Trefoil Peptide - an overview

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43.3.1.3 Trefoil Peptides

The mucous gel contains trefoil peptides, a class of protein that is important to mucosal defense. The trefoil gene family included three peptides expressed in the gastrointestinal tract (TFF1, TFF2, and TFF3) All trefoil gene products are 7–12 kDa peptides and share a common structure of three internal cysteine bonds that yield the signature “trefoil” structure of three internal loops, and all are stored in mucous secretory cells and secreted in parallel with mucins.34 The stomach contains TFF1 stored in the gastric pit and surface mucous cells, and TFF2 is present in the gastric gland mucous cells. In the duodenum, TFF3 is stored in the small and large intestinal goblet cells.35–38 Secreted TFF1 and TFF2 are expressed in the adherent mucous gel layer, with the concentration of TFF2 in rat gastric mucus estimated at 10 μM.36 The coordinated localization of trefoil peptides and mucins in the normal gastrointestinal epithelium and in the ulcer-associated cell lineage suggests that they may organize the protection and repair of the gastrointestinal mucosa.37,39 Trefoil peptides are markedly protease resistant, prolonging their survival in the gastric lumen.40 Addition of TFF2 to mucin solutions significantly increases viscosity and elasticity, in which the mucin solutions are turned into a gel-like state.41 These effects on the physical properties of mucus, however, are only one aspect of trefoil peptide actions, which include regulation of gastric acid secretion and of cellular differentiation. TFF2−/− (knockout) mice have modest changes in tissue morphology in unstressed animals (shorter gastric pits and gastric glands with decreased epithelial migration with no detected change in mucous cell number) with unaffected TFF1 mRNA abundance. TFF2−/− animals had increased basal net acid output (decreased bicarbonate secretion or increased acid secretion), and a fourfold increase in the number of lesions after 12 hour exposure to the non-selective cyclooxygenase (COX) inhibitor indomethacin.42 Chronic treatment with a proton-pump inhibitor (omeprazole) increased TFF2 concentration in gastric secretions, thereby promoting repair and preventing injury to the gastric mucosa in response to luminal noxious agents.43 Evidence also suggests that trefoil peptides promote epithelial restitution across damaged epithelium (see also Chapter 42). Using in vivo confocal microscopy, 3–5 gastric epithelial cells were photodamaged using a two-photon laser with cellular restitution and surface pH (pHs) monitored. Increased pHs over the injured area was observed only in control mice, mitigated by exogenous TFF2 (Figure 43.2). Injury-associated extracellular alkalinization, but not repair, was blocked by luminal application of the anion exchange inhibitor H2DIDS or in apical anion exchanger SLC26A9 knockout mice, consistent with alkalinization mediated by the SLC26A9 anion exchanger. Exogenous PGE2 alkalinized pHs in control, but not in SLC26A9 mice. From these studies it was concluded that the SLC26A9 anion exchanger is the dominant mechanism whereby the gastric surface layer is alkalinized in response to microinjury.10 Recombinant TFF accelerated the healing rate of burn stress ulcers in rats.44,45 Thus, results suggest multiple physiologic functions for TFF2 in the promotion of mucosal healing.

Figure 43.2. Time course of changes in damaged area after laser-induced photodamage (LPD) in (A) control mice, (B) trefoil factor 2 knockout (TFF2-KO) mice, and (C) TFF2-KO mice after topical addition of recombinant rat TFF3 (rTFF3).

(Left) In each panel, eight images from a representative time course experiment. In those images, the white rectangle in the first image of the series shows the gastric surface region exposed to LPD (7034 mm2) at time zero, and all other images indicate the time of imaging the same tissue location after LPD. (Right) results are compiled for the indicated number of experiments (each collected from at least three animals) with values presented as mean ± SEM.

(Modified and adapted from 9.)

