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Cellular retinoic acid-binding protein II (CRABP-II) is an intracellular lipid-binding protein that associates with retinoic acid with a subnanomolar affinity. We previously showed that CRABP-II enhances the transcriptional 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 mechanisms underlying the effects of CRABP-II on the transcriptional activity of RAR and the functional consequences of these effects were studied. We show that CRABP-II, a predominantly cytosolic protein, massively undergoes nuclear 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 data establish that potentiation of the transcriptional activity of RAR stems directly from the ability of CRABP-II to channel retinoic acid to the receptor. We demonstrate further that overexpression of CRABP-II in MCF-7 mammary 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, the data unequivocally establish the function of CRABP-II in modulating the RAR-mediated biological activities of retinoic acid.
The vitamin A metabolite retinoic acid (RA) regulates multiple biological processes, including cell proliferation and differentiation, by virtue of its ability to modulate the rate of transcription of numerous target genes. The transcriptional activities of this hormone are mediated by two members of the nuclear hormone receptor superfamily: the retinoid X receptor (RXR), which is activated by the 9-cis isomer of RA, and the retinoic acid receptor (RAR), which responds to both 9-cis and all-trans RA. These retinoid receptors bind to specific response elements in the promoter regions of target genes and function as ligand-inducible transcription factors (5, 23, 25). RXR can bind to DNA and activate transcription as a homodimer. In contrast, tight binding to DNA and transcriptional activation by RAR usually occurs through heterodimerization with RXR (12, 17, 23, 34). Upon ligand binding, RAR-RXR heterodimers recruit multicomponent coactivator complexes that, in turn, remodel chromatin and bridge to the general transcription 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 proteins (CRABP-I and CRABP-II). These proteins are members of the family of intracellular lipid-binding proteins which also includes the cellular retinol-binding proteins and nine known isotypes of fatty acid-binding proteins (26, 27). Intracellular lipid-binding proteins share a highly conserved three-dimensional structure which allows them to bind specific hydrophobic ligands within an antiparallel β-barrel constructed of two orthogonal, five-stranded β-sheets. Interestingly, the crystal structures of holo-CRABP-I and CRABP-II indicate that access to the entrance of the ligand-binding pockets of these proteins is restricted (21). These observations suggest that significant conformational changes may be required to enable the release of RA from the binding pocket, and they raise the question of how such rearrangements are induced to allow the ligand to reach its sites of metabolism or action. The two CRABPs exhibit distinct patterns of expression across different cells and developmental stages. In the adult, CRABP-I is widely expressed, while the expression of CRABP-II is restricted to skin (27), testis, uterus and ovary (30, 35, 36), and the choroid plexus (33). Both CRABPs are widely expressed in the embryo, but they do not usually coexist in the same cells (24). These differential expression profiles, combined with the high 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 to solubilize and transport their lipophilic ligand in the aqueous phase of cytosol. Some studies suggest, however, that in addition to these general functions the CRABPs may have specific roles in mediating RA action. It was reported that elevated CRABP-I expression in F9 teratocarcinoma cells enhances the rate of formation of polar metabolites of RA and decreases the sensitivity of these cells to RA-induced differentiation. It was consequently proposed that CRABP-I acts to moderate the cellular response to RA by enhancing the activity of an enzyme(s) that catalyzes RA degradation (1, 2). On the other hand, available information implicates CRABP-II in a functional interplay with the nuclear receptor with which it shares a common ligand, RAR.
In support of such a function for CRABP-II, we and others recently reported that overexpression of this isotype enhances the transcription of a reporter gene driven by a RAR response element in cells (8, 9). We showed further that this activity is specific for CRABP-II and is not shared by CRABP-I (9). To investigate the molecular basis for the functional differences between the two proteins that allow CRABP-II, but not CRABP-I, to augment the transcriptional activity of RAR, we examined the kinetic parameters of the process by which RA moves from either binding protein to the receptor. These in vitro studies revealed that the mechanisms by which RA transfers from the two CRABPs to RAR are fundamentally different. Movement of RA from CRABP-I was found to proceed by dissociation of the ligand from the binding protein to the aqueous phase, followed by its association with RAR. In contrast, the data indicated that RA transfers from CRABP-II to RAR through collision-mediated channeling, a process that bypasses the bulk aqueous phase and that results in facilitation of the formation of the holo-receptor. We thus suggested that ligand channeling mediated by CRABP-II underlies the ability of this protein to potentiate the transcriptional activity of RAR (9). Additional studies have focused on structural determinants that underlie CRABP-II to facilitate the delivery of RA to RAR. These studies identified a surface region, positioned just above the entrance to the ligand-binding pocket of CRABP-II, that displays an electric potential which is remarkably 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 of RA from CRABP-II to RAR and for enhancing the transcriptional activity of the receptor. The patch thus comprises the RAR interaction domain of CRABP-II (4).
