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The trefoil factor family (TFF) is a relatively new family of peptides which bear the three-loop trefoil domain. They are mainly synthesised and secreted by mucin secreting epithelial cells lining the gastrointestinal tract and have a close association with mucins. They are highly conserved during evolution and are heat, acid and enzyme resistant. Their abundant expression in distinct patterns in the normal physiological state and ectopic expression in various ulcerative conditions suggests an important role in mucosal defence and repair. Expression of TFF peptides in neoplasia has stimulated interest in deducing the biological role of these peptides in tumour progression. The underlying molecular mechanism of TFF peptide action is still unknown, but their physical properties, and the biological activities of these peptides as motogens may prove to be useful as therapeutic agents in ulcerative conditions, including inflammatory bowel disease where present treatment is far from ideal.
In humans, three trefoil peptides or trefoil factors (TFF) are known. The first trefoil peptide discovered was pS2/TFF1 or breast cancer oestrogen inducible gene— discovered during a search for oestrogen induced mRNAs from the mammary carcinoma cell line MCF7 in 1982.1 In the same year, TFF2 (formerly spasmolytic polypeptide, SP) was purified and extracted from porcine pancreas during the preparation of porcine insulin.2-4 Several years later, it was noticed that these peptides share a common novel sequence motif,5 ,6 later named the trefoil domain7 or P-domain (fig 1).8 The third mammalian protein in the family, ITF/TFF3 (previously called intestinal trefoil factor, ITF or hP1.B) was later discovered as a rat cDNA sequence in 19919 and the human cDNA sequence was reported in 1993.10-12 These peptides are resistant to thermal and enzymatic digestion, largely because the TFF domain shows a structure tightly held together by three pairs of disulphide bonds, which is encoded by shuffled modules highly conserved during evolution from amphibians to mammals (fig 1). TFF1 and TFF3 contain one trefoil domain, TFF2 has two and in the amphibianXenopus, there are molecules containing multiple trefoil domains.13-16 All three human trefoil genes are clustered on chromosome 21q22.3.17-21 The present nomenclature is based on the work of a group at a Conférence Philippe Laudat in which pS2 was named TFF1, spasmolytic polypeptide TFF2 and intestinal trefoil factor TFF3. It was recommended that different species be indicated by a lower case letter prefixed to the peptide name—for example, hTFF1 for human pS2, rTFF3 for rat ITF, etc.22 This article will mainly focus on recent proposals for the role of these peptides in epithelial inflammation, repair, and neoplasia in the gastrointestinal tract.
Expression in normal and pathological conditions
Table 1 summarises the sites of expression of the trefoil factors in the normal gastrointestinal tract and in pathological states. The predominant site of expression of all three mammalian trefoil factors is in the gastrointestinal tract. TFF1 is produced mainly in the stomach, in superficial cells of the body and antral mucosa. TFF2 is abundant in the mucous neck cells in the body and in antral glands of the stomach,23 ,24 and the acini and distal ducts of Brunner’s gland in the duodenum. TFF3 is expressed throughout the intestine and has recently been found to be abundant in salivary glands (Devine et al, unpublished data). It has been shown that both TFF1 and TFF2 are expressed by mucous neck cells in the corpus of the stomach,25 in which the secretory phenotype is remarkably similar to that of a reparative lineage in the gut—the ulcer associated cell lineage (UACL).26
TFF1 was first identified in human breast carcinoma cell lines by virtue of its regulation by oestrogen1 ,27; indeed TFF1 mRNA can be detected in 68% of breast tumours.28 Since then, TFF1 was found to be expressed in a variety of other carcinomas including those of the stomach, pancreas, lung, endometrium, ovary (particularly mucinous carcinomas), prostate, bladder, cervix, and pancreas, and in medullary carcinoma of the thyroid and mucinous carcinoma of the skin.29-49 In breast carcinoma, expression is significantly associated with oestrogen receptor status, responsiveness to hormone therapy and favourable prognosis.29-34 Interestingly, TFF2 is not expressed in breast carcinomas,23 but TFF3 expression is also induced in breast carcinomas in a hormone dependent manner.36
TFF1 protein expression is lower in gastric adenomas and carcinomas than in the adjacent normal gastric mucosa and hyperplastic polyps,37 and its expression is lost in 50% of gastric carcinomas.38-41 Expression of TFF1 is associated with the diffuse histological type of gastric carcinoma, and although some studies have shown no correlation with prognosis, others have shown a significant association between TFF1 expression and tumour staging.40-42 Immunoreactive TFF3 expression has been detected in neoplastic human colonic mucosa, co-localising with neutral mucin production,43 but loss of TFF3 expression is associated with tumour necrosis and advanced Dukes’ stage. Immunoreactive TFF1 has been found in advanced adenocarcinoma of the lung, particularly the bronchioalveolar subtype, and is associated with a poor prognosis.44 ,45 In prostate cancer, TFF1 has been detected in close association with neuroendocrine differentiation, as assessed by chromogranin A immunoreactivity, and was reported to be a useful diagnostic marker of neuroendocrine differentiation in prostate cancer.29 TFF1 is expressed both in the sporadic and hereditary form of human medullary thyroid carcinoma (MTC)48 and also in C-cell hyperplasia, in all probability a precursor of familial MTC,50 suggesting possible involvement in the pathogenesis of MTC. Interestingly, TFF1 protein in neuroendocrine cells was first described in the mucosal endocrine cell hyperplasia which accompanies small bowel Crohn’s disease.51 The importance of TFF1 expression in neuroendocrine cells in several different tissues is yet to be uncovered.
