Direct Channeling of Retinoic Acid between Cellular Retinoic Acid-Binding Protein II and Retinoic...

MOLECULAR AND CELLULAR BIOLOGY, Apr. 2002, p. 2632–2641 Vol. 22, No. 80270-7306/02/$04.00�0 DOI: 10.1128/MCB.22.8.2632–2641.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Direct Channeling of Retinoic Acid between Cellular RetinoicAcid-Binding Protein II and Retinoic Acid Receptor Sensitizes

Mammary Carcinoma Cells to Retinoic Acid-Induced Growth ArrestAnuradha S. Budhu and Noa Noy*

Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853

Received 10 September 2001/Returned for modification 11 October 2001/Accepted 8 January 2002

Cellular retinoic acid-binding protein II (CRABP-II) is an intracellular lipid-binding protein that associateswith retinoic acid with a subnanomolar affinity. We previously showed that CRABP-II enhances the transcrip-tional activity of the nuclear receptor with which it shares a common ligand, namely, the retinoic acid receptor(RAR), and we suggested that it may act by delivering retinoic acid to this receptor. Here, the mechanismsunderlying the effects of CRABP-II on the transcriptional activity of RAR and the functional consequences ofthese effects were studied. We show that CRABP-II, a predominantly cytosolic protein, massively undergoesnuclear localization upon binding of retinoic acid; that it interacts with RAR in a ligand-dependent fashion;and that, in the presence of retinoic acid, the CRABP-II–RAR complex is a short-lived intermediate. The dataestablish that potentiation of the transcriptional activity of RAR stems directly from the ability of CRABP-IIto channel retinoic acid to the receptor. We demonstrate further that overexpression of CRABP-II in MCF-7mammary carcinoma cells dramatically enhances their sensitivity to retinoic acid-induced growth inhibition.Conversely, diminished expression of CRABP-II renders these cells retinoic acid resistant. Taken together, thedata unequivocally establish the function of CRABP-II in modulating the RAR-mediated biological activitiesof retinoic acid.

The vitamin A metabolite retinoic acid (RA) regulates mul-tiple biological processes, including cell proliferation and dif-ferentiation, by virtue of its ability to modulate the rate oftranscription of numerous target genes. The transcriptionalactivities of this hormone are mediated by two members of thenuclear hormone receptor superfamily: the retinoid X receptor(RXR), which is activated by the 9-cis isomer of RA, and theretinoic acid receptor (RAR), which responds to both 9-cis andall-trans RA. These retinoid receptors bind to specific responseelements in the promoter regions of target genes and functionas ligand-inducible transcription factors (5, 23, 25). RXR canbind to DNA and activate transcription as a homodimer. Incontrast, tight binding to DNA and transcriptional activationby RAR usually occurs through heterodimerization with RXR(12, 17, 23, 34). Upon ligand binding, RAR-RXR het-erodimers recruit multicomponent coactivator complexes that,in turn, remodel chromatin and bridge to the general transcrip-tion machinery to modulate gene expression (16, 32).

In addition to retinoid receptors, RA binds to two small (ca.15 kDa) intracellular proteins termed cellular RA-binding pro-teins (CRABP-I and CRABP-II). These proteins are membersof the family of intracellular lipid-binding proteins which alsoincludes the cellular retinol-binding proteins and nine knownisotypes of fatty acid-binding proteins (26, 27). Intracellularlipid-binding proteins share a highly conserved three-dimen-sional structure which allows them to bind specific hydrophobicligands within an antiparallel �-barrel constructed of two or-thogonal, five-stranded �-sheets. Interestingly, the crystal

structures of holo-CRABP-I and CRABP-II indicate that ac-cess to the entrance of the ligand-binding pockets of theseproteins is restricted (21). These observations suggest thatsignificant conformational changes may be required to enablethe release of RA from the binding pocket, and they raise thequestion of how such rearrangements are induced to allow theligand to reach its sites of metabolism or action. The twoCRABPs exhibit distinct patterns of expression across differentcells and developmental stages. In the adult, CRABP-I iswidely expressed, while the expression of CRABP-II is re-stricted to skin (27), testis, uterus and ovary (30, 35, 36), andthe choroid plexus (33). Both CRABPs are widely expressed inthe embryo, but they do not usually coexist in the same cells(24). These differential expression profiles, combined with thehigh level of interspecies conservation of the two isotypes,suggest that despite the similarity of their RA-binding affinities(9) the two CRABPs have distinct functions in RA biology.Similar to the proposed function of other intracellular lipid-binding proteins, it is usually believed that CRABPs serve tosolubilize and transport their lipophilic ligand in the aqueousphase of cytosol. Some studies suggest, however, that in addi-tion to these general functions the CRABPs may have specificroles in mediating RA action. It was reported that elevatedCRABP-I expression in F9 teratocarcinoma cells enhances therate of formation of polar metabolites of RA and decreases thesensitivity of these cells to RA-induced differentiation. It wasconsequently proposed that CRABP-I acts to moderate thecellular response to RA by enhancing the activity of an en-zyme(s) that catalyzes RA degradation (1, 2). On the otherhand, available information implicates CRABP-II in a func-tional interplay with the nuclear receptor with which it sharesa common ligand, RAR.

* Corresponding author. Mailing address: 225 Savage Hall, CornellUniversity, Ithaca, NY 14853. Phone: (607) 255-2490. Fax: (607) 255-1033. E-mail: [email protected].

