Constitutively Express RXR and RXR mRNA

As a first step in the analysis of the RARE, we decided to complete the retinoid receptor inventory in S91 cells by analyzing the nature of the expressed RXR species. Poly(A) RNA was isolated from cells that were untreated (0 h) or treated with 1 µM RA for the indicated time period (Fig. 1) and analyzed by Northern blotting. As a positive control, we probed the blot with RAR cDNA. In agreement with published reports(32, 34) , levels of RAR mRNA increase from barely detectable in untreated cells, to readily observable after only 2 h of treatment. Levels continue to increase largely over the first 24 h, after which maximum levels appear to have been reached (10-12-fold over untreated cells). The band in Fig. 1denoted as RAR represents the product of the RAR2 gene, because a polymerase chain reaction-generated probe containing the RAR2-specific N-terminal sequence hybridizes with the same band (not shown). Interestingly, the RXR probe detects three transcripts in treated and untreated cells that are weakly up-regulated (about 2-fold) by RA. Of these, only the smallest transcript (5 kb) appears to have the size reported by Mangelsdorf et al.(14) for RXR. The larger transcripts may be due to deregulated expression and/or chromosomal rearrangements in these cells. RXR expression is also observed in untreated cells and likewise is only slightly up-regulated by RA. The size of the (single) transcript is as expected (14) . In contrast, RXR could not be detected (data not shown). Thus, in the absence of high doses of RA, S91 cells contain mRNAs for RAR, RAR(32, 34) , RXR, RXR, and very low levels of RAR. As shown in Fig. 1, all receptors are constitutively expressed except for RAR, which is induced by RA.


Figure 1: RXR and RXR mRNA are constitutively expressed, in contrast to RAR, which is strongly induced by RA. Poly(A) RNA (1 µg) from cells treated with 10M RA for the indicated time periods was subjected to electrophoresis, blotted, and hybridized with the indicated probes on the left. RNA size markers are indicated at the right.



Regulation of Protein Complexes Binding to the RARE

Next, we wished to determine the nature of the nuclear proteins that mediate RA-regulation of RAR gene transcription. For this purpose, we examined the binding of proteins, present in nuclear extracts obtained from cells undergoing the same treatment as described above, to a labeled DNA probe containing the minimal RARE (see Fig. 5B) in EMSA. The results are shown in Fig. 2. In untreated cells, a single retarded complex is observed (Complex I), which essentially remains unchanged over the first 24 h of RA treatment, although there appears to be more binding at the 8 h time point. However, after 48 h of RA-treatment, Complex I has completely disappeared. Interestingly, a new complex (Complex II) with a higher mobility becomes apparent at the 24 h time point, and after 48 h it has greatly increased in intensity and is the only remaining complex. Binding of both these complexes is specific, as illustrated by the DNA competition studies with the 24 h time point sample (both Complexes I and II are present in this extract). Fig. 2shows that both complexes can be effectively competed by a 50-fold excess of cold RARE but not by two unrelated oligonucleotides representing the CCAAT-box transcription factor binding site and the minimal promoter region of the Herpes simplex virus thymidine kinase gene. Interestingly, DR1, which can bind RARRXR and RXRRXR dimers, also competes. These results indicate that the RARE could be occupied by a different set of retinoid receptors in untreated cells (Complex I) than in RA-treated cells (Complex II). In agreement with this, we find that addition of 9-cis-RA and RA slightly enhances the mobility of Complex I and II, respectively, without significantly affecting the binding efficiency (not shown). These possibilities are explored in the next section.


Figure 5: Chemical modification interference assay of Complexes I and II binding to the RARE (-61/-29) show differences in binding. As a source of Complex I, nuclear extract from untreated cells (0 h) was used; for Complex II, the 48 h RA-treated cells were used. Circles refer to protected guanine residues (dimethyl sulfate, DMS); squares refer to protected thymine residues (KMnO). Opensymbols denote weak protection (intensity between 30 and 70% of corresponding band in free probe lane); solidsymbols denote strong protection (intensity less than 30% of the corresponding band in the free probe lane). A, autoradiograms of chemical modification interference experiments (see ``Materials and Methods''). Coding and noncoding strands are indicated above the gel (see also below). F and B indicate free and bound probe, respectively. For orientation, the upstream and downstream half-sites are indicated by an open, and solidblackarrow, respectively, alongside the gel. Protected nucleotides are indicated in relation to the half-sites. B, summary of interference patterns shown above.