Critical questions remain regarding the hypothesis that CRABP-II functions to potentiate the transcriptional activity of RAR and that it does so through direct channeling of RA to the receptor. Importantly, a requisite of this hypothesis is that CRABP-II and RAR colocalize in the same cellular compartment at least under some conditions. The activities of RAR are exerted in the nucleus. However, although it has been suggested that a fraction of both CRABP-I and CRABP-II may be present in the nuclei of various cells (14), these proteins are usually thought to be predominantly cytoplasmic (27). Another difficulty in establishing the proposed role of CRABP-II is that our attempts to capture the putative complex between CRABP-II and RAR failed despite utilization of multiple experimental approaches, including coprecipitation assays, covalent cross-linking, and fluorescence anisotropy studies (9). We thus suggested that the CRABP-II-RAR complex that mediates RA channeling is a short-lived intermediate that rapidly dissociates following completion of transfer (9). It should be noted that our failure to visualize a CRABP-II-RAR complex conflicts with the report that CRABP-II and RARα could be coimmunoprecipitated from whole-cell extracts (8). However, in that report, the interactions between CRABP-II and RAR appeared to have occurred in a ligand-independent fashion, and similar interactions were observed between CRABP-II and RXR, a receptor that does not share a ligand with this binding protein (13). The significance of these ostensible interactions is thus unclear. In any event, it was suggested based on these observations that CRABP-II acts as a coactivator for retinoid receptors (8), a suggestion that implies that the protein may enhance the transcriptional activity of RAR by binding to the receptor and perhaps recruiting additional accessory proteins. Hence, it remains to be clarified whether the ability of CRABP-II to channel RA to RAR is key in allowing this protein to augment the receptor's activity, or whether the binding protein functions by a different mechanism. The observations that CRABP-II enhances transcriptional activation by RA also raise important questions regarding the biological significance of this activity. This issue is especially pertinent in view of the report that CRABP-II-null mice are viable and fertile and that they show only minor defects in limb development, implying that the protein is dispensable under standard laboratory conditions (22). Nevertheless, considering its strict evolutionary conservation, it is reasonable to presume that the activity of CRABP-II will be essential at least under some physiological conditions that are yet to be defined.
The study reported here was undertaken in order to elucidate the molecular mechanisms through which CRABP-II augments the transcriptional activity of RAR and to clarify the significance of the presence of this protein in cells for the biological activities of RA. We show that CRABP-II is predominantly cytosolic in the absence of ligand, but that it undergoes 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-lived enough to allow its visualization only in the presence of a ligand that does not readily move from the binding protein to the receptor. The data further establish that augmentation of the transcriptional activity of RAR stems directly from the ability of CRABP-II to channel ligands to the receptor and that this potentiation activity is important only when cellular levels of either RAR or RA are limiting, at least in some cells. Finally, we demonstrate that CRABP-II governs the sensitivity of a mammary carcinoma line to RA-induced growth inhibition. Taken together, these observations unequivocally establish the function of CRABP-II in modulating the RAR-mediated biological activities of RA.
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 in amber vials at −80°C.
Antibodies against mCRABP-I were purchased from Affinity Bioreagents (Golden, Colo.), and anti-mCRABP-II (5CRA3B3) antibodies were a gift from Pierre Chambon (IGMCB, Strasbourg, France). Antibodies against actin were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Anti-mouse and anti-goat immunoglobulin horseradish peroxidase-conjugated antibodies were from Amersham (Arlington Heights, Ill.). Primary and secondary antibodies 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.).
hRARα-ligand-binding domain (hRARα-LBD) (amino acids 145 to 390) was prepared by PCR amplification using full-length hRARα as a template. The PCR product was subcloned into the NdeI-XhoI sites of the bacterial expression vector pET28a. The plasmid was amplified in Escherichia coli strain DH5α and transfected into E. coli BL21 for protein expression. E. coli cells harboring 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-β-d-thiogalactopyranoside, and cells were grown for an additional 3 h and pelleted by centrifugation. Following lysis, protein was purified by metal-chelating affinity chromatography. Purified hRARα-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 concentration determinations. The bacterial expression vector for bCRABP-II was a generous gift of David Ong (Vanderbilt University). This protein was purified as previously described (19).
MCF-7 cells were purchased from the American Type Culture Collection (Manassas, Va.). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Stably transfected cell lines were also supplemented with 200 μg of G418/ml (Clontech, Palo Alto, Calif.) and/or 100 μg of hygromycin/ml (Invitrogen, Carlsbad, Calif.). During transactivation experiments, MCF-7 cells overexpressing CRABP-II were cultured in DMEM supplemented with 1 μg of doxycyline/ml.