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In support of such a function for CRABP-II, we and othersrecently reported that overexpression of this isotype enhancesthe transcription of a reporter gene driven by a RAR responseelement in cells (8, 9). We showed further that this activity isspecific for CRABP-II and is not shared by CRABP-I (9). Toinvestigate the molecular basis for the functional differencesbetween the two proteins that allow CRABP-II, but notCRABP-I, to augment the transcriptional activity of RAR, weexamined the kinetic parameters of the process by which RAmoves from either binding protein to the receptor. These invitro studies revealed that the mechanisms by which RA trans-fers from the two CRABPs to RAR are fundamentally differ-ent. Movement of RA from CRABP-I was found to proceed bydissociation of the ligand from the binding protein to theaqueous phase, followed by its association with RAR. In con-trast, the data indicated that RA transfers from CRABP-II toRAR through collision-mediated channeling, a process thatbypasses the bulk aqueous phase and that results in facilitationof the formation of the holo-receptor. We thus suggested thatligand channeling mediated by CRABP-II underlies the abilityof this protein to potentiate the transcriptional activity of RAR(9). Additional studies have focused on structural determi-nants that underlie CRABP-II to facilitate the delivery of RAto RAR. These studies identified a surface region, positionedjust above the entrance to the ligand-binding pocket ofCRABP-II, that displays an electric potential which is remark-ably different from that of the homologous region in CRABP-I.Mutagenesis analyses demonstrated that this surface patch,consisting of amino acid residues GLN75, PRO81, and LYS102,is necessary and sufficient both for facilitating the transfer ofRA from CRABP-II to RAR and for enhancing the transcrip-tional activity of the receptor. The patch thus comprises theRAR interaction domain of CRABP-II (4).

Critical questions remain regarding the hypothesis thatCRABP-II functions to potentiate the transcriptional activityof RAR and that it does so through direct channeling of RA tothe receptor. Importantly, a requisite of this hypothesis is thatCRABP-II and RAR colocalize in the same cellular compart-ment at least under some conditions. The activities of RAR areexerted in the nucleus. However, although it has been sug-gested that a fraction of both CRABP-I and CRABP-II may bepresent in the nuclei of various cells (14), these proteins areusually thought to be predominantly cytoplasmic (27). Anotherdifficulty in establishing the proposed role of CRABP-II is thatour attempts to capture the putative complex betweenCRABP-II and RAR failed despite utilization of multiple ex-perimental approaches, including coprecipitation assays, cova-lent cross-linking, and fluorescence anisotropy studies (9). Wethus suggested that the CRABP-II–RAR complex that medi-ates RA channeling is a short-lived intermediate that rapidlydissociates following completion of transfer (9). It should benoted that our failure to visualize a CRABP-II–RAR complexconflicts with the report that CRABP-II and RAR� could becoimmunoprecipitated from whole-cell extracts (8). However,in that report, the interactions between CRABP-II and RARappeared to have occurred in a ligand-independent fashion,and similar interactions were observed between CRABP-IIand RXR, a receptor that does not share a ligand with thisbinding protein (13). The significance of these ostensible in-teractions is thus unclear. In any event, it was suggested based

on these observations that CRABP-II acts as a coactivator forretinoid receptors (8), a suggestion that implies that the pro-tein may enhance the transcriptional activity of RAR by bind-ing to the receptor and perhaps recruiting additional accessoryproteins. Hence, it remains to be clarified whether the ability ofCRABP-II to channel RA to RAR is key in allowing thisprotein to augment the receptor’s activity, or whether the bind-ing protein functions by a different mechanism. The observa-tions that CRABP-II enhances transcriptional activation byRA also raise important questions regarding the biologicalsignificance of this activity. This issue is especially pertinent inview of the report that CRABP-II-null mice are viable andfertile and that they show only minor defects in limb develop-ment, implying that the protein is dispensable under standardlaboratory conditions (22). Nevertheless, considering its strictevolutionary conservation, it is reasonable to presume that theactivity of CRABP-II will be essential at least under somephysiological conditions that are yet to be defined.

The study reported here was undertaken in order to eluci-date the molecular mechanisms through which CRABP-II aug-ments the transcriptional activity of RAR and to clarify thesignificance of the presence of this protein in cells for thebiological activities of RA. We show that CRABP-II is pre-dominantly cytosolic in the absence of ligand, but that it un-dergoes a dramatic nuclear localization upon binding of RA.We also show that CRABP-II interacts with RAR in a ligand-dependent fashion, but that the resulting complex is long-livedenough to allow its visualization only in the presence of aligand that does not readily move from the binding protein tothe receptor. The data further establish that augmentation ofthe transcriptional activity of RAR stems directly from theability of CRABP-II to channel ligands to the receptor and thatthis potentiation activity is important only when cellular levelsof either RAR or RA are limiting, at least in some cells.Finally, we demonstrate that CRABP-II governs the sensitivityof a mammary carcinoma line to RA-induced growth inhibi-tion. Taken together, these observations unequivocally estab-lish the function of CRABP-II in modulating the RAR-medi-ated biological activities of RA.

MATERIALS AND METHODS

Ligands. RA was purchased from ICN Pharmaceuticals Inc. (Costa Mesa,Calif.). CD270 was a gift from Uwe Reichert (Galderma, Sophia Antipolis,France). Stock solutions were made in dimethyl sulfoxide (DMSO) and stored inamber vials at �80°C.

Antibodies. Antibodies against mCRABP-I were purchased from AffinityBioreagents (Golden, Colo.), and anti-mCRABP-II (5CRA3B3) antibodies werea gift from Pierre Chambon (IGMCB, Strasbourg, France). Antibodies againstactin were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Anti-mouse and anti-goat immunoglobulin horseradish peroxidase-conjugated anti-bodies were from Amersham (Arlington Heights, Ill.). Primary and secondaryantibodies for Western blotting were diluted 1:1,000 and 1:3,000, respectively.Texas Red-X-goat anti-mouse antibody was purchased from Molecular Probes(Eugene, Oreg.).

Proteins. hRAR�–ligand-binding domain (hRAR�-LBD) (amino acids 145 to390) was prepared by PCR amplification using full-length hRAR� as a template.The PCR product was subcloned into the NdeI-XhoI sites of the bacterial ex-pression vector pET28a. The plasmid was amplified in Escherichia coli strainDH5� and transfected into E. coli BL21 for protein expression. E. coli cellsharboring the vector were grown at 37°C to an optical density at 600 nm (OD600)of 0.6, protein expression was induced with 0.5 mM isopropyl-�-D-thiogalacto-pyranoside, and cells were grown for an additional 3 h and pelleted by centrif-ugation. Following lysis, protein was purified by metal-chelating affinity chroma-

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tography. Purified hRAR�-LBD bound to Ni�2-nitriloacetic acid (NTA)-agarose beads (Qiagen, Valencia, Calif.) was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for purity and concentrationdeterminations. The bacterial expression vector for bCRABP-II was a generousgift of David Ong (Vanderbilt University). This protein was purified as previouslydescribed (19).

