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The receptor for epidermal growth factor is present in human fetal kidney, liver and lung
Regulatory Peptides, 26 (1989) 1-8
1
Elsevier REGPEP 00828
The receptor for epidermal growth factor is present in human fetal kidney, liver and lung E b b a N e r o 1 and Niels Kryger-Baggesen 2 i Department of Clinical Chemistry and 2Department of Gynaecology and Obstetrics, Central Hospital, Hillerod (Denmark) (Received 12 December 1988; revised version received21 March 1989; accepted 18 April 1989) Key words: Epidermal growth factor; Fetal; Human; Kidney; Liver; Lung;
Placenta; Receptor
Summary In animals a pharmacological dosis of the growth-promoting peptide epidermal growth factor (EGF) has an effect on the growth and/or maturation of several organs such as the lung, the kidney, the liver and the gastrointestinal tract. Since E G F elicits its function via binding to specific cellular receptors the presence of these receptors predicts a possible physiological role for E G F and E G F agonists. We have studied the presence of the EGF-receptor on human fetal membrane preparations from the kidney, the liver, the lung and the placenta (gestational age 13-20 weeks). The 4 membrane preparations all bind labeled E G F thus allowing us to calculate the apparent affinity constant and the number of receptors present per mg of membrane protein. The apparent affinity constant (gestational age 13-20 weeks) varies between 0.5 and 3.5 x 109 mol- 1, median 1.3 x 109 tool- 1 (n = 40). No difference is observed for the 4 tissues examined, and no difference is found as a function of the gestational age. The number of receptors present per mg of membrane protein (gestational age 16-20 weeks) are (range and (median) 90-220 (130) fmol, n -- 10 for the kidney, 80-480 (250) fmol, n = 9 for the liver, 90-690 (300) fmol, n = 10 for the lung, and 2100-4200 (3400) fmol, n -- 7 for the placenta. Results for a fetus of gestational age 13 weeks show high values for kidney receptors (240 fmol) and lung receptors (800 fmol) and low values for the placenta receptors (410 fmol).
Correspondence: E. Next, Department of Clinical Chemistry, Central Hospital, DK-3400 Hillered,
Denmark. 0167-0115/89/$03.50 © 1989 Elsevier Science Publishers B.V. (BiomedicalDivision)
Introduction
Epidermal growth factor (EGF) is a growth-promoting peptide f'n'stisolated from the submandibular glands of mice due to its ability to induce precocious eyelid opening and tooth eruption in the newborn mouse [1 ]. In the fetus EGF-like activity has been identified in whole fetuses [2], and in several fetal organs including the submandibular gland, the kidney and the lung [3-5]. In experimental animals injections of pharmacological doses of EGF have suggested it to play a role for the growth and/or maturation of the fetal liver, kidney, lung and gastrointestinal tract [6]. Some years ago an EGF agonist, transforming growth factor alpha (TGFct), was isolated from tumour cells [7] and later mRNA for TGF~ has been visualized in the fetal rat [8]. EGF and TGF~ elicit their function via binding to the EGF receptor, a protein kinase present on a number of tissues including the human placenta [9]. If EGF or TGF~ is to play a role during human fetal development, the cellular receptor for EGF has to be present. So far the presence of the EGF receptor has been studied only for the human placenta and for the human intestine [ 10,11 ]. Since animal studies have indicated a role for EGF also in other organs we wanted to examine the occurrence of the receptor for EGF on selected organs from human fetuses as a function of gestational age.
Materials and Methods
Fetal tissue Women (age range 21-37 years, n = 11) referred for legal abortion on socioeconomic indication gave informed consent. Labor was induced by installation of 140 mg prostaglandin-F2~ (Dinoprost, Upjohn, U.S.A.) in the amniotic cavity. The fetus was delivered between 7 and 33 h after induction of labor. Only male fetuses were employed. The length of the foot was measured and gestational week was calculated according to the formula (length of foot in mm + 24) x 4/11 equals the gestational week [12]. The following 11 fetuses were studied: 1 (gestational age 13 weeks), 4 (gestational age 16 weeks), 1 (gestational age 17 weeks), 2 (gestational age 18 weeks), 2 (gestational age 19 weeks) and 1 (gestational age 20 weeks). The placenta, the liver, the kidneys and the lungs were removed and kept at - 80 °C until further analyzed. Biochemical analyses Preparation of membranes. Approximately 1 g of tissues was homogenized in 5 mi 0.25 M sucrose, 0.025 M "Iris buffer, pH 7.4 per g of tissue and membranes were prepared as previously described [10]. The membranes were stored in 0.05 M "Iris buffer, pH 7.4 and kept at - 20 ° C until employed. Binding isotherms were performed employing 50 #1 membrane preparation (2.5 mg protein/ml or less), 50/zl 125I-labeled human urinary EGF (approx. 30.000 cpm, 50 fmol) and 50 #1 unlabeled EGF (0-950 fmol). The assay buffer was 0.1 M phosphate, 0.1 ~ albumin (human albumin, Behringwerke, F.R.G.), pH 8.0. The mixtures
