Distinct Functions for Retinoic Acid Receptors During Differentiation of P19 Embryonal Carcinoma Cells
 
Gert E. Folkers, and Paul T. van der Saag
 
Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, the Netherlands
 
submitted for publication
 

Abstract

We have used the P19-embryonal carcinoma (EC) cell line as a model system to study the role of retinoids in early development. Upon retinoic acid (RA) treatment these cells can differentiate into derivatives of all three germ layers. We have previously shown that RAC65 cells, an RA-resistant P19-EC cell derivative, express a truncated dominant-negative retinoic acid receptor (RAR). To identify the function of the individual RARs in EC cell differentiation we have made stable transfectants in RAC65 cells overexpressing either RAR or RAR. Overexpression of RAR permits RA-dependent differentiation of these cells, which is accompanied with a change in the expression of several RA-target genes. Contrary to this RAR overexpressing cells still cannot differentiate upon RA-treatment. Instead an increased RA-dependent growth repression and extensive cell death was observed. Suggesting that RAR is directly involved in the RA-dependent differentiation process, while RAR is required for negative growth-regulation by retinoids. This was further confirmed in P19-EC cells overexpressing RAR. These cells can differentiate when only RAR is activated (by AM80), while if both RAR and RAR are activated extensive apoptosis was observed. Analysis of the expression of RAR in normal P19-EC cells indicated that this gene product is present at the time when maximal RA-induced apoptosis is occurring. With the use of double labeling we present evidence for a direct role of RAR in the induction of apoptosis. Based on these data we propose that RAR is involved in RA-induced differentiation, while RAR which is induced directly after RA-treatment of EC cells, is involved in growth regulation and in cell death of those cells that have failed to differentiate.

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Introduction

Excess as well as lack of vitamin A (retinol) during embryonal development can lead to severe malformations (54). After birth vitamin A is essential for growth, reproduction and vision. Retinoic acid (RA), the active metabolite of vitamin A, exerts its effect through two families of retinoid receptors: retinoic acid receptors (RAR), and retinoid X receptors (6). Targeted disruption in mice of individual receptors, as well as compound mutants, further support the view that retinoids, fulfill an important role in embryonal development through their receptors (36). These receptors belong to the steroid/ thyroid hormone receptor superfamily which all have a similar organization (18,42). Embryonal carcinoma (EC) cell lines are useful to investigate early processes in embryonal development (47), since they resemble inner cell mass cells of the blastocyst and can, when injected into a host blastocyst, participate in development (25,47). Depending on the culture conditions, these cell lines can differentiate towards cells resembling endoderm, mesoderm or neurons in the presence of RA (17,34,76). Treatment of EC cells with RA causes drastic changes in their growth characteristics: decreased proliferation, increase in the fraction of cells in G1 phase of the cell cycle and loss of the malignant phenotype as illustrated by the loss of the capacity for anchorage-independent growth (44,56,67,68). Furthermore gene expression of various genes is changed both positively and negatively during differentiation of EC cells (23,27). Finally it was recently shown that treatment of EC cells with RA can lead to programmed cell death or apoptosis (for review: 28,81) (1,24,30,61,75), as was observed earlier in breast cancer cells (83), hematopoietic progenitor cells, (21) and myeloblastic leukemia cells (59) which all have been reported to undergo apoptosis after RA treatment.

To investigate which receptor, receptor isoforms or combination of receptors are required for the RA-dependent effects in EC cell differentiation and/or apoptosis, various type of experiments can be performed. (1) Targeted disruption of individual receptors by homologous recombination. (2) Introduction of mutated receptors that interfere with the normal function of RAR and/or RXR. (3) Overexpression of functional receptors in cell lines lacking these receptors or containing mutated receptors. (4) Using synthetic retinoids, either agonists or antagonists, that are specific for a one receptor form or class of receptors. Although each of these experimental approaches has its limitations, together such experiments will eventually permit delineation of specific role(s) for individual receptors in the EC cell differentiation process. Inactivation of RAR in F9-EC cells caused a delay in morphological differentiation by RA and aberrant expression of some but not all target genes (4), supporting the notion that individual receptors regulate different target genes (58). This is further supported by F9-EC cells lacking RAR, which are not impaired in their differentiation characteristics; however regulation of a subset of the target genes is altered furthermore an increase in retinoid-metabolism was observed (5). The results from these experiments already indicated that each receptor fulfills specific functions, with RAR as being most critical for RA-dependent differentiation of F9-EC cells, although these experiments have also shown that differentiation of F9 RAR^-/- cells can be taken over by RAR (4,79). The dominant role for RAR in differentiation of F9-EC cells is further supported by the use of receptor-specific synthetic retinoids (69). Moreover it was recently shown that inactivation of RXR prevents differentiation of F9 EC cells by RA (11). With the use of synthetic ligands it was further shown that RXR is directly involved in the differentiation process and not only functioning as a silent (dimerization) partner for RARs (11). Combination of low concentrations RAR and RXR-specific ligands leads to differentiation and synergistic activation of target genes while these ligands alone where not sufficient for both functions (11,69).