Cell lines. MCF-7 cells were purchased from the American Type CultureCollection (Manassas, Va.). Cells were cultured in Dulbecco’s modified Eagle’smedium (DMEM) supplemented with 10% fetal bovine serum (FBS). Stablytransfected cell lines were also supplemented with 200 �g of G418/ml (Clontech,Palo Alto, Calif.) and/or 100 �g of hygromycin/ml (Invitrogen, Carlsbad, Calif.).During transactivation experiments, MCF-7 cells overexpressing CRABP-II werecultured in DMEM supplemented with 1 �g of doxycyline/ml.

Generation of GFP-CRABP constructs. To generate hCRABP-II andhCRABP-I tagged with green fluorescence protein (GFP), EcoRI and BamHIsites were introduced by PCR and the resulting products were ligated intopEGFP-C2 (Clontech).

Fluorescence microscopy. COS-7 cells were seeded on glass coverslip cham-bers (Nalge Nunc Intl., Vernon Hill, Ill.) and transfected with GFP-taggedhCRABP (1 �g). Twenty-four hours after transfection, cells were treated withDMSO (control), RA (20 nM), or CD270 (20 nM). Three hours following ligandaddition, images of GFP-tagged proteins were collected by using an OlympusBX-50 fluorescent microscope (20� objective) using the Metamorph acquisitionand image analysis software package from Universal Imaging. For RA-pulseexperiments, cells were treated with RA for 3 h and then incubated in freshmedium without RA for an additional 3-h period. For immunofluorescencestaining, COS-7 cells were seeded on glass coverslip chambers (Nalge Nunc Intl.)and after 24 h they were treated with DMSO (control) or RA (20 nM) for 3 h.Culture medium was removed, cells were rinsed with phosphate-buffered saline(PBS), fixed in 3.7% formaldehyde at 25°C for 10 min, and permeabilized with0.2% Triton X-100 at 25°C for 10 min. Cells were then washed with PBS andincubated with antibodies against CRABP-I or -II (dilution, 1/50) at 37°C for 30min. After three washes in PBS (10 min each), cells were incubated with TexasRed-X-goat anti-mouse immunoglobulin G (dilution, 1/200) at 37°C for 30 min.Following three washes with PBS (10 min each), cells were analyzed by fluores-cence microscopy as detailed above.

Coprecipitation assays. Purified bCRABP-II (7.5 �M) was precomplexed withRA or CD270 for 5 min at 4°C. The holo-protein was then incubated withhRAR�-LBD (7.5 �M) immobilized on Ni�2-NTA-agarose beads for 15 min at4°C. Mixtures were centrifuged, pelleted beads were extensively washed with abuffer containing 50 mM Tris (pH 7.5), 0.3 M NaCl, 2 mM phenylmethylsulfonylfluoride, 10 �g of leupeptin/ml, and 10 �g of aprotinin/ml, and proteins wereresolved by SDS-PAGE and visualized by Coomassie blue staining.

Transactivation assays. COS-7 cells were seeded on six-well plates and trans-fected with a pSG5 vector harboring the cDNA for hCRABP-I or -II (1 �g)together with a DR5-tk-luciferase reporter plasmid (1 �g) and pCH110 (internalstandard, 0.3 �g). In some experiments, cells were also cotransfected with pSG5expression vectors for hRXR� and hRAR� (0.1 �g each). At 24 h after trans-fection, RA (20 nM) or CD270 (100 nM) was added singly or in combination tocells. In other experiments, MCF-7 parental cells as well as stable clones ex-pressing either diminished levels of CRABP-II or overexpressing CRABP-IIwere seeded on six-well plates and transfected with a DR5-tk-luciferase reporterplasmid (1 �g) and pCH110 (internal standard, 0.3 �g). At 24 h after transfec-tion, RA (2, 20, 200, or 2,000 nM) was added and cells were incubated at 37°Cfor 24 h. Expression of the reporter was measured by the activity of luciferaseusing the luciferase assay system (Promega), following the instructions of themanufacturer. Luciferase activity was corrected for transfection efficiency basedon the activity of �-galactosidase as measured by standard procedures.

Generation of cell lines stably expressing antisense constructs. To generateantisense constructs for CRABP-I or -II, EcoRI and HindIII sites were intro-duced to the respective cDNAs by PCR and the resulting product was ligated inthe reverse orientation to the mammalian expression vector pJ4H. To generatecell lines stably expressing the antisense constructs, MCF-7 cells were transfectedwith the respective antisense CRABP construct along with a pTK-Hyg vector(Clontech) at a 10:1 (CRABP/Hyg) ratio. Cells harboring these constructs wereselected in DMEM supplemented with 10% FBS and 100 �g of hygromycin/ml.Individual clones were screened by Western blotting to select those displayingminimal expression of CRABP-I or -II.

Generation of stable cell lines that conditionally overexpress CRABP. Condi-tional overexpressing cell lines for CRABP-I or CRABP-II were generated usingClontech’s pTET-ON system. Briefly, BamHI and XbaI sites were introduced toCRABP-I or -II by PCR and the resulting product was ligated to the pTRE2vector. MCF-7 cells were transfected with the pTET-ON vector, and cells har-

boring this vector were selected in DMEM supplemented with 10% FBS and 200�g of G418/ml. pTET-ON clones were selected by transient transfection withpTRE2-luciferase followed by measurement of reporter gene activity by theluciferase assay system. Clones showing over 20-fold induction of luciferaseactivity in the presence of 1 �g of doxycycline/ml were selected and cotransfectedwith either CRABP-I–pTRE2 or CRABP-II–pTRE2 along with pTK-Hyg(CRABP-pTRE2/pTK-Hyg ratio, 10:1), followed by selection in DMEM con-taining 10% FBS, 200 �g of G418/ml, and 100 �g of hygromycin/ml. Each clonewas grown on 100-mm plates in the absence or presence of 1 �g of doxycycline/mlfor 48 h, followed by cell lysis. Clones were screened by Western blotting fordoxycycline-induced overexpression of either CRABP-I or CRABP-II.

