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Epidermal growth factor (EGF)
3 Epidermal
growth factor (EGF)
ROBERT A. GOODLAD NICHOLAS A. WRIGHT Despite the wealth of information and detailed knowledge about the epidermal growth factor (EGF) gene and the structure of EGF and its receptor, its role in vivo is still not clear. EGF was first isolated from the submandibular salivary glands of mice and found to stimulate precocial eyelid opening and tooth eruption. EGF also stimulated the proliferation and maturation of several other tissues, including the epidermis, pulmonary epithelium and corneum and was shown to be a very potent mitogen in vitro (Carpenter, 1981). Human EGF was originally known as urogastrone, where it was identified by its mitogenic and anti-ulcer actions in the urine of pregnant women. EGF is formed as a pre-pro molecule of 1217 amino acid residues (Bell et al, 1986), which is processed by the salivary glands to EGF l-53, has a relative molecular mass of 6215 and includes six cysteine residues that form three disulphide bridges (Campbell et al, 1990). EGF’s location in the salivary glands and in duodenal Brunner’s glands (Heitz et al, 1978), and its presence in a variety of biological fluids, such as saliva, plasma and milk (Carpenter, 1980, 1981) all suggested that it had an important role to play in gastrointestinal homeostasis. EGF can stimulate the proliferation and maturation of the pre- and postnatal intestine (Malo and Menard, 1982; Weaver and Walker, 1988). Postnatally EGF is first found in Brunner’s glands, then in the Paneth cells, and finally in the submandibular glands (Raaberg et al, 1988). Interest in EGF was furthered by reports that saliva itself was trophic to the intestine (Li et al, 1983) and that EGF could have a ‘cytoprotective’ effect on the gut (Kirkegaard et al, 1983). An additional action of EGF was to inhibit gastric acid secretion (Bower et al, 1975; Koffman et al, 1982). Thus EGF, like many growth factors has more than one action. Molecular biological techniques have been used to produce recombinant synthetic EGF (Smith et al, 1982), which has an identical structure to the natural EGF so enabling the detailed study of its biological effects (Gregory, 1985). Several ligands bind to the EGF receptor, and it may therefore be better to talk of the EGF family of peptides rather than of EGF itself. Furthermore, it is possible, and also quite likely, that some of the effects seen after administration of EGF may actually be mimicking the natural actions of other members of this extended family. For Bailli&-e’s Clinical GastroenterologyVol. 10, No. 1, March 1996 ISBN O-7020-2002-8 0950-3528/96/010033 + 15 $12.00/00
33 Copyright 0 1996, by Baillike All rights of reproduction in any form
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example, the distribution of transcripts for EGF and transforming growth factor-a (TGF-a) in the intestine led to the suggestion that the true ligand of the EGF receptor in the intestine is TGF-a (Koyama and Podolsky, 1989).
THE EGF RECEPTOR The EGF receptor (EGFR) was the first receptor protein identified as a tyrosine specific protein kinase, and is a ‘single pass’ transmembrane protein of around 1200 amino acid residues. The EGFR has a large glycosylated extracellular domain that binds EGF and (eventually) leads to the activation of an intracellular tyrosine kinase domain, which in turn transfers phosphate from APT on selected tyrosine side-chains (both on the receptor and on specific cellular proteins). Over 50 such receptor tyrosine kinases, belonging to 14 receptor families, have now been identified (Lemnon and Schlessinger, 1994). The importance of this signalling system is demonstrated by its being purloined by the proto-oncogene c-e&B, which codes for the EGFR. Mutation can delete the extracellular domain and turn c-&B into a tumour producing oncogene that constantly produces an intracellular proliferative signal. The question of how this signalling is transmitted via a single transmembrane protein was answered by the finding that receptors can dimerize after ligand binding, thus allowing the internal domains to cross-phosphorylate (or autophosphorylate). These can then act as ‘docking sites’ to set off a signalling cascade (Alberts et al, 1994); see Figure 2 in the Introduction to this issue. After binding the receptors accumulate in coated pits, and rather than be recycled like some groups of receptor, most EGFRs are degraded in the lysosomes, thus receptor binding leads to decreased receptor concentration (receptor downregulation). EGF is not the only ligand for the EGFR, as TGF-a, amphiregulin, heparin-binding EGF-like growth factor and betacellulin all bind to the receptor. Furthermore, there is homology with at least three viral proteins including vaccinia virus growth factors (VVGF: Burgess, 1989). TGF-a is found in high levels throughout the gut in the adult (and in even greater quantities in the juvenile) and transcripts for TGF-a but not EGF are found in intestinal cells. Furthermore, it has been reported that the intestine of a 19-week-old human foetus contained 10 times more EGF receptor-binding substance than EGF (Miettinen et al, 1989). The above data has led to the suggestion that the true ligand for the EGFR may be TGF-a (Koyama and Podolsky, 1989). TGF-a over-expression is associated with intestinal hyperplasia (Sandgren et al, 1990) and TGF-a is also trophic to the intestine of parenterally fed rats (Goodlad et al, 1990) but to a lesser extent (Goodlad et al, 1990; Sandgren et al, 1990). The similarities and differences in the response of the gut to EGF and TGF-a have recently been extensively reviewed (Barnard et al, 1995), and this led to the advancement of the hypothesis that while the two peptides bind the same receptor with equal affinity, they nevertheless bind differently, and in
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addition, there is a morphological compartmentalization of the two peptides. One consequence of this multiple ligand binding is that there is a considerable degree of redundancy in the EGF family, which permits substitution should one factor be missing or have been ‘knocked out’. Recent reports of an EGFR knock out mouse confirm the body’s ability to function without a major growth factor receptor, as the mice can survive, albeit briefly, for about a week after birth, but they then succumb to respiratory distress or to a necrotising enterocolitis-like syndrome (Miettinen et al, 1995). ACTIONS
OF EGF
Cytoprotection Early reports indicated that topical EGF was ‘cytoprotective’ as a result of a mechanism independent of gastric secretion inhibition or prostaglandin formation, but related to the increase in DNA synthesis in oxyntic mucosa (Konturek et al, 1981). The trophic effects of EGF will be discussed later. The submandibular glands influence healing of chronic gastric ulcers and this has led to the suggestion that EGF participates in healing of chronic gastric ulcers (Olsen et al, 1986). Lower levels of salivary EGF immunoreactivity have been reported in patients with a peptic ulcer (Ohmura et al, 1987). Recent studies have also suggested that the systemic EGF may also reduce mucosal damage and inflammation in a model of colitis (Procaccino et al, 1994). Some of these protective mechanisms may be through the stimulation of goblet cells to release mucus (Ishikawa et al, 1994), and further cytoprotective effects may be attributable to increased gastric blood flow (Hui et al, 1993). Acid inhibition EGF, like other trophic agents for the gut such as enteroglucagon (Goodlad and Wright, 1990) can inhibit gastric acid (and intrinsic factor) secretion (Rackoff et al, 1988). There is some evidence that this occurs by way of a separate mechanism to the mitogenic one (Dembinski et al, 1982), perhaps by different signal transducing mechanisms (Gamer, 1992). Furthermore, EGF can stimulate the transcription of gas&in, by a gastrin response element in the gash-in promoter (Merchant et al, 1991). There is also evidence that EGF may play a role in the maintenance of parietal cell function at much lower concentrations than those required for inhibition of acid secretion, indicating that EGF modulates parietal cell function by multiple signalling pathways (Chew et al, 1994). Trophic
