ARTICLE |
Correspondence to: Cécile RochetteEgly, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), BP 163, 67404 Illkirch Cedex, C.U. de Strasbourg, France..
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Summary |
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Apart from the retinoic acid nuclear receptor family, there are two low molecular weight (15 kD) cellular retinoic acid binding proteins, named CRABPI and II. Mouse monoclonal and rabbit polyclonal antibodies were raised against these proteins by using as antigens either synthetic peptides corresponding to amino acid sequences unique to CRABPI or CRABPII, or purified CRABP proteins expressed in E. coli. Antibodies specific for mouse and/or human CRABPI and CRABPII were obtained and characterized by immunocytochemistry and immunoblotting. They allowed the detection not only of CRABPI but also of CRABPII in both nuclear and cytosolic extracts from transfected COS-1 cells, mouse embryos, and various cell lines. (J Histochem Cytochem 46:11031111, 1998)
Key Words: cellular retinoic acid binding proteins, antibodies, immunodetection, nuclear localization
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Introduction |
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Retinoic acid (RA), the major active derivative of vitamin A, is indispensable for vertebrate development and homeostasis (
In addition to RA nuclear receptors, there exist two low molecular mass (~15 kD) cytoplasmic RA binding proteins named cellular retinoic acid binding proteins I and II (CRABPI and II). These proteins are found in all vertebrates (for reviews see
Up to now, the expression and distribution of CRABPs have been studied mainly at the mRNA level by in situ hybridization. Perhaps because of the difficulty in obtaining high-titer sensitive antisera to CRABPs, which are highly conserved among species, few studies report the expression of the corresponding proteins (
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Materials and Methods |
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Synthesis of Peptides, Preparation of Antisera, and Monoclonal Antibodies
Synthetic peptides corresponding to amino acids 95108 from mouse (m) CRABPI (SPB 63, CTQTLLEGDGPKTY) and CRABPII (SPB 64, CEQRLLKGEGPKTS) (
Expression Vectors and Transfections
The mCRABPI and mCRABPII pSG5 expression vectors (
Immunocytochemistry
Transfected COS cells were grown on Leighton tubes (Costar; Cambridge, MA), washed in PBS, pH 7, and fixed with either paraformaldehyde (2% PFA, pH 7.4) or Bouin's fixative (10% PFA, 5% acetic acid, 2% picric acid) for 4 min at room temperature (RT). Cells were incubated overnight at RT in a humidified chamber with the monoclonal antibodies against either CRABPI (4CRA3B1) or CRABPII (5CRA3B3) purified using caprylic acid and ammonium sulfate precipitation (
Cells, Extracts, and Immunoblotting
Mouse embryonal carcinoma cells (P19 cell line), human colorectal carcinoma cells (Caco-2 cell line), and human breast pleural metastasis cells (MCF7 cell line), were grown on petri dishes in appropriate medium (
Extracts were fractionated by SDS-PAGE (15% acrylamide) and electrotransferred onto nitrocellulose (NC) filters. After blocking in PBS3% nonfat powdered milk, the filters were immunoprobed with the specific antibodies for 2 h at 37°C. The polyclonal antibodies were diluted 1:200 and the monoclonal antibodies 1:1000. After extensive washing in PBS containing 0.05% Tween-20, the filters were then incubated for 30 min at RT with peroxidase-labeled anti-mouse immunoglobulins or protein A, diluted 1:10,000 (Amersham, Poole, UK). Specific complexes were revealed by chemiluminescence detection according to the manufacturer's protocol (Amersham). The specificity of the reactions was checked by depleting the antisera from the specific antibodies by incubation with the corresponding peptide (
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Results |
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Preparation and Characterization of Mouse Monoclonal Antibodies Against CRABPI and II
