©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Protein Phosphatases 1 and 2A Regulate the Transcriptional and DNA Binding Activities of Retinoic Acid Receptors (*)

Philippe Lefebvre (1)(§), Marie-Pierre Gaub (2), Ali Tahayato (1), Cécile Rochette-Egly (2), Pierre Formstecher (1)

From the (1) From CJF INSERM 92-03, Laboratoire de Biochimie Structurale Faculté de Médecine de Lille 1, place de Verdun, 59045 Lille Cédex and (2) Institut de Génétique et de Biologie Moléculaire et Cellulaire, INSERM U184, Laboratoire de Genétique Moleculaire des Eukaryotes du CNRS Parc d'Innovation, BP 163, 67404 Illkirch Cedex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To determine which factors may regulate the DNA binding and transcriptional properties of retinoic acid receptors (RARs and RXRs), we investigated the sensitivity of reporter genes bearing various retinoic acid response elements (RAREs) to protein phosphatases (PPases) inhibition. PPases inhibition by okadaic acid led to an increase of the reporter genes activity in a RARE-dependent and ligand-independent manner and was dependent on the type of response element used. Overexpression of protein phosphatases 2A and 1 (PP2A and PP1) decreased the inducibility of the reporter genes tested. Nuclear extracts from okadaic acid-treated COS cells displayed an 2-5-fold increased level of receptor binding to RAREs in vitro, suggesting that PPases inhibition increased the DNA binding activity of retinoid receptors. Treatment of receptors extracted from COS cells by alkaline phosphatase and partially purified PP1 and PP2A decreased their DNA binding activity, but heterodimers bound to DNA were not sensitive to phosphatase treatment. Reconstitution experiments showed that phosphorylation of both receptors increased the DNA binding activity of RXR/RAR heterodimers. Taken together, these data show that the modulation of the phosphorylation state of RARs and RXRs represents an other level of regulation of the retinoid signaling pathway.


INTRODUCTION

All- trans-retinoic acid (ATRA)() receptors , , and (RAR, RAR, and RAR) and 9- cis-retinoic acid receptors (RXR, , and ) are ligand-inducible transcription factors that belong to the nuclear receptor superfamily (1, 2) . The heterodimerization properties of RARs and RXRs, as well as their relative affinity for retinoids, can account for the multiple effects of retinoids observed in vivo (reviewed in (3) ). Binding of RAR-containing heterodimers to cognate DNA binding sequences (retinoic acid-responsive elements or RAREs) is required to observe transcriptional activation of ATRA-controlled promoters. The recognition code is less stringent than for steroid hormone receptors, since natural RAREs have a half-site spacing which ranges from 2 to 5 bases (4) , and RAR/RXR heterodimers can bind to half-sites arranged into palindromes, inverted palindromes (5) , and direct or inverted repeats. In addition, their cis-acting properties vary according to the promoter context (4, 6) and transcriptional activation may require additional ``bridging'' factors like E1A (7, 8) .

An additional level of control of the transcriptional activity of these receptors may also be provided by extracellular signals, as reported for the progesterone receptor (9) and the estrogen receptor (10) . Most of the nuclear receptors have been shown to be phosphoproteins (11) , including RAR, RAR, and RAR (12, 13, 14) . Treatment of COS cells with retinoic acid did not induce detectable changes in the phosphorylation state of overexpressed mRAR, mRAR, and mRAR2. On the contrary, mRAR1 and mRAR3 are strongly phosphorylated upon agonist treatment (12) , like steroid receptors (11) . In addition, we have recently shown that the protein kinase C pathway is involved in the regulation of retinoid-induced transcription (15) , and Huggenvik et al.(16) reported that the cAMP-dependent protein kinase pathway alters the transcriptional response to ATRA. Although these observations suggest that phosphorylation processes could be regulating retinoid receptors functions, it is not yet clear by which mechanism(s) kinases and phosphatases modulate their trans-activating function.

The phosphorylation state of a given protein is the result of both phosphorylation and dephosphorylation reactions. The tumor promoter okadaic acid (OA) is a complex fatty acid polyketal that specifically inhibits protein phosphatases PP1 and PP2A, both in vitro and in cultured cells (17) . PP2A is responsible for a significant protein phosphatase activity in various tissues and has a broad spectrum of substrates in vitro. Significant amounts of PP1 and PP2A are located in the nucleus (18, 19) . PP1 has a 5-fold higher specific activity in the nuclear compartment than in the cytosolic fraction (19) , and is associated with chromatin (see (18) and references therein). Inhibition of PP1 and PP2A by OA has also been shown to cause hormone-independent activation of progesterone (9, 20) and glucocorticoid-regulated reporter genes (21) . However, the strong effect on transcription could not be correlated with detectable alterations of the phosphorylation state of PR (22) and GR (21) , and was, in the latter case, attributed to a post-translational modification of a putative GR-associated factor.

We therefore used OA to oppose the activity of protein kinases constitutively active in COS cells, which were transfected with different RA-responsive reporter genes and expression vectors coding for RAR and RXR. The role of basal phosphorylation was further tested by an in vitro approach in which RAR or RXR were selectively treated with phosphatases and tested for their DNA binding and heterodimerization activities.


MATERIALS AND METHODS

Cell Culture

COS and HeLa cells were grown in Dulbecco's modified Eagle medium (ICN-Flow Laboratories, Scotland) supplemented with penicillin/streptomycin and 10% fetal calf serum. Sf9 cells were propagated in suspension in Grace's insect medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum, 1 mM glutamine, 50 mg/ml gentamicin, and 2.5 mg/ml amphotericin B.

Nuclear Extract Preparation

Nuclear extracts from Sf9, COS, and HeLa cells were performed as described previously (23) with minor modifications. Nonidet P-40 was omitted from the cell lysis buffer and phosphatases inhibitors were added to the extraction buffer (10 mM sodium molybdate, 10 mM sodium pyrophosphate, and 10 mM sodium vanadate). Protein concentration was estimated by the Bradford assay (24) and usually found to be in the 3-5 mg/ml range.

