ARTICLE |
Correspondence to: Denis Barritault, Laboratoire de Recherche sur la Croissance Cellulaire, la Réparation et la Régénération Tissulaires, Unité CNRS Associée 1813, Université Paris XII, Ave. du Général de Gaulle, 94010 Créteil Cedex, France.
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Summary |
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The heparin affin regulatory peptide (HARP) growth factor, also known as pleiotrophin, is a developmentally regulated protein that displays biological functions during cell growth and differentiation. To study the physiological role of this protein, we investigated the cellular distribution of HARP mRNA and protein in the resting human mammary gland. In situ hybridization histochemistry revealed that HARP mRNA was localized in alveolar myoepithelial cells, whereas alveolar epithelial cells were negative. In the stroma, HARP mRNA was localized in endothelial cells and smooth muscle cells of blood vessels. Interestingly, HARP protein and mRNA were not always co-localized. HARP protein immunocytochemistry staining was observed in an area including both alveolar myoepithelial and epithelial cells, although epithelial cells do not express HARP transcript. In contrast, the distribution of HARP protein is parallel to that of HARP mRNA in endothelial and vascular smooth muscle cells. In the light of these results, the putative role of HARP in controlling the proliferation and/or differentiation of the different mammary cell types is proposed and discussed. (J Histochem Cytochem 45:1239-1245, 1997)
Key Words: HARP, pleiotrophin, breast, myoepithelial cell, endothelial cell, in situ hybridization immunocytochemistry
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Introduction |
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A NUMBER of studies have demonstrated that the growth and differentiation of normal breast are regulated by hormonal factors. After interaction with their specific receptors, these hormones induce the synthesis of regulatory peptides that either positively or negatively control the homeostatic maintenance of the mammary gland. Using in vitro as well as in vivo experiments, various regulatory peptides have been identified and implicated in breast tissue growth, including transforming growth factors and ß, epidermal growth factor, insulin-like growth factors I and II, platelet-derived growth factors A and B, vascular endothelial growth factor, fibroblast growth factors (for review see
Distribution of HARP mRNA using in situ hybridization in embryonic as well as adult rat tissues has been previously studied (
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Materials and Methods |
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Tissue Samples
Human normal breast tissue biopsy specimens were obtained immediately after surgery from the Department of Pathology of the Institut Curie (Paris, France) and the Henri Mondor hospital (Créteil, France). They were taken from areas opposite to invasive adenocarcinomas (n = 11) and from reduction mammoplasties (n = 6). Frozen tissue sections 5 µm thick were applied to SuperFrost/Plus glass slides (Kindler; Freiburg, Germany) and fixed for 15 min in acetone at 4C for immunocytochemistry. For in situ hybridization, sections were fixed in 4% neutral-buffered formalin at room temperature (RT) for 15 min and dehydrated through graded ethanols. Fixed sections were then stored at -20C until used.
Production of Anti-HARP Antibody
Polyclonal antibodies against HARP were raised in rabbits as follows: One hundred µg of purified human recombinant HARP diluted in PBS containing 5 mg/ml of heparin was emulsified with Freund's complete adjuvant (1:1) and injected SC in multiple sites. The rabbits were then boosted every 4 weeks with approximately 50 µg of human recombinant HARP in the presence of 5 mg/ml of heparin in Freund's incomplete adjuvant. The presence of anti-HARP antibodies was followed by enzyme-linked immunosorbent assay (ELISA). The antiserum used in this study was obtained after three SC injections followed by a final IV injection of 100 µg of recombinant HARP diluted in PBS. The resulting immunoglobulins (Ig) were prepared from preimmune and immune sera using protein A-Sepharose affinity chromatography. HARP-specific Igs were isolated using HARP-HiTrap affinity chromatography according to the manufacturer's instructions (Pharmacia; Uppsala, Sweden).
Western Blotting Assay
Frozen biopsy specimens of human mammary glands were disrupted in 20 mM Hepes, pH 7.4, 1 µg/ml aprotinin, leupeptin, and pepstatin (Sigma; St Louis, MO), 0.1 mM PMSF, 3 mM EDTA (lysis buffer) containing 2 M NaCl using an Ultra-Turrax T25 (Jankle & Kunkel; Staufen, Germany). The clarified supernatant from the centrifugation step was diluted fivefold with lysis buffer and incubated overnight at 4C with heparin-Sepharose CL-6B beads (Pharmacia) on a rotating rack. The beads were washed twice with 20 mM Hepes, pH 7.4, 0.5 M NaCl, and once with 20 mM Hepes pH 7.4. Heparin-Sepharose-bound molecules were then eluted with electrophoresis sample buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 0.02% bromophenol blue, 2% SDS, and 5% ß-mercaptoethanol) at 95C for 5 min.