Experiments described above were all performed in F9-EC cells which can differentiate into extra-embryonic endoderm cells only, and form primitive, parietal or visceral endoderm, depending on the culture conditions . P19-EC cells however can differentiate into derivatives from all three germ layers (17,34). Experiments using receptor specific retinoids in P19-EC cells have shown that both RAR and RAR can induce differentiation (69). Furthermore introduction of a dominant negative RAR or RXR in P19 EC cells prevents RA-dependent differentiation, showing that functional receptors are required for differentiation of these cells (13, 51; unpublished results). In agreement with this RAC65 cells, a retinoic acid-resistant derivative of P19 cells, is resistant to RA, which is caused by the insertion of a transposon-like sequence within the activation domain present in the C-terminal region of the RAR ligand-binding domain. This insertion abolishes the transactivation domain AF-2, while dimerization and DNA-binding functions are retained (39,65). Also the RA-dependent expression of most RA target genes in this cell line is lost (66,85). Stable expression of a functional RAR in this resistant cell line resulted in a transfectant that resembled P19-EC cells with respect to morphological differentiation, growth inhibition, expression of extracellular markers, and target gene expression (39,85).

Differentiation of P19 EC cells as well as F9 cells is accompanied by an induction of RAR2, expression is maximal after 3-5 days, then starts to decline and is absent in fully differentiated endoderm cells as shown by the lack of expression in the END2 cells, an endoderm-like derivative of P19-EC (35,37,39). This led us to hypothesize that RAR fulfills an important role in early EC cell differentiation. To directly investigate this we overexpressed RAR in the RA-resistant RAC65 cells and compared the growth and differentiation characteristics of these transfectants with RAR-overexpressing clones. In contrast to RAR-expressing clones RAR clones do not differentiate, while instead an increase in growth repression was observed. We further show that overexpression of RAR prevents P19-EC cells from RA-dependent differentiation, leading to extensive cell death through apoptosis. With the use of a RAR2 promoter-LacZ containing P19-EC cell stable transfectant we observed that after 2-4 days RA treatment expression of RAR2 is restricted to only a subset of non-differentiated cells, which eventually die. We propose that RAR is required for differentiation of P19-EC cells, whereas RAR is involved in growth control.

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Materials and methods
 

Cell culture, RNA isolation, Northern blotting and RNase protection

RNA was isolated using the acid-phenol single step method as described by Chomczynski and Sacchi (10). Ten to twenty µg of total RNA was denaturated at 65 ^oC for 10 minutes in running buffer (20 mM MOPS, 1 mM EDTA and 5 mM sodium acetate) containing 50% (w/v) formamide and 2.2 M formaldehyde. Loadingbuffer ( 0.5% (w/v) LMP-agarose, 10% (w/v) sucrose) was added and RNA was separated by electrophoresis in a 0.8% (w/v) agarose/ 2.2 M formaldehyde gel and subsequently blotted onto Hybond C-extra (Amersham) by capillary transfer using 20x SSC. Hybridization was performed in 50% (w/v) formamide, 5x SSC, 0.5% (w/v) SDS, 10% (w/v) dextran sulfate, 1x Denhardt solution, 0.1 mg/ml salmon sperm DNA for 14-24 hrs at 42^oC. Probes were labeled using a multiprime labelingkit and ³²P-dCTP (Amersham). After hybridization blots were washed twice in 2x SSC, 0.1% (w/v) SDS, twice in 0.5x SSC, 0.1% (w/v) SDS and twice in 0.1x SSC, 0.1% (w/v) SDS. If required additional washes at 42^o or 65^oC with 0.1xSSC, 0.1% (w/v) SDS were performed. Blots were exposured for 2-10 days to Fuji RX films using an intensifying screen. Probes were removed by submerging the filters in boiling solution containing 0.1x SSC, 0.1% (w/v) SDS.RNase protection was performed as described before (35) using the probes described by Vanderleede (80) and Jonk (35). In short, 10 µg total RNA was hybridized overnight at 45^oC with 150,000 cpm RAR or RAR and 30,000 cpm GAPDH (labeled to a ten-fold lower specific activity) as probes in 25 µl buffer containing 80% (v/v) formamide, 40 mM Pipes, pH 6.4, 0.4 M NaCl, 1 mM EDTA. After hybridization samples were digested for 30 min at 37^oC with 20 µg RNase A (Boehringer Mannheim) in 350 µl digestion buffer (10 mM Tris pH 7.5, 300 mM NaCl 5 mM EDTA). Subsequently Proteinase K treatment, phenol-chloroform extraction and ethanol precipitation in the presence of 10 µg tRNA were performed. RNA was resuspended in formamide loading buffer and electrophoresed on a 5% (w/v) polyacrylamide, 7 M urea, TBE gel. Dried gels were exposed for 5 to 10 days to Fuji RX films using an intensifying screen.
 