Overexpression of hRAR and hRXR in Bacteria and Sf9 Cells

hRAR and A/B hRXR were expressed in the bacterial strain BL21(DE3) pLysS transformed with plasmid pET3DRAR and pET31-RXR. Bacteria were grown in chloramphenicol and ampicillin-containing LB medium and induced at mid-log phase (OD = 0.6) by addition of isopropylthio--D-galactoside to 0.5 mM final. After 4 h of induction, cells were pelleted and resuspended in 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 10% glycerol, 5 mM dithiothreitrol, 0.4 M NaCl, 10 µg/ml aprotinin and pepstatin A, and 1 mM phenylmethylsulfonyl fluoride (25) . Phosphatases inhibitors were also included: 10 mM sodium molybdate, 10 mM sodium pyrophosphate, and 10 mM sodium vanadate. Lysozyme was added to 150 µg/ml, and cells were finally lysed by addition of deoxycholate to a final concentration of 0.05%. Cells debris were pelleted at 60,000 g, 4 °C for 2 h. Supernatant usually contained 2-5 mg/ml protein.

Sf9 cells were infected with a recombinant baculovirus encoding for the hRAR. Nuclear extracts from infected cells were prepared as described above. Purified A/B RXR and A/B RAR were a kind gift from H. Gronemeyer.

Phosphatase Treatment

Extracts were treated with either agarose-immobilized calf intestine alkaline phosphatase (CIP) or soluble CIP (Sigma). When agarose immobilized-CIP was used (Figs. 5 and 6), 75 µg of COS cell extracts were incubated for 15 min at 37 °C with 22 units of enzyme in 150 µl of EMSA buffer. Beads were spun down and supernatants brought to 20 mM di-sodium hydrogen phosphate (NaHPO) and 5 mM NaVO. When soluble CIP was used (Figs. 4 and 7), 25 µl of extracts (75 µl) were diluted 4-fold in EMSA buffer and incubated 20 min at 30 °C with 50 units of the enzyme. The reaction was stopped by transferring samples at 4 °C and bringing the mix to 20 mM NaMoO, 20 mM NaVO, and 20 mM NaPO. These conditions were found to reduce CIP activity by more than 95% as assayed by p-nitrophenylphosphate hydrolysis.

EMSA

Oligonucleotides containing the various response elements (see below) were end-filled with the Klenow fragment of DNA polymerase. Typically, 40 µg of protein was incubated with 20 fmol of the labeled probe, in the presence of 2.5 µg of salmon sperm DNA and a binding buffer giving a final concentration of 20 mM HEPES, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1 mM dithiothreitrol, and 10% glycerol. When purified Escherichia coli RAR or RXR were used, 0.05-0.1 µg of purified receptor was added to 4 µg of CIP-treated or control extracts and incubated 15 min on ice prior to the binding reaction. Control experiments were performed with heat-inactivated phosphatase (5 min at 100 °C) according to the same protocol. This treatment allowed for a complete inactivation of CIP as judged by its lack of activity on 5`-labeled probes (data not shown). DNA binding reactions were for 30 min on ice or 15 min at 20 °C, in a final volume of 20 or 40 µl. When required, 1 µl of mouse monoclonal ascites fluid Ab9()F directed against RAR (13) or 4RX-1D12 directed against each type of RXR, was added for a further 15-min incubation as described previously (13) . ProteinDNA complexes were then resolved on a 5% nondenaturing polyacrylamide gel run at 150 V for 3 h at 4 °C. The running buffer was 0.5 TAE (1 TAE is 40 mM Tris acetate, pH 7.5, 2 mM EDTA) (Figs. 3, 4, 7, and 8). Alternatively, gels were run in 0.5 TBE at room temperature (45 mM Tris base, 45 mM boric acid, ans 2 mM EDTA) (Figs. 5 and 6).

Transient Transfections, -Galactosidase, and CAT Assays

COS cells were transfected by the calcium phosphate precipitation method as follows: 10 cells were plated per 35-mm dish. The following day, cells were fed with 1 ml of fresh medium, and calcium-DNA coprecipitate was added 4 h later. The mixture contained 1 µg pSV-gal plasmid (pCH110, Pharmacia), 1 µg of the reporter gene, 1 µg of pSG5-hRAR (26) and pSG5-mRXR expression vector (6) . The DNA concentration was adjusted to 10 µg final with carrier DNA. Incubation with the coprecipitate was for 16 h, after which cells were glycerol-shocked. Cells were incubated for 10 h in complete medium and then submitted to various treatments as indicated in the text. -Galactosidase and CAT assays were performed as previously reported (15, 27) .

Western Blotting Procedure and Antibodies

Immunoblotting of RAR was performed using the IBI Enzygraphic Web system (15) or I-protein A (13) . The anti-hRAR polyclonal antibody IS39 was raised against synthetic peptides from the F domain of hRAR. AntiRAR monoclonal antibody Ab9(F) and antiRXR monoclonal antibodies 4RX-1D12 and 1RX-6G12 are described elsewhere (13, 28) .

Protein Phosphatase Purification

PP1 and PP2A were partially purified from rat livers. Purification steps were performed as described by Silberman et al.(29) . Fractions eluting from a DEAE-Sepharose column (6 10 cm) between 0.2 and 0.35 M NaCl were pooled, concentrated on a Centricon 10 filter, and dialyzed against buffer PPB (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 20% glycerol, 5 mM DTT, and 100 mM NaCl). The catalytic activity of the preparation was estimated by its alkaline phosphatase activity with p-nitrophenyl phosphate as a substrate. The specific activity of the preparation used in the presented experiments was 85 units/mg protein, and protein concentration was around 12 mg/ml. Inhibition by okadaic acid was used to identify the phosphatase activity as PP1 and PP2A.