effects
Early reports on the actions of EGF in vivo were somewhat confusing, as several studies were contradictory. Some groups found increased DNA
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synthesis in the gastric mucosa, but not the duodenal and colonic mucosa (Johnson and Guthrie, 1980), while others reported that the actions of EGF were more dramatic in the caecum, colon and rectum than in the small intestine (Scheving et al, 1980). Al-Nafussi and Wright (1982) found that EGF was trophic to the duodenum, jejunum, ileum and colon but not the stomach, and that the response differed at different times after the injection. Other groups only found trophic effects in starved (Dembinski et al, 1982) or undernourished (Majumdar, 1984) animals. Some of these above differences can be attributed to the use of inappropriate assays, others most likely reflect the use of different animal models, and we will briefly discuss these essential points. Proliferative
assays
While there are several published methods for the study of intestinal epithelial cell proliferation, many of these have serious flaws, especially those based on the gross uptake of tritiated thymidine (Goodlad and Wright, 1982; Maurer, 1981), and even the more acceptable methods can be misleading if the three-dimensional nature of the gut is not considered. We strongly advocate the use of a stathmokinetic drug, such as vincristine, to quantify the rate of entry of arrested metaphase cells into mitosis, which defines the crypt cell production rate (CCPR). This is the most productive and robust method for the study of gastrointestinal cell proliferation in animals. A great advantage of the technique is that results can be expressed on a per crypt basis and thus avoids the several pitfalls associated with scoring crypt sections. Moreover using the crypt as the denominator means that there is no need to count non-dividing cells. The technique, unlike almost all others, also accounts for all the factors involved in cell renewal, i.e. phase duration, growth fraction and compartment size (Goodlad, 1994). If more detailed information on the mechanisms underlying altered cell kinetics is required, the study of the distribution of DNA synthesizing (S-phase) cells, using tritiated thymidine and/or bromodeoxyuridine can yield valuable information, providing that the interpretation takes into account the three-dimensional nature of the crypts (Goodlad, 1992). For human studies, ethical considerations prevent the use of metaphase arrest or nucleotide labelling, nevertheless, the number of native mitoses per (microdissected) crypt can give valid results (Goodlad et al, 1991a). This method is superior in many ways to the several in vitro methods available for the assessment of biopsies. Many groups favour the use of antibody-based techniques to locate dividing cells, but these can also have several problems as the reaction products are difficult to standardize and antigens can be expressed anomalously, for example, expression of the so-called proliferating cell nuclear antigen (PCNA) can occur without any concomitant changes in proliferation (Hall et al, 1994). Finally, all section-based techniques cannot escape the many problems associated with studying three-dimensional structures in two dimensions.
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models
Many groups have used starved animals, presumably to reduce the effects of endogenous secretions, maximize the response of the gut and ‘standardize’ the animals. However, starved rodents are not in a steady state of cell renewal. A better model for the study of potential proliferative agents on the gastrointestinal tract is seen in the hypoplastic intestine of rats maintained by total parenteral nutrition (TPN). The intestine of TPN rats is in a steady state of basal cell proliferation, as are the endogenous secretions and gut hormones. Furthermore, the several variables associated with altered food intake and food intake patterns are removed. The TPN model has the further advantage that bolus or continuous administration of the agent can readily be achieved, and if a two-channel system is used, intra-intestinal infusion is also practical (Figure 1). The cannulae exit through flexible
Figure 1. The total parenteralnutrition(TPN)rat.
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tethers and special fluid swivel joints, so that the rat is fully conscious and relatively unrestrained. In vivo studies (animal)
The TPN rat model has shown that recombinant human EGF, at doses that should not inhibit gastric acid secretion, is a very potent stimulator of intestinal epithelial cell proliferation (Goodlad et al, 1985; Bragg et al, 1990). This trophic effect occurred throughout the gastrointestinal tract, but was especially prominent in the stomach and colon (Goodlad et al, 1987). Higher doses of EGF increased proliferation in the stomach by over four times, by four and a half times in the colon and doubled proliferation in the small intestine (Figure 2). Whilst EGF was very effective when given intravenously, it had no effect, even at massive doses, if given intragastrically. These proliferative effects of EGF were not associated with increased plasma levels of the hormone enteroglucagon, which has often been linked (albeit by circumstantial evidence) with increased proliferation in the gut, indicating that the two agents probably exert their proliferative effects through separate mechanisms (Goodlad et al, 1989a,b). There are several means by which a crypt can increase its rates of cell production, namely by altering the cell cycle time, increasing the proportion of the crypt actively involved in proliferation (the growth fraction) or increasing the size of the crypt. We have shown that the main effect of EGF is to increase the size of the crypt, especially its length. One outcome of this is that few differences in proliferative indices were seen between the orally fed and TPN groups, despite the large difference in cell production and tissue mass, this being the consequence of the concomitant changes in crypt population, as proliferative indices are the ratio of dividing cells to total cell population. The number of mitoses and labelled cells/crypt, and thus the crypt cell production rates, were therefore all significantly decreased in the TPN group when compared to the orally fed. EGF increased both the proliferative indices and the number of dividing cells/crypt. The growth fraction was actually reduced in the EGF-treated group, which was a consequence of the very large increase in crypt length (Goodlad et al, 1992). In vivo studies (human)
While there have been several studies demonstrating the trophic effects of EGF on human explants (Menard et al, 1988), and on acid secretion (Gregory, 1985) and even some estimations of the crypt cell production rate in cultured explants, using a stathmokinetic technique and crypt microdissection (Challacombe and Wheeler, 1991), there have been few published reports on its action in the human in vivo. The first use of EGF to try and stimulate epithelial renewal was in a neonate with congenital microvillus atrophy. That study established that EGF was also a very potent mitogen in the human intestine (Walker-Smith et al, 1985). This finding was later confirmed (Drumm et al, 1988).
