THEMES
Lessons From Genetically Engineered Animal Models
X. Trefoil peptide and EGF receptor/ligand transgenic mice*

Andrew S. Giraud

Department of Medicine, University of Melbourne, Western Hospital, Footscray 3011, Australia


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The use of genetically engineered mice with both gain-of-function and loss-of-function mutations has been particularly informative about the normal and pathophysiological actions of a number of regulatory peptides of the gastrointestinal tract. This review highlights some of the major findings pertinent to the epidermal growth factor (EGF) receptor and its ligands, particularly the major gut ligand transforming growth factor-alpha , as well as the trefoil peptides. Both of these peptide families have important local actions in maintaining tissue homeostasis and repair after injury, and when mechanisms governing their regulation are disrupted they may contribute to disease progression. Future applications of transgenic technology to these areas are likely to be productive in furthering our understanding of the biology of these peptides in health and disease.

epidermal growth factor receptor; transforming growth factor-alpha ; transgenic mice


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THE ADVENT OF GENETIC ENGINEERING and advances in the application of molecular biology methods to problems in gastrointestinal physiology and disease have advanced our understanding of the underlying cellular processes of digestion, as well as mechanisms of ulceration and inflammation, in Crohn's disease, ulcerative colitis, and gastrointestinal cancer.

Maintenance of the normal physiological function of the gastrointestinal mucosa is continually compromised by the harsh extracellular (luminal) environment of the gut. As a result, there are sophisticated reparative mechanisms in place that have the capacity to rapidly restore mucosal integrity and digestive function. These include restitution, in which local epithelial cells migrate to cover denuded areas, as well as increased mucus production and blood flow, followed by proliferation and differentiation programs. The epidermal growth factor (EGF) receptor (EGF-r) ligands [transforming growth factor (TGF)-alpha , EGF, amphiregulin, betacellulin, heparin-binding (HB)-EGF, and epiregulin] and the trefoil peptides [TFF1 (pS2), TFF2 (spasmolytic polypeptide; SP), and TFF3 (intestinal trefoil factor; ITF)] are two distinct families of polypeptides that are important cellular mediators of both normal tissue homeostasis and repair. They act locally, are multifunctional in nature, and when inappropriately expressed may contribute to cancer progression.

The relatively recent application of transgenic animal technology to investigation of the function of the EGF-r ligands and trefoil peptides has provided important new information about their normal physiological roles and specialized functions in gastrointestinal maturation and repair and in disease. This approach has been instructive despite the extensive functional redundancy exemplified by both polypeptide families, further underlining their biological importance. This article attempts to summarize what we have learned so far and where application of these models could be fruitful in the future.


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The main endogenous ligand for the EGF-r in the gut is TGF-alpha . TGF-alpha is expressed by gastric parietal cells, intestinal villus epithelial cells, surface crypt epithelial cells of the colon, pancreatic ductular cells, and hepatocytes (2). Betacellulin, a potent mitogen originally isolated from conditioned medium of an insulinoma cell line derived from the transgenic RIP-tag2 mouse is synthesized in the pancreas, liver, and intestine (24). Epiregulin, characterized from a tumorigenic fibroblast line (3T3/T7), is a low-affinity ligand for the EGF-r and is weakly expressed in the small bowel and colon but strongly expressed by many colon cancer cell lines (30). Amphiregulin is expressed mainly in the colon and pancreas and is a weak ligand for the EGF-r. EGF itself is expressed only at low levels in the normal pancreas and small intestine, and HB-EGF is not present at appreciable levels in the gut (32). All these ligands bind the EGF-r, resulting in homodimerization or heterodimerization with members of the erbB receptor family (erbB2, -3, and -4) of which the EGF-r, or erbB1, is also a member. The natural ligands for erbB3 and -4 particularly are the heregulins. Because the erbB family are tyrosine kinases, ligand binding leads to autophosphorylation followed by a well-characterized signaling cascade via Ras and activation of mitogen-activated protein kinases to transduce the functional signal.


    TREFOIL PEPTIDES IN THE GUT
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The trefoil peptides are a family of abundant 7- to 14-kDa polypeptides, which are secreted by mucus cells predominantly of the gut. There are three mammalian members of the family: a gastric peptide TFF1 (alternative nomenclature pS2), expressed by surface and pit mucus cells; TFF2/SP, a gastric peptide found in mucous neck and glandular mucus cells as well as Brunner's glands of the proximal duodenum; and TFF3/ITF, expressed by goblet cells of the intestine and colon. Individual peptides have homology in cysteine-rich regions of 38 or 39 amino acids known as trefoil motifs, in which the component cysteine residues form disulfide bonds in an invariable 1-5, 2-4, 3-6 design that results in a compact triple-loop or trefoil structure (28). TFF-1 and -3 have a single trefoil motif and form bioactive homodimers linked by a disulfide bond, whereas TFF-2 has two motifs. The trefoil motif is extremely stable and is the basis for the extraordinary resistance of these peptides to acid hydrolysis and proteolysis, making them well suited for action in the harsh luminal environment of the gut.

It is now well established that trefoil peptides are cytoprotective and promote healing in response to gastrointestinal damage. Both oral and subcutaneous TFF2/SP or TFF3/ITF can protect the gastric epithelium from a variety of damaging agents and/or processes including ethanol, nonsteroidal anti-inflammatory drugs, and restraint stress, and luminal application of TFF2/SP in a rat model of inflammatory bowel disease was shown to accelerate healing and reduce inflammation (31). However, the cellular mechanisms by which TFF2/SP and TFF3/ITF accomplish this are unclear, as is whether their effects are receptor mediated. Primarily in vitro evidence supports a role for the trefoil peptides in both extracellular and intracellular actions, including stimulation of cell migration, inhibition of apoptosis in certain cells, and increasing the barrier function of mucus; all of these are commensurate with a role in protection and repair. Of interest is the large body of evidence that trefoils are also ectopically expressed in many epithelial cancers (although not in others), and it is possible that their inappropriate expression under these circumstances may contribute to the inhibition of programmed cell death and metastatic capacity of these cells.


    APPLICATION OF TRANSGENIC MOUSE MODELS TO DETERMINATION OF TREFOIL PEPTIDE FUNCTION
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Before the development of the first loss-of-function or gain-of-function trefoil peptide transgenics, the evidence for a direct role for these peptides in injury repair was circumstantial. The separate publication of descriptions of mouse phenotypes lacking TFF1/pS2 (16) and TFF3/ITF (19), as well as the TFF1/pS2 gain-of-function phenotype (21), showed that the trefoil peptides were essential for normal wound repair and that they also have likely roles in gastrointestinal cell migration and maturation programs. To date, TFF2/SP transgenics have not been described. However, given the demonstrable importance of TFF2/SP in repair programs of the stomach and the colon, the gastric localization of this trefoil peptide to the developmentally pivotal mucous neck/antral gland/Brunner's gland cell, and the instructive TFF1/pS2 and TFF3/ITF transgenic phenotypes, it is likely that TFF2/SP transgenic animals will also be informative about trefoil function.

Trefoil peptides are essential for normal gastrointestinal wound repair. Generation of TFF3/ITF -/- mice showed that they were phenotypically identical to wild-type animals and developed normally but were extremely sensitive to otherwise mildly damaging concentrations of ingested dextran sulfate given in drinking water, so that 50% of treated mice died and others showed high morbidity with diarrhea and rectal bleeding. Histology confirmed a marked colitis. Moreover, the severity of colitis, when induced by the topical irritant acetic acid could be reduced by intracolonic application of recombinant TFF3/ITF (19). Further application of this model (3) to the clinical problem of intestinal mucositis brought about by chemotherapy or radiotherapy showed that animals lacking TFF3/ITF had more severe symptoms (bloody diarrhea, weight loss, and intestinal permeability) than wild-type controls and that these symptoms could be reduced by giving oral ITF.

Conversely, when the TFF3/ITF gene, driven by the rat fatty acid binding protein promoter, was overexpressed in the villi of the mouse jejunum, the mucosa of this region alone was now less susceptible to indomethacin-induced villus shortening (18). These data suggest that endogenous TFF3/ITF helps progress repair functions in the intestine and colon and that when endogenous reparative mechanisms are overwhelmed, luminal administration of high concentrations of TFF3/ITF, either orally or rectally depending on the location of the lesion, may be effective in accelerating reepithelialization and mucosal function. Conclusive evidence in support of this proposal has come from the recent application of recombinant TFF2/SP in a rat model of colitis that showed markedly accelerated colonic mucosal rebuilding and reduced inflammatory indexes after intrarectal administration (31), raising the possibility that trefoils heal by multiple mechanisms. Together these observations suggest that trefoil peptides or their mimetics may be useful as clinical therapeutics in the future, particularly because they are not mitogens for a variety of transformed and nontransformed cells (9) and therefore are unlikely to initiate unscheduled DNA synthesis.

In mice with a TFF1/pS2 null mutation, by 5 mo of age the distal stomach has become hyperplastic, with all animals having circumferential adenoma formation and in some cases apparent dysplasia (16). These observations have been interpreted as evidence that TFF1/pS2 acts as a gastric-specific tumor suppressor; however, the lack of penetrance in carcinoma development and the elevated expression of TFF1/pS2 in other epithelial cancers make it more likely that the observed phenotype comes about either because repair mechanisms are now more frequently compromised in the distal stomach of null mice during the normal course of day-to-day repair or because TFF1/pS2 exerts an inhibitory brake on proliferation in the antrum. Evidence that the latter explanation may have some credence has recently been published (5).

Trefoil peptides participate in gastrointestinal maturation. TFF1/pS2 -/- mice exhibit an increased mitotic index, with reduced migration to other glandular compartments, resulting in elongated gastric pits lacking mucus in the foveolar cells (16). TFF3/ITF -/- mice also have an expanded stem cell/proliferative zone so that normal migration and cell turnover patterns from crypt to villus are disrupted (19). These observations suggest that the trefoil peptides participate in maturation events including cell migration, differentiation, and possibly apoptosis. Experiments using in vitro systems confirmed that trefoils act as weak motogens for epithelial cells (9) and inhibit apoptosis in cancer cell lines (26). They also show a late and sustained induction of expression in the regenerating gastrointestinal mucosa in in vivo models of repair (1, 7), temporally commensurate with the differentiation and cell positioning programs of many epithelial cell types (15). Experiments in cell lines in which differentiation pathways are defined and that are amenable to transfection with trefoil peptide constructs, treatment of late-gestation fetal gut explants with recombinant peptides, or antisense constructs and antisera may help to clarify trefoil regulation of differentiation pathways. This approach, or the development of triple mutants (TFF1/pS2, TFF2/SP, and TFF3/ITF null mice) is required to overcome the overlapping expression and function of trefoil genes in the maturation of the gastrointestinal mucosa.

Trefoil peptide and EGF-r ligand expression are functionally linked. There appears to be an important link between the expression of the trefoil peptides and the main EGF-r ligands of the gut, particularly TGF-alpha in the regenerating gastrointestinal tract. It has been proposed (35) that epithelial rebuilding takes place as a consequence of the induction of a new cell lineage (ulcer-associated cell lineage; UACL) that arises at the periphery of the ulcerated mucosa. EGF-r ligands and all three trefoil peptides have been proposed to be expressed sequentially, with the latter being induced by the former and presumably both contributing to the overall reepithelialization. Support for this hypothesis has come from in vitro and in vivo studies demonstrating synergy between EGF and TFF3/ITF in promoting cell migration and in protecting the stomach against indomethacin/restraint-induced injury (6, 21) and from unpublished data demonstrating increased sequential protein expression of TGF-alpha and then TFF2/SP and TFF3/ITF in the stomach during restitution and glandular reepithelialization after deep ulceration. An added dimension to this relationship comes from the observation that trefoil peptides are able to transactivate the EGF-r (27) and perhaps other members of the erbB family.


    APPLICATION OF TRANSGENIC MODELS TO DETERMINATION OF FUNCTION OF EGF-R AND ITS LIGANDS IN THE GUT
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The application of transgenic models to the determination of function of EGF-r ligands in the gut (as opposed to other organs) has mainly been restricted to overexpressing TGF-alpha under the control of promoters that allow tissue-specific and temporal regulation. In particular, the metallothionein promoter/enhancer (MT)-TGF-alpha mouse, in which the transgene can be activated by application of cadmium or zinc salts in adult life, has been very useful in elucidating and confirming much of the physiology and pathophysiology of TGF-alpha . This has been particularly instructive for understanding liver disease and regeneration, the latter being the subject of a previous review in this series (13).

Mice engineered to be TGF-alpha null, or mutants found to be so by genetic analysis after comparison of coat phenotype with TGF-alpha knockouts (10), were initially thought to be relatively uninformative with respect to gut function. However, careful analysis of the gastrointestinal repair phenotype has revealed some unexpected insights.

TGF-alpha regulates differentiation and repair programs. Ectopic TGF-alpha expression in the stomach has been reported in the MT-TGF-alpha mouse (8, 23). A major consequence of this is that the fundic surface mucous cell compartment is greatly expanded, effectively increasing the length of the pits and compressing the progenitor zone to the gland bases. This process has been termed "antralization," presumably because of the phenotypic and structural similarity of the glands to the gastric antrum (14), and appears to be brought about by a block in the terminal differentiation of parietal and chief/zymogen cells once gastric TGF-alpha reaches a certain threshold level of expression. Expansion of the foveolar compartment also results in an increase in TFF1/pS2 (14) but also TFF2/SP (23) expression, suggesting either that foveolar cells have a mixed phenotype or that a metaplastic repair phenotype (UACL-like) has been induced. The extent of expression of TFF3/ITF in this model would help resolve the issue and would also better define the relationship between TGF-alpha and trefoil gene expression.

In addition to the stomach, TGF-alpha is highly expressed by both columnar epithelial and goblet cells of the colon, in which it is likely to be involved in both maintenance and repair functions. Direct evidence for this has come from the engineered TGF-alpha -/- mouse and the mutant waved-1 (Wa-1) mouse, which is deficient in TGF-alpha . Although cutaneous wound healing appears to proceed normally [as does gross macroscopic gastric ulcer healing (7)], these mice develop more severe and extensive colonic injury after dextran sulfate ingestion than wild-type controls, which can be ameliorated by giving recombinant hTGF-alpha (11). Conversely, MT-TGF-alpha -overexpressing mice are protected against dextran sulfate ulceration and inflammation (12). The compelling conclusion from these studies is that TGF-alpha is a prime regulator of repair and cytoprotective mechanisms in the colon and that these effects are region specific. It may also be no coincidence that the reparative effects of TGF-alpha are most pronounced in that region of the gut with the highest bacterial antigen load (12), because the presence of luminal bacteria is necessary for the progression of many animal models of colitis, especially those with a marked ulcerative component.

Menetrier's disease: a consequence of inappropriate expression of TGF-alpha ? The gastric phenotype of MT-TGF-alpha transgenic mice is very similar to that of human Menetrier's disease, a relatively uncommon precancerous condition marked by foveolar hyperplasia, hypochlorhydria (lack of HCl), and increased mucus synthesis and secretion. Menetrier's patients have high TGF-alpha expression (8), and this along with the characteristic morphology makes it likely that increased TGF-alpha , or inappropriate induction of expression in conjunction with other cofactors, might contribute to the pathogenesis of this disease.

TGF-alpha overexpression results in pancreatic fibrosis and transdifferentiation and may contribute to multistage carcinogenesis. In the MT-TGF-alpha transgenic mouse TGF-alpha is highly expressed in pancreatic acini, and early studies showed a progressive pancreatic enlargement with age caused by interstitial fibrosis along with a redifferentiation of acinar to ductular cells and, ultimately, tubular complexes (4). Subsequent work in the EL-TGF-alpha -hGF transgenic line demonstrated that there is progressive dysplasia in the tubular structures accompanied by increases in EGF-r and p53 expression (33) and that 20% of mice over 1 yr old had cystic papillary tumors. Acinar cell carcinomas have also been induced in double-transgenic mice expressing oncogenes of either viral (SV40 T antigens) or cellular (c-myc) origin and TGF-alpha . In these animals the TGF-alpha transgene alone was not oncogenic; however, its presence accelerated the development of tumors produced by either oncogene (22). The results from these experiments, which could not have been carried out in any other system, point to a role for TGF-alpha in promoting multistage carcinogenesis in conjunction with accumulated mutations as well as tumor growth. It is likely that future multiple transgenics based on TGF-alpha and known facilitators of tumor development such as Ras will also be informative in this respect.

Multiple EGF-r ligand knockouts emphasize importance of redundancy in EGF-r signaling. To circumvent the compensatory mechanisms involved in EGF-r activation, a gene targeting strategy in which the three main upper gut EGF-r ligands were knocked out was carried out (17). Surprisingly, there were no morphological abnormalities in the gut, although the triple-null mutant neonates did not thrive and showed growth retardation, compared with wild-type animals, because of an inadequate milk supply. However, the fact that most mutants reached sexual maturity and exhibited apparently normal gut function suggests that further redundancy in EGF-r ligand binding or transactivation via other erbB receptors exists, either by known or uncharacterized EGF-r ligands.

Contributions from EGF-r null mutant and conditionally immortalized mice and derived cell lines. The importance of the EGF-r in transducing signaling by a multiplicity of ligands involved in proliferation, differentiation, and migration is underscored by the fact that EGF-r null mice invariably die either in utero because of defects in placentation or early in postnatal life because of multiorgan failure, depending on the genetic background (20, 25, 29). Mouse mutants bred on a CD-1 or C57 background had impaired gut proliferation with a reduced stem cell zone and disorganized mucosal architecture. The C57 background phenotype was most severe, exhibiting hemorrhagic lesions and villus destruction characteristic of the premature human infant gut condition necrotizing enterocolitis (20).

Unlike EGF-r null animals, waved-2 mice with a naturally occurring mutation in the tyrosine kinase domain of the EGF-r that results in impaired signaling (10) are viable and have apparently normal gut morphology. This suggests that even attenuated EGF-r function is sufficient to maintain tissue homeostasis or that transactivation by other ligands assumes greater importance under these circumstances.

Because conditional EGF-r knockouts have not yet been reported and EGF-r null mice do not live until sexual maturity, it has not been possible to fully assess the contribution of this pivotal receptor in gastrointestinal function. However, the establishment of a conditionally immortalized transgenic mouse strain (H-2Kb-tsA58) in which the SV40 tsA58 thermolabile transgene is driven by an interferon-gamma -inducible H-2Kb promoter has provided a source of gastrointestinal cell lines that are neither transformed nor tumorigenic yet proliferate when grown at the permissive temperature of 33°C and express many of the differentiated markers of the tissues from which they are derived (34), including the EGF, HGF, TGF-beta , and TNF-alpha receptors. To date, colonic (YAMC) and small intestinal (MSIE) cell lines have been derived and characterized (34) as well as a conditionally immortalized colonic cell line derived from rare mature EGF-r -/- and H-2Kb-tsA58 crosses (R. Whitehead, personal communication) that lacks the EGF-r. These developments show great promise for elucidating mechanisms of receptor activation, gene regulation, and cell motility of nontransformed colonic epithelial cells with or without the EGF-r.


    THE FUTURE FOR GENETICALLY ENGINEERED MICE IN TREFOIL AND EGF-R/LIGAND FUNCTION
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In the last five years, the use of genetically engineered mice has confirmed the pivotal role that the trefoil peptides play in gastrointestinal repair and the likelihood that this is brought about in part by their motogenic actions and ability to promote differentiation during the extensive mucosal rebuilding after ulceration. An unexpected finding was the specific and potentially detrimental adenoma-carcinoma progression at the gastric pylorus in TFF1/pS2 null animals, suggesting an otherwise unpredicted inhibitory role for this peptide. This must be followed up, possibly in conjunction with the generation of a TFF2/SP-only null mouse, especially because 70% of null TFF1/pS2 mutants also lack gastric but not pancreatic TFF2/SP (16).

When interpreting data from the next generation of trefoil null mice in which multiple trefoil genes are inactivated simultaneously, it will be important to ask questions not just about wound healing in the gut but also about the implications of a lack of active trefoil expression in other tissues like the lung, uterus, kidney, and eye in which mucus secretion occurs and in which trefoil expression has been detected. In addition, we have recently shown (6a) that a population of unidentified immunocytes present in spleen, thymus, lymph node, and bone marrow synthesize TFF2/SP and TFF3/ITF, raising the possibility that they may be involved in some general aspect of immune regulation. Because application of recombinant trefoils can reduce inflammatory indexes in animal models of colitis and TFF1/pS2 knockout mice have intestinal inflammation it is possible that this is an important function for these peptides and one that requires a whole animal response to be tested. This could be accomplished by inducing experimental inflammation in trefoil loss-of-function and gain-of-function transgenics and comparing and quantifying the inflammatory response in a tissue-specific way.

Because EGF-r null mice usually die in utero or in early postnatal life, the elucidation of EGF-r function using transgenic animals has been less informative. What is now required is the production of conditional knockouts so that the contribution of EGF-r and other members of the erbB family to gut homeostasis can be fully assessed. This can be accomplished in several ways, including use of the Cre/loxP site-specific recombination system, in which mice expressing the enzyme Cre recombinase driven by a tissue-specific promoter are crossed with mutants expressing the gene of interest engineered with flanking loxP sites. Cre cleaves at the loxP sites, resulting in gene deletion only where it is expressed. This means that target genes can be knocked out in a cell-, tissue-, or time-dependent manner, allowing exact definition of gene function. This system is especially useful for genes that are normally widely expressed or those that are developmentally lethal.

Unlike the situation with the EGF-r, the broad gut organ expression of TGF-alpha and its contribution to differentiation, repair, and multistage carcinogenesis in a tissue-specific pattern when conditionally overexpressed in adult tissues have been very instructive. The contribution to these processes by other EGF-r ligands expressed in the gut such as betacellulin, amphiregulin, and epiregulin now must be addressed.


    ACKNOWLEDGEMENTS

A. S. Giraud was supported by grants from the National Health and Medical Research Council of Australia.


    FOOTNOTES

* Tenth in a series of invited articles on Lessons From Genetically Engineered Animal Models.

Address for reprint requests and other correspondence: A. S. Giraud, Dept. of Medicine, Univ. of Melbourne, Western Hospital, Footscray 3011, Australia (E-mail: ag{at}medicine.unimelb.edu.au).


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Am J Physiol Gastrointest Liver Physiol 278(4):G501-G506
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