Oligonucleotides and Plasmids

The following oligonucleotides and their complements, flanked by BamHI sites, were synthesized: (i) a DR5 retinoic acid response element from the promoter P2 of the RAR- gene (30) , (ii) a TREpal thyroid response element (31) , (iii) a RXRE from the rat CRBPII gene promoter (32) , and (iv) a DR2 from the mouse CRABPII gene promoter (4) . Sequences of these oligonucleotides are as follows: (i) -RARE (DR5), gatcGGGTAGGGTTCACCGAAAGTTCACTCG; (ii) TREpal, gatcTTCAGGTCATGACCTGAA; (iii) rCRBPII-RXRE, gatcTGAACTGTGACCTGTGACCTCTGACCTGTGACAGCA; and (iv) mCRBPII-DR2, gatcGTACAGGTCATCAGGTCAAG. These response elements were cloned into pBLCAT2 (33) , and this plasmid is referred to as RAREtk-CAT in the text. p(RARE)tk CAT was obtained by inserting two copies of the DR5 oligonucleotide into the BamHI site of RAREtk-CAT. A similar procedure was used to clone the TREpal and DR2 sequences as tandem copies into the same reporter gene, whereas the rCRBPII-RXRE was cloned as a single element between the BamHI and HindIII sites. The SV40-based expression vectors pSG5-RAR and pSG5-RXR are described elsewhere (6, 26, 34) . pCMV5-PP2A was kindly provided by Dr. M. Mumby (University of Texas, Southwestern Medical Center, Dallas, TX). pCMV5-PP1 was created by inserting the PP1 cDNA as an EcoRI- BamHI fragment in pCMV5. The PP1 cDNA was amplified from pRSET-PP1 (35) to generate a 1000-base pair DNA using the following primers: 5`-CCGGAATTCGCCACCATGTCCGACAGC-3` (upstream primer) and 5`-CGCGGATCCCTATTTCTTGGCTTTGGC-3` (downstream primer).


RESULTS

Inhibition of PP1 and PP2A Alters Retinoic Acid-induced Transcription

The effect of OA, an inhibitor of phosphatases PP1 and PP2A (18) on retinoid-induced transcription, was investigated in a cotransfection assay (Fig. 1). Various RA-inducible reporter genes were introduced in COS cells, in the presence or absence of expression vectors coding for RAR and RXR. We have used deliberately high amounts of DNA (1 µg) in these experiments. Although this does not allow for a clear detection of a synergy between RAR and RXR, it allowed the detection of reporter gene activity in the presence of only one overexpressed receptor or both, thereby reflecting preferentially the transcriptional activity of homodimers or heterodimers. At lower DNA concentrations (25 ng), a synergy was observed.() In preliminary experiments, we noted that the OA effect increased with concentration up to 100 nM and became cytotoxic above 150 nM. We therefore used OA at a concentration of 100 nM to selectively block PP1 and PP2A activities in transfected cells. Moreover, this concentration did not affect the activity of the parental vector pBLCAT2, designed RAREtkCAT thereafter (Fig. 1). Cells, with or without OA treatment, were treated with 50 nM ATRA or 50 nM 9- cis-RA. 50 nM ATRA exclusively activates RARs while 9- cis-RA activates both RARs and RXRs, due to its high affinity for both receptors (36) .


Figure 1: Effect of okadaic acid on RAR and RXR-mediated transcription. COS cells were cotransfected with 0.5 µg of the indicated reporter gene and with or without 1 µg of pSG5hRAR and pSG5RXR. Cells were treated with 100 nM OA for 16 h 24 h after transfection, either with vehicle (MeSO, empty bars), 50 nM ATRA ( dotted bars), or 50 nM 9- cis-RA. CAT activity was assayed and normalized to -galactosidase activity as described under ``Materials and Methods.'' CAT activity is expressed as a percentage of the ATRA-induced transcription level for cells in the absence of overexpressed receptors, except for the TREpal-CAT construct. In this latter case, 100% CAT activity is the level of enzymatic activity detected in cells transfected with pSG5-hRAR alone and treated with 50 nM ATRA. Values represent the mean of at least three independent experiments performed with triplicate assays, and standard deviations did not exceed 15% of the mean values. Values are indicated in CAT activity normalized to -galactosidase activity .



We first examined the effect of inhibition of protein phosphatases on the RAREtkCAT (DR5) reporter gene activity. This reporter gene is bearing a DR5 response element that can be activated by the low level of endogenous RARs and RXRs present in COS cells (Fig. 1 A). Overexpression of RAR did not significantly increase the promoter activity in response to ATRA or 9- cis-RA, whereas overexpression of RXR increased the -RARE CAT activity by 3-4-fold. Coexpression of both receptors did not further increase the level of activation of the promoter in response to 9- cis-RA treatment, although we noted that ATRA yielded a lower CAT activity than in the presence of RXR alone. This could suggest that RAR has a moderate inhibitory effect under these conditions, a result comparable to the one obtained with the rCRBPII-RXRE construct (Fig. 1 C). In no case did addition of 100 nM OA significantly modify the detected CAT activity either in the presence or the absence of ligand.

A similar analysis was performed with the TREpal-CAT construct, which is not inducible in the absence of overexpressed RAR or RXRs (Fig. 1 B). Overexpression of RAR, RXR, or both receptors conferred a significant inducibility by retinoic acid on this promoter, indicating that the observed activation is due solely to transfected receptors. This result is in agreement with previously reported results and reflects the low concentrations of RAR and RXR in nontransfected COS cells (37) . 9- cis-RA was a better activator than ATRA, showing that RXR activation increases the promoter activity. In the absence of transfected receptors, OA displayed virtually no effect on the TREpal-CAT activity. On the contrary, OA caused a clear increase in TREpal-CAT expression in response to ATRA (2-fold) and 9- cis-RA (1.3-fold) in the presence of RAR. More strikingly, overexpression of RXR alone caused a ligand-independent activation by OA of the reporter gene to a level similar to that achieved in the presence of 50 nM ATRA or 9- cis-RA alone. The ligand-induced transcription, in the presence of OA, was boosted to a similar extent (3-4-fold). Coexpression of RAR and RXR increased the inducibility of the TREpal-CAT promoter, when compared to the level reached upon overexpression of a single receptor, as previously reported (38) . Phosphatases inhibition caused a further increase in CAT activity similar to that observed with RXR alone.

The rCRBPII-RXRE-CAT construct, containing five repeats of the sequence AGGTCA spaced by one nucleotide, is poorly activated in the absence of transfected receptors or in the presence of RAR alone. OA was nevertheless able to increase the CRBPII promoter activity in the absence of receptors or in the presence of RAR, and it increased the basal activity to a level similar to the one observed with ATRA or 9- cis-RA. This effect was noticeable, although the CAT activity was much lower than that seen in the presence of RXR. Indeed, this reporter gene became highly inducible in the presence of transfected RXR, to reach a 25-30-fold higher activity in the presence of both ATRA and 9- cis-RA (Fig. 1 C). Initially described as a RXRE, the rCRBPII-RARE is, in our experimental conditions, activated by a RAR-specific ligand, indicating that RAR is a component of the activation complex. The activation of a CRBPII-driven promoter by ATRA was also reported, although this could be due, in the reported conditions, to a metabolic conversion of ATRA to 9- cis-RA (32) . Thus our results suggest that this particular response element can be activated by hRAR, in opposition to a ``true'' DR1 (39) . Coexpression of RAR and RXR lowered the rCRBPII-CAT promoter activity, in agreement with the proposed inhibitory role of RXR transactivation by RAR (32) . Phosphatases inhibition by OA increased CRBPII-CAT activity in the presence of overexpressed RXR, albeit to a lower extent, and counteracted the inhibitory activity of RAR in the presence of RXR.