Figure 2: Two different protein complexes bind specifically, but with different kinetics, to the RARE. Nuclear extracts were prepared from cells treated with 10M RA for the time points indicated above the gel and analyzed by EMSA, using the RARE as probe. Cold competitor DNA, as indicated below the gel, was added in 50-fold excess (see text). Arrows indicate the presence of Complexes I and II. P, probe alone.



Immunological Characterization of Complexes I and II Reveals the Presence of RXR and RXR, and of RXR and RAR, Respectively

To establish the identity of complexes I and II, we made use of a battery of RAR- and RXR-specific antibodies. When used in the EMSA with in vitro translated receptors, these antibodies can ``supershift'' retarded complexes if the epitope is accessible and specifically change their mobility in the gel to a position with lower mobility (data not shown). For simplicity, we focused our attention on the 0 and 48 h time points, inasmuch as they each contain just one complex (either I or II, respectively). For a fair comparison of the proteins in Complexes I and II in this experiment, we used 3 µg of the 0 h time point and 1.5 µg of the 48 h time point nuclear extract, respectively, because these amounts give equal levels of shifted probe in both complexes.

First, we used two different RAR antibodies: RARc, which cross-reacts with the three major RAR isoforms, and a RAR-specific antibody (RAR). The results are shown in Fig. 3A. Contrary to our expectations, none of the RAR antibodies affected the mobility of Complex I in the untreated extract, indicating that they either do not contain RARs or that the epitope is inaccessible. In contrast, both RAR antibodies completely supershifted Complex II at the 48 h timepoint, whereas preimmune serum and a nonrelevant antibody had no effect. This shows that Complex II contains at least RAR.


Figure 3: Characterization of Complex I and II binding to the RARE in the EMSA using antibody supershifts with various specific RARRXR antisera. As a source of Complex I, nuclear extract from untreated cells (0 h) was used; for Complex II, the 48 h RA-treated cells were used, as indicated above the gels. The arrows with associated roman numerals point to Complex I and II. Supershifted complexes are also indicated on the left by arrows, marked SS. Antibodies are added as indicated below the gel; -, no antibody added; *, nonspecific, serum-induced binding. A, detection of RARs. Antibodies used were as follows: Gal, anti--galactosidase; Pre, preimmune antisera; RAR, specific RAR antibody; RARc, nonisoform-specific RAR antibody. B and C, detection of RXRs in Complexes I (B) and II (C). Antibodies used were as follows: RXR (H), RXR-specific antibody raised against hinge-region epitope; RXR(N), RXR-specific antibody raised against N-terminal epitope; RXR and RXR, antibodies raised against N-terminal epitopes in RXR and RXR, respectively. P, probe alone.



Next, we used a battery of RXR antibodies, consisting of two different RXR antibodies (designated H and N, that recognize epitopes in the hinge region, or in the N terminus, respectively), as well as RXR and RXR antibodies (all raised against N-terminal epitopes). Fig. 3B shows the following results. Complex I can be partially supershifted by both RXR antibodies and slightly less well by the RXR antibody. Addition of more antibodies did not increase the amount of shifted probe (data not shown). However, the combination of both RXR (type H) and RXR antibodies quantitatively supershifts the entire complex, whereas no shift is observed with two different preimmune sera or the RXR antibody. (Note that all our non-IgG-purified antisera give rise to a nonspecific, serum-dependent shifted band that migrates with a lower mobility than the antibody-specific supershifted complex. It is nonspecific because nonrelated antisera give the same result, they cannot be competed with the immunizing peptide, and there is no concomitant reduction in the intensity of the complexes of interest.) This suggests that Complex I consists of, at least, RXR and RXR.

