PERSPECTIVE
Trefoil Peptides in Airway Epithelium
An Important Addition to the Plethora of Peptides

Steven R. White

Section of Pulmonary and Critical Care Medicine, The University of Chicago, Chicago, Illinois

An increasing number of regulatory peptides have been demonstrated to be expressed in airway epithelium; these modulate various aspects of proliferation, motility and secretory function. These recognized peptides include epidermal growth factor (EGF), transforming growth factor beta (TGF-), the insulin-like growth factor family, the fibroblast growth factor family, a few other seemingly unrelated regulatory peptides, such as hepatocyte growth factor, platelet-derived growth factor, and various interleukins, interferons and tumor necrosis factor-related proteins. Many of these peptides have roles in other epithelial structures such as intestine and breast, and insights gained in these tissues have been useful in understanding airway function. Over the past twenty years, one family of peptides has been identified as an important regulator of gastric and intestinal epithelial repair and motility. Known as the trefoil factor family (TFF), their abundant expression in both normal and neoplastic tissues has stimulated substantial interest in understanding their regulatory roles in cell motility and mucous secretion.

The trefoil family shares a variable number of conserved motifs, usually 39 to 43 residues each, based on a compact three-dimensional trefoil structure of six cysteine residues of almost invariant spacing held together by three pairs of disulfide bonds (1, 2). This structure gives the peptide a characteristic three-loop shape (Figure 1) and provides for exceptional resistance to acid digestion and proteolytic degradation. Other conserved residues have been shown to form a hydrophobic patch on the surface of the trefoil domain which may provide a binding cleft for either a sugar group or an aromatic amino acid side-chain during intermolecular interactions. Three trefoil peptides are currently recognized in humans: TFF1 (also known as pS2) and TFF3 (intestinal trefoil factor), which each have a single trefoil motif and both of which homodimerize, and TFF2 (human spasmolytic polypeptide, hSP), which has two motifs and does not require dimerization for its activity (Williams and Wright, 1997). Each map to the same cluster on chromosome 21 (3, 4). TFF2 is glycosylated via an N-linkage, and TFF2 polymorphisms are known to exist (5). The trefoil motifs in TFF2 form a tight structure compared to the loose structure formed by the dimerization of TFF1; this may lead to differences in binding and function (6).

View larger version (114K): [in this window] [in a new window]   Figure 1.   Representation of TFF1 (pS2), demonstrating the three characteristic loops created by disulfide bonds that provide the molecule with its characteristic shape. These bonds also provide resistance to acid and proteolytic digestion. The C-terminal end links to its counterpart in a homodimer, giving the dimer its flexibility. From reference (42).

The trefoil peptides localize predominately to the gastrointestinal tract (7). Each of the peptides are found in the intestinal tract within the ductal lumenal cells of Brunner's glands and in goblet cells near the surface of crypts. Both TFF1 and TFF2 are also found within the stomach in mucous neck cells of the antrum and fundus and in the epithelium of the pancreas and gall bladder. Peptides are packaged by the Golgi apparatus in mucin-producing cells and secreted with mucous into the protective layer above the epithelium. TFF3 also co-localizes with oxytocin-producing cells in the hypothalamus (8) and is found in epithelial cells of the female genitourinary tract (7). TFF1 is expressed in rat hippocampus, cortex, and cerebellum (9), in cultured mouse astrocytes (10) and normal breast ductal epithelium (11). TFF2 has not been reported to be expressed in normal tissues outside the gastrointestinal tract.

TFF1 was originally isolated from human breast carcinoma cell lines (12, 13). Pathologic expression of this trefoil peptide since has been demonstrated in a variety of adenocarcinomas, especially those with a mucinous histology, in breast, lung, endometrium, ovary, pancreas, stomach and intestine (summarized in 14). Expression in breast carcinoma is associated with estrogen receptor presence and a favorable clinical outcome (15). Expression in lung adenocarcinoma is common, though expression in non-malignant adenomas, squamous cell carcinoma, and small cell carcinoma is not seen (18). Expression in lung adenocarcinoma, in contrast to breast adenocarcinoma, has been associated with a poorer prognosis (19). The abundance of trefoil peptides in gastrointestinal malignancies is exactly opposite that seen with breast and lung malignancies. TFF1 abundance is decreased in gastric carcinomas and is lost completely in about 50% of these neoplasms (20). A similar decrease in trefoil peptide abundance has been demonstrated in colonic tumors, though complete disappearance is rare (21). The reasons for the wide discrepancies in abundance and prognosis await exploration.