DR2 response elements have been shown, by random selection of binding sites for RAR/RXR heterodimers, to bind heterodimers with a lower affinity than a DR5 (40) . However, the mCRABPII DR2 conferred a significant inducibility to the thymidine kinase promoter in response to ATRA and 9- cis-RA in the absence of transfected receptors (Fig. 1 D). 9- cis-RA was in all cases a better inducer than ATRA in the absence of OA, and RAR overexpression yielded a higher level of CAT activity than RXR overexpression. Coexpression of RAR and RXR did not significantly increase the CRABPII-CAT promoter activity. In that promoter context, OA increased the basal level of CAT activity, which reached values similar to that obtained in the presence of ligand alone when RXR was overexpressed. At the specified concentrations, ligand and OA effects on the promoter activity were cumulative. This result is analogous to that of TREpal-CAT (see Fig. 1 B).

This set of experiments demonstrates several interesting features of retinoid-induced transcription in response to OA treatment in COS cells: (i) the observed effects are specific for RARE-containing promoters since the OA effect was not detected when the parental reporter gene RAREtkCAT was used or when the TREpal-CAT plasmid was used in the absence of overexpressed receptors, indicating that the thymidine kinase promoter activity is not significantly altered upon phosphatases inhibition. Furthermore, we() and others (9, 20, 21, 22) did not detect any effect of OA on Rous sarcoma virus or SV40 promoter-controlled genes. (ii) OA did not increase the activity of the -RARE CAT construct, whatever combination of receptors and ligands was used. (iii) The TREpal and DR2-driven promoters, which can be considered to be equally activated by agonists in the presence of overexpressed RAR or RXR, and which have a lower affinity for RAR/RXR heterodimers in vitro than the -RARE, are strongly activated by OA. Remarkably, phosphatases inhibition was able to bring transcription, in the absence of ligand, to a level similar to that induced by retinoids in the presence of overexpressed RXR. (iv) The CRBPII-RXRE-CAT construct is highly inducible upon expression of RXR. The OA effect was less marked in the presence of overexpressed RXR than in the presence of overexpressed RAR, and the inhibitory effect of RAR on RXR-mediated transcription could be relieved by OA treatment of transfected cells. These results suggest that phosphatases inhibition is able to activate low affinity RARE-driven promoters to a maximum activity in a ligand-independent manner.

Overexpression of PP2A and PP1 Inhibits Retinoic Acid-induced Transcription

Although okadaic acid is a valuable tool to study the role of PP1 and PP2A in various cellular processes, its use has some potential drawbacks such as its cellular toxicity (41) . Thus we analyzed the ability of each enzyme to modulate retinoic acid-induced transcription from each type of reporter gene (Fig. 2). Exponentially growing COS cells were transfected with a RARE-containing reporter plasmid or the parental reporter gene RARE tkCAT, expression vectors coding for both RAR and RXR and increasing amounts of PP2A or PP1 expression vectors. As it could be expected, PP2A and PP1 overexpression markedly and specifically reduced the inducibility by ATRA of RARE-driven reporter genes in a dose-dependent manner. PP2A overexpression inhibited the ATRA-induced CAT activity of all reporter genes, although the RXRE-CAT construct appeared consistently less sensitive. The basal level of CAT activity for the DR2 and RXRE-driven reporter genes was also lowered upon PP2A overexpression, although low levels of enzymatic activity made quantitation of the results difficult for the -RARE and TREpal constructs. PP1 also inhibited the activity of all reporter genes except that of the RARE-tk CAT reporter gene. Furthermore, RARE-driven reporter genes displayed a differential sensitivity to each catalytic subunit of these enzymes (compare left and right columns, for the 5 µg of plasmid concentration). Thus the retinoic acid inducibility of each reporter gene was differentially affected by the type of protein phosphatase used in this assay.


Figure 2: PP2A and PP1 overexpression decrease the transcriptional activity of RARE-driven promoters. COS cells were transfected with 0.5 µg of the indicated reporter gene and 1 µg of each expression vector coding for RAR and RXR, with increasing amounts of CMV-PP2A ( left column) or CMV-PP1 ( right column) plasmids. Cells were then treated with vehicle ( open circles) or with 1 µM ATRA ( filled circles). Levels of CAT activity as a percentage of the CAT activity detected in COS cells transfected without PP1 or PP2A expression vectors and treated with 1 µM ATRA. Graphic data are averaged from four independent experiments.



OA Treatment Increases the DNA Binding Affinity of RAR/RXR Heterodimers

Nuclear extracts from COS cells, treated or untreated with OA and transfected with both RAR and RXR expression vectors, were used to perform in vitro RARE-binding assays (Fig. 3). As shown by Western blot analysis, OA treatment did not modify the receptor content of the extracts (Fig. 3 B). However, specific binding to each response elemen t tested was found to be increased upon OA treatment of COS cells. This increase in DNA binding activity was especially apparent for the TREpal and DR2 probes (3-4-fold increase), but less obvious for the -RARE (DR5) and RXRE probes (1.5-2-fold). The latter probe yielded two specific complexes. The upper band migrated with a mobility similar to RXR homodimers, whereas the fastest species migrated as RAR/RXR heterodimers. Since RAR and RXR have been shown to be the only RARE-binding proteins in COS cells extracts (34) , we conclude that phosphatases inhibition led to an increased DNA binding activity of RAR and RXR in vitro. This is in agreement with transient transfection results which showed a clear increase of the DR2-CAT and TREpal-CAT expression in the presence of 100 nM OA (see Fig. 1). This observation prompted us to further investigate the role of basal phosphorylation in the in vitro DNA-binding properties of RAR/RXR heterodimers.