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Trefoil Factor Family Proteins

TFF proteins are small 7- to 12-kDa, highly stable proteins that are produced by mucin-secreting epithelial cells found throughout the body (709–713,753–756). Synthesized as precursors containing an N-terminal signal sequence, TFF proteins are copackaged into mucous granules within the Golgi apparatus and eventually secreted onto the epithelial cell surface (713). Although trefoil peptides are principally expressed within the gastrointestinal tract, their expression has also been observed in a variety of other tissues including conjunctiva, uterus/endocervix, salivary glands, respiratory tract, nonlactating breast, gallbladder, pituitary, hypothalamus, hippocampus, frontal cortex, and cerebellum (757–769). Human serum contains approximately 150 pg/ml TFF1, whereas human urine has a TFF1 concentration of 14 ng/mg creatinine (770–772). The concentration of TFF2 in gastric juice has been reported to be between 1 and 20 μg/ml with dramatic diurnal variation (773).

Within the gastrointestinal tract, trefoil proteins display cell–specific patterns of expression that are both temporally and spatially regulated (753). TFF1 and TFF2 are considered “gastric” peptides showing complementary expression within the pit mucous cells and mucous neck cells of the stomach, respectively (753,758,764,774–777). TFF2 expression is also observed within Brunner's glands of the duodenum (776,778,779). In contrast, expression of TFF3 appears “intestinal,” being primarily limited to goblet cells of the small intestine and colon (740,780–782). These cell–specific patterns of TFF expression result in a regional colocalization of specific TFF proteins and secretory mucins, thereby providing a potential molecular basis for the functional mucosa specificity.

TFF expression occurs early in embryonic development, with all three TFF proteins coordinately expressed within the gastrointestinal tract (753,783,784). As development proceeds, the expression of the individual TFF peptides becomes progressively more restricted, resulting in the cell–specific patterns observed in the adult. Similar developmental regulation of TFF expression has also been observed in the brain (767). It is important to note that there is a large degree of species variation in the expression patterns of TFF proteins.

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Overview and Discovery of Trefoil Factor Family

Trefoil factor family (TFF) represents a relatively new class of biologically active peptides that appear critical in maintaining gastrointestinal epithelial integrity under normal and pathologic conditions (709–713). TTF peptides are differentially synthesized wherever mucin secretion occurs, being prominently expressed within the gastrointestinal mucosa. TFF proteins are constituents of mucous gels and have been shown to rapidly modulate mucosal restitution through a combination of motogenic, cell–scattering, antiapoptotic, and anti-inflammatory activities. Several members of the trefoil family have also been implicated as gastrointestinal-specific tumor-suppressor genes, whereas the coordinate expression of TFF peptides within neural tissues suggests a potential neuromodulatory role for these proteins both within the brain and peripheral targets including the gastrointestinal tract.

The trefoil, or P domain, is a conserved 38- to 39-amino-acid motif containing 6 cysteine residues held together by 3 pairs of disulfide bonds configured 1-5, 2-4, and 3-6 (714). This domain produces a compact “three-leafed clover” protein structure that has marked thermal and enzymatic stability, a property that is most likely critical to the function of trefoil proteins within the harsh environment of the gastrointestinal tract (715–719). The trefoil domain has been highly conserved through evolution and is found in numerous species from Xenopus to humans (709). Three distinct trefoil peptides have been identified in mammals. TFF1, originally designated pS2, was isolated from an estrogen-sensitive breast cancer cell line (720). TFF2, or spasmolytic polypeptide, was identified in the early 1980s during purification of porcine insulin (715), whereas intestinal trefoil factor, or TFF3, was isolated from rat intestine in 1991 (721). TFF1 and TFF3 each contain a single-trefoil domain, whereas TFF2 contains two-trefoil motifs as a result of genomic duplication (721–723).