Western blotting. Cells were cultured in 100-mm plates to confluency, washedwith PBS, and lysed in lysis buffer (10 mM potassium phosphate [pH 7.5], 0.5%[wt/vol] Triton X-100, 10 �g of leupeptin/ml, 10 �g of aprotinin/ml). Followingcentrifugation, protein concentrations in cell lysates were determined by theBradford assay (Bio-Rad). Equivalent total protein amounts were loaded in eachlane of an SDS–12% PAGE gel, followed by transfer and blocking in 5% nonfatdry milk. Blots were probed with appropriate primary antibodies, followed byincubation with horseradish peroxidase-conjugated secondary antibody. To stripblots, membranes were incubated in stripping solution (62.5 mM Tris [pH 6.7],100 mM �-mercaptoethanol, 2% [wt/vol] SDS) for 30 min at 50°C followed byextensive washes in TBST (50 mM Tris [pH 7.5], 0.9% NaCl, 1% Tween 20) andreprobed with the appropriate primary and secondary antibodies. Antibody-antigen complexes were detected by enhanced chemiluminescence according tothe manufacturer’s protocol (Amersham).

MTT assays. Cells were seeded on 96-well plates (1,000 cells/well) and allowedto grow for 24 h. DMSO (control) or RA was added at various concentrationsand supplemented to fresh media every 48 h. On the day of the assay, cells ineach well were treated with 10 �l of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide (MTT) reagent and incubated for an additional 4-h period,followed by overnight incubation with 100 �l of solubilization buffer (RocheDiagnostics). Absorbance at 570 nm was measured using an enzyme-linkedimmunosorbent assay reader.

RESULTS

CRABP-II, but not CRABP-I, localizes to the nucleus in thepresence of ligand. A prediction of the hypothesis thatCRABP-II enhances the transcriptional activity of RARthrough direct protein-protein interactions is that the two pro-teins will colocalize in the same cellular compartment underconditions where RAR becomes activated. The observationsthat CRABP-II channels RA to RAR, while CRABP-I doesnot (9), further suggest that cocompartmentalization withRAR may be a specific property of the former but not thelatter binding protein. To explore these predictions, the sub-cellular localization of the two CRABP isotypes was deter-mined. GFP-tagged constructs of CRABP-I and -II were gen-erated and transfected into COS-7 cells, and their location wasdetermined by direct fluorescence microscopy. GFP–CRABP-Iwas predominantly present in the cytoplasm, both in the ab-sence and presence of RA. In fact, this protein appeared to beexcluded from the nucleus (Fig. 1a and b). In contrast,CRABP-II which, similarly, was confined to the cytoplasm inthe absence of ligand, massively located into the nucleus uponaddition of RA (Fig. 1e and f). Removal of RA from themedium resulted in repartitioning of CRABP-II into the cy-tosol (Fig. 1g), indicating that movement of CRABP-II into thenucleus is a reversible process regulated by the availability ofligand. To examine whether the nuclear translocation ofCRABP-II is a specific response to RA, a high-affinity RARligand, or whether it is a consequence of the ligation ofCRABP-II, we utilized a synthetic ligand, termed CD270, con-taining a substituted benzo[b]thiophene carboxylic acid. Thisligand was reported to interact with CRABP with a high affinitybut to be a poor RAR� ligand (15). Similarly to RA, treatment

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of the cells with CD270 did not affect the subcellular localiza-tion of CRABP-I (Fig. 1d) but efficiently induced movement ofCRABP-II into the nucleus (Fig. 1h). To verify that the ob-served ligand-induced nuclear translocation of CRABP-II isnot an artifact emanating from the addition of the GFP tag, thesubcellular location of endogenous CRABP-II in COS-7 cellswas also examined by immunofluorescence microscopy using

CRABP type-selective antibodies. The data of these experi-ments showed that, similarly to the behavior of ectopicallyexpressed GFP-tagged proteins, endogenous CRABP-I wascytoplasmic both in the absence and presence of RA (Fig. 2aand c), while endogenous CRABP-II translocated from thecytoplasm to the nucleus in the presence of RA (Fig. 2b and d).

A complex between CRABP-II and RAR is stabilized in thepresence of CD270. Our previous observations provided ki-netic evidence for direct interactions between CRABP-II andRAR�. However, our attempts to capture the CRABP-II–RAR complex failed despite the application of multiple exper-imental approaches. We thus suggested that CRABP-II andRAR interact transiently and that, following movement of RAfrom the binding protein to the receptor, the complex rapidlydissociates (9). In other words, holo-CRABP-II associates withapo-RAR, and the ensuing movement of RA from the bindingprotein to the receptor (accompanied by dramatic conforma-tional changes in RAR [28, 31]) results in rapid dissociation ofthe ligand-depleted CRABP-II from the liganded receptor. Ifso, it is possible that the CRABP-II–RAR complex would bestabilized in the presence of a ligand that does not readilychannel between CRABP-II and RAR. To examine this pos-tulate, we used the synthetic ligand CD270, which binds toCRABP with a Kd of 6 nM, displaying more than 2 orders ofmagnitude higher affinity for the binding protein versus thatfor RAR (15). Due to these binding characteristics, CD270 willnot readily partition from CRABP-II to RAR, allowing a sig-nificant fraction of the binding protein to remain liganded inthe presence of apo-RAR, resulting in stabilization of theCRABP-II–RAR complex and perhaps enabling its visualiza-tion.

Potential physical interactions between CRABP-II withRAR were investigated by coprecipitation assays that werecarried out in the absence or presence of either RA or CD270.In these experiments, RAR-LBD, the region of the receptorthat mediates its interaction with CRABP-II, was used as a baitto capture CRABP-II. Bacterially expressed histidine-taggedRAR-LBD was immobilized on Ni�2-NTA-agarose beads andincubated with CRABP-II in the absence or presence of li-gands. Beads were centrifuged and washed, and precipitatedproteins were analyzed by SDS-PAGE and visualized by Coo-

FIG. 1. GFP-tagged CRABP-II, but not CRABP-I, localizes to thenucleus in response to ligand. COS-7 cells were transfected with GFP–CRABP-I (a to d) or GFP–CRABP-II (e to h). Images of GFP-taggedproteins were collected following a 3-h incubation with the ligands.Treatments were as follows: panels a and e, DMSO (vehicle); b and f,1 �M RA; c and g, cells treated with 1 �M RA for 3 h followed by a3-h chase with fresh media devoid of ligand (RA pulse/chase); d and h,1 �M CD270.