Trefoil factors are expressed in a wide variety of ulcerative conditions of the gastrointestinal tract, from Barrett’s oesophagus52 to gastric53 ,54 and duodenal ulcers55 (fig 2), and also in the small and large intestine in Crohn’s disease,51 ,56 ,57 which emphasises their importance as molecules involved in the repair of gastrointestinal mucosa.58
Functions of trefoil factors
For several years after their isolation, no function was ascribable to these molecules, although their increased expression in chronic inflammatory bowel disease and around experimental and peptic ulcers presaged a role in mucosal defence and healing. The close association of trefoil factor expression with mucus secreting cells suggested that they could also possibly be important for the stabilisation of the mucous lining of the gastrointestinal tract.
An important development in the study of TFF peptide function has been the production of recombinant peptides inEscherichia coli or yeast: recombinant hTFF1, hTFF2 and hTFF3, and rTFF3 have all been produced and have been made freely available. It is now clear that all three mammalian trefoil factors are motogens, namely able to promote cell migration without promoting cell division, and are all upregulated at sites of mucosal injury and stimulate the repair process. Thus they participate in mucosal repair by stimulating the migration of surviving cells from the edge of the damaged region over the denuded area, a process called epithelial restitution, and essential for the repair of both minor and more extensive lesions. They are also morphogens: murine mammary carcinoma cell lines transfected with human TFF1 show an enhanced dispersed growth pattern in three dimensional collagen gels.59 TFF2 and TFF3 act as motogens when cell monolayers are wounded, in a transforming growth factor (TGF) β independent manner.60 ,61 Interestingly, the established gut repair peptides epidermal growth factor (EGF) and TGF-α are mitogens and also act as motogens, but here the effect is mediated through TGF-β receptors on the basolateral side of the epithelium.53
TFF peptides are expressed by mucus producing cells throughout the normal gastrointestinal tract in a site specific manner, and there is some evidence to suggest that trefoil factors might be involved in mucus polymerisation. There is indeed a strong association between the expression of trefoil factors and mucins, and both contribute to mucosal defence. In general, TFF1 is associated with MUC6 expression, TFF2 with MUC5AC, and TFF3 with MUC2 (Longman et al, personal communication). Several trefoil motifs inXenopus laevis are encoded by genes that also bear mucin core sequences,62 but in mammals, no such homologues are yet known. In this respect, a goblet cell specific enhancer element is present in the promoter region of the rat TFF3 gene, and is bound by a goblet cell nuclear protein, thus providing a link between the goblet cell phenotype and the expression of TFF3.63 ,64
Thus there could be a direct interaction between TFF and mucins, either through the oligosaccharide side chains or the core protein at exposed sites, but there is no direct evidence of this proposal as yet. All three TFFs are co-expressed in humans in gut regions where chronic ulceration has occurred. It has been suggested that the interaction between TFF2 and mucins inhibits, both in vivo and in vitro, proton permeation through the mucus layer.65 Indeed, the addition of trefoil factors to purified mucin preparations leads to a rapid increase in optical density and viscosity and the combination acts synergistically in cell migration assays.66 Hence TFFs may participate in the stabilisation of the mucous gel that overlies the gastrointestinal epithelium, and exert their effects in this way.
In spite of numerous in vitro and in vivo studies, it has never been shown that TFF1 enhances epithelial cell growth. TFF2 is occasionally found to be mitogenic, possibly as a result of an increase in the availability of glutathione within the cells.67 So far it seems that trefoil factors are motogens, possibly morphogens, but not mitogens.
