THE JOURNAL OF EXPERIMENTAL ZOOLOGY 258336-343 (1991)
Expression of Epidermal Growth Factor (EGF) and the EGF Receptor in Human Tissues RYUICHI FUKUYAMA AND NOBUYOSHI SHIMIZU Department of Molecular Biology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160, Japan ABSTRACT
The EGF-EGF receptor system has been widely examined for signal transduction, control of cell growth and differentiation, and in vivo physiological function and carcinogenesis. The localization of EGF receptors in vivo led to the idea that the system is operative in proliferation and differentiation of cells and tissues. However, a consensus for its distribution and function in human tissues has not yet been determined because of discrepancies in the reported results. Using a highly specific monoclonal antibody against the EGF receptor, we examined various tissues of a n infant and adults as well as embryonal carcinoma. We observed restricted localization of EGF receptors in basal cells of epithelial tissues and duct cells of secretory tissues. Fibroblasts express a high level of EGF receptors when they are rapidly growing. Using the monoclonal anti-EGF antibodies, we observed that EGF is localized to differentiated cells rather than to stem cells such as glandular tissues. We also observed that some cells express both EGF and the EGF receptor. All histochemical results indicated that in epidermis and various glandular tissues, EGF may be expressed in differentiating cells derived from the stem cells expressing EGF receptors.
Since the discovery of EGF (Cohen, '62, '861, charide moieties of the EGF receptors. Therefore, its role in physiological function has been widely we have investigated human tissues using a studied, especially in rodents, in the development monoclonal EGF receptor antibody which recogof intestine (Malo and Menard, '82; Oka et al., nizes a peptide moiety of the extracellular domain '831, oocyte (Downs et al., '88) and lung (Catterton (Amagai et al., '88; Behzadian and Shimizu, '85; et al., '79) as well as in eyelid opening and tooth Gamou et al., '88; Ozawa et al., '87). We tested growth (Cohen, '62, '86). The ligand binding stud- various tissues from a 1year old infant and adults ies suggested that the EGF receptor is localized and a testicular embryonal carcinoma. The amount of EGF in body fluids increases unon epithelial cell surface. To date, several monoclonal antibodies against human EGF receptors til about 9 years old and decreases thereafter (Joh (Behzadian and Shimizu, '85; Shimizu, '84; Wa- et al., '86; Uchihashi et al., '82). The histology of terfield et al., '82) and synthetic oligopeptides embryonal carcinoma includes immature and/or (Gullick et al., '86) were obtained. Using these adult teratoid structures as well as ectoblastic monoclonal antibodies, localization of EGF recep- and trophoblastic elements (Teilium, '65). Thus, tors in human normal and cancer tissues (Adam- infant and embryonal carcinoma tissues are useson and Rees, '81; Amagai et al., '88; Damjanov ful for EGF and EGF receptor localization. By imet al., '86; Gullick et al., 86; Nanney et al., '84a, munohistochemical staining, we observed that 84b; Ozawa et al., '87) has been examined t o eluci- the localization of EGF and the EGF receptor is date the possible role of the EGF-EGF receptor correlated with differentiated cells and their stem system in these physiological states. The EGF cells, respectively. We postulate that the expresreceptor was detected in both proliferating and sion of EGF and the EGF receptor switches durdifferentiating cells, and, hence, EGF was pos- ing the development and evolution of human epitulated t o affect both cell growth and differen- thelial tissues. tiation. However, the results obtained with these antibodies were often inconsistent between groups and with the results obtained using conReceived April 30, 1990; revision accepted September 26, 1990. ventional autoradiography with 1251-labeledEGF Address reprint requests to Nobuyoshi Shimizu, Department of Mo(Green et al., '85). This discrepancy may be due lecular Biology, Keio University School of Medicine, 35 Shinanoto the fact that most antibodies recognize polysac- machi, Shinjuku-ku, Tokyo 160, Japan. 0 1991 WILEY-LISS, INC.
EGFANDEGFRECEPT()R IN HUMAN TISSUES
MATERIALS AND METHODS Cells and tissues A431 cells were grown on glass slides under previously described culture conditions (Gamou et al., '88). Tissues of testicular embryonal carcinoma were obtained from surgical specimens. One year old infant and adult tissues were obtained from autopsied material. A431 cells grown on glass slides were fixed with cold acetone for 10 minutes and air dried. All the tissues obtained were trimmed, frozen immediately with liquid nitrogen, and stored at -70°C until use. Cryostat sectioned tissues (4-6 Fm) mounted on albumin-coated glass slides were fixed for 10 minutes with absolute methanol for EGF staining or acetone for EGF receptor staining. Some slides were processed for staining without fixation.
Incorporation of BrdU to human embryonal carcinoma tissues Small pieces (about 5 mm in diameter) of embryonal carcinoma tissues were immediately immersed in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 400 FM BrdU (Ikeda Rika, Osaka, Japan) as previously described (Amagai et al., '88). Incorporation of BrdU was processed for 3 hours under pressure 2 kg w/cm2 at 37°C with shaking followed by fixation with Carnoy's solution and embedded in paraffin. Antibodies The B4G7 monoclonal antibody recognizes a peptide moiety of the extracellular domain of the EGF receptor (Behzadian and Shimizu, '85). Monoclonal antibodies against mouse EGF (mEGF) and human EGF (hEGF), designated KEM-9 and HA, were generous gifts from Wakunaga Pharmaceutical Co. Ltd. (Hiroshima, Japan) and Ikuta et al. (Ikuta et al., '85), respectively. All three antibodies are IgG and are used after 1:400 dilution from a 1mg/ml stock solution. For detection of nuclei incorporating BrdU, monoclonal anti-BrdU antibody (Becton Dickinson, Mountain View, CA) was used in a 1:100 dilution. FITC-conjugated anti-mouse IgG and biotinylated anti-mouse IgG were purchased from SILENOUS Laboratories (Victoria, Australia). Avidin-biotin-peroxidase complex made just before use was obtained from Vector Laboratories (Burlingame, CA). We used non-immune mouse Ig as a control.