were incubated for 1 h at room temperature and overnight at 4 ° C. Free and bound EGF were separated by vacuum filtration through 0.2 #m filters (SM 11107 Sartorius, GOttingen, F.R.G.). The bound fraction, the filters were counted in a multichannel gammacounter (Molsg~d, Denmark). The fraction of labeled peptide bound varied between 0.10 and 0.60 when no unlabeled peptide was added. Non-specific binding obtained by including 60 pmol of human EGF in the assay mixture accounted for a fraction around 0.03 of added counts. All samples were analyzed in duplicate and non-specific binding was subtracted prior to analysis of the data. The data were analyzed according to Scatchard [ 13]. For analyses of association and dissociation curves membranes from liver and placenta were employed. To construct the association curve labeled EGF (100 #1, 50 fmol) and membranes (50 #1) corresponding to approximately 20 fmol EGF binding sites were incubated for time intervals between 30 min and 24 h. To construct the dissociation curve 60 pmol of human EGF was added to membranes preincubated for 24 h with labeled EGF and the reaction was terminated at intervals between 30 min and 24 h after addition of unlabeled EGF. To test the pH dependence of binding between EGF and its receptor, 90 #1 assay buffer adjusted to pH between 6.0 and 11.0 was mixed with 10 #1 ~25I-EGF (50 fmol) and 50 #1 membrane preparation (approx. 20 fmol EGF binding sites). Human EGF purified from urine by immunoaffinity chromatography and pure as judged by SDS polyacrylamid gel electrophoresis, and N-terminal sequence was employed for preparation of calibration standards, and for preparation of 125I-labeled peptide [ 14,15]. The specific activity varied between 500 and 1000 cpm/fmol. Protein was determined by the Lowry method [16] employing human albumin as calibrator.
Results
High affinity receptors for EGF are present on membranes prepared from human fetal kidney, liver, lung and placenta. All the tissues studied show one class of receptors as judged from the Scatchard plots (Fig. 1). The apparent affinity constants are between 0.5 and 3.5 x 10 9 mol- 1 (median 1.3 x 109 mol- 1) (n --- 40) (Fig. 2). No systematic change occurs in the affinity constant with the age of the fetus and no difference is observed between the apparent affmity constant of the 4 tissues examined, nor do the affinity constants obtained on tissues from the same fetus show any characteristic pattern. Studies performed on membranes from the liver show the same association rate and dissociation rate as membranes from the placenta. Also the sensitivity towards acid pH is the same for membranes obtained from the liver, the lung and the placenta (Fig. 3). The number of EGF binding sites present on the 4 tissues is given in Fig. 4. The pattern is unique for the 13-week-old fetus. The lung contains more receptors per mg of membranes than the placenta. Compared to the absolute values obtained for fetuses 16-20 weeks of age, the 13 week placenta contains a small number of receptors (410 fmol), while the lung and the kidney contain the highest number of receptors found
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Fig. 1. Binding of EGF to membranes from human fetal kidney, liver, lung and placenta. The figure shows binding data calculated according to Scatchard. The organs were obtained from fetuses of gestational age 18 weeks (kidney), 19 weeks (liver), 20 weeks (lung) and 16 weeks (placenta). Abcissa, EGF bound (fmol); ordinate, Bound/free x 10- 6 liter.
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Fig. 2. Apparent affinity constant for binding of EGF to membranes obtained from human fetal kidney, liver, lung and placenta• Fetuses gestational age 13 (*), 16 ( ~ 7 V A A), 17 (o), 18 ( [ ] I ) , 19 ( Q O ) and 20 ( x ) weeks were examined. Individual results are given. The horizontal lines indicate the medians.
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Fig. 3. Binding oflabeled EGF to human fetai membrane preparations. A: association curve. Labeled EGF and membranes from liver (O) or placenta (O) were mixed at the time zero. The reaction was terminated at the time intervals shown. Abcissa, time after addition of labeled EGF; ordinate, fraction of label bound relative to the maximal amount of label bound. B: dissociation curve. Labeled EGF and membranes from liver (O) or placenta (©) was incubated overnight. At time zero excess unlabeled EGF was added and the reaction was terminated at the time intervals shown. Abcissa, time after addition of unlabeled EGF; ordinate, fraction of label bound relative to label bound at time zero. C: pH optimum. Labeled EGF and membranes wore incubated with membranes from liver (O), lung ( x ) or placenta (O) at different pH. Abcissa, pH of assay mixture; ordinate: fraction of label bound relative to maximum binding.