Growth inhibition assay

Effect of RA on the growth was determined using a [³H]-thymidine incorporation assay. Cells (5,000-15,000) were plated in a 24-wells plate in 1 ml DF medium containing 7.5% (v/v) FCS, and were treated for 72 hrs with the indicated concentrations RA. 18 hrs prior to harvesting 0.5 µCi [³H]-thymidine (Amersham) was added to each well. After extensive washing (3-4 times) with PBS cells were fixed with methanol for 10 min. After incubation of the cells with 400 µl 0.1M NaOH at 37^oC for 30 min, the amount of incorporated [³H]-thymidine was determined by counting extracts in a liquid scintillation counter (Topcount, Packard Instuments Inc.). Each treatment was performed in triplicate. RA-dependent growth repression was determined as the percentage incorporated [³H] thymidine relative to untreated cells. Results were the mean (+/- S.D) calculated from 3-4 independent experiments.

Growth in soft agar

To determine the ability of the various cell lines to grow in soft agar after retinoic acid treatment (1 µM), 5,000-20,000 cells were added to DF medium containing 13.5% (w/v) FCS and 0.375% (w/v) agar and plated onto a base layer of 0.5% (w/v) agar as described before (56). Percentage growth was determined by comparing the number of colonies observed after ten days of culture, both in the presence or absence of RA.

Cell culture, differentiation, isolation of the stable cell transfectants

Cells were cultured as described before (37,38). The ability of the various cell lines to differentiate in monolayer was determined by growing the cells for 2 to 5 days in 1µM RA. Morphology of transfectants was compared with P19-EC cells and/or the RA resistant cell-line RAC65. Neuronal differentiation was investigated by aggregating cells in 1-2 ml medium (1-2x 10⁵ cells/ml) in bacterial dishes in the presence or absence of 1 µM RA for 3 days. Thereafter 10-50 colonies were plated (without addition of RA) in 6-well tissue culture dishes, and aggregates were microscopically analyzed for the presence of neurites after 6 days.

The RAC65 cells overexpressing RAR have been described before (39). For the isolation of stable transfectants, cells were transfected as described before (39) with a plasmid containing an SV40 promoter-driven RAR2 expression construct and a neomycin resistance gene driven by the same promoter. Twenty four hrs after removal of the calcium-phosphate precipitate the cells were replated in several dilutions (1:2 to 1:10) in 6-well plates in the presence of geneticin (200 µg/ml) (G418, Life Technologies). Medium containing G418 was refreshed every two days and colonies were isolated 7-10 days later. Cells were maintained in the presence of G418 during all experiments performed with stable transfectants. Cell lines expressing sufficient amounts of RAR, both at protein and RNA level were subsequently analyzed.
 

Plasmids

pSG5-neo was made by cloning a SV40 driven neo cassette in the SalI site of pSG5. For cloning purposes a poly-linker was inserted in the blunt EcoRI site of pSG5. The SstI-BamHI RAR-containing fragment derived from pSG5 HA-RAR2 (19) was cloned into the corresponding sites of pSG5 neo. RAR AB deletion was made by cloning the HindIII-BamHI fragment of the HA-RAR AB construct (19). The VP A RAR was constructed by cloning the PCR generated EcoRV fragment containing the transactivation domain of VP16 (aa414-491), in the SmaI site at the N-terminus of HA-RAR A (19). Constructs were controlled by sequencing and expression was confirmed by western blotting as described before (19) using either a HA-tag antibody or a polyclonal anti RAR antibody.
 

