Vol. 35, No. 10, pp. 823-828, 1990 Printed in Great Britain. All rights reserved
0003-9969/90 $3.00 + 0.00 Copyright 0 1990 Pergamon Press plc
Archs oral Bid.
OF EPIDERMAL GROWTH FACTOR IN HUMAN BUCCAL MUCOSA
S.-L. WAI~G,’ M. MILLFS,~ C.-Y. WU-WANG,’ J. LIU,’ A. SLOMIANY’and B. L. SLOMIANY’ ‘Research Center and 2Department of Oral and Maxillofacial Surgery, New Jersey Dental School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103-2400, U.S.A. (Accepted 1 May 1990)
Summary-EGF receptor was identified and its binding characteristics were determined. Buccal mucosa was obtained from 12 healthy volunteers (6 males and 6 females) and assayed individually for [ ‘251]-EGF binding. The specific binding of [ ‘251]-EGF to the receptor ranged from 2.85 to 6.12 fmol/mg protein. There was no significant difference in binding between male and female (4.31 k 0.61 versus 3.94 i 0.53 fmol/mg protein; mean + SEM). Individual tissue homogenates were pooled for Scatchard analysis and cross-linking experiments. Scatchard analysis produced curvilinear plots with a Kd of 0.71 nM and B,,, of 0.024 pmol/mg protein for the high-affinity binding sites, and Kd of 435 nM and B,, of 9.92 pmol/mg, protein for the low-affinity binding sites. To determine the molecular weight of the EGF receptor, the [ ‘251]-EGF and receptor complex were cross-linked by DSS and subjected to SDS-PAGE. The autorachogram of the gel revealed one major protein band of 160 K and a minor band of 170 K, characteristics shared with the EGF receptors in other tissues. The study is thought to be the first to demonstrate the presence of the EGF receptor in human buccal tissue and to show its biochemical features. Key words: epidermal growth factor, receptor, buccal mucosa, human.
Epidermal growth ITactor is a well-characterized, 53amino acid peptide mitogen which, as first observed by Cohen (1962), causes premature eruption of teeth and early eyelid opening in newborn mice (Carpenter and Cohen, 1979). The peptide is secreted primarily by the granular convoluted tubule cells of the submandibular glands ‘ofthe mouse and rat (Gresik, van der Noen and Barka, 1979; Barka, 1980; Murphy et al., 1980). In humans, the major sources of EGF are salivary glands, duodenum and pancreas (Konturek et al., 1989). Various reports indicate that EGF stimulates proliferation of numerous cell types in vitro and of epithelial cells in uiuo (Cohen, 1962; Carpenter, 1987) and, in addition to its mitogenic effect, influences various functions during differentiation, such as hormone synthesis and secretion, cell morphology, phosphorylation 01’ cellular proteins (Fox, Linsley and Wrann, 1982; Carpenter, 1987). The factor interacts with the targel: cells by binding to its receptors on the cellular membrane. The binding of EGF to the cell surface receptor is considered to be the first step in a chain of reactions that culminate in mitosis (Carpenter, 1987). This growth factor also has biological effects throughout the emire gastrointestinal tract (Marti, Abbreviufions:
BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulphoxide; DSS, disuccinimidyl suberate; EGF, epidermal growth factor; PMSF, phenylmethylsulphonyl fluoride; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis.