Fig. 3C shows that Complex II, on the other hand, can only be partially supershifted with the hinge region-epitope RXR antibody (RXR H) but not at all by the RXR antibody raised against the N-terminal epitope (RXR N). Neither one of the other RXR antibodies, or preimmune sera, affected the mobility of the complex. This not only suggests that Complex II contains RXR, but also that the N-terminal epitope of RXR is now inaccessible, unlike the epitope in Complex I. Again, addition of more antisera did not change the amount of retarded probe, suggesting that these effects are not due to limiting amounts of antibodies (not shown), but may be due to different intra-dimer protein-protein interactions between the RXR dimers and the RXRRAR heterodimers. However, we cannot exclude the possibility that there are also other unknown nuclear protein(s) present in both complexes, although antibodies against thyroid hormone receptors and , chick ovalbumin upstream activator transcription factor, estrogen receptor, glucocorticoid receptor, and even a monoclonal antibody against RAR did not affect the mobility of either complex (data not shown). However, there is no support in the literature for higher order RXRRAR complexes on the RARE. It is also possible that the receptors could undergo posttranslational modification(s) upon treatment with RA, which could cause epitope masking, or as yet uncharacterized isoforms could be induced, but again, we unaware of any studies giving credence to these hypotheses. Together, these data indicate that, with respect to retinoid receptors, Complex I consists mainly, if not completely, of RXR and RXR, whereas Complex II at least contains RXRRAR heterodimers.

Our data may also explain the disappearance of Complex I because the RXRs in that complex may be recruited into a heterodimer complex with the induced RAR in Complex II, which should bind with higher affinity. At the 48 h time point, there may be enough RAR protein produced that all RXRs are titrated, such that Complex II completely replaces Complex I. This interpretation is further strengthened by the results of the Western blot shown in Fig. 4. Proteins from 10 µg of nuclear extract of cells treated with RA for a period for up to 48 h were separated by SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose membrane, and probed with a RAR antiserum. A specific band, migrating at about the expected mobility for RAR, is induced between 8 and 24 h of treatment. The kinetics of appearance of the RAR protein, therefore, match the observed DNA-binding activity by RAR in Complex II in the EMSA. Interestingly, as shown before, the mRNA for RAR can already be detected within 2 h of treatment. Thus, according to our detection methods, translation of RAR mRNA lags at least 6 h behind transcription of the gene.


Figure 4: Western blot showing induction of RAR protein between 8 and 24 h of treatment with RA. 10 µg of nuclear extract of cells treated for the indicated time periods was electrophoresed through 10% SDS slabgel, electroblotted onto nitrocellulose, and probed with RAR antisera (see ``Materials and Methods''). The arrow denotes the position of the RAR protein; the asterisk a nonspecific band.



Chemical Modification Interference Assays of Complex I and II Show Differential Protection of Bases in the RARE

The RARE is an element with dyad symmetry, and RARsRXRs are known to bind as (homo/hetero)dimers. Our results suggest that RXRRXR and RXRRXR homodimers, perhaps in combination with RXRRXR heterodimers, are the receptors that occupy the RARE in untreated cells. This is surprising, since this element is only very weakly bound by RXRs in vitro(21, 27, 28, 42, 43) , in contrast to RARRXR heterodimers(17, 18, 20, 21, 22, 23) . If this interpretation is true, it is possible that Complexes I and II form different contact points with the bases in the RARE oligonucleotide. To test this hypothesis, we performed a chemical modification interference assay of both complexes (time points 0 and 48 h) using dimethyl sulfate and KMnO to identify protected G and T residues, respectively. The results, shown in Fig. 5, show that Complex I does not protect any T residues and only weakly protects a few G residues at -41 in the coding strand and at -38, -47, -49, -60, and -61 in the noncoding strand. Except for the latter two bases, they appear to cluster around the two half-sites in the probe (indicated by arrowsabove the sequence in Fig. 5B). In contrast, Complex II gives a very clear and much stronger protection pattern of both G and T residues. Particularly strong protection occurs at residues -41, -52, and -53 in the coding strand and at -37, -38, -48, and -49 in the noncoding strand. Weaker protection is also observed between the two half-sites on this strand. Thus, the contact sites for Complexes I and II are close to the two established half-sites, but protection of bases is much stronger in Complex II. These data are consistent with the hypothesis that there are RXR dimers in Complex I that bind specifically, but with low affinity (as reported for in vitro translated receptors) to the RARE, and that the RARRXR heterodimers in Complex II bind with much higher affinity to this probe. In agreement with this, the pattern of protection of this element by RARRXR heterodimers in RA-induced P19 embryonic carcinoma (EC) cells is essentially the same as that in Complex II (44) . Because it is reasonable to assume that other types of RARRXR heterodimers would also give strong protection, this argues against the possibility that there are significant amounts of RARs in Complex I that escaped our detection.