To generate hCRABP-II and hCRABP-I tagged with green fluorescence protein (GFP), EcoRI and BamHI sites were introduced by PCR and the resulting products were ligated into pEGFP-C2 (Clontech).
COS-7 cells were seeded on glass coverslip chambers (Nalge Nunc Intl., Vernon Hill, Ill.) and transfected with GFP-tagged hCRABP (1 μg). Twenty-four hours after transfection, cells were treated with DMSO (control), RA (20 nM), or CD270 (20 nM). Three hours following ligand addition, images of GFP-tagged proteins were collected by using an Olympus BX-50 fluorescent microscope (20× objective) using the Metamorph acquisition and image analysis software package from Universal Imaging. For RA-pulse experiments, cells were treated with RA for 3 h and then incubated in fresh medium without RA for an additional 3-h period. For immunofluorescence staining, 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 with 0.2% Triton X-100 at 25°C for 10 min. Cells were then washed with PBS and incubated with antibodies against CRABP-I or -II (dilution, 1/50) at 37°C for 30 min. After three washes in PBS (10 min each), cells were incubated with Texas Red-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 fluorescence microscopy as detailed above.
Purified bCRABP-II (7.5 μM) was precomplexed with RA or CD270 for 5 min at 4°C. The holo-protein was then incubated with hRARα-LBD (7.5 μM) immobilized on Ni+2-NTA-agarose beads for 15 min at 4°C. Mixtures were centrifuged, pelleted beads were extensively washed with a buffer containing 50 mM Tris (pH 7.5), 0.3 M NaCl, 2 mM phenylmethylsulfonyl fluoride, 10 μg of leupeptin/ml, and 10 μg of aprotinin/ml, and proteins were resolved by SDS-PAGE and visualized by Coomassie blue staining.
COS-7 cells were seeded on six-well plates and transfected with a pSG5 vector harboring the cDNA for hCRABP-I or -II (1 μg) together with a DR5-tk-luciferase reporter plasmid (1 μg) and pCH110 (internal standard, 0.3 μg). In some experiments, cells were also cotransfected with pSG5 expression vectors for hRXRα and hRARα (0.1 μg each). At 24 h after transfection, RA (20 nM) or CD270 (100 nM) was added singly or in combination to cells. In other experiments, MCF-7 parental cells as well as stable clones expressing either diminished levels of CRABP-II or overexpressing CRABP-II were seeded on six-well plates and transfected with a DR5-tk-luciferase reporter plasmid (1 μg) and pCH110 (internal standard, 0.3 μg). At 24 h after transfection, RA (2, 20, 200, or 2,000 nM) was added and cells were incubated at 37°C for 24 h. Expression of the reporter was measured by the activity of luciferase using the luciferase assay system (Promega), following the instructions of the manufacturer. Luciferase activity was corrected for transfection efficiency based on the activity of β-galactosidase as measured by standard procedures.
To generate antisense constructs for CRABP-I or -II, EcoRI and HindIII sites were introduced to the respective cDNAs by PCR and the resulting product was ligated in the reverse orientation to the mammalian expression vector pJ4H. To generate cell lines stably expressing the antisense constructs, MCF-7 cells were transfected with the respective antisense CRABP construct along with a pTK-Hyg vector (Clontech) at a 10:1 (CRABP/Hyg) ratio. Cells harboring these constructs were selected in DMEM supplemented with 10% FBS and 100 μg of hygromycin/ml. Individual clones were screened by Western blotting to select those displaying minimal expression of CRABP-I or -II.
Conditional overexpressing cell lines for CRABP-I or CRABP-II were generated using Clontech's pTET-ON system. Briefly, BamHI and XbaI sites were introduced to CRABP-I or -II by PCR and the resulting product was ligated to the pTRE2 vector. MCF-7 cells were transfected with the pTET-ON vector, and cells harboring this vector were selected in DMEM supplemented with 10% FBS and 200 μg of G418/ml. pTET-ON clones were selected by transient transfection with pTRE2-luciferase followed by measurement of reporter gene activity by the luciferase assay system. Clones showing over 20-fold induction of luciferase activity in the presence of 1 μg of doxycycline/ml were selected and cotransfected with either CRABP-I-pTRE2 or CRABP-II-pTRE2 along with pTK-Hyg (CRABP-pTRE2/pTK-Hyg ratio, 10:1), followed by selection in DMEM containing 10% FBS, 200 μg of G418/ml, and 100 μg of hygromycin/ml. Each clone was grown on 100-mm plates in the absence or presence of 1 μg of doxycycline/ml for 48 h, followed by cell lysis. Clones were screened by Western blotting for doxycycline-induced overexpression of either CRABP-I or CRABP-II.