337
Immunohistochemistry Indirect immunofluorescence with FITC-conjugated secondary antibody was used t o detect B4G7. The ABC method (Hsu et al., '81) was employed for immunohistochemical staining of both human frozen and paraffin-embedded tissues. Color was developed with a solution of 0.025% 3' ,3-diaminobenzidine (Dojindo, Japan) and 0.03% H,02. A431 cells were incubated with antibody B4G7 as a positive control for staining efficiency.
RESULTS EGF receptors in A431 cells were localized on the cell surface using monoclonal antibody B4G7 (Fig. la,b). The sensitivity of EGF receptor detection under these conditions was very high. Although the whole cell surface was stained, dense staining was obtained in areas of cell-to-cell contact. Embryonal carcinoma tissues were stained positive with B4G7 on the cell surface of single and stratified endodermal epithelia (Fig. lc) as well as mesenchymal cells accumulating around the mature endodermal glands (Fig. Id). The cytoplasm of goblet cells from endodermal glands was stained positive for EGF expression when analyzed with the EGF-specific antibody HA (Fig. le,f). EGF or a n EGF-like substance was discharged into the lumen from glandular cells. Such apparently mature endodermal epithelia displayed low BrdU incorporation into nuclei (Fig. lg), whereas stromal cells showed high incorporation of BrdU into .nuclei (24%) (Fig. lg, dark nuclei). Figure 2 shows infant and adult skin tissues including epidermis and dermis, sweat gland, hair outer root sheath, and sebaceous gland which were stained with anti-EGF receptor antibody B4G7 (Fig. 2a-c,e) or with anti-EGF antibody HA (Fig. 2d,f). All basal cells of the epidermis, sweat gland duct, and excretory duct of sebaceous glands, from which the differentiated progenies arise, were clearly stained with anti-receptor antibodies, whereas stratum corneum, the inner cells of the sweat gland duct, and sebaceous gland were negative for the EGF receptor. Sweat gland cells were stained in their cytoplasm with antiEGF antibody (Fig. 2d). EGF-staining in the cytoplasm of sebaceous gland cells was faint and granular (Fig. 2f, arrowheads). Figure 3 shows tissues derived from an adult and a 1year old infant, including liver, bronchial gland, prostate gland, epididymis, and a ganglion
338
R. FUKUYAMA AND N. SHIMIZU
Fig. 1. Immunohistochemical staining of EGF and the EGF receptor expressed on various cells and tissues. a: A431 cells stained with anti-EGF receptor antibody B4G7. Spindleshaped cells were mouse A9 cells cocultured as a n internal negative control. b: A431 cells reacted with non-immune mouse Ig. Original magnification (O.M.) x 640. c , d Tissues of embryonal carcinoma stained with B4G7. e: Endodermal gland of embryonal carcinoma stained with anti-EGF antibody HA. f: Adjacent sections of (e) reacted with non-immune mouse Ig. These specimens were counterstained with hematoxylin. g: Tissues of embryonal carcinoma stained with antiBrdU antibody. O.M. x 320.
E G F AND EGF RECEPTOR IN HUMAN TISSUES
339
Fig. 2. Immunohistochemical staining of EGF and the EGF receptor expressed on skin tissues of adult (b)and infant (a,c-f ). a-c,e: Stained with anti-EGF receptor antibody B4G7; d,f: stained with anti-EGF antibody HA. a,b: Epider-
mis and dermis; c: sweat gland duct; d: sweat gland; e,f sebaceous gland and hair. Arrowheads (f) indicate immunoreactive substances detected with anti-EGF antibody in sebaceous gland. Original magnification x 320.
stained with B4G7 (Fig. 3a,c-f) and with HA (Fig. 3b). The cell membranes of the hepatocytes were distinctly stained. Enlargement of sinuses appears as an artifact of frozen sectioning. The cytoplasm of hepatocytes reacted with anti-EGF antibody HA. Intercalated ducts of the bronchial glands showed intense staining for the EGF receptor, whereas the glands were negative (Fig. 3c, arrowheads). Basal cells of epididymis were again posi-
tive for EGF receptors; staining intensity of the luminal cells was weaker than that of the basal cells (Fig. 3e). In the ganglion tissues, the perineurium surrounding parenchyma was distinctly stained. Schwann cell bodies (Fig. 3f) were also positive, whereas neurons were completely negative (Fig. 3f, arrowheads). These results obtained from 1 year old infant tissues as well as those of embryonal carcinoma are summarized in Table 1 together with previously published data.
R. FUKUYAMA AND N. SHIMIZU
340
Fig. 3. Immunohistochemical staining of EGF and the EGF receptor expressed on internal tissues of adult (d) and infant (a-c,e,f). a,c-f: Stained with anti-EGF receptor antibody B4G7; b: stained with anti-EGF antibody HA. a,b: Liver; c: bronchial gland and duct; d: prostate glands; e: epididymis; f: peripheral nerve ganglion. Original magnification x 320.