RAR Gene Expression Is More Rapidly Induced by 9-cis-RA Than by RA

The above described results indicate that RXR dimers mediate the retinoid-dependent induction of the RAR gene, instead of RARRXR heterodimers. This hypothesis can be tested by comparing the effects of RA and 9-cis-RA on RAR induction. RARs bind RA and 9-cis-RA with high affinity, but RXRs bind 9-cis-RA with much higher affinity than RA(45, 46) . If RARRXR heterodimers are involved, it might be expected that the effects of RA and 9-cis-RA will be of comparable magnitude, because both ligands activate RARs. If, on the other hand, RXR (homo)dimers are the activating species, then it might be expected that 9-cis-RA will generate a quicker response than RA. To test the hypothesis, in this experiment the cells were grown in media that was stripped of thyroid/steroid hormones to exaggerate the effects of both ligands. S91 cells still grow and differentiate in this media upon retinoid treatment (data not shown). Total RNA was isolated from cells that were untreated (0 h) or treated with RA or 9-cis-RA at the indicated time points in Fig. 6. Fifteen µg of total RNA was subjected to electrophoresis through an agarose gel, and analyzed by Northern blotting (Fig. 6). The results show that, in cells treated with RA, RAR mRNA induction becomes apparent between 4 and 6 h, which is about 2 h slower than cells grown in regular serum. In contrast, RAR mRNA levels show a more rapid and potent increase (between 2 and 4 h) after 9-cis-RA treatment. These results, therefore, are supportive of our model of RXR-mediated RAR induction by retinoids. The classic RA response (see Fig. 1) may be due to metabolic conversion of RA to the 9-cis-RA stereoisomer, which is how 9-cis-RA was identified(45, 46) .


Figure 6: 9-cis-RA is a more potent inducer of the RAR gene than RA. A, Total RNA (15 µg) from cells, which were grown in thyroid/steroid hormone-depleted media and treated with 10M RA for the indicated time periods, were subjected to electrophoresis, blotted, and hybridized with the indicated probes on the left. B, quantitation of this Northern blot by PhosphorImager scanning. The number of counts at time point 0 (no RA treatment) for the RAR probe was arbitrarily set at 1. All other values are normalized to this value. The cyclophilin probe was used to normalize the data for loading/transfer differences.




DISCUSSION

The S91 melanoma cell line is a useful model system to study retinoid-induced growth arrest and differentiation and may provide important clues to the transformation of melanocytes in disease. However, the molecular basis of the retinoid-induced effects is still largely unknown. There are two classes of retinoid receptors, and it is likely that RARs and RXRs are at the top of the cascade of events that lead to the arrested growth and morphological changes in these cells. Previously, it was shown that these cells constitutively express RAR and RAR and very small amounts, if any, of RAR. Of these RAR isoforms, only the latter is up-regulated by retinoids(32, 34) , which we confirmed in this study. We completed the inventory of retinoid receptors in these cells by performing Northern blots with RXR-specific probes, and we showed that S91 cells also constitutively express RXR and RXR, but no RXR.

It was also previously observed that induction of RAR appears to correlate with the induction of growth arrest. This particular isoform has been suggested to have an important function in neoplastic cells, i.e. its absence or aberrant expression has been observed in many tumors and tumor cell lines and appears to correlate with malignancy(3) . As a first step in understanding the role of RAR, we wished to determine which RARs and/or RXRs mediate the retinoid-dependent activation of the RAR2 promoter. Although the RARE in this promoter has been well characterized (35, 36, 37) , the nature of the endogenous, intracellular receptors that bind to this element has not been established. We employed EMSA with the RARE as probe incubated with nuclear extracts of cells that were treated for various periods of time with RA and analyzed the retarded complexes with a battery of RAR- and RXR-specific antibodies. Our analysis shows a single retarded complex (Complex I) in melanoma cells in their fully undifferentiated, malignant state. This complex can be quantitatively supershifted with a combination of antibodies against RXR and RXR, but it does not react at all with two different RAR antibodies. This observation can be most easily explained by assuming that this complex consists of RXRRXR and RXRRXR homodimers or perhaps in combination with RXRRXR heterodimers (this cannot currently be determined). However, we cannot exclude the possibility that other unknown nuclear protein(s) can form heterodimers with RXRs, or could be part of the RXR dimer complex, although this has never been reported to occur on the RARE. In any case, our observations suggest that binding to the RARE, and, therefore, probably transcriptional activation of the RAR gene is largely mediated by RXRs rather than RARs or RARRXR heterodimers.