Burwen and Jones, 1989) and its receptors are found in gastric glandular tissue (Forque-Lafitte, Kobari and Gespach, 1984), small intestinal mucosa (Blay and Brown, 1985; Gallo-Payet and Hugon, 1985), and several carcinoma cell lines of gastrointestinal origin (St. Hilaire, Gospodarowicz and Kim, 1980; Bans-Schlegel and Quintero, 1986). There is also evidence that EGF inhibits gastric acid secretion (Gregory and Willshire, 1975; Adamson and Rees, 1981), enhances epidermal proliferation and wound healing (Sakamoto, Swierczek and Ogden, 1985; Schultz et al., 1987), and plays a role in mucosal protection (Sarosiek et al., 1988; Slomiany et nl., 1989). Although the mouth is the first part of the alimentary tract and is exposed to a variety of physical, chemical and microbial insults, little is known about the function of EGF receptors in the soft oral tissues. Nevertheless, it has been suggested that EGF may play a significant role in the development and maintenance of oral tissue integrity. Histological studies have indicated high labelling with [ 125 I]-EGF of various tissues in the mouth, such as the enamel of the rat incisor (McKee, Martineau and Warshawsky, 1986), the apical tissue of the developing root and dental follicle mesenchyme in the mouse (Thesleff, Partanen and Rihtniemi, 1987; Partanen and Thesleff, 1987), human dental follicle (Thesleff, 1987), oral epithelium, papillary cells of the enamel periodontal ligament fibroblasts and organ, preosteoblasts (Cho, Lee and Garant, 1988). We have now sought to characterize the binding properties of EGF receptor in human buccal mucosa and assess its biochemical characteristics. 823
S.-L. WANGet al.
Table 1. Specific binding of [ ‘2sI]-EGF to human buccal mucosal homogenate Specific binding ( fmol/mg protein) Male 4.31 2 0.61 (n = 6)
Female 3.94 * 0.53 (n = 6)
Values are the mean f SEM of duplicate analyses performed on the individual samples. MATERIALS
protein standard marker, DMSO and urethane were from Sigma Chemical Co., St Louis, MO. Chemicals for SDS-PAGE were purchased from Bio-Rad, Hempstead, NY. The [ ‘2SI]-EGF (sp. act. 100 Ci/pg) was obtained from Amersham Co., Arlington Heights, IL. The DMEM was from Gibco, Grand Island, NY, and the BCA protein assay kit and DSS were purchased from Pierce Biochemicals, Rockford, IL.
Bovine serum albumin fraction V, porcine insulin, mouse submaxillary EGF, Nonidet P-40, prestained
Fig. 1. Scatchard plots of [ iz51]-EGF binding to human buccal mucosal homogenate. The standard binding assay was conducted in duplicate in the presence of 0, 0.0025, 0.005, 0.01, 0.05, 0.1, 0.25, 0.5, 0.75 and l.OpM of unlabelled EGF. The B/F ratio is plotted as a function of EGF bound to the cell membrane.
Collection of human buccal mucosa
Twelve healthy volunteers (6 females and 6 males; aged 22-26 yr) participated in the study after giving informed consent. The specimens of their oral mucosa were obtained by a biopsy technique in which a 15 x 10 mm eliptical incision was made in the mid portion of the cheek mucosa adjacent to the second premolar/first molar teeth under local anaesthesia with 2% lidocaine containing epinephrine, 1: 100,000. Immediately after collection the specimens were rinsed with cold DMEM and stored in liqud nitrogen until use.
A standard [ i2’I]-EGF binding assay was conducted as previously by Wang et al. (1989). The tissue homogenate was incubated in duplicate with [‘2sI]EGF (40,000 cpm, 0.18 nM) and EGF receptor binding assay buffer (5 mM tris-HCl, 125 mM sucrose, 75 mM NaCl, 0.5 mM Ca2+ and 0.5% BSA, pH 7.0) in a final volume of 200 ,ul for 1 h at 4°C for the measurement of total binding. Non-specific binding was determined in the presence of 0.25 PM unlabelled EGF from mouse submaxillary gland. The reaction was terminated by addition of 1 ml of ice-cold 10 mM tris-HCl, pH 7.0, containing 0.5% BSA. Membranebound [‘251]-EGF was separated immediately by centrifugation at 10,OOOgfor 10 min in a Beckman microcentrifuge at 4”C, followed by aspiration of the supernatant. The pellet was washed once more with the same buffer and separated as described. Specific binding was obtained by subtracting the radioactivity of non-specific binding from the total binding and expressed as a ratio of the specific binding versus unbound [ ‘251]-EGF (B/F) or mol EGF bound/mg protein.
