ARTICLE |
Correspondence to: Margot M. Ip, Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. E-mail: mip@sc3101.med.buffalo.edu
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Summary |
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Studies were undertaken to examine the natural role of ErbB2, ErbB3, and ErbB4 during the development of normal rat mammary epithelial cells (MECs) in vivo and in vitro. Immunohistochemical analysis demonstrated that mammary gland terminal end buds expressed abundant ErbB2 and ErbB4 but limited ErbB3 in pubescent rats, whereas luminal epithelial cells in nulliparous rats expressed ErbB2, ErbB3, and/or ErbB4. During pregnancy, ductal epithelial cells and stromal cells expressed abundant ErbB3 but limited ErbB2. Although ErbB2 and ErbB3 were downregulated throughout lactation, both receptors were re-expressed during involution. In contrast, ErbB4 was downregulated throughout pregnancy, lactation, and involution. Immunoblotting and immunoprecipitation studies confirmed the developmental expression of ErbB2 and ErbB3 in the mammary gland and the co-localization of distinct ErbB receptors in the mammary gland of nulliparous rats. In agreement with our in vivo findings, primary culture studies demonstrated that ErbB2 and ErbB3 were expressed in functionally immature, terminally differentiated and apoptotic MECs, and downregulated in functionally differentiated MECs. ErbB receptor signaling was required for epithelial cell growth, functional differentiation, and morphogenesis of immature MECs, and the survival of terminally differentiated MECs. Finally, ErbB4 expression did not interfere with functional differentiation and apoptosis of normal MECs. (J Histochem Cytochem 48:6380, 2000)
Key Words: mammary, breast, ErbB2, her-2/neu, ErbB3, ErbB4, EGF receptor, immunohistochemistry, immunocytochemistry
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Introduction |
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The ErbB FAMILY of transmembrane tyrosine kinase receptors includes epidermal growth factor receptor (EGFR/ErbB1), ErbB2 (her2/neu), ErbB3, and ErbB4 (
The mammary gland undergoes cyclic postnatal development and regression during each estrous cycle and during pregnancy, lactation, and involution ( (TGF
), amphiregulin, and neuregulin-
during distinct developmental stages (
The roles played by ErbB2, ErbB3, and ErbB4 in normal mammary gland development and the early stages of breast cancer progression are unclear. Unfortunately, transgenic mice lacking ErbB2 (
To test the hypothesis that select ErbB receptors play natural, non-oncogenic roles in regulating growth, differentiation, and/or apoptosis of normal mammary epithelial cells (MECs), immunohistochemistry studies were undertaken to define the cell type-specific and developmental stage-dependent profile of ErbB2, ErbB3, and ErbB4 in serial sections of rat mammary glands during puberty, sexual maturation, pregnancy, lactation, and involution. Immunoblotting and immunoprecipitation studies were used to confirm the specificity of the ErbB-selective antibodies and the developmental expression of ErbB2 and ErbB3. A primary culture model system (
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Materials and Methods |
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Materials
Progesterone, hydrocortisone, insulin, ascorbic acid, fatty acid-free fraction V bovine serum albumin (BSA), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), phenylmethylsulfonyl fluoride (PMSF), 3,3'-diaminobenzidine (DAB), transferrin, fetal bovine serum (FBS), and phenol red-free F12/DMEM medium were purchased from Sigma (St Louis, MO). Grade II dispase was obtained from BoehringerMannheim Biochemicals (Indianapolis, IN). Gentamycin and porcine trypsin were purchased from Gibco BRL Life Technologies (Grand Island, NY). Culture grade human recombinant TGF was acquired from Collaborative Research (Bedford, MA). Ovine prolactin (NIDDK oPRL-19 and -20) was a gift of the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases. PD158780 was generously provided by Dr. David Fry at Parke-Davis Pharmaceutical Research (Ann Arbor, MI). Sterile tissue culture plastic flasks and plates were purchased from Becton Dickenson Labware (Franklin Lakes, NJ).