Analysis of Apoptosis

For DNA fragmentation assays cells were cultured in the presence or absence of RA for the indicated period in 150 cm² dishes. In each experiment also gelatin coated glass coverslips were present in the culture dishes for microscopical analysis. Supernatant and cells scraped in PBS were combined and genomic DNA was isolated using the Qiagen genomic DNA columns as described by the manufacturer. 7.5 µg of genomic DNA was separated on a 1.5% (w/v) agarose/TBE gel. For Hoechst staining and TUNEL assay glass coverslips were fixed in 2% (w/v) paraformaldehyde in PBS for 30 min, washed three times during 30 min in PBS, thereafter stained for 15 min at 37 ^oC with 0.1 µg/ml Hoechst 33258 in PBS and washed three times with PBS. Alternatively first a TUNEL assay was performed as described by Blaschke et al (2) and thereafter stained with Hoechst dye and photographed on a fluorescence microscope. The percentage apoptotic cells determined by TUNEL assay was determined by counting the amount of apoptotic cells divided by the number of cells (stained nuclei) per microscopical field (75-400 cells/field). At least three different fields were counted and the percentage of apoptotic cells was determined as the mean percentage apoptotic cells of these fields. Results are the mean (+/- SEM) of three independent experiments.

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Results
 

RAC65 cells stably expressing RAR and RAR

We have previously reported that introduction of RAR in the RA-resistant P19 derivative RAC65 restored its ability to respond to RA, as shown by induction of target genes (85), growth repression and morphological differentiation (39). Knock out experiments in mice with individual RARs have led to the idea that receptors could be redundant in their function (36). To investigate whether RAR and RAR are functionally redundant during EC cell differentiation we produced cell lines derived from RAC65 cells expressing RAR or RAR. Therefore a pSG5-neo-HA-RAR2 expression construct was transfected in RAC65 cells. Seventeen clones were isolated, all of which expressed various levels of RAR (data not shown) and six were selected for further characterization. Expression of RAR in these clones both at RNA (Fig 1B) and protein level (Fig. 1D) was clearly detectable as judged by RNAse protection and Western blot, respectively. Simultaneously, ten control RAC65 clones were made, only containing an empty SV40 neomycin resistance gene construct (pSG5-neo), three of which were chosen for further analysis. The RNA and protein expression of the previously isolated RAR expressing RAC65 cells (39) is also shown in Figure 1A,C).
 

Characterization of the RAC65 clones

We first established the RA sensitivity of the neo controls, by subjecting RAR and RAR expressing RAC65 cells to [³H]thymidine incorporation assays. As shown in Figure 2 P19-EC cells were strongly growth-repressed by addition of RA. On the other hand RAC65 cells were completely resistant to RA treatment. Similarly, none of the mock transfectants were significantly growth-inhibited by the addition of RA (Fig. 2A). Clones expressing RAR however were clearly growth-repressed, depending on the expression levels of RAR (Fig. 2B). All of the RAR expressing clones were growth-repressed, significantly stronger than in RAR overexpressing clones (Fig. 2C). None of the RAR or RAR expressing clones was growth inhibited to the same extent as P19-EC cells; this most likely is caused by the presence of the truncated RAR in the RAC65 cells interfering with the function of the introduced RAR or RAR.

Next we investigated the RA-dependent differentiation of these clones in monolayer cultured for 4 days in the presence or absence of 1µM RA. As shown in Figure 3, wild-type P19-EC cells morphologically change after RA treatment from small round cells to big, flat endoderm-like cells (Fig. 3). RAC65 cells, like control clones, remain undifferentiated, morphologically resembling undifferentiated EC cells. In the absence of RA all stable transfectants resembled RAC65 or the untreated P19-EC cells (except RAR4). However, upon RA treatment in RAC65 clones RAR3, RAR10 and to a lesser extent RAR4, differentiation similar as described for wild type P19-EC cells was observed (Fig. 3, data not shown). The RAR4 clone showing already a change in morphology, in the absence of RA and displayed further changes upon RA treatment, although these cells did not resemble the RA-treated P19-EC cells (data not shown). Contrary to RAR-expressing RAC65 cells, addition of RA to RAR clones however, did not cause morphological differentiation. Upon RA treatment a decrease in cell number was observed, cells were irregularly shaped, many round cells were observed (Fig. 3) and in the culture medium extensive cell death was detected (data not shown). We further investigated the ability of the clones to differentiate into the neuronal direction. RA-dependent differentiation after aggregation and replating resulted in the formation of neurites and endodermal cells in P19-EC cells and the RAR transfectants but not in RAC65 cells, neo clones and RAR overexpressing cells (Table 1). Finally the ability of the RAR expressing cells to grow in soft agar was studied. RA-treated P19-EC cells are unable to grow in soft agar (56), whereas RAC65 cells and control cells are able to grow in soft agar, both in the presence and absence of RA. RAR expressing clones however were unable to grow in soft agar in the presence of RA and also without RA less colonies were found, when compared with P19-EC and RAC65 cells, RAR clones or neo controls (Table 1).