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GROWTH FACTOR (EGF)
Stomach 600, 500 400, 300-
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However, the defect in congenital microvillus atrophy is a structural one in the glycocalyx and although stimulating proliferation was palliative, it was not curative. A second use of EGF in extremis was in a moribund baby with necrotising enteritis (Sullivan et al, 1991). In this case EGF administration was associated with significantly altered proliferation, increased mitotic activity (and decreased crypt branching). Fortunately, these actions were also associated with a highly favourable clinical outcome (Cooper, 1992). Polyamines EGF can induce the first, rate-limiting, enzyme involved in the synthesis of polyamines, ornithine decarboxylase (Feldman et al, 1978). Ornithine decarboxylase activities in the small bowel mucosa rose within 12 hours of EGF administration in the atrophic small intestine of rats fed an elemental diet, indicating that EGF may stimulate mucosal proliferation through polyamine metabolism (Tsujikawa et al, 1990). This was substantiated by our finding that difluoromethylornithine, an inhibitor of the enzyme ornithine decarboxylase, significantly reduced the proliferative effect of EGF in the stomach, small intestine and colon, but the inhibition in the colon was less pronounced (Goodlad et al, 1989a). Although polyamines are involved, and are probably essential for effecting the proliferative response, there is still doubt as to whether they represent control points. Enzyme activity The effects of EGF on the enzyme activities of rats at different stages of development are somewhat complicated, depending on the specific enzyme concerned, the site within the small intestine and the timing of the treatment (Bradbury et al, 1992). In the adult animal EGF prevented the decrease in brush border enzyme activity seen in the TPN rat, but the response differed depending on the intestinal location, with the specific activities of some enzymes being higher than those seen in orally fed rats. Some of the variability can be attributed to the old problem of choosing the right denominator with which to express results, as if EGF is increasing both tissue mass and activity, specific activities can be misleading. It may well be better to express results per gut or per centimetre of gut. Nevertheless, there is no doubt that EGF can increase the functional capacity of the intestine by mechanisms separate from its trophic effect (Bragg et al, 1990; Goodlad et al, 1991b). EGF and cancer There is much interest on the autocrine stimulation of gastrointestinal carcinoma growth. Elevated levels of EGFR, EGF or related mRNAs, have been demonstrated in many gastric carcinomas. Human gastric cancer xenografts grown in nude mice increased their growth following EGF infusion and their growth was inhibited by sialoadenectomy (Okuda et al, 1994). The growth of certain gastrointestinal cell lines can be inhibited by
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antibodies against EGF, and by antisense oligonucleotides based on EGF mRNA (Baldwin and Whitehead, 1994). EGF or TGF-a and EGFR staining positively can be inversely related to prognosis in gastric cancers, implying that the autocrine mechanism between EGFITGF-a and EGFR may play an important role in the progression of gastric cancer, and that when such a mechanism becomes operative, prognosis may be poor (Yonemura et al, 1991, 1992). High levels of EGFR expression have been reported in a variety of cancer cell lines including liver, ovarian and colon (Siegall et al, 1989), nevertheless, others have reported lower EGFR levels in the carcinomatous colon (Koenders et al, 1992). This may perhaps reflect the extent of differentiation, as aggressively growing and poorly differentiated colon cells may not respond to EGF alone, while less aggressively growing and more differentiated cells respond with increased growth, so that downregulation of EGF-R may occur in aggressive colon tumour cells (Wan et al, 1988). While the involvement of the EGFR in these tumours suggests various therapeutic possibilities, the extent of ligand redundancy in the EGF family has led some authors to conclude that therapies aimed at blocking the action of a single factor is unlikely to influence the growth of most tumours (Garner, 1992). An interesting possibility has been raised by a few reports of prolongation of survival time by EGF, which may be the result of EGF inhibition of tumour cell metastasis (Amagase et al, 1990). The ulcer-associated
cell lineage
In several conditions where endodermal damage occurs, a newly recognized cell lineage, the ulcer-associated cell lineage (UACL), can develop (Wright et al, 1990). The UACL is seen associated with peptic ulceration, ulcerative colitis and Crohn’s disease. The phenotype of the cells of this lineage varies depending upon the cell’s position, expressing various peptides such as hSP, pS2 (see Chapter 8) and EGF, producing neutral mucin, and showing a unique lectin-binding profile and immunophenotype. The lineage also stains intensely with the antibody to TGF-a. This is of interest because the TGF-a gene can be induced by EGF (Miettinen, 1993). The lineage grows from the bases of existing crypts to open through a pore onto the surface. All gastrointestinal stem cells seem able to produce this lineage following mucosal ulceration. The secretion of EGF (and the other peptides) should then be able to stimulate cell proliferation, regeneration and healing. The EGF paradox It is of interest that we found no effect of large doses of EGF when given luminally (Goodlad et al, 1987). However, others have reported luminal effects in the small bowel (Ulshen et al, 1986) or in the colon (Polack et al, 1987). O’Loughlin et al (1994) reported that oral EGF treatment in resected rats did not alter mucosal proliferation but did stimulate maltase specific activity and caused a three to four-fold increase in glucose transport and
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phlorizin binding, which reflects the density of Na+ dependent glucose carriers. Dembinski et al (1982) found that EGF infused subcutaneously, but not intragastrically, inhibited spontaneous gastric acid and pepsin secretion. EGF injected intraperitoneally at 8 hourly intervals for 24 hours significantly stimulated DNA synthesis in the gastroduodenal mucosa and the pancreas, whereas when administered intragastrically it stimulated DNA synthesis only in the gastroduodenal mucosa but not in the pancreas. Ulshen et al (1986) found that intraluminal administration of EGF stimulated a mucosal proliferative response in the small intestine. It is of interest that they infused EGF into the ileum, but found a response in both the ileum and the jejunum (Ulshen et al, 1986), which implies a systemic mechanism. Thompson et al (1989) used implanted minipumps to investigate several routes of EGF delivery, and found that the greatest response was with intravenous EGF followed by subcutaneous, and then intraluminal EGF; these authors did, however, use a model of renewal (neomucosal patches) which can be regarded as damaged. Bamba et al (1993), working on the hypothesis that the EGFR was on the microvillous membrane of the enterocytes, investigated various routes of administration and were surprised to find that intraperitoneal not intraluminal EGF stimulated growth. With regard to the cytoprotection story there is also some debate, but several groups have found that oral EGF has no biological activity on the pathophysiology of the stomach (Kuwahara et al, 1990). Some of these discrepancies could be explained by the hypothesis that luminal EGF is only effective if the mucosa is damaged, and luminal EGF can indeed be protective in such models (Ishikawa et al, 1994). Another hypothesis concerns the question as to what form of EGF is present. A derivative of human epidermal growth factor, EGF148, has the same acid inhibitory actions as the full molecule (l-53) (Guglietta and Lesch, 1993), leading to the suggestion that this was the biologically active form. Recent work, however, has suggested that 148 is less active as a mitogen (Goodlad et al, unpublished results). Previous studies had indicated that EGF was stable in acid/pepsin (B&ton et al, 1988). These studies used size exclusion chromatography to determine stability, which is not a very precise technique. More recent work has suggested that the C-terminal five amino acid residues of EGF can be cleaved in acid/pepsin and that a relatively small change in this region has a marked effect on receptor binding (Araki et al, 1989). EGF has been shown to be digested to EGF1-49 and EGF1-46 by pepsin, but only when the pH was below pH 4, these forms of EGF had only 25% of the biological activity of native EGF (Playford et al, 1995). While EGF is digested by pancreatic enzymes in fasting intestinal juice, it may be preserved when food proteins block the active sites of these enzymes (Playford et al, 1993). These studies have raised the possibility that the addition of enzymeinhibiting proteins, or alternative substrates, such as casein, may preserve intestinal integrity and function. This would tie in well with our previous results, which have shown that systemic but not oral EGF causes intestinal proliferation in parenterally fed
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animals (Goodlad et al, 1987), as the virtually empty gut of the TPN rat would contain very few alternative substrates for acid and pepsin, so that EGF would probably be destroyed. The preservation of EGF in achlorhydric stomachs raised the intriguing suggestion that the mode of action of acid suppressant drugs may include the preservation of luminal EGF (Playford et al, 1995), and reduced EGF levels have been observed in the stomachs of peptic ulcer patients (Maccini and Veit, 1990). Nevertheless, EGF cannot be the total answer as antacids are still effective in ulcer models in sialoadenectomised rats (Konturek et al, 1990). An alternative explanation for our lack of response to oral EGF is that EGF may need to be present continuously for a mitogenic response, rather than, as in our previous study, given as a bolus three times a day. Our most recent studies using a two-channel swivel joint-TPN system have in fact shown a direct trophic response to luminal EGF, but only in the duodenum, not on the more distal regions of the bowel, probably reflecting rapid proteolysis of the EGF by luminal proteases. In addition there was also evidence for EGF effects in the more distal sites using soya bean trypsin inhibitor to protect the luminal EGF (Marchbank et al, 1995). A third factor to consider in terms of luminal EGF action concerns the continuing debate as to whether the enterocytes have apical (luminal) receptors. One well-cited report found the EGFR on the microvillus membrane and, thus, suggested a mechanism for the intraluminal regulation of enterocyte proliferation (Thompson, 1988). However, other groups only found the EGFRs to be basolateral (Scheving et al, 1989) or on both microvillar and basolateral surfaces of enterocytes (Kelly et al, 1992). Caco-2 cell monolayers bound EGF to the basolateral membrane with two to three times more affinity than the apical membrane (Hidalgo et al, 1989). On the other hand autoradiographical studies found no binding sites on the brush border of human fetal gut. In addition, the greatest concentration of EGF binding sites was found in regions of high proliferative activity suggesting that, under normal circumstances, systemic but not luminal EGF has free access to its specific receptor (Menard and Pothier, 1991). A similar basolateral location of EGFK was reported for the parietal cell (Mori et al, 1987). Later work by Thompson et al (1993) using more sensitive techniques, demonstrated that while intraluminal EGF stimulates rapid tyrosine phosphorylation of the EGF receptor in vivo, the localization of the EGF receptor was on the basolateral membrane suggesting that EGF is rapidly transported across the intestinal epithelium. SUMMARY Despite the wealth of information concerning EGF and its related peptides, its precise role in the control of gastrointestinal functions is still not fully resolved. However, there is no doubt that it can have some very potent effects on the gastrointestinal tract. These may be related to the control of growth and development and to the regular control of cell renewal. Nevertheless, in the adult, EGF may only be active in response to luminal
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damage and repair, and furthermore this may also only occur if the luminal EGF is protected from proteolytic degradation. Notwithstanding this, ‘EGF’-like responses may be evoked in the gut by intestinal TGF-a. The possible therapeutic use of EGF and members of its family in ulcer therapy will be discussed in later Chapters of this volume, other potential uses are in the control of necrotising enteritis and in the alleviation of the mucositis associated with cancer treatment.