Affinity-purified rabbit polyclonal anti-peptide antibodies against EGFR (SC-03), ErbB2 (SC-284), ErbB3 (SC-285), and ErbB4 (SC-283), as well as ErbB receptor-specific peptides (SC-03P for EGFR, SC-284P for ErbB2, SC-285P for ErbB3, and SC-283P for ErB4) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A mouse monoclonal antibody against ErbB3 (clone 2F12, UBI 05-390) and a sheep polyclonal antibody against EGFR (UBI 06-129) were acquired from Upstate Biotechnology (Lake Placid, NY). NeoMarkers (Freemont, CA) was the source of rabbit polyclonal antibodies against either ErbB2 (Ab-1; clone 21N, cat # RB-103) or ErbB4 (Ab-2; cat # RB-284), the synthetic Ab-1 ErbB2 peptide (cat # PP-103) as well as mouse monoclonal IgG1 anti-ErbB2 Ab-9 (clone B10; cat # MS-326) or Ab-17 (a mixture of clones e2-4001 + 3B5; cat # MS-730). Chromopure rabbit IgG, chromopure mouse IgG1, and preabsorbed F(ab')2 fragments of affinity-purified donkey anti-rabbit IgG or anti-mouse IgG conjugated to either biotin or horseradish peroxidase were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Mouse myeloma IgG2a and the streptavidinhorseradish peroxidase conjugate were purchased from Zymed (San Francisco, CA). ImmunoPure Immobilized Protein G was acquired from Pierce (Rockford, IL). Immobilon P membrane was obtained from Millipore (Bedford, MA) and the enhanced chemiluminescence (ECL) reagent was acquired from Amersham (Arlington Heights, IL). Peroxidase In Situ ApopTag Plus Detection Kit was obtained from Oncor (Gaithersburg, MD).
Animals
Pubescent nulliparous and untimed pregnant female SpragueDawley CD rats (Crl:CDBR) were purchased from Charles River (Kingston, NY) or Taconic Laboratories (Germantown, NY). Nulliparous rats were used at 5054 or 8385 days of age. Pregnant rats were allowed to advance to mid-pregnancy (days 1416 of gestation), lactation (57, 15, or 21 days after birth), or involution (34 or 610 days after weaning 21-day-old pups). CD2F1 mice, purchased from NCI Frederick Cancer Research Facility, Biological Testing Branch (Frederick, MD), were used to carry the EngelbrethHolmSwarm (EHS) sarcoma. Animals were fed rat or mouse chow diets (Teklad, Madison, WI), ad libitum and had free access to water. Animal rooms were temperature- and humidity-controlled. Light cycles were 14 hr on/10 hr off for rats and 12 hr on/12 hr off for mice. The care and use of animals were in accordance with National Institute of Health guidelines and Institute Animal Care and Use Committee regulations.
Isolation of Mammary Epithelial Cells and Fibroblasts
Abdominal and inguinal mammary glands, excised from six to 12 5054-day-old female SpragueDawley rats were mechanically and enzymatically disaggregated. Mammary epithelial organ-like fragments (organoids) were isolated as previously described (
Mammary fibroblasts were recovered from the 60-µm filtrate by a 10-min, 500 x g centrifugation at 4C, suspended in F12/DMEM medium with 10% (v/v) FBS and 50 µg/ml gentamicin, and cultured on tissue culture plastic for 2 hr at 37C. The nonadherent cells were removed from the flasks and a relatively homogenous population of fibroblasts was expanded and passaged.
Primary Culture Conditions for Mammary Epithelial Organoids
The reconstituted basement membrane (RBM) used for the primary culture studies was extracted from the EHS mouse sarcoma as previously described ( medium (F12/DMEM medium with 10 µg/ml insulin, 1 µg/ml prolactin, 1 µg/ml progesterone, 1 µg/ml hydrocortisone, 5 µg/ml apotransferrin, 880 ng/ml ascorbic acid, 1 mg/ml fatty acid-free BSA, 50 µg/ml gentamycin, and 10 ng/ml human recombinant TGF
). Medium was changed every 3.5 days. The pyridopyrimidine analogue PD158780 (
medium from a 10-mM stock stored at -80C in 100% DMSO. Each treatment condition in the primary culture studies was carried out in at least two and as many as eight separate experiments.
Culture Conditions for Preadipocytes, Fibroblasts, NMU Cells, and RBA Cells
Rat mammary preadipocytes were isolated from the abdominal and inguinal mammary glands from eight to 12 5054-day-old female SpragueDawley rats and retained a fibroblast-like morphology when cultured in F12/DMEM medium with 10% (v/v) FBS and 50 µg/ml gentamicin (
Immunolocalization of ErbB2, ErbB3, and ErbB4
ErbB localization was examined in serial sections of formalin-fixed, paraffin-embedded abdominal mammary glands from at least three SpragueDawley rats during puberty (5054 days of age), at sexual maturation (8385 days of age), at days 1416 of pregnancy, during lactation (57 and 15 days after birth), and during involution (34 and 610 days after weaning), or in MEOs cultured for up to 21 days in 100-mm dishes in two primary culture studies. For the developmental series, one large assay was carried out for the detection of each ErbB receptor to allow intensity levels and localization of that receptor to be compared in the different sections. ErbB4 but not ErbB2 and ErbB3 detection required a PBS microwave antigen retrieval incubation. ErbB2 localization and specificity were evaluated using a 2-hr room temperature (RT) incubation with either rabbit ErbB2 antibody, ErbB2 antibody preincubated with ErbB2 peptide or EGFR peptide, rabbit IgG, or PBS only. ErbB4 localization and specificity were analyzed using a 2-hr RT incubation with either rabbit ErbB4 antibody, ErbB4 antibody preincubated with ErbB4 peptide or ErbB2 peptide, rabbit IgG, or PBS. The two rabbit primary antibodies were used at 1 µg/ml and preincubated overnight at 4C with either 0 or 10 µg/ml of the appropriate peptide before use. ErbB3 localization and specificity were examined using a 2-hr RT incubation with 10 µg/ml of either mouse IgG2a monoclonal ErbB3 antibody or the isotype control, mouse IgG2a. Reactive proteins were visualized using preabsorbed F(ab')2 fragments of affinity-purified donkey anti-rabbit or anti-mouse antibody conjugated with biotin, a streptavidinhorseradish peroxidase conjugate, and DAB, and sections were counterstained with hematoxylin. Color photographs were taken of identical fields in serial sections using a Nikon FX-35A camera and an Olympus BH-2 microscope, photographs were scanned using an AGFA Argus II flatbed scanner, and images were processed using Adobe Photoshop (Adobe Systems; San Jose, CA) and printed with a Kodak DS 8650 PS printer.