Samples were loaded and run on an SDS-polyacrylamide gel before transfer to Immobilon-P membrane (Millipore; Saint-Quentin-Yvelines, France) in 10 mM CAPS, pH 11, 10% MeOH using a Sartorius electrophoretic transfer system. Nonspecific binding was prevented by incubating the membrane in PBS-T [phosphate-buffered saline (PBS), 0.2% Tween-20] plus 3% gelatin. The membrane was then incubated overnight at 4C with rabbit anti-HARP Ig at 1:500 dilution in PBS-T plus 1.5% normal goat serum. After washes in PBS-T, bound antibodies were visualized with peroxidase-conjugated goat anti-rabbit IgG (Sanofi Diagnostic Pasteur; Marnes Coquette, France) and ECL (Amersham Life Science; Poole, UK) as substrate. The membrane was then processed for autoradiography by exposure to Kodak X-Omat film for 3-30 min.
Immunocytochemistry
Tissue sections were rehydrated in PBS. Nonspecific binding of antibodies was prevented by incubating the slides with PBS-T containing 1.5% normal goat serum and 1% gelatin (Buffer A) at RT for 30 min. After a blocking step with an avidin-biotin blocking kit (Vector Laboratories; Burlingame, CA), sections were incubated for 30 min at 37C in Buffer A containing 5 µg/ml of affinity-purified anti-HARP Ig. After two washes in PBS-T, a second incubation for 30 min at RT using biotinylated anti-rabbit IgG (Vector Laboratories) was carried out at a dilution of 1:200 in Buffer A. After two washes in PBS-T, sections were incubated for 30 min at RT with an avidin-biotin-alkaline phosphatase complex and red coloration and fluorescent staining was obtained using the Vector red substrate (Vector Laboratories). Specific staining controls included omission of the first antibody, incubation with the first antibody supplemented with a 50-fold molar excess of FGF-2 or EGF, and replacement of the purified anti-HARP Ig with the HARP-HiTrap affinity-chromatography unbound Ig used at 5 µg/ml. Immunocytochemical staining with mouse antiserum against human Factor VIII-related antigen (Dako; Glostrup, Denmark) was performed in the same way as that used for HARP Ig, except that the second incubation was performed with an alkaline phosphatase (AP)-conjugated anti-mouse IgG (Dako). All sections were counterstained with Harris's hematoxylin before mounting in Aquamount (BDH; Nottingham, UK). Sections were then viewed in a Zeiss microscope and photographed on Kodak 100 ASA film.
In Situ Hybridization
All reagents were purchased from Boehringer Mannheim (Mannheim, Germany) except those for the polymerase chain reaction (PCR), which were from Promega (Madison, WI). Detection of HARP mRNA was undertaken using a digoxigenin (DIG)-11-dUTP-labeled probe according to Caruelle (unpublished procedure). Sense and anti-sense DIG-11-dUTP-labeled probes were prepared as follows. A linear 1200-bp cDNA containing the open reading frame of the HARP gene was subjected to 35 cycles in a standard PCR using 1 µM of sense 5'-GAAAATTTGCAGCTGCCTT-3' and anti-sense 5'-CTTCTCCTGTTTCTTGCCT-3' primer (Eurogentec; Seraing, Belgium) and 0.2 mM dATP, dCTP, dGTP, dTTP; 50 mM KCl, 10 mM Tris-HCl, pH 8.3; 1.5 mM MgCl2; 0.01% gelatin; 1.25 U of Taq polymerase. The 464-BP amplified product was separated from the primers and unincorporated nucleotides. Unidirectional PCR was then performed using 100 ng of the 464-BP double-stranded DNA and a typical primer ratio of 50:1 with 40 nM of the limited primer. The conditions are the same as above, except that DIG-labeled 11-dUTP is added at a ratio of 0.13 mM dTTP:0.07 mM DIG 11-dUTP. Amplified DIG-labeled DNA probes were precipitated with absolute ethanol, washed with 70% ethanol, and resuspended in hybridization buffer (4 x SSC, 50% deionized formamide, 1 x Denhardt's solution, 5 x dextran sulfate, 0.5 mg/ml denatured salmon sperm DNA, 0.25 mg/ml yeast tRNA) to a final concentration of 0.5 µg/ml. For efficient penetration and hybridization, single-stranded DNA probes were boiled for 60 min at 100C. The specificity of the DIG-labeled cDNA probes was checked against HARP, MK, and FGF-2 cDNA in a Southern blotting assay.
