Spasmolytic Polypeptide Research Articles

High-resolution solution structure of human intestinal trefoil factor and functional insights from detailed structural comparisons with the other members of the trefoil family of mammalian cell motility factors.

The secreted proteins intestinal trefoil factor (ITF, 59 residues), pS2 (60 residues), and spasmolytic polypeptide (SP, 106 residues) form a small family of trefoil domain-containing mammalian cell motility factors, which are essential for the maintenance of all mucous-coated epithelial surfaces. We have used 1H NMR spectroscopy to determine the high-resolution structure of human ITF, which has allowed detailed structural comparisons with the other trefoil cell motility factors. The conformation of residues 10-53 of hITF is determined to high precision, but the structure of the N- and C-terrminal residues is poorly defined by the NMR data, which is probably indicative of significant mobility. The core of the trefoil domain in hITF consists of a two-stranded antiparallel beta-sheet (Cys 36 to Asp 39 and Trp 47 to Lys 50), which is capped by an irregular loop and forms a central hairpin (loop 3). The beta-sheet is preceded by a short alpha-helix (Lys 29 to Arg 34), with the majority of the remainder of the domain contained in two loops formed from His 25 to Pro 28 (loop 2) and Ala 12 to Arg 18 (loop 1), which lie on either side of the central hairpin. The region formed by the surface of loop 2, the cleft between loop 2 and loop 3, and the adjacent face of loop 3 has previously been proposed to form the functional site of trefoil domains. Detailed comparisons of the backbone conformations and surface features of the family of trefoil cell motility factors (porcine SP, pS2, and hITF) have identified significant structural and electrostatic differences in the loop 2/loop 3 regions, which suggest that each trefoil protein has a specific target or group of target molecules.

Trefoil Peptides Promote Restitution of Wounded Corneal Epithelial Cells

The ocular surface shares many characteristics with mucosal surfaces. In both, healing is regulated by peptide growth factors, cytokines, and extracellular matrix proteins. However, these factors are not sufficient to ensure most rapid healing. Trefoil peptides are abundantly expressed epithelial cell products which exert protective effects and are key regulators of gastrointestinal epithelial restitution, the critical early phase of cell migration after mucosal injury. To assess the role of trefoil peptides in corneal epithelial wound healing, the effects of intestinal trefoil factor (ITF/TFF3) and spasmolytic polypeptide (SP/TFF2) on migration and proliferation of corneal epithelial cells were analyzed. Both ITF and SP enhanced restitution of primary rabbit corneal epithelial cells in vitro. While the restitution-enhancing effects of TGF-α and TGF-β were both inhibited by neutralizing anti-TGF-β-antibodies, trefoil peptide stimulation of restitution was not. Neither trefoil peptide significantly affected proliferation of primary corneal epithelial cells. ITF but not SP or pS2 mRNA was present in rabbit corneal and conjunctival tissues. In summary, the data indicate an unanticipated role of trefoil peptides in healing of ocular surface and demand rating their functional actions beyond the gastrointestinal tract.

Augmented Intestinal Trefoil Factor (TFF3) and Loss of pS2 (TFF1) Expression Precedes Metaplastic Differentiation of Gastric Epithelium