Lysate Preparation
Lysates were prepared of intact mammary glands, isolated MEOs, and various types of cultured cells using ice-cold lysis buffer [50 mM Tris, pH 8.0, at 4C, with 150 mM NaCl, 2 mM EDTA, 10 mM Na2HP04, 10 mM Na4P2O710H2O, 5 mM Na3VO4, 1% (v/v) Triton X-100, 0.1% (w/v) Na-dodecyl sulfate (SDS), 0.5% (w/v) Na deoxycholate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 100 µg/ml soybean trypsin inhibitor, and 20 µg/ml leupeptin]. Rat liver or intact mammary glands excised either from nulliparous adult (85-day-old or 6-month-old) rats or from rats at Day 21 of lactation were harvested in 5 ml lysis buffer per gram of tissue. MEOs isolated from 50-day-old nulliparous rats were harvested in 1 ml of lysis buffer per 2 x 107 MECs. Finally, lysates were prepared of cultured cells, using 1 ml of lysis buffer per T175 flask containing PBS-washed cells and scraping. Each lysate was sonicated three times on ice for 10 sec using a Tekmar sonic disruptor, vortexed for 10 min at 4C, and microfuged for 15 min at ~12,000 x g at 4C. Mammary gland samples were homogenized with three 30-sec bursts of a Polytron on ice before the sonication step. Supernatants were quickly frozen in liquid nitrogen and stored at -20C.
Alternatively, Trizol extracts were prepared of MEOs isolated from the mammary glands of rats during puberty (5052 days of age), pregnancy (Days 1416 of gestation), lactation (67 days after birth), and involution (7 days after weaning 21-day-old pups) as previously described (
Immunoblot Detection of EGFR, ErbB2, ErbB3, and ErbB4
Lysates mixed with reducing and denaturing sample buffer were separated on 420% polyacrylamide gradient gels and transferred to Immobilon P membranes. Immunoblot analysis was performed on membranes using an overnight 4C incubation either with 0.1 µg/ml of rabbit antibodies against EGFR (SC-03), ErbB2 (SC-284), or ErbB3 (SC-285) or with 4 µg/ml of NeoMarkers' ErbB4 Ab-2. Alternatively, membranes were incubated with 2 µg/ml of either rabbit IgG or rabbit anti-ErbB2 Ab-1, or with 0.5 µg/ml of either mouse IgG1 or mouse anti-ErbB2 Ab-17. Membranes were then incubated for 1 hr at RT with preabsorbed F(ab')2 fragments of affinity-purified donkey anti-rabbit IgG or anti-mouse IgG conjugated to horseradish peroxidase. Immunoreactive bands were visualized on X-ray film using ECL reagent, exposed films were scanned, and images were processed as described above and printed with a Kodak DS 8650 printer.
Immunoprecipitation of EGFR, ErbB2, ErbB3, and ErbB4
Mammary gland lysates (100 µl/reaction) from nulliparous adult (85-day-old) rats or rat liver lysates (100 µl/reaction) were precleared with protein G/agarose and incubated overnight at 4C with 1 µg of rabbit IgG, or affinity-purified rabbit antibodies against EGFR (SC-03), ErbB2 (SC-284), ErbB3 (SC-285), or ErbB4 (SC-283). Alternatively, precleared lysates were incubated overnight with 10 µg of either rabbit IgG or rabbit anti-ErbB2 Ab-1 (clone 21N), or with 2 µg of either mouse IgG1, mouse anti-ErbB2 Ab-9, or mouse anti-ErbB2 Ab-17. Samples were then incubated with protein G/agarose for 90 min at 4C. Immune complexes were precipitated with protein G/agarose, washed four times with ice-cold lysis buffer, and dissociated from the protein G/agarose by boiling in 50 µl of 2 x sample buffer [62.5 mM Tris-HCl, pH 8.8, at RT with 2% (w/v) SDS, 2.5% (v/v) glycerol, 5% (v/v) ß-mercaptoethanol, and 0.0125% (w/v) bromophenol blue]. Thirty µl of each sample was separated on replicate 420% polyacrylamide gradient gels and transferred to Immobilon P membranes. Individual membranes were incubated overnight at 4C with 0.1 µg/ml of affinity-purified rabbit polyclonal antibody to EGFR (SC-03), ErbB2 (SC-284), ErbB3 (SC-285), or ErbB4 (SC-283) and then for 1 hr at RT with preabsorbed F(ab')2 fragments of affinity-purified donkey anti-rabbit IgG conjugated to horseradish peroxidase. Immunoreactive bands were visualized and processed as indicated above.