Figure 3: OA treatment increases the affinity of retinoic acid receptors for various retinoic acid response elements. A, nuclear extracts were prepared from untreated or OA-treated COS cells transfected with RAR and RXR expression vectors. Their ability to form specific complexes on a -RARE, TRE-pal, RXRE, or DR2 response element was tested by electrophoretic mobility shift assay. Nuclear extracts (20 µg of protein) from untreated cells were used for competition experiments in which a 50-fold excess of the same oligonucleotide ( Spec. 1, lanes 2), of the -RARE probe ( Spec. 2, lanes 3) or the DR2 probe when the -RARE was the labeled probe, and of a palindromic glucocorticoid response element ( Non Spec., lanes 4) were used. Increasing amounts of nuclear extracts from untreated cells ( lanes 5-8) or OA-treated cells ( lanes 9-12) were incubated with the indicated probe and resolved on a 5% nondenaturing acrylamide gel. B, Western blot analysis of RAR and RXR in control and OA-treated COS cell nuclear extracts. 100 µg of protein was resolved on a 12% SDS-PAGE and immunodetected with the anti-RAR polyclonal antibody IS39 or the anti-RXR monoclonal antibody 1RX-6G12.



In Vitro Phosphatase Treatment of Nuclear Extracts Prevents Specific Binding to a Retinoic Acid Response Element

Crude extracts containing overexpressed hRAR in E. coli, Sf9 cells or RAR and RXR in COS cells were submitted to a DNA-binding assay before and after treatment with calf intestine alkaline phosphatase ( CIP, Fig. 4). Since alkaline phosphatase has a broad substrate specificity, its use can be compared favorably with that of PP1 and PP2A. No -RARE-specific binding activity was detected in mock transformed bacteria, infected Sf9 cells, or in nuclear extracts from native COS cells (Fig. 4, upper panel, lanes 2, 8, and 14MDRV). Upon introduction of a vector coding for hRAR in E. coli and Sf9 cells or vectors coding for RAR and RXR in COS cells, proteinDNA complexes were formed specifically on the -RARE oligonucleotide ( lanes 3, 9, and 15). The specific DNA binding activity detected in bacterial extracts, resulting from RAR homodimer formation onto the -RARE ( lane 3) was not sensitive to phosphatase treatment ( lane 6). Western blot analysis ( lower panel) of native or phosphatase-treated E. coli extracts ( lanes 3 and 6, respectively) identified a single immunoreactive species revealed by a polyclonal anti-hRAR antibody, with a molecular mass of 52 kDa. No difference in SDS-PAGE electrophoretic mobility was detected after CIP treatment, indicating that the E. coli-expressed hRAR is not phosphorylated. ()


Figure 4: Phosphatase treatment of crude nuclear extracts inactivates DNA binding activity of hRAR synthesized in eukaryotic cells. E. coli, Sf9 cells, or COS cells nuclear extracts were tested for their DNA binding activity by EMSA. Samples from nuclear extracts containing 20 µg of protein were incubated with 20 fmol of labeled -RARE oligonucleotide ( lanes C), with a 50-fold excess of cold -RARE ( lanes S), or with a 100-fold excess of a nonspecific oligonucleotide ( lanes NS). The same amount of extract was either treated with 50 units of native calf intestine alkaline phosphatase ( CIP) or with CIP in the presence of inhibitors ( CIP, i). F (free) lane, DNA alone; M (mock) lanes: DNA probe incubated with nontransformed ( E. coli), noninfected Sf9 cells, or nontransfected COS cells extracts. Lower panel, mock, control or phosphatase-treated extracts were analyzed in parallel for their content in hRAR. Forty µg of nuclear extract from E. coli, Sf9 cells, or COS cells nuclear extracts was resolved on a 8% SDS-PAGE and blotted onto a nitrocellulose membrane. Immunodetection was performed using the antiRAR polyclonal antibody IS39. Molecular masses (in kDa) are indicated on the right.



In contrast, the DNA binding activity of hRAR synthesized in eukaryotic cells (Sf9 and COS) appeared to be sensitive to phosphatase treatment (compare lane 9 to lane 12 and lane 15 to lane 18). A 75-90% decrease in DNA binding activity was consistently observed which was concomitant with an increase of the electrophoretic mobility of RAR in SDS-PAGE of the phosphatase-treated sample ( lower panel). This increased mobility is indicative of the removal of several phosphate groups from the RAR molecule. We showed previously that treatment of P-labeled RAR in COS cells led to a significant, but not complete, loss of phosphate groups (13) . Similar results were obtained with RXRs. The effect of phosphatase treatment on the DNA binding activity was also observed using potato acid phosphatase and agarose-immobilized alkaline phosphatase, and for nuclear extracts from HEL, HL-60, and HeLa cells.() The DNA binding activity was not affected when the phosphatase was inhibited by 10 mM inorganic phosphate, 10 mM sodium molybdate and vanadate (Fig. 4, lanes 13 and 19). Thus hRAR is a phosphoprotein when synthesized in eukaryotic cells, and phosphatase treatment of extracts strongly decreased its DNA binding affinity under these conditions. The concomitant dephosphorylation of hRAR implies that phosphorylation of the receptor is required for DNA binding. Alternatively, this could mean that an inhibitory activity was unmasked after CIP treatment. This hypothesis can be ruled out, however, since when E. coli RAR and RXR are combined to native or CIP-treated COS cells extract, they bind to DNA with a similar efficiency. The apparent molecular masses of native RAR synthesized in Sf9 and COS cells were identical. In each case, RAR migrated as a doublet and was detected as 54- and 58-kDa polypeptides, and both forms appeared to be sensitive to CIP treatment. As shown by Western blot analysis of the extracts, overexpression of RAR in each system yielded similar amounts of RAR polypeptide. Receptors extracted from these cells bound ATRA with a similar dissociation constant (3 nM) and yielded an equivalent amount of ATRA-binding sites (5-10 pm receptor/mg protein, data not shown). This indicates that whatever the system used, receptors have similar properties and stability. We cannot, however, be sure in this system that hRAR is the only substrate for the alkaline phosphatase, which could also dephosphorylate other proteins necessary for the DNA binding activity of the receptor.