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247. Heitz
P, Kasper M, Van Noorden S et al (1978) Immunohistochemical localisation of urogastrone to human duodenal and submandibular glands. Gut 19: 408-413. Hidalgo IJ, Kato A & Borchardt RT (1989) Binding of epidermal growth factor by human colon carcinoma cell (Caco-2) monolayers. Biochemical Biophysical Research Communications 160:
317-324. Hui WM, Chen BW, Kung AW et al (1993) Effect of epidermal growth factor on gastric blood flow in rats: possible role in mucosal protection. Gastroenterology 104: 1605-1610. Ishikawa S, Cepinskas G, Specian RD et al (1994) Epidermal growth-factor attenuates jejunal mucosal injury-induced by oleic-acid-role of mucus. American Journal of Physiology 30: G1067-G1077. Johnson LR & Guthrie PD (1980) Stimulation of rat oxyntic gland mucosal growth by epidermal growth factor. American Journal of Physiology 238: G45-G49. Kelly D, McFadyen M, King TP et al (1992) Characterisation and autoradiographic localisation of the epidermal growth factor receptor in the jejunum of neonatal and weaned pigs. Reproductive Fertility and Development 4: 183-l 91. Kirkegaard P, Olsen PS, Poulsen SS et al (1983) Epidermal growth factor inhibits cysteamine-induced duodenal ulcers. Gustroenterology 85: 1277-1283. Koenders PG, Peters WH, Wobbes T et al (1992) Epidermal growth factor receptor levels are lower in carcinomatous than in normal colorectal tissue. Brifish Journal of Cancer 65: 189-192.
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Koffman CG, Elder JB, Ganguli PC et al (1982) Effect of urogastrone on gastric secretion and serum gastrin concentration in patients with duodenal ulceration. Gut 23: 951-956. Konturek SJ, Brzozowski T & Piastucki et al (1981) Role of mucosal prostaglandins and DNA synthesis in gastric cytoprotection by luminal epidermal growth factor. Gut 22: 927-932. Konturek SJ, Brzozowski T & Drozdowicz D et al (1990) Healing of chronic gastroduodenal ulcerations by antacids. Role of prostaglandins and epidermal growth factor. Digestive Diseases and Sciences 35: 1121-l 129. Koyama SY & Podolsky DK (1989) Differential expression of transforming growth factors alpha and beta in rat intestinal epithelial cells. Journal of Clinical Investigation 83: 17681773. Kuwahara Y, Sunagawa Y & Imoto Y et al (1990) Effects of orally administered human epidermal growth factor on natural and delayed healing of acetic acid induced gastric ulcers in rats. Japanese Journal of Pharmacology 52: 164-166. Lemnon MA & Schlessinger J (1994) Regulation of signal transduction and signal diversity by receptor oligomerization. Trends in Biological Science 19: 459-463. Li AK, Schattenkerk ME & Huffman RG et al (1983) Hypersecretion of submandibular saliva in male mice: trophic response in small intestine. Gastroenterology 84: 949-955. Maccini DM & Veit BC (1990) Salivary epidermal growth factor in patients with and without acid peptic disease. American Journal of Gustroenterology 85: 1102-l 104. Majumdar AP (1984) Postnatal undernutrition: effects of epidennal growth factor on growth and function of gastrointestinal tract in rats. Journal of Paediatric Gastroenterology and Nutrition 3: 618-625. Malo C & Menard D (1982) Influence of epidermal growth factor on the development of suckling mouse intestinal mucosa. Gastroenterology 83: 28-35. Marchbank T, Goodlad RA & Lee CY et al (1995) Luminal epidermal growth-factor is trophic to the small-intestine of parenterally fed rats. Clinical Science 89: 117-120. Maurer HR (1981) Potential pitfalls of 3H-thymidine technique to measure cell proliferation. Cell and Tissue Kinetics 14: 11l-120. Menard D & Pothier P (199 1) Radioautographic localisation of epidermal growth factor receptors in human fetal gut. Gastroenterology 101: 640-649. Menard D, Arsenault P & Pothier P (1988) Biologic effects of epidermal growth factor in human fetal jejunum. Gustroenterology 94: 656-663. Merchant JL, Demediuk B & Brand SJ (1991) A GC-rich element confers epidermal growth factor responsiveness to transcription from the gas&in promoter. Molecular and Cellular Biology 11: 2686-2696. Miettinen PJ (1993) Transforming growth factor-alpha and epidermal growth factor expression in human fetal gastrointestinal tract. Paediatric Research 33: 481-486. Miettinen PJ, Perheentupa J, Otonkoski T et al (1989) EGF- and TGF-alpha-like peptides in human fetal gut. Puediutric Research 26: 25-30. Miettinen PJ, Berger JE, Meneses J et al (1995) Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor. Nature 376: 337-441. Mori S, Morishita Y, Sakai K et al (1987) Electron microscopic evidence for epidermal growth factor receptor (EGF-R)-like immunoreactivity associated with the basolateral surface of gastric parietal cells. Acta Pathology Japan 37: 1909-1917. O’Loughlin E, Winter M, Shun A et al (1994) Structural and functional adaptation following jejunal resection in rabbits: effect of epidermal growth factor. Gastroenterology 107: 87-93. Ohmura E, Emoto N, Tsushima T et al (1987) Salivary immunoreactive human epidermal growth factor (IR-hEGF) in patients with peptic ulcer disease. Hepatogastroenterology 34: 160163. Okuda T, Onda M, Tokunaga A et al (1994) Stimulatory effect of EGF and inhibitory effect of sialoadenectomy on growth of an EGF receptor-hyper producing gastric cancer xenograft in nude mice. Surgery Today 24: 725-733. Olsen PS. Poulsen SS. Therkelsen K et al (1986) Effect of sialoadenectomy and synthetic human urogastrone on healing of chronic gastric ulcers in rats. Gut 27: 1443-1449. Playford RJ, Woodman AC, Clark P et al (1993) Effect of luminal growth factor preservation on intestinal growth. Lancet 341 (8849): 843-848. Playford RJ, Marchbank T, Calnan DP et al (1995) Epidermal growth factor is digested to smaller, less active forms in acidic gastric juice. Gustroenterology 108: 92-101. Polack PF, Goda T, Colony PC et al (1987) Effects of enterally fed EGF on the small and large intestine of the suckling rat. Regulatory Peptides 17: 121-132.