Together these data demonstrate that the differentiation characteristics of RAC65 cells

Expression of RA-target genes in the stable cell lines

The differences in morphology after RA treatment between the RAR- and RAR-expressing RAC65 cells led us to investigate whether also activation of target genes by RA had been altered. One obvious RA target gene is RAR itself. We therefore transfected a RAR2 promoter luciferase reporter construct (19) in the various cell lines and determined activation of this promoter by RA. As shown in Figure 4A, none of the cell lines activated the RAR2 promoter as efficient as P19-EC cells, while hardly any activation by RA was observed with mock transfectants or RAC65 cells. The RAR2 promoter was activated to similar extent in RAR or RAR overexpressing cells, varying from 20 to 40-fold activation. Similarly also RA-dependent upregulation of RAR expression in the RAR or RAR transfectants was observed by Northern blotting (data not shown).

We next performed Northern blot analysis with RNA isolated from cells cultured for two days in the presence or absence of RA. We observed that expression of target genes which are activated or repressed by RA treatment in P19-EC cells was not influenced by addition of RA in both RAC65 cells and in the mock transfectant (neo 10) (Fig. 4B). Expression of target genes in the RAR expressing clone was comparable with P19-EC cells although generally lower expression was observed in the former (with the exception of c-jun; similar oservations have been made before (66,85). For unknown reasons, down-regulation of Oct4 was not observed in the RAR transfectants (Fig. 4B, data not shown). Besides weak activation of CRABPII, no activation of target genes by addition of RA was observed in the RAR expressing clones 10, 25 (Fig. 4B, data not shown). The reason for this lack of activation of target genes in the RAR expressing clones is unclear. Possibly the observed RA-induced growth arrest/cell death in these clones could be the cause of the lack of target gene activation.

Overexpression of RAR in RAC65 allows the cells to differentiate and permits activation of target genes which are normally upregulated during EC cell differentiation, whereas the RAR overexpressing cells do not differentiate and most target genes are poorly activated upon RA-treatment while the cells are strongly growth repressed, and will eventually die. These data together demonstrate that RAR and RAR fulfill different functions during EC cell differentiation, causing receptor-dependent changes in RA-target gene expression.
 

AF-1 of RAR2 is not required for growth repression

RAR expression in P19-EC cells

RA-induced apoptosis during EC-cell differentiation

RAR overexpressing RAC65 cells are strongly growth-repressed by RA which is accompanied by an increase in cell death (Fig. 3). The above described in situ RAR2 promoter activity further support the idea that RAR could be involved in the growth repression and cell death. We therefore investigated whether RAR is involved in the RA-induced apoptosis or cell death. Apoptosis is an active process leading to cell death, which is accompanied with typical morphological changes including change in nuclear morphology, chromatic condensation, nuclear fragmentation and formation of apoptotic bodies (28,81). Recently it has been shown that EC cells treated with RA are undergoing apoptosis (see Introduction). If overexepression of RAR is the cause of the observed cell death, a correlation between RAR expression and high levels of apoptosis is expected. Above we have shown that RAR expression is maximal between 2-4 days after RA treatment of P19-EC cells (Fig. 6) and therefore if RAR2 is directly involved, maximal apoptosis can be expected within this period. Morphological nuclear changes characteristic for cells undergoing apoptosis were observed (see also Fig. 9), including the formation of apoptotic bodies, being maximal after two days of RA treatment. Apoptotic ladder formation is a widely used indicator for apoptosis. In the absence of RA no apoptotic ladders were observed, while after one day of RA treatment ladders appeared, reaching a maximum after two days, and which was after four days only barely detectable (Fig. 7B).