MTT Assay to Quantify Viable Cell Numbers
Cell numbers were quantified in triplicate culture wells per treatment for each of the different time points using an MTT assay (
Evaluation of MEC-specific Functional Differentiation
Casein accumulation, used as an indicator of MEC functional differentiation, was monitored in triplicate culture wells per treatment for each of the different time points. Culture medium was removed and lysates of RBM without or with MEOs were prepared at 4C with ice-cold lysis buffer containing 0.1 mM PMSF, 100 ng/ml soybean trypsin inhibitor, and 20 ng/ml leupeptin. Each lysate was sonicated three times on ice for 10 sec, vortexed, and microfuged for 15 min at ~12,000 x g at 4C. Supernatants were quickly frozen in liquid nitrogen and stored at -20C. Casein levels were then quantified in these lysates using a previously described noncompetitive ELISA (
Microscopic Examination of and Apoptosis Detection in Cultured MEOs
An Olympus CK2 microscope mounted with a Nikon FX-35A camera was used for time-lapse light microscopic examination and photography of individual colonies. Three culture wells of living organoids for each treatment group were repeatedly examined to identify changes in colony number, size, coloring, and/or shape during the course of the 21-day culture period. Cultured MEOs in 100-mm dishes were exposed to TGF medium with 0 or 0.5 µM PD158780 from Days 1820 of the study, fixed with formalin, embedded in paraffin, and sectioned at 45 µm. Sections were processed using Oncor's ApopTag Peroxidase In Situ Apoptosis detection kit as described by the manufacturer, and then analyzed and photographed using an Olympus BH-2 microscope and a Nikon FX-35A camera. Images were processed using Adobe Photoshop and printed using a Kodak DS 8650 PS printer.
Statistics
Data are presented as mean ± SEM. Statistical significance was evaluated using a one-way analysis of variance (ANOVA) with the StudentNewmanKeuls test for pairwise multiple comparisons. p<0.05 was judged to be statistically significant.
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Results |
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Localization of ErbB2, ErbB3, And ErbB4 During Rat Mammary Gland Development
If select ErbB receptors play natural, non-oncogenic roles in regulating growth, differentiation, apoptosis, and/or remodeling in normal mammary glands, then these receptors should be differentially expressed in mammary epithelial and/or stromal cells during stages of extensive growth (puberty and pregnancy), differentiation (pregnancy and lactation), apoptosis (involution), and/or tissue remodeling (puberty and involution). This hypothesis was examined using an immunohistochemical study and the results are summarized in Table 1. During puberty, terminal end buds expressed a high level of ErbB2 (Figure 1A) and ErbB4 (Figure 1C), but limited ErbB3 (Figure 1B). Most fibroblasts and adipocytes surrounding these end buds exhibited ErbB2 (Figure 1A), ErbB3 (Figure 1B), and/or ErbB4 (Figure 1C) staining.
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Specificity of ErbB2 reactivity was demonstrated using rabbit IgG (not shown) and by selective competition of the rabbit ErbB2 antibody SC-284 with its immunogenic peptide (Figure 1D) but not the EGFR peptide (not shown). Moreover, rat sweat glands, skeletal muscle, salivary gland, and kidney were positive controls, whereas rat liver was the negative control tissue for the ErbB2 immunolocalization studies (not shown). The absence of brown staining in mammary sections exposed to mouse IgG2a isotype control antibody confirmed the specificity of the ErbB3 monoclonal antibody clone 2F12 (Figure 1E). The immunizing peptide was not available for peptide competition of this antibody. Specificity of ErbB4 staining was determined by selective competition of the rabbit ErbB4 antibody (SC-283) with its immunizing ErbB4 peptide (Figure 1F), and using rabbit IgG (not shown). Infiltrating macrophages were found to nonspecifically react with the ErbB4 antibody (Figure 1F, arrowhead). It should also be noted that rat brain and epidermis were the positive control tissues for the ErbB3 and ErbB4 immunohistochemistry assays (not shown).