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Fig. 4. Number of binding sites for EGF present on membranes obtained from human fetal kidney, liver, lung and placenta. Human fetuses gestationai age 13 (*), 16 ( V V A A ) , 17 (.), 18 ( V l l ) , 19 ( O O ) and 20 ( x ) weeks were examined. Individual results are given. The horizontal lines indicate the medians.
on these two tissues, that is respectively 800 and 240 fmol/mg protein. No variation with age is found for the number of receptors present on the 4 tissues obtained from fetuses aged 16-20 weeks. The absolute values given as range and median for the receptors expressed per mg of membrane protein are approximately 10 times higher for the placenta (2100-4200 (3400) fmol, n -- 7) than for the lung (90-690 (300) fmol, n = 10) and the liver (80-480 (250) fmol, n = 9). The kidney display the lowest values for the receptor (90-220 (130) fmol, n = 10).
Discussion
We report that human fetal kidney, liver and lung possess receptors for EGF from at least gestational week 13. Our finding of receptors on the fetal human lung is in accord with previous studies on rabbit and mouse fetuses [ 17,18 ]. In rabbits the number of receptors is considerably higher during prenatal development than after birth [19]. EGF given to premature fetuses (sheep and rabbit) accelerates the maturation of the lung and prevents respiratory distress syndrome [20,21 ]. We have recently demonstrated EGF immunoreactivity to occur in the pneumocytes II of the rat lung a few days before birth [5]. Together, the above results strongly suggest a physiological role for EGF in the maturation of the human lung. Also the fetal liver from mice and rats expose receptors for EGF [ 18,22], and in the fetal sheep exogenous EGF stimulates the growth of the liver [23]. In the human fetus the number of receptors present on the liver is comparable to the values obtained for the kidney and the lung. In the adult rat the liver is the tissue that contains the highest amount of receptors for EGF, most likely because the liver plays a role in removal of EGF from the circulation [24]. The kidney synthesizes EGF in fetal life and throughout postnatal life, and large amounts of EGF are present in the urine [3,25]. Previous work has indicated that injected EGF stimulates growth of the fetal kidney [23]. As the lung, the kidney contains receptors for EGF and synthesizes EGF. It is thus possible that EGF also plays a role in the physiological development of the human kidney. The human placenta is one of the first tissues on which receptors for EGF have been observed, and no other non-pathological source is known that contains as many receptors for EGF per mg of protein [ 11 ]. Previous work has indicated the number of receptors to increase with gestational age [26]. We find that the week 13 foetus contains few receptors per mg of membrane protein compared to fetuses of gestational age 16 weeks, but no increase in the number of receptors occurs between gestational ages 16 and 20 weeks. The role for the EGF receptor on placental tissues is not known. Recently, mRNA for the EGF agonist TGF~ has been localized in maternal decidua of the rat [8]. This, together with the observation that EGF induces differentiation of the cytothrophoblast to form the syncytothrophoblast, suggests an autocrine role for TGF~ in the placenta [28]. TGF~ occurs early during fetal development, and it peaks around mid-term in both mouse and rat [8,27]. In contrast, EGF immunoreactivity occurs relatively late during
fetal d e v e l o p m e n t [3,5] a n d m R N A coding for the synthesis o f E G F occurs only after birth in the m o u s e [29]. It is thus likely that the s a m e r e s p o n s e system, the E G F receptor, is stimulated by one peptide, T G F ~ , during early d e v e l o p m e n t a n d by another, E G F , during f'mal fetal development. In conclusion the present p a p e r d e m o n s t r a t e s h u m a n fetal kidney, liver a n d lung to p o s s e s s receptors for E G F . T h e receptors have the s a m e atTmity t o w a r d s E G F as the receptors present on the h u m a n placenta, but the quantity is about 1/10 o f the a m o u n t present on placenta.
Acknowledgement T h e technical a s s i s t a n c e o f M a r i a n n e Rye H a n s e n a n d K a m m a Velin is w a r m l y acknowledged. This w o r k was s u p p o r t e d by T h e D a n i s h C a n c e r Society (87-042, 88-090).