DISCUSSION Several conclusions emerge from the distribution of EGF and the EGF receptor obtained from this study and past studies (Table 1).Basal cells (generally stem cells in the stratified and/or pseudostratified epithelium) of endodermal or ectoderma1 origin produce EGF receptor but not EGF. These include epidermis, hair follicles, other skin
appendages, bronchus, prostate, epididymis, alimentary tract, endometrium, and chorionic villi. When surface epithelial cells expressing the EGF receptor differentiate into glandular cells, they lose the EGF receptor and start t o express EGF. These include sebaceous gland, sweat gland, pancreatic gland, mammary gland, Brunner’s gland, submandibular gland, and endometrial gland. Basal cells of the connecting duct between the lu-
341
EGF AND EGF RECEPTOR IN HUMAN TISSUES TABLE 1 . Summary ofEGF and the EGF receptor (EGFR) distribution in human tissues Tissue ~
.~
Eetodermal Epidermis Hair follicle
Stem cell
Sebaceous gland Sweat gland Dental tissue
Basal cell Outer hair sheet basal cell Basal cell Basal cell of duct Basal cell
Mammary gland
Lactiferous duct
Peripheral nervous system Schwann cell Endodermal and mesodermal Basal cell Esophagus Surface and Stomach foveolar cell Surface and Duodenum foveolar cell Small and large intestine Mucosa1,cell Basal cell Bronchus Bronchial gland Duct cell Liver Bile duct Secretory duct Pancreas Kidney Basal cell Urethra Urinary bladder Urothelial basal cell Basal cell Prostate Epididymis Basal cell Basal cell Cervix Endometrial gland Endometrium (proliferating phase) Cytotrophoblast Placenta
EGFR
~.
EGF
Differentiating cell
EGFR
EGF
References"
-
ps, 4, 5, 10, 11, 15 ps, 4, 6, 10, 11
~
+ + + + + +
+
Upper 4 layers Inner hair sheet and hair Sebocyte Glandular cell Ameloblast odontoblast Alveolus non-lactating lactating Neuron Schwann cell
+
+
Cells of luminal layer Fundic and pyloric gland
+
Brunner's gland
+ + +
Mucosal cell Other luminal cells Alveolar cell Hepatocyte Duct cell Secretory gland Cells of urinary tubule Other luminal cells Other luminal cells
+ + + + + + + +
Corpus luteum Connective tissue Striated muscle
Other luminal cells Other luminal cells Upper layer Endometrial gland (secretory phase) Syncytiotrophoblast Syncytiotrophoblast (mid-term) Luteal cell (mid-secretory phase) Fibroblast and blood vessels of embryonal carcinoma stroma Heart muscle
-
+ + nd nd
~.
ps, 4, 10, 15 ps, 4, 10, 15 3, 16
4, 6, 7
+ -
-
+ + +
PS PS,
15
13, 15 7, 15
4, 14, 15
nd nd
4, 14, 15, 17 6, 7, 15 PS, 6, 7 PS, 3, 4, 6 PS, 4, 6 PS, 4, 7 ps, 6, 7, 14, 15 6 4, 12, 15
-
PS, 4,
+ + + -
+ +
-
+ + + +
7, 9, 15
PS, 4 15 4, 15
2, 7 2, 7 1, 8
-
PS
-
PS
aps: present study; n d not determined. References cited are 1, Ayyagari and Khan-Dawood, '87; 2, Chegini and Rao, '85; 3, Cohen, '86; 4, Damjanov et al., '86; 5, Green et al., '85; 6, Gusterson et al., '84; 7, Kasselberg et al., '85; 8, Khan-Dawood, '87; 9, Maddy et al., '87; 10, Nanney et al., '84a; 11, Nanney et al., '84b; 12, Neal et al., '85; 13, Ozawa et al., '87; 14, Poulsen et al., '86; 15, Real et al., '86; 16, Thesleff et al., '87 and 17, Wright et al., '90.
minal epithelium and the glandular portion, which are proliferating and differentiate into luminal and secretory cells, express EGF receptor. These include sweat and sebaceous gland ducts, mammary duct, pancreatic duct, and bronchial gland duct. Parenchymal cells, if they continue proliferating, under certain conditions express the EGF receptor. These include hepatocytes, urinary tubule cells, and Schwann cell. Parenchymal cells, even those that are derived from ectoderm or endoderm, cease expression of the EGF recep-
tor when they completely lose their capacity for proliferating. These include neurons in the central and peripheral nervous system. Mesenchyma1 cells start t o express EGF receptor when they rapidly proliferate. These include immature blood vessels and fibrous stroma of embryonal carcinoma. Both EGF and the EGF receptor can be produced in the same cell, including hepatocytes, urinary tubule cells, and syncytiotrophoblasts. In human gastrointestinal tissues, the opposite relationship between EGF and EGF receptor ex-
342
R. FUKUYAMA AND N. SHIMIZU
pression was documented for the mucosal ulcer (Wright et al., '90). In rodent mammary gland tissue, EGF was shown to be a mitogen for mammary gland duct cells and it affected the morphogenesis of the tissue (Coleman et al., '88). No discrepancy of EGF receptor distribution between adult and infant tissues were observed, suggesting that the expression of the EGF receptor may be fixed since 1 year of age in humans. TGF-a is expressed in embryonal tissues (Wilcox and Derynk, '88). In the adult normal and psoriatic epidermis, TGF-a is expressed, suggesting the importance of this growth factor for the adult tissues (Elder et al., '89) as well as embryonal tissues. Further examination is needed t o determine which is physiologically important, EGF or TGFa,for the growth control of stem cells. To date, no reports except for the developing mouse embryo (Partanen and Thesleff, '87) demonstrated the expression of the EGF receptor in stromal cells such as testicular embryonal carcinoma examined in this report (Fig. Id). The monoclonal antibody, B4G7, detected no EGF receptors on stromal fibroblasts in Go in normal human tissues (for example, Figs. 2 and 31, whereas in the embryonal carcinoma tissues, as indicated by BrdU incorporation (Fig. lg), fibroblasts which are rapidly pioliferating produce high levels of the EGF receptor. In our hypothesis, stem cells express the EGF receptor, especially in the skin as reported by Lever and Schaumburg-Lever, '83. Distribution of the NGF receptor in human tissues (Chesa et al., '88) and distribution of the neu oncogene product in rat tissues (Kokai et al., '87) are very similar to the EGF receptor distribution in human tissues. EGF inhibits mesothelial differentiation (Connell and Rheinwald, '83). An inverse relationship exists between differentiation of melanocytes and granulosa cells and production of EGF receptors (Back and Schomberg, '88; Real et al., '86). These facts suggest that stem cells in adult tissues have a set of growth factor receptors and that their differentiation is inhibited as a result of growth promotion by growth factors. An in situ hybridization study utilizing an EGF receptor cDNA probe is underway to evaluate the hypothesis proposed here for human tissue development.