This result is surprising because the RARE represents a DR5 element, which is very poorly bound by RXR dimers in vitro but strongly bound by RARRXR heterodimers, as illustrated in our chemical modification interference assays. Since the RNAs for all three RAR isoforms (including RAR), and that of two RXRs can be detected by Northern blot analysis, we wondered why we did not observe any RARRXR heterodimers binding to the RARE. The mechanism that we propose is that in untreated S91 cells there is an excess of RXRs over endogenous RARs such that RXR dimers are formed and are able to bind the RARE because there is no other competitor complex (the RARRXR heterodimer) present. For instance, it was recently shown that in human skin there is 5 times more total RXR than total RAR protein(47) , and in S91 cells this difference could conceivably be even higher. This explanation would be consistent with published in vitro binding data, which show that RXR homodimers can bind to the RARE with low affinity; binding is only enhanced if RARs are mixed in(21, 28, 42) . This would also explain our observations in the EMSA in Fig. 2. The RXR dimer complex (Complex I), which binds the RARE in untreated cells, remains virtually unchanged up to 24 h of RA treatment, although the 8 h time point shows more intense binding. This may be expected, as the RXR mRNAs are only weakly regulated by RA (Fig. 1). Meanwhile, RAR mRNA is being transcribed and translated, and RAR protein begins to accumulate. Between 8 and 24 h of treatment with RA, RARRXR heterodimers become visible as Complex II, and by 48 h of treatment, all endogenous RXRs are titrated into the RARRXR heterodimer complex, leading to the complete disappearance of Complex I. This would more satisfactorily explain the abrupt change in complexes at the 48 h time point than assuming that the RXRs are suddenly degraded or inactivated. This also implies that Complex II not only consists of RARRXR but also contains RARRXR heterodimers, since both RXRs were identified in Complex II.

However, we are unable to prove this formally, because the N-terminal epitope, at least in RXR, is inaccessible to our antibody in the heterodimer complex (see Fig. 3C), in contrast to that in the non-RAR containing RXR complex (Fig. 3B). As our RXR antibody recognizes an N-terminal epitope and both types of RXRs can be expected to form similar complexes with RARs, it probably explains why we did not observe any supershifts with this antibody on Complex II but only with Complex I. Again, we favor this explanation over that of assuming that only RXR somehow is no longer able to bind DNA between 24 and 48 h of treatment with RA. Interestingly, all RXR antibodies are able to supershift quantitatively RARRXR heterodimers using proteins that were obtained through the rabbit reticulocyte lysate translation system, suggesting that either intracellular receptors are different from their in vitro counterparts or contain other, as yet unknown, nuclear partners. Also, the mobility of these in vitro translated heterodimers is quite different from those of Complex II (data not shown).