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Procaccino F, Reinshagen M, Hoffmann P et al (1994) Protective effect of epidermal growth factor in an experimental model of colitis in rats. Gastroenterology 107: 12-17. Raaberg L, Nexo E, Damsgaard MJ et al (1988) Immunohistochemical localisation and developmental aspects of epidermal growth factor in the rat. Histochemistry 89: 351-356. Rackoff PJ, Zdon MJ, Tyshkov M et al (1988) Epidermal growth factor (EGF) inhibits both intrinsic factor secretion and acid secretion in histamine-stimulated isolated gastric glands. Regulatory Peptides 21: 279-287. Sandgren EP, Luetteke NC, Paliter RD et al (1990) Overexpression of TGFa in transgenic mice: induction of epithelial hyperplasia, pancreatic metaplasia and carcinoma of the breast. Cell 61: 1121-l 135. Scheving LA, Yeh YC, Tsai TH et al (1980) Circadian phase-dependent stimulatory effects of epidermal growth factor on deoxyribonucleic acid synthesis in the duodenum, jejunum, ileum, caecum, colon, and rectum of the adult male mouse. Endocrinology 106: 1498-1503. Scheving LA, Shiurba RA, Nguyen TD et al (1989) Epidermal growth factor receptor of the intestinal enterocyte. Localisation to laterobasal but not brush border membrane. Journal of Biological Chemistry 264: 1735-1741. Siegall CB, Xu YH, Chaudhary VK et al (1989) Cytotoxic activities of a fusion protein comprised of TGF alpha and Pseudomonas exotoxin. FASEB Journal 3: 2647-2652. Smith J, Cook E, Fotheringham I et al (1982) Chemical synthesis and cloning of a gene for human P-urogastrone. Nucleic Acid Research 10: 4467-4482. Sullivan PB, Brueton MJ, Tabara Z et al (1991) Epidermal growth factor in necrotising enteritis, Lancet 338: 53-54. Thompson JF (1988) Specific receptors for epidermal growth factor in rat intestinal microvillus membranes. American Journal of Physiology 254: G4294435. Thompson JS, Saxena SK, Greaton C et al (1989) The effect of the route of delivery of urogastrone on intestinal regeneration. Surgery 106: 45-5 1. Thompson JF, Lamprey RM & Stokkers PC (1993) Orogastric EGF enhances c-neu and EGF receptor phosphorylation in suckling rat jejunum in vivo. American Journal of Physiology 265: G63-G72. Tsujikawa T, Bamba T & Hosoda S (1990) The trophic effect of epidermal growth factor on morphological changes and polyamine metabolism in the small intestine of rats. Gastroenterology Japan 25: 328-334. Ulshen MH, Lyn CLE & Raasch RH (1986) Effects of intraluminal epidermal growth factor on mucosal proliferation in the small intestine of adult rats. Gastroenterology 91: 1134-l 140. Walker-Smith JA, Phillips AD, Walford N et al (1985) Intravenous epidermal growth factor/urogastrone increases small intestinal cell proliferation in congenital microvillous atrophy. Lancet 11: 1239-1240. Wan CW, McKnight MK, Brattain DE et al (1988) Different epidermal growth factor growth responses and receptor levels in human colon carcinoma cell lines. Cancer Letters 43: 139-143. Weaver LT & Walker WA (1988) Epidermal growth factor and the developing human gut. Gastroenterology 94: 845-847. Wright NA, Pike C & Elia G (1990) Induction of a novel epidermal growth factor-secreting cell lineage by mucosal ulceration in human gastrointestinal stem cells. Nature 343 (6253): 82-85. Yonemura Y, Sugiyama K, Fushida S et al (1991) Tissue status of epidermal growth factor and its receptor as an indicator of poor prognosis in patients with gastric cancer. Analytical Cell Pathology 3: 343-350. Yonemura Y, Takamura H, Ninomiya I et al (1992) Interrelationship between transforming growth factor-alpha and epidermal growth factor receptor in advanced gastric cancer. Oncology 49: 157-161.
growth factor (EGF)
ROBERT A. GOODLAD NICHOLAS A. WRIGHT Despite the wealth of information and detailed knowledge about the epidermal growth factor (EGF) gene and the structure of EGF and its receptor, its role in vivo is still not clear. EGF was first isolated from the submandibular salivary glands of mice and found to stimulate precocial eyelid opening and tooth eruption. EGF also stimulated the proliferation and maturation of several other tissues, including the epidermis, pulmonary epithelium and corneum and was shown to be a very potent mitogen in vitro (Carpenter, 1981). Human EGF was originally known as urogastrone, where it was identified by its mitogenic and anti-ulcer actions in the urine of pregnant women. EGF is formed as a pre-pro molecule of 1217 amino acid residues (Bell et al, 1986), which is processed by the salivary glands to EGF l-53, has a relative molecular mass of 6215 and includes six cysteine residues that form three disulphide bridges (Campbell et al, 1990). EGF’s location in the salivary glands and in duodenal Brunner’s glands (Heitz et al, 1978), and its presence in a variety of biological fluids, such as saliva, plasma and milk (Carpenter, 1980, 1981) all suggested that it had an important role to play in gastrointestinal homeostasis. EGF can stimulate the proliferation and maturation of the pre- and postnatal intestine (Malo and Menard, 1982; Weaver and Walker, 1988). Postnatally EGF is first found in Brunner’s glands, then in the Paneth cells, and finally in the submandibular glands (Raaberg et al, 1988). Interest in EGF was furthered by reports that saliva itself was trophic to the intestine (Li et al, 1983) and that EGF could have a ‘cytoprotective’ effect on the gut (Kirkegaard et al, 1983). An additional action of EGF was to inhibit gastric acid secretion (Bower et al, 1975; Koffman et al, 1982). Thus EGF, like many growth factors has more than one action. Molecular biological techniques have been used to produce recombinant synthetic EGF (Smith et al, 1982), which has an identical structure to the natural EGF so enabling the detailed study of its biological effects (Gregory, 1985). Several ligands bind to the EGF receptor, and it may therefore be better to talk of the EGF family of peptides rather than of EGF itself. Furthermore, it is possible, and also quite likely, that some of the effects seen after administration of EGF may actually be mimicking the natural actions of other members of this extended family. For Bailli&-e’s Clinical GastroenterologyVol. 10, No. 1, March 1996 ISBN O-7020-2002-8 0950-3528/96/010033 + 15 $12.00/00
33 Copyright 0 1996, by Baillike All rights of reproduction in any form
Tindall reserved
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example, the distribution of transcripts for EGF and transforming growth factor-a (TGF-a) in the intestine led to the suggestion that the true ligand of the EGF receptor in the intestine is TGF-a (Koyama and Podolsky, 1989).