References 1 Cohen, S., Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal, J. Biol. Chem., 237 (1962) 1555-1562. 2 Nexo, E., Hollenberg, M.D., Figueroa, A. and Pratt, R.M., Detection of epidermal growth factorurogastrone and its receptor during fetal mouse development, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 2782-2785. 3 Poulsen, S.S., Nexo, E., Olsen, P.S., Hess, J. and Kirkegaard, P., Immunohistochemical localization of epidermal growth factor in rat and man, Histochemistry, 85 (1986) 389-394. 4 Yamahara, M., Fujito, T., Ishikawa, T., Shimosato, T., Yokozaki, H., Yasui, W. and Tahara, E., Phenotypic expression of human epidermal growth factor in foetal submandibular gland and pleomorphic adenoma of salivary gland, Virchow's Arch. A Pathol. Anat. Histopathol., 412 (1988) 301-306. 5 Raaberg, L., Nexo, E., Damsgaard Mikkelsen, J. and Seier Poulsen, S., Immunohistochemical localisation and developmental aspects of epidermal growth factor in the rat, Histoehemistry, 89 (1988) 351-356. 6 Gluckman, P.D., The role of pituitary hormones, growth factors and insulin in the regulation of fetal growth. In J. R. Clarke (Ed.), Oxford Reviews of Reproductive Biology, Vol. 8, Clarendon, Oxford, 1986, pp. 1-60. 7 DeLarco, J.E. and Todaro, G.J., Sarcoma growth factor: specific binding to epidermal growth factor (EGF) membrane receptors, J. Cell. Physiol., 102 (1980) 267-277. 8 Lee, D.C., Rochford, R., Todaro, G.J. and Villarreal, L.P., Developmental expression of rat transforming growth factor-alpha mRNA, Mol. Cell. Biol., 5 (1985) 3644-3646. 9 Cohen, S., Epidermal growth factor, Biosci. Rep., 6 (1986) 1017-1028. 10 Hock, R.A. and Hoilenberg, M.D., Characterization of the receptor for epidermal growth factorurogastrone in human placenta membranes, J. Biol. Chem., 255 (1980) 10731-10736. 11 Pothier, P. and M6nard, D., Presence and characteristics of epidermal growth factor receptors in human fetal small intestine and colon, FEBS Lett., 228 (1988) 113-117. 12 Trolle, D., Age of foetus determined from its measures, Acta Obst. Gynecol. Scand., 27 (1948) 327-337. 13 Scatchard, G., The attractions of proteins for small molecules and ions, Ann. N.Y. Aead. Sci., 51 (1949) 660-672. 14 Nexo, E., Olsen, P.S. and Hansen, H.F., Purification of human epidermal growth factor-urogastrone by immunoaffinity chromatography. In H. Peeters (Ed.), Protides of the Biological Fluids XXXII, Pergamon, Oxford, 1984, pp. 1113-1115.
15 Nexe, E. and Hansen, H. F., Binding of epidermal growth factor from man, rat and mouse to the human epidermal growth factor receptor, Biochim. Biophys. Acta, 843 (1985) 101-106. 16 Lowry, O.H., Rosenbrough, W.J., Farr, A.R. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 17 Sadiq, H.F. and Devaskar, U.P., Glucocorticoids increase pulmonary epidermal growth factor receptors in female and male fetal rabbit, Biochem. Biophys. Res. Commun., 119 (1984) 408-414. 18 Adamson, E.D. and Meek, J., The ontogeny of epidermal growth factor receptors during mouse development, Dev. Biol., 103 (1984) 62-70. 19 Devaskar, U. P., Epidermal growth factor receptors in fetal and maternal rabbit lung, Biochem. Biophys. Res. Commun., 107 (1982) 714-720. 20 Catterton, W.Z., Escobedo, M.B., Sexson, W.R., Gray, M.E., Sundell, H.W. and Stahlman, M.T., Effect of epidermal growth factor on lung maturation in fetal rabbits, Pediatr. Res., 13 (1979) 108-118. 21 Sundell, H. W., Gray, M.E., Serenius, F. S., Escobedo, M.B. and Stahlman, M.T., Effects of epidermal growth factor on lung maturation in fetal lambs, Am. J. Pathol., 100 (1980) 707-719. 22 Heath, S.B., Pickens, W.L., Bucuvalas, J.C. and Suchy, F.J., Characterizations of hepatic epidermal growth factor receptors in the developing rat, Biochim. Biophys. Acta, 930 (1987) 107-113. 23 Thorburn, G.D., Waters, M.J., Young, I.R., Dolling, M., Buntine, D. and Hopkins, P.S., Epidermal growth factor: a critical factor in fetal maturation? In Ciba Foundation Symposium Vol. 86, The Fetus and Independent Life, Pitman, London, 1981, pp. 172-198. 24 Jorgensen, P., Poulsen, S. S. and Nexe, E., Distribution ofintravanously administered epidermal growth factor in the rat, Regul. Pept., 23 (1988) 161-169. 25 Mattila, A.-L., Perheentupa, J., Pesonen, K. and Viinikka, L., Epidermal growth factor in human urine from birth to puberty, J. Clin. Endocrin. Metab., 61 (1985) 997-1000. 26 Carson, S.A., Chase, R., Ulep, E., Scommegna, A. and Benveniste, R., Ontogenesis and characteristics of epidermal growth factor receptors in human placenta, Am. J. Obstet. Gynecol., 147 (1983) 932-939. 27 Twardzik, D. R., Differential expression transforming growth factor-alpha during prenatal development of the mouse, Cancer Res., 45 (1985) 5413-5416. 28 Morrish, D.W., Bhardwaj, D., Dabbagh, L.K., Marusyk, H. and Siy, O., Epidermal growth factor induces differentiation and secretion of human chorionic gonadotropin and placental lactogen in normal human placenta, J. Clin. Endocrinol. Metab., 65 (1987) 1282-1290. 29 Popliker, M., Shatz, A., Avivi, A., Ullrieh, A., Schlessingcr, J. and Webb, C.G., Onset of endogenous synthesis of epidermal growth factor in neonatal mice, Dev. Biol., 119 (1987) 38-44.
