ARTICLE |
Correspondence to: Edward W. Gresik, Dept. of Cell Biology and Anatomical Sciences, City University of New York Medical School, New York, NY 10031.
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Summary |
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Branching morphogenesis of the fetal mouse submandibular gland (SMG) can be modulated in vitro by stimulation or inhibition of the epidermal growth factor receptor (EGFR). Because the mRNAs for EGF and EGFR are detectable in RNA of SMG rudiments isolated directly from fetuses, the EGF system probably operates physiologically as a regulator of SMG morphogenesis. However, neither EGFR protein nor its precise cellular localization has been characterized in the fetal SMG. Here we show EGFR protein in fetal mouse SMG by immunoprecipitation, affinity labeling, ligand-induced autophosphorylation, and immunohistochemistry. SMGs from E16 fetuses (day of vaginal plug = E0) were labeled with [35S]-cysteine/methionine and homogenized. After addition of specific antibody to EGFR, the immunoprecipitate was isolated, resolved by polyacrylamide gel electrophoresis, and detected by autoradiography. A single band of 170 kD was detected, corresponding to the EGFR protein. Affinity labeling with [125I]-EGF of the membrane fraction of E18 SMG also revealed a prominent band at 170 kD, showing that this EGFR protein can bind specifically to its ligand. Incubation of SMG membranes from E18 fetuses with EGF in the presence of [-32P]-ATP, followed by immunoprecipitation with anti-phosphotyrosine antibody also showed a single band at 170 kD, demonstrating autophosphorylation of the EGFR in response to binding of its ligand. Immunohistochemical localization of the cellular sites of EGFR in the fetal SMG required use of a catalyzed signal amplification procedure, with biotinyltyramide as the amplifying agent. EGFR was localized predominantly, if not exclusively, in cell membranes of epithelial cells of the rudiment, whereas staining of mesenchymal cells was equivocal. Staining was strongest on duct cells, and weak on cells of the end-pieces. These findings clearly show that a functional EGFR protein is expressed in fetal SMG chiefly, if not exclusively, on epithelial cells. (J Histochem Cytochem 45:1651-1657, 1997)
Key Words: mouse, fetal, submandibular gland, EGF receptor
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Introduction |
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A VARIETY of growth factor systems play important roles during fetal organogenesis (EGF and transforming growth factor-
(TGF
)
influence fetal development of the lung (
We have previously shown that the mRNAs for EGF and EGFR are expressed in the SMG throughout late fetal development (
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Materials and Methods |
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Animals
Timed-pregnant CD-1 mice were purchased from Charles River Labs (Wilmington, MA). Day of discovery of the vaginal plug was taken as day 0 (E0). The mice were sacrificed by cervical dislocation and fetuses were collected aseptically and staged according to the criteria of
Metabolic Labeling
E16 SMG rudiments (11 paired glands, all from a single litter) were isolated in sterile BGJb medium (GIBCO BRL; Grand Island, NY). They were metabolically labeled with [35S]-cysteine/methionine as described previously (
Immunoprecipitation of Metabolically Labeled EGFR
35S-labeled SMGs were thawed and homogenized in a glass-Teflon mortar and pestle in 0.8 ml of Buffer A (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 1 µg/ml leupeptin, 1 µg/ml antipain, 1 mM AEBSF ([4-(2-amino-ethyl)]-benzenesulfonylfluoride, a protease inhibitor equivalent to phenylmethylsulfonylfluoride, PMSF) (Calbiochem; La Jolla, CA). Membranes were dispersed by incubation at 4C for 2 hr and the homogenate was cleared by centrifugation (14,000 x g, 20 min). Levels of incorporation of [35S]-cysteine/methionine were determined by trichloroacetic acid precipitation. Aliquots of the cleared supernatant containing 1.5 x 106 cpm were preabsorbed overnight with 100 µl of protein G-Agarose (50% solution; GIBCO BRL) and then incubated overnight with 7 µl of anti-EGFR (monoclonal anti-human EGFR, E-3138, Clone F4; Sigma Chemicals, St Louis, MO). Then 40 µl of protein G-Agarose beads was added and the mixture incubated for 1 hr. The beads with the bound immune complexes were pelleted at 16,000 x g for 5 min, washed three times with Buffer B (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Nonidet P-40, 0.25% gelatin, 1 mM EDTA), resuspended in 20 µl of SDS sample buffer [50 mM Tris-HCl buffer, pH 6.8, 100 mM dithiotreitol, 10 mM EDTA, 1% sodium dodecyl sulfate (SDS), 10% glycerol, 0.05% bromphenol blue], and boiled for 3 min. The boiled mixture was subjected to electrophoresis in a 7.5% SDS-PAGE and the resolved radiolabeled immunoprecipitates were visualized by autoradiography of the dried gel by exposure to Kodak XAR film (Eastman Kodak; Rochester, NY) for 4 days at -80C.
