[Frontiers in Bioscience 4, d87-101, January 15, 1999]

HOME
Current Issue
VITAL LINKS
INDEX
PDF FILE
ACCESS PUBMED
REPRINTS
CAVEAT LECTOR

Received: 9/30/98
Accepted: 12/18/98

Send correspondence to:

Daniel Ménard, Ph.D.,
Professor of Cell Biology,
Département d’anatomie et de biologie cellulaire,
Faculté de médecine,
Université de Sherbrooke,
Sherbrooke (Quebec) Canada J1H 5N4

Tel: +1 819-564-5278,
Fax:+1 819-564-5320,
E-mail: dmenard@courrier. usherb.ca

KEY WORDS

EGF receptor; EGF binding; Human fetal gut; Intestinal mucosa; Gastric mucosa; Cell proliferation; Brush border hydrolases; Gastric enzymes

SEARCH FBS

Copyright © Frontiers in Bioscience, 1995

ONTOGENY OF EGF RECEPTORS IN THE HUMAN GUT

Pierre Chailler and Daniel Ménard

Groupe du CRM sur le Développement Fonctionnel et la Physiopathologie du Tube Digestif, Département d’anatomie et de biologie cellulaire, Faculté de médecine, Université de Sherbrooke, Sherbrooke (Québec) Canada

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Identification and biochemistry of EGF receptor
3.1. Gene and protein structure
3.2. Mechanism of activation
3.3. General distribution
3.4. Ligands
4. Expression in human fetal gut segments
4.1. Small intestine
4.2. Colon
4.3. Stomach
5. Comparison with animal models
6. Integration and perspectives
7. Acknowledgements
8. References

1. ABSTRACT

Epidermal growth factor and related substances mediate their effects on epithelial cells through binding to high-affinity receptors (EGF-R) at their basolateral surface and it is hypothesized that this growth factor system play a major role in gut morphogenesis and maintenance. The current review emphasizes on analyzing the expression and the biochemical characteristics of EGF-R in human fetal gut segments and correlating the biological actions of EGF-R ligands. They appear to be primarily involved in the local regulation of epithelial cell proliferation in which EGF-R are abundant. Alternatively, EGF-R ligands exert some precocious maturative effects by increasing intestinal lactase activity and decreasing brush border hydrolases in colon while they down modulate the expression of segment-specific markers of terminal differentiation such as sucrase, trehalase and glucoamylase in the intestine and chief cell lipase in the stomach. Such effects are consistent with the identification of receptors at the surface of all epithelial cell types, illustrating the modulatory role of EGF on differentiated gut epithelial cells. Comparison with animal models illustrates similar biochemical properties of receptors and underlines physiological aspects specific to human gut development. The relevance for ligand heterogeneity is also discussed and tentatively associated with different delivery pathways or physiological responses.

2. INTRODUCTION

Epidermal growth factor or EGF is one of the best characterized growth factors that was initially isolated by Stanley Cohen as a contaminating material in nerve growth factor preparations promoting the growth of embryonic neurons (1,2). Early on, it was found to be abundant in some body fluids i.e. saliva, serum, milk and urine (3-6). Accordingly, radioimmunoassay analysis in conjunction with immunohistochemical studies demonstrated that the submaxillary salivary glands, duodenal Brunner's glands, mammary glands and metanephric kidneys represented major sources of the EGF peptide (7-9). EGF-related material (transforming growth factor-alpha or TGFalpha) was also extracted from the amniotic fluid (0.25-4.3 ng/ml; refs 9-11) and found to be produced by extraembryonic membranes (trophoectoderm, placenta) in animals and humans (12-15) although a fraction of this material is likely to derive from developing kidneys (9). Taken altogether, these findings lead to the hypothesis that EGF or related forms released from these sources could be involved in embryonic development and gut morphogenesis or the maintenance of gut tissues in the adult. A major breakthrough was then achieved when EGF mRNA transcripts and EGF-like substance/ activity were detected in lower amounts in various tissues (16-18). Thus, it was proposed that the growth factor could act in vivo as a local paracrine factor in addition to its role as a long-distance acting hormone.

Concerning the characterization of the biological action of EGF in tissues, pioneering studies (19,20) using subcutaneous injection protocols in neonatal mice helped to recognize the maturing epidermis and its appendages as important targets of EGF action. It was initially observed that the factor accelerated eyelid opening and incisor eruption (thus accounting for the 'tooth lid factor' denomination used in the early 1960's). Ulterior experiments showed that EGF was also able to stimulate hair growth and epidermal cell proliferation and keratinization (21). The mitogenic effect of EGF was then rigourously studied in vitro on fibroblasts (22-25) where the predicted presence of EGF surface receptors was verified for the first time using iodinated (125I)EGF binding techniques (24,26). As will be discussed further, the presence of surface binding sites was also demonstrated in mesodermal and endodermal derivatives, especially in gut epithelial compartments, and the gene encoding the EGF-R protein in rodents and man was successfully cloned and sequenced (see reviews 27-29).

In 1983, an oncogenic protein, termed v-erbB1, was identified as a product of avian erythroblastosis virus (30) which causes the abnormal presence of immature blood cells (erythroblasts) into systemic circulation in chicken. Viral erbB1 in fact represents a truncated and constitutively-active form of the EGF-R (30); a cellular homolog oncogene named c-erbB1 was identified in embryonic and tumor cells in rodents (31), and then found to be identical to the mouse EGF-R. Such discoveries and biochemical/immunological data from pathological studies permitted to establish a functional relationship between the expression of EGF-R and the initiation of cancer, as proposed for the gut in particular, where the relative abundance of EGF-R serves as a prognostic factor in esophageal, gastric and colon tumorigenesis (32-34). Enhanced expression of EGF-R may also be a marker for the increased risk of colon cancer in inflammatory bowel disease (35).

In the following years, the expression pattern, the mechanism of activation, the recycling through the endocytic pathway as well as the involvement of EGF-R in the biological action of EGF were extensively studied in normal tissues and cultured cells originating from animals and man. As examplified in basic reviews (2,36-40) integrating all aspects of EGF/EGF-R biochemistry and physiology, the expression and function of this growth factor system seem to be primarily associated with the involvement of EGF as a competence growth factor stimulating the survival, the migration and the mitosis of stem cells and pre-differentiated cells, including those of the gastrointestinal tract giving rise to mature epithelial cells. The more recent development of gene knock-out and promoter engineering strategies which enable the generation of mouse lineages carrying either EGF/TGFalpha/EGF-R genes inactivated by defective mutations or genes which expression is forced by an active promoter (mutations integrated in the whole organism or targeted to specific organs) indeed supports the latter concept. Overexpression of the TGFalpha transgene (41-46) primarily alters epithelial proliferative compartments and ultimately leads to a desequilibrium between pre-differentiated and differentiated cell populations: hyperplasia and rarefication of differentiated phenotypes. Alternatively, null mutations of TGFalpha/EGF-R genes (47-50) reveal that they exert a complex function since their deficiencies not only modify cell proliferation but perturb a wide range of developmental activities (branching morphogenesis, epithelial cell differentiation or maturation, skin architecture). In accordance with the latter observations, other pioneering studies suggest that EGF itself or EGF acting in synergism with other growth factors plays a role in the differentiation of specific cell species such as keratinocytes (as mentioned above; ref.19-21), palatal cells (51), surfactant-secreting pneumocytes (52,53), cervical cells (54), nasal epithelial cells (55), ameloblasts/odontoblasts (56), trophoblasts (57) as well as enterocytes (58-60). These observations jointly reinforces the physiological importance of EGF and EGF-related molecules as cell inducers and stresses the complexity of regulatory processes involved in organogenesis and tissue maintenance. In this context, a growth factor system may play an epigenetic role by stimulating distinct functions in different cell types at specific developmental stages when the ordered sequence of survival, proliferation and differentiation is normally progressing. The following sections describe the general properties of the EGF-R as well as its expression pattern and biological significance in specific segments of the human fetal gastrointestinal tract.

3. IDENTIFICATION AND BIOCHEMISTRY OF EGF RECEPTOR

3.1. Gene and protein structure

The nucleotide sequence of the EGF-R, which is termed HER-1 in human (61), predicts a 1186-1210 aa backbone with a molecular weight of 134-135 kilodaltons (kD)(see reviews 36,38). The mature polypeptide is glycosylated at 11-12 asparagine residues, its molecular mass is estimated to ~170 kilodaltons in various tissues and it is characterized by intrinsic tyrosine kinase activity (62-65). In addition, the extracellular region contains 51 cysteine residues which are concentrated in two domains that cooperate to form a high affinity EGF binding site (66,67; see figure 1). Drosophila and Caenorhabditis EGF-R homologs contain the same functional domains (68,69) thus revealing a high degree of evolutionary conservation. Although a single human EGF-R gene exists, two spliced variants were revealed in A431 cells (64,70,71) which served in many laboratories as a model for studying EGF-R expression and subcellular trafficking. The physiological significance of this process remains uncertain however since this epidermoid carcinoma cell line produces aberrant forms of the receptor which are absent in nonmalignant tissues (64,72,73). Only a few data concerning the possible existence of alternative splicing mechanism in human carcinomas (71) and developing kidney (74) were obtained in the past. Presumably, this subject has not retained much attention because the respective contributions of 10- and 5.6-kb transcripts for protein expression have not been examined. Nonetheless the recent discovery of a new spliced mRNA species in human placenta with a lower number of base pairs (75) may lead the way to a new comprehension of post-transcriptional regulatory processes controlling the tertiary conformation and function of the EGF-R.

Figure 1. Tertiary structure and consensus motifs of the EGF-R and its common ligands, EGF, TGFalpha and HB-EGF. The extracellular portion of the receptor contains two domains enriched in cysteine residues (black bars). The intracellular portion contains 1) a catalytic domain with tyrosine kinase activity and 2) several phosphorylation sites at threonine (T) and tyrosine (Y) residues.

3.2. Mechanism of activation

Early studies have demonstrated that EGF binding to the extracellular domain of EGF-R stimulates its tyrosine kinase activity causing increased phosphorylation of protein substrates as well as autophosphorylation of tyrosine residues near the C-terminus region (76). Moreover, threonine phosphorylation of the EGF-R by protein kinase C reduces its binding affinity and negatively regulates tyrosine kinase activity (77). Indeed the functionality of EGF-R varies according to the cell status; high- and low-affinity binding sites were identified in many cell types (78,79) that could be the result of post-translational modifications (phosphorylation, interaction with other proteins)(80,81).

Biochemical data showed in the past that an orderly sequence of receptor dimerization, clustering and internalization is normally activated following EGF binding (82) allowing the cellular response and the endocytic recycling of EGF/EGF-R complexes, as for many receptor systems. In fact, the physical aggregation of multiple EGF/EGF-R complexes at the level of the plasma membrane appears to be required for subsequent activation of tyrosine kinase activity (83,84). Numerous studies have demonstrated over the years that the latter process leads 1) to activation of intracellular effectors associated to EGF-R and which are organized into multimeric complexes (src, ras, phospholipase C-g , phosphatidylinositol 3-kinase, etc.) and 2) to various nuclear events required for mitogenesis, transformation and cell differentiation (reviewed in 85,86). Recent data even suggest that the receptor aggregation process as well as the optimal phosphorylation of associated effectors are operated through a cooperation with the integrin/matrix apparatus (87). This assumption remains to be verified specifically for the EGF/EGF-R system in the gut.

3.3. General distribution

Putative EGF-R were initially identified and characterized using 125I-EGF binding techniques in a wide range of cell types. Radioautographic data soon established that they are preferentially localized on the plasma membrane of epithelial cells, and at their basolateral pole (reviews 36-40). Moreover their number is high in proliferative cells, neoplastic cells and carcinoma cell lines (27,88). In A431 cells for example, EGF-R are overexpressed by 20 to 50-fold compared to normal tissues (27,64,72). Using the same strategy, receptors were successfully detected in embryonic/fetal and extraembryonic tissues of rodents (36,38,40) where they generally increase in number during gestation while their affinity for the ligand EGF somewhat decreases (89). Also, when fetal organs are explanted and cultured with EGF, the incorporation of (3H)thymidine into DNA is usually stimulated (90).

Then with the advance of immunological techniques, anti-EGF-R antibodies were produced that helped to confirm the precise localization of the protein at the tissular and subcellular levels. The general distribution of EGF-R in human fetal tissues (91-93) was found to be quite similar, as expected. In comparison with rodents however, the number of receptors tend to decrease before birth probably reflecting the more advanced degree of organ maturation that is achieved in utero. One interesting new discovery made on cultured isolated cells pertains to the concentration of EGF-R and associated effectors of signal transduction into adhesion plaques or cytoskeleton-membrane focal adhesions (94,95).

3.4. Ligands

EGF is a 53 amino acid polypeptide, with a molecular weight of 6045 daltons, which possesses three intrachain disulfide bonds. It is the prototypical member of a family of growth factors that also includes human pancreatic secretory trypsin inhibitor (96), transforming growth factor-alpha (TGFalpha)(97,98), vaccinia growth factor (99,100), schwannoma-derived growth factor (SDGF) or amphiregulin (AR) (101,102), betacellulin (103), cripto-1 (CR-1)(104), heparin-binding EGF-like factor (HB-EGF)(105), the neu differentiation factor (NDF)(106), the heregulins (107), and two homeotic gene products, the drosophila Notch protein (108) and the lin-12 protein of the nematode Caenorhabditis elegans (109). EGF-related molecules derive from large membrane-bound precursors which are released by proteolytic cleavage (110). PreproEGF, for example, comprises 1217 aa, seven EGF-like repeats in addition to the mature EGF coding region (111,112), and a portion of the molecule exhibits limited homology with the low density lipoprotein (LDL) receptor, suggesting that the EGF precursor and the LDL receptor descended from a common ancestral transmembrane protein (113,114). TGFalpha derives from a shorter 160 aa precursor (115) and it exhibits 33-44% sequence homology with mouse or human EGF (12 residues are strictly conserved). Of interest, its discovery in the culture medium of established cell lines contributed to elaborate the autocrine hypothesis of cell growth control (116,117). In comparison, HB-EGF is characterized by a longer N-terminal arm with a specific structural motif that confers sensitivity to sulfated glycosaminoglycans and proteoglycans (118). All three peptides represent potent agonists of the EGF-R in several normal (non transformed) tissues and cell lines.

4. EXPRESSION IN HUMAN FETAL GUT SEGMENTS

4.1. Small intestine

The presence of EGF-R in human fetal small intestine was demonstrated for the first time in 1988 by binding measurements (119). In this study, classical 125I-EGF binding procedures were performed at 22° C for 45 minutes on epithelial cell preparations that were obtained by manual shaking (in EDTA/NaCl solution) of everted segments of small intestine. For Scatchard analysis, cells were incubated with the iodinated ligand plus increasing concentrations of unlabelled EGF(10-11 to 10-7 M) and non-linear plots were analyzed according to the two-site model. The pattern of 125I- EGF binding between 12 and 17 weeks (postfertilization) shows that binding was significantly higher (2.5-fold) in 12 to 14-week fetuses than in older fetuses, as illustrated in figure 2. In percentage terms, specific binding in youger fetuses represented more than 16.5% of labeled EGF/mg cell protein whereas 17-week-old specimens bound less than 6%. Low- and high-affinity binding sites were identified with association constants of K1 = 1.90 ± 0.45 x 10-9M-1 and K2 = 0.033 ± 0.016 x 10-9M-1 respectively. This binding was also specific; no cross-competition for EGF binding sites was observed with insulin, ACTH, thyroxine, hydrocortisone, IGF-1, IGF-2 or dexamethasone when they were added in excess concentration together with EGF. Quantitative autoradiography of 125I-EGF binding (120) reveals extensive accumulation in undifferentiated cells of the crypt and at the base of the villus, as well as in the inner circular layer of the muscularis externa bordering the submucosa (some labeling was detected in mesenchymal and vascular elements of the lamina propria). A gradient of silver grain density was clearly established along the crypt-villus axis towards the regions of high proliferative activity. Epithelial cells in the deep portion of the crypt showed the highest density (9.2 grains/m m2), which gradually decreased in the upper crypt (6.5) and the lower villus (3.9), with very little labeling in the upper third of the villus (0.4). The cellular distribution of silver grains in epithelial cells of the lower villus revealed a polarization of labeling in the basolateral infranuclear region (6.0 grains/m m2 versus 0.7 in the supranuclear/brush border compartment). The last observations support the hypothesis that EGF would access to its receptors on epithelial cells from the serosal side, not from the gut lumen, under normal non-pathological conditions. Confirming this assumption, the same study (120) demonstrated that labeled EGF could not access to its receptors when infused into the lumen of jejunal segments, either at 22° C or 4° C.

Figure 2. Developmental pattern (A) and Scatchard analyses (B,C) of iodinated EGF binding to epithelial cells isolated from human fetal small intestine and colon. Indices in graph A represent the number of specimens for each age (adapted from ref 119).

Of particular interest, the small intestine undergoes extensive morphogenesis between 8 and 12 weeks of gestation with the onset of villus and crypt formation and subsequent transformation from pseudostratified columnar to simple columnar epithelium (121). Labeling indices within this epithelium are also at their highest value between 8 and 10 weeks (approximately 26-30%) and decrease markedly during the next 4-6 weeks (122). It is thus during this critical period of morphogenesis and remodeling that the intestinal epithelium exhibits its highest EGF binding capabilities. Moreover, Poulsen and co-workers (9) have demonstrated the presence of immunoreactive EGF in intestinal Paneth cells of the 20-week-old fetus suggesting that this cell type represents an endogenous source of the peptide. Paneth cells normally appears at 11-12 weeks (122) which again correlates with the high number of EGF-R at this early age. The possibility that EGF from Paneth cells may influence the neibourghing undifferentiated cells has yet to be explored. In subsequent studies using the immunohistochemistry approach (123,124), the presence of EGF-R at the basolateral cellular pole, their consistent absence in brush border preparations and their rarefication with increasing age were verified in intestinal epithelium of fetuses, children and adults.

Two colon carcinoma cell lines, namely Caco-2 and HT-29, exhibit human enterocyte-like features in culture and are commonly used as in vitro models to study the role of putative regulators. The capacity of these cells to bind EGF has been demonstrated (125) as well as the involvement of an EGF-like factor in their autonomous growth (126,127) and migration behavior over laminin substrate (128). Recent data indeed show that antisens EGF-R expression has an antiproliferative effect on HT-29 cells (129). While both Caco-2 and HT-29 cells express a minor pool of EGF-R at their apical surface, only basolateral membrane stimulation with EGF increased tyrosine kinase activity and enhanced proliferation (130,131), in accordance with the proposed model of EGF action on epithelial cells. EGF not only stimulates DNA synthesis and proliferation of Caco-2 cells but reduces sucrase activity markedly, by affecting its processing in the endoplasmic reticulum and mRNA synthesis (132). The same study reveals that EGF maintains Caco-2 cells in a poorly-differentiated phenotype thus suggesting that the growth factor would act as a mitogen and a repressor of terminal differentiation. Interestingly, ulterior experiments showed that parental HT-29 and sublines lose their proliferative response to EGF ligands as they spontaneously differentiate in culture (133): they are mitogenic for undifferentiated cells but inhibit the growth of more differentiated cells. Changes in the signaling machinery rather than the modulation of EGF-R expression appear to be involved in this process.

The study of EGF-R expression and ligand activity in primary cultures of human small intestinal cells still awaits the development of a suitable experimental system. Nonetheless, the biological effects of EGF have been carefully examined in organ cultures of fetal jejunum (11 to 14 week of gestation) (134). While the addition of exogenous EGF did not modify the morphology of intestinal explants, lactase activity was significantly increased and the rise in sucrase, trehalase and glucoamylase activities that normally occurs during culture was repressed in the presence of increasing concentrations of EGF. DNA synthesis and labeling index dropped drastically (within 24 hours of culture) in this model, thus recalling the response of differentiated cultured cell lines. These results clearly suggest that the influence of EGF on the regulation of small intestinal epithelium development depends upon the differentiation status of target cells.

4.2. Colon

Binding of 125I-EGF to colonic epithelial cells isolated from 12 to 17-week fetuses was assessed using the same methodology and optimal conditions as for intestinal epithelial cells (119; figure 2). Compared to the small intestine, specific binding in the colon was substantially higher (1.4 to 2.4-times) at all ages and also decreased with advancing age (figure 2). High-affinity (K1 = 1.78 ± 0.83 x 10-9 M-1) and low-affinity (K2 = 0.014 ± 0.005 x 10-9 M-1) binding sites were similarly demonstrated by Scatchard plot. Their cellular distribution in smooth muscle fibers as well as in developing villi and crypts of the fetal colon was identical to that found in small intestine, being concentrated in regions of high proliferative activity and tritiated thymidine uptake while being absent from the brush border of villus cells (120).

The overall pattern of EGF binding in the human fetal colon is notheworthy since colonic epithelial cells exhibited even higher binding than their intestinal counterpart. This difference can be attributed partly to a greater concentration of EGF-R at the cell surface. Moreover, since the onset of villus formation and epithelial differentiation in the developing colon occurs 3-4 weeks after initiation of morphogenesis in the small intestine, there may exist a relation between the fact that levels of EGF binding in the colon at 17 weeks are equivalent to peak levels observed in the 12-14 week-old intestine (figure 2). It is also known that the expression of intestinal-type markers are down-regulated and that fetal colonic villi are transformed into adult-type colonic crypts before birth. Whether the expression and binding kinetics of EGF-R are modified during this ontogenic process, when epithelial cell proliferation contributes to tissue remodeling, remains to be determined.

As mentioned previously for the small intestine, the influence of EGF was studied in organ cultures of human fetal colon (135) where it did not alter the overall morphology of explants. Biochemical data, however, suggest that EGF may participate in the maturation process (loss of villus markers) since it caused a decrease of DNA synthesis rates and brush border sucrase, maltase and alkaline phosphatase activity levels. Illustrating the significance and the specificity of EGF action, hydrocortisone generated no effect in this model (135).

4.3. Stomach

The biological relevance of EGF action at the level of the stomach has been demonstrated early on. The molecule called urogastrone, an inhibitor of gastric acid secretion produced by the human kidney and present in urine, was proven to be identical to the mature EGF peptide (136). The presence of EGF-R in the adult gastric mucosa was also verified and their increased expression in foveolar and surface epithelial compartments (compared to the base of glands) supports a pivotal role for this growth factor system in the maintenance of gastric mucosal integrity (protection, healing)(137-139). However the developmental aspects of EGF-R physiology in the human fetal stomach were only studied recently.

The presence of the EGF-R immunoreactive protein was first suggested in a study analyzing its widespread expression in digestive tract epithelia and pancreatic tissues (123). Using indirect immunofluorescence and autoradiographic localization of 125I-EGF binding sites, it was then demonstrated that the pattern of EGF-R expression in developing stomach significantly differed from the adult situation (140). In fact, EGF-R were detected as soon as 10-11 weeks of gestation when the gastric mucosa is lined by a stratified undifferentiated epithelium. At subsequent stages (12 to 20 weeks; figure 3), all epithelial cell types of the developing pit/gland structure (undifferentiated, mucous, endocrine, parietal and chief cells) were immunoreactive and the three major functional compartments e.g. the surface epithelium, the pit/neck region and the gland contained comparable numbers of binding sites. Thus, when compared to the adult stomach, it seems plausible that EGF-R and its ligands exert a broad range of developmental activities in the fetal organ due to their ubiquitous expression in all gastric epithelial cell species. As expected also, the cellular distribution of EGF binding was concentrated in the infranuclear region of epithelial cells (140). Reflecting the highly polarized localization of EGF-R and, possibly, the early compartmentalization of EGF-R expression in the pit/gland unit, an intensive immunostaining was seen at the basolateral pole of surface mucous cells in 20-week specimens (figure 3).

Figure 3. Localization of immunoreactive EGF-R protein in human fetal stomach mucosa at 12 (A) and 20 (B) weeks of gestation. Apical staining on surface cells represents non specific binding of antibody to mucus (from ref.140).

In the past, only a few studies were aimed at studying the role of the EGF/TGFalpha/EGF-R system in cultured human gastric tissues. One report (141) helped to confirm that EGF-R are present on epithelial cells isolated from adult stomach and showed that exogenous EGF stimulates the release of an angiogenic growth factor. Results obtained with human fetal gastric tissue maintained in organ culture (140) demonstrated that EGF regulates the proliferative and differentiative functions of the developing epithelium. The factor was able to stimulate DNA synthesis in mitogenically-competent cells and the incorporation of tritiated glucosamine into newly-synthesized glycoproteins (in mucous cells). Moreover, EGF down-regulated lipase activity in this model without affecting pepsin, two digestive enzymes co-localized in human glandular chief cells. The latter observations are consistent with the ubiquitous expression of EGF-R in all epithelial cells of the fetal gastric mucosa and further reinforce the assumption that associated ligands might control a variety of developmental activities contributing to the differentiation and/or the maintenance of gastric epithelial lineages. These results also emphasize the uncoupled regulatory process for lipase and pepsin, and enlighten the role of EGF in the modulation of lipolytic activity of the human gastric mucosa. Recently, it has been shown that EGF exerts its down-regulatory action on gastric lipase at the mRNA level (142).

5. COMPARISON WITH ANIMAL MODELS

On a general basis, the characteristics of EGF receptors in both human fetal intestinal and colonic epithelial cells in terms of binding kinetics, ligand specificity and binding affinities (Kd = 10-10 to 10-9 M) are typical of those found in rodent intestinal epithelial cells using the same protocols (143-145). As seen for the human, the concentration of EGF-R was higher in mouse developing colon than in small intestine at the same postnatal age (146).

While there is a general agreement about the tissular localization of EGF-R and the growth-promoting effect of EGF-related factors on the adult gastrointestinal tract of several species, discrepancies between humans and rodents have been reported regarding its implication in the functional differentiation of intestinal epithelia during ontogeny (refer to comprehensive reviews 147-149). For example, EGF promotes in organ culture the maturation of the rough endoplasmic reticulum and some brush border enzymes of mouse small intestine at the fetal stage (15-17 days)(150-152) in accordance with its suggested role as an inducer of enterocyte differentiation with a specific and restricted action. In suckling (8 days) animals however, injections of EGF stimulate proliferation in all gut segments, including small intestine, and simultaneously increase all brush border hydrolase activities in a dose-dependent manner (153). Therefore, EGF does not only act as a fetal promoting agent but seems to be one of the few factors involved in re-differentiation of epithelial cells during postnatal adaptation of intestinal function at weaning, a process which is highly coordinated along the entire digestive tract in rodents (147). Discrepancies were even observed between mouse and rat as exogenous EGF triggered limited effects on some brush border enzymic activities (154) or had no influence (155,156) in the latter species. In addition, EGF administered orogastrically but not intraperitoneally induced precocious maturation of intestinal disaccharidase activities in suckling rabbits (157). Many explanations can be put forward to explain these variations such as the amount of EGF used, route of administration, rapid uptake of exogenous EGF by the liver, phase of circadian rhythm, nutritional state, length of the experimental period, injection schedule and developmental stages as well as the possible masking effect of endogenous EGF ligands. Eventhough animal models are necessary for the identification of involved growth factors and characterization of their biological roles, it is clear that effects induced by EGF in rodent small intestine cannot be directly extrapolated to the human gut.

There are several evidences that EGF, TGFalpha and related substances may exert a similar influence on developing stomach of rodents (rat, rabbit, guinea pig) and humans (reviewed in 158). They stimulate epithelial proliferation and mucus synthesis while they act negatively on parietal cell differentiation. Moreover targeted overexpression of TGF-alpha gene in mice causes alterations of the gastric mucosa that ressemble Ménétrier's disease (46,159,160), a premalignant disorder characterized by foveolar hyperplasia, hypochlorhydia and increased mucus. Thus it appears that this hormonal system would assume a general and important role in development of the mucigenic gastric lineage. The earlier finding by Dembinski and Johnson (161) that EGF significantly increased weight of the whole stomach and DNA content of the oxyntic glands in unweaned rats but had no effect on pepsinogen synthesis/secretion led to the conclusion that EGF is a candidate inducer of oxyntic mucosal growth which does not participate in the developmental regulation of gastric zymogen expression. Our recent observation that this growth factor specifically influences gastric lipase expression in human chief cells (an enzyme absent in rodents) without affecting pepsinogen (140) stresses again the caution to be taken in directly extrapolating concepts established in animal models to humans.

6. INTEGRATION

In 1976, David Wingate formulated a new global theory of gastrointestinal hormone action, called the eupeptide system (162), which stated that the established model of endocrine function - the concept of endocrine glands secreting at a distance from target tissues - was inappropriate for the context of gut physiology. This theory proposed that local 'paracrine' polypeptides may be no less important and may cooperate with 'endocrine' true hormones or mediate some of their effects in the control of nutrient absorption, smooth muscle contraction and tissue maintenance. One must realized today that the discovery and the study of the EGF/EGF-R system has greatly contributed to the conceptual enlightenment of the former theory. As suggested by the variety of its biological actions and its widespread expression in developing and adult gut segments, this growth factor system seems to be involved in the global and intrinsic control of gut morphogenesis and homeostasis.

The current review emphasized on analyzing the expression of EGF-R in human fetal gut and correlating the biological actions of EGF-R ligands. The data presented reveal the ubiquitous distribution of the receptor in the developing mucosa of gut segments as well as in the various epithelial cell types. In comparison, its expression is known to become more restricted in the corresponding adult tissues. We may therefore propose an important and pleiotropic role for the EGF-R and its ligands in the context of gut epithelium development. Firstly, the growth factor system appears to be involved in the local regulation of epithelial cell proliferation in which EGF-R are abundant. Depending upon the specific segment and its differentiation status, cell proliferation would be stimulated or decreased by agonists of EGF-R; the growth factor acts as a mitogen for gastric explants and as an inducer of precocious maturation in small intestine (lactase activity increased) and colon (intestinal enzymes down-regulated). A second function of this system in utero may be to counter-regulate the terminal differentiation of human digestive epithelia; it negatively modulates the expression of segment-specific functional markers such as brush border sucrase, trehalase and glucoamylase in the intestine and chief cell lipase in the stomach. In the latter organ, the relative abundance of EGF-R in surface epithelium and the effect of EGF supplementation in culture also argues for a role in mucus synthesis. In addition, this hormonal system is involved in the normal maintenance of adult gut tissues (plus inhibition of gastric acid secretion) and it is deregulated in cancer (148,149). These facts illustrate that EGF-R and EGF-related factors still exert significant developmental activities in the more mature digestive tract.

According to the literature, multiple ligands for the EGF-R are present in tissues, including fetal organs. What would be the relevance of such a heterogeneity in the context of gut epithelium development and maintenance? Probably to serve in different delivery pathways or in different physiological responses (see figure 4). TGFalpha, for example, likely represents the fetal and paracrine ligand, which is synthesized in proliferative compartments of human fetal gut (123,163,164) and which locally regulates epithelial proliferation/differentiation. This factor activates EGF-R and mitogenesis as potently as EGF in vitro (165). It might be of some significance in this context that TGFalpha acts more strongly than EGF on cell proliferation, motility and branching morphogenesis in specific experimental systems (166 plus several studies cited in 148,149) due to differences in ligand processing, receptor down-regulation and, possibly, binding efficiency. The global expression of both TGFalpha and cognate EGF-R are simultaneously down-regulated in maturing organs while EGF expression increases at the end of gestation and experiments realized in mouse provided evidences that these developmental changes are under the control of thyroid hormones (167,168). However, expression of the TGFalpha protein persists in a number of mature tissues and recent data demonstrate that it represents the main form locally produced in proliferative compartments of the adult gastrointestinal tract e.g. intestinal crypts and gastric glands (123,169-171). There are new evidences that HB-EGF, another agonist of EGF-R, might be the autocrine form expressed by epithelial cells in response to environmental factors. It is expressed together with EGF or TGFalpha in many tissues (172) and it is rapidly up-regulated by EGF-related ligands themselves (173) and by Helicobacter pylori in gastric cells (174). Its expression greatly increases in gut-associated carcinomas (175,176). HB-EGF thus represents an immediate early gene candidate. Also at the specific level of the human stomach, HB-EGF appears to be the main form produced by fundic parietal cells and gastrin cells of pyloric glands (177). Concerning EGF, the prototypical growth factor, it likely represents the long-distance acting form released into the gut lumen (a lumone) and one current hypothesis is that it would assume a surveillance role by maintaining the integrity of gut tissues upon mucosal damage (stimulation of epithelial restitution and proliferation)(178,179). It is interesting to remind, in this regard, earlier data from Wright and collaborators (180) showing that ulceration of the mucosal epithelium in the human gastrointestinal tract induced the development of a novel cell lineage that produced neutral mucin and abundant immunoreactive EGF. It is true that EGF secreted into the lumen is processed into smaller and less active forms (181,182) and that its stability is positively influenced by ingestion of food proteins (183,184). In the specific context of gut epithelial development in the human fetus and infant, it should be pointed out that EGF may remain bioactive for long periods in gastric and intestinal fluids due to the immaturity of pancreatic enzymic function (185) and the protective effect of milk proteins (186). However, the biological significance and efficiency of luminal binding on epithelial cells (especially in immature gut), the controversial identification of low- or high-affinity binding sites on apical membranes as well as the involvement of a transcytosis mechanism (serosal towards luminal or luminal towards serosal) remain to be clarified (148,187).

Figure 4. Theoretical model of EGF action on the EGF-R expressing gut epithelum. EGF-related substances (EGF, TGFalpha, HB-EGF) derived from various sources would access to EGF-R at sites of epithelial damage or would be released in a paracrine/autocrine fashion at the epithelio-mesenchymal interface to participate in normal regeneration and differentiation.

7. PERSPECTIVES

The overall characteristics of EGF/EGF-R physiology in the developing human gastrointestinal tract do not entirely correlate with those found in animal models. The expression of the receptor or its ligands may also be altered or subjected to abnormal regulatory events in adenocarcinoma cell lines. For these reasons, it will be necessary to establish normal human epithelial cell models in order to further our understanding of the specific developmental actions of this growth factor system and its modulation during successive stages of epithelial differentiation.

Concerning the molecular properties of EGF-R activation and regulation, which are determined by the level of EGF-R expression and modulated by intracellular kinases, it now appears that they depend upon heterodimerization events with EGF-R-related receptors expressed in epithelial cells. HER-2, HER-3 and HER-4 (erbB2, erbB3 and erbB4 in mouse) are known to bind neuregulin-like substances and differential heterodimerization of EGF-R with each partner receptor might account for distinct responses (stimulatory or inhibitory) observed in given cell types (188,189). Interestingly, these EGF-R-related proteins seem to be abundantly expressed in human fetal gut compared to the adult (190,191) and some evidence for their dynamic expression in relation with epithelial morphogenesis was recently reported (192). At the light of experiments exploring MAP kinase activation kinetics in the presence of growth factors alone or growth factors with integrin ligands (87), it is now obvious that EGF-R can also synergize with receptors to extracellular matrix proteins for inducing an optimal response. Confirming this theory, the growth of human colon carcinoma cells was differentially stimulated by TGFalpha on plastic and collagen substratum while the induced-formation of crypt-like structures in 3-dimensional culture increased the expression of specific integrins (193). Future studies must be oriented to enlighten the intracellular events leading to the biological effects and to characterize the molecular events underlying the synergism between EGF-R and integrin ligands specific to human gastrointestinal epithelial cells.

7. ACKNOWLEDGEMENTS

Daniel Ménard (Ph.D.) is director of the research group on the Functional Development and Physiopathology of the Digestive Tract (Développement Fonctionnel et Physiopathologie du Tube Digestif) supported by the Medical Research Council (MRC) of Canada. Pierre Chailler (Ph.D.) is a MRC group research associate. The authors wish to thank Drs Francis Jacot and Claude Poulin of the 'Département de la Santé Communautaire' for their helpful and long-lasting collaboration in providing human fetal specimens.

8. REFERENCES

1. Cohen, S.: Epidermal growth factor (Lecture for the Nobel Foundation 1987, Stockholm). In Vitro Cell Dev Biol 23, 239-246 (1987)

2. Carpenter, G. & S. Cohen: Epidermal growth factor. Annu Rev Biochem 48, 193-216 (1979)

3. Gregory, H. & I.R. Willshire: The isolation of the urogastrones - inhibitors of gastric acid secretion from human urine. Hoppe Seylers Z Physiol Chem 356, 1765-1774 (1975)

4. Gregory, H., S. Walsh & C.R. Hopkins: The identification of urogastrone in serum, saliva and gastric juice. Gastroenterology 77, 313-318 (1979)

5. Carpenter, G.: Epidermal growth factor is a major growth-promoting agent in human milk. Science 210, 198-199 (1980)

6. Beardmore, J.M. & R.C. Richards: Concentrations of epidermal growth factor in mouse milk throughout lactation. J Endocrinol 96, 283-292 (1983)

7. Heitz, P.U., M. Kasper, S. Van Noorden, J. M. Polak, H. Gregory & A.G.E. Pearse: Immunohistochemical localisation of urogastrone to human duodenal and submandibular glands. Gut 19, 408-413 (1978)

8. Kasselberg, A.G., D.N. Orth, M.E. Gray & M.T. Stahlman: Immunocytochemical localization of human epidermal growth factor/urogastrone in several human tissues. J Histochem Cytochem 33, 315-322 (1985)

9. Poulsen, S.S., E. Nexo, P. Skov Olsen, J. Hess & P. Kirkegaard: Immunohistochemical localization of epidermal growth factor in rat and man. Histochemistry 85, 389-394 (1986)

10. Barka, T., H. Van der Noen, E.W. Gresik & T. Kerenyi: Immunoreactive epidermal growth factor in human amniotic fluid. Mt Sinai J Med 45, 679-684 (1978)

11. D'Souza, S.W., R. Haigh, L. Micklewright, P. Konnai & A. Keys: Amniotic fluid epidermal growth factor and placental weight at term. Lancet II, 272-273 (1985)

12. Vaughan, T.J., P.S. James, J.C. Pascall & K.D. Brown: Expression of the genes for TGFalpha, EGF and the EGF receptor during early pig development. Development 116, 663-669 (1992)

13. Shigeta, H., M. Taga, A. Katoh & H. Minaguchi: Ontogenesis and distribution of epidermal growth factor immunoreactivity and binding activity in the mouse fetal and neonatal tissues. Endocrine J 40, 641-647 (1993)

14. Chia, C.M., R.M.L. Winston & A.H. Handyside: EGF, TGF-alpha and EGFR expression in human preimplantation embryos. Development 121, 299-307 (1995)

15. Stromberg, K., D.A. Pigott, J.E. Ranchalis & D.R. Twardzik: Human term placenta contains transforming growth factors. Biochem Biophys Res Commun 106, 354-361 (1982)

16. Byyny, R.L., D.N. Orth & S. Cohen: Radioimmunoassay of epidermal growth factor. Endocrinology 90, 1261-1266 (1972)

17. Hirata, Y. & D.N. Orth: Epidermal growth factor (urogastrone) in human tissues. J Clin Endocrinol Metab 48, 667-679 (1979)

18. Rall, L.B., J. Scott, G.I. Bell, R.J. Crawford, J.D. Penshow, H.D. Niall & J.P. Coghlan: Mouse prepro-epidermal growth factor synthesis by the kidney and other tissues. Nature 313, 228-231 (1985)

19. Cohen, S.: Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the new-born animal. J Biol Chem 237, 1555-1562 (1962)

20. Cohen, S. & G. Carpenter: Human epidermal growth factor: isolation and chemical and biological properties. Proc Natl Acad Sci USA 72, 1317-1321 (1975)

21. Cohen, S. & G.A. Elliott: The stimulation of epidermal keratinization by a protein isolated from the submaxillary gland of the mouse. J Invest Dermatol 40, 1-5 (1963)

22. Armelin, H.: Pituitary extracts and steroid hormones in the control of 3T3 cell growth. Proc Natl Acad Sci USA 70, 2702-2706 (1973)

23. Rose, S.P., R.M. Pruss & H.R. Herschman: Initiation of 3T3 fibroblast cell division by epidermal growth factor. J Cell Physiol 86, 593-598 (1975)

24. Hollenberg, M.D. & P. Cuatrecasas: Insulin and epidermal growth factor - human fibroblast receptors related to deoxyribonucleic acid synthesis and amino acid uptake. J Biol Chem 250, 3845-3853 (1975)

25. Carpenter, G. & S. Cohen: Human epidermal growth factor and the proliferation of human fibroblasts. J Cell Physiol 88, 227-238 (1976)

26. Carpenter, G., K.J. Lembach, M. Morrison & S. Cohen: Characterization of the binding of 125I-labeled epidermal growth factor to human fibroblasts. J Biol Chem 250, 4297-

4304 (1975)

27. Carpenter, G. & J.G. Zendegui: Epidermal growth factor, its receptor, and related proteins. Exp Cell Res 164, 1-10 (1986)

28. Mroczkowski, B. & G. Carpenter: Epidermal growth factor. In: Hormones, Cell Biology, and Cancer: Perspectives and Potentials, Alan R. Liss, New York, 207-216 (1988)

29. Adamson, E.D.: Oncogenes in development. Development 99, 449-471 (1987)

30. Yamamoto, T., T. Nishida, N. Miyajima, S. Kawai, T. Ooi et al.: The erbB gene of avian erythroblastosis virus is a member of the src gene family. Cell 35, 71-78 (1983)

31. Müller, R.: Differential expression of cellular oncogenes during murine development and in teratocarcinoma cell lines. Cold Spring Harb Conf Cell Prolif 10, 451-468 (1983)

32. Yasui, W., H. Sumiyoshi, J. Hata, T. Kameda, A. Ochiai, H. Ito & E. Tahara: Expression of epidermal growth factor receptor in human gastric and colonic carcinomas. Cancer Res 48, 137-141 (1988)

33. Ozawa, S., M. Ueda, N. Ando, O. Abe & N. Shimizu: Epidermal growth factor receptors in cancer tissues of esophagus, lung, pancreas, colorectum, breast and stomach. Jpn J Cancer Res 79, 1201-1207 (1988)

34. Stemmermann,G., S.C. Heffelfinger, A. Noffsinger, Y.Z. Hui, M.A. Miller & C.M. Fenoglio-Preiser: The molecular biology of esophageal and gastric cancer and their precursors: oncogenes, tumor suppressor genes, and growth factors. Hum Pathol 25, 968-981 (1994)

35. Alexander, R.J., A. Panja, E. Kaplan-Liss, L. Mayer & R.F. Raicht: Expression of growth factor receptor-encoded mRNA by colonic epithelial cells is altered in inflammatory bowel disease. Dig Dis Sci 40, 485-494 (1995)

36. Adamson, E.D. & A.R. Rees: Epidermal growth factor receptors. Molec Cell Biochem 34, 129-152 (1981)

37. Gospodarowicz, D.: Growth factors and their action in vivo and in vitro. J Pathol 141, 201-233 (1983)

38. Gill, G.N., P.J. Bertics & J.B. Santon: Epidermal growth factor and its receptor. Molec Cell Endocrinol 51, 169-186 (1987)

39. Burgess, A.: Epidermal growth factor and transforming growth factor alpha. Brit Med Bull 45, 401-424 (1989)

40. Adamson, E.D.: Developmental activities of the epidermal growth factor receptor. Curr Top Dev Biol 24, 1-55 (1990)

41. Dominey, A.M., X.J. Wang, L.E. King jr, L.B. Nanney, T.A. Gagne, K. Sellheyer, D.S. Bundman, M.A. Longley, J.A. Rothnagel, D.A. Greenhalgh et al.: Targeted overexpression of transforming growth factor alpha in the epidermis of transgenic mice elicits hyperplasia, hyperkeratosis, and spontaneous, squamous papillomas. Cell Growth Differ 4, 1071-1082 (1993)

42. Webber, E.M., J.C. Wang, L. Wang, G. Merlino & N. Fausto: Overexpression of transforming growth factor-alpha causes liver enlargement and increased hepatocyte proliferation in transgenic mice. Am J Pathol 145, 398-408 (1994)

43. Acra, S.A., N. Bulus, G. Bogatcheva, R.J. Coffey & J.A. Barnard: Increased intestinal epithelial proliferation in metallothioneine-transforming growth factor alpha transgenic mice. Regul Pept 74, 105-112 (1998)

44. Sharp, R., M.W. Babyatsky, H. Takagi, S. Tagerud, T.C. Wang, D.E. Bockman, S.J. Brand & G. Merlino: Transforming growth factor alpha disrupts the normal program of cellular differentiation in the gastric mucosa of transgenic mice. Development 121, 149-161 (1995)

45. Bockman, D.E., R. Sharp & G. Merlino: Regulation of terminal differentiation of zymogenic cells by transforming growth factor alpha in transgenic mice. Gastroenterology 108, 447-454 (1995)

46. Goldenring, J.R., G.S. Ray, C.J. Soroka, J. Smith, I.M. Modlin, K.S. Meise & R.J. Coffey Jr: Overexpression of transforming growth factor-alpha alters differentiation of gastric cell lineages. Dig Dis Sci 41, 773-784 (1996)

47. Mann, G.B., K.J. Fowler, A. Gabriel, E.C. Nice, R.L. Williams & A.R. Dunn: Mice with a null mutation of the TGF alpha gene have abnormal skin architecture, wavy hair, and curly whiskers and often develop corneal inflammation. Cell 23, 249-261 (1993)

48. Helmrath, M.A., C.R. Erwin & B.W. Warner: A defective EGF-receptor in waved-2 mice attenuates intestinal adaptation. J Surg Res 69, 76-80 (1997)

49. Hansen, L.A., N. Alexander, M.E. Hogan, J.P. Sundberg, A. Dlugosz, D.W. Threadgill, T. Magnuson & S.H. Yuspa: Genetically null mice reveal a central role for epidermal growth factor receptor in the differentiation of the hair follicle and normal hair development. Am J Pathol 150, 1959-1975 (1997)

50. Miettinen, P.J., D. Warburton, D. Bu, J.S. Zhao, J.E. Berger, P. Minoo, T. Koivisto, L. Allen, L. Dobbs, Z. Werb & R. Derynck: Impaired lung branching morphogenesis in the absence of functional EGF receptor. Dev Biol 186, 224-236 (1997)

51. Pratt, R.M.: Involvement of hormones and growth factors in the development of the secondary palate. In: Development in mammals, vol.4. Ed: Johnson, M.H., Elsevier/North-Holland Biomedical Press, New York, 203 (1980)

52. Catterton, W.Z., M.B. Escobedo, W.R. Sexson, M.E. Gray, H.W. Sundell & M.T. Stahlman: Effect of epidermal growth factor on lung maturation in fetal rabbits. Pediatr Res 13, 104-108 (1979)

53. Messmer, T.O., R. Armour & R.W. Holley: Factors influencing the growth of alveolar type II epithelial cells isolated from rat lungs. Exp Cell Res 142, 417-426 (1982)

54. Stanley, M.A. & Dahlenburg: The effect of choleragen and epidermal growth factor on proliferation and maturation in vitro of human ectocervical cells. In Vitro 20, 144-151 (1985)

55. Wu, K., J. Yankaskas, E. Cheng, M.R. Knowles & R. Boucher: Growth and differentiation of human nasal epithelial cells in culture. Am Rev Respir Dis 132, 311-320 (1985)

56. Topham, R.T., D.J. Chiego, V.H. Gattone II, D.A. Hinton & R.M. Klein: The effect of epidermal growth factor on neonatal incisor differentiation in the mouse. Dev Biol 124, 532-543 (1987)

57. Morrish, D.W., D. Bhardwaj, L.K. Dabbagh, H. Marusyk & O. Siy: Epidermal growth factor induces differentiation and secretion of human chorionic gonadotropin and placental lactogen in normal human placenta. J Clin Endocrinol Metab 65, 1282-1290 (1987)

58. Calvert, R., J.F. Beaulieu & D. Ménard: Epidermal growth factor (EGF) accelerates the maturation of fetal mouse intestinal mucosa in utero. Experientia 38, 1096-1097 (1982)

59. Malo, C. & D. Ménard: Influence of epidermal growth factor on the development of suckling mouse intestinal mucosa. Gastroenterology 83, 28-35 (1982)

60. Oka, Y., F.K. Ghisham, H.L. Green & D.N. Orth: Effects of mouse epidermal growth factor/urogastrone on the functional maturation of rat intestine. Endocrinology 112, 940-944 (1983)

61. Prigent, S.A. & N.R. Lemoine: The type 1 (EGFR-related) family of growth factor receptors and their ligands. Prog Growth Factor Res 4, 1-24 (1992)

62. Ushiro, H & S. Cohen: Identification of phosphotyrosine as a product of epidermal growth factor-activated protein kinase in A-431 cell membranes. J Biol Chem 255, 8363-8365 (1980)

63. Cohen, S., H. Ushiro, C. Stoschek & M. Chinkers: A native 170,000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J Biol Chem 257, 1523-1531 (1982)

64. Ullrich, A., L. Coussens, J.S. Hayflick, T.J. Dull, A. Gray, A.W. Tam, J. Lee, Y. Yarden, T.A. Libermann, J. Schlessinger, J. Downward, E.L.V. Mayes, N. Whittle, M. D. Waterfield & P.H. Seeburg: Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature 309, 418-425 (1984)

65. Hunter, T. & J.A. Copper: Protein-tyrosine kinases. Annu Rev Biochem 54, 897-930 (1985)

66. Weber, W., P.J. Bertics & G.N. Gill: Immunoaffinity purification of the epidermal growth factor receptor. J Biol Chem 259, 14631-14636 (1984)

67. Gill, G.N., M.G. Rosenfeld, W.S. Chen, P.J. Bertics & C.S. Lazar: Analysis of functional domains in the epidermal growth factor receptor using site-directed mutagenesis. Adv Exp Med Biol 234, 91-103 (1988)

68. Livneh, E., L. Glazer, D. Segal, J. Schlessinger & B.Z. Shilo: The drosophila EGF receptor gene homolog: conservation of both hormone binding and kinase domains. Cell 40, 599-607 (1985)

69. Aroian, R.V., M. Koga, J.E. Mendel, Y. Oshima & P.W. Sternberg: The let-23 gene necessary for Caenorhabditis elegans vulval induction encodes a tyrosine kinase of the EGF receptor subfamily. Nature 348, 693-699 (1990)

70. Xu, Y.H., S. Ishii, A.J.L. Clark, M. Sullivan, R.K. Wilson, D.P. Ma, B.A. Roe, G.T. Merlino & I. Pastan: Human epidermal growth factor receptor cDNA is homologous to a variety of RNAs overproduced in A431 carcinoma cells. Nature 309, 806-810 (1984)

71. Xu, Y.H., N. Richert, S. Ito, G.T. Merlino & I. Pastan: Characterization of epidermal growth factor receptor gene expression in malignant and normal human cell lines. Proc Natl Acad Sci USA 81, 7308-7312 (1984)

72. Merlino, G.T., Y.H. Xu, S. Ishii, A.J.L. Clark, K. Semba, K. Toyoshima, T. Yamamoto & I. Pastan: Amplification and enhanced expression of the epidermal growth factor receptor gene in A431 human carcinoma cells. Science 224, 417-419 (1984)

73. Stoscheck, C.M. & L.E. King, Jr.: Functional and structural characteristics of EGF and its receptor and their relationship to transforming proteins. J Cell Biochem 31, 135-152 (1986)

74. Goodyer, P.R., J. Fata, L. Mulligan, D. Fisher, R. Fagan, H.J. Guyda & C.G. Goodyer: Expression of transforming growth factor-alpha and epidermal growth factor receptor in human fetal kidneys. Molec Cell Endocrinol 77, 199-206 (1991)

75. Ilekis, J.V., B.C. Stark & B. Scoccia: Possible role of variant RNA transcripts in the regulation of epidermal growth factor receptor expression in human placenta. Mol Reprod Dev 41, 149-156 (1995)

76. Hunter, T. & J.A. Cooper: Epidermal growth factor induces rapid tyrosine phosphorylation of proteins in A431 human tumor cells. Cell 24, 741-752 (1981)

77. Cochet, C., G.N. Gill, J. Meisenhelder, J.A. Cooper & T. Hunter: C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity. J Biol Chem 259, 2553-2558 (1984)

78. King, A.C. & P. Cuatrecasas: Resolution of high and low affinity epidermal growth factor receptors. J Biol Chem 257, 3053-3060 (1982)

79. Kawamoto, T., J.D. Sato, A. Le, J. Polikoff, G.H. Sato & J. Mendelsohn: Growth stimulation of A431 cells by epidermal growth factor: identification of high-affinity receptors for epidermal growth factor by an anti-receptor monoclonal antibody. Proc Natl Acad Sci USA 80, 1337-1341 (1983)

80. Lin, C.R., W.S. Chen, C.S. Lazar, C.D. Carpenter, G.N. Gill, R.M. Evans & M.G. Rosenfeld: Protein kinase C phosphorylation at Thr 654 of the unoccupied EGF receptor and EGF binding regulate functional receptor loss by independant mechanisms. Cell 44, 839-848 (1986)

81. Sargent, D.F. & R. Schwyzer: Membrane lipid phase as catalyst for peptide-receptor interaction. Proc Natl Acad Sci USA 83, 5774-5778 (1986)

82. Schlessinger, J., Y. Schechter, M.C. Willingham & I. Pastan: Direct visualization of binding, aggregation, and internalization of insulin and epidermal growth factor on living fibroblastic cells. Proc Natl Acad Sci USA 75, 2659-2663 (1978)

83. Schechter, Y., L. Hernaez, J. Schlessinger & P. Cuatrecases: Local aggregation of hormone-receptor complexes is required for activation by epidermal growth factor. Nature 278, 835-838 (1979)

84. Schreiber, A.B., T.A. Libermann, I. Lax, Y. Yarden & J. Schlessinger: Biological role of epidermal growth factor-receptor clustering. J Biol Chem 258, 846-853 (1983)

85. Schlessinger, J. & A. Ullrich: Growth factor signaling by receptor tyrosine kinases. Neuron 9, 383-391 (1992)

86. Hunter, T.: Tyrosine phosphorylation: past, present and future. Biochem Soc Trans 24, 307-327 (1996)

87. Miyamoto, S., H. Teramoto, J.S. Gutkind & K.M. Yamada: Integrins can collaborate with growth factors for phosphorylation of receptor tyrosine kinases and MAP kinase activation: roles of integrin aggregation and occupancy of receptors. J Cell Biol 135, 1633-1642 (1996)

88. Velu, T.J.: Structure, function and transforming potential of the epidermal growth factor receptor. Mol Cell Endocrinol 70, 205-216 (1990)

89. Adamson, E.D. & J. Meek: The ontogeny of epidermal growth factor receptors during mouse development. Dev Biol 103, 62-71 (1984)

90. Adamson, E.D., M.J. Deller & J.B. Warshaw: Functional EGF receptors on embryonic cells. Nature 291, 656-659 (1981)

91. Damjanov, I., B. Mildner & B.B. Knowles: Immunohistochemical localization of the epidermal growth factor receptor in normal human tissues. Lab Invest 55, 588-592 (1986)

92. Oliver, A.M.: Epidermal growth factor receptor expression in human foetal tissues is age-dependent. Br J Cancer 58, 461-463 (1988)

93. Fukuyama, R. & N. Shimizu: Expression of epidermal growth factor (EGF) and the EGF receptor in human tissues. J Exp Zool 258, 336-343 (1991)

94. Landreth, G.E., L.K. Williams & G.D. Rieser: Association of the epidermal growth factor receptor kinase with the detergent-insoluble cytoskeleton of A431 cells. J Cell Biol 101, 1341-1350 (1985)

95. van Bergen en Henegouwen, P.M., J.C. den Hartigh, P. Romeyn, A.J. Verkleij & J. Boonstra: The epidermal growth factor receptor is associated with actin filaments. Exp Cell Res 199, 90-97 (1992)

96. Hunt, L.T., W.C. Barker & M.O. Dayhoff: Epidermal growth factor: internal duplication and probable relationship to pancreatic secretory trypsin inhibitor. Biochem Biophys Res Commun 60, 1020-1028, 1974

97. DeLarco, L.E. & G.J Todaro: Growth factors from murine sarcoma virus-transformed cells. Proc Natl Acad Sci USA 75, 4001-4005 (1978)

98. Massagué, J.: Epidermal growth factor-like transforming growth factor. J Biol Chem 258, 13606-13613 (1983)

99. Brown, L.P., D.R. Twardzik, H. Marquardt & G.J. Todaro: Vaccinia virus encodes a polypeptide homologous to epidermal growth factor and transforming growth factor. Nature 313, 491-492 (1989)

100. Stroobant, P., R.P. Rice, W.J. Gullick, D.J. Cheng, I.M. Kerr & M.D. Waterfield: Purification and characterization of vaccinia virus growth factor. Cell 42, 383-393 (1985)

101. Shoyab, M., G.D. Plowman, V.L. McDonald, J.G. Bradley & G.J. Todaro: Structure and function of human amphiregulin: a member of the epidermal growth factor family. Science 243, 1074-1076 (1989)

102. Kimura, H., W.H. Fisher & D. Schubert: Structure, expression and function of a Schwannoma-derived growth factor. Nature 348, 257-260 (1990)

103. Sasada, R., Y. Ono, Y. Taniyama, Y. Shing, J. Folkman & K. Igarashi: Cloning and expression of cDNA encoding human betacellulin, a new member of the EGF family. Biochem Biophys Res Commun 190, 1173-1179 (1993)

104. Ciccodicola, A., R. Dono, S. Obici, A. Simeone, M. Zollo & M.G. Persico: Molecular characterization of a gene of the 'EGF family' expressed in undifferentiated human NTERA2 teratocarcinoma cells. EMBO J 8, 1987-1991 (1989)

105. Higashiyama, S., J.A. Abraham, J. Miller, J.C. Fiddes & M. Klagsbrun: A heparin-bindign growth factor secreted by macrophage-like cells that is related to EGF. Science 251, 936-939 (1991)

106. Wen, D., E. Peles, R. Cupples, S.V. Suggs, S.S. Bacus, Y. Luo, G. Trail, S. Hu, S.M. Silbiger, R.B. Levy, R.A. Koski, H.S. Lu & Y. Yarden: Neu differentiation factor: a transmembrane glycoprotein containing an EGF domain and an immunoglobulin homology unit. Cell 69, 559-572 (1992)

107. Holmes, W.E., M.X. Sliwkowski, R.W. Akita, W.J. Henzel, J. Lee, J.W. Park, D. Yansura, N. Abadi, H. Raab, G.D. Lewis, H.M. Shepard, W.J. Kuang, W.I. Wood, D.V. Goeddel & R.L. Vandlen: Identification of heregulin, a specific activator of p185erbB2. Science 256, 1205-1210 (1992)

108. Wharton, K.A., K.M. Johansen, T. Xu & S. Artavanis-Tsakonas: Nucleotide sequence from the neurogenic locus notch implies a gene product that shares homology with proteins containing EGF-like repeats. Cell 43, 567-581 (1985)

109. Greenwald, I.: lin-12, a nematode homeotic gene, is homologous to a set of mammalian proteins that includes epidermal growth factor. Cell 43, 583-590 (1985)

110. Burmeister, M., A. Avivi, J. Schlessinger & H. Soreq: Production of EGF-containing polypeptides in Xenopus oocytes microinjected with submaxillary gland mRNA. EMBO J 3, 1499-1505 (1983)

111. Doolittle, R.F., D.F. Feng & M.S. Johnson: Computer-based characterization of epidermal growth factor precursor. Nature 307, 558-560 (1984)

112. Gray, A., T.J. Dull & A. Ullrich: Nucleotide sequence of epidermal growth factor cDNA predicts a 128,000-molecular weight protein precursor. Nature 303, 722-725 (1983)

113. Russell, D.W., W.J. Schneider, T. Yamamoto, K.L. Luskey, M.S. Brown & J.L. Goldstein: Domain map of the LDL receptor: sequence homology with the epidermal growth factor precursor. Cell 37, 577-585 (1984)

114. Yamamoto, T., C.G. Davis, M.S. Brown, W.J. Schneider, M.L. Casey, J.L. Goldstein & D.W. Russell: The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA. Cell 39, 27-38 (1984)

115. Derynck, R., A.B. Roberts, M.E. Winkler, E.Y. Chan & D.V. Goeddel: Human transforming growth factor-alpha: precursor structure and expression in E.coli. Cell 38, 287-297 (1984)

116. de Larco, J.E. & G.J. Todaro: Growth factors from murine sarcoma virus-transformed cells. Proc Natl Acad Sci USA 75, 4001-4005 (1978)

117. Todaro, G.J., J.E. deLarco, C. Fryling, P.A. Johnson & M.B. Sporn: Transforming growth factors (TGFs): properties and possible mechanisms of action. J Supramol Struct Cell Biochem 15, 287-301 (1981)

118. Cook, P.W., D. Damm, B.L. Garrick, K.M. Wood, C.E. Karkaria, S. Higashiyama, M. Klagsbrun & J.A. Abraham: Carboxyl-terminal truncation of leucine76 converts heparin-binding EGF-like factor from a heparin-enhancible to a heparin-suppressible growth factor. J Cell Physiol 163, 407-417 (1995)

119. Pothier, P. & D. Ménard: Presence and characteristics of epidermal growth factor receptors in human fetal small intestine and colon. FEBS Lett 228, 113-117 (1988)

120. Ménard, D. & P. Pothier: Radioautographic localization of epidermal growth factor receptors in human fetal gut. Gastroenterology 101, 640-649 (1991)

121. Moxey, P.C. & J.S. Trier: Specialized cell types in the human fetal small intestine. Anat Rec 191, 269-286 (1978)

122. Arsenault, P. & D. Ménard: Cell proliferation in developing human jejunum. Biol Neonate 51, 297-304 (1987)

123. Hormi, K. & T. Lehy: Developmental expression of transforming growth factor-alpha and epidermal growth factor receptor proteins in the human pancreas and digestive tract. Cell Tissue Res 278, 439-450 (1994)

124. Playford, R.J., A.M. Hanby, S. Gschmeissner, L.P. Peiffer, N.A. Wright & T. McGarrity: The epidermal growth factor receptor (EGF-R) is present on the basolateral, but not the apical, surface of enterocytes in the human gastrointestinal tract. Gut 39, 262-266 (1996)

125. Hidalgo, I.J., A. Kato & R.T. Borchardt: Binding of epidermal growth factor by human colon carcinoma cell (Caco-2) monolayers. Biochem Biophys Res Commun 160, 317-324 (1989)

126. Culouscou, J.M., F. Garrouste, M. Remacle-Bonnet, D. Bettetini, J. Marvaldi & G. Pommier: Autocrine secretion of a colorectum-derived growth factor by HT-29 human colon carcinoma cell line. Int J Cancer 42, 895-901 (1988)

127. Anzano, M.A., D. Rieman, W. Prichett, D.F. Bowen-Pope & R. Greig: Growth factor production by human colon carcinoma cell lines. Cancer Res 49, 2898-2904 (1989)

128. Basson, M.D., I.M. Modlin & J.A. Madri: Human enterocyte (Caco-2) migration is modulated in vitro by extracellular matrix composition and epidermal growth factor. J Clin Invest 90, 15-23 (1992)

129. Wang, H.M., S. Rajagopal, S. Chakrabarty: Inhibition of human colon cancer malignant cell behavior and growth by antisens epidermal growth factor receptor expression vector. Anticancer Res 18, 2297-2300 (1998)

130. Lehmann, M., M. Remacle-Bonnet, F. Garrouste, J. Luis, C. Rabenandrasana, J. Marvaldi & G. Pommier: Surface distribution of the EGF receptor during differentiation of the human colon carcinoma cell line HT29-D4. J Recept Res 14, 319-333 (1994)

131. Bishop, W.P. & J.T. Wen: Regulation of Caco-2 cell proliferation by basolateral membrane epidermal growth factor receptors. Am J Physiol 267, G892-G900 (1994)

132. Cross, H.S. & A. Quaroni: Inhibition of sucrase-isomaltase expression by EGF in the human colon adenocarcinoma cells Caco-2. Am J Physiol 261, C1173-C1183 (1991)

133. Huang, F., S. Sauma, Z. Yan & E. Friedman: Colon absorptive epithelial cells lose their proliferative response to TGF alpha as they differentiate. Exp Cell Res 219, 8-14 (1995)

134. Ménard, D., P. Arsenault & P. Pothier: Biological effects of epidermal growth factor in human fetal jejunum. Gastroenterology 94, 656-663 (1988)

135. Ménard, D., L. Corriveau & P. Arsenault: Differential effects of epidermal growth factor and hydrocortisone in human fetal colon. J Pediatr Gastroenterol Nutr 10, 13-20 (1990)

136. Gregory, H.: The isolation and structure of urogastrone and its relationship to epidermal growth factor. Nature 257, 325-327 (1975)

137. Olsen, P.S., S.S. Poulsen, P. Kirkegaard & E. Nexo: Role of submandibular saliva and epidermal growth factor in gastric cytoprotection. Gastroenterology 87, 103-108 (1984)

138. Orsini, B., A. Calabro, S. Milani, C. Grappone, H. Herbst & C. Surrenti: Localization of epidermal growth factor/transforming growth factor-alpha receptor in the human gastric mucosa. Virchows Archiv A Pathol Anat 423, 57-63 (1993)

139. Konturek, J.W., T. Brzozowski & S.J. Konturek: Epidermal growth factor in protection, repair, and healing of gastroduodenal mucosa. J Clin Gastroenterol 13(suppl 1), S88-S97 (1991)

140. Tremblay, E., S. Monfils & D. Ménard: Epidermal growth factor influences cell proliferation, glycoproteins, and lipase activity in human fetal stomach. Gastroenterology 112, 1188-1196 (1997)

141. Takahashi, M., K. Ogura, S. Maeda, K. Mori, K. Mafune, Y. Mikami, A. Terano & M. Omata: Promoters of epithelialization induce expression of vascular endothelial growth factor in human gastric epithelial cells in primary culture. FEBS Lett 418, 115-118 (1997)

142. Ménard, D. & E. Tremblay: Regulation of human gastric lipase (HGL) expression in organ culture by epidermal growth factor (EGF). [Abstract, AGA meeting] Gastroenterology 114, A894 (1998)

143. Gallo-Payet, N. & J.S. Hugon: Epidermal growth factor receptors in isolated adult mouse intestinal cells: studies in vivo and in organ culture. Endocrinology 116, 194-201 (1985)

144. Gallo-Payet, N., P. Pothier & J.S. Hugon: Ontogeny of EGF receptors during postnatal development of mouse small intestine. J Pediatr Gastroenterol Nutr 6:114-120 (1987)

145. Forgue-Laffitte, M.E., M. Laburthe, M.C. Chamblier, A.J. Moody & G. Rosselin: Demonstration of specific receptors for EGF-urogastrone in isolated rat intestinal epithelial cells. FEBS Lett 114, 243-246 (1980)

146. Ménard, D., P. Pothier & N. Gallo-Payet: Epidermal growth factor receptors during postnatal development of the mouse colon. Endocrinology 121, 1548-1554 (1987)

147. Ménard, D. & R. Calvert: Fetal and postnatal development of the small and large intestine: patterns and regulation. In: Growth of the Gastrointestinal tract: gastrointestinal hormones and growth factors. Eds: Morisset, J. & Solomon T.E., CRC Press, Boca Raton, 159-174 (1991)

148. Podolsky, D.K.: Peptide growth factors in the Gastrointestinal Tract. In: Physiology of the Gastrointestinal Tract. Ed: Johnson, L.R., 3rd ed., Raven Press, New York, 129-168 (1994)

149. Karnes, W.E. Jr: Epidermal growth factor and transforming growth factor-alpha. In: Gut peptides. Biochemistry and Physiology (Comprehensive Endocrinology Serie). Eds: Walsh, J.H. & Dockray G.J., Raven Press, New York, 553-586 (1994)

150. Beaulieu, J.F. & R. Calvert: The effect of epidermal growth factor (EGF) on the differentiation of the rough endoplasmic reticulum in fetal mouse small intestine in organ culture. J Histochem Cytochem 29, 765-770 (1981)

151. Beaulieu, J.F., D. Ménard & R. Calvert: Influence of epidermal growth factor on the maturation of the fetal mouse duodenum in organ culture. J Pediatr Gastroenterol Nutr 4, 476-481 (1985)

152. Conteas, C.N., J.M. DeMorrow & A.P. Majumdar: Effect of epidermal growth factor on growth and maturation of fetal and neonatal rat small intestine in organ culture. Experientia 42, 950-952 (1986)

153. Malo, C. & D. Ménard: Influence of epidermal growth factor on the development of suckling mouse intestinal mucosa. Gastroenterology 83, 28-35 (1982)

154. Oka, Y., F.K. Ghishan, H.L. Greene & D.N. Orth: Effect of mouse epidermal growth factor/urogastrone on the functional maturation of rat intestine. Endocrinology 112, 940-944 (1982)

155. Simon, P.M., M. Kedinger, F. Raul, J.F. Grenier & K. Haffen: Organ culture of suckling rat intestine: comparative study of various hormones on brush border enzymes. In Vitro 18, 339-346 (1982)

156. Majumdar, A.P.N.: Postnatal undernutrition effect of epidermal growth factor on growth and function of the gastrointestinal tract in rats. J Pediatr Gastroenterol Nutr 3, 618-625 (1984)

157. O'Loughlin, E.V., M. Chung, M. Hollenberg, J. Hayden, I. Zahavi & D.G. Gall: Effect of epidermal growth factor on ontogeny of the gastrointestinal tract. Am J Physiol 249, G674-G678 (1985)

158. Coffey, R.J.Jr, M. Romano & J. Goldenring: Roles of transforming growth factor-alpha in the stomach. J Clin Gastroenterol 21(suppl.1),S36-S39 (1995)

159. Dempsey, P.J., J.R. Goldenring, C.J. Soroka, I.M. Modlin, R.W. McClure, C.D. Lind, D.A. Ahlquist, M.R. Pittelkow, D.C. Lee, E.P. Sandgren, D.L. Page & R.J. Coffey: Possible role for transforming growth factor alpha in the pathogenesis of Ménétrier's disease, supportive evidence from humans and transgenic mice. Gastroenterology 103, 1950-1963 (1992)

160. Takagi, H., C. Jhappan, R. Sharp & G. Merlino: Hypertrophic gastropathy resembling Menetrier's disease in transgenic mice overexpressing transforming growth factor alpha in the stomach. J Clin Invest 90, 1161-1167 (1992)

161. Dembinski, A.B. & L.R. Johnson: Effect of EGF on the development of the rat gastric mucosa. Endocrinology 116, 90-94 (1985)

162. Wingate, D.: The eupeptide system: a general theory of gastrointestinal hormones. The Lancet 1, 529-532 (1976)

163. Yasui, W., J. Zhong-Qiang, H. Kuniyasu, A. Ayhan, H. Yokozaki, H. Ito & E. Tahara: Expression of transforming growth factor alpha in human tissues: immunohistochemical study and Northern blot analysis. Virchows Archiv A Pathol Anat 421, 513-519 (1992)

164. Miettinen, P.J.: Transforming growth factor-alpha and epidermal growth factor expression in human fetal gastrointestinal tract. Pediatr Res 33, 481-486 (1993)

165. Todaro, G.J., C. Fryling & J.E. DeLarco: Transforming growth factors produced by certain tumor cells: polypeptides that interact with epidermal growth factor receptors. Proc Natl Acad Sci USA 77, 5258-5262 (1980)

166. Barros, E.J.G., O.F.P. Santos, K. Matsumoto, T. Nakamura & S.K. Nigam: Differential tubulogenic and branching morphogenetic activities of growth factors: implications for epithelial tissue development. Proc Natl Acad Sci USA 92, 4412-4416 (1995)

167. Salido, E.C., J. Lakshamanan, S. Koy, L. Barajas & D.A. Fisher: Effect of thyroxine administration on the expression of epidermal growth factor in the kidney and submandibular gland of neonatal mice. An immunocytochemical and in situ hybridization study. Endocrinology 127, 2263-2269 (1990)

168. North, D., J. Lakshmanan, A. Reviczky, M. Kaser & D.A. Fisher: Ontogeny of epidermal growth factor, transforming growth factor-alpha, epidermal growth factor receptor, and thyroid hormone receptor RNA levels in rat kidney and changes in those levels indiced by early thyroxine treatment. Pediatr Res 31, 330-334 (1992)

169. Thomas, D.M., M.M. Nasim, W.J. Gullick & M.R. Alison: Immunoreactivity of transforming growth factor alpha in the normal adult gastrointestinal tract. Gut 33, 628-631 (1992)

170. Cartlidge, S.A. & J.B. Elder: Transforming growth factor alpha and epidermal growth factor levels in normal human gastrointestinal mucosa. Br J Cancer 60, 657-660 (1989)

171. Borlinghaus, P., S. Wieser & R. Lamerz: Epidermal growth factor, transforming growth factor-alpha, and epidermal growth factor receptor content in normal and carcinomatous gastric and colonic tissue. Clin Invest 71, 903-907 (1993)

172. Vaughan, T.J., J.C. Pascall & K.D. Brown: Tissue distribution of mRNA for heparin-binding epidermal growth factor. Biochem J 287, 681-684 (1992)

173. Barnard, J.A., R. Deal-Graves, M.R. Pittelkow, R. DuBois, P. Cook, G.W. Ramsey, P.R. Bishop, L. Damstrup & R.J. Coffey: Auto- and cross-induction within the mammalian epidermal growth factor-related peptide family. J Biol Chem 269, 22817-22822 (1994)

174. Romano, M., V. Ricci, A. Popolo, P. Sommi, C. Del Vecchio Blanco, C.B. Bruni, U. Ventura, T.L. Cover, M.J. Blaser, R.J. Coffey & R. Zarrilli: Helicobacter pylori upregulates expression of epidermal growth factor-related peptides, but inhibits their proliferative effect in MKN 28 gastric mucosal cells. J Clin Invest 101, 1604-1613 (1998)

175. Inui, Y., S. Higashiyama, S. Kawata, S. Tamura, J. Miyagawa, N. Taniguchi & Y. Matsuzawa: Expression of heparin-binding epidermal growth factor in human hepatocellular carcinoma. Gastroenterology 107, 1799-1804 (1994)

176. Naef, M., M. Yokoyama, H. Friess, M.W. Buchler & M. Korc: Co-expression of heparin-binding EGF-like growth factor and related peptides in human gastric carcinoma. Int J Cancer 66, 315-321 (1996)

177. Murayama, Y., J.I. Miyagawa, S. Higashiyama, S. Kondo, M. Yabu, K. Isozaki, S. Kanayama, Y. Shinomura, N. Taniguchi & Y. Matsuzawa: Localization of heparin-binding epidermal growth factor-like growth factor in human gastric mucosa. Gastroenterology 109, 1051-1059 (1995)

178. Playford, R.J. & N.A. Wright: Why is epidermal growth factor present in the gut lumen? Gut 38, 303-305 (1996)

179. Goodlad, R.A. & N.A. Wright: Epidermal growth factor and transforming growth factor-alpha actions on the gut. Eur J Gastroenterol Hepatol 7, 928-932 (1995)

180. Wright, N.A., C. Pike & G. Elia: Induction of a novel epidermal growth factor-secreting cell lineage by mucosal ulceration in human gastrointestinal stem cells. Nature 343, 82-85 (1990)

181. Britton, J.R., C. George-Nascimento & O. Koldovsky: Luminal hydrolysis of recombinant human epidermal growth factor in the rat gastrointestinal tract: segmental and developmental differences. Life Sci 43, 1339-1347 (1988)

182. Playford, R., T. Marchbank, D.P. Calnan, J. Calam, P. Royston, J.J. Batten & H.F. Hansen: Epidermal growth factor is digested to smaller, less active forms in acidic gstric juice. Gastroenterology 108, 92-101 (1995)

183. Playford, R.J., A.C. Woodman, P. Clark, P. Watanapa, D. Vesey, P.H. Deprey, R.C.N. Williamson & J. Calam: Effect of luminal growth factor preservation on intestinal growth. Lancet 341, 843-848 (1993)

184. Marchbank, T., R.A. Goodlad, C.Y. Lee & R.J. Playford: Luminal growth factor is trophic to the small intestine of parenterally fed rats. Clin Sci 89, 117-120 (1995)

185. Rao, R.K., O. Koldovsky, M. Korc, P.F. Pollack, S. Wright & T.P. Davis: Processing and transfer of epidermal growth factor in developing rat jejunum and ileum. Peptides 11, 1093-1102 (1990)

186. Rao, R.K., R.D. Baker & S.S. Baker: Bovine milk inhibits proteolytic degradation of epidermal growth factor in human gastric and duodenal lumen. Peptides 19, 495-504 (1998)

187. Podolsky, D.K.: Peptide growth factors and regulation of growth in the intestine. In: Gut Peptides. Biochemistry and Physiology (Comprehensive Endocrinology Serie). Eds: Walsh, J.H. & Dockray, G.J., Raven Press, New York, 803-824 (1994)

188. Spivak-Kroizman, T., D. Rotin, D. Pinchasi, A. Ullrich, J. Schlessinger & I. Lax: Heterodimerization of c-erbB2 with different epidermal growth factor receptor mutants elicits stimulatory or inhibitory responses. J Biol Chem 267, 8056-8063 (1992)

189. Graus-Porta, D., R.R. Beerli, J.M. Daly & N.E. Hynes: ErbB-2, the preferred heterodimerization partner of all erbB receptors, is a mediator of lateral signaling. EMBO J 16, 1647-1655 (1997)

190. Mori, S., T. Akiyama, Y. Yamada, Y. Morishita, I. Sugawara, K. Toyoshima & T. Yamamoto: C-erbB-2 gene product, a membrane protein commonly expressed on human fetal epithelial cells. Lab Invest 61, 93-97 (1989)

191. Press, M.F., C. Cordon-Cardo & D.J. Slamon: Expression of the HER-2/neu proto-oncogene in normal human adult and fetal tissues. Oncogene 5, 953-962 (1990)

192. Schroeder, J.A. & D.C. Lee: Dynamic expression and activation of ERBB receptors in the developing mouse mammary gland. Cell Growth Differ 9, 451-464 (1998)

193. Liu, D., G. Gagliardi, M.R. Alison, T. Oates, E.N. Lalani, G.W. Stamp & M. Pignatelli: TGF-alpha can act as morphogen and/or mitogen in a colon-cancer cell line. Int J Cancer 56, 603-608 (1994)