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.



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.

AOB wIti*

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.

I 0.1


Bovine serum albumin fraction V, porcine insulin, mouse submaxillary EGF, Nonidet P-40, prestained


0.2 Bound



I 0.4


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).

binding assay

Table 2. Scatchard analysis: affinity (&) and binding site (B,,,) of EGF receptor in human buccal mucosal homogenate High affinity

Male Female Combined*

Low affinity

& (nM)

Brnll (pmol/mg protein)

Ki (t&f)

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


180 k

116 k


58 k

48.5 k

Plate 1




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.


Gregory H. and Willshire I. R. (1975) The isolation of the urogastrone inhibitors of gastric acid secretion from human urine. Hoppe-Seyler’s Z. physiol. Chem. 356, 1765-1774. Gresik E. W., van der Noen H. and Barka T. (1979) Epidermal growth factor-like material in rat submandibular gland. Am. J. Anat. 156, 83-90. Hirata T. and Orth D. N. (1979) Concentration of epiderma1 growth factor and submandibular gland renin in male and female mouse tissue and fluids. Endocrinology 105, 1382-1387. Jacober L., Wang S. L., Wu-Wang C. Y., Slomiany A. and Slomiany B. L. (1990) Effect of ethanol on oral mucosa epidermal growth factor receptor. J. dent. Res. 69. 197. Konturek J. W., Bielanski W., Konturek S. J., Bogdal J. and Oleksv J. (1989) Distribution and release of eoidermal growth factor in man. Gut 30, 1194-1200. _ Marti U., Burwen S. J. and Jones A. L. (1989) Biological effects of epidermal growth factor with emphasis on the gastrointestinal tract and liver: an update. Hepatology 9, 126138.

REFERENCES Adamson E. D. and Rees A. R. (1981) Epidermal growth factor receptors. M,vlec. Cell Biochem. 34, 129-152. Bans-Schlegel S. P. and Quintero J. (1986) Human esophageal carcinoma cells have fewer, but higher affinity epidermal growth factor receptors. J. biol. Chem. Ml, 43594362.Barka T. (1980) Biologically active polypeptides in submandibular alands. J. Hisiochem. Cvrochem. 28.836-859. Blay J. and Brown K. D. (1985) Functional receptors for epidermal growth factor in an epithelial-cell line derived from the rat small intestine. Biochem. J. 225, 85-94. Byyny R. L., Orth D. N., Cohen S. and Doyne E. S. (1974) Epidermal growth factor: effects of androgens and adrenergic agents. Endoc,rinology 95, 776782. Carpenter G. (1987) Receptors for epidermal growth factor and other polypeptide mitogens. A. Rev. Biochem. 56, 881-914.

Carpenter G. and Cohen S. (1979) Epidermal growth factor. A. Rev. Biochem. CB, 193-216. Carpenter G., Lembach K. J., Morrison M. M. and Cohen S. (1975) Characterization of binding of ‘251-labeled epidermal growth factor to human fibroblasts. J. biol. Chem. 250, 42974304.

Cho M.-I., Lee Y. L. and Garant P. R. (1988) Radioautographic demonstration of receptors for epidermal growth factor in various cells of the oral cavity. Anat. Rec. 222, 191-200.

Cohen S. (1962) Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid open&g in the newborn anima.1. J. biol. Chem. 237. 1555-1562. Cohen S., Ushiro H., Stoscheck C. and Chinkers M. (1982) A native 170,000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J. biol. Chem. 257, 1523-1531. Collins M. K. L., Sinnett-Smith J. W. and Rozengart E. (1983) Platelet-derived growth factor treatment decreases the affinity of the ~epidermal growth factor receptors of Swiss 3T3 cells. J. biol. Chem: 258, 11,68%11,693. Foraue-Lafitte M. E.. Kobari L. and Gesnach C. (1984) Characterization and repartition of epihermal growth factor urogastrone receptor in gastric glands isolated from young and adult guinea pigs. Biochim. biophys. Acta 798, 192-198.

Fox C. F., Linsley P. S. and Wrann M. (1982) Receptor remodelling and regulation in the actions of epidermal growth factor. Celibl, 2988-2994. Gallo-Pavet N. and Huaon J. S. (1985) Eoidermal erowth factor receptors in isolated adult mouse intestinal cells: studies in uiuo and in organ culture. Endocrinology 116, 194-201.