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1 Introduction

The trefoil factors (TFF) are soluble proteins, comprising of small (12–22 kD) peptides, which have a common three leaved/looped structure formed by inter-chain disulphide bond. The first member of this family was discovered around thirty years back in 1980s, and was named as PSP (pancreatic spasmolytic peptide).1 The mammalian trefoil factor family consists of three peptides, TFF1, TFF2 and TFF3, which are secreted mainly from mucous epithelia with mucus gel.2 The predominant secretion of TFF occurs from gastric mucosal lining, small and large intestine, oral mucosal cells, and salivary glands.3 They are also expressed within the brain.4 The trefoil structures form homo- or hetero-dimers, either with the same peptide or with other trefoil peptide,5 and this dimerization is responsible for proteolysis and acid resistance against harsh internal environment.6 Different members of the family are expressed by different cells, such as TFF1 is expressed mainly from gastric mucosal lining,3 while TFF2 is expressed mainly from Brunner's gland of the duodenum.2 TFF3 is expressed mainly by intestinal goblet cells.3 In 1982, TFF2 was the first member of this family discovered during purification of insulin from the porcine pancreatic cells. Initially, it was named as PSP, as it showed the inhibitory effect on gastric secretion.2 The second member discovered of this family was TFF1 and was initially named as human breast cancer associated peptide 2, as it was discovered unintentionally with the gene regulated by estrogen in breast cancer cells (MCF-7).7 TFF3 is the last known member of this family, which was cloned from rat intestinal epithelial cells and was named as intestinal trefoil factor.8 The human homologue was then cloned and named hP1B.9

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8.5.3 Trefoil Factor Family Proteins

TFF proteins are small 7–12 kDa, highly stable proteins that are produced by mucin-secreting epithelial cells found throughout the body.709–713,753–756 Synthesized as precursors containing an N-terminal signal sequence, TFF proteins are copackaged into mucous granules within the Golgi apparatus and eventually secreted onto the epithelial cell surface.713 Although trefoil peptides are principally expressed within the gastrointestinal tract, their expression has also been observed in a variety of other tissues including conjunctiva, uterus/endocervix, salivary glands, respiratory tract, non-lactating breast, gallbladder, pituitary, hypothalamus, hippocampus, frontal cortex, and cerebellum.757–769 Human serum contains approximately 150 pg/ml TFF1, whereas human urine has a TFF1 concentration of 14 ng/mg creatinine.770–772 The concentration of TFF2 in gastric juice has been reported to be between 1–20 μg/ml with dramatic diurnal variation.773

Within the gastrointestinal tract, trefoil proteins display cell-specific patterns of expression that are both temporally and spatially regulated.753 TFF1 and TFF2 are considered “gastric” peptides showing complementary expression within the pit mucous cells and mucous neck cells of the stomach, respectively.753,758,764,774–777 TFF2 expression is also observed within Brunner’s glands of the duodenum.776,778,779 In contrast, expression of TFF3 appears “intestinal”; it is primarily limited to goblet cells of the small intestine and colon.740,780–782 These cell-specific patterns of TFF expression result in a regional colocalization of specific TFF proteins and secretory mucins, providing a potential molecular basis for the functional mucosa specificity.

TFF expression occurs early in embryonic development, with all three TFF proteins coordinately expressed within the gastrointestinal tract.753,783,784 As development proceeds, the expression of the individual TFF peptides becomes progressively more restricted, resulting in the cell-specific patterns observed in the adult. Similar developmental regulation of TFF expression has also been observed in the brain.767 It is important to note that there is a large degree of species variation in the expression patterns of TFF proteins.

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Abstract

Trefoil factor family (TFF), composed of TFF1, TFF2, and TFF3, is a cluster of secreted peptides characterized by trefoil domain (s) and C-terminal dimerization domain. TFF1, a gastric tumor suppressor, is a single trefoil peptide originally detected in breast cancer cell lines but expressed mainly in the stomach; TFF2, a candidate of gastric cancer suppressor with two trefoil domains, is abundant in the stomach and duodenal Brunner's glands; and TFF3 is another single trefoil peptide expressed throughout the intestine which can promote the development of gastric carcinoma. According to multiple studies, TFFs play a regulatory function in the mammals' digestive system, namely in mucosal protection and epithelial cell reconstruction, tumor suppression or promotion, signal transduction and the regulation of proliferation and apoptosis. Action mechanisms of TFFs remain unresolved, but the recent demonstration of a GKN (gastrokine) 2-TFF1 heterodimer implicates structural and functional interplay with gastrokines. This review aims to encapsulate the structural and biological characteristics of TFF.