Dephosphorylation of Both RAR and RXR Reduces the Affinity of RAR/RXR Heterodimers for DNA

To further determine the importance of the phosphorylation state of each partner in the heterodimerization process, RAR and RXR were expressed either in E. coli, in a nonphosphorylated form, or in COS cells in which polypeptides are fully processed. Nuclear extracts from COS cells containing RAR (Fig. 5 A) or RXR (Fig. 5 B) were treated with CIP for 1 or 15 min, mixed with purified A/B RXR or A/B RAR, respectively, and analyzed by EMSA. Heterodimeric complexes formed on the -RARE probe were identified by supershifts using monoclonal anti-RAR antibody ( 9, lanes 2, 6, 10, and 14), an antiRXR antibody ( 4RX, lanes 3, 7, 11, and 15), or both ( 9+4RX, lanes 4, 8, 12, and 16). Complexes were totally supershifted in the presence of both antibodies ( lanes 4, 8, 12, and 18 in both panels), showing that these complexes are made of RAR and RXR. CIP treatment of COS cell extract for 15 min caused a strong decrease in the amount of RARRXRDNA complexes, regardless of which receptor was present in the CIP-treated extract (compare lanes 4 to lanes 8 in panels A and B). Interestingly, we also noted that heat inactivation of CIP was not sufficient to prevent a partial loss of the DNA binding activity (compare lanes 12 to lanes 16 in both panels), in opposition to phosphatases inhibitors (sodium phosphate, sodium molybdate, and vanadate, see Fig. 4). This is suggestive of the presence of an endogenous phosphatases activity in COS extracts that remains to be identified. Thus, phosphorylation of both RAR and RXR appeared to be important for the DNA binding activity of RAR/RXR heterodimers. However, this post-translational modification is not an absolute requisite for dimerization since RAR and RXR produced in E. coli can generate similar complexes (see below), suggesting that phosphorylation may increase the affinity of one receptor for its dimerization partner.


Figure 5: Alkaline phosphatase treatment impaired the DNA binding activities of both RAR and RXR overexpressed in COS cells. Whole cell extracts from COS cells transfected either with RAR ( panel A) or RXR expression vectors ( panel B) were treated with active ( panels A and B, lanes 1-8) or heat-inactivated CIP ( lanes 9-16) for 1 or 15 min at 37 °C. Treated COS extracts containing RAR ( panel A) and RXR ( panel B) were then mixed with purified A/B RXR or A/B RAR, respectively, and resolved by EMSA. The protein composition of the retarded complexes ( arrow 1) was determined by supershift experiments using monoclonal antibodies directed against RAR ( 9) or RXR ( 4RX). Supershifted complexes are indicated by arrow 2. The empty arrowhead indicates nonspecific complexes.



Bacterially Expressed RAR and RXR Are Less Efficient at Forming Heterodimers than RAR and RXR Extracted from Eukaryotic Cells

Receptors synthesized in eukaryotic cells and in bacteria were used in EMSA experiments to further answer the question whether post-translational modifications alter the ability of RAR/RXR heterodimers to form on a RARE. To test this hypothesis, we performed experiments comparing the ability of RAR and RXR, expressed either in bacteria or in COS cells, to generate heterodimers on the -RARE. The amount of RAR or RXR extracted from E. coli or COS cells extracts necessary to generate an equivalent amount of receptorDNA complex in the presence of its nonphosphorylated dimerization partner was titrated by EMSA. As shown in Fig. 6, none of the individual components of the binding reaction bound to DNA by itself, when used at the indicated concentrations ( lanes 13- 21). When COS or E. coli RAR1 was added to a constant amount of non phosphorylated ( i.e. from E. coli extracts) A/B RXR (Fig. 6 A), an identical level of heterodimer formation could be reached with both type of extracts (compare lanes 1- 6 and lanes 7-12). However, quantification of RAR by Western blotting revealed that a much higher amount (8-10-fold) of the RAR polypeptide was present in the bacterial extract (Fig. 6 B, compare lane 1 to lane 7), indicating that a higher concentration of nonphosphorylated RAR is necessary to yield an identical level of heterodimer binding to DNA. Quantification of the retarded bands formed for each condition (Fig. 6 C) demonstrated that E. coli or COS-expressed RAR yielded an equal amount of receptorDNA complexes, suggesting that the binding of RAR/RXR heterodimers to DNA is not affected by post-translational modifications once the heterodimers are formed.


Figure 6: RAR synthesized in bacteria forms RAR/RXR heterodimers less efficiently than RAR extracted from COS cells. A, increasing amounts of whole cell extracts from COS cells ( lanes 6 to 1) or from E. coli ( lanes 12 to 7) overexpressing RAR were added to a constant amount of A/B RXR purified from E. coli. ProteinDNA complexes formed on the -RARE probe were analyzed by EMSA. The ability of each component to bind to this probe was also assessed in a similar manner ( COS RAR, lanes 15 to 13; E. coliRAR, lanes 18 to 16; purified RXR, lanes 21 to 19). Protein concentration is given in µg/20 µl of EMSA mix. Arrow 1 indicates RAR/RXR heterodimers, whereas arrow 2 points to RAR homodimers ( lane 16). The empty arrowhead shows nonspecific complexes. B, quantification of RAR in COS and E. coli extracts by Western blot. Extracts used to run the gel retardation assay were analyzed for their content in RAR as described under ``Materials and Methods.'' The membrane was probed with the polyclonal antibody Rp(F). C, quantification of the retarded bands shown in panel A from lanes 1 to 12. The autoradiogram was scanned using a PhosphorImager and data plotted as a graph showing the variation of the amount of the retarded complexes versus the protein concentration of RAR-containing extracts.