The physiological functions of the trefoil peptides are several, and can be divided into two broad categories: mucosal surface protection and repair after injury. Trefoil peptides interact with mucins (22) and regulate mucous viscosity (23), and via these actions may enhance the protective capabilities of the mucosal defense barrier. Both glycosylated and non-glycosylated TFF2 is found in gastric secretions with a diurnal variation, highest at night, suggesting that the maximum cytoprotective effects of trefoil peptides occur at this time (24). Using a yeast two-hybrid system to seek TFF1-interacting proteins, Tomasetto and colleagues isolated clones which corresponded to the murine counterpart of fragments of MUC2 and MUC5AC mucins (23). Mutagenesis studies showed that TFF1 interacts by binding to the von Willebrand Factor (VWBF)C1 and VWBFC2 domains of these mucins, which are cysteine-rich. The TFF motif contains a hydrophobic binding pocket which could be a binding site for a protein side chain, for specific sugar residues, or a specific receptor. Binding the VWFC domain of mucins is one important step in the formation of mucus gel of high viscosity. One function of trefoil peptides, then, may be cooperating with mucins to regulate the protective function of the mucous gel over epithelial surfaces.

In gastrointestinal cells, each TFF enhances cell migration in vitro, and epithelial restitution and mucosal injury after injury in vivo (25, 26). Trefoil peptides are expressed in high concentrations in the ulcer-cell associated stem cell lineage at sites of mucosal ulceration in the stomach, and are thought to have a prominent role in ulcer repair (27). TFF1 must dimerize in order to elicit cell migration (28), and its effect is independent of neutralizing antibodies for TGF-. Generation of TFF3 -/- mice demonstrates an essential role for this peptide after chemical injury to the colon (29) or radiation injury to the intestinal tract (30); deficient mice had a higher degree of injury, delayed repair and increased mortality. In both models, providing exogenous TFF3 reduced both symptoms and mortality. Conversely, mice overexpressing TFF2 are protected against indomethacin-induced mucosal injury (31). The molecular basis for the mitogenic effect in intestinal cells involves the mitogen-associated protein kinase pathway (32) and phosphorylation of both -catenin and the EGF receptor (33). This suggests a receptor-mediated response as well as protection based on mucosal defense. The trefoil peptides may also have a role in cell proliferation and differentiation. Targeted disruption of the murine TFF1 gene led to the development of antral and pyloric mucosal thickening and the occurrence of large adenomas, compared to control mice (34). A significant proportion of these tumors contained foci of carcinoma. In contrast, the colons of these mice were normal.

Trefoil peptides have functional roles outside of the gastrointestinal tract, though these functions have not been explored in detail. As one example, injections of synthetic TFF3 into the rat amygdala is anxiolytic at a low dose and anxiogenic at a higher dose (35). The role of the trefoil peptides in tumorigenesis is less clear.

New reports now suggest that the trefoil peptides can be localized to the normal airway epithelium and may have a functional role similar to that seen in the gastrointestinal tract. In a recent study, Wiede and associates demonstrated the presence of TFF3 in airway mucosa and in the sputum of subjects with chronic bronchitis (36). In contrast, neither TFF1 nor TFF2 could be identified in the airways. As predicted from studies of the gastrointestinal tract, TFF3 could be localized to mucous cells in the acini of submucosal glands where it co-localized with MUC5B and MUC8, in goblet cells of the submucosal glands where it co-localized with MUC5AC, and to the surface epithelium. This co-localization is an important clue to the potential function of trefoil peptides in airways: the cooperation of trefoil peptides with these mucins may confer surface protection to the mucosa. Disruption of either set of peptides then may alter mucous viscosity and protection. Whether this indeed occurs in airway diseases associated with changes in mucous rheology, such as chronic bronchitis, cystic fibrosis, and asthma, is not known.

Trefoil peptides may also regulate epithelial cell motility in airways. In the present issue of the Journal, Oertell and colleagues demonstrate that both human recombinant TFF2 and TFF3 stimulate migration of human airway epithelial cells in chemotactic and two-dimensional wound repair assays (37). This effect could be enhanced by EGF, but the EGF receptor was not phosphorylated by the addition of trefoil peptide. This suggests that the two peptides exert their effects by mechanisms independent of EGF. This stands in contrast to intestinal cells where the EGF receptor is phosphorylated upon exposure to TFF3 (33). Other studies examining the interaction of trefoil peptides with growth factors and cytokines have demonstrated trefoil expression in gastrointestinal epithelium is not regulated by EGF, TGF-, IL-1, or IL-2 (38). As in the gastrointestinal tract, then, both function and expression of trefoil peptides may be independent of mediators and growth factors commonly considered in inflammatory airway diseases.