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TFF-1 Loss

Loss of the trefoil peptide TFF1(pS2), a stable 3-loop molecule synthesized in mucus-secreting cells, has been described in approximately 50% of GCs.68 The biologic significance of this loss was reported in a knockout mouse model of gastric antral neoplasia.69 Additionally, expression of TFF1 was observed to be lower in some gastric intestinal metaplasia and gastric adenomatous lesions compared with adjacent normal or hyperplastic mucosa.70 TFF1 resides on chromosome 21q22, a region noted to be deleted in some GCs in LOH studies.71 Overexpression of TFF1 in the GC cell line, AGS, inhibited its growth.72 Furthermore, the overwhelming majority of GCs studied had absent to minimal transcript levels compared with normal gastric mucosa.73 Moreover, C/EBP-β was found to be overexpressed in most GCs in a corresponding manner and bind to the promoter of TFF1, suggesting a regulatory factor role.74 Finally, cytokine signaling through the coreceptor gp130, with increased STAT-3 expression, has also been observed to decrease TFF1 and lead to gastric lesions in mice.75 Additionally, characterization of gastrokine (GKN) 2, a stomach-specific tumor suppressor protein, was found to be structurally and physically related to TFF1.76 Noteworthy, loss of GKN1 and GKN2 expression was found to occur in gastric adenocarcinoma and portend an overall shorter survival.77

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Acquired somatic genetic/molecular alterations

TFF-1 Loss

Loss of the trefoil peptide TFF1(pS2), a stable 3-loop molecule synthesized in mucus-secreting cells, has been described in approximately 50% of GCs.68 The biologic significance of this loss was reported in a knockout mouse model of gastric antral neoplasia.69 Additionally, expression of TFF1 was observed to be lower in some gastric intestinal metaplasia and gastric adenomatous lesions compared with adjacent normal or hyperplastic mucosa.70 TFF1 resides on chromosome 21q22, a region noted to be deleted in some GCs in LOH studies.71 Overexpression of TFF1 in the GC cell line, AGS, inhibited its growth.72 Furthermore, the overwhelming majority of GCs studied had absent to minimal transcript levels compared with normal gastric mucosa.73 Moreover, C/EBP-β was found to be overexpressed in most GCs in a corresponding manner and bind to the promoter of TFF1, suggesting a regulatory factor role.74 Finally, cytokine signaling through the coreceptor gp130, with increased STAT-3 expression, has also been observed to decrease TFF1 and lead to gastric lesions in mice.75 Additionally, characterization of gastrokine (GKN) 2, a stomach-specific tumor suppressor protein, was found to be structurally and physically related to TFF1.76 Noteworthy, loss of GKN1 and GKN2 expression was found to occur in gastric adenocarcinoma and portend an overall shorter survival.77

E-Cadherin Alteration

In addition to germline mutations leading to HDGC described previously, several sporadic diffuse GCs have displayed altered E-cadherin. E-cadherin is a transmembrane, calcium ion–dependent adhesion molecule important in epithelial cell homotypic interactions, which, when decreased in expression, is associated with invasive properties.78 Reduced E-cadherin expression determined by immunohistochemical analysis was noted in most GCs, 92% of 60 cases, when compared with their adjacent normal tissue.79 Genetic abnormalities of the E-cadherin gene and transcripts were demonstrated in a third to one-half of diffuse GCs.23,80 E-cadherin splice site alterations producing exon deletion and skipping, large deletions including allelic loss, and point mutations mostly of the missense nature have been demonstrated in diffuse-type cancers, some even exhibiting alterations in both alleles.81 Methylation of the E-cadherin promoter region was found in 16 (26%) of 61 GCs studied.61