An analogous titration experiment was done to compare the capacity of nonpurified E. coli and COS RXR to interact with RAR present in crude E. coli extracts (Fig. 7). In this experiment, we first estimated the concentration of RXR present in both type of extracts by Western blot analysis. RXR concentration in E. coli extracts was 10-fold higher in this typical experiment, since 40 µg of protein from COS cell extracts had to be loaded to yield a signal equivalent to that observed with 4 µg of E. coli extracts, as shown by Western blot analysis (Fig. 7 B). However, both types of RXR were able to form heterodimers on the -RARE oligonucleotide, as shown by supershift experiments (Fig. 7, lanes 9 and 11, arrow 2). COS RXR was able to generate a higher amount of heterodimeric complexes than E. coli RXR despite its lower concentration in COS cell extracts (Fig. 7 A, compare lanes 1-4 to lanes 5-8). Quantification of the results showed that COS RXR formed heterodimeric complexes with a 10-15-fold higher efficiency than E. coli RXR. Thus it appeared from the experiments presented in Figs. 6 and 7 that nonphosphorylated RAR and RXR bound to DNA, in the presence of their dimerization partner, with a lower efficiency (at least 10-fold) when compared to the fully processed polypeptide. This lower efficiency could be overcome by using a higher amount of the nonphosphorylated receptor form, which have, by all other criteria, the same functionality. Indeed, receptors were expressed at the same rate in E. coli and COS cells (see Fig. 4) and bound ATRA() and DNA (Fig. 6 C) with similar affinities. Moreover, RAR homodimer formation on the same RARE was not compromised by the lack of post-translational modifications (see Fig. 4), suggesting that its affinity for DNA in the presence of its dimerization partner is decreased with respect to that of the fully processed polypeptide in COS cells.


Figure 7: RXR synthesized in bacteria forms RAR/RXR heterodimers less efficiently than RXR extracted from COS cells. A, increasing amounts (indicated in µg of proteins) of A/B RXR extracted from bacteria ( E. coliRXR) or full-length RXR extracted from COS cells was incubated in the presence of a constant amount of hRAR and proteinDNA complexes were analyzed by EMSA. Supershift experiments were performed using an antiRXR monoclonal antibody in the presence ( lane 9) or the absence ( lane 10) of nuclear extracts. Similarly, a polyclonal antiRAR antibody was used in the presence ( lane 11) or the absence of nuclear extract ( lane 12). In these experiments, the amount of nuclear extract was similar to that used in lane 4. Arrows 1 indicate the retarded complexes, whereas arrows 2 indicate complexes supershifted by either a polyclonal antiRAR antibody ( lane 11) or a monoclonal antiRXR antibody ( lane 9). B, assay of the RXR content of COS and E. coli extracts. 2.5, 5, 10, or 40 µg of COS extracts or 0.25, 0.5, 1, and 4 µg of bacterial extracts were analyzed by Western blotting. The full-length mRXR migrated as a 54 kDa species, whereas the A/BRXR migrated as a 42-kDa polypeptide. The nitrocellulose membrane was probed with the monoclonal antiRXR antibody 1RX-6G12.



PP1 Efficiently Inhibits RAR/RXR Binding to -RARE in Vitro

Transient transfection experiments showed that protein phosphatases exert a noteworthy influence on the inducibility of RARE-driven reporter genes. In vitro DNA binding experiments showed that both RAR and RXR are the target for phosphatase action. To address the question as to whether PP1 or PP2A are equally active in abolishing the DNA binding activity of RAR/RXR heterodimers, we partially purified PP1 and PP2A and used them as the source of dephosphorylating enzymes. The PP1/PP2A mix turned out to be as efficient as alkaline phosphatase (and potato acid phosphatase) to abolish the formation of RARE-receptor complexes (Fig. 8, panel A). When OA was added at various concentrations to selectively block PP2A and PP1, we noted that the DNA binding activity was preserved for OA concentrations of 10-50 nM. Since PP2A and PP1 are inhibited by I of 0.2 and 20 nM, respectively (18) , we infer that PP1 is, under these specific conditions, the most likely candidate as a receptor-dephosphorylating enzyme.


Figure 8: Okadaic acid prevents the loss of DNA binding activity of RAR/RXR complexes treated with purified PP1 and PP2A in vitro. A, nuclear extracts from HeLa cells were treated with partially purified PP2A and PP1 for 1 h at 37 °C in the presence of the indicated concentration of OA. Samples were then analyzed by EMSA for their ability to bind to the -RARE probe. B, decay of the RARRXR complexes in the presence of PP1 and PP2A. HeLa nuclear extracts were submitted to DNA-binding conditions in the presence of labeled -RARE, then treated for 1 h in the presence or absence of PP1 and PP2A at 37 °C, as described above. Samples were transferred at 4 °C, and a 200-fold excess of cold probe was added. Samples were loaded on a 5% nondenaturing gel at the indicated times. Results were quantified by excision of the radioactive bands and scintillation counting. Results are expressed as a percentage of total probe input.



The loss of binding to DNA upon phosphatase treatment can be potentially explained by two modes of action for these enzymes: (i) they increase the dissociation rate of the ternary complex RARRXRRARE, or (ii) phosphatase treatment lowers the on-rate of the association of heterodimers with RAREs. To test the first hypothesis, we preassembled complexes on the -RARE, treated them with or without with PP1 and PP2A, and followed the dissociation of RARRXR complexes by addition of an excess of the same radioinert probe (Fig. 8 B). The off-rate was similar, indicating that while RAR and RXR in solution are highly sensitive to phosphatases action assembled heterodimers do not display such a sensitivity.


DISCUSSION

Molecular mechanisms controlling cellular fate determination and proliferation are subject to various levels of regulation. These processes are either triggered by molecules binding to membrane receptors or by liposolubles hormones (vitamin A and derivatives, vitamin D, and steroid hormones) that bind to intracellular receptors. Both types of signals affect, directly or indirectly, the expression rate of key regulatory genes. An understanding of interactions occurring between these two signaling pathways is therefore required to decipher cellular responses at the nuclear level in response to mitogenic or differentiating signals.