Cells were cultured in 100-mm plates to confluency, washed with PBS, and lysed in lysis buffer (10 mM potassium phosphate [pH 7.5], 0.5% [wt/vol] Triton X-100, 10 μg of leupeptin/ml, 10 μg of aprotinin/ml). Following centrifugation, protein concentrations in cell lysates were determined by the Bradford assay (Bio-Rad). Equivalent total protein amounts were loaded in each lane of an SDS-12% PAGE gel, followed by transfer and blocking in 5% nonfat dry milk. Blots were probed with appropriate primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibody. To strip blots, membranes were incubated in stripping solution (62.5 mM Tris [pH 6.7], 100 mM β-mercaptoethanol, 2% [wt/vol] SDS) for 30 min at 50°C followed by extensive washes in TBST (50 mM Tris [pH 7.5], 0.9% NaCl, 1% Tween 20) and reprobed with the appropriate primary and secondary antibodies. Antibody-antigen complexes were detected by enhanced chemiluminescence according to the manufacturer's protocol (Amersham).
Cells were seeded on 96-well plates (1,000 cells/well) and allowed to grow for 24 h. DMSO (control) or RA was added at various concentrations and supplemented to fresh media every 48 h. On the day of the assay, cells in each well were treated with 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent and incubated for an additional 4-h period, followed by overnight incubation with 100 μl of solubilization buffer (Roche Diagnostics). Absorbance at 570 nm was measured using an enzyme-linked immunosorbent assay reader.
A prediction of the hypothesis that CRABP-II enhances the transcriptional activity of RAR through direct protein-protein interactions is that the two proteins will colocalize in the same cellular compartment under conditions where RAR becomes activated. The observations that CRABP-II channels RA to RAR, while CRABP-I does not (9), further suggest that cocompartmentalization with RAR may be a specific property of the former but not the latter binding protein. To explore these predictions, the subcellular localization of the two CRABP isotypes was determined. GFP-tagged constructs of CRABP-I and -II were generated and transfected into COS-7 cells, and their location was determined by direct fluorescence microscopy. GFP-CRABP-I was predominantly present in the cytoplasm, both in the absence and presence of RA. In fact, this protein appeared to be excluded from the nucleus (Fig. 1a and b). In contrast, CRABP-II which, similarly, was confined to the cytoplasm in the absence of ligand, massively located into the nucleus upon addition of RA (Fig. 1e and f). Removal of RA from the medium resulted in repartitioning of CRABP-II into the cytosol (Fig. (Fig.1g),1g), indicating that movement of CRABP-II into the nucleus is a reversible process regulated by the availability of ligand. To examine whether the nuclear translocation of CRABP-II is a specific response to RA, a high-affinity RAR ligand, or whether it is a consequence of the ligation of CRABP-II, we utilized a synthetic ligand, termed CD270, containing a substituted benzo[b]thiophene carboxylic acid. This ligand was reported to interact with CRABP with a high affinity but to be a poor RARα ligand (15). Similarly to RA, treatment of the cells with CD270 did not affect the subcellular localization of CRABP-I (Fig. (Fig.1d)1d) but efficiently induced movement of CRABP-II into the nucleus (Fig. (Fig.1h).1h). To verify that the observed ligand-induced nuclear translocation of CRABP-II is not an artifact emanating from the addition of the GFP tag, the subcellular location of endogenous CRABP-II in COS-7 cells was also examined by immunofluorescence microscopy using CRABP type-selective antibodies. The data of these experiments showed that, similarly to the behavior of ectopically expressed GFP-tagged proteins, endogenous CRABP-I was cytoplasmic both in the absence and presence of RA (Fig. 2a and c), while endogenous CRABP-II translocated from the cytoplasm to the nucleus in the presence of RA (Fig. 2b and d).
Our previous observations provided kinetic evidence for direct interactions between CRABP-II and RARα. However, our attempts to capture the CRABP-II-RAR complex failed despite the application of multiple experimental approaches. We thus suggested that CRABP-II and RAR interact transiently and that, following movement of RA from the binding protein to the receptor, the complex rapidly dissociates (9). In other words, holo-CRABP-II associates with apo-RAR, and the ensuing movement of RA from the binding protein to the receptor (accompanied by dramatic conformational changes in RAR [28, 31]) results in rapid dissociation of the ligand-depleted CRABP-II from the liganded receptor. If so, it is possible that the CRABP-II-RAR complex would be stabilized in the presence of a ligand that does not readily channel between CRABP-II and RAR. To examine this postulate, we used the synthetic ligand CD270, which binds to CRABP with a Kd of 6 nM, displaying more than 2 orders of magnitude higher affinity for the binding protein versus that for RAR (15). Due to these binding characteristics, CD270 will not readily partition from CRABP-II to RAR, allowing a significant fraction of the binding protein to remain liganded in the presence of apo-RAR, resulting in stabilization of the CRABP-II-RAR complex and perhaps enabling its visualization.