The trefoil peptides spasmolytic polypeptide (SP), intestinal trefoil factor (ITF), and pS2 show lineage-specific expression in the normal gut and are strongly induced after mucosal injury. We assessed the relationship between this induction and the development of the regenerative epithelial lineage over time in the rat stomach and verified these observations in the metaplastic and dysplastic human stomach. Antral or colonic ulcers were induced in Wistar rats by application of serosal acetic acid and tissues harvested 2 hours to 125 days later. Human endoscopic biopsies or gastric resection specimens were also assessed. Tissues were examined by radioimmunoassay, immunoblotting, or immunohistochemistry for ITF, SP, and transforming growth factor α (rat) or ITF and pS2 (human) expression. ITF and SP mRNA in antral ulcer margins was localized by in situ hybridization. ITF and SP peptide expression rose steadily in ulcer margins after 4 days, with the rise in ITF being more pronounced. By 40 days, several hundred-fold elevations in ITF levels were present, with a field effect in uninvolved mucosa. Hyperproliferative, elongated glands of undifferentiated cells expressing abundant trefoil peptides and acid sulfomucins were present after day 12 and persisted after ulcer healing. ITF mRNA was aberrantly expressed in basal and mid-regions of these regenerative glands. In contrast, transforming growth factor α peptide expression rose promptly after injury then fell to baseline levels with healing. Seven months after injury, gastric atrophy, intestinal metaplasia, and severe dysplasia with conserved ITF expression were seen. ITF was also induced in human intestinal metaplasia and conserved in all gastric cancers, whereas expression of the gastric peptide pS2 was progressively reduced in the progression from metaplasia to dysplasia. Persistent, selective overexpression of ITF, possibly acting in an autocrine fashion, is a feature of regeneration after antral ulceration, and may provide insight into the nature of metaplastic phenotypes arising from chronic gastric injury. The loss of pS2 expression in metaplasia and cancer supports a role for this protein in gastric tumor suppression.

Induction of scattering and cellular invasion by trefoil peptides in src- and RhoA-transformed kidney and colonic epithelial cells.

Trefoil factors (TFFs) are protease-resistant peptides that promote epithelial cell migration and mucosal restitution during inflammatory conditions and wound healing in the gastrointestinal tract. To date, the molecular mechanism of TFFs action and their possible role in tumor progression are unclear. In the present study, we observed that premalignant human colonic PC/AA/C1 and canine kidney MDCK epithelial cells are not competent to invade collagen gels in response to exogenously added TFFs (pS2, spasmolytic polypeptide, and intestinal trefoil factor). In contrast, activated src and RhoA exert permissive induction of invasion by the TFFs that produce similar parallel dose-response curves in src-transformed MDCKts.src and PCmsrc cells (EC50=20-40 nM). Cell scattering is also induced by TFFs in MDCKts.src cells. Stable expression of the pS2 cDNA promotes constitutive invasiveness in MDCKts.src-pS2 cells and human colonic HCT8/S11-pS2 cells established from a sporadic tumor. Furthermore, we found that TFF-mediated cellular invasion is dependent of several signaling pathways implicated in cell transformation and survival, including phosphoinositide PI3'-kinase, phospholipase C, protein kinase C, and the rapamycin target TOR. Constitutive and intense expression of pS2 was revealed by Western blot analyses and immunohistochemistry in human colorectal tumors and their adjacent control mucosa during the neoplastic progression, from the adenoma to the liver metastases. Our studies indicated that TFFs can be involved in cell scattering and tumor invasion via autocrine loops and may serve as potential targets in the control of colon cancer progression.

Generation and charaterization of a spasmolytic polypeptide (TFF2) knockout mouse

Generation and charaterization of a spasmolytic polypeptide (TFF2) knockout mouse

Differential expression of human spasmolytic polypeptide (trefoil factor family-2) in pancreatic carcinomas, ampullary carcinomas, and mucin-producing tumors of the pancreas.

Human spasmolytic polypeptide (hSP) is a member of the trefoil peptide group, thought to be involved in mucin production and cell growth. It has been reported that hSP protein is expressed in digestive cancers but not in normal pancreas. The expression of hSP in pancreatic neoplasms has not been investigated in detail. The immunohistochemical expression of hSP protein was investigated in pancreatic carcinomas, ampullary carcinomas, mucin-producing tumors, serous cystadenomas and islet cell tumors of the pancreas. hSP was expressed in 23% of pancreatic duct cell carcinomas, and hSP protein was more frequently detected in cases of early-stage or histologically low-grade duct cell carcinomas than in cases of late-stage or histologically high-grade carcinomas. Patients with hSP protein expression showed a better prognosis than did those with negative hSP expression. hSP expression was detected in 92% of mucin-producing tumors, but was not detected in serous cystadenoma or islet cell tumors. Immunohistochemical hSP expression is related to differentiation and a better prognosis in pancreatic duct cell carcinomas. Furthermore, hSP protein is related to the pathogenesis and clinical characteristics of mucin-producing tumors of the pancreas.