FIG. 2. Endogenous CRABP-II, but not CRABP-I, localizes to thenucleus in response to ligand. COS-7 cells were treated with DMSO(vehicle; a and c) or 1 �M RA (b and d) for 3 h. Cells were immuno-stained for CRABP-I (a and b) or CRABP-II (c and d) as detailed inMaterials and Methods.



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massie blue staining (Fig. 3). In agreement with the results ofour previous studies carried out using RAR�AB as a bait,CRABP-II did not coprecipitate with the RAR-LBD either inthe absence of ligand (Fig. 3, lane 3) or in the presence ofsaturating concentrations of RA (Fig. 3, lanes 4 and 5). How-ever, CRABP-II associated with the receptor in the presenceof CD270 and did so in a dose-dependent manner (Fig. 3, lanes6 and 7). The stabilization of the CRABP-II–RAR complex inthe presence of CD270 demonstrates that CRABP-II interactswith RAR in a ligand-dependent fashion. Importantly, theseobservations provide strong support for the notion that theinability to visualize this complex in the presence of RAstemmed from the short half-life of the complex when inducedto form by a ligand that is rapidly channeled from the bindingprotein to the receptor. Furthermore, the data reveal that theinteraction between CRABP-II and RAR are mediated via theLBD of RAR, establishing a novel role for this region of thereceptor.

CRABP-II does not potentiate the transcriptional activity ofRAR in the presence of CD270. The kinetic evidence thatCRABP-II channels RA to RAR suggested that this activitymay underlie the ability of the binding protein to enhance thetranscriptional activity of the receptor. It could be argued,however, that CRABP-II functions by a different mechanism,for example, by binding to the receptor and recruiting addi-tional accessory proteins; that is, that it acts in a mannersimilar to known transcriptional coactivators. Utilization of the

ligand CD270 provides a unique opportunity to further clarifythe molecular mechanism underlying CRABP-II function. Thiscompound induces nuclear localization of CRABP-II (Fig. 1h),gives rise to a CRABP-II–RAR complex (Fig. 3), and alsobinds to RAR, albeit at a low affinity (15). However, due to itsbinding characteristics, CD270 does not appear to efficientlymove from CRABP-II to RAR. It was thus of interest toexamine whether the RAR-potentiating activity of CRABP-II,which was observed in the presence of RA, will be retained inthe presence of CD270.

To address this issue, transactivation assays were carried out.COS-7 cells were cotransfected with CRABP-II and a lucif-erase reporter construct driven by a RAR response element.Cells were treated with RA, or CD270, or a combination of thetwo ligands and assayed for ligand-induced expression of thereporter (Fig. 4). RA enhanced the transcriptional activity ofRAR, and this activity was markedly augmented upon cotrans-fection of CRABP-II. CD270 functioned as an activator ofRAR although at lower efficiency than RA. In contrast with theeffect of CRABP-II on the transcriptional activity of RARinduced by RA, cotransfection of the binding protein had noeffect on CD270-induced transactivation. Furthermore, CD270abolished the ability of CRABP-II to enhance the transcrip-tional activity of RAR in the presence of RA. Similar resultswere obtained at various RA/CD270 ratios in the 5/1 to 50/1range (data not shown). The loss of the RAR-potentiatingactivity of CRABP-II in the presence of CD270 clearly dem-onstrates that the ability to channel ligands to RAR is criticalfor the effect of the binding protein on transcriptional rates.

CRABP-II augments the transcriptional activity of RAR inCOS-7 cells only when cellular levels of either RA or RAR arelimiting. An important question that arises from the observa-tions that CRABP-II potentiates RA-induced, RAR-mediatedtranscriptional activation relates to the physiological condi-tions under which this activity becomes important. To addressthis question, the effect of CRABP-II on the RA-dependentactivity of RAR was investigated by transactivation assays car-

FIG. 3. CRABP-II stably interacts with RAR�-LBD in the pres-ence of CD270. Coprecipitation assays were conducted as described inMaterials and Methods. (a) Proteins were resolved by SDS-PAGE andvisualized by Coomassie blue staining. Arrows indicate the positions ofCRABP-II and RAR�-LBD. Lanes: M, molecular weight marker; 1,CRABP-II at 50% of total input; 2, RAR�-LBD immobilized onNi�2-NTA-agarose beads; 3, immobilized RAR�-LBD incubated withCRABP-II in the absence of ligand; 4 and 5, immobilized RAR�-LBDincubated with CRABP-II in the presence of 7.5 and 18.75 �M RA,respectively; 6 and 7, immobilized RAR�-LBD incubated withCRABP-II in the presence of 7.5 and 18.75 �M CD270, respectively.(b) Quantitation of bands shown in Fig. 2a.

FIG. 4. CRABP-II does not enhance the transcriptional activity ofRAR in the presence of CD270. Cells were cotransfected with a lucif-erase reporter construct driven by a RAR responsive element, apCH110 vector (internal standard), and an empty expression vector, oran expression vector for either CRABP-I or CRABP-II. Cells weretreated with RA (20 nM), CD270 (100 nM), or a combination of thetwo ligands for 24 h. Luciferase activity normalized to the activity of�-galactosidase is shown. Data are means � standard errors of themean (SEM; n 3).