To generate specific mouse monoclonal antibodies, two peptides localized in the central part of mCRABPI (SPB 63) and mCRABPII (SPB 64) proteins ( except for 5CRA3B3, which was IgG2a
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The monoclonal antibodies were tested for their ability to detect by Western blotting the recombinant mCRABP proteins overexpressed in COS-1 cells. Figure 1A shows that 1CRA4C9 and 5CRA3B3 recognize mCRABPII (Figure 1, Lanes 2 and 4) as a single band migrating close to 15 kD, the expected molecular weight predicted from the cDNA sequence. No crossreactions were seen with extracts from mCRABPI-transfected COS-1 cells (Figure 1, Lanes 1 and 3), confirming that these antibodies are specific for CRABPII. In contrast, 3CRA10F5 and 4CRA3B1 recognize specifically mCRABPI (Figure 1, Lanes 58). Note that 3CRA10G2 recognize both mCRABPI and II proteins (Figure 1, Lanes 9 and 10). The fact that the 16 amino-acid sequence of mCRABPI, between amino acids 95 and 108 (SPB 63), differs from that of mCRABPII (SPB 64) by eight amino acids may explain such a crossreactivity, thus suggesting the existence of a common epitope between both proteins. The specificity of the signals was corroborated by using either extracts from COS-1 cells transfected with a control expression vector or antisera immunoabsorbed with the corresponding peptide (data not shown).
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Immunoblotting Detection of CRABPs in Mouse Embryos and Cultured Cell Lines Using the Monoclonal Antibodies
The monoclonal antibodies were used for detection of endogenous CRABP proteins by immunoblotting. First, to check the specificity of these antibodies, cytosolic extracts from 13.5 dpc CRABPI-/-/CRABPII-/- double mutant mice (
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Interestingly, both antibodies revealed a strong signal corresponding to mCRABPI or II, not only in the cytosol but also in the nucleus of 13.5 dpc mouse embryos (Figure 2B, upper panels; Lanes 6, 7, 13, and 14). Such a nuclear signal is specific because it was absent from nuclear extracts of CRABPI-/-/CRABPII-/- double mutant mice (data not shown). In addition, these signals disappeared when the monoclonal antibodies were immunodepleted with the cognate peptide (Figure 2B, lower panels).
1CRA4C9 and 3CRA10F5 also detected CRABPII and CRABPI proteins, respectively, in both cytoplasmic and nuclear extracts from mouse P19 cells (Figure 2B, Lanes 25 and 912). The signal corresponding to CRABPII was increased on treatment with RA (10-7 M) in both the cytosolic (Figure 3B, Lanes 2 and 4) and nuclear extracts (Figure 2B, Lanes 3 and 5), in agreement with RNA studies and the characterization of an RA response element in the promoter region of the mCRABPII gene (
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Then we checked whether these monoclonal antibodies were also able to recognize human CRABPs by using extracts from the human Caco-2 (Figure 3A and Figure 3B) and MCF7 (Figure 3C) cell lines. Unfortunately, 1CRA4C9 did not recognize human CRABPII in these cells (Figure 3A, Lane 2; and data not shown). However, 5CRA3B3, also raised against CRABPII, was able to detect the human protein in cytoplasmic extracts from Caco-2 cells (Figure 3A, Lane 4, and 3B, Lane 2). The monoclonal antibodies raised against CRABPI, 3CRA10F5, and 4CRA3B1 also recognized the human protein in Caco-2 cells (Figure 3A, Lane 6, and 3B, Lane 5). Finally, 3CRA10G2, which was found to recognize both mouse CRABPI and II, was also able to detect endogenous human CRABPs in these cells (Figure 3A, Lane 8). It must be stressed that, with our antibodies, both CRABPI and CRABPII proteins were confined to the cytoplasm of Caco-2 cells and were undetectable in the nuclei of these cells (Figure 3B, Lanes 2, 3, 5, and 6). In contrast, CRABPII was present in both cytoplasmic and nuclear extracts from MCF7 cells (Figure 3C, Lanes 2 and 3). However, no CRABPI was detectable in MCF7 cells with either 3CRA10F5 (Figure 3C, Lanes 5 and 6) or 4CRA3B1 (data not shown).