1
Elsevier REGPEP 00828
The receptor for epidermal growth factor is present in human fetal kidney, liver and lung E b b a N e r o 1 and Niels Kryger-Baggesen 2 i Department of Clinical Chemistry and 2Department of Gynaecology and Obstetrics, Central Hospital, Hillerod (Denmark) (Received 12 December 1988; revised version received21 March 1989; accepted 18 April 1989) Key words: Epidermal growth factor; Fetal; Human; Kidney; Liver; Lung;
Placenta; Receptor
Summary In animals a pharmacological dosis of the growth-promoting peptide epidermal growth factor (EGF) has an effect on the growth and/or maturation of several organs such as the lung, the kidney, the liver and the gastrointestinal tract. Since E G F elicits its function via binding to specific cellular receptors the presence of these receptors predicts a possible physiological role for E G F and E G F agonists. We have studied the presence of the EGF-receptor on human fetal membrane preparations from the kidney, the liver, the lung and the placenta (gestational age 13-20 weeks). The 4 membrane preparations all bind labeled E G F thus allowing us to calculate the apparent affinity constant and the number of receptors present per mg of membrane protein. The apparent affinity constant (gestational age 13-20 weeks) varies between 0.5 and 3.5 x 109 mol- 1, median 1.3 x 109 tool- 1 (n = 40). No difference is observed for the 4 tissues examined, and no difference is found as a function of the gestational age. The number of receptors present per mg of membrane protein (gestational age 16-20 weeks) are (range and (median) 90-220 (130) fmol, n -- 10 for the kidney, 80-480 (250) fmol, n = 9 for the liver, 90-690 (300) fmol, n = 10 for the lung, and 2100-4200 (3400) fmol, n -- 7 for the placenta. Results for a fetus of gestational age 13 weeks show high values for kidney receptors (240 fmol) and lung receptors (800 fmol) and low values for the placenta receptors (410 fmol).
Correspondence: E. Next, Department of Clinical Chemistry, Central Hospital, DK-3400 Hillered,
Denmark. 0167-0115/89/$03.50 © 1989 Elsevier Science Publishers B.V. (BiomedicalDivision)
Introduction
Epidermal growth factor (EGF) is a growth-promoting peptide f'n'stisolated from the submandibular glands of mice due to its ability to induce precocious eyelid opening and tooth eruption in the newborn mouse [1 ]. In the fetus EGF-like activity has been identified in whole fetuses [2], and in several fetal organs including the submandibular gland, the kidney and the lung [3-5]. In experimental animals injections of pharmacological doses of EGF have suggested it to play a role for the growth and/or maturation of the fetal liver, kidney, lung and gastrointestinal tract [6]. Some years ago an EGF agonist, transforming growth factor alpha (TGFct), was isolated from tumour cells [7] and later mRNA for TGF~ has been visualized in the fetal rat [8]. EGF and TGF~ elicit their function via binding to the EGF receptor, a protein kinase present on a number of tissues including the human placenta [9]. If EGF or TGF~ is to play a role during human fetal development, the cellular receptor for EGF has to be present. So far the presence of the EGF receptor has been studied only for the human placenta and for the human intestine [ 10,11 ]. Since animal studies have indicated a role for EGF also in other organs we wanted to examine the occurrence of the receptor for EGF on selected organs from human fetuses as a function of gestational age.
Materials and Methods
Fetal tissue Women (age range 21-37 years, n = 11) referred for legal abortion on socioeconomic indication gave informed consent. Labor was induced by installation of 140 mg prostaglandin-F2~ (Dinoprost, Upjohn, U.S.A.) in the amniotic cavity. The fetus was delivered between 7 and 33 h after induction of labor. Only male fetuses were employed. The length of the foot was measured and gestational week was calculated according to the formula (length of foot in mm + 24) x 4/11 equals the gestational week [12]. The following 11 fetuses were studied: 1 (gestational age 13 weeks), 4 (gestational age 16 weeks), 1 (gestational age 17 weeks), 2 (gestational age 18 weeks), 2 (gestational age 19 weeks) and 1 (gestational age 20 weeks). The placenta, the liver, the kidneys and the lungs were removed and kept at - 80 °C until further analyzed. Biochemical analyses Preparation of membranes. Approximately 1 g of tissues was homogenized in 5 mi 0.25 M sucrose, 0.025 M "Iris buffer, pH 7.4 per g of tissue and membranes were prepared as previously described [10]. The membranes were stored in 0.05 M "Iris buffer, pH 7.4 and kept at - 20 ° C until employed. Binding isotherms were performed employing 50 #1 membrane preparation (2.5 mg protein/ml or less), 50/zl 125I-labeled human urinary EGF (approx. 30.000 cpm, 50 fmol) and 50 #1 unlabeled EGF (0-950 fmol). The assay buffer was 0.1 M phosphate, 0.1 ~ albumin (human albumin, Behringwerke, F.R.G.), pH 8.0. The mixtures