THE EGF RECEPTOR The EGF receptor (EGFR) was the first receptor protein identified as a tyrosine specific protein kinase, and is a ‘single pass’ transmembrane protein of around 1200 amino acid residues. The EGFR has a large glycosylated extracellular domain that binds EGF and (eventually) leads to the activation of an intracellular tyrosine kinase domain, which in turn transfers phosphate from APT on selected tyrosine side-chains (both on the receptor and on specific cellular proteins). Over 50 such receptor tyrosine kinases, belonging to 14 receptor families, have now been identified (Lemnon and Schlessinger, 1994). The importance of this signalling system is demonstrated by its being purloined by the proto-oncogene c-e&B, which codes for the EGFR. Mutation can delete the extracellular domain and turn c-&B into a tumour producing oncogene that constantly produces an intracellular proliferative signal. The question of how this signalling is transmitted via a single transmembrane protein was answered by the finding that receptors can dimerize after ligand binding, thus allowing the internal domains to cross-phosphorylate (or autophosphorylate). These can then act as ‘docking sites’ to set off a signalling cascade (Alberts et al, 1994); see Figure 2 in the Introduction to this issue. After binding the receptors accumulate in coated pits, and rather than be recycled like some groups of receptor, most EGFRs are degraded in the lysosomes, thus receptor binding leads to decreased receptor concentration (receptor downregulation). EGF is not the only ligand for the EGFR, as TGF-a, amphiregulin, heparin-binding EGF-like growth factor and betacellulin all bind to the receptor. Furthermore, there is homology with at least three viral proteins including vaccinia virus growth factors (VVGF: Burgess, 1989). TGF-a is found in high levels throughout the gut in the adult (and in even greater quantities in the juvenile) and transcripts for TGF-a but not EGF are found in intestinal cells. Furthermore, it has been reported that the intestine of a 19-week-old human foetus contained 10 times more EGF receptor-binding substance than EGF (Miettinen et al, 1989). The above data has led to the suggestion that the true ligand for the EGFR may be TGF-a (Koyama and Podolsky, 1989). TGF-a over-expression is associated with intestinal hyperplasia (Sandgren et al, 1990) and TGF-a is also trophic to the intestine of parenterally fed rats (Goodlad et al, 1990) but to a lesser extent (Goodlad et al, 1990; Sandgren et al, 1990). The similarities and differences in the response of the gut to EGF and TGF-a have recently been extensively reviewed (Barnard et al, 1995), and this led to the advancement of the hypothesis that while the two peptides bind the same receptor with equal affinity, they nevertheless bind differently, and in
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addition, there is a morphological compartmentalization of the two peptides. One consequence of this multiple ligand binding is that there is a considerable degree of redundancy in the EGF family, which permits substitution should one factor be missing or have been ‘knocked out’. Recent reports of an EGFR knock out mouse confirm the body’s ability to function without a major growth factor receptor, as the mice can survive, albeit briefly, for about a week after birth, but they then succumb to respiratory distress or to a necrotising enterocolitis-like syndrome (Miettinen et al, 1995). ACTIONS
OF EGF
Cytoprotection Early reports indicated that topical EGF was ‘cytoprotective’ as a result of a mechanism independent of gastric secretion inhibition or prostaglandin formation, but related to the increase in DNA synthesis in oxyntic mucosa (Konturek et al, 1981). The trophic effects of EGF will be discussed later. The submandibular glands influence healing of chronic gastric ulcers and this has led to the suggestion that EGF participates in healing of chronic gastric ulcers (Olsen et al, 1986). Lower levels of salivary EGF immunoreactivity have been reported in patients with a peptic ulcer (Ohmura et al, 1987). Recent studies have also suggested that the systemic EGF may also reduce mucosal damage and inflammation in a model of colitis (Procaccino et al, 1994). Some of these protective mechanisms may be through the stimulation of goblet cells to release mucus (Ishikawa et al, 1994), and further cytoprotective effects may be attributable to increased gastric blood flow (Hui et al, 1993). Acid inhibition EGF, like other trophic agents for the gut such as enteroglucagon (Goodlad and Wright, 1990) can inhibit gastric acid (and intrinsic factor) secretion (Rackoff et al, 1988). There is some evidence that this occurs by way of a separate mechanism to the mitogenic one (Dembinski et al, 1982), perhaps by different signal transducing mechanisms (Gamer, 1992). Furthermore, EGF can stimulate the transcription of gas&in, by a gastrin response element in the gash-in promoter (Merchant et al, 1991). There is also evidence that EGF may play a role in the maintenance of parietal cell function at much lower concentrations than those required for inhibition of acid secretion, indicating that EGF modulates parietal cell function by multiple signalling pathways (Chew et al, 1994). Trophic
effects
Early reports on the actions of EGF in vivo were somewhat confusing, as several studies were contradictory. Some groups found increased DNA
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synthesis in the gastric mucosa, but not the duodenal and colonic mucosa (Johnson and Guthrie, 1980), while others reported that the actions of EGF were more dramatic in the caecum, colon and rectum than in the small intestine (Scheving et al, 1980). Al-Nafussi and Wright (1982) found that EGF was trophic to the duodenum, jejunum, ileum and colon but not the stomach, and that the response differed at different times after the injection. Other groups only found trophic effects in starved (Dembinski et al, 1982) or undernourished (Majumdar, 1984) animals. Some of these above differences can be attributed to the use of inappropriate assays, others most likely reflect the use of different animal models, and we will briefly discuss these essential points. Proliferative
assays
While there are several published methods for the study of intestinal epithelial cell proliferation, many of these have serious flaws, especially those based on the gross uptake of tritiated thymidine (Goodlad and Wright, 1982; Maurer, 1981), and even the more acceptable methods can be misleading if the three-dimensional nature of the gut is not considered. We strongly advocate the use of a stathmokinetic drug, such as vincristine, to quantify the rate of entry of arrested metaphase cells into mitosis, which defines the crypt cell production rate (CCPR). This is the most productive and robust method for the study of gastrointestinal cell proliferation in animals. A great advantage of the technique is that results can be expressed on a per crypt basis and thus avoids the several pitfalls associated with scoring crypt sections. Moreover using the crypt as the denominator means that there is no need to count non-dividing cells. The technique, unlike almost all others, also accounts for all the factors involved in cell renewal, i.e. phase duration, growth fraction and compartment size (Goodlad, 1994). If more detailed information on the mechanisms underlying altered cell kinetics is required, the study of the distribution of DNA synthesizing (S-phase) cells, using tritiated thymidine and/or bromodeoxyuridine can yield valuable information, providing that the interpretation takes into account the three-dimensional nature of the crypts (Goodlad, 1992). For human studies, ethical considerations prevent the use of metaphase arrest or nucleotide labelling, nevertheless, the number of native mitoses per (microdissected) crypt can give valid results (Goodlad et al, 1991a). This method is superior in many ways to the several in vitro methods available for the assessment of biopsies. Many groups favour the use of antibody-based techniques to locate dividing cells, but these can also have several problems as the reaction products are difficult to standardize and antigens can be expressed anomalously, for example, expression of the so-called proliferating cell nuclear antigen (PCNA) can occur without any concomitant changes in proliferation (Hall et al, 1994). Finally, all section-based techniques cannot escape the many problems associated with studying three-dimensional structures in two dimensions.