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ried out under two sets of conditions: in the absence of ectopicexpression of RAR, i.e., relying on the (limiting) endogenouslevel of RAR in the cells; and upon overexpression of thereceptor. In the latter set of experiments, cells were cotrans-fected with expression vectors for both hRXR� and hRAR�.The data (Fig. 5a) show that, in the presence of endogenouslevels of RAR, CRABP-II markedly enhanced the receptor’stranscriptional activity. As expected, overexpression of retinoidreceptors significantly increased the transcriptional activity.However, under these conditions, CRABP-II did not enhancethe transcriptional activity of RAR further. Similar resultswere obtained when cells were transfected with an expressionvector for hRAR�, but not with the hRXR� expression vector(data not shown). In another set of transactivation assays, therange of RA doses in which the activity of RAR is responsiveto the presence of CRABP-II was studied. The results of theseexperiments (Fig. 5b) revealed that expression of CRABP-IImarkedly enhanced the transcriptional activity of RAR at lowconcentrations of RA, but that the ability of the protein toaugment transcriptional rates was rapidly lost when the con-centration of the ligand was raised. It could be argued that, at

high ligand concentrations, CRABP-II did not affect transcrip-tional rates because the reporter vector activity became satu-rated. However, the data indicate that CRABP-II markedlylost its potentiating activity upon elevation of RA concentra-tions even under conditions that are far from the observedmaximal activity (Fig. 5b, inset). It may also be argued that theeffect of CRABP-II at low RA concentrations is due simply toits ability to solubilize the hydrophobic ligand, thereby prevent-ing its accumulation in membranes and maintaining its acces-sibility to the receptor. However, if this were the case, anothercellular protein with a similar affinity for RA, such asCRABP-I, would have a similar enhancing effect, a predictionthat is not borne out by the data (Fig. 5a). These results thussubstantiate that, in COS-7 cells, CRABP-II is efficacious inenhancing RAR activity but only at low, physiological (nano-molar) ligand concentrations.

CRABP-II is critical for the transcriptional activity of RARin MCF-7 mammary carcinoma cells. The effects of CRABP-IIon the transcriptional activity of RAR were further investi-gated using MCF-7 cells, a mammary carcinoma cell line whichis known to be responsive to the antiproliferative activity ofRA. Western blot analyses showed that both CRABP-I andCRABP-II are present in MCF-7 cells (Fig. 6a). Several deriv-atives of the parental MCF-7 cells were then generated. Theexpression of either CRABP-I or CRABP-II was minimized bygenerating lines that stably express an antisense construct foreither of these isotypes (see Materials and Methods for de-tails). Western blotting of the newly generated lines indicatedthat the antisense constructs successfully reduced the expres-sion of the respective CRABPs to undetectable levels. Theseanalyses also verified that the constructs acted with strict spec-

FIG. 5. CRABP-II enhances the transcriptional activity of RAR inCOS-7 cells only under limiting cellular levels of RAR or RA. Cellswere cotransfected with a luciferase reporter construct driven by aRAR responsive element, a pCH110 vector (internal standard), and anempty expression vector or an expression vector for either CRABP-I orCRABP-II. Cells were treated with RA for 24 h and luciferase activitywas measured and normalized for �-galactosidase activity. Data arepresented as fold induction relative to cells treated with vehicle alone.Bars represent means � SEM (n 3). (a) The effect of CRABP-II onRA-induced reporter gene expression in the presence of endogenousretinoid receptors, or upon ectopic coexpression of hRXR� andhRAR�. (b) The effect of CRABP-II on RA-induced reporter geneexpression at various RA concentrations. The inset to panel b depictsthe fold activation observed upon cotransfection of CRABP-II relativeto fold activation in the absence of ectopic binding protein at thevarious RA concentrations.

FIG. 6. MCF-7 derivatives that stably under- or overexpress CRABP.(a) Western blot analyses of parental MCF-7 cells (MCF-7) and oftheir derivatives expressing antisense constructs for either CRABP-I orCRABP-II (anti-I and anti-II). Fifty micrograms of total cell lysateprotein was loaded in each lane, and blots were also probed for actinto ensure equivalent loading. Data from two independent clones areshown. (b) Western blot analyses of MCF-7 lines conditionally over-expressing either CRABP-I or CRABP-II. Cells were assayed in theabsence of doxycycline or following treatment with 1 �g of doxycycline/ml. Ten micrograms of total cell lysate protein was loaded in each lane,and blots were probed for actin to ensure equivalent loading. Datafrom two independent clones are shown.



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ificity to inhibit only the expression of the CRABP isotypetowards which they were directed (Fig. 6a). In addition, MCF-7cell lines that overexpress either CRABP-I or CRABP-II weregenerated. In these lines, overexpression of the respectivebinding proteins could be induced at will by addition of doxy-cycline (see Materials and Methods). Western blotting verifiedthat expression levels of the two CRABPs in the cells stablyharboring the constructs were similar to those observed in theparent MCF-7 cells in the absence of doxycycline and thatexpression of the respective CRABPs was dramatically ele-vated upon treatment with doxycycline (Fig. 6b).

The effects of varying the expression level of CRABP-II onthe transcriptional activity of RAR were then studied by trans-activation assays carried out in the parental MCF-7 cells andtheir derivatives that either under- or overexpress this protein(Fig. 7). The transcriptional activity of RAR in the parentalMCF-7 cells was enhanced in a dose-dependent fashion uponaddition of RA. In contrast, in cells with diminished levels ofCRABP-II, RAR was inactive even at RA concentrations ashigh as 2 �M. The activity of RAR in cells that stably overex-press CRABP-II was dramatically enhanced compared to thatobserved in the parental cell line. In contrast with COS-7 cells,the ability of CRABP-II to augment RA-induced, RAR-medi-ated transcriptional rates in MCF-7 cells was not diminishedupon increasing ligand concentration, suggesting importantdifferences in RA homeostasis between COS-7 and MCF-cells(see Discussion).

CRABP-II sensitizes MCF-7 cells to RA-induced growth in-hibition. The augmentation of RAR activity by CRABP-IIraises the possibility that this protein will sensitize cells toRAR-mediated biological activities of RA. To explore thispossibility, we investigated the role of CRABP-II in modulat-ing an important activity of RA which is known to be mediatedby retinoid receptors, namely, the induction of growth arrest in

mammary carcinoma cells (7, 11, 18). To this end, the ability ofRA to induce growth arrest in the various MCF-7 cell lines thatexpress different levels of CRABP was examined by MTTgrowth assays (Fig. 8). RA inhibited the growth of the parentalMCF-7 cells in a dose-dependent manner with a 50% effectiveconcentration (EC50) of about 30 nM (Fig. 8). Cell lines thateither underexpress (Fig. 8a) or overexpress (Fig. 8b)CRABP-I responded to RA in a manner similar to that ob-served in the parental cells. In contrast, the RA dose-responsecurve in cell lines in which the expression of CRABP-II wasdiminished was markedly shifted to higher concentrations (Fig.8a). These cells thus displayed a pronounced RA resistancecompared to the parental MCF-7 cells. Clones harboring theconditional constructs for CRABP-II behaved similarly to theparental cells in the absence of doxycycline (data not shown).However, in the presence of doxycycline, the RA dose re-sponse of these cells was dramatically shifted to lower concen-trations and displayed an EC50 of 2 nM RA, i.e., about anorder of magnitude lower than the value observed in the pa-