We present here evidence that alteration of the intracellular equilibrium between phosphorylation and dephosphorylation processes leads to the modulation of the activity of different RA-responsive reporter genes in COS cells. The effects of OA (Fig. 1) and that of PP1 and PP2A (Fig. 2) were variable depending on the response element used. In contrast to the high affinity -RARE (DR5) sequence, the TREpal and mCRABP2-CAT (DR2) constructs were activated by OA in a ligand-independent, RXR-dependent manner to a level equal to that reached in the presence of ATRA or 9- cis-RA. This activation was also detected for the rCRBP2 RXRE-CAT construct in the presence of overexpressed RAR, and OA relieved the inhibitory effect of RAR upon RXR-mediated transcription. PP2A and PP1 therefore play a role in the modulation of the activity of RARE-driven promoters, and this control appeared to be dependent upon the type of RARE and the ratio between the intracellular concentration of RAR versus that of RXR. Conversely, overexpression of the PP2A and PP1 catalytic subunit significantly lowered the inducibility of all the reporter genes tested. The sensitivity varied for each phosphatase in the order TREpal > -RARE > DR2 > RXRE (PP2A), DR2 = RXRE > TREpal -RARE (PP1). The lack of sensitivity of the -RARE construct to PP1 overexpression was unexpected considering the effect of this enzyme on the in vitro DNA binding activity of receptors, but it has been reported that overexpression of these enzymes yield mostly insoluble proteins (42) . It is therefore likely that the phosphatase to receptor ratio was not identical in these two experiments. Thus, the cis-acting properties of a RARE could be affected by physiological conditions that alter endogenous PP1 and PP2A expression. In that respect, we note that the expression of PP2A is decreased upon ATRA-induced differentiation of HL-60 cells (43) , and we hypothesize that this down-regulation could potentially favor the activation of RA-regulated genes directly implicated in the differentiation process. Similarly, PP1 and PP2A activities are regulated by insulin in rat skeletal muscle cells in a differentiation-dependent manner (44) . Our data therefore suggest that, in target cells, subset(s) of RA-controlled genes will be differentially affected by the phosphorylation state of retinoic acid receptors.

Reports from various laboratories identified RXRs in mammalian cells (34, 38, 45, 46) as the primary dimerization partners of all- trans-retinoic acid receptors. Our in vitro DNA-binding experiments showed that (i) OA treatment increased the DNA binding activity of RAR/RXR dimers and (ii) that RAR and RXR dephosphorylation is detrimental to RAR/RXR heterodimers binding to RAREs, lowering the heterodimerDNA complex formation efficiency by at least 10-fold. While this work was under review, Bhat and colleagues (47) reported similar values for heterodimer formation of TR/RXR in solution. Taken together, these data suggest that while phosphorylation of RXR or RAR is required neither for homodimer nor for heterodimer formation per se, it may modulate their heterodimerization properties when one partner is present in limiting concentrations.

OA is a powerful pharmacological tool which has been used to demonstrate the involvement of phospho/dephosphorylation processes in the regulation of the transactivating potential of transcription factors. Initial reports from B. O'Malley's (9, 20) laboratory established the importance of phosphorylation processes in PR-mediated transcription using this compound and other modulators of kinases. OA was able to induce ligand-free activation of the PR, and to potentiate the ligand-inducible transcription by GR (21, 48) . Because of the lack of correlation between OA treatment and the phosphorylation state of GR, it has been proposed that phosphorylation of coactivators involved in GR-mediated transcription could be responsible for this potentiation. Alternatively, processes such as the nuclear/cytoplasmic shuttling of receptors could be modified as well (49) . The difficulty to establish a clear role for phosphorylation of steroid receptors is undoubtedly linked to the multiplicity of the experimental systems used, as well as technical limitations. For example, PP1 and PP2A have recently been reported to be histidine phosphatases (50) . Phosphohistidine residues are present in proteins in quantities comparable to that of phosphoserine and phosphothreonine. However, phosphohistidine residues are acid labile and thus not detected by the standard procedures of phosphoamino acid analysis. Indeed, multiple phosphorylation sites have been identified and mutated in the GR, without strongly altering its transactivating potential (51) . On the contrary, critical serine or threonine residues have been identified in v-erb-A (52) and the estrogen receptor (53) . Lin and colleagues (54) have also demonstrated a correlation between OA-induced hyperphosphorylation of hTR-1 and an increase in transcriptional activity of this receptor. This effect has been recently attributed to a more efficient homodimerization of hTR-1 (55) . Our observations further substantiate the regulatory role of the phosphorylation state of dimerization partners of retinoid receptors and demonstrated its role on their transactivating potential. Given the intricacy of the retinoid signaling pathway, which is controlled by specific ligands and regulated by a delicate balance between heterodimer and homodimer formation (56) which can either potentiate (reviewed in (3, 57) ) or inhibit (37, 58, 59, 60, 61, 62) RAR activity, establishing a well-defined role for a given phosphorylated amino acid from each receptor will be necessary.

Our observations thus show that phosphorylation of RARs and RXRs provides another level of regulation of the function of these receptors, in addition to that already provided by multiple receptors isoforms, multiple dimerization partners, distinct response elements, diverse promoter contexts, and ligand variety.


FOOTNOTES

*
This work was supported by grants from INSERM, the Association pour la Recherche sur le Cancer, the Federation Nationale des Centres de Lutte contre le Cancer, the Universite de Lille II, C.H.U de Lille, and the Conseil Regional du Nord-Pas-de-Calais. 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.

§
To whom correspondence should be addressed. Tel.: 33-20-62-68-87; Fax: 33-20-62-68-68.

The abbreviations used are: ATRA, all- trans-retinoic acid; RA, retinoic acid; OA, okadaic acid; RAR, all-trans retinoic acid receptor; RXR, 9- cis-retinoic acid receptor; PP1, protein phosphatase 1, PP2A, protein phosphatase 2A; DR, direct repeat retinoic acid response element with a spacer of n bases; CRABP, cellular retinoic acid-binding protein; CRBP, cellular retinol-binding protein; EMSA, electrophoretic mobility shift assay; RARE, retinoic acid response element; PAGE, polyacrylamide gel electrophoresis; CAT, chloramphenicol acetyltransferase; CIP, calf intestine alkaline phosphatase; EMSA, electrophoretic mobility shift assay.

P. Lefebvre, unpublished observations.

A. Tahayato and P. Lefebvre, unpublished observations.

C. Rochette-Egly, manuscript in preparation.

P. Lefebvre and A. Tahayato, unpublished observations.

B. Lefebvre, personal communication.


ACKNOWLEDGEMENTS

We are indebted to Dr. J. Grippo (Hoffman-Laroche) for 9- cis-RA, to Dr. B. Sablonniere who supplied us with anti-RAR polyclonal antibody IS39, and Dr. H. Gronemeyer who provided us with purified RAR and RXR. We also acknowledge Pr. P. Chambon for stimulating discussions and Dr. J. Clifford for critically reading the manuscript. We are also grateful to Drs. B. Wadzinski and L. Peruski for the gift of the PP1 cDNA and to Dr. M. Mumby who provided us with the CMV-PP2a construct.


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