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models
Many groups have used starved animals, presumably to reduce the effects of endogenous secretions, maximize the response of the gut and ‘standardize’ the animals. However, starved rodents are not in a steady state of cell renewal. A better model for the study of potential proliferative agents on the gastrointestinal tract is seen in the hypoplastic intestine of rats maintained by total parenteral nutrition (TPN). The intestine of TPN rats is in a steady state of basal cell proliferation, as are the endogenous secretions and gut hormones. Furthermore, the several variables associated with altered food intake and food intake patterns are removed. The TPN model has the further advantage that bolus or continuous administration of the agent can readily be achieved, and if a two-channel system is used, intra-intestinal infusion is also practical (Figure 1). The cannulae exit through flexible
Figure 1. The total parenteralnutrition(TPN)rat.
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tethers and special fluid swivel joints, so that the rat is fully conscious and relatively unrestrained. In vivo studies (animal)
The TPN rat model has shown that recombinant human EGF, at doses that should not inhibit gastric acid secretion, is a very potent stimulator of intestinal epithelial cell proliferation (Goodlad et al, 1985; Bragg et al, 1990). This trophic effect occurred throughout the gastrointestinal tract, but was especially prominent in the stomach and colon (Goodlad et al, 1987). Higher doses of EGF increased proliferation in the stomach by over four times, by four and a half times in the colon and doubled proliferation in the small intestine (Figure 2). Whilst EGF was very effective when given intravenously, it had no effect, even at massive doses, if given intragastrically. These proliferative effects of EGF were not associated with increased plasma levels of the hormone enteroglucagon, which has often been linked (albeit by circumstantial evidence) with increased proliferation in the gut, indicating that the two agents probably exert their proliferative effects through separate mechanisms (Goodlad et al, 1989a,b). There are several means by which a crypt can increase its rates of cell production, namely by altering the cell cycle time, increasing the proportion of the crypt actively involved in proliferation (the growth fraction) or increasing the size of the crypt. We have shown that the main effect of EGF is to increase the size of the crypt, especially its length. One outcome of this is that few differences in proliferative indices were seen between the orally fed and TPN groups, despite the large difference in cell production and tissue mass, this being the consequence of the concomitant changes in crypt population, as proliferative indices are the ratio of dividing cells to total cell population. The number of mitoses and labelled cells/crypt, and thus the crypt cell production rates, were therefore all significantly decreased in the TPN group when compared to the orally fed. EGF increased both the proliferative indices and the number of dividing cells/crypt. The growth fraction was actually reduced in the EGF-treated group, which was a consequence of the very large increase in crypt length (Goodlad et al, 1992). In vivo studies (human)
While there have been several studies demonstrating the trophic effects of EGF on human explants (Menard et al, 1988), and on acid secretion (Gregory, 1985) and even some estimations of the crypt cell production rate in cultured explants, using a stathmokinetic technique and crypt microdissection (Challacombe and Wheeler, 1991), there have been few published reports on its action in the human in vivo. The first use of EGF to try and stimulate epithelial renewal was in a neonate with congenital microvillus atrophy. That study established that EGF was also a very potent mitogen in the human intestine (Walker-Smith et al, 1985). This finding was later confirmed (Drumm et al, 1988).
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GROWTH FACTOR (EGF)
Stomach 600, 500 400, 300-
/
'A
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Dose EGF @g/rat/day) Figure 2. Epidermal growth factor (EGF) dose responses. SI, small intestine.
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However, the defect in congenital microvillus atrophy is a structural one in the glycocalyx and although stimulating proliferation was palliative, it was not curative. A second use of EGF in extremis was in a moribund baby with necrotising enteritis (Sullivan et al, 1991). In this case EGF administration was associated with significantly altered proliferation, increased mitotic activity (and decreased crypt branching). Fortunately, these actions were also associated with a highly favourable clinical outcome (Cooper, 1992). Polyamines EGF can induce the first, rate-limiting, enzyme involved in the synthesis of polyamines, ornithine decarboxylase (Feldman et al, 1978). Ornithine decarboxylase activities in the small bowel mucosa rose within 12 hours of EGF administration in the atrophic small intestine of rats fed an elemental diet, indicating that EGF may stimulate mucosal proliferation through polyamine metabolism (Tsujikawa et al, 1990). This was substantiated by our finding that difluoromethylornithine, an inhibitor of the enzyme ornithine decarboxylase, significantly reduced the proliferative effect of EGF in the stomach, small intestine and colon, but the inhibition in the colon was less pronounced (Goodlad et al, 1989a). Although polyamines are involved, and are probably essential for effecting the proliferative response, there is still doubt as to whether they represent control points. Enzyme activity The effects of EGF on the enzyme activities of rats at different stages of development are somewhat complicated, depending on the specific enzyme concerned, the site within the small intestine and the timing of the treatment (Bradbury et al, 1992). In the adult animal EGF prevented the decrease in brush border enzyme activity seen in the TPN rat, but the response differed depending on the intestinal location, with the specific activities of some enzymes being higher than those seen in orally fed rats. Some of the variability can be attributed to the old problem of choosing the right denominator with which to express results, as if EGF is increasing both tissue mass and activity, specific activities can be misleading. It may well be better to express results per gut or per centimetre of gut. Nevertheless, there is no doubt that EGF can increase the functional capacity of the intestine by mechanisms separate from its trophic effect (Bragg et al, 1990; Goodlad et al, 1991b). EGF and cancer There is much interest on the autocrine stimulation of gastrointestinal carcinoma growth. Elevated levels of EGFR, EGF or related mRNAs, have been demonstrated in many gastric carcinomas. Human gastric cancer xenografts grown in nude mice increased their growth following EGF infusion and their growth was inhibited by sialoadenectomy (Okuda et al, 1994). The growth of certain gastrointestinal cell lines can be inhibited by
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antibodies against EGF, and by antisense oligonucleotides based on EGF mRNA (Baldwin and Whitehead, 1994). EGF or TGF-a and EGFR staining positively can be inversely related to prognosis in gastric cancers, implying that the autocrine mechanism between EGFITGF-a and EGFR may play an important role in the progression of gastric cancer, and that when such a mechanism becomes operative, prognosis may be poor (Yonemura et al, 1991, 1992). High levels of EGFR expression have been reported in a variety of cancer cell lines including liver, ovarian and colon (Siegall et al, 1989), nevertheless, others have reported lower EGFR levels in the carcinomatous colon (Koenders et al, 1992). This may perhaps reflect the extent of differentiation, as aggressively growing and poorly differentiated colon cells may not respond to EGF alone, while less aggressively growing and more differentiated cells respond with increased growth, so that downregulation of EGF-R may occur in aggressive colon tumour cells (Wan et al, 1988). While the involvement of the EGFR in these tumours suggests various therapeutic possibilities, the extent of ligand redundancy in the EGF family has led some authors to conclude that therapies aimed at blocking the action of a single factor is unlikely to influence the growth of most tumours (Garner, 1992). An interesting possibility has been raised by a few reports of prolongation of survival time by EGF, which may be the result of EGF inhibition of tumour cell metastasis (Amagase et al, 1990). The ulcer-associated