Affinity Labeling of EGFR
Microsomal membranes were prepared from SMGs pooled from 13 E18 fetuses (about 150 mg tissue) by Polytron homogenization in 3 ml of 50 mM HEPES buffer, pH 7.4, 250 mM sucrose, 5 mM EDTA, and 1 mM PMSF. The homogenate was centrifuged at 12,000 x g for 20 min. The resulting supernatant was then centrifuged at 100,000 x g for 45 min. The microsomal pellet was suspended in the above buffer without EDTA and centrifuged at 100,000 x g for 45 min. The pellet was resuspended in the above buffer without EDTA, and the protein concentration was measured by the method of
Immunoprecipitation of Autophosphorylated EGFR
SMG rudiments were pooled from 25 fetuses from two litters at E18, and a microsomal membrane fraction was prepared essentially as above. The microsomal membranes were pelleted (100,000 x g, 45 min, 4C), washed twice with homogenization buffer, and solubilized in 200 µl 50 mM HEPES, pH 7.4, 1% TritonX-100, 10% glycerol, 2 mg/ml bacitracin, 1 mM PMSF with gentle shaking for 1 hr at 4C. The solubilized membrane fraction was collected by centrifugation at 100,000 x g for 45 min at 4C, and protein content was determined with a Protein Assay Kit (BioRad; Hercules, CA).
The phosphorylation was performed according to -32P]-ATP/µl). After incubation at 0C for 30 min, the reaction was terminated by addition of 40 µl 100 mM Tris-HCl, pH 6.8, 4% SDS, and boiling for 5 min. A 15-µl aliquot of the reaction mixture was diluted to 600 µl with 50 mM Tris-HCl, pH 7.4, 150 mM NaCl. Five µg of monoclonal antibody against phosphotyrosine (PY20; Wako Pure Chemical Industries, Osaka, Japan) was then added and the mixture was incubated for 3 hr at 4C. The immune complexes were then reacted with 20 µl protein G-Agarose for 1 hr at 4C. The agarose beads with bound immune complexes were pelleted at 16,000 x g for 5 min and washed three times with 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Nonidet P-40, 0.25% gelatin, 1 mM EDTA. The final pellets were boiled in 20 µl SDS sample buffer, and the immunoreacted proteins were analyzed by SDS-PAGE, as above.