We next performed a TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay which determines in situ DNA strands breaks and which is a relative early event in the apoptotic process. In the absence of RA no apoptosis could be detected (Fig. 8) with this assay. After one day of RA treatment a few positive cells were observed, while after 2-3 days (Fig. 8 and data not shown) large numbers of positive cells were found. These apoptotic cells contained the characteristically altered nuclear morphology as determined by Hoechst staining (Fig. 8, lower panel), which is absent in non-apoptotic cells. After 4 days cells are almost completely differentiated, and only small numbers of positive cells were still found, mostly as fragmented apoptotic bodies or as cellular debris. The typical bigger flat endoderm-like cells with large nuclei were negative in the TUNEL assay (Fig. 8, data not shown). Finally we quantified the number of apoptotic cells by counting the percentage positively stained cells. Again, maximal apoptosis was observed after two days of RA treatment, and at four days a decrease became apparent (Fig. 9). From these data it is clear that apoptosis is taking place around the period of maximal RAR2 expression.

RAR overexpression causes growth repression accompanied by apoptosis

The correlation between RAR expression and appearance of apotosis during RA- dependent differentiation suggested that the RAR expressing cells are undergoing apoptosis. To test this hypothesis directly we made stable P19-EC cells overexpressing RAR. The constructs described above, were used to make P19 cells stably expressing either RAR or the empty expression vector (pSG5-Neo). Two RAR expressing clones and one mock transfectant were chosen for further characterization. RAR expression of the P19-EC cell clones is relatively low as compared with the expression observed in the RAC65 stable RAR transfectants (Fig. 10A), which can possibly be explained by the possibility that high expression in the absence of RA could cause growth inhibition. As determined by [³H]-thymidine incorporation assay, these cells were ten-fold more sensitive to RA treatment than wild-type P19-EC cells or the mock transfectant (Fig. 10B). This was accompanied with formation of rounded cells and the loss of adherence, possibly leading to cell death (data not shown).
We next investigated the differentiation characteristics of these clones upon RA treatment. In the absence of RA the neo clone as well as the two RAR clones resembled P19-EC cells in morphology (Fig. 11). Treatment of P19-EC cells and the mock transfectant with 10^-7 M RA (but not 10^-9 M) caused differentiation of these cells towards endoderm like cells (Fig. 11). The RAR overexpressing clones however, did not differentiate, but instead after 4-5 days only a few cells were left and the surviving cells were irregularly shaped, not resembling differentiated cells. Treatment of these cells with 1 nM RA did not cause differentiation, morphological alterations or significant cell death.

To investigate whether these cells had lost the ability to differentiate we took advantage of a synthetic ligand AM80, which at low concentrations is selective for RAR (26,69). In P19-EC cells 10^-9 M AM80 (but not RA) can activate RAR at approximately 60% of the maximal response. At this concentration both RA and AM80 are unable to significantly activate RAR. At 10^-7 M both RA and AM80 activate both RAR and RAR maximally (data not shown). After treatment of RAR overexpressing cells with 10^-7 M AM80 extensive cell death occurred, resulting in even lower number of surviving cells, possibly caused by a higher stability of this synthetic ligand. Treatment of these cells with 10^-9 M AM80 which does not activate RAR, no cell death is observed, but instead a morphologically differentiated culture was formed which resembled the mock transfectant or P19-EC cells, although differentiation was not complete at this concentration of AM80 (Fig. 11).

To further confirm that the RAR overexpressing cells are indeed undergoing apoptosis we analyzed the nuclei of P19-EC cells and one of the RAR expressing clones (37). After two days of RA treatment less cells were present in the RAR expressing cells as compared with P19-EC cells. Surviving cells were morphologically different from the P19-EC cells, and examination of the nuclei stained with Hoechst dye confirmed a higher percentage of cells undergoing apoptosis, while surviving cells all had irregularly shaped nuclei and mostly showed typical apoptotic characteristics, including formation of apoptotic bodies and nuclear condensation (Fig. 12A). These experiments clearly demonstrate that the RAR overexpressing cells undergo cell death upon RA treatment, although they have not lost the ability to differentiate, as shown in Fig. 11. Finally in order to be able to directly correlate cell death with RAR overexpression we performed a combined Xgal, Hoechst staining and TUNEL assay. During the first days of differentiation most cells express RAR and during this period no correlation between RAR expression and apoptosis was found. However in almost completely differentiated cultures (3-4 days), where only few, high RAR expressing cells were left, a correlation was observed. Cells expressing RAR are either highly positive in the TUNEL assay, or weakly positive, showing changes in the nuclear morphology (Fig 12B). The big flat endoderm-like cells, were negative for both RAR expression and apoptosis, as determined by Xgal staining and TUNEL assay, respectively. Finally, small particles were found staining positively in the TUNEL assay which are most likely apoptotic bodies after degradation of the cell membrane. These particles were only seen in regions were cell death is taking place and not in regions were differentiation was complete. Based on these data we propose that RA-treated P19-EC cells that did not differentiate, are overexpressing RAR, and subsequently die as the consequence of apoptosis.