McKee M. D., Martineau B. and Warshawsky H. (1986) Penetration of various molecular weight proteins into the enamel organ and enamel of the rat incisor. Archs oral Biol. 31, 287-296. Moore J. B. (1978) Purification and partial characterization of epidermal growth factor isolated from the male rat submaxillary gland. Archs biochem. Biophys. 189, 1-7. Munson P. J. and Rodbard D. (1980) Ligand: a versatile computerized approach for characterization of ligandbinding systems. Analyt. Biochem. 107, 220-229. Murphy R. A., Watson A. Y., Metz J. and Forssman W. G. (1980) The mouse submandibular gland: an exocrine organ for growth factors. J. Histochem. Cytochem. 28, 890-902.

Osborne C. K., Hamilton B. and Nover M. (1982) Receptor binding and processing of epidennal growth factor by human breast cancer cells. J. clin. Endocr. Metab. 55, 8693.

Partanen A.-M. and Thesleff I. (1987) Localization and quantitation of “%epidermal growth factor binding in mouse embryonic tooth and other embryonic tissues at different developmental stages. Deu. Biol. 120, 186197. Phillips P. D., Kuhnle E. and Cristofalo V. J. (1983) [‘*‘I]-EGF binding ability is stable throughout the reolicative life-man on WI-38 cells. J. Cell Phvsiol. 114. 31’1-316. Sakamoto T., Swierczek J. S. and Ogden W. D. (1985) Cytoprotective effect of pentagastrin and epidermal growth factor on stress ulcer formation. Possible role of somatostatin. Ann. Surg. 301, 290-295. Sarosiek J., Bilski J., Murty V. L. N., Slomiany A. and Slomiany B. L. (1988) Role of salivary epidennal growth factor in the maintenance of physicochemical characteristics of oral and gastric mucosal mucus coat. Biochem. biophys. Res. Commun. 152, 1421-1427.

Scatchard G. (1949) Attractions of uroteins for small molecules and ions. Ann N. Y. Acad. ‘Sci. 51, 66&672. Schultz G. S., White M., Mitchell R., Brown G., Lynch J., Twardzik D. R. and Todaro G. J. (1987) Epithelial wound healing enhanced by transforming growth factor-a and vaccinia growth factor. Science 235, 35&352. Slomiany B. L., Murty V. L. N., Piotrowski J. and Slomiany A. (1989) Effect of antiulcer agent on the ohvsicochemical pro’pertids of gastric mucus. 1;: Mucus and Related Topics (Edited bv Chantler E. and Ratcliffe N. A.) DD. 179-191. Company of Biologists, Cambridge. ’ _St. Hilaire R. J.. Gosoodarowicz D. and Kim Y. S. (1980) Epidermal growth factor: effect on the growth of human colon adenocarcinoma cell line Gastroenterology 78, 127 1 (Abstr.).


S.-L. WANG et al.

Thesleff I. (1987) Epithelial cell rests of Malassez bind epidermal growth factor intensely. J. periodont. Res. 22, 419-421.

Thesleff I., Partanen A. M. and Rihtniemi L. (1987) Localization of epidermal growth factor receptors in mouse incisors and human premolars during eruption. Eur. J. Orrhop. 9, 24-32.

Wang S.-L., Shiverick K. T., Ogilvie S., Dunn W. A. and Raizada M. K. (1989) Characterization of epidermal

growth factor receptors in astrocytic ghal and neuronal cells in primary culture. Endocrinology 124, 24&247. Wang S.-L., Wu-Wang C.-Y., Slomiany A. and Slomiany B. L. (1990) Characterization of epidermal growth factor receptor in rat buccal mucosal cells. Znt. J. Biochem. 22, 669-675.

Webber W., Bertics P. J. and Gill G. N. (1984) Immunoaffinity purification of the epidermal growth factor. J. biol. Chem. 259, 14,631-14,636.

Identification of epidermal growth factor receptor in human buccal mucosa.

EGF receptor was identified and its binding characteristics were determined. Buccal mucosa was obtained from 12 healthy volunteers (6 males and 6 fema...
791KB Sizes 0 Downloads 0 Views