Multiple questions remain to be answered in understanding the role of trefoil peptides in the airway, both in terms of epithelial repair after injury and in the regulation of mucous secretion. Unlike the gastrointestinal tract, no distinct lineage of reparative cells has been demonstrated, and denuded basement membrane is thought to be repopulated by proliferation and migration of basal cells at the edge of the injury site (39). Are trefoil peptides expressed by these cells at and near injury sites in the airway, and if so, are they associated with active cell migration? Do the trefoil peptides have a protective role for the epithelium? If so, can a deficiency either in expression or function of these peptides be demonstrated in airway diseases? And very importantly, is the motility of epithelial cells as regulated by trefoil peptides dependent in part on the ability of these peptides to regulate mucous viscosity? The report of Wiede suggests that there were no differences in TFF concentration in sputum collected in a preliminary review of subjects with asthma, cystic fibrosis, bronchiectasis and chronic bronchitis (36). A more detailed analysis of trefoil peptide function in patients with airway diseases may delineate pathological differences that may be exploited in future therapies.

Delineation of the mechanisms of trefoil peptide function has advanced the understanding of several gastrointestinal disorders. Application of this knowledge to airway diseases may yield a richer understanding of airway epithelial repair after injury and mucin regulation. Since the trefoil peptides can function on the lumenal side of the mucosa, they may find a therapeutic role in enhancing repair and mucosal protection in airway inflammatory diseases. The apparent independence of trefoil expression and function apart from other growth factors and cytokines presents a unique opportunity to regulate these processes.

	Footnotes

Address correspondence to: Steven R. White, M.D., The University of Chicago, 5841 S. Maryland Ave., MC 6076, Chicago, IL 60637. E-mail: swhite{at}medicine.bsd.uchicago.edu

(Received in original form August 22, 2001).

Acknowledgments: This review was supported by Grants HL-60531 and HL-63300 from the National Heart, Lung and Blood Institute.

	References

1. De, A., D. G. Brown, M. A. Gorman, M. D. Carr, M. R. Sanderson, and P. S. Freemont. 1994. Crystal structure of a disulfide-linked "trefoil" motif found in a large family of putative growth factors. Proc. Natl. Acad. Sci. USA 91: 1084-1088 [Abstract/Free Full Text].

2. Polshakov, V. I., M. A. Williams, A. R. Gargaro, T. A. Frenkiel, B. R. Westley, M. P. Chadwick, F. E. B. May, and J. Feeney. 1997. High-resolution solution structure of human pNR-2/pS2: a single trefoil motif protein. J. Mol. Biol. 267: 418-432 [Medline].

3. Chinery, R., J. Williamson, and R. Poulsom. 1996. The gene encoding human intestinal trefoil factor (TFF3) is located on chromosome 21q22.3 clustered with other members of the trefoil peptide family. Genomics 32: 281-284 [Medline].

4. Schmitt, H., I. Wundrack, S. Beck, P. Gott, C. Welter, H. Shizuya, M. I. Simon, and N. Blin. 1996. A third P-domain peptide gene (TFF3), human intestinal trefoil factor, maps to 21q22.3. Cytogenet. Cell Genet. 72: 299-302 [Medline].

5. May, F. E. B., J. I. Semple, J. L. Newton, and B. R. Westley. 2000. The human two domain trefoil protein, TFF2, is glycosylated in vivo in the stomach. Gut 46: 454-459 [Abstract/Free Full Text].

6. Williams, M. A., B. R. Westley, F. E. B. May, and J. Feeney. 2001. The solution structure of the disulphide-linked homodimer of the human trefoil protein TFF1. FEBS Lett. 493: 70-74 [Medline].

7. Williams, G. R., and N. A. Wright. 1997. Trefoil factor family domain peptides. Virchows Arch. 431: 299-304 [Medline].

8. Jagla, W., A. Wiede, K. Dietzmann, K. Rutkowski, and W. Hoffmann. 2000. Co-localization of TFF3 peptide and oxytocin in the human hypothalamus. FASEB J. 14: 1126-1131 [Abstract/Free Full Text].