Preparation of tissue homogenate Individual specimens were homogenized with 1.5 ml of 0.25 M sucrose, 25 mM tris-HCl buffer, pH 7.4, containing 1 mM PMSF, 100 kU/ml aprotinin and 1 pgg/ml leupeptin. The homogenate was centrifuged at 600g for 15 min and the supernatant was used for the experiment. The protein concentration of the homogenate was determined by the BCA protein
assay kit using BSA as the standard (Pierce Biochem. Inc., protein assay manual).
Table 2. Scatchard analysis: affinity (&) and binding site (B,,,) of EGF receptor in human buccal mucosal homogenate High affinity
Male Female Combined*
Brnll (pmol/mg protein)
Bnlar (pmol/mg protein)
0.64 0.86 0.71
0.021 0.032 0.024
515 194 435
4.7 2.1 9.9
*The data derived through re-analysis of raw data of male and female combined.
Plate 1 Fig. 2. Autoradiogram of SDS-PAGE characterizing the EGF receptor covalently cross-linked with [ iz51]-EGF. Human buccal tissue homogenate was incubated with [ ‘*‘I]-EGF in the absence (-EGF) and the presence (+ EGF) of excess unlabelled EGF.
Growth factor receptor in buccal mucosa
WANG et ai
To elucidate the binding properties, such as the affinity and the binding site of the EGF receptors, individual homogenates from male or female vohmteers were pooled for Scatchard analysis. The analysis was done with a standard binding assay, except that various concentrations of unlabelled EGF (O-l PM) were added to the reaction mixtures. The Scatchard plot was analysed by a computer program ‘LIGAND, based on the mathematical vector analysis for two-site receptor (Scatchard, 1949; Munson and Rodbard, 1980). [ “-‘I]-EGF cross -linking The cross-linking study was made according to a modified method of Blay and Brown (1985), as described by Wang et al. (1989, 1990). Pooled homogenate (male and female) was incubated with 10 ng of [‘251]-EGF in the absence of presence of 1 PM unlabelled EGF in a final volume of 350 ~1 for 1 h at 4°C. The EGF-bound pellet was centrifuged, extensively washed, resuspended in 1 ml of cold phosphate-buffered saline containing 0.9 mM Ca2+ and 0.5 mM Mg2+ and then cross-linked using 3 mM DSS in 30 ~1 DMSO at 4°C for 15 min. The cross-linking reaction was quenched by replacing the solution with 1 ml of cold 0.15 M tris-HCI, pH 7.4. The pellet was lysed in 100 ~1 of lysis buffer (10 mM NaCI/l.S mM MgCl/l% Nonidet P-40/10 mM tris-HCl, pH 7.4) at 25°C for 10min. The solubilized samples were then analysed by SDS-PAGE. The gels were dried on filter paper under vacuum, and exposed to a Kodak XAR-5 film cassette with standard intensifying screen at -70°C. Results were expressed as means + SEM. Student’s t-test was used to determine significance, and p values of 0.05 or less were considered significant. RESULTS
binding to buccal mucosa
The specific binding of [ ‘25I]-EGF to the receptor ranged from 2.85 to 6.12 fmol/mg protein. Table 1 shows the specific binding of [ i2’I]-EGF to the receptor prepared from male and female buccal mucosa; there was no significant difference for this binding between the sexes. Scatchard plot analysis of [‘251]EGF specific binding to buccal mucosa demonstrated a typical curvilinear plot for both male and female (Text Fig. 1). Analysis of the data by the ‘LIGAND’ computer program indicated the presence of highand low-affinity binding sites in the buccal mucosa. The affinities (&) and binding capacities (B,,,) of high- and low-affinity receptors from male and female buccal mucosa are summarized in Table 2. Since the best-fit curve was obtained by re-analysing the combined data from male and female subjects, the combined data of Kd and B,,, are also included. Identl$cation of the EGF receptor To identify the EGF receptor on buccal mucosa, the membrane receptor were covalently labelled with [ ‘251]-EGF using DSS, analysed by SDS-PAGE under reducing conditions, and autoradiographed. The autoradiogram in Plate Fig. 2 shows the crosslinking of [ “‘I]-EGF to mucosal EGF receptor. One