cell lineage
In several conditions where endodermal damage occurs, a newly recognized cell lineage, the ulcer-associated cell lineage (UACL), can develop (Wright et al, 1990). The UACL is seen associated with peptic ulceration, ulcerative colitis and Crohn’s disease. The phenotype of the cells of this lineage varies depending upon the cell’s position, expressing various peptides such as hSP, pS2 (see Chapter 8) and EGF, producing neutral mucin, and showing a unique lectin-binding profile and immunophenotype. The lineage also stains intensely with the antibody to TGF-a. This is of interest because the TGF-a gene can be induced by EGF (Miettinen, 1993). The lineage grows from the bases of existing crypts to open through a pore onto the surface. All gastrointestinal stem cells seem able to produce this lineage following mucosal ulceration. The secretion of EGF (and the other peptides) should then be able to stimulate cell proliferation, regeneration and healing. The EGF paradox It is of interest that we found no effect of large doses of EGF when given luminally (Goodlad et al, 1987). However, others have reported luminal effects in the small bowel (Ulshen et al, 1986) or in the colon (Polack et al, 1987). O’Loughlin et al (1994) reported that oral EGF treatment in resected rats did not alter mucosal proliferation but did stimulate maltase specific activity and caused a three to four-fold increase in glucose transport and
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phlorizin binding, which reflects the density of Na+ dependent glucose carriers. Dembinski et al (1982) found that EGF infused subcutaneously, but not intragastrically, inhibited spontaneous gastric acid and pepsin secretion. EGF injected intraperitoneally at 8 hourly intervals for 24 hours significantly stimulated DNA synthesis in the gastroduodenal mucosa and the pancreas, whereas when administered intragastrically it stimulated DNA synthesis only in the gastroduodenal mucosa but not in the pancreas. Ulshen et al (1986) found that intraluminal administration of EGF stimulated a mucosal proliferative response in the small intestine. It is of interest that they infused EGF into the ileum, but found a response in both the ileum and the jejunum (Ulshen et al, 1986), which implies a systemic mechanism. Thompson et al (1989) used implanted minipumps to investigate several routes of EGF delivery, and found that the greatest response was with intravenous EGF followed by subcutaneous, and then intraluminal EGF; these authors did, however, use a model of renewal (neomucosal patches) which can be regarded as damaged. Bamba et al (1993), working on the hypothesis that the EGFR was on the microvillous membrane of the enterocytes, investigated various routes of administration and were surprised to find that intraperitoneal not intraluminal EGF stimulated growth. With regard to the cytoprotection story there is also some debate, but several groups have found that oral EGF has no biological activity on the pathophysiology of the stomach (Kuwahara et al, 1990). Some of these discrepancies could be explained by the hypothesis that luminal EGF is only effective if the mucosa is damaged, and luminal EGF can indeed be protective in such models (Ishikawa et al, 1994). Another hypothesis concerns the question as to what form of EGF is present. A derivative of human epidermal growth factor, EGF148, has the same acid inhibitory actions as the full molecule (l-53) (Guglietta and Lesch, 1993), leading to the suggestion that this was the biologically active form. Recent work, however, has suggested that 148 is less active as a mitogen (Goodlad et al, unpublished results). Previous studies had indicated that EGF was stable in acid/pepsin (B&ton et al, 1988). These studies used size exclusion chromatography to determine stability, which is not a very precise technique. More recent work has suggested that the C-terminal five amino acid residues of EGF can be cleaved in acid/pepsin and that a relatively small change in this region has a marked effect on receptor binding (Araki et al, 1989). EGF has been shown to be digested to EGF1-49 and EGF1-46 by pepsin, but only when the pH was below pH 4, these forms of EGF had only 25% of the biological activity of native EGF (Playford et al, 1995). While EGF is digested by pancreatic enzymes in fasting intestinal juice, it may be preserved when food proteins block the active sites of these enzymes (Playford et al, 1993). These studies have raised the possibility that the addition of enzymeinhibiting proteins, or alternative substrates, such as casein, may preserve intestinal integrity and function. This would tie in well with our previous results, which have shown that systemic but not oral EGF causes intestinal proliferation in parenterally fed
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animals (Goodlad et al, 1987), as the virtually empty gut of the TPN rat would contain very few alternative substrates for acid and pepsin, so that EGF would probably be destroyed. The preservation of EGF in achlorhydric stomachs raised the intriguing suggestion that the mode of action of acid suppressant drugs may include the preservation of luminal EGF (Playford et al, 1995), and reduced EGF levels have been observed in the stomachs of peptic ulcer patients (Maccini and Veit, 1990). Nevertheless, EGF cannot be the total answer as antacids are still effective in ulcer models in sialoadenectomised rats (Konturek et al, 1990). An alternative explanation for our lack of response to oral EGF is that EGF may need to be present continuously for a mitogenic response, rather than, as in our previous study, given as a bolus three times a day. Our most recent studies using a two-channel swivel joint-TPN system have in fact shown a direct trophic response to luminal EGF, but only in the duodenum, not on the more distal regions of the bowel, probably reflecting rapid proteolysis of the EGF by luminal proteases. In addition there was also evidence for EGF effects in the more distal sites using soya bean trypsin inhibitor to protect the luminal EGF (Marchbank et al, 1995). A third factor to consider in terms of luminal EGF action concerns the continuing debate as to whether the enterocytes have apical (luminal) receptors. One well-cited report found the EGFR on the microvillus membrane and, thus, suggested a mechanism for the intraluminal regulation of enterocyte proliferation (Thompson, 1988). However, other groups only found the EGFRs to be basolateral (Scheving et al, 1989) or on both microvillar and basolateral surfaces of enterocytes (Kelly et al, 1992). Caco-2 cell monolayers bound EGF to the basolateral membrane with two to three times more affinity than the apical membrane (Hidalgo et al, 1989). On the other hand autoradiographical studies found no binding sites on the brush border of human fetal gut. In addition, the greatest concentration of EGF binding sites was found in regions of high proliferative activity suggesting that, under normal circumstances, systemic but not luminal EGF has free access to its specific receptor (Menard and Pothier, 1991). A similar basolateral location of EGFK was reported for the parietal cell (Mori et al, 1987). Later work by Thompson et al (1993) using more sensitive techniques, demonstrated that while intraluminal EGF stimulates rapid tyrosine phosphorylation of the EGF receptor in vivo, the localization of the EGF receptor was on the basolateral membrane suggesting that EGF is rapidly transported across the intestinal epithelium. SUMMARY Despite the wealth of information concerning EGF and its related peptides, its precise role in the control of gastrointestinal functions is still not fully resolved. However, there is no doubt that it can have some very potent effects on the gastrointestinal tract. These may be related to the control of growth and development and to the regular control of cell renewal. Nevertheless, in the adult, EGF may only be active in response to luminal
44
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damage and repair, and furthermore this may also only occur if the luminal EGF is protected from proteolytic degradation. Notwithstanding this, ‘EGF’-like responses may be evoked in the gut by intestinal TGF-a. The possible therapeutic use of EGF and members of its family in ulcer therapy will be discussed in later Chapters of this volume, other potential uses are in the control of necrotising enteritis and in the alleviation of the mucositis associated with cancer treatment.
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