ACKNOWLEDGMENTS The authors thank Dr. T. Fuwa (Wakunaga Pharmaceutical Co., Ltd.) for providing monoclonal antibody to EGF and Ms. H. Harigai for her assistance in manuscript preparation. This work
was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan.
LITERATURE CITED Adamson, E.D., and A.R. Rees (1981) Epidermal growth factor receptors. Mol. Cell. Biochem., 34:129-152. Amagai, M., S. Ozawa, M. Ueda, T. Nishikawa, 0. Abe, and N. Shimizu (1988) Distribution of EGF receptor expressing and DNA replicating epidermal cells in psoriasis vulgaris and Bowen's disease. Br. J . Dermatol., 119:661-668. Ayyagari, R.R., and F.S. Khan-Dawood (1987) Human corpus luteum: Presence of epidermal growth factor receptors and binding characteristics. Am. J . Obstet. Gynecol., 156: 942-946. Behzadian, M.A., and N. Shimizu (1985) Monoclonal antibody that immunoreacts with a subclass of human receptors for epidermal growth factor. Cell. Struct. Funct., 10:219-232. Back, P.A., and D.W. Schomberg (1988) ['251]-epidermal growth factor binding and mitotic responsiveness of porcine granulosa cells are modulated by differentiation and follicle-stimulating hormone. Endocrinology, 122:28-33. Catterton, W.Z., M.B. Escobedo, and W.R. Sexson (1979) Effect of EGF on lung maturation in fetal rabbits. Pediatr. Res., 13:104-108. Chegini, N., and C.V. Rao (1985) Epidermal growth factor binding to human amnion, chorion, decidua and placenta from mid- and term-pregnancy: Quantitative light microscopic autoradiographic study. J. Clin. Endocrinol. Metab., 6:529-535. Chesa, P.G., W.J. Rettig, T.M. Thomson, L.T. Old, and M.R. Melonred (1988) Immunohistochemical analvsis of nerve growth factor receptor expression in normal and malignant human tissues. J. Histochem. Cytochem., 4:383-389. Cohen, S. (1962) Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal. J. Biol. Chem., 237:1555-1562. Cohen, S. (1986) Epidermal growth factor. Biosci. Rep., 6: 1017-1028. Coleman, S., G.B. Silberstein, and C.W. Daniel (1988) Ductal morphogenesis in the mouse' mammary gland Evidence supporting a role for epidermal growth factor. Dev. Biol., 127:304-315. Connell, N.D., and J.G. Rheinwald (1983) Regulation of the cytoskeleton in mesothelial cells: Reversible loss of keratin and increase in vimentin during rapid growth in culture. Cell, 34:245-253. Damjanov, I., B. Mildner, and B.B. Knowles (1986) Immunohistochemical localization of the epidermal growth factor receptor in normal human tissue. Lab. Invest., 55.588-592. Downs, S.M., S.A.J. Daniel, and J. Eppig (1988) Induction of maturation in cumulus cell enclosed mouse oocyte by follicle-stimulating hormone and epidermal growth factor: Evidence for a positive stimulus of somatic cell origin. J . Exp. ZOO^., 245:86-96. Elder, J.T., G.T. Fisher, P.B. Lindquist, G.L. Bennett, M.R. Pittelkov, R.J. Coffey, Jr., L. Ellingsworth, R. Derynck, and J.J. Voorhees (1989) Overexpression of transforming growth factor in psoriatic epidermis. Science, 243:811-814. Gamou, S., M. Hirai, K. Rikimaru, S . Enomoto, and N. Shimizu (1988) Biosynthesis of the epidermal growth factor receptor in human squamous cell carcinoma lines: Secretion of the truncated receptor is not common to epidermal growth factor receptor-hyperproducing cells. Cell Struct. Funct., 13:25-38.