FIG. 7. CRABP-II enhances the transcriptional activity of RAR inMCF-7 cells. Transactivation assays were carried out in parentalMCF-7 cells, in MCF-7 clones that stably express CRABP-II antisense(anti-II), or in cells that stably overexpress CRABP-II (over-II). Cellswere cotransfected with a luciferase reporter construct driven by aRAR responsive element along with a pCH110 vector (internal stan-dard). Cells were treated with the denoted concentrations of RA for24 h, and luciferase activity was measured and normalized for �-ga-lactosidase activity. Data are presented as fold induction relative tocells treated with vehicle alone. Data are means � SEM (n 3).

FIG. 8. CRABP-II sensitizes MCF-7 cells to RA-induced growthinhibition. Parental MCF-7 cells and their derivatives that displayminimized expression of CRABP-I (anti-I) or CRABP-II (anti-II) (a)or that overexpress CRABP-I (over-I) or CRABP-II (over-II) (b) weretreated with RA for 5 days. To induce overexpression of CRABP, cellswere also treated with doxycycline. Viable cell number was scored withthe MTT assay as described in Materials and Methods. Data shownrepresent cell growth as a percent of vehicle-treated cell growth (con-trol) and are the mean � SEM (n 5).



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rental cells (Fig. 8b). Hence, while CRABP-I does not affectthe RA responsiveness of MCF-7 cells, CRABP-II plays animportant role in modulating the response of these cells to theantiproliferative effects of RA.

DISCUSSION

We previously showed that, despite the high homology andthe similarities of the RA-binding affinities of CRABP-I andCRABP-II, the latter, but not the former, enhances the tran-scriptional activity of the nuclear receptor RAR. In vitro ki-netic analyses further demonstrated that RA moves to RARfrom CRABP-II (but not from CRABP-I) in a collision-medi-ated process that facilitates the formation of the liganded re-ceptor. We thus suggested that ligand channeling by CRABP-IImay underlie the effects of the binding protein on RAR-me-diated transcriptional regulation (9). Here, we set out to in-vestigate the mechanism by which CRABP-II augments thetranscriptional activity of RAR and the functional conse-quences of the presence of this protein in cells for the biolog-ical activities of RA. Taken together, the data establish thatCRABP-II functions through the sequence of events schemat-ically depicted in Fig. 9. The protein is predominantly cytosolicin the absence of ligand. While in cytosol, CRABP-II binds RAwhen it becomes available (either by influx from serum or

through the activity of retinal dehydrogenases intracellularly).Ligand binding induces nuclear localization of CRABP-II. Inthe nucleus, holo-CRABP-II associates with apo-RAR to forma complex that mediates direct channeling of RA and facili-tates the ligation of the receptor. Subsequently, the ligand-depleted CRABP-II rapidly dissociates from holo-RAR, re-leasing the receptor to its transcriptional activities.

The observations that the cellular distribution of CRABP-IIis dramatically shifted into the nucleus upon exposure to RAraise interesting questions relating to the mechanism by whichCRABP-II is targeted to the nucleus when liganded. Analysisof the sequence of this protein fails to identify an obviousnuclear localization signal. It is possible that, due to its smallsize, CRABP-II enters the nucleus by simple diffusion. How-ever, the translocation may involve accessory proteins thatregulate either the nuclear exclusion of the apo-CRABP-II orthe nuclear entry of the holo-protein. How the subcellulardistribution of CRABP-II is regulated thus remains to be elu-cidated.

Although our previously reported kinetic analyses providedstrong evidence for direct interactions between CRABP-II andRAR, our attempts to visualize a CRABP-II–RAR complexfailed despite utilization of multiple experimental approaches,including coimmunoprecipitation of endogenous proteins (9).

FIG. 9. A model for the mechanism of action of CRABP-II. CRABP-II is predominantly cytosolic in the absence of ligand. Upon ligandbinding, holo-CRABP-II relocates into the nucleus where it associates with apo-RAR to form a complex that mediates direct channeling of RAand facilitates the ligation of the receptor. The ligand-depleted CRABP-II rapidly dissociates from holo-RAR, releasing the receptor to itstranscriptional activities. RALDH, retinal dehydrogenase.



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We therefore suggested that the complex is a short-lived in-termediate (9), i.e., that holo-CRABP-II binds to apo-RAR,and that following movement of RA from the binding proteinto the receptor (accompanied by dramatic conformationalchanges in RAR [28, 31]), the ligand-depleted CRABP-II rap-idly dissociates from the liganded receptor. To examine thispostulate, we used the synthetic ligand CD270, which displaysa 200-fold-lower affinity towards RAR� than does CRABP(15). Due to these binding characteristics, CD270 does notreadily partition from CRABP-II to RAR, allowing for reten-tion of a significant fraction of holo-CRABP-II in the presenceof apo-RAR. Coprecipitation experiments demonstrated that,although the CRABP-II–RAR–LBD complex was not ob-served in the presence of RA, a robust complex formed uponincubation of the two proteins with CD270 and it did so in adose-dependent manner (Fig. 3). The stabilization of theCRABP-II–RAR complex in the presence of CD270 demon-strate that CRABP-II indeed interacts with RAR in a ligand-dependent fashion. Importantly, these observations also pro-vide strong support for the notion that the inability to visualizethis complex in the presence of RA stems from the shorthalf-life of the CRABP-II–RAR complex when induced toform by a ligand that is rapidly channeled.