Immunolocalization of CRABPs in Transfected COS-1 Cells Using the Monoclonal Antibodies
The monoclonal antibodies were also tested for their ability to recognize, by immunocytochemistry, mouse CRABPs overexpressed in COS-1 cells (Figure 4). When fixation was performed with PFA (2%, pH 7.4), 4CRA3B1 and 5CRA3B3 revealed a strong signal in nuclei of cells transfected with CRABPI and CRABPII, respectively (Figure 4A1 and 4A4). In the positive nuclei, CRABPs were evenly localized, excluding nucleoli. It must be stressed that about 10% of the labeled cells also displayed a weak punctate staining in the cytoplasmic compartment. Cells with a cytosolic signal always displayed nuclear labeling. In other words, among the labeled cells examined here, none excluded CRABPs from the nucleus. In contrast, when Bouin's fixative was used (acidic pH), these antibodies revealed a strong signal in the cytoplasm only (Figure 4B1, and 4B4). This staining was homogeneous and excluded nuclei.
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With both fixation procedures, none of these antibodies showed any crossreaction towards the noncorresponding CRABP (Figure 4A2, 4A3, 4B2, and 4B3). Similar results were obtained with 3CRA10F5 and 1CRA4C9 (data not shown). According to these results, one can speculate that PFA and Bouin's fixative preferentially reveal the epitopes of CRABPs confined in the nuclei or the cytoplasm, respectively. Such a hypothesis is corroborated by the observation that, in transfected COS cells, CRABPs are detected in both the cytosolic and nuclear compartments by Western blot with the same antibodies (data not shown). Nevertheless, it cannot be excluded that Bouin's fixation (acidic conditions) promotes the diffusion of CRABPs from nuclei into the cytoplasm.
Rabbit Polyclonal Antibodies Against mCRABPI and II
Rabbits were also immunized with the synthetic peptides SPB63 and SPB64. Two rabbit polyclonal antisera were obtained with SPB 63 (antisera 204 and 206) and two with SPB 64 (antisera 207 and 208).
As described above for the monoclonal antibodies, these antisera were tested by immunoblotting for their ability to specifically detect recombinant mCRABPI and/or mCRABPII proteins overexpressed in transfected COS-1 cells. Figure 1B shows that antisera 207 and 208 recognize mCRABPII (Figure 1B, Lanes 3 and 4) and not mCRABPI (Figure 1B, Lanes 7 and 8), whereas antiserum 206 recognizes specifically CRABPI (Figure 1B, Lanes 2 and 6). However, antiserum 204 recognized both CRABPI and CRABPII (Figure 1B, Lanes 1 and 5), as did the monoclonal antibodies 3CRA10G2 (also raised against SPB 63), suggesting again that there is a common epitope in mCRABPI and mCRABPII. Antisera 206 and 208 also recognized the human CRABPI and CRABPII proteins respectively (data not shown).
By immunocytochemistry, antisera 206 and 208 recognized mouse CRABPI and CRABPII, respectively, in transfected COS cells (data not shown). As described above for the monoclonal antibodies, both CRABPs could be detected in the nuclei after PFA (pH 7.4) fixation, whereas they were excluded from this cell compartment when Bouin's fixative was used (acidic pH) (data not shown). Note that antisera 206 and 208 gave weaker signals than the monoclonal antibodies.