E G F AND EGF RECEPTOR IN HUMAN TISSUES Green, M.R., D. Phil, and J.R. Couchman (1985) Differences in human skin between the epidermal growth factor receptor distribution detected by EGF binding and monoclonal antibody recognition. J. Invest. Dermatol., 85~239-245. Gullick, W.J., J.J. Marsden, N. Whittle, B. Ward, L. Bobrow, and M.D. Waterfield (1986) Expression of epidermal growth factor receptors on human cervical, ovarian, and vulva1 carcinoma. Cancer Res., 46:285-292. Gusterson, B., G. Cowley, J.A. Smith, and B. Ozanne (1984) Cellular localization of epidermal growth factor receptors. Cell Biol. Int. Rep., 8:649-658. Hsu, S.M., L. Raine, and H. Fanger (1981) Use of avidinbiotin-peroxidase complex (ABC) in immunoperoxidase technique: A comparison between ABC unlabeled antibody (PAP) procedures. J . Histochem. Cytochem., 29.577480. Ikuta, S., Y. Yamada, Y. Toshida, and K. Nishikawa (1985) Production and properties of a monoclonal antibody against human epidermal growth factor. Biochem. Int., 10~251258. Joh, T., M. Ito, K. Katumi, Y. Yokoyama, T. Takeuchi, T. Kato, Y. Wada, and R. Tanaka (1986) Physiological concentration of human epidermal growth factor in biological fluids. Use of a sensitive enzyme immunoassay. Clin. Chim. Acta., 158:81-90. Kasselberg, A.G., D.N. Orth, M.E. Gray, and M.T. Stahlman (1985) Immunocytochemical localization of human epidermal growth factorhrogastrone in several human tissues. J. Histochem. Cytochem., 4:315-322. Khan-Dawood, F.S. (1987) Human corpus luteum: Immunocytochemical localization of epidermal growth factor. Fertil. Steril., 47:916-919. Kokai, Y., J.A. Cohen, J.A. Drebin, and M.I. Greene (1987) Stage- and tissue-specific expression of the neu oncogene in rat development. Proc. Natl. Acad. Sci. U S A . , 84:84988501. Lever, W.F., and G. Schaumburg-Lever (1983) Embryology of the skin, Ed. 6. In: Histopathology of the Skin. W.F. Lever, ed. J.B. Lippincott, Philadelphia, pp. 3-7. Maddy, S.Q., G.D. Chisholm, R.A. Hawkins, and F.K. Habib (1987) Localization of epidermal growth factor receptors in the human prostate by biochemical and immunocytochemical methods. J . Endocrinol., 113:147-153. Malo, C., and D. Menard (1982) Influence of epidermal growth factor on the development of suckling mouse intestinal mucosa. Gastroenterology, 83:28-35. Nanney, L.B., M. Magid, C.M. Stroscheck, and L.E. King (1984a) Comparison of epidermal growth factor binding and receptor distribution in normal human epidermis and epidermal appendages. J. Invest. Dermatol., 83:385-393. Nanney, L.B., J.A. Mckanna, C.M. Stoscheck, G. Carpenter, and L.E. King (1984b) Visualization of epidermal growth
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factor receptors in human epidermis. J . Invest. Dermatol., 82:165-169. Neal, D.E., C. Marsh, M.K. Bennet, P.D. Abel, R.R. Hall, J.R.C. Saumburg, and A.L. Harris (1985) Epidermalgrowth-factor receptors in human bladder cancer: Comparison of invasive and superficial tumors. Lancet, 1:366-368. Oka, Y., F.K. Ghishan, H.L. Greene, and D.N. Orth (1983) Effect of mouse epidermal growth factorhrogastrone on the functional maturation of rat intestine. Endocrinology, 112:940-944. Ozawa, S., M. Ueda, N. Ando, 0. Abe, and N. Shimizu (1987) High incidence of EGF receptor hyperproduction in esophageal squamous-cell carcinoma. Int. J. Cancer, 39~333-337. Partanen, A.M., and I. Thesleff (1987) Localization and quantitation of ‘251-epidermal growth factor binding in mouse embryonic tooth and other embryonic tissues at a different developmental stage. Dev. Biol., 120~186-197. Poulsen, S.S.,E. Nexo, P.S. Olsen, J. Hess, and P. Kirkegaard (1986) Immunohistochemical localization of epidermal growth factor in rat and man. Histochemistry, 85:389-394. Real, F.X., W.J. Reetig, P.G. Chesa, M.R. Melamed, L.J. Old, and J. Medelson (1986) Expression of epidermal growth factor receptor in human cultured cells and tissues; relationship t o cell lineage and stage of differentiation. Cancer Res., 46:4726-4731. Shimizu, N. (1984) Cell genetic analysis of the receptor systems for bioactive polypeptides. In: Receptors and Recognition. P. Goodfellow, ed. Chapman and Hall, London, Series B, Vol. 16, pp. 109-142. Teilum, G. (1965) Classification of endodermal sinus tumor (mesoblastoma Vitallium) and so-called “embryonal carcinoma” of the ovary. Acta Pathol. Microbiol. Scand., 64: 407-429. Thesleff, I., A.M. Partanen, and L. Rihtniemi (1987) Localization of epidermal growth factor receptors in mouse incisors and human premolars during eruption. Eur. J. Orthodont., 9:24-32. Uchihashi, M., Y. Hirata, T. Fujita, and S. Matsukawa (1982) Age-related decrease of urinary excretion of human epidermal growth factor (hEGF). Life Sci., 31 ~679-683. Waterfield, M.D., E.L.V. Mayes, P. Stroobant, P.L.P. Bennet, S.Young, P.N. Goodfellow, G.S. Banting, and B. Ozanne (1982) A monoclonal antibody to the human epidermal growth factor receptor. J. Cell. Biochem., 20:149-161. Wilcox, J., and R. Derynck (1988) Developmental expression of transforming growth factor alpha and beta in mouse fetus. Mol. Cell Biol., 8:3415-3422. Wright, N.A., C. Pike, and G. Elia (1990) Induction of a novel epidermal growth factor-secreting cell lineage by mucosal ulceration in human gastrointestinal stem cells. Nature. 343:82-85.
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Expression of Epidermal Growth Factor (EGF) and the EGF Receptor in Human Tissues RYUICHI FUKUYAMA AND NOBUYOSHI SHIMIZU Department of Molecular Biology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160, Japan ABSTRACT
The EGF-EGF receptor system has been widely examined for signal transduction, control of cell growth and differentiation, and in vivo physiological function and carcinogenesis. The localization of EGF receptors in vivo led to the idea that the system is operative in proliferation and differentiation of cells and tissues. However, a consensus for its distribution and function in human tissues has not yet been determined because of discrepancies in the reported results. Using a highly specific monoclonal antibody against the EGF receptor, we examined various tissues of a n infant and adults as well as embryonal carcinoma. We observed restricted localization of EGF receptors in basal cells of epithelial tissues and duct cells of secretory tissues. Fibroblasts express a high level of EGF receptors when they are rapidly growing. Using the monoclonal anti-EGF antibodies, we observed that EGF is localized to differentiated cells rather than to stem cells such as glandular tissues. We also observed that some cells express both EGF and the EGF receptor. All histochemical results indicated that in epidermis and various glandular tissues, EGF may be expressed in differentiating cells derived from the stem cells expressing EGF receptors.