Sections were rehydrated, washed in 10 mM Tris-HCl, pH 7.5, incubated for 30 min at 37C with 10 µg/ml of proteinase K in 20 mM Tris-HCl, pH 7.5, 2 mM CaCl2, and postfixed in 4% neutral-buffered formalin for 15 min at RT. After washing in PBS and 4 x SSC, sections were prehybridized for 4 hr at 37C with hybridization buffer in a humid chamber. Hybridization was performed overnight at 37C with 25 µl/section of DIG-labeled sense or anti-sense probe. Posthybridization washes were for 30 min each as follows: 2 x SSC, RT; 1 x SSC, RT; 0.5 x SSC, 37C; and finally 0.5 x SSC, RT. Sections were then preincubated in 3% normal sheep serum and 1% Boehringer blocking reagent (BBR) in 100 mM Tris-HCl, pH 7.4, 150 mM NaCl (7.4 AP buffer) for 45 min at RT, followed by an incubation with 1:200 diluted AP-conjugated anti-digoxigenin in 7.4 AP buffer containing 1% BBR, 0.3% Triton X-100 for 90 min at RT. The sections were washed twice in 7.4 AP buffer and once in 100 mM Tris-HCl, pH 9.5, 150 mM NaCl, 50 mM MgCl2 (9.5 AP buffer). Color was developed by incubation in 9.5 AP buffer containing 0.33 mg/ml nitroblue tetrazolium chloride (NBT) and 0.17 mg/ml of 5-bromo-1-chloro-3-indolyl phosphate (BCIP) for 2-3 hr at RT. Reaction was achieved by washing the sections in 7.4 AP buffer. Sections were mounted in Aquamount (BDH), then viewed in a Zeiss microscope and photographed on Kodak 100 ASA film.
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Results |
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Cellular Distribution of HARP mRNA in NHMG
In situ hybridization histochemistry showed that HARP mRNA staining was entirely confined to alveolar myoepithelial cells surrounding the glandular structure, whereas alveolar epithelial cells did not display positive HARP mRNA hybridization (Figure 1C and Figure 1D). Negative control sections incubated with sense probe showed no staining, demonstrating the specificity of the DIG-labeled probe system (Figure 1B). In addition, HARP transcripts were also strongly expressed in blood vessels present in the stroma of NHMG. More precisely, and as shown in Figure 1E, the staining occurred in capillaries, since adjacent serial section of the same capillary was positively stained by an anti-factor VIII-related antigen serum (Figure 1F) and negatively by an anti--smooth actin-related antigen serum (not shown), thus demonstrating that endothelial cells expressed HARP mRNA. Moreover, strong staining was also seen in a cross-section of an arteriole, indicating that HARP mRNA was synthesized by vascular smooth muscle cells (Figure 1G).
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Validation of Anti-HARP Antibodies and Immunochemical Staining
Specificity of the rabbit affinity-purified antibodies was confirmed by Western blotting analysis of purified HARP, MK, FGF-2, and EGF proteins. Figure 2 shows that affinity-purified Ig bound to HARP but failed to recognize MK, FGF-2, and EGF molecules, indicating no crossreactivity. Western blotting of heparin-purified cell extracts of human normal mammary gland with anti-HARP Ig produced only a unique immunoreactive signal that co-migrated with the 18-kD HARP protein.
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Immunocytochemical Staining of HARP Protein in NHMG
Immunocytochemical staining using specific rabbit antibodies to HARP protein showed this growth factor to be localized in a region that included both alveolar epithelial and myoepithelial cells (Figure 1H and Figure 1I). In the stroma, the matrix compartment was clearly negative. No staining was observed when negative control sections were incubated either with affinity chromatography-unbound Ig (Figure 1J) or with PBS-T in replacement of the first antibody (not shown). Moreover, no modification of labeling was seen when the slides were incubated with the first antibody supplemented with a 50-fold molar excess of FGF-2 or EGF (not shown). In agreement with the mRNA localization, immunostaining was observed in capillaries (Figure 1K) and arterioles (Figure 1L).
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Discussion |
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The strong expression of HARP mRNA and protein originally observed during postnatal development of rat brain suggested that HARP may play important functions in the growth and differentiation of neuronal cells (
The presence of HARP mRNA in biopsy specimens from both normal and tumoral human mammary gland has been previously described using reverse transcrip-tase-polymerase chain reaction (RT-PCR) (
HARP mRNA and protein were also detected both in the endothelial and smooth-muscle cells of blood vessels. Several studies have demonstrated that purified human recombinant HARP was mitogenic for capillary endothelial cells and possessed angiogenic activity (
Interestingly, it is noteworthy that HARP and fibroblast growth factor-2 (FGF-2) are co-localized in myoepithelial cells and in blood vessels (
In conclusion, we have now collected more information on HARP in normal mammary tissues showing that myoepithelial, endothelial, and vascular smooth muscle cells expressed both HARP mRNA and protein. It is noteworthy that this mRNA and protein pattern of localization was observed in biopsies derived from an area opposite to adenocarcinoma (n = 11) as well as from mammoplasties (n = 6). In any event, it now appears of greatest interest to perform the same type of investigation in pathological mammary tissues to evaluate whether this molecule could be a major factor in the malignant progression of breast cancer, as has been previously suggested (
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Acknowledgments |
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Supported by grants from the Association pour la Recherche sur le Cancer (no. 6595), the Ministère de l'Éducation Nationale (DRED), the Ligue Nationale Contre le Cancer, and Naturalia et Biologia.
We are grateful to Dr Bagot and Dr Charu (Henri Mondor hospital, Créteil, France) and Dr Zafrani and Dr. Sastre (Institut Curie, Paris, France) for providing breast biopsies.
Received for publication July 30, 1996; accepted March 18, 1997.
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