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Discussion
 

We show here that RAR but not RAR is involved in the differentiation of P19-EC cells since differentiation characteristics as well as RA target gene expression were restored in RAC65 cells expressing RAR, but not RAR. RAR however inhibits the growth of these cells, and only a subset of target genes were activated by this receptor. We also observed that (over)expression of RAR in the presence of RA caused an increase in apoptosis as shown in RAR overexpressing cell lines and by a correlation during differentiation between RAR expression and the occurance of apoptosis as determined by several criteria, e.g. nuclear morphology, DNA fragmentation and in situ detection of strand breaks.
 

Different roles for RAR and RAR in EC cell differentiation

The observed differences after RA treatment of RAR or RAR overexpressing RAC65 cell leading to differentiation and growth arrest respectively, suggest that RAR and RAR also normally fulfill different functions. This is substantiated by differential activation of target genes, differences in differentiation characteristics in monolayer, aggregates and growth in monolayer and soft agar by comparing RAR versus RAR expressing RAC65 cells (summarized in Table 1). Distinct functions for different RAR subtypes have also been reported in the human embryonal carcinoma cell NTera2/clone D1 (52). Furthermore different functions were observed for the N-terminal parts of RAR1 and RAR1 (as RAR/TR chimera) in regenerating newt limb blastema cells, the first being involved in growth inhibition while the RAR1-TR chimera-induced expression of target genes was found to be associated with differentiation (64,72). This proposed functional difference for individual receptor forms is in agreement with experiments using receptor-specific retinoids, showing that RAR and to a lesser extent RAR, but not RAR or RXR-specific ligands can induce differentiation of P19-EC cells (79). Furthermore experiments in the RAR^-/- and RAR^-/- F9-EC cells indicate that inactivation of RAR has a more profound effect on target gene expression and differentiation than disruption of RAR (5,78). Overexpression of RAR or RAR in these RAR^-/- cells permits differentiation while overexpression of RAR did not restore the ability to differentiate (78). Similarly as we observed in the RAC65 cells overexpressing RAR or RAR, also in this situation the RA-induced expression of some target genes is different between the F9 RAR^-/- clones overexpressing RAR or RAR versus RAR (78). Although these cellular knockout experiments in F9-EC cells are also indicative for the existence of compensatory mechanisms (69,78,79), it is clear that in these cells also receptor-specific functions are observed for individual receptors (5,79).

In RAR expressing RAC65 cells almost all RA target genes tested are regulated as in wild type P19-EC cells, although the levels of expression are lower. This can be explained by the presence of a dominant negative RAR in these cells, competing for RXRs as well as preventing functional RAR/RXR heterodimers from binding to retinoid-response elements. Support for this idea comes from the RAR expressing RAC65 clone 4 which expresses a much higher level of RAR (Fig.1). In this case levels of most target genes are enhanced and some (c-jun, Hox-b1, CRABPII) are present in the absence of added RA. Furthermore these cells show already a more differentiated phenotype in the absence of RA (data not shown). We propose that as a consequence of overexpression of RARs, these cells are no longer capable in regulating the activity of the receptors in a negative fashion. Recently several groups have cloned proteins that negatively regulate the activity of RARs: N-CoR (29) and SMRT (7). Expression of N-CoR was reported to be relatively low in P19-EC cells (29) possibly explaining the RA-independent expression of target genes as well as the differentiated phenotype of the RAR4 clone in the absence of RA. These findings suggest that when receptors are overexpressed in cells that express low levels of negative co-regulators (NCor/SMART) possibly a higher level of active receptors are present in the absence of ligand. In line with this a model has recently been proposed suggesting that ligand-free receptors can be repressed when bound to a co-repressor. When ligand is bound to the receptor co-repressors are removed, permitting basal transcription. Activated transcription is dependent on the binding of positive cofactors to the receptor (8,73). In the RAR expressing cell lines however, despite the high expression, only a subset of target genes were marginally upregulated while others were not affected by RA in these cells. Possibly activation of these genes is receptor form-specific as observed before for some RA-responsive promoters (58).

