Spasmolytic Peptide Research Articles

Elevated Protein Kinase A Activity in Stomach Mesenchyme Disrupts Mesenchymal-epithelial Crosstalk and Induces Preneoplasia.

Background & AimsMesenchymal-epithelial crosstalk (MEC) in the stomach is executed by pathways such as bone morphogenetic protein (BMP) and extracellular signal-regulated kinase (ERK). Mis-regulation of MEC disrupts gastric homeostasis and causes tumorigenesis. Protein Kinase A (PKA) crosstalks with BMP and ERK signaling; however, PKA function(s) in stomach development and homeostasis remains undefined.MethodsWe generated a novel Six2-Cre+/-PKAcαRfl/wt (CA-PKA) mouse in which expression of constitutive-active PKAcαR was induced in gastric mesenchyme progenitors. Lineage tracing determined spatiotemporal activity of Six2-Cre in the stomach. For phenotyping CA-PKA mice histological, co-immunofluorescence, immunoblotting, mRNA sequencing, and bioinformatics analyses were performed.ResultsLineage tracing showed that Six2-Cre activity in the stomach is restricted to the mesenchymal compartment. CA-PKA mice showed disruption of gastric homeostasis characterized by aberrant mucosal development and epithelial hyperproliferation; ultimately developing multiple features of gastric corpus preneoplasia including decreased parietal cells, mucous cell hyperplasia, spasmolytic peptide expressing metaplasia with intestinal characteristics, and dysplastic and invasive cystic glands. Furthermore, mutant corpus showed marked chronic inflammation characterized by infiltration of lymphocytes and myeloid-derived suppressor cells along with the upregulation of innate and adaptive immune system components. Striking upregulation of inflammatory mediators and STAT3 activation was observed. Mechanistically, we determined there is an activation of ERK1/2 and downregulation of BMP/SMAD signaling characterized by marked upregulation of BMP inhibitor gremlin 1.ConclusionsWe report a novel role of PKA signaling in gastric MEC execution and show that PKA activation in the gastric mesenchyme drives preneoplasia by creating a proinflammatory and proproliferative microenvironment associated with the downregulation of BMP/SMAD signaling and activation of ERK1/2.

Covering the Cover

Covering the Cover

Trefoil factor 3 (TFF3) from human breast milk activates PAR-2 receptors, of the intestinal epithelial cells HT-29, regulating cytokines and defensins.

Trefoil factors are effector molecules in gastrointestinal tract physiology. Each one improves healing of the gastrointestinal tract. Trefoil factors may be grouped into three classes: the gastric peptides (TFF1), spasmolytic peptide (TFF2) and intestinal trefoil factor (TFF3). Significant amounts of TFF3 are present in human breast milk. Previously, we have reported that trefoil factor 3 isolated from human breast milk produces down regulation of cytokinesand promotes human beta defensins expression in intestinal epithelial cells. This study aimed to determine the molecular mechanism involved. Here we showed that the presence of TFF3 strongly correlated with protease activated receptors 2 (PAR-2) activation in human intestinal cells. Intracellular calcium ((Ca2+)i)mobilization was induced by the treatment with: 1) TFF3, 2) synthetic PAR-2 agonist peptide. The co-treatment with a synthetic PAR-2 antagonist peptide and TFF3 eliminates the latter's effect. Additionally, we demonstrated the existence of interactions amongTFF3 and PAR-2 receptors through far Western blot and co-precipitation. Finally, down regulationof PAR-2 by siRNAresulted in a decrease of TFF3 induced intracellular (Ca2+)i mobilization, cytokine regulation and defensinsexpression. These findings suggest that TFF3 activates intestinal cells through PAR-2 (Fig. 4, Ref. 19).