Kinases/Phosphatases

Activation of protein kinases by tyrosine kinases and phosphatases has been shown to affect many cellular activities, including cell growth, differentiation, and survival.82 Noteworthy, a novel KGF-R2 phosphorylation inhibitor, Ki23057, decreased proliferation of 2 scirrhous GC cell lines with K-sam amplification.83 Phosphatidylinositol 3-kinases (PI3K) are lipid kinases that regulate pathways for neoplastic proliferation, adhesion, survival and motility.84 Mutations in PIC3A, which encodes a catalytic subunit of PI3K, have been characterized in several cancers including gliobastoma, colon, and up to 25% of GCs analyzed.85 Further attempts at identifying mutations in PIC3A have reported a lower prevalence of 4.3% in GCs; however, this study also reported increased expression of this gene product in gastric tumors and a specific expression profile associated with this upregulation.86 Moreover, increased PI3K/AKT signaling from nuclear factor kappaB (NF-kB) has been shown to activate transcription of HuR, which has proliferative and antiapoptotic effects on GC cells.87 Furthermore, a kinase involved in chromosome segregation and stability, STK15 (BTAK, Aurora2), was found to be amplified and overexpressed in GCs.88

Wang and colleagues89 identified 83 somatic mutations in human protein tyrosine phosphatases (PTPs), most affecting colon cancer, but also found them in 17% of GCs analyzed. The most commonly altered PTP, protein-tyrosinase phosphatase-receptor type (PTPRT) was found to decrease its activity, suggesting a role as a tumor suppressor gene. However, an additional polymerase chain reaction–based study suggests a much smaller role for alterations in PTPRT in gastric tumorigenesis, being found in only 1% of cases.90 Overexpression of DARP32 and a novel truncated isoform was found in most GCs.91 Moreover, an antiapoptotic effect of this overexpression of DARP32 and t-DARP was observed in vitro.92

Overexpression of the MET gene, which encodes a tyrosine kinase receptor for the hepatocyte growth factor, has been reported to have prognostic value to indicate poorer survival in multivariate analysis.93 There have been numerous reports in the literature indicating that the MET gene is amplified in approximately 15% and its expression elevated in up to 50% of GCs.94

Methylation Silencing Alterations (Epigenetic Alteration)

A significant number (41%) of GCs exhibited CpG island methylation in a study of the promoter region of p16.61 Many of these cases with hypermethylation of promoter regions displayed the MSI-H phenotype with multiple sites of methylation, including the MLH1 promoter region.95

RUNX3, a tumor suppressor gene, appears to suppress gastric epithelial cell growth by inducing p21 (WAF1/Cip1) expression in cooperation with TGF-β–activated SMAD.96 RUNX3 was found to be altered in 82% of GCs through either gene silencing or protein mislocalization to the cytoplasm.97 Multiple other tumor suppressor genes and candidates, including protocadherin 10 (PCDH10), CDKN2A, GSTP1, APC, MGMT, DAKP, XAF1, THBS-1, RUNX1, and CDH1, have been shown to be methylated in GCs.98

Apoptosis Signaling Alterations

Mutations of the BAX gene, believed to be a promoter of apoptosis, have been identified in up to 33% of GCs, as well as in colorectal and endometrial cancers.99 Another study supporting the role of the BAX gene in tumor suppression linked low expression levels of the protein Bax-interacting factor-1 (Bif-1) in GCs, suggesting another breakdown in the pathway of regulating apoptosis.100 The bcl-2 homolog BAK, another promoter of apoptosis, was found to have missense mutations in 12.5% of GCs analyzed.101 Defects in cell surface receptors of the apoptosis pathway have also been observed in gastric tumors, such as in the death receptors DR4 and DR5.102

Other Alterations

The p53 gene has consistently been demonstrated to be altered in GCs, both by allelic loss in more than 60% of cases and mutations identified in approximately 30% to 50% of cases.103 The spectrum of mutations in this gene is similar to other cancers, with a predominance of base transitions, especially at CpG dinucleotides.