were incubated for 1 h at room temperature and overnight at 4 ° C. Free and bound EGF were separated by vacuum filtration through 0.2 #m filters (SM 11107 Sartorius, GOttingen, F.R.G.). The bound fraction, the filters were counted in a multichannel gammacounter (Molsg~d, Denmark). The fraction of labeled peptide bound varied between 0.10 and 0.60 when no unlabeled peptide was added. Non-specific binding obtained by including 60 pmol of human EGF in the assay mixture accounted for a fraction around 0.03 of added counts. All samples were analyzed in duplicate and non-specific binding was subtracted prior to analysis of the data. The data were analyzed according to Scatchard [ 13]. For analyses of association and dissociation curves membranes from liver and placenta were employed. To construct the association curve labeled EGF (100 #1, 50 fmol) and membranes (50 #1) corresponding to approximately 20 fmol EGF binding sites were incubated for time intervals between 30 min and 24 h. To construct the dissociation curve 60 pmol of human EGF was added to membranes preincubated for 24 h with labeled EGF and the reaction was terminated at intervals between 30 min and 24 h after addition of unlabeled EGF. To test the pH dependence of binding between EGF and its receptor, 90 #1 assay buffer adjusted to pH between 6.0 and 11.0 was mixed with 10 #1 ~25I-EGF (50 fmol) and 50 #1 membrane preparation (approx. 20 fmol EGF binding sites). Human EGF purified from urine by immunoaffinity chromatography and pure as judged by SDS polyacrylamid gel electrophoresis, and N-terminal sequence was employed for preparation of calibration standards, and for preparation of 125I-labeled peptide [ 14,15]. The specific activity varied between 500 and 1000 cpm/fmol. Protein was determined by the Lowry method [16] employing human albumin as calibrator.
Results
High affinity receptors for EGF are present on membranes prepared from human fetal kidney, liver, lung and placenta. All the tissues studied show one class of receptors as judged from the Scatchard plots (Fig. 1). The apparent affinity constants are between 0.5 and 3.5 x 10 9 mol- 1 (median 1.3 x 109 mol- 1) (n --- 40) (Fig. 2). No systematic change occurs in the affinity constant with the age of the fetus and no difference is observed between the apparent affmity constant of the 4 tissues examined, nor do the affinity constants obtained on tissues from the same fetus show any characteristic pattern. Studies performed on membranes from the liver show the same association rate and dissociation rate as membranes from the placenta. Also the sensitivity towards acid pH is the same for membranes obtained from the liver, the lung and the placenta (Fig. 3). The number of EGF binding sites present on the 4 tissues is given in Fig. 4. The pattern is unique for the 13-week-old fetus. The lung contains more receptors per mg of membranes than the placenta. Compared to the absolute values obtained for fetuses 16-20 weeks of age, the 13 week placenta contains a small number of receptors (410 fmol), while the lung and the kidney contain the highest number of receptors found
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Fig. 1. Binding of EGF to membranes from human fetal kidney, liver, lung and placenta. The figure shows binding data calculated according to Scatchard. The organs were obtained from fetuses of gestational age 18 weeks (kidney), 19 weeks (liver), 20 weeks (lung) and 16 weeks (placenta). Abcissa, EGF bound (fmol); ordinate, Bound/free x 10- 6 liter.
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Fig. 2. Apparent affinity constant for binding of EGF to membranes obtained from human fetal kidney, liver, lung and placenta• Fetuses gestational age 13 (*), 16 ( ~ 7 V A A), 17 (o), 18 ( [ ] I ) , 19 ( Q O ) and 20 ( x ) weeks were examined. Individual results are given. The horizontal lines indicate the medians.
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Fig. 3. Binding oflabeled EGF to human fetai membrane preparations. A: association curve. Labeled EGF and membranes from liver (O) or placenta (O) were mixed at the time zero. The reaction was terminated at the time intervals shown. Abcissa, time after addition of labeled EGF; ordinate, fraction of label bound relative to the maximal amount of label bound. B: dissociation curve. Labeled EGF and membranes from liver (O) or placenta (©) was incubated overnight. At time zero excess unlabeled EGF was added and the reaction was terminated at the time intervals shown. Abcissa, time after addition of unlabeled EGF; ordinate, fraction of label bound relative to label bound at time zero. C: pH optimum. Labeled EGF and membranes wore incubated with membranes from liver (O), lung ( x ) or placenta (O) at different pH. Abcissa, pH of assay mixture; ordinate: fraction of label bound relative to maximum binding.