During puberty (5054 days of age; Figure 2A2I) and at sexual maturation when proliferation has decreased (8385 days of age; not shown), luminal epithelial cells in mammary gland ducts and alveoli exhibited ErbB2 (Figure 2A and Figure 2B), ErbB3 (Figure 2D2F), and/or ErbB4 (Figure 2G and Figure 2H) staining either along the apical cell membrane or in both the cytoplasm and along apical and/or basolateral cell membranes. Myoepithelial cells surrounding epithelial ducts and alveoli were positive for ErbB2 (Figure 2B) and/or expressed moderate ErbB3 (Figure 2E) and minimal ErbB4 (Figure 2G and Figure 2H) reactivity. Mammary fibroblasts and adipocytes surrounding ducts and alveoli exhibited ErbB2 (Figure 2A2C) and ErbB3 (Figure 2D2F) staining, and either limited or no ErbB4 reactivity (Figure 2G2I).
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During pregnancy (Days 1416), ductal epithelial cells expressed a low level of cytoplasmic and apical membrane-associated ErbB2 (Figure 3A) and extensive ErbB3 reactivity (Figure 3D). Alveolar epithelial cells did not exhibit ErbB2 (Figure 3B) nor ErbB3 (Figure 3E) staining along the cell membrane. ErbB2 expression in myoepithelial and stromal cells was either limited or below detection limits (Figure 3A3C). In contrast, most of the remaining fibroblasts (Figure 3D) and adipocytes (Figure 3F) exhibited strong ErbB3 staining, whereas myoepithelial cells either failed to express or expressed moderate ErbB3 reactivity (Figure 3D and Figure 3E). ErbB4 staining was almost completely absent in mammary epithelial and stromal cells at this time point in pregnancy (Figure 3G3I).
During lactation, ErbB2 staining was downregulated in the functionally differentiated epithelial cells within ducts (Figure 3J) and alveoli (Figure 3K). Modest ErbB3 staining was observed in the apical cytoplasm and on the cell membrane of luminal epithelial cells (Figure 3M and Figure 3N). Weak ErbB4 staining was detected in certain luminal epithelial cells, and the localization was supranuclear (not shown). Although myoepithelial cells, fibroblasts, and adipocytes surrounding functionally differentiated MEC did not exhibit ErbB2 (Figure 3J3L) or ErbB4 (not shown) reactivity, a number of these cells displayed distinct ErbB3 staining (Figure 3M and Figure 3O). There was no difference in the intensity or localization of ErbB2, ErbB3, or ErbB4 staining within the various cell types in mammary glands from rats at Days 57 compared with Day 15 of lactation (not shown).
ErbB receptor expression was examined at Days 34 (Figure 3P3V) of postweaning involution, and ductal MECs displayed distinct ErbB2 (Figure 3P) and ErbB3 (Figure 3T) reactivity that was cytoplasmic, supranuclear, and/or cell membrane-associated. Luminal epithelial cells in alveoli (Figure 3Q and Figure 3U) and residual epithelial cells in apoptotic alveoli (Figure 3R and Figure 3V) exhibited grainy ErbB2 (Figure 3Q and Figure 3R) and ErbB3 (Figure 3U and Figure 3V) staining. Limited supranuclear ErbB4 staining was observed in a number of healthy epithelial cells as well as in apoptotic alveoli (not shown). Myoepithelial cells and fibroblasts exhibited ErbB2 (Figure 3P3R) and/or ErbB3 (Figure 3T3V) staining but not ErbB4 reactivity (not shown). Mature adipocytes displayed limited staining for ErbB2 (Figure 3S) and prominent ErbB3 reactivity (not shown) but were not ErbB4-reactive (not shown). Mammary epithelial and stromal cells continued to express ErbB2 and ErbB3 through at least Day 10 of involution (not shown).