RA-dependent apoptosis during differentiation of EC-cells.

The induction of apoptosis by RA has been reported before in a number of cell types, including EC-cells (see Introduction). Here we show that the level of apoptosis is increased during the differentiation process and when cells are fully differentiated into endoderm-like cells hardly any apoptotic cells can be found (Fig. 10). While this manuscript was in preparation, Horn et al (30) observed, in agreement with our results, maximal apoptosis after 48 hr RA treatment of P19-EC cells although 72 hr was their latest time-point, at which differentiation is not yet complete. Here we show that overexpression of RAR is the cause for this RA-dependent apoptosis. This is demonstrated by: (1) the increased RA-dependent growth inhibition in RAR (over)-expressing clones (Fig. 2, 10), (2) increased apoptosis in the RAR expressing P19-EC cells (Fig. 12), (3) the correlation between the number of apoptotic cells and RAR expression observed both by Western blot (Fig 6, 9) and by in situ double staining (Fig 12B). The involvement of RAR in growth repression has been reported before in breast tumor cells (43,45,74) as well as in HeLa cells where RAR2-mediated growth repression was observed (63). Interestingly, RAR2 expression is present in normal breast but is lost in tumor cells (77). Lack of RAR expression or its loss during tumor progression has also been observed in other solid tumors (22,31,60,82). An observation further strengthening the role of RAR in the regulation of growth and cell death is the increased expression of RAR2 after serial passages in senescing cells (41,77). Furthermore lung cancer cells that overexpress RAR are less tumorigenic in nude mice (32), whereas mice expressing an antisense RAR2 transgene are more susceptible to lung tumor formation (2) indicating that RAR may function as a tumor suppressor gene (32). Altogether these observations suggest that RAR has an important role in growth regulation, partially by promoting apoptosis. Recently however evidence was presented supporting a role for RXR in RA-dependent apoptosis in F9 cells (11) and in HL-60 cells (48,59). Possibly also here RAR may be required although also cell-specific differences could be the cause for different receptor requirements between the various cell lines. Furthermore, recently evidence was presented for an active role of liganded RXR by the RAR/RXR heterodimer (9,40,69), which may explain why RXR could be directly involved in this process.

In situ hybridization experiments have shown that among the retinoid receptors RAR is most commonly expressed during embryonal development, while expression of RAR is much more restricted (15,16,70,71), possibly underlying the functional difference between RAR and RAR. Programmed cell death (apoptosis) is an important process during embryonal development (33). Based on the expression of RAR during embryonal development, a role for this receptor has been proposed in digit formation by causing cell death in the interdigital regions (15,50). Furthermore maternal RA-treatment caused upregulation of RAR in mouse embryonic facial structures which is accompanied with excessive cell death in this region (55,62). Targeted disruption of RAR and RAR2 however show that this receptor is not essential (46,49). Also mice lacking RAR or RAR have a relatively mild phenotype in comparison with double mutants lacking two receptors; these mice are severely affected at various stages of development, depending on which specific genes are disrupted (36). This type of experimental evidence indicates that receptors are at least partial functionally redundant (36). On the other hand these observations are in contrast with experiments described here and performed in other tissue culture cells where specific functions for individual receptors were observed (5,12,43,48,78,79).

Whether the normal function of RAR is indeed specifically involved in growth control awaits the elucidation of the downstream targets of RAR. Based on the data presented here we propose that the function of RAR is to control the growth rate of the differentiating cells and to remove by apoptosis, cells that did not differentiate. This is performed by an elegant mechanism whereby RAR expression in P19-EC cells is induced immediately after addition of RA (35,37), through the RARE present in the RAR2 promoter (14), after which expression is increased, probably involving induction of RAR expression by an autoregulatory loop. Cells that are differentiated loose the ability to express RAR through loss of expression of specific cofactors and appearance of COUP-TF (35,38,53; Folkers et al submitted). Thus when RA and receptors are present, RAR expression increases until differentiation occurs; those cells that are not differentiated will eventually express high levels of RAR causing apoptosis, through a mechanism to be identified.
 

Acknowledgements

We are grateful to P. Chambon for expression plasmids and antibodies and to K. Shudo for AM80. The authors thank Johan van Burgsteden for performiMw initial experiments, Stieneke van den Brink, Astrid de Greeff and Patricia Swanink for technical assistance. Ferdinand Vervoordeldonk and Jaap Heinen for preparing photography. This work is supported by the Dutch Cancer Society.

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