Trefoil factor 3 (TFF3) expression is regulated by insulin and glucose

Introduction: Trefoil factors are effector molecules in gastrointestinal tract physiology. They are classified into three groups: the gastric peptides (TFF1), spasmolytic peptide (TFF2) and intestinal trefoil factor (TFF3). Previous studies have shown that trefoil factors are located and expressed in human endocrine pancreas suggesting that TFF3 play a role in: a) pancreatic cells migration, b) β-cell mitosis, and c) pancreatic cells regeneration. We speculated that the presence of TFF3 in pancreas, could be associated to a possible regulation mechanism by insulin and glucose. To date, there are not reports whether the unbalance in carbohydrate metabolism observed in diabetes could affect the production or expression of TFF3.Methods: We determined the TFF3 levels and expression by immunoassay (ELISA) and semi-quantitative RT-PCR technique respectively, of intestinal epithelial cells (HT-29) treated with glucose and insulin. Also,Real Time-PCR (RTq-PCR) was done.Results: Increasing concentrations of glucose improved TFF3 expression and these levels were further elevated after insulin treatment. Insulin treatment also led to the up-regulation of human sodium/glucose transporter 1 (hSGLT1), which further increases intracellular glucose levels. Finally, we investigated theTFF3 levels in serum of diabetes mellitus type 1 (T1DM) and healthy patients. Here we shown that serum TFF3 levels were down-regulated in T1DM and this levels were up-regulated after insulin treatment. Also, the TFF3 levels of healthy donors were up-regulated 2 h after breakfast.Conclusion: Our fi ndings suggest for the fi rst time that insulin signaling is important for TFF3 optimal expression in serum and intestinal epithelial cells.

Trefoil factor 3 isolated from human breast milk downregulates cytokines (IL8 and IL6) and promotes human beta defensin (hBD2 and hBD4) expression in intestinal epithelial cells HT-29

Trefoil factors (TFF) are secretory products of mucin producing cells. They play a key role in the maintenance of the surface integrity of oral mucosa and enhance healing of the gastrointestinal mucosa by a process called restitution. TFF comprises the gastric peptides (TFF1), spasmolytic peptide (TFF2), and the intestinal trefoil factor (TFF3). They have an important and necessary role in epithelial restitution within the gastrointestinal tract. Significant amounts of TFF are present in human milk. This study aimed to determine a possible correlation between TFF3 isolated from human breast milk and levels of cytokines (IL8 and IL6) and defensins (hBD2 and hBD4) in intestinal epithelial cells HT-29 treated with trefoil. Samples of human milk were collected within 2-4 weeks postpartum from healthy human mothers (18-30-years-old) by manual breast massage, and TFF3 was purified by ammonium sulfate precipitation, isoelectric precipitation, DEAE-chromatography, and gel filtration. In this work we measured the concentrations and mRNA levels of cytokines and defensins by immunoassay (ELISA) and semiquantitative RT-PCR technique, respectively. Also we measured the peroxidase activity. We present the first evidence of human milk TFF3 purification. Here we show that the presence of TFF3 isolated from milk strongly correlates with downregulation of IL8 and IL6 in human intestinal epithelial cells. On the other hand, TFF3 activated the epithelial cells in culture to produce beta defensins 2 (hBD2) and beta defensins 4 (hBD4). These findings suggest that TFF can activate intestinal epithelial cells and could actively participate in the immune system of breastfed babies by inducing the production of peptides related to innate defence, such as defensins.

Trefoil Family Factor 2 Is Expressed in Murine Gastric and Immune Cells and Controls both Gastrointestinal Inflammation and Systemic Immune Responses