RAR transcription is induced in many cells, in particular EC cells. Is the same mechanism for RXR-mediated activation that we propose here operative in those cells as well? Minucci et al.(44) studied RA-induced differentiation of P19 EC cells. In the absence of RA, very little binding is observed to the RARE in EMSA, with no protection of residues using in vivo genomic footprinting techniques. In the presence of RA, binding is strongly enhanced (with virtually identical protection patterns as our Complex II), concomitant with induction of RAR gene expression. We propose that constitutively expressed RARs ( and ) may be involved in the initial activation in a heterodimer complex with RXR. However, without the use of antibodies, this has not yet been proven conclusively. It is also interesting, that they too observe similar binding of two complexes to the RARE, of which only the lower complex (corresponding to our Complex II) increases significantly in intensity after RA treatment. Thus, RXRs might also play some role in activation of the RAR gene in P19 EC cells. The observation that, in RAC65 cells that are RA-resistant P19 EC cells due to a truncation of the RAR gene (48) or in F9 EC cells in which the RAR gene has been deleted(49) , RA-induced RAR expression is virtually unchanged is certainly not at odds with this interpretation. On the other hand, Clifford et al.(32) , who used synthetic retinoids like 4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)1E-propenylbenzoic acid (TTNPB) and [4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl)2-naphtalenylcarbamoyl]benzoic acid (Am80), which are specific for RARs, showed induction of the RAR gene in S91-C2 cells, although only treatment with TTNPB reached the same induction level as treatment with RA. A possible explanation for this apparent discrepancy may be that at the very high doses that were used (1 µM), these retinoids are less specific in their activities and also activate RXRs.

It is possible that there are very low amounts of RARRXR heterodimers present in untreated cells, which are below our detection levels and which may be able to activate the RAR gene. Also, it is even possible that Complex I contains RARRXR heterodimers with inaccessible epitopes, perhaps due to posttranslational modifications or the association of (unknown) additional protein(s). However, it appears that RXRs are the key mediators of retinoid-induced transcription of the RAR gene in S91 cells. Nonetheless, it would be interesting to look at the kinetics of induction by TTNPB and Am80 compared with 9-cis-RA as we did between RA and 9-cis-RA in Fig. 6. Also, the availability of new RXR-specific retinoids would be helpful to these studies(50) .

Another interesting aspect is that RAR mRNA levels plateau after about 24 h of treatment with RA (Fig. 1), whereas between 24 and 48 h of treatment relatively large amounts of RARRXR heterodimers begin to appear (Fig. 2), and the concentration of RAR protein also continues to increase (Fig. 4). Thus, if heterodimers completely replace RXR dimers by 48 h of treatment, one could expect an increase in transcription between 24 and 48 h of treatment, since the RARE is much more potently activated by RARRXR heterodimers than RXR dimers. A possible explanation for this phenomenon may be that the cells have already differentiated to a certain extent by 24 h of treatment, such that they no longer sustain transcription of certain genes. A similar phenomenon was observed in embryonic carcinoma cells. After RA-induced differentiation, an adenovirus EIA-like activity, which is required for RAR expression, disappears(51, 52, 53) . For instance, we found that the DNA-binding activity of octamer binding factor 1 (Oct1) disappears between 8 and 24 h of treatment with RA (not shown). It is conceivable that Oct1, as well as other nuclear factors, plays an important role in these processes, and may contribute to the decline or transcriptional arrest of the RAR gene after 24 h of RA treatment.

Our results suggest that a mechanism of sequential occupation of the RARE takes place after treatment with retinoids in S91 cells. We believe that in the melanoma state (untreated cells), RXRs alone occupy the RARE, and are mainly responsible for transcriptional activation of the RAR gene. Between 24 and 48 h of treatment, when the cells have essentially been converted to a benign, melanocytic state, there appears to be a switch in receptor occupancy so that the RXR dimer complex is replaced by a RARRXR heterodimer. It will be interesting to see whether similar switches also occur at other RAREs and whether our observations also apply to other cell types.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Surgery, Division of Surgical Oncology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115. To whom correspondence should be addressed: G. W. Thorn Research Bldg., Rm. 303, Brigham and Women's Hospital, 20 Shattuck St., Boston, MA 02115.

The abbreviations used are: RAR, retinoic acid receptor; RA, all-trans-retinoic acid; RXR, retinoid X receptor; RARE, retinoic acid response element; DR, direct repeat; EMSA, electrophoretic mobility shift assay; EC, embryonic carcinoma; TTNPB, 4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)1E-propenylbenzoic acid; Am80, [4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl)2-naphtalenylcarbamoyl]benzoic acid.

B. Neel, personal communication.


ACKNOWLEDGEMENTS

We thank Dr. Pierre Chambon for providing the pSG5RAR plasmids, Dr. Ronald Evans for the pBSRXR plasmids and the RXR (H) antibody, and Dr. Benjamin Neel for the RAR antibodies.


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