9. Hirota, M., H. Awatsuji, Y. Sugihara, S. Miyashita, Y. Furukawa, and K. Hayashi. 1995. Expression of pS2 gene in rat brain. Biochem. Mol. Biol. Int. 35: 1079-1084 [Medline].

10. Hirota, M., S. Miyashita, H. Hayashi, Y. Furukawa, and K. Hayashi. 1994. pS2 gene especially expressed in the late G1/S phase of mouse astrocytes. Neurosci. Lett. 171: 49-51 [Medline].

11. Piggott, N. H., J. A. Henry, F. E. B. May, and B. R. Westley. 1991. Antipeptide antibodies against the pNR-2 oestrogen-regulated protein of human breast cancer cells and detection of pNR-2 expression in normal tissues by immunohistochemistry. J. Pathol. 163: 95-104 [Medline].

12. Masiakowski, P., R. Breathnach, F. Bloch, F. Gannon, A. Krust, and P. Chambon. 1982. Cloning of cDNA sequences of hormone-regulated genes from the MCF-7 human breast cancer cell line. Nucleic Acids Res. 10: 7895-7903 [Abstract/Free Full Text].

13. May, F. E. B., and B. R. Westley. 1986. Cloning of estrogen-regulated messenger RNA sequences from human breast cancer cells. Cancer Res. 46: 6034-6040 [Medline].

14. Henry, J. A., M. K. Bennett, N. H. Piggott, D. L. Levett, F. E. B. May, and B. R. Westley. 1991. Expression of the pNR-2/pS2 protein in diverse human epithelial tumours. Br. J. Cancer 64: 677-682 [Medline].

15. Foekens, J. A., M.-C. Rio, P. Seguin, W. L. van Putten, J. Fauque, M. Nap, J. G. Klijn, and P. Chambon. 1990. Prediction of relapse and survival in breast cancer patients by pS2 protein status. Cancer Res. 50: 3832-3837 [Abstract/Free Full Text].

16. Henry, J. A., S. Nicholson, C. Hennessy, T. W. Lennard, F. E. May, and B. R. Westley. 1989. Expression of the estrogen-regulated pNR-2 mRNA in human breast cancer: relation to estrogen receptor mRNA levels and response to tamoxifen therapy. Br. J. Cancer 61: 32-38 .

17. Pallud, C., V. Le Dousal, M.-F. Pichon, J.-F. Prud'homme, K. Hacene, and E. Milgrom. 1993. Immunohistochemistry of pS2 in normal human breast and in various histological forms of breast tumours. Histopathology 23: 249-256 [Medline].

18. Higashiyama, M., O. Doi, K. Kodama, H. Yokuchi, H. Inaji, and R. Tateishi. 1996. Estimation of serum pS2 protein in patients with lung adenocarcinoma. Anticancer Res. 16: 2351-2356 [Medline].

19. Higashiyama, M., O. Doi, K. Kodama, H. Yokouchi, H. Inaji, S. Nakamori, and R. Tateishi. 1994. Prognostic significance of pS2 protein expression in pulmonary adenocarcinoma. Eur. J. Cancer [A] 30: 792-797 .

20. Machado, J. C., F. Carneiro, N. Blin, and M. Sobrinho-Simoes. 1996. Pattern of pS2 protein expression in premalignant and malignant lesions of gastric mucosa. Eur. J. Cancer Prev. 5: 169-179 [Medline].

21. Taupin, D., K. Ooi, N. Yeomans, and A. Giraud. 1996. Conserved expression of intestinal trefoil factor in the human colonic adenoma-carcinoma sequence. Lab Invest. 75: 25-32 [Medline].

22. Kindon, H., C. Pothoulakis, L. Thim, K. Lynch-Devaney, and D. K. Podolsky. 1995. Trefoil peptide protection of intestinal epithelial barrier function: co-operative interaction with mucin glycoprotein. Gastroenterology 109: 516-523 [Medline].

23. Tomasetto, C., R. Masson, J.-L. Linares, C. Wendling, O. Lefebvre, M.-P. Chenard, and M.-C. Rio. 2000. pS2/TFF1 interacts directly with the VWF cysteine-rich domains of mucins. Gastroenterology 118: 70-80 [Medline].

24. Semple, J. I., J. L. Newton, B. R. Westley, and F. E. B. May. 2001. Dramatic diurnal variation in the concentration of the human trefoil peptide TFF2 in gastric juice. Gut 48: 648-655 [Abstract/Free Full Text].

25. Podolsky, D. K.. 1997. Healing the epithelium: solving the problem from two sides. J. Gastroenterol. 32: 122-126 [Medline].