Two alternative hypotheses may be postulated for under-standing the molecular mechanism by which CRABP-II aug-ments the transcriptional activity of RAR. It is possible thatthis activity stems from the transient association between thetwo proteins which mediates channeling of RA and facilitatesligation of RAR. Alternatively, in view of the observations thatCRABP-II associates with RAR in a ligand-dependent fash-ion, it could be argued that this protein acts by recruitingadditional accessory proteins that, in turn, activate transcrip-tion, i.e., that CRABP-II potentiates the activity of RAR by amechanism similar to those of bona fide transcriptional coac-tivators. To distinguish between these possibilities, we againused the synthetic ligand CD270, a compound that inducesnuclear localization of CRABP-II (Fig. 1), promotes the asso-ciation of the binding protein with RAR (Fig. 3), and functionsas a RAR activator (Fig. 4). Nevertheless, in the presence ofCD270, CRABP-II was unable to enhance the transcriptionalactivity of RAR. We note that the binding characteristics ofCD270 dictate that, in its presence, a larger fraction ofCRABP-II will remain liganded, prolonging the lifetime of theCRABP-II–RAR complex. Hence, if CRABP-II functioned asa coactivator, it would be expected that CD270 would enhancerather than inhibit the ability of the protein to augment tran-scriptional rates. The inhibitory effect of CD270 on CRABP-IIactivity hence unequivocally demonstrates that ligand channel-ing followed by rapid dissociation of the CRABP-II–RARcomplex are key in allowing CRABP-II to potentiate the ac-tivity of RAR.

The augmentation of the transcriptional activity of RAR byCRABP-II raises the important question of under what phys-iological conditions this activity becomes important. The ques-tion is further accentuated by the report that CRABP-II-nullmice are viable and fertile and that they show only minordevelopmental defects (22). It should be noted in regard to thisthat our data do not indicate that CRABP-II is absolutelyessential for RA-induced RAR transcriptional activity. Clearly,RAR can be activated by its ligand in the absence of this

binding protein, at least in some cells (Fig. 4). The observa-tions described here do however demonstrate that the poten-tiating activity of CRABP-II is especially significant when cel-lular levels of either RA or RAR are limiting, i.e., underconditions that necessitate enhanced efficiency of ligand deliv-ery (Fig. 5). The results therefore suggest that while CRABP-IIis dispensable at high RA concentrations, it may become es-sential for RAR activity at nanomolar concentrations (Fig. 5b).A prediction that can be derived from our observations is thatmice lacking CRABP-II will display a revealing phenotypeunder conditions of RA deficiency.

In contrast with COS-7 cells, CRABP-II appears to be crit-ical for enabling RAR in MCF-7 cells to properly respond toRA even at high RA concentrations (Fig. 7). The basis for thisremarkable difference between the two cell types remains to beclarified but one possibility is that, unlike in COS-7 cells, RA inMCF-7 cells is either rapidly degraded or rapidly secreted. Insuch a situation, CRABP-II may rescue RA activity by shut-tling it to the nucleus, thereby removing it from compartmentswhere it can be metabolized and protecting it from plasmamembrane transporters. It is worth noting in regard to this thatvarious carcinomas, including MCF-7 cells (10), develop drugresistance which is due, in part, to the up-regulation of energy-dependent efflux pumps that are members of the family ofABC transporters. In such cells, the ability of CRABP-II torapidly transport RA into the nucleus may be critical for main-taining the cellular response towards this hormone.

To explore the physiological consequences of the function ofCRABP-II revealed by these studies, the effect of the proteinon the ability of RA to induce growth arrest in mammarycarcinoma cell lines was investigated. This important biologicalactivity of RA is known to be mediated by retinoid receptors(7, 11, 18) and may therefore respond to the presence ofCRABP-II. The effect of varying the level of CRABP expres-sion on RA-induced growth inhibition was investigated usingthe RA-sensitive mammary carcinoma cell line MCF-7 (6).The data demonstrated that, unlike the parental counterpart,MCF-7 cells in which the expression of CRABP-II is dimin-ished by antisense methodology display a marked RA resis-tance. On the other hand, MCF-7 cells were dramatically sen-sitized to RA-induced growth arrest upon overexpression ofthe binding protein. Hence, the ability of CRABP-II to poten-tiate the activity of RAR has important consequences for thebiological functions of this receptor in cells. Interestingly, twostudies previously pointed at possible relationships betweenCRABP-II and the antiproliferative activities of RA in carci-noma cells. It was reported that ectopic expression of thisprotein enhances RA-induced transcriptional activation andcellular response to RA in some mammary carcinoma cell lines(20). It was also shown that decreased expression of CRABP-IIin SCC25 cells renders these cells less sensitive to RA-medi-ated inhibition of proliferation (29). The results of the presentwork shed light on the mechanisms that underlie these effects.It remains to be seen whether CRABP-II similarly affects bi-ological activities of RA other than growth inhibition, for ex-ample, induction of cell differentiation. It is worth noting thatit has been reported that, during embryonal development, ex-pression of CRABP-II is elevated in cells that actively synthe-size RA (3, 33, 35). In view of the present findings, theseobservations appear to reflect that the activity of RA during



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development requires both an increase in RA synthesis and anup-regulation of CRABP-II, allowing for rapid activation ofRAR.

While CRABP-II dramatically modulated the growth inhib-itory activity of RA in MCF-7 cells, neither overexpression norabolishment of CRABP-I had any discernible effects on thisactivity. These observations were somewhat surprising in viewof the reports that, in F9 cells, increased expression ofCRABP-I is accompanied by faster rates of RA degradationand a lowered sensitivity to RA-induced differentiation (1, 2).The subcellular location of CRABP-I supports the notion thatthis protein acts in the extranuclear milieu, and the observa-tions that its overexpression somewhat inhibited the transcrip-tional activity of RAR are in accordance with the hypothesisthat it functions to moderate cellular responses to RA. Nev-ertheless, the lack of effect on RA-induced inhibition of growthof MCF-7 cells upon alteration of the level of CRABP-I raisesthe possibility that this protein may play a different role inMCF-7 than in F9 cells. Hence, while the mechanism of actionof CRABP-II is clearly established by the present study, thefunction(s) of CRABP-I remains to be elucidated.

ACKNOWLEDGMENTS

We are very grateful to Uwe Reichert for providing the RA deriv-ative CD270 and to David Ong and Pierre Chambon for constructs andantibodies.

This work was supported by grant CA68150 from the NIH. A.B. wassupported by grant 5-T32-DK07158 from the NIH.

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