The human epidermal growth factor receptor 2 (HER2/neu/ERBB2) has been demonstrated to be overexpressed in many types of cancer, including GC; and to be associated with metastasis. HER2 was shown to interact with CD44 and upregulated CXCR4 through epigenetic silencing of microRNA (miRNA)-139 in GC cells.104 Several of the vascular endothelial growth factor (VEGF) gene polymorphisms were found to be independent prognostic markers for patients with GC and the analysis of VEGF gene polymorphisms may help identify patients at greatest risk for poor outcomes.105

Evidence of tumor suppressor loci on chromosome 3p has accumulated from a variety of studies, including allelic loss in primary GCs (46%) and homozygous deletion in a GC cell line (KATO III), as well as xenografted tumors.106 The FHIT gene was isolated from the common fragile site FRA3B region at 3p14.2 and found to have abnormal transcripts with deleted exons in 5 of 9 GCs.107 Furthermore, loss of FHIT protein expression was demonstrated immunohistochemically in most GCs.108 One somatic missense mutation was identified in exon 6 of the FHIT gene during a coding region analysis of 40 GCs.109 Additional studies are needed to determine the role that breakpoints in this region of 3p have in GC development.

Somatic mutations in the chromatin remodeling gene, ARIDIA, occurred in several GCs as well as other tumors.110 Only 1 of 35 GCs contained an intragenic mutation of SMAD4 along with allelic loss, suggesting this gene is infrequently altered in sporadic gastric tumorigenesis.96,111 Additionally, only one missense change of uncertain functional significance in the LKB1 (STK11) gene was noted in a study of 28 sporadic GCs.112

Members of the Wnt signaling pathway, APC and β-Catenin, have had several somatic alterations noted in a few cases of GC.113 Missense somatic mutations in β-Catenin, which also has connections with the cell adhesion complex involving E-cadherin, as well as Lcf/LEF transcription regulation, were identified in a few cases of intestinal-type GC.114 Elevated expressions of the hedgehog target genes, human patched gene 1 (PTCH1) or Glil, were reported in 63 of 99 primary GCs. Interestingly, treatment of GC cells with KAAD-cyclopamine, a hedgehog signaling inhibitor, decreased expression of Glil and PTCH1, resulting in cell growth inhibition and apoptosis.115 Additionally, cyclopamine decreased the growth of GC cell lines that expressed high levels of spermine oxidase.116

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General Considerations

Advances in recombinant peptide technology now allow virtually pure peptide to be produced at moderate cost. This removes the concern of contamination with infectious agents (as seen in some patients treated with purified growth hormone who develop Creutzfeldt– Jakob disease) and the development of antibodies to purified peptides from other species (as seen with porcine insulin). The most widely prescribed recombinant peptide used by gastroenterologists is probably interferon-α for the treatment of infectious liver disease. The use of growth factors for hollow organ gastroenterology is, however, at a much earlier stage. One of the major areas of concern regarding the use of peptides such as EGF in the clinical setting is the worry that systemic administration of growth factors may act to promote tumor growth elsewhere in the body. Oral, as opposed to systemic, administration of growth factors provides one possible approach to this problem. However, for EGF and the trefoil peptides, the oral doses required to treat gastrointestinal damage may be up to 1000 times greater than when the peptide is given systemically, making oral therapy economically unrealistic. Stabilizing molecules against digestion in the small intestine is likely to be more difficult due to the many different proteolytic enzymes produced by the pancreas. Possible strategies include administering the peptide along with nonspecific serine protease inhibitors or administering the peptide in a site-specific release formulation to overcome these problems.

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prostaglandins (PGE2 in particular; its synthesis is blocked by non-steroidal anti-inflammatory drugs, majorly contributing to their ulcerogenicity)

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epidermal growth factors (e.g. heparin-binding epidermal growth factors, amphiregulin)

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‘trefoil’ peptides which are secreted into the lumen and may monitor for damage and protect the mucosa.

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