Since the discovery of EGF (Cohen, '62, '861, charide moieties of the EGF receptors. Therefore, its role in physiological function has been widely we have investigated human tissues using a studied, especially in rodents, in the development monoclonal EGF receptor antibody which recogof intestine (Malo and Menard, '82; Oka et al., nizes a peptide moiety of the extracellular domain '831, oocyte (Downs et al., '88) and lung (Catterton (Amagai et al., '88; Behzadian and Shimizu, '85; et al., '79) as well as in eyelid opening and tooth Gamou et al., '88; Ozawa et al., '87). We tested growth (Cohen, '62, '86). The ligand binding stud- various tissues from a 1year old infant and adults ies suggested that the EGF receptor is localized and a testicular embryonal carcinoma. The amount of EGF in body fluids increases unon epithelial cell surface. To date, several monoclonal antibodies against human EGF receptors til about 9 years old and decreases thereafter (Joh (Behzadian and Shimizu, '85; Shimizu, '84; Wa- et al., '86; Uchihashi et al., '82). The histology of terfield et al., '82) and synthetic oligopeptides embryonal carcinoma includes immature and/or (Gullick et al., '86) were obtained. Using these adult teratoid structures as well as ectoblastic monoclonal antibodies, localization of EGF recep- and trophoblastic elements (Teilium, '65). Thus, tors in human normal and cancer tissues (Adam- infant and embryonal carcinoma tissues are useson and Rees, '81; Amagai et al., '88; Damjanov ful for EGF and EGF receptor localization. By imet al., '86; Gullick et al., 86; Nanney et al., '84a, munohistochemical staining, we observed that 84b; Ozawa et al., '87) has been examined t o eluci- the localization of EGF and the EGF receptor is date the possible role of the EGF-EGF receptor correlated with differentiated cells and their stem system in these physiological states. The EGF cells, respectively. We postulate that the expresreceptor was detected in both proliferating and sion of EGF and the EGF receptor switches durdifferentiating cells, and, hence, EGF was pos- ing the development and evolution of human epitulated t o affect both cell growth and differen- thelial tissues. tiation. However, the results obtained with these antibodies were often inconsistent between groups and with the results obtained using conReceived April 30, 1990; revision accepted September 26, 1990. ventional autoradiography with 1251-labeledEGF Address reprint requests to Nobuyoshi Shimizu, Department of Mo(Green et al., '85). This discrepancy may be due lecular Biology, Keio University School of Medicine, 35 Shinanoto the fact that most antibodies recognize polysac- machi, Shinjuku-ku, Tokyo 160, Japan. 0 1991 WILEY-LISS, INC.
EGFANDEGFRECEPT()R IN HUMAN TISSUES
MATERIALS AND METHODS Cells and tissues A431 cells were grown on glass slides under previously described culture conditions (Gamou et al., '88). Tissues of testicular embryonal carcinoma were obtained from surgical specimens. One year old infant and adult tissues were obtained from autopsied material. A431 cells grown on glass slides were fixed with cold acetone for 10 minutes and air dried. All the tissues obtained were trimmed, frozen immediately with liquid nitrogen, and stored at -70°C until use. Cryostat sectioned tissues (4-6 Fm) mounted on albumin-coated glass slides were fixed for 10 minutes with absolute methanol for EGF staining or acetone for EGF receptor staining. Some slides were processed for staining without fixation.
Incorporation of BrdU to human embryonal carcinoma tissues Small pieces (about 5 mm in diameter) of embryonal carcinoma tissues were immediately immersed in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 400 FM BrdU (Ikeda Rika, Osaka, Japan) as previously described (Amagai et al., '88). Incorporation of BrdU was processed for 3 hours under pressure 2 kg w/cm2 at 37°C with shaking followed by fixation with Carnoy's solution and embedded in paraffin. Antibodies The B4G7 monoclonal antibody recognizes a peptide moiety of the extracellular domain of the EGF receptor (Behzadian and Shimizu, '85). Monoclonal antibodies against mouse EGF (mEGF) and human EGF (hEGF), designated KEM-9 and HA, were generous gifts from Wakunaga Pharmaceutical Co. Ltd. (Hiroshima, Japan) and Ikuta et al. (Ikuta et al., '85), respectively. All three antibodies are IgG and are used after 1:400 dilution from a 1mg/ml stock solution. For detection of nuclei incorporating BrdU, monoclonal anti-BrdU antibody (Becton Dickinson, Mountain View, CA) was used in a 1:100 dilution. FITC-conjugated anti-mouse IgG and biotinylated anti-mouse IgG were purchased from SILENOUS Laboratories (Victoria, Australia). Avidin-biotin-peroxidase complex made just before use was obtained from Vector Laboratories (Burlingame, CA). We used non-immune mouse Ig as a control.
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Immunohistochemistry Indirect immunofluorescence with FITC-conjugated secondary antibody was used t o detect B4G7. The ABC method (Hsu et al., '81) was employed for immunohistochemical staining of both human frozen and paraffin-embedded tissues. Color was developed with a solution of 0.025% 3' ,3-diaminobenzidine (Dojindo, Japan) and 0.03% H,02. A431 cells were incubated with antibody B4G7 as a positive control for staining efficiency.