major radioactive band with molecular weight of 160 kDa and a minor band of 170 kDa were observed. No radioactive bands were detected in the presence of excess amount of unlabelled EGF. DISCUSSION
We here provide evidence for the presence of the EGF receptor in human bucccal mucosa and describe its binding kinetics. The receptor binding was reversible, with curvilinear plots from Scatchard analysis similar to those described for EGF receptor binding in other types of cells and tissues, and with similar high-affinity dissociation constants, 0.71 x 10m9 versus 0.1-1.0 x 10m9 M, (Carpenter et al., 1975; Osborne, Hamilton and Nover, 1982; Collins et al., 1983; Phillips, Kuhnle and Cristofalo, 1983; Blay and Brown, 1985; Carpenter, 1987; Wang et al., 1989). The Kd value of the low-affinity binding site was higher than those reported before, 4.35 x lo-’ versus 5.0 x lo-’ M (Blay and Brown, 1985; Collins er al., 1983), which might be due to the different sample source or methods of membrane preparation. The biochemical evidence for the presence of EGF receptor in human buccal mucosa was further substantiated by the data obtained from the experiments with [ I25I]-EGF cross-linking. The covalent crosslinking of [ ‘2SI]-EGF to buccal mucosa revealed one major radioactive band with molecular weight of 160 K which is slightly lower than the molecular weight of EGF receptor in rat buccal mucosa cells (Wang ef al., 1990). Blay and Brown (1985) found that the EGF receptor in an epithelial cell line derived from the rat small intestine was a 160 K protein. A minor radioactive band with molecular weight of 170 K was also observed. This band may actually represent the intact form of EGF receptor, as it has been reported that the 160 K protein arises through proteolysis of the 170 K protein (Cohen et al., 1982; Webber, Bertics and Gill, 1984; Blay and Brown, 1985). Various proteases may have been present in the crude homogenate used in our experiments and some of them could be resistant to protease inhibitors (PMSF, aprotinin and leupeptin) included in the homogenization buffer. Because of the limited amount of human buccal mucosal tissue used, we were not able to ascertain the nature of protease responsible for this effect. However, the [ I25I]-EGF binding to 160 kDa receptor protein was competitively displaced by the unlabelled EGF as evidenced by the disappearance of the radioactive band in the presence of excess amount of unlabelled EGF. This observation suggests that the 160 K protein is EGF-specific. EGF is present in high concentrations in the salivary glands of animals (Moore, 1978; Gresik et al., 1979) and humans (Konturek et al., 1989), and is secreted with saliva (Byyny et al., 1974; Hirata and Orth, 1979; Konturek et al., 1989). Other than the well-known stimulation of incisor eruption by EGF (Cohen, 1962), there are indications that salivary EGF is essential for the protection of oral and gastric epithelium through the maintenance of mucus-coat dimension and its integrity (Sarosiek er al., 1988; Slomiany et al., 1989). Our recent investigation (Jacober et al., 1990) of the effect of ethanol on EGF
Growth factor receptor in buccal mucosa
receptor in rat buccal mucosa indicated an ethanolrelated decrease of EGF binding and implicated a relationship between the EGF receptor and ethanolinduced disease in the mouth. Our demonstration of the presence of specific EGF receptor in buccal mucosa now provides further evidence in favour of a cellular mechanism by which salivary EGF affects epithelial cell function in the mouth. This receptor has biochemical properties similar to the EGF receptors in different tissues. The identification of EGF receptor in buccal mucosa may open new avenues in understanding the mechanism of oral mucosal defence. Acknowledgements-Supported by USPHS Grant No. DE05666-12 from the National Institute of Dental Research and Grant No. AA05858-08 from the National Institute of Alcoholism and Alcohol Abuse, NIH.
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