Differential Expression of Select ErbB Receptors in Rat Mammary Cells and Tissues
The observation that the ErbB2, ErbB3, and ErbB4 antibodies recognized distinct cell types during select developmental stages in rat mammary glands strongly supports the contention that these antibodies did not have overlapping reactivity. However, to confirm the specificity of these ErbB antibodies, lysates prepared from different rat mammary cells and tissues were evaluated by immunoblot analysis (Figure 4). This experiment demonstrated that the EGFR, ErbB2, and ErbB3 antibodies each detected distinct proteins. First, Lanes 1 and 2 in Figure 4A4C indicate that in RBA and NMU rat mammary tumor cells, the proteins detected by each antibody were of a different molecular weight (165, 185, and 180 kDa for EGFR, ErbB2 and ErbB3, respectively). Second, although EGFR and ErbB2 were detected in mammary fibroblasts (MFCs) and mammary preadipocytes (MPAs), ErbB3 was not, demonstrating that the EGFR and ErbB2 antibodies did not crossreact with ErbB3 (Lanes 3 and 4 in Figure 4A4C). Third, although EGFR was detected in rat liver, ErbB2 and ErbB3 were not, showing that the EGFR antibody did not crossreact with ErbB2 and ErbB3 (Lane 5 in Figure 4A4C). Fourth, EGFR, ErbB2 and ErbB3 were all detected in normal rat mammary gland lysates from 85-day-old virgin female rats (V85-MG) and rats at Day 21 of lactation (Lanes 6 and 7 in Figure 4A4C). Finally, we found that the ErbB4 antibody SC-283 (not shown) and Ab-2 (Figure 4D) were not suitable for direct immunoblot detection. It should be noted that
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Confirmation of ErbB2 Expression in Mammary Glands of Nulliparous Rats
Although Dati and co-workers (1996) used an immunofluorescence assay to demonstrate that ErbB2 was expressed in the mammary glands of virgin and early pregnant rats but not of late pregnant and lactating rats, their immunoblot study indicated that high levels of ErbB2 were detected in mammary gland lysates from rats at late pregnancy and lactation but not from virgin rats. To further bolster our data that ErbB2 is present in the mammary gland of nulliparous rats, immunoblot and immunoprecipitation studies were carried out using different ErbB2-reactive antibodies including Ab-1 (clone 21N) used by Dati and co-workers. Figure 5A demonstrates that full-length ErbB2 was readily detected with the ErbB2 selective antibodies Ab-17 (clones e2-4001+3B5), SC-284, and Ab-1 (clone 21N) in a mammary gland lysate from 85-day-old virgin female rats (MG) but not in the negative control rat liver lysate. In addition, mouse IgG1 (Lanes 1 and 2 in Figure 5A) and rabbit IgG (Lanes 7 and 8 in Figure 5B) did not detect a 185-kD protein in either lysate. Incubation of Ab-1 with its immunogenic peptide competed the 185-kD reactive protein but not the lower molecular weight nonspecific proteins (compare Lanes 1 and 3 in Figure 5B).
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An immunoprecipitation study demonstrated that full-length ErbB2 was detected in the pellet recovered when a mammary gland lysate from 85-day-old female rats was immunoprecipitated with the ErbB2-selective antibodies SC-284, Ab-1 (clone 21N), Ab-9 (clone B10), or Ab-17 (clones e2-4001+3B5) but not with rabbit IgG or mouse IgG1 (Figure 6A). Furthermore, ErbB2 was depleted in the supernatant fraction recovered when another aliquot of the same lysate was immunoprecipitated with any of the four ErbB2-selective antibodies but not when immunoprecipitated with rabbit IgG or mouse IgG1 (Figure 6B). Finally, rat liver lysate was used as a negative control to demonstrate that none of the ErbB2-selective antibodies, rabbit IgG, or mouse IgG1 was able to immunoprecipitate a 185-kD protein (Figure 6C).
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An additional immunohistochemical study was carried out to determine whether Ab-1 and/or Ab-17 could reliably detect ErbB2 in formalin-fixed, paraffin-embedded rat salivary gland, kidney, and mammary gland, but not in liver. Although Ab-1 showed strong reactivity in salivary gland, kidney, and mammary gland, the staining was not competed when Ab-1 was preincubated with its immunogenic peptide (not shown), presumably because of the nonspecific protein with which this antibody also reacts (Figure 5B). In addition, Ab-17 did not show positive staining in any of the tissues tested (not shown). In this study, SC-284 staining was observed in rat salivary gland and kidney as well as in mammary glands from nulliparous, pregnant, and postlactational rats but not in rat liver or mammary glands from lactating rats.
Relative Epithelial Expression of ErbB2 And ErbB3 During Mammary Gland Development
Our immunohistochemical studies demonstrated that ErbB2 and ErbB3 levels were decreased in the lactating animals compared with the rats at other developmental stages (Figure 1 Figure 2 Figure 3). To examine this more definitively, epithelial cell-specific changes in ErbB2 and ErbB3 were examined in lysates of epithelial organoids isolated from the mammary glands of rats during puberty, pregnancy, lactation, and involution. Both mechanical and enzymatic steps were employed to dissociate the epithelium from the surrounding stroma. This resulted in detection of multiple immunoreactive proteins in these lysates (Figure 7). Figure 7A illustrates the detection of three immunoreactive proteins using the ErbB2-selective antibody SC-284 in lysates of isolated mammary epithelial organoids. The 125- and 145-kD proteins are believed to represent proteolytic fragments of full-length ErbB2 (
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Immunoprecipitation of Distinct ErbB Receptor Heterodimers
The coincident localization of the various ErbB receptors in the mammary glands of virgin rats (Figure 1 and Figure 2) suggested the possibility that these receptors formed heterodimers. In addition, the ErbB ligands EGF, TGF, amphiregulin, and neuregulin-
have previously been shown to be expressed in mammary epithelial and stromal cells (
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Role of ErbB Receptors in Normal MECs
The above data suggested that ErbB receptors may be important in rat mammary gland development during puberty, pregnancy, and involution and at sexual maturation but not during lactation. Because embryonic lethality prevented the study of mammary gland development in ErbB2 (-stimulated ErbB receptor-dependent effects were identified using the pyridopyrimidine analogue PD158780 (
On the first day of culture, immature organoids were small, lobular, and heterogenous in their expression of cytoplasmic and cell membrane-associated ErbB2, ErbB3, and/or ErbB4 (not shown). After 7 days in culture, most MECs developed into complex multilobular alveolar organoids, with the individual lobes being primarily composed of a monolayer of well polarized epithelial cells organized around a distended central lumen (Figure 9A, Figure 9D, and Figure 9G) (
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To evaluate the role of ErbB receptor signaling in the development of functionally immature MECs, newly isolated epithelial organoids were cultured in TGF-containing medium for 4 or 7 days and exposed to 0.5 µM PD158780 from Days 04 or 47 of the study, respectively. Exposure of MEOs to 0.5 µM PD158780 during the first week of culture inhibited epithelial cell growth (Figure 10A) and casein accumulation (Figure 10B). In the presence of PD158780, the epithelial organoids at Day 4 or Day 7 of the study resembled organoids at Day 0 of the study (not shown). These findings support the hypothesis that ErbB receptor signaling was required for the induction of epithelial growth, functional differentiation, and organoid morphogenesis and invasion into the surrounding RBM.