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PLACENTA
Fig. 4. Number of binding sites for EGF present on membranes obtained from human fetal kidney, liver, lung and placenta. Human fetuses gestationai age 13 (*), 16 ( V V A A ) , 17 (.), 18 ( V l l ) , 19 ( O O ) and 20 ( x ) weeks were examined. Individual results are given. The horizontal lines indicate the medians.
on these two tissues, that is respectively 800 and 240 fmol/mg protein. No variation with age is found for the number of receptors present on the 4 tissues obtained from fetuses aged 16-20 weeks. The absolute values given as range and median for the receptors expressed per mg of membrane protein are approximately 10 times higher for the placenta (2100-4200 (3400) fmol, n -- 7) than for the lung (90-690 (300) fmol, n = 10) and the liver (80-480 (250) fmol, n = 9). The kidney display the lowest values for the receptor (90-220 (130) fmol, n = 10).
Discussion
We report that human fetal kidney, liver and lung possess receptors for EGF from at least gestational week 13. Our finding of receptors on the fetal human lung is in accord with previous studies on rabbit and mouse fetuses [ 17,18 ]. In rabbits the number of receptors is considerably higher during prenatal development than after birth [19]. EGF given to premature fetuses (sheep and rabbit) accelerates the maturation of the lung and prevents respiratory distress syndrome [20,21 ]. We have recently demonstrated EGF immunoreactivity to occur in the pneumocytes II of the rat lung a few days before birth [5]. Together, the above results strongly suggest a physiological role for EGF in the maturation of the human lung. Also the fetal liver from mice and rats expose receptors for EGF [ 18,22], and in the fetal sheep exogenous EGF stimulates the growth of the liver [23]. In the human fetus the number of receptors present on the liver is comparable to the values obtained for the kidney and the lung. In the adult rat the liver is the tissue that contains the highest amount of receptors for EGF, most likely because the liver plays a role in removal of EGF from the circulation [24]. The kidney synthesizes EGF in fetal life and throughout postnatal life, and large amounts of EGF are present in the urine [3,25]. Previous work has indicated that injected EGF stimulates growth of the fetal kidney [23]. As the lung, the kidney contains receptors for EGF and synthesizes EGF. It is thus possible that EGF also plays a role in the physiological development of the human kidney. The human placenta is one of the first tissues on which receptors for EGF have been observed, and no other non-pathological source is known that contains as many receptors for EGF per mg of protein [ 11 ]. Previous work has indicated the number of receptors to increase with gestational age [26]. We find that the week 13 foetus contains few receptors per mg of membrane protein compared to fetuses of gestational age 16 weeks, but no increase in the number of receptors occurs between gestational ages 16 and 20 weeks. The role for the EGF receptor on placental tissues is not known. Recently, mRNA for the EGF agonist TGF~ has been localized in maternal decidua of the rat [8]. This, together with the observation that EGF induces differentiation of the cytothrophoblast to form the syncytothrophoblast, suggests an autocrine role for TGF~ in the placenta [28]. TGF~ occurs early during fetal development, and it peaks around mid-term in both mouse and rat [8,27]. In contrast, EGF immunoreactivity occurs relatively late during
fetal d e v e l o p m e n t [3,5] a n d m R N A coding for the synthesis o f E G F occurs only after birth in the m o u s e [29]. It is thus likely that the s a m e r e s p o n s e system, the E G F receptor, is stimulated by one peptide, T G F ~ , during early d e v e l o p m e n t a n d by another, E G F , during f'mal fetal development. In conclusion the present p a p e r d e m o n s t r a t e s h u m a n fetal kidney, liver a n d lung to p o s s e s s receptors for E G F . T h e receptors have the s a m e atTmity t o w a r d s E G F as the receptors present on the h u m a n placenta, but the quantity is about 1/10 o f the a m o u n t present on placenta.
Acknowledgement T h e technical a s s i s t a n c e o f M a r i a n n e Rye H a n s e n a n d K a m m a Velin is w a r m l y acknowledged. This w o r k was s u p p o r t e d by T h e D a n i s h C a n c e r Society (87-042, 88-090).