Trefoil peptide TFF2 (spasmolytic polypeptide) potently accelerates healing and reduces inflammation in a rat model of colitis

BACKGROUNDThe trefoil peptides are major secretory products of mucus cells of the gastrointestinal tract and show increased expression after inflammatory or ulcerative damage. Recombinant human TFF2 (spasmolytic polypeptide) has been...

The trefoil gene family are coordinately expressed immediate-early genes: EGF receptor- and MAP kinase-dependent interregulation.

The trefoil gene family of mucus cell-secreted proteins is a critical mediator of gastrointestinal mucosal restitution. Transcription of trefoil genes is induced during mucosal repair, but the regulatory mechanisms involved are unknown. Mice deficient in the intestine-specific peptide intestinal trefoil factor (ITF), in which colonic restitution is lethally impaired, showed reduced expression of the gastric trefoil genes SP and pS2, suggesting that trefoil peptides may individually regulate transcription of the entire family. In gastric cell lines, the trefoils were shown to act in a manner suggestive of immediate-early genes capable of auto- and cross-induction through cis-acting regulatory regions. Trefoil-mediated transcriptional regulation required activation of the Ras/MEK/MAP kinase signal transduction pathway. EGF receptor (EGF-R) activation was also necessary for trefoil auto- and cross-induction, and both spasmolytic polypeptide (SP) and ITF stimulation of gastric cell lines led to phosphorylation of EGF-R. Nevertheless, ITF and ITF-thioredoxin cell surface binding at 4 degrees C colocalized not with EGF-R, but with CD71, which is found in clathrin-coated pits, suggesting that integration of trefoil peptide responses may occur after internalization. As EGF-R expression is itself strongly induced after mucosal damage, the trefoil/EGF-R relationship may be pivotal in the generation and maintenance of the mucosal repair phenotype.

Mutated epithelial cadherin is associated with increased tumorigenicity and loss of adhesion and of responsiveness to the motogenic trefoil factor 2 in colon carcinoma cells.

Epithelial (E)-cadherin and its associated cytoplasmic proteins (alpha-, beta-, and gamma-catenins) are important mediators of epithelial cell-cell adhesion and intracellular signaling. Much evidence exists suggesting a tumor/invasion suppressor role for E-cadherin, and loss of expression, as well as mutations, has been described in a number of epithelial cancers. To investigate whether E-cadherin gene (CDH1) mutations occur in colorectal cancer, we screened 49 human colon carcinoma cell lines from 43 patients by single-strand conformation polymorphism (SSCP) analysis and direct sequencing. In addition to silent changes, polymorphisms, and intronic variants in a number of the cell lines, we detected frameshift single-base deletions in repeat regions of exon 3 (codons 120 and 126) causing premature truncations at codon 216 in four replication-error-positive (RER+) cell lines (LS174T, HCT116, GP2d, and GP5d) derived from 3 patients. In LS174T such a mutation inevitably contributes to its lack of E-cadherin protein expression and function. Transfection of full-length E-cadherin cDNA into LS174T cells enhanced intercellular adhesion, induced differentiation, retarded proliferation, inhibited tumorigenicity, and restored responsiveness to the migratory effects induced by the motogenic trefoil factor 2 (human spasmolytic polypeptide). These results indicate that, although inactivating E-cadherin mutations occur relatively infrequently in colorectal cancer cell lines overall (3/43 = 7%), they are more common in cells with an RER+ phenotype (3/10 = 30%) and may contribute to the dysfunction of the E-cadherin-catenin-mediated adhesion/signaling system commonly seen in these tumors. These results also indicate that normal E-cadherin-mediated cell adhesion can restore the ability of colonic tumor cells to respond to trefoil factor 2.

Tissue interaction regulates expression of a spasmolytic polypeptide gene in chicken stomach epithelium.