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After 14 days in culture, epithelial cell numbers continued to increase (Figure 10C) and the multilobular alveolar organoids were primarily composed of functionally differentiated epithelial cells that produced and accumulated extensive casein (Figure 10D) and intracellular lipid (Figure 9B, Figure 9E, and Figure 9H). ErbB2 (Figure 9B) and ErbB3 (Figure 9H) stainings were significantly downregulated in these functionally differentiated epithelial cells. In contrast, distinct ErbB4 staining was noted in the cytoplasm and/or along the cell membrane (Figure 9H). Exposure of MEOs to PD158780 from Days 1114 of culture did not inhibit epithelial cell growth (Figure 10C), casein accumulation (Figure 10D), or the morphologic appearance of the organoids. This supports the hypothesis that ErbB receptors are not required to maintain functional differentiation of MECs.
In this study, viable cell numbers (Figure 10C) and casein accumulation (Figure 10D) continued to increase during the third week of culture. Exposure of MEOs to 0.5 µM PD158780 from Days 1821 of culture did not affect casein accumulation (Figure 10D) but cell numbers were decreased (Figure 10C). The decrease occurred as large numbers of cells along the periphery of many of these organoids broke away and died (Figure 10F). Cell death was judged microscopically by the inability of these cells to convert the yellow MTT dye to purple formazan crystals. Furthermore, apoptotic DNA can be readily seen in the lumen of organoids exposed to PD158780 from Day 1821 (Figure 9C, Figure 9F, and Figure 9I). It should be noted that exposure of MEOs to 0.5 µM PD158780 before Day 18 of culture did not induce morphological evidence of apoptosis. Moreover, MEOs cultured with 0 (not shown) or 0.5 µM (Figure 9C, Figure 9F, and Figure 9I) PD158780 from Days 1821 expressed a similar level of cytoplasmic and cell membrane-associated ErbB2 (Figure 9C), ErbB3 (Figure 9F), and ErbB4 (Figure 9I). The latter finding confirmed that suppression of ErbB signaling was not required for the re-expression of ErbB2 and ErbB3 in terminally differentiated MECs.
An additional primary culture study was carried out to quantify the apoptotic response observed when MEOs were exposed to 0.5 µM PD158780 starting on Day 18 of culture. PD158780 exposure for 48 but not for 24 hr starting on Day 18 of culture induced 75% of the luminal epithelial cells within individual organoids to undergo apoptosis (Figure 10G). Analysis of serial sections of these organoids confirmed that apparently healthy and apoptotic MECs exhibited extensive ErbB2, ErbB3, and ErbB4 staining (not shown).
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Discussion |
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This report presents novel and distinct localization and expression profiles for ErbB2, ErbB3, and ErbB4 in the rat mammary gland during puberty, pregnancy, lactation, and involution. ErbB2 localization was detected immunohistochemically using antibody SC-284, levels were examined by immunoblot analysis using three different antibodies (SC-284, Ab-1, and Ab-17), and immunoprecipitation was accomplished using four different antibodies (SC-284, Ab-1, Ab-9, and Ab-17). ErbB3 localization was identified using a mouse monoclonal anti-ErbB3 antibody, and antibody SC-285 was used for ErbB3 immunoblotting and immunoprecipitation. SC-283 was the only ErbB4-reactive antibody shown to detect ErbB4 in formalin-fixed, paraffin-embedded rat mammary glands or to immunoprecipitate ErbB4 from rat mammary gland lysates.