26. Wong, W. M., R. Poulsom, and N. A. Wright. 1999. Trefoil peptides. Gut 44: 890-895 [Free Full Text].

27. Wright, N.A.. 1998. Aspects of the biology of regeneration and repair in the human gastrointestinal tract. Phil. Trans. R. Soc. Lond. B 353: 925-933 [Medline].

28. Marchbank, T., B. R. Westley, F. E. B. May, D. P. Calnan, and R. J. Playford. 1998. Dimerization of human pS2 (TFF1) plays a key role in its protective/healing effects. J. Pathol. 185: 153-158 [Medline].

29. Mashimo, H., D.-C. Wu, D. K. Podolsky, and M. C. Fishman. 1996. Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor. Science 274: 262-265 [Abstract/Free Full Text].

30. Beck, P.L., and D. K. Podolsky. 1999. Intestinal trefoil factor reduces the severity of chemotherapy and radiotherapy-induced intestinal mucositis. Gastroenterology 116: G2133 .

31. .Playford, R. J., T. Marchbank, R. A. Goodlad, R. A. Chinery, R. Poulsom, and A. M. Hanby. 1996. Transgenic mice that overexpress the human trefoil peptide pS2 have an increased resistance to intestinal damage. Proc. Natl. Acad. Sci. USA 93: 2137-2142 [Abstract/Free Full Text].

32. Kanai, M., C. Mullen, and D. K. Podolsky. 1998. Intestinal trefoil factor induces inactivation of extracellular signal-regulated protein kinase in intestinal epithelial cells. Proc. Natl. Acad. Sci. USA 95: 178-182 [Abstract/Free Full Text].

33. Liu, D., I. El-Hariry, A. J. Karayiannakis, J. Wilding, R. Chinery, W. Kmiot, P. D. McCrea, W. J. Gullick, and M. Pignatelli. 1997. Phosphorylation of -catenin and epidermal growth factor receptor by intestinal trefoil factor. Lab. Invest. 77: 557-563 [Medline].

34. Lefebvre, O., M.-P. Chenard, R. Masson, J. Linares, A. Dierich, M. LeMeur, C. Wendling, C. Tomasetto, P. Chambon, and M.-C. Rio. 1996. Gastric mucosa abnormalities and tumorigenesis in mice lacking the pS2 trefoil protein. Science 274: 259-262 [Abstract/Free Full Text].

35. Schwarzberg, H., H. Kalbacher, and W. Hoffmann. 1999. Differential behavioral effects of TFF-peptides: injections of synthetic TFF3 into the rat amygdala. Pharmacol. Biochem. Behav. 62: 173-178 [Medline].

36. Wiede, A., W. Jagla, T. Welte, T. Köhnlein, H. Busk, and W. Hoffmann. 1999. Localization of TFF3, a new mucus-associated peptide of the human respiratory tract. Am J. Respir. Crit. Care Med. 159: 1330-1335 [Abstract/Free Full Text].

37. Oertel, M., A. Graness, L. Thim, F. Bühling, H. Kalbacher, and W. Hoffman. 2001. Trefoil factor family-peptides promote migration of human bronchial epithelial cells: synergistic effect with epidermal growth factor. Am. J. Respir. Cell Mol. Biol. 25: 418-424 [Abstract/Free Full Text].

38. Ogata, H., and D. K. Podolsky. 1997. Trefoil peptide expression and secretion is regulated by neuropeptides and acetylcholine. Am. J. Physiol. 273: G348-G354 [Abstract/Free Full Text].

39. Keenan, K., J. W. Combs, and E. M. McDowell. 1982. Regeneration of hamster tracheal epithelium after mechanical injury. III. Large and small lesions: comparative stathmokinetic and single pulse and continuous thymidine labeling autoradiographic studies. Virchows Arch. [Cell Pathol.] 41: 231-252 .

40. Erjefält, J. S., I. Erjefält, F. Sundler, and C. G. A. Persson. 1995. In vivo restitution of airway epithelium. Cell Tissue Res. 281: 305-316 [Medline].

41. Kim, J. S., V. S. McKinnis, and S. R. White. 1997. Proliferation and repair of guinea pig tracheal epithelium after neuropeptide depletion and injury in vivo. Am. J. Physiol. 273: L1235-L1241 [Abstract/Free Full Text].

42. Plaut, A. G.. 1997. Trefoil peptides in the defense of the gastrointestinal tract. N. Engl. J. Med. 336: 506-507 [Free Full Text].