Other important features of trefoil factor expression
TFF1 is expressed in a wide variety of human carcinomas as mentioned earlier. Interestingly, in a TFF1 knockout mouse model68 all mice showed loss of expression of gastric mucus and showed notably elongated pits, occupying the whole mucosa. Epithelial cells exhibited severe hyperplasia and high grade dysplasia, adenomas develop in the antrum of the stomach, and some of these progress into frankly invasive carcinomas. It was therefore proposed that TFF1 may be a specific tumour suppressor gene for the stomach.
TFF2 mRNA levels increase within 30 minutes after mucosal injury induced by a cryoprobe on the serosal surface of the rat stomach.54 TFF2 increases cell migration in in vitro models of cell wounding60 ,61 and it also acts as a cytoprotective agent in rats treated with indomethacin.61 ,69 Thus TFF2 may be regarded as one of the rapid response peptides, upregulated very quickly after mucosal damage.70 Selective TFF2 knockout or transgenic mice have yet to be made, but it is interesting that no tumours have been found to express abundant TFF2.
TFF3 also plays an important role in the repair and healing of the gastrointestinal tract. In TFF3 knockout mice71 there is impaired mucosal healing and death from extensive colitis after oral administration of dextran sulphate sodium, an agent that only causes mild to severe epithelial injury in wild type mice. Repletion of TFF3 deficient mice by luminal supply of recombinant TFF3 resulted in restoration of normal healing and enhanced epithelial migration after acetic acid induced mucosal injury. Although the expression of the two other gastrointestinal trefoil factors seemed to be normal, the TFF3 deficient mice still had poor epithelial regeneration after injury, indicating that TFF1 and TFF2 cannot compensate for this deficiency in this model, as we might expect from their site specificity of expression (but see later).
THE ULCER ASSOCIATED CELL LINEAGE
The importance of the trefoil factors in mucosal repair and healing are well illustrated in the ulcer associated cell lineage (UACL; fig 3). All trefoil factors are expressed the UACL developing in humans in response to chronic ulceration.10 ,26 ,56 ,58 In situ hybridisation shows TFF2 mRNA in the acini and lower duct cells, whereas TFF1 mRNA and protein are localised in large amounts in the upper duct and all surface cells. TFF3 is expressed throughout the UACL with some glands showing stronger expression than others.10 Thus the UACL produces trefoil peptides which participate actively in mucosal repair. The mucous cells adjacent to the UACL express TFF1, with TFF1 again co-packaged with mucous granules. We have mentioned earlier that neuroendocrine cells adjacent to the UACL in Crohn’s disease also express TFF1.51 The function of these cell lineages is very different. Mucus is released into the lumen where it has lubricating and protective functions, whereas endocrine secretions release various regulatory peptides into the local circulation that act via paracrine or autocrine mechanisms to produce manifold effects on the gut. The expression of TFF1 by two different lineages suggests that it may have a dual mechanism of action—one is through the interaction with the overlying mucus layer and the other through an action on putative trefoil receptors on the basolateral surface of the epithelial cells (see later).
Recently, an animal model of UACL was reported by Hanbyet al in which UACL was induced in the distal oesophagus by oesophago-jejunal anastomosis.72 UACL buds appear in crypt bases in the adjacent jejunal mucosa. TFF3 mRNA is localised patchily throughout the UACL, whereas TFF1 mRNA was found in the upper portions of the lineage and TFF2 mRNA and its product in the acini—essentially similar to that of early human UACL. Thus this model will allow a detailed study of the histogenesis and the origin and temporal progression of the UACL.