Our immunohistochemical studies demonstrate that the naturally proliferative and invasive parenchymal cells within terminal end buds co-express a high level of ErbB2 and ErbB4 but not ErbB3, whereas luminal epithelial cells within ducts and alveoli of nulliparous (pubescent and adult) rats coordinately co-express ErbB2, ErbB3, and/or ErbB4. By Days 1416 of pregnancy, the epithelium undergoes extensive proliferation, differentiation, and alveolar morphogenesis. At this stage, ductal epithelial cells maintain a high level of ErbB3 but ErbB2 is markedly reduced and ErbB4 expression is lost completely. Concurrently, limited or no ErbB receptors were detected along the cell membrane of alveolar epithelium. Although all three ErbB receptors are downregulated in MECs throughout lactation, high levels of ErbB2 and ErbB3 but not ErbB4 are re-expressed in rat mammary glands during involution. In comparison with the epithelial cells, mammary stromal cells express a high level of ErbB2 during puberty, at sexual maturation, and during involution, but this receptor is downregulated and then lost during pregnancy and lactation, respectively. In contrast, ErbB3 is expressed in mammary fibroblasts and/or adipocytes throughout the four developmental stages, whereas stromal cells surrounding terminal end buds, but not ducts or alveoli, express a high level of ErbB4. These studies lay the groundwork for future studies in which activation and signaling of the ErbB receptors can be investigated in the various mammary cell types during these distinct developmental stages. Our immunoblot and immunoprecipitation studies demonstrate the specificity and/or selectivity of the different ErbB receptor antibodies in various types of rat tissues and cells and confirm the developmental expression of ErbB2 and ErbB3 in rat mammary glands.
Our laboratory recently described selective changes in EGFR (ErbB1) expression and localization during rat mammary gland development (
The individual ErbB receptors have been shown to possess unique promoters, catalytic activities, cellular routing, transmodulation domains, and cytoplasmic binding sites for a particular subset of tyrosine kinase substrates (
Expression and Localization of ErbB2
Similar to our studies in rat, a high level of ErbB2 was detected in mammary gland lysates from nulliparous mice (
Expression and Localization of ErbB3
ErbB3 expression was demonstrated by immunoblot and immunohistochemical analysis in mouse mammary glands during pregnancy, lactation, and involution but not during puberty (
Expression and Localization of ErbB4
Recently, functionally immature epithelial cells and myoepithelial cells in normal human breast tissue were shown to express cytoplasmic and cell membrane-associated ErbB4 (
Implications of the Primary Culture Studies
Our primary culture studies not only examined the relationship between ErbB receptor expression and function but also tested the hypothesis that mammary stromal cells are not required for functionally immature and apoptotic MEC to express ErbB2, ErbB3, and/or ErbB4 and for the downregulation of all three receptors in functionally differentiated MECs. The culture studies demonstrate that EGFR (
In summary, the immunohistochemistry study demonstrates high level co-expression of ErbB2 and ErbB4 but not ErbB3 in the proliferative and invasive mammary gland terminal end buds, of ErbB2, ErbB3, and/or ErbB4 in luminal epithelial cells in mammary glands from nulliparous rats, and of ErbB2 and ErbB3 in mammary epithelial and stromal cells during involution. The immunoblotting data indicate that the ErbB receptors were selectively expressed in distinct rat tissues and cells and confirmed the developmental expression of ErbB2 and ErbB3 in the rat mammary gland. All six ErbB receptor heterodimers could be immunoprecipitated from lysates of functionally immature mammary glands from nulliparous rats. These findings, along with the primary culture data, support the hypothesis that select ErbB receptors regulate growth, differentiation, survival, and/or remodeling in the normal mammary gland. These characteristics would enable breast cancer cells that overexpress these receptors to be highly aggressive and invasive. It appears unlikely that this receptor family plays a major role in regulating milk production, secretion, or transportation in rats during lactation because all the ErbB receptors are downregulated in epithelial cells throughout this developmental stage. Finally, additional studies are required to precisely define the factors that regulate ErbB receptor expression, localization, activation, signaling, and/or function in normal and tumor cells in the mammary gland. It is hoped that this information will allow the optimization of strategies to effectively target breast cancer cells that overexpress this receptor family.
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Acknowledgments |
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Supported by DAMD17-94-J-4159, NIH CA33240, and CA64870, and by the shared resources of the NIH Cancer Center Support grant CA16056.
We thank Lawrence Mead, Wendy SheaEaton, Nannette StangleCastor, and Dr Patricia MassoWelch for providing information and discussing issues related to this project, and for helping collect some of the samples for the immunohistochemistry study. Special thanks go to Suzanne M. Shoemaker for her technical expertise in quantifying casein accumulation in the cultured MECs, and to Dr Ping-Ping H. Lee and Dr Patricia MassoWelch for their critical review of this manuscript.
Received for publication March 15, 1999; accepted August 10, 1999.
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