Trefoil family factor 2 (TFF2), also known as spasmolytic peptide, is a low-molecular-weight protein that is upregulated in gastric tissues infected with Helicobacter or having other inflammatory conditions, but a precise function is yet to be elucidated. The role of TFF2 in the development of gastritis, colitis, and inflammatory cytokine responses was examined both in vivo and in vitro using wild-type and TFF2 knockout mice. TFF2 knockout and wild-type mice were infected with Helicobacter felis (H. felis) to induce gastritis. Colitis was induced in TFF2 knockout and wild-type mice by administering dextran sodium sulfate (DSS) in drinking water. Histopathology, clinical disease (colitis), and antibody levels (H. felis) were examined. TFF2 expression in tissues was determined by reverse transcriptase PCR, and the inflammatory and proliferative responses of TFF2-expressing macrophages and spleen cells were examined by cytokine enzyme-linked immunosorbent assay, thymidine incorporation, and gene array studies. TFF2 knockout mice have increased susceptibility to H. felis-induced gastritis, with enhanced gastric inflammation. They were also more susceptible to DSS-induced colitis, with prolonged colonic hemorrhage and persistent weight loss. Remarkably, TFF2 expression was not limited to the gastrointestinal tract, as suggested in previous studies, but was also present in macrophages and lymphocytes. The inflammatory and proliferative responses of these immune cell types were dysregulated in TFF2 knockout mice. TFF2-/- cells were hyperresponsive to interleukin 1 beta stimulation but showed normal responses to lipopolysaccharide, suggesting a specific role for TFF2 in interleukin 1 receptor but not Toll-like receptor 4 signaling via their Toll-interleukin 1 resistance domains. TFF2-/- lymphocytes also produced higher levels of interleukin 2 than wild-type cells. Thus, TFF2 was expressed in the gastrointestinal cells and in immune cells and was a negative regulator of gastrointestinal inflammation and immune cell cytokine responses. Our studies suggest that TFF2 not only controls gastrointestinal repair but also regulates mononuclear cell inflammatory responses.

Gastrin transcriptionally regulates trefoil family factor 2

The trefoil family factor 2, also know as spasmolytic peptide containing a dual-trefoil domain, is secreted by mucous neck cells of stomach and proposed to be the principal cytoprotective tretbil peptide. ]'he antral hormone gastrin was known tot its acid stimulating properties and inducing proliferation of the acid secreting mucosa. The recent TFF2-deficient mice demonstrating a decreased thickness and proliferation rate of the gastric mucosa prompt us investigating whether gastrin is a mediator of TFF2. In gastrin-deficient mice, TFF2 is lound to he siguificant decreased expression in fundus of the stomach, as determined by immunohistochemistry with an antibody specifically recognizing the whole mouse TFF2 protein. Furthermore, TFF2 mRNA expression was rapidly and potently induced by gastrin in AGS ceils that are stablely introduced the gastrin/CCKB receptor. A series of deletion mutants containing mouse promoter sequence are constructed in a dual-luciferase reporter to map gastrin t~spons~ve elements. Cells co-transfected with prompter constructs and internal control, treated with 10-7M of gastrin for 18 h are subsequently measured the ludterase activities. The results indicate that gastrin-treated cells show much higher hiciferase acti~aty while had little effect on the reporter vector itself. P-46 and P-306, containing 46bp and 306bp of upstream sequence of the transcriptional start site, respectively, have a 38 and 59 fold increase in luciferase activity' over those non-treated AGS cells. The increases in lucilerase activity are largely abolished when 10-6 M of YF476, a chemical antagonist of gastrin/CCKg receptor were added with gastrin, suggesting that increase of TFF2 promoter activity is gastrin-specific mediation through its receptor. These results indicate that multiple gastrin responsive elements are located within these regions. In conclusion, our data suggest that gasmn is manscriptionally regulating TFF2 thus may play an important role in mediating the physiological function of trefoil family factors.

Activation of genes for spasmolytic peptide, transforming growth factor alpha and for cyclooxygenase (COX)-1 and COX-2 during gastric adaptation to aspirin damage in rats.