RESULTS EGF receptors in A431 cells were localized on the cell surface using monoclonal antibody B4G7 (Fig. la,b). The sensitivity of EGF receptor detection under these conditions was very high. Although the whole cell surface was stained, dense staining was obtained in areas of cell-to-cell contact. Embryonal carcinoma tissues were stained positive with B4G7 on the cell surface of single and stratified endodermal epithelia (Fig. lc) as well as mesenchymal cells accumulating around the mature endodermal glands (Fig. Id). The cytoplasm of goblet cells from endodermal glands was stained positive for EGF expression when analyzed with the EGF-specific antibody HA (Fig. le,f). EGF or a n EGF-like substance was discharged into the lumen from glandular cells. Such apparently mature endodermal epithelia displayed low BrdU incorporation into nuclei (Fig. lg), whereas stromal cells showed high incorporation of BrdU into .nuclei (24%) (Fig. lg, dark nuclei). Figure 2 shows infant and adult skin tissues including epidermis and dermis, sweat gland, hair outer root sheath, and sebaceous gland which were stained with anti-EGF receptor antibody B4G7 (Fig. 2a-c,e) or with anti-EGF antibody HA (Fig. 2d,f). All basal cells of the epidermis, sweat gland duct, and excretory duct of sebaceous glands, from which the differentiated progenies arise, were clearly stained with anti-receptor antibodies, whereas stratum corneum, the inner cells of the sweat gland duct, and sebaceous gland were negative for the EGF receptor. Sweat gland cells were stained in their cytoplasm with antiEGF antibody (Fig. 2d). EGF-staining in the cytoplasm of sebaceous gland cells was faint and granular (Fig. 2f, arrowheads). Figure 3 shows tissues derived from an adult and a 1year old infant, including liver, bronchial gland, prostate gland, epididymis, and a ganglion
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R. FUKUYAMA AND N. SHIMIZU
Fig. 1. Immunohistochemical staining of EGF and the EGF receptor expressed on various cells and tissues. a: A431 cells stained with anti-EGF receptor antibody B4G7. Spindleshaped cells were mouse A9 cells cocultured as a n internal negative control. b: A431 cells reacted with non-immune mouse Ig. Original magnification (O.M.) x 640. c , d Tissues of embryonal carcinoma stained with B4G7. e: Endodermal gland of embryonal carcinoma stained with anti-EGF antibody HA. f: Adjacent sections of (e) reacted with non-immune mouse Ig. These specimens were counterstained with hematoxylin. g: Tissues of embryonal carcinoma stained with antiBrdU antibody. O.M. x 320.
E G F AND EGF RECEPTOR IN HUMAN TISSUES
339
Fig. 2. Immunohistochemical staining of EGF and the EGF receptor expressed on skin tissues of adult (b)and infant (a,c-f ). a-c,e: Stained with anti-EGF receptor antibody B4G7; d,f: stained with anti-EGF antibody HA. a,b: Epider-
mis and dermis; c: sweat gland duct; d: sweat gland; e,f sebaceous gland and hair. Arrowheads (f) indicate immunoreactive substances detected with anti-EGF antibody in sebaceous gland. Original magnification x 320.
stained with B4G7 (Fig. 3a,c-f) and with HA (Fig. 3b). The cell membranes of the hepatocytes were distinctly stained. Enlargement of sinuses appears as an artifact of frozen sectioning. The cytoplasm of hepatocytes reacted with anti-EGF antibody HA. Intercalated ducts of the bronchial glands showed intense staining for the EGF receptor, whereas the glands were negative (Fig. 3c, arrowheads). Basal cells of epididymis were again posi-
tive for EGF receptors; staining intensity of the luminal cells was weaker than that of the basal cells (Fig. 3e). In the ganglion tissues, the perineurium surrounding parenchyma was distinctly stained. Schwann cell bodies (Fig. 3f) were also positive, whereas neurons were completely negative (Fig. 3f, arrowheads). These results obtained from 1 year old infant tissues as well as those of embryonal carcinoma are summarized in Table 1 together with previously published data.
R. FUKUYAMA AND N. SHIMIZU
340
Fig. 3. Immunohistochemical staining of EGF and the EGF receptor expressed on internal tissues of adult (d) and infant (a-c,e,f). a,c-f: Stained with anti-EGF receptor antibody B4G7; b: stained with anti-EGF antibody HA. a,b: Liver; c: bronchial gland and duct; d: prostate glands; e: epididymis; f: peripheral nerve ganglion. Original magnification x 320.
DISCUSSION Several conclusions emerge from the distribution of EGF and the EGF receptor obtained from this study and past studies (Table 1).Basal cells (generally stem cells in the stratified and/or pseudostratified epithelium) of endodermal or ectoderma1 origin produce EGF receptor but not EGF. These include epidermis, hair follicles, other skin
appendages, bronchus, prostate, epididymis, alimentary tract, endometrium, and chorionic villi. When surface epithelial cells expressing the EGF receptor differentiate into glandular cells, they lose the EGF receptor and start t o express EGF. These include sebaceous gland, sweat gland, pancreatic gland, mammary gland, Brunner’s gland, submandibular gland, and endometrial gland. Basal cells of the connecting duct between the lu-
341
EGF AND EGF RECEPTOR IN HUMAN TISSUES TABLE 1 . Summary ofEGF and the EGF receptor (EGFR) distribution in human tissues Tissue ~
.~
Eetodermal Epidermis Hair follicle
Stem cell
Sebaceous gland Sweat gland Dental tissue
Basal cell Outer hair sheet basal cell Basal cell Basal cell of duct Basal cell
Mammary gland
Lactiferous duct
Peripheral nervous system Schwann cell Endodermal and mesodermal Basal cell Esophagus Surface and Stomach foveolar cell Surface and Duodenum foveolar cell Small and large intestine Mucosa1,cell Basal cell Bronchus Bronchial gland Duct cell Liver Bile duct Secretory duct Pancreas Kidney Basal cell Urethra Urinary bladder Urothelial basal cell Basal cell Prostate Epididymis Basal cell Basal cell Cervix Endometrial gland Endometrium (proliferating phase) Cytotrophoblast Placenta
EGFR
~.
EGF
Differentiating cell
EGFR
EGF
References"
-
ps, 4, 5, 10, 11, 15 ps, 4, 6, 10, 11
~
+ + + + + +
+
Upper 4 layers Inner hair sheet and hair Sebocyte Glandular cell Ameloblast odontoblast Alveolus non-lactating lactating Neuron Schwann cell
+
+
Cells of luminal layer Fundic and pyloric gland
+
Brunner's gland
+ + +
Mucosal cell Other luminal cells Alveolar cell Hepatocyte Duct cell Secretory gland Cells of urinary tubule Other luminal cells Other luminal cells
+ + + + + + + +
Corpus luteum Connective tissue Striated muscle
Other luminal cells Other luminal cells Upper layer Endometrial gland (secretory phase) Syncytiotrophoblast Syncytiotrophoblast (mid-term) Luteal cell (mid-secretory phase) Fibroblast and blood vessels of embryonal carcinoma stroma Heart muscle
-
+ + nd nd
~.
ps, 4, 10, 15 ps, 4, 10, 15 3, 16
4, 6, 7
+ -
-
+ + +
PS PS,
15
13, 15 7, 15
4, 14, 15
nd nd
4, 14, 15, 17 6, 7, 15 PS, 6, 7 PS, 3, 4, 6 PS, 4, 6 PS, 4, 7 ps, 6, 7, 14, 15 6 4, 12, 15
-
PS, 4,
+ + + -
+ +
-
+ + + +
7, 9, 15
PS, 4 15 4, 15
2, 7 2, 7 1, 8
-
PS
-
PS
aps: present study; n d not determined. References cited are 1, Ayyagari and Khan-Dawood, '87; 2, Chegini and Rao, '85; 3, Cohen, '86; 4, Damjanov et al., '86; 5, Green et al., '85; 6, Gusterson et al., '84; 7, Kasselberg et al., '85; 8, Khan-Dawood, '87; 9, Maddy et al., '87; 10, Nanney et al., '84a; 11, Nanney et al., '84b; 12, Neal et al., '85; 13, Ozawa et al., '87; 14, Poulsen et al., '86; 15, Real et al., '86; 16, Thesleff et al., '87 and 17, Wright et al., '90.
minal epithelium and the glandular portion, which are proliferating and differentiate into luminal and secretory cells, express EGF receptor. These include sweat and sebaceous gland ducts, mammary duct, pancreatic duct, and bronchial gland duct. Parenchymal cells, if they continue proliferating, under certain conditions express the EGF receptor. These include hepatocytes, urinary tubule cells, and Schwann cell. Parenchymal cells, even those that are derived from ectoderm or endoderm, cease expression of the EGF recep-
tor when they completely lose their capacity for proliferating. These include neurons in the central and peripheral nervous system. Mesenchyma1 cells start t o express EGF receptor when they rapidly proliferate. These include immature blood vessels and fibrous stroma of embryonal carcinoma. Both EGF and the EGF receptor can be produced in the same cell, including hepatocytes, urinary tubule cells, and syncytiotrophoblasts. In human gastrointestinal tissues, the opposite relationship between EGF and EGF receptor ex-
342
R. FUKUYAMA AND N. SHIMIZU
pression was documented for the mucosal ulcer (Wright et al., '90). In rodent mammary gland tissue, EGF was shown to be a mitogen for mammary gland duct cells and it affected the morphogenesis of the tissue (Coleman et al., '88). No discrepancy of EGF receptor distribution between adult and infant tissues were observed, suggesting that the expression of the EGF receptor may be fixed since 1 year of age in humans. TGF-a is expressed in embryonal tissues (Wilcox and Derynk, '88). In the adult normal and psoriatic epidermis, TGF-a is expressed, suggesting the importance of this growth factor for the adult tissues (Elder et al., '89) as well as embryonal tissues. Further examination is needed t o determine which is physiologically important, EGF or TGFa,for the growth control of stem cells. To date, no reports except for the developing mouse embryo (Partanen and Thesleff, '87) demonstrated the expression of the EGF receptor in stromal cells such as testicular embryonal carcinoma examined in this report (Fig. Id). The monoclonal antibody, B4G7, detected no EGF receptors on stromal fibroblasts in Go in normal human tissues (for example, Figs. 2 and 31, whereas in the embryonal carcinoma tissues, as indicated by BrdU incorporation (Fig. lg), fibroblasts which are rapidly pioliferating produce high levels of the EGF receptor. In our hypothesis, stem cells express the EGF receptor, especially in the skin as reported by Lever and Schaumburg-Lever, '83. Distribution of the NGF receptor in human tissues (Chesa et al., '88) and distribution of the neu oncogene product in rat tissues (Kokai et al., '87) are very similar to the EGF receptor distribution in human tissues. EGF inhibits mesothelial differentiation (Connell and Rheinwald, '83). An inverse relationship exists between differentiation of melanocytes and granulosa cells and production of EGF receptors (Back and Schomberg, '88; Real et al., '86). These facts suggest that stem cells in adult tissues have a set of growth factor receptors and that their differentiation is inhibited as a result of growth promotion by growth factors. An in situ hybridization study utilizing an EGF receptor cDNA probe is underway to evaluate the hypothesis proposed here for human tissue development.
ACKNOWLEDGMENTS The authors thank Dr. T. Fuwa (Wakunaga Pharmaceutical Co., Ltd.) for providing monoclonal antibody to EGF and Ms. H. Harigai for her assistance in manuscript preparation. This work
was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan.
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