References 1 Cohen, S., Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal, J. Biol. Chem., 237 (1962) 1555-1562. 2 Nexo, E., Hollenberg, M.D., Figueroa, A. and Pratt, R.M., Detection of epidermal growth factorurogastrone and its receptor during fetal mouse development, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 2782-2785. 3 Poulsen, S.S., Nexo, E., Olsen, P.S., Hess, J. and Kirkegaard, P., Immunohistochemical localization of epidermal growth factor in rat and man, Histochemistry, 85 (1986) 389-394. 4 Yamahara, M., Fujito, T., Ishikawa, T., Shimosato, T., Yokozaki, H., Yasui, W. and Tahara, E., Phenotypic expression of human epidermal growth factor in foetal submandibular gland and pleomorphic adenoma of salivary gland, Virchow's Arch. A Pathol. Anat. Histopathol., 412 (1988) 301-306. 5 Raaberg, L., Nexo, E., Damsgaard Mikkelsen, J. and Seier Poulsen, S., Immunohistochemical localisation and developmental aspects of epidermal growth factor in the rat, Histoehemistry, 89 (1988) 351-356. 6 Gluckman, P.D., The role of pituitary hormones, growth factors and insulin in the regulation of fetal growth. In J. R. Clarke (Ed.), Oxford Reviews of Reproductive Biology, Vol. 8, Clarendon, Oxford, 1986, pp. 1-60. 7 DeLarco, J.E. and Todaro, G.J., Sarcoma growth factor: specific binding to epidermal growth factor (EGF) membrane receptors, J. Cell. Physiol., 102 (1980) 267-277. 8 Lee, D.C., Rochford, R., Todaro, G.J. and Villarreal, L.P., Developmental expression of rat transforming growth factor-alpha mRNA, Mol. Cell. Biol., 5 (1985) 3644-3646. 9 Cohen, S., Epidermal growth factor, Biosci. Rep., 6 (1986) 1017-1028. 10 Hock, R.A. and Hoilenberg, M.D., Characterization of the receptor for epidermal growth factorurogastrone in human placenta membranes, J. Biol. Chem., 255 (1980) 10731-10736. 11 Pothier, P. and M6nard, D., Presence and characteristics of epidermal growth factor receptors in human fetal small intestine and colon, FEBS Lett., 228 (1988) 113-117. 12 Trolle, D., Age of foetus determined from its measures, Acta Obst. Gynecol. Scand., 27 (1948) 327-337. 13 Scatchard, G., The attractions of proteins for small molecules and ions, Ann. N.Y. Aead. Sci., 51 (1949) 660-672. 14 Nexo, E., Olsen, P.S. and Hansen, H.F., Purification of human epidermal growth factor-urogastrone by immunoaffinity chromatography. In H. Peeters (Ed.), Protides of the Biological Fluids XXXII, Pergamon, Oxford, 1984, pp. 1113-1115.
15 Nexe, E. and Hansen, H. F., Binding of epidermal growth factor from man, rat and mouse to the human epidermal growth factor receptor, Biochim. Biophys. Acta, 843 (1985) 101-106. 16 Lowry, O.H., Rosenbrough, W.J., Farr, A.R. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 17 Sadiq, H.F. and Devaskar, U.P., Glucocorticoids increase pulmonary epidermal growth factor receptors in female and male fetal rabbit, Biochem. Biophys. Res. Commun., 119 (1984) 408-414. 18 Adamson, E.D. and Meek, J., The ontogeny of epidermal growth factor receptors during mouse development, Dev. Biol., 103 (1984) 62-70. 19 Devaskar, U. P., Epidermal growth factor receptors in fetal and maternal rabbit lung, Biochem. Biophys. Res. Commun., 107 (1982) 714-720. 20 Catterton, W.Z., Escobedo, M.B., Sexson, W.R., Gray, M.E., Sundell, H.W. and Stahlman, M.T., Effect of epidermal growth factor on lung maturation in fetal rabbits, Pediatr. Res., 13 (1979) 108-118. 21 Sundell, H. W., Gray, M.E., Serenius, F. S., Escobedo, M.B. and Stahlman, M.T., Effects of epidermal growth factor on lung maturation in fetal lambs, Am. J. Pathol., 100 (1980) 707-719. 22 Heath, S.B., Pickens, W.L., Bucuvalas, J.C. and Suchy, F.J., Characterizations of hepatic epidermal growth factor receptors in the developing rat, Biochim. Biophys. Acta, 930 (1987) 107-113. 23 Thorburn, G.D., Waters, M.J., Young, I.R., Dolling, M., Buntine, D. and Hopkins, P.S., Epidermal growth factor: a critical factor in fetal maturation? In Ciba Foundation Symposium Vol. 86, The Fetus and Independent Life, Pitman, London, 1981, pp. 172-198. 24 Jorgensen, P., Poulsen, S. S. and Nexe, E., Distribution ofintravanously administered epidermal growth factor in the rat, Regul. Pept., 23 (1988) 161-169. 25 Mattila, A.-L., Perheentupa, J., Pesonen, K. and Viinikka, L., Epidermal growth factor in human urine from birth to puberty, J. Clin. Endocrin. Metab., 61 (1985) 997-1000. 26 Carson, S.A., Chase, R., Ulep, E., Scommegna, A. and Benveniste, R., Ontogenesis and characteristics of epidermal growth factor receptors in human placenta, Am. J. Obstet. Gynecol., 147 (1983) 932-939. 27 Twardzik, D. R., Differential expression transforming growth factor-alpha during prenatal development of the mouse, Cancer Res., 45 (1985) 5413-5416. 28 Morrish, D.W., Bhardwaj, D., Dabbagh, L.K., Marusyk, H. and Siy, O., Epidermal growth factor induces differentiation and secretion of human chorionic gonadotropin and placental lactogen in normal human placenta, J. Clin. Endocrinol. Metab., 65 (1987) 1282-1290. 29 Popliker, M., Shatz, A., Avivi, A., Ullrieh, A., Schlessingcr, J. and Webb, C.G., Onset of endogenous synthesis of epidermal growth factor in neonatal mice, Dev. Biol., 119 (1987) 38-44.