Potential physical interactions between CRABP-II with RAR were investigated by coprecipitation assays that were carried out in the absence or presence of either RA or CD270. In these experiments, RAR-LBD, the region of the receptor that mediates its interaction with CRABP-II, was used as a bait to capture CRABP-II. Bacterially expressed histidine-tagged RAR-LBD was immobilized on Ni+2-NTA-agarose beads and incubated with CRABP-II in the absence or presence of ligands. Beads were centrifuged and washed, and precipitated proteins were analyzed by SDS-PAGE and visualized by Coomassie blue staining (Fig. (Fig.3).3). In agreement with the results of our previous studies carried out using RARΔAB as a bait, CRABP-II did not coprecipitate with the RAR-LBD either in the absence of ligand (Fig. (Fig.3,3, lane 3) or in the presence of saturating concentrations of RA (Fig. (Fig.3,3, lanes 4 and 5). However, CRABP-II associated with the receptor in the presence of CD270 and did so in a dose-dependent manner (Fig. (Fig.3,3, lanes 6 and 7). The stabilization of the CRABP-II-RAR complex in the presence of CD270 demonstrates that CRABP-II interacts with RAR in a ligand-dependent fashion. Importantly, these observations provide strong support for the notion that the inability to visualize this complex in the presence of RA stemmed from the short half-life of the complex when induced to form by a ligand that is rapidly channeled from the binding protein to the receptor. Furthermore, the data reveal that the interaction between CRABP-II and RAR are mediated via the LBD of RAR, establishing a novel role for this region of the receptor.
The kinetic evidence that CRABP-II channels RA to RAR suggested that this activity may underlie the ability of the binding protein to enhance the transcriptional 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 additional accessory proteins; that is, that it acts in a manner similar to known transcriptional coactivators. Utilization of the ligand CD270 provides a unique opportunity to further clarify the molecular mechanism underlying CRABP-II function. This compound induces nuclear localization of CRABP-II (Fig. (Fig.1h),1h), gives rise to a CRABP-II-RAR complex (Fig. (Fig.3),3), and also binds to RAR, albeit at a low affinity (15). However, due to its binding characteristics, CD270 does not appear to efficiently move from CRABP-II to RAR. It was thus of interest to examine whether the RAR-potentiating activity of CRABP-II, which was observed in the presence of RA, will be retained in the presence of CD270.
To address this issue, transactivation assays were carried out. COS-7 cells were cotransfected with CRABP-II and a luciferase reporter construct driven by a RAR response element. Cells were treated with RA, or CD270, or a combination of the two ligands and assayed for ligand-induced expression of the reporter (Fig. (Fig.4).4). RA enhanced the transcriptional activity of RAR, and this activity was markedly augmented upon cotransfection of CRABP-II. CD270 functioned as an activator of RAR although at lower efficiency than RA. In contrast with the effect of CRABP-II on the transcriptional activity of RAR induced by RA, cotransfection of the binding protein had no effect on CD270-induced transactivation. Furthermore, CD270 abolished the ability of CRABP-II to enhance the transcriptional activity of RAR in the presence of RA. Similar results were obtained at various RA/CD270 ratios in the 5/1 to 50/1 range (data not shown). The loss of the RAR-potentiating activity of CRABP-II in the presence of CD270 clearly demonstrates that the ability to channel ligands to RAR is critical for the effect of the binding protein on transcriptional rates.