The polyclonal antibodies 206 and 208 were also able to specifically detect endogenous CRABPI and II proteins, respectively, in mouse embryos by immunoblotting. Figure 5A shows that CRABPs were readily detected in extracts from wild-type embryos (13.5 dpc; Figure 5A, Lanes 1 and 5), whereas no signal was detected in the double CRABPI-/-/CRABPII-/- mutants (Figure 5A, Lanes 2 and 6), thus corroborating their specificity. Then cytosolic and nuclear extracts from mouse embryos at different days post coitum (10.517.5 dpc) were immunoprobed with antiserum 208 or 206 (Figure 5B). According to our results, both CRABPs were detectable in cytosolic extracts as early as 10.5 dpc. However, CRABPII was detectable only up to 13.5 dpc and CRABPI up to 16.5 dpc. Nevertheless, both proteins were barely detectable at 17.5 dpc. These results which are in agreement with the in situ hybridization studies (
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Similar observations were made with P19 cells. As shown in Figure 5C, CRABPI was detectable in both cytosolic and nuclear extracts from P19 cells with antiserum 206 (Figure 5C, Lanes 25, lower panel), whereas CRABPII remained confined to the cytoplasm of these cells when antiserum 208 was used (Figure 5C, Lanes 25, upper panel). However, an increase in CRABPII was observed on RA treatment (Figure 5C; compare Lanes 2 and 4).
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Discussion |
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In this study we have described the preparation and the characterization of mouse monoclonal and rabbit polyclonal antibodies specific for CRABPI and CRABPII proteins. These antibodies recognize their cognate protein either by immunocytochemistry or by Western blotting analysis. In addition, their mouse and/or human specificity has been determined. These results are summarized in Table 1.
Interestingly, the present antibodies enabled us to demonstrate that both CRABPI and CRABPII can be detected not only in the cytoplasm but also in nuclei of transfected COS cells, mouse embryos, and various cell lines by Western blotting and immunocytochemistry.
The nuclear localization of CRABPI could be seen with any of our polyclonal or monoclonal antibodies either by immunoblotting or by immunocytochemistry, thus confirming previous reports (
The interesting discovery from our work presented here is the evidence for a nuclear localization of CRABPII. This nuclear localization of CRABPII has been seen by immunoblotting in mouse embryos and in the various cell lines described herein. A similar nuclear localization has been also observed with our antibodies in hematopoietic cells (Delva et al., unpublished observations). Nevertheless, such a nuclear localization appears to depend on the cell type, CRABPII accumulating in the nucleus of some cells and being excluded from the nuclei of other cells, as recently reported by
By immunocytochemistry, both CRABPI and CRABPII can be detected in the nuclei of transfected COS cells with our antibodies. Again, stronger signals were obtained with the monoclonals. However, according to our results, the fixation procedure appears to be crucial for the detection of nuclear CRABPs. Indeed, in the present study, PFA fixation (at neutral pH) allows the detection of both CRABPs (CRABPI and CRABPII) in nuclei, as recently reported by
These observations point out the importance of the fixation procedure for detection of CRABPs in nuclei with a given antibody. Fixation type and pH may also explain the heterogeneity of the cytosolic labeling (punctate or homogeneous) observed under both fixation conditions. However, the origin and the titer of the antibodies, as well as the incubation conditions, may also affect the distribution of labelings and should be taken into account.
Although the low molecular mass of CRABPs suggests that these proteins would freely enter the nucleus, it cannot be excluded that accessory factors, expressed in a cell-specific fashion, may regulate the subcellular compartmentalization of these proteins (
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Acknowledgments |
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Supported by funds from the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), the Hôpital Universitaire de Strasbourg (HUS), the Collège de France, The Association pour la Recherche sur le Cancer (ARC), the Fondation pour la Recherche Médicale (FRM), the Ligue Nationale contre le Cancer, the Human Frontier Science Program, and Bristol Myers Squibb.
We are grateful to V. Schultz and N. Young for their help in the preparation of the monoclonal antibodies, to G. Duval for the rabbit injections, and to P. Oberling for preparation of the synthetic peptides. We are indebted to J.L. Vonesch for confocal analysis. We also wish to thank the cell culture group for maintaining and providing cells, M. Lemeur for providing embryos, and C. Werlé and B. Boulay for art work. We thank Dr J.J. Voorhees for the gift of purified human CRABPI and CRABPII proteins overexpressed in E. coli.
Received for publication April 8, 1998; accepted June 9, 1998.
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