Mechanism of action
The mechanism by which trefoil factors bring about mucosal protection and healing may result from their mucus stabilisation effect or motogenic actions, or possibly a combination of the two. But it is interesting that both orally administrated peptide69 as well as a much smaller amount of subcutaneously infused peptide61 are both apparently active in mucosal protection. The protective effect of subcutaneous TFF2 is seen at infused doses as small as 25 μg/kg/h, substantially less than that needed if given orally (1–15 mg/rat).69 This strongly suggests the presence of basolateral receptors or of transporting proteins which mediate the action of TFF2.61 TFF3 mediates epithelial chloride transport after application to the basolateral surface of monolayers of colorectal carcinoma cells or of rat jejunum.73 Furthermore, TFF3 seems to bind a protein present in membrane preparations of colorectal epithelial cells, accompanied by phosphorylation of tyrosine, and the phosphorylation occurs within 10 seconds—redolent of a receptor mediated response.74 Autoradiographic study of frozen rat gastrointestinal tissue using 125I-labelled rTFF3 showed the presence of a high density of specific 125I-rTFF3 binding sites in the gastric, colonic and jejunal mucosal glands.74 Receptors for TFF2 have also been proposed in epithelial membranes that activate adenlyate cyclase.75The trefoil domain is highly conserved during evolution from amphibians to mammals: nuclear magnetic resonance or xray crystallographic analysis of TFF1 and TFF2 shows that the TFF domain has a compact structure containing a binding site which looks potentially suitable for mucin sugar side chains, but equally it may also be argued that it represents a binding site for receptors.76-78
If trefoil factors act as cross-linkers in the mucus gel, then at least two trefoil domains are required for this action, only apparently possible in one of the mammalian trefoil factors—TFF2 as it contains two trefoil domains. However, both TFF179 ,80 and TFF381 are capable of dimerising and are potentially able to form heterodimers, and thus single domain trefoil factors could interact with the mucus gel. Furthermore, Marchbank and colleagues82 have shown that the dimeric form of TFF2, which forms dimers via Cys,58 is more potent than the monomeric form in preventing indomethacin induced gastric damage and also a stronger stimulant of the rate of migration of cells at the leading edge of wounded monolayers, suggesting that the Cys58 residue may play an important role in the biological function of TFF1.80 The dimerisation of TFF1 could also be necessary to facilitate binding to any putative receptors as a disulphide bond itself can have a significant effect on receptor binding.83
It is thus clear that all trefoil factors are powerful motogens in the gastrointestinal tract. But how do they act at the molecular level? It has been shown that recombinant rTFF3 causes tyrosine phosphorylation of β-catenin and the EGF receptor (EGFR) in the HT29 colonic carcinoma cell line: tyrosine phosphorylation of β-catenin was associated with reduced membranous E-cadherin expression, with consequent perturbation of intercellular adhesion, and promotion of cell motility (fig 4).85 Recently, it has also been shown, in the HT29 cell line, which harbours anadenomatous polyposis coli (APC) mutation but has a normal E-cadherin–catenin complex, that rTFF3 leads to downregulation of E-cadherin, decreased cell–cell and cell–substratum adhesion, downregulation of expression of APCand α- and β-catenin, translocation of APC from the cytoplasm to the nucleus, and the induction of apoptotic changes.86 The effects of rTFF3 are prevented by inhibition of protein tyrosine kinases. Thus TFF3 may act through a signalling pathway involving the APC–catenin and E-cadherin–catenin complexes, mediated via a putative receptor through tyrosine phosphorylation. Moreover, TFF3 has been shown to decrease extracellular signal related protein kinase (ERK) activity. ERK is a member of the mitogen activated protein kinases (MAPK) family.87 The effect of TFF3 on ERK activity was blocked by a tyrosine phosphatase inhibitor, indicating that abrogation of the MAPK pathway by TFF3 is mediated by tyrosine phosphatase or a dual specific phosphatase. It is still unclear, however, whether these effects operate through cell surface receptors. Furthermore, the close association of TFF3 with EGFR is shown by a synergistic action of TFF3 on electrogenic chloride transport,73 cell migration and cytoprotection.88 Hence trefoil peptides may act closely with EGFR, APC, the E-cadherin–catenin complex and the MAPK family.
Trefoil factors seem to play a central role in gastrointestinal mucosal defence and repair. In vitro studies show that they are effective motogens, able to stimulate epithelial restitution, but their exact mechanisms at the cellular/molecular level are still unclear; they may act via a receptor or by interaction with mucus or by both mechanisms. The ectopic expression of trefoil factors in various carcinomas may prove to be a useful prognostic marker, but the biological effects of these proteins in tumour progression are still not known. Further studies in these areas are urgently required as they may shed light on the role of trefoil peptides in malignancy and the possible therapeutic use of trefoil factors in ulcerative conditions of the gastrointestinal tract.
This work was supported by the Imperial Cancer Research Fund and a Croucher Foundation Fellowship to WMW.
- trefoil factor family
- spasmolytic polypeptide
- intestinal trefoil factor
- ulcer associated cell lineage
- medullary thyroid carcinoma
- transforming growth factor
- epidermal growth factor
- adenomatous polyposis coli
- extracellular signal related protein kinase
- mitogen activated protein kinases
- hepatocyte growth factor