The primitive epithelium of embryonic chicken proventriculus (glandular stomach) differentiates, after day 6 of incubation, into luminal epithelium, which faces the lumen and abundantly secretes mucus, and glandular epithelium, which invaginates into mesenchyme and later expresses embryonic chicken pepsinogen (ECPg). So far it is not well understood how undifferentiated epithelial cells differentiate into these two distinct cell populations. Spasmolytic polypeptide (SP) is known to be expressed in surface mucous cells of mammalian stomach. In order to obtain the differentiation marker for proventricular luminal epithelial cells, we cloned a cDNA encoding chicken SP (cSP). Sequence analysis indicated that cSP has the duplicated cysteine-rich domain characteristic of SP. Examination of the spatial and temporal expression pattern of cSP gene revealed that, during embryogenesis, cSP was expressed in lumina epithelial cells of the proventriculus, gizzard, small intestine, and lung, but not the esophagus. In the proventriculus, cSP mRNA was first detected on day 8 of incubation and was localized to differentiated luminal epithelial cells. By using cSP as a molecular marker, the effects of mesenchyme on the differentiation of epithelium were analyzed in vitro. On the basis of these data, a model is presented concerning the differentiation of proventricular epithelium.

Growth factors and the gastrointestinal tract.

This paper reviews areas of interest in gut mucosal growth factor physiology. Several epidermal growth factor (EGF)-like peptides (EGF, transforming growth factor [TGF]-alpha, heparin-binding EGF-like peptide, amphiregulin, and betacellulin) have been identified in the gut, EGF is produced by the salivary glands and is present in milk. It may act on the mucosa from the lumen as a surveillance peptide promoting mucosal repair. A stem-cell-derived "ulcer-associated cell lineage" develops adjacent to ulcers and produces EGF, which may play a role in ulcer healing. TGF-alpha is expressed by villus enterocytes and may have an important role in mucosal healing. The Trefoil peptides (pS2, spasmolytic polypeptide, intestinal trefoil factor) are protease resistant molecules secreted by mucin cells throughout the gut, with a role in mucosal healing. The TGF-beta family inhibit cell proliferation, and promote cell differentiation. TGF-beta has a gradient of expression along the crypt villus axis, with maximum production at the villus tip. It is suspected that it may prevent cell proliferation and support differentiation of villus enterocytes. Hepatocyte growth factor is a multifunctional growth factor expressed in many tissues, including the gastrointestinal tract. It has a role in organogenesis. Intestinal adaptation is highly dependent on enteral nutrition, and it is likely that growth factors are involved in adaptation. Little is known, however, about interactions between nutrients and growth factors. Milk contains a range of potentially important growth factors. Their biological significance is uncertain, and this is an area of active research.

Distribution and metabolism of intravenously administered trefoil factor 2/porcine spasmolytic polypeptide in the rat

Background—Trefoil peptides are secreted by mucus producing cells in the gastrointestinal tract and are supposed to be involved in oligomerisation processes of the mucin glycoproteins in the lumen. Endocrine functions...

Establishment and characterization of two cell lines from N-methyl-N-nitrosourea-induced mouse glandular stomach carcinomas.

We previously reported the induction with N‐methyl‐N‐nitrosourea (MNU) of mouse glandular stomach carcinomas showing a gastric phenotype but variation in histologic appearance, as with human gastric carcinomas. In the present study, we established two cell lines, designated MGT‐40 and MGT‐93, from MNU‐induced mouse glandular stomach carcinomas. These cell lines are keratin‐positive and grow as epithelial monolayers in culture, requiring transforming growth factor α, epidermal growth factor or insulin/transferrin for optimal growth in addition to 10% fetal bovine serum. Retention of the differentiated phenotype for gastric surface mucous cells has been confirmed by cathepsin E immunohistochemistry and reverse transcriptase‐polymerase chain reaction for mouse spasmolytic polypeptide. Neither transplantability in nude mice nor colony formation on soft agar was observed, except in one subline. Chromosome analysis revealed aneuploidy with modal chromosome numbers ranging from 58 to 78 and no specific structural abnormalities. This is the first report of cell lines derived from mouse glandular stomach carcinomas. They should prove useful for studies of the mechanisms of regulation of growth and differentiation.

Oral human spasmolytic polypeptide protects against aspirin-induced gastric injury in rats.

Spasmolytic polypeptide (SP) is a member of the trefoil peptide family; gut peptides that participate in the protection and repair of the gastric mucosa. Previous studies have failed to agree on the mode of action of human SP (hSP). We investigated the effect of orally administered human SP on the protection and repair of rat gastric mucosa in an established in vivo model of damage induced by the non-steroidal anti-inflammatory drug aspirin (ASA). The integrity of the gastric mucosa was quantified in four ways: the temporal change in transmucosal potential difference (PD), area of macroscopic damage by planimetry, relative area of microscopic damage by histological morphometry, and the number of deep erosions per centimetre of mucosa sectioned. Human SP (200 micromol/L) administered orally before, or in combination with ASA significantly reduced the fall in PD, the area of microscopic damage, and the number of deep erosions (P < 0.05). The area of macroscopic damage was significantly reduced only in rats where hSP (200 micromol/L) was given in conjunction with ASA (P < 0.05). Human spasmolytic polypeptide (70 or 200 micromol/L) administered after ASA failed to hasten the re-establishment of PD or stimulate the repair of the gastric mucosa in the 90 min following injury (P > 0.05, compared with ASA alone). We conclude that hSP prevents gastric mucosal damage by its topical actions, probably by a rapid interaction with luminal mucins or epithelial cells, but fails to stimulate early restitution in the injured gastric mucosa.

Human spasmolytic polypeptide (hSP) significantly enhances the cholesterol crystal nucleating properties of mucin

Human spasmolytic polypeptide (hSP) significantly enhances the cholesterol crystal nucleating properties of mucin

Mice lacking secretory phospholipase A 2 show altered apoptosis and differentiation with Helicobacter felis infection

Background & Aims: Infection with Helicobacter pylori uniformly leads to a chronic superficial gastritis that may progress to atrophic gastritis, a premalignant process. A mouse model of Helicobacter felis infection was used to study possible genetic determinants of the response to infection. Methods: Three inbred mouse strains with known secretory phospholipase A 2 (sPLA 2) genotypes [BALB/c (+/+), C3H/HeJ (+/+), and C57BL/6 (−/−)] were orally infected with H. felis and examined longitudinally using routine histology, immunocytochemistry, electron microscopy, proliferating cell nuclear antigen, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling, and Northern and Western blot studies. Results: Only the C57BL/6 strain showed increased gastric fundic proliferation and apoptosis in response to infection. In addition, the C57BL/6 mouse showed a marked loss of parietal and chief cells, along with a marked expansion of an aberrant gastric mucous cell lineage that stained positive for spasmolytic polypeptide. In contrast, no significant change in these cell types was observed in BALB/c and C3H/HeJ strains. Increased expression of sPLA 2 was observed in BALB/c and C3H/HeJ after H. felis infection, whereas sPLA 2 expression was absent in C57BL/6 mice. Conclusions: H. felis infection leads to increased apoptosis and altered cellular differentiation in the C57BL/6 mouse, a strain that lacks gastric sPLA 2 expression. Because sPLA 2 has been identified recently as the MOM1 (modifier of MIN) locus that influences polyp formation in the colon, these studies suggest that sPLA 2 may also influence the gastric epithelial response to Helicobacter infection. GASTROENTEROLOGY 1998;114:675-689

Intestinal trefoil factor (TFF 3) and pS2 (TFF 1), but not spasmolytic polypeptide (TFF 2) mRNAs are co-expressed in normal, hyperplastic, and neoplastic human breast epithelium.

pS2-TFF 1 is expressed in breast cancers and has been investigated as a potential prognostic factor reflecting oestrogen dependence. The relationship to the expression of other trefoil peptides, human spasmolytic polypeptide (hSP-TFF 2) and intestinal trefoil factor (hITF/hPI.B-TFF 3) is documented here. Fifty-seven breast specimens were selected from surgical pathology archives and included five normal breasts (two lactating), seven benign proliferative lesions, 11 ductal carcinomas in situ (DCIS), three lobular carcinomas in situ (LCIS), 24 invasive ductal carcinomas (IDC), and seven invasive lobular carcinomas (ILC). The comparative distribution of trefoil mRNAs was assessed by in situ hybridization using 35S-labelled riboprobes and immunohistochemical staining for pS2-TFF 1 and hSP-TFF 2. pS2-TFF 1 and hITF/hPI.B-TFF 3 mRNA were focally present at low signal intensity in normal and benign breast. Both pS2-TFF 1 and hITF/hPI.B-TFF 3 were expressed in all DCIS, LCIS and ILC, and 21/24 IDC. Overall, expression patterns of pS2-TFF 1 and hITF/hPI.B-TFF 3 coincided, but hITF/hPI.B-TFF 3 mRNA was usually found in a greater proportion of cells. Expression of hSP-TFF 2 peptide or mRNA was not detected in any of these cases. MCF 7 breast carcinoma cells also expressed hITF/hPI.B-TFF 3 and pS2-TFF 1 mRNAs but not hSP-TFF 2. hITF/hPI.B-TFF 3 co-expression with pS2-TFF 1 may act as a prognostic factor, but also raises questions about the regulatory pathway for pS2-TFF 1 hITF/hPI.B-TFF 3. Trefoil factors have effects on cell motility and spreading in vitro, and co-expression of hITF/hPI.B-TFF 3 with pS2-TFF 1 could be functionally significant if they form a heterodimer or compete for receptor binding. Absence of hSP-TFF 2 expression may be of equal relevance to tumour cell biology.

Trefoil proteins: their role in normal and malignant cells.

The three human trefoil proteins pS2, human intestinal trefoil factor (hITF), and human spasmolytic polypeptide (hSP) are expressed principally in the mucosa of the gastrointestinal tract. They are also expressed in a variety of other normal tissues and tumours. This review discusses the pattern of expression of trefoil proteins in cancer, current views on the biological functions of trefoil proteins, and the way in which the expression of trefoil proteins may influence the behaviour of cancer cells.

Review: Trefoil Proteins: Their Role in Normal and Malignant Cells

The three human trefoil proteins pS2, human intestinal trefoil factor (hITF), and human spasmolytic polypeptide (hSP) are expressed principally in the mucosa of the gastrointestinal tract. They are also expressed in a variety of other normal tissues and tumours. This review discusses the pattern of expression of trefoil proteins in cancer, current views on the biological functions of trefoil proteins, and the way in which the expression of trefoil proteins may influence the behaviour of cancer cells. © 1997 by John Wiley & Sons, Ltd.

Pancreatic spasmolytic polypeptide protects the gastric mucosa but does not inhibit acid secretion or motility.

The objectives of these studies were to examine whether the trefoil peptide porcine pancreatic spasmolytic polypeptide (PSP) had gastric mucosal protectant properties similar to its human equivalent human spasmolytic polypeptide (hSP) and to confirm the antisecretory and antimotility action of the peptide. PSP and recombinant hSP reduced gastric mucosal damage caused by a combination of subcutaneous indomethacin and restraint stress in the conscious rat. At a dose of 500 micrograms/kg bolus plus 500 micrograms.kg-1.h-1 sc, PSP significantly reduced the total area of damage by 58%. PSP at a dose of 150 micrograms/kg iv had no inhibitory effect on pentagastrin-stimulated gastric acid secretion in the perfused stomachs of anesthetized rats. This lack of antisecretory activity was confirmed in vitro using an isolated stomach preparation from the immature rat. PSP and hSP at concentrations up to 800 nM did not inhibit electrically or chemically evoked contractions of the guinea pig ileum and duodenum in vitro. Thus antisecretory and antimotility actions do not underlie the mucosal protectant properties of PSP. PSP did, however, stimulate cell migration, and this may, at least in part, account for its protectant properties.