Immunohistochemistry for EGFR
Fetal tissues were fixed overnight at room temperature (RT) in Carnoy's solution or Bouin's solution and embedded in paraffin. Fetuses at E13 and E14 were fixed in toto; older fetuses were cut across the thorax, and only the cranial portion was processed for microscopy. Paraffin sections (6 µm) were affixed to TESPA-coated glass slides (TESPA = 3-aminopropyl-triethoxysilane; Sigma) and dried overnight in a 55C oven. The primary antibody was a sheep antiserum against human EGFR (GIBCO), applied at RT for 1 hr at a dilution of 1:2000 in 0.5% BSA in PBS. The sparsity of EGFR required use of the Catalyzed Signal Amplification (CSA) System (DAKO; Carpinteria, CA), which was used according to the manufacturer's protocol except that 1:200 biotinylated anti-sheep IgG (Vector Labs; Burlingame, CA) was used in place of the first immunological reagent of the kit. Final detection was achieved by exposing the sections to diaminobenzidine/H2O2 (
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Results |
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Immunoprecipitation of EGFR
Immunoprecipitation by a specific antibody against the EGFR revealed a single radiolabeled band at 170 kD (Figure 1), typical of the EGFR (
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Affinity Labeling of the EGFR with [125I]-EGF
SDS-PAGE of fetal SMG membrane fractions after crosslinking of [125I]-EGF disclosed a predominant band at 170 kD and a minor band at 150 kD (Figure 2, Lane 1), also typical of the behavior of the EGFR (
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Immunoprecipitation of Autophosphorylated EGFR
Exposure of the membrane fraction of fetal SMGs to EGF in the presence of [-32P]-ATP, followed by immunoprecipitation with anti-phosphotyrosine antibodies and resolution of the immunoprecipitate by SDS-PAGE, demonstrated a specifically labeled band at 170 kD, representing the autophosphorylated EGFR (Figure 3, Lane 4). An extremely faint band at 170 kD was also seen in membrane fractions that were not exposed to exogenous EGF; this band was so light that it could not be reproduced photographically (Figure 3, Lane 2), and is an indication of a very low level of EGFR autophosphorylation owing to endogenous EGF in the fetal rudiments.
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Immunohistochemical Localization of EGFR
At all fetal ages, specific staining for the EGFR was localized to membranes of epithelial structures (Figure 4). Strongest staining was seen in developing ducts, and weak staining was seen in end-pieces. Questionable staining was seen on the surfaces of some mesenchymal cells. At earlier stages of development, staining was seen on all surfaces of epithelial cells (Figure 4A-C), but at later stages it appeared to be more prominent on basolateral surfaces of cells in contact with the mesenchyme, especially in developing duct cells (Figure 4B-E). Replacement of the primary antibody by anti-EGFR preabsorbed with A431 membranes abolished the strong staining of the ducts and the weak but specific staining of the end-pieces, but had equivocal effects on the staining of mesenchymal cells (Figure 4F).
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Discussion |
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These findings clearly establish that functional EGFR is expressed in the fetal SMG. Previously, we showed that mRNA for EGFR is present in this developing organ and that it increases with fetal age, but questions remained as to whether and when this mRNA was translated into protein. Our immunohistochemical findings show that EGFR protein is present throughout fetal development of the gland. Moreover, immunoprecipitation and affinity labeling demonstrate that the EGFR present in the gland is of the same size as that in adult tissues (
EGFR is expressed primarily by the epithelial cells and principally in the ducts of the developing gland. Expression by mesenchymal cells is apparently absent or is minimal at best, in agreement with the autoradiographic localization of iodinated EGF by 6-integrin subunit (
The EGF system not only is a potent mitogen but is also an effective regulator of cytodifferentiation in many tissues (6-integrin subunit proteins in their basal regions more strongly than do the end-pieces (
In other developing tissues, the EGFR may be expressed on epithelial cells or on mesenchymal cells, or on both cell types, and the pattern of expression may vary with developmental age (
Although the in vitro findings of Nogawa and colleagues ( does not, implying that EGF or proEGF is the physiological ligand for this receptor in this gland (
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Acknowledgments |
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Supported by NIH Grant DE10858, by the Kitasato University Fund for International Exchange Programmes, and by a Fogarty International Fellowship RC29637043 (EG), and by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (no. 08670031 to SY and no 08670033 to YK).
We thank Osamu Katsumata and Cindy Zhang for expert technical help with the immunohistochemical preparations.
Received for publication February 28, 1997; accepted June 25, 1997.
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