An important question that arises from the observations that CRABP-II potentiates RA-induced, RAR-mediated transcriptional activation relates to the physiological conditions under which this activity becomes important. To address this question, the effect of CRABP-II on the RA-dependent activity of RAR was investigated by transactivation assays carried out under two sets of conditions: in the absence of ectopic expression of RAR, i.e., relying on the (limiting) endogenous level of RAR in the cells; and upon overexpression of the receptor. In the latter set of experiments, cells were cotransfected with expression vectors for both hRXRα and hRARα. The data (Fig. (Fig.5a)5a) show that, in the presence of endogenous levels of RAR, CRABP-II markedly enhanced the receptor's transcriptional activity. As expected, overexpression of retinoid receptors significantly increased the transcriptional activity. However, under these conditions, CRABP-II did not enhance the transcriptional activity of RAR further. Similar results were obtained when cells were transfected with an expression vector for hRARα, but not with the hRXRα expression vector (data not shown). In another set of transactivation assays, the range of RA doses in which the activity of RAR is responsive to the presence of CRABP-II was studied. The results of these experiments (Fig. (Fig.5b)5b) revealed that expression of CRABP-II markedly enhanced the transcriptional activity of RAR at low concentrations of RA, but that the ability of the protein to augment transcriptional rates was rapidly lost when the concentration of the ligand was raised. It could be argued that, at high ligand concentrations, CRABP-II did not affect transcriptional rates because the reporter vector activity became saturated. However, the data indicate that CRABP-II markedly lost its potentiating activity upon elevation of RA concentrations even under conditions that are far from the observed maximal activity (Fig. (Fig.5b,5b, inset). It may also be argued that the effect of CRABP-II at low RA concentrations is due simply to its ability to solubilize the hydrophobic ligand, thereby preventing its accumulation in membranes and maintaining its accessibility to the receptor. However, if this were the case, another cellular protein with a similar affinity for RA, such as CRABP-I, would have a similar enhancing effect, a prediction that is not borne out by the data (Fig. (Fig.5a).5a). These results thus substantiate that, in COS-7 cells, CRABP-II is efficacious in enhancing RAR activity but only at low, physiological (nanomolar) ligand concentrations.
The effects of CRABP-II on the transcriptional activity of RAR were further investigated using MCF-7 cells, a mammary carcinoma cell line which is known to be responsive to the antiproliferative activity of RA. Western blot analyses showed that both CRABP-I and CRABP-II are present in MCF-7 cells (Fig. (Fig.6a).6a). Several derivatives of the parental MCF-7 cells were then generated. The expression of either CRABP-I or CRABP-II was minimized by generating lines that stably express an antisense construct for either of these isotypes (see Materials and Methods for details). Western blotting of the newly generated lines indicated that the antisense constructs successfully reduced the expression of the respective CRABPs to undetectable levels. These analyses also verified that the constructs acted with strict specificity to inhibit only the expression of the CRABP isotype towards which they were directed (Fig. (Fig.6a).6a). In addition, MCF-7 cell lines that overexpress either CRABP-I or CRABP-II were generated. In these lines, overexpression of the respective binding proteins could be induced at will by addition of doxycycline (see Materials and Methods). Western blotting verified that expression levels of the two CRABPs in the cells stably harboring the constructs were similar to those observed in the parent MCF-7 cells in the absence of doxycycline and that expression of the respective CRABPs was dramatically elevated upon treatment with doxycycline (Fig. (Fig.6b6b).
The effects of varying the expression level of CRABP-II on the transcriptional activity of RAR were then studied by transactivation assays carried out in the parental MCF-7 cells and their derivatives that either under- or overexpress this protein (Fig. (Fig.7).7). The transcriptional activity of RAR in the parental MCF-7 cells was enhanced in a dose-dependent fashion upon addition of RA. In contrast, in cells with diminished levels of CRABP-II, RAR was inactive even at RA concentrations as high as 2 μM. The activity of RAR in cells that stably overexpress CRABP-II was dramatically enhanced compared to that observed in the parental cell line. In contrast with COS-7 cells, the ability of CRABP-II to augment RA-induced, RAR-mediated transcriptional rates in MCF-7 cells was not diminished upon increasing ligand concentration, suggesting important differences in RA homeostasis between COS-7 and MCF-cells (see Discussion).
The augmentation of RAR activity by CRABP-II raises the possibility that this protein will sensitize cells to RAR-mediated biological activities of RA. To explore this possibility, we investigated the role of CRABP-II in modulating an important activity of RA which is known to be mediated by retinoid receptors, namely, the induction of growth arrest in mammary carcinoma cells (7, 11, 18). To this end, the ability of RA to induce growth arrest in the various MCF-7 cell lines that express different levels of CRABP was examined by MTT growth assays (Fig. (Fig.8).8). RA inhibited the growth of the parental MCF-7 cells in a dose-dependent manner with a 50% effective concentration (EC50) of about 30 nM (Fig. (Fig.8).8). Cell lines that either underexpress (Fig. (Fig.8a)8a) or overexpress (Fig. (Fig.8b)8b) CRABP-I responded to RA in a manner similar to that observed in the parental cells. In contrast, the RA dose-response curve in cell lines in which the expression of CRABP-II was diminished was markedly shifted to higher concentrations (Fig. (Fig.8a).8a). These cells thus displayed a pronounced RA resistance compared to the parental MCF-7 cells. Clones harboring the conditional constructs for CRABP-II behaved similarly to the parental cells in the absence of doxycycline (data not shown). However, in the presence of doxycycline, the RA dose response of these cells was dramatically shifted to lower concentrations and displayed an EC50 of 2 nM RA, i.e., about an order of magnitude lower than the value observed in the parental cells (Fig. (Fig.8b).8b). Hence, while CRABP-I does not affect the RA responsiveness of MCF-7 cells, CRABP-II plays an important role in modulating the response of these cells to the antiproliferative effects of RA.
We previously showed that, despite the high homology and the similarities of the RA-binding affinities of CRABP-I and CRABP-II, the latter, but not the former, enhances the transcriptional activity of the nuclear receptor RAR. In vitro kinetic analyses further demonstrated that RA moves to RAR from CRABP-II (but not from CRABP-I) in a collision-mediated process that facilitates the formation of the liganded receptor. We thus suggested that ligand channeling by CRABP-II may underlie the effects of the binding protein on RAR-mediated transcriptional regulation (9). Here, we set out to investigate the mechanism by which CRABP-II augments the transcriptional activity of RAR and the functional consequences of the presence of this protein in cells for the biological activities of RA. Taken together, the data establish that CRABP-II functions through the sequence of events schematically depicted in Fig. Fig.9.9. The protein is predominantly cytosolic in the absence of ligand. While in cytosol, CRABP-II binds RA when it becomes available (either by influx from serum or through the activity of retinal dehydrogenases intracellularly). Ligand binding induces nuclear localization of CRABP-II. In the nucleus, holo-CRABP-II associates with apo-RAR to form a complex that mediates direct channeling of RA and facilitates the ligation of the receptor. Subsequently, the ligand-depleted CRABP-II rapidly dissociates from holo-RAR, releasing the receptor to its transcriptional activities.
The observations that the cellular distribution of CRABP-II is dramatically shifted into the nucleus upon exposure to RA raise interesting questions relating to the mechanism by which CRABP-II is targeted to the nucleus when liganded. Analysis of the sequence of this protein fails to identify an obvious nuclear localization signal. It is possible that, due to its small size, CRABP-II enters the nucleus by simple diffusion. However, the translocation may involve accessory proteins that regulate either the nuclear exclusion of the apo-CRABP-II or the nuclear entry of the holo-protein. How the subcellular distribution of CRABP-II is regulated thus remains to be elucidated.
Although our previously reported kinetic analyses provided strong evidence for direct interactions between CRABP-II and RAR, our attempts to visualize a CRABP-II-RAR complex failed despite utilization of multiple experimental approaches, including coimmunoprecipitation of endogenous proteins (9). We therefore suggested that the complex is a short-lived intermediate (9), i.e., that holo-CRABP-II binds to apo-RAR, and that following movement of RA from the binding protein to the receptor (accompanied by dramatic conformational changes in RAR [28, 31]), the ligand-depleted CRABP-II rapidly dissociates from the liganded receptor. To examine this postulate, we used the synthetic ligand CD270, which displays a 200-fold-lower affinity towards RARα than does CRABP (15). Due to these binding characteristics, CD270 does not readily partition from CRABP-II to RAR, allowing for retention of a significant fraction of holo-CRABP-II in the presence of apo-RAR. Coprecipitation experiments demonstrated that, although the CRABP-II-RAR-LBD complex was not observed in the presence of RA, a robust complex formed upon incubation of the two proteins with CD270 and it did so in a dose-dependent manner (Fig. (Fig.3).3). The stabilization of the CRABP-II-RAR complex in the presence of CD270 demonstrate that CRABP-II indeed interacts with RAR in a ligand-dependent fashion. Importantly, these observations also provide strong support for the notion that the inability to visualize this complex in the presence of RA stems from the short half-life of the CRABP-II-RAR complex when induced to form by a ligand that is rapidly channeled.
Two alternative hypotheses may be postulated for understanding the molecular mechanism by which CRABP-II augments the transcriptional activity of RAR. It is possible that this activity stems from the transient association between the two proteins which mediates channeling of RA and facilitates ligation of RAR. Alternatively, in view of the observations that CRABP-II associates with RAR in a ligand-dependent fashion, it could be argued that this protein acts by recruiting additional accessory proteins that, in turn, activate transcription, i.e., that CRABP-II potentiates the activity of RAR by a mechanism similar to those of bona fide transcriptional coactivators. To distinguish between these possibilities, we again used the synthetic ligand CD270, a compound that induces nuclear localization of CRABP-II (Fig. (Fig.1),1), promotes the association of the binding protein with RAR (Fig. (Fig.3),3), and functions as a RAR activator (Fig. (Fig.4).4). Nevertheless, in the presence of CD270, CRABP-II was unable to enhance the transcriptional activity of RAR. We note that the binding characteristics of CD270 dictate that, in its presence, a larger fraction of CRABP-II will remain liganded, prolonging the lifetime of the CRABP-II-RAR complex. Hence, if CRABP-II functioned as a coactivator, it would be expected that CD270 would enhance rather than inhibit the ability of the protein to augment transcriptional rates. The inhibitory effect of CD270 on CRABP-II activity hence unequivocally demonstrates that ligand channeling followed by rapid dissociation of the CRABP-II-RAR complex are key in allowing CRABP-II to potentiate the activity of RAR.
The augmentation of the transcriptional activity of RAR by CRABP-II raises the important question of under what physiological conditions this activity becomes important. The question is further accentuated by the report that CRABP-II-null mice are viable and fertile and that they show only minor developmental defects (22). It should be noted in regard to this that our data do not indicate that CRABP-II is absolutely essential 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. (Fig.4).4). The observations described here do however demonstrate that the potentiating activity of CRABP-II is especially significant when cellular levels of either RA or RAR are limiting, i.e., under conditions that necessitate enhanced efficiency of ligand delivery (Fig. (Fig.5).5). The results therefore suggest that while CRABP-II is dispensable at high RA concentrations, it may become essential for RAR activity at nanomolar concentrations (Fig. (Fig.5b).5b). A prediction that can be derived from our observations is that mice lacking CRABP-II will display a revealing phenotype under conditions of RA deficiency.
In contrast with COS-7 cells, CRABP-II appears to be critical for enabling RAR in MCF-7 cells to properly respond to RA even at high RA concentrations (Fig. (Fig.7).7). The basis for this remarkable difference between the two cell types remains to be clarified but one possibility is that, unlike in COS-7 cells, RA in MCF-7 cells is either rapidly degraded or rapidly secreted. In such a situation, CRABP-II may rescue RA activity by shuttling it to the nucleus, thereby removing it from compartments where it can be metabolized and protecting it from plasma membrane transporters. It is worth noting in regard to this that various carcinomas, including MCF-7 cells (10), develop drug resistance which is due, in part, to the up-regulation of energy-dependent efflux pumps that are members of the family of ABC transporters. In such cells, the ability of CRABP-II to rapidly transport RA into the nucleus may be critical for maintaining the cellular response towards this hormone.
To explore the physiological consequences of the function of CRABP-II revealed by these studies, the effect of the protein on the ability of RA to induce growth arrest in mammary carcinoma cell lines was investigated. This important biological activity of RA is known to be mediated by retinoid receptors (7, 11, 18) and may therefore respond to the presence of CRABP-II. The effect of varying the level of CRABP expression on RA-induced growth inhibition was investigated using the 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 diminished by antisense methodology display a marked RA resistance. On the other hand, MCF-7 cells were dramatically sensitized to RA-induced growth arrest upon overexpression of the binding protein. Hence, the ability of CRABP-II to potentiate the activity of RAR has important consequences for the biological functions of this receptor in cells. Interestingly, two studies previously pointed at possible relationships between CRABP-II and the antiproliferative activities of RA in carcinoma cells. It was reported that ectopic expression of this protein enhances RA-induced transcriptional activation and cellular response to RA in some mammary carcinoma cell lines (20). It was also shown that decreased expression of CRABP-II in SCC25 cells renders these cells less sensitive to RA-mediated inhibition of proliferation (29). The results of the present work shed light on the mechanisms that underlie these effects. It remains to be seen whether CRABP-II similarly affects biological activities of RA other than growth inhibition, for example, induction of cell differentiation. It is worth noting that it has been reported that, during embryonal development, expression of CRABP-II is elevated in cells that actively synthesize RA (3, 33, 35). In view of the present findings, these observations appear to reflect that the activity of RA during development requires both an increase in RA synthesis and an up-regulation of CRABP-II, allowing for rapid activation of RAR.
While CRABP-II dramatically modulated the growth inhibitory activity of RA in MCF-7 cells, neither overexpression nor abolishment of CRABP-I had any discernible effects on this activity. These observations were somewhat surprising in view of the reports that, in F9 cells, increased expression of CRABP-I is accompanied by faster rates of RA degradation and a lowered sensitivity to RA-induced differentiation (1, 2). The subcellular location of CRABP-I supports the notion that this protein acts in the extranuclear milieu, and the observations that its overexpression somewhat inhibited the transcriptional activity of RAR are in accordance with the hypothesis that it functions to moderate cellular responses to RA. Nevertheless, the lack of effect on RA-induced inhibition of growth of MCF-7 cells upon alteration of the level of CRABP-I raises the possibility that this protein may play a different role in MCF-7 than in F9 cells. Hence, while the mechanism of action of CRABP-II is clearly established by the present study, the function(s) of CRABP-I remains to be elucidated.
We are very grateful to Uwe Reichert for providing the RA derivative CD270 and to David Ong and Pierre Chambon for constructs and antibodies.
This work was supported by grant CA68150 from the NIH. A.B. was supported by grant 5-T32-DK07158 from the NIH.