NSAIDs, such as aspirin (ASA), cause widespread mucosal damage, but repeated ASA insults appear to induce mucosal tolerance (adaptation) to this injury. The mechanism of the gastric adaptation to the damage induced by ASA has not been fully explained. To determine the role of the mucosal gene expression for spasmolitic peptide (SP) (a member of trefoil peptides) and transforming growth factor alpha (TGF alpha) as well as for cyclooxygenase (COX)-1 and COX-2 during gastric adaptation to ASA in rats. Gastric lesions were produced by ASA (100 mg/kg in 1.5 mL of 0.2 M HCl) applied intragastrically (i.g.) as a single dose. every day for 5 days. Control rats were given 1.5 mL of vehicle (0.2 M HCl i.g.) as a single dose, during 5 consecutive days. Gastric blood flow (GBF) was measured by H2-gas clearance technique and gastric mucosal specimens were taken for the assessment of cell proliferation rate in gastric mucosa by bromodeoxyuridine (BrdU) uptake, mucosal generation of prostaglandin E2 measured by radioimmunoassay, and for expression of SP, TGF alpha COX-1 and COX-2 mRNA as determined by RT-PCR. To quantify the relative amounts of mRNA for SP and TGF alpha, southern blotting analysis of the PCR products was performed and the intensity of PCR products was compared with that of beta-actin used as a standard. ASA applied once produced numerous gastric erosions, but with repeated ASA doses the adaptation to this NSAID developed, the area of gastric lesions being reduced by 86% after six consecutive ASA insults. This adaptation to ASA was accompanied by approximately a 90% reduction in prostaglandin E2 biosynthesis, by a significant rise in BrdU uptake by glandular cells predominantly in the neck region of gastric glands and by expression of SP (SP/beta-actin ratio; 0.96 +/- 0.08 in ASA-adapted mucosa vs. 0.38 +/- 0.05 in the control mucosa) and TGF alpha (TGF alpha/beta-actin ratio: 0.97 +/- 0.07 in ASA-adapted mucosa vs. 0.77 +/- 0.06 in the control mucosa). COX-1 expression was detected in vehicle-control gastric mucosa and after single exposure to ASA or after six consecutive ASA insults, while COX-2 mRNA was not detected in vehicle-control gastric mucosa, but appeared after single ASA insult and was sustained after subsequent ASA doses. (i) Gastric adaptation to aspirin injury involves enhanced cell proliferation which appears to be mediated by increased expression of SP and TGF alpha, and (ii) rapid upregulation of COX-2 expression following single and repeated ASA insults may represent a compensatory response to suppression of prostaglandin generation by this NSAID.

Reversible induction of mucous cell hyperplasia and parietal cell loss in rats dosed with DMP-777

Parietal cell loss and mucous cell hyperplasia are important histological components of a spectrum of gastric mucosal diseases including Menetrier's disease and fundic H. pylori gastritis. Recent investigations using transgenic mouse strains have highlighted the importance of intrinsic mucosal growth factors such as TGFct in the differentiation of specific cell lineages in the gastric mucosa. Unfortunately, experiments in transgenic mice have been hampered by the irreversibility of atrophic-gastritis phenotypes. In three-month toxicity studies in rats, oral dosing with DMP-777 (50-200 mg/kg/day), a neutrophil elastase inhibitor, resulted in an unexpected phenotype of gastric mucous cell hyperplasia and parietal cell loss associated with hypergastrinemia. Routine histological staining (diastase-resistant PAS) and immunocytochemistry for BrdU, H/K-ATPase, spasmolytic peptide, gastrin, somatostatin and chromogranin A were utilized to determine alterations in mucosal lineages. Rats did not demonstrate any significant inflammatory infiltrate. Fundic mucosa demonstrated a prominent hyperplasia of surface mucous cells, 4 to 8-fold increases in isthmus zone BrdU-labeling index and a profound decrease in the numbers of parietal ceils, chief cells, somatostatin ceils and ECL ceils after 9 days of dosing with 200 mg/kg/day. The numbers of mucous neck cells were unaffected by drug treatment. Hyperplasia of gastrin cells was seen in the gastric antrum, but there was no hyperplasia of antral mucous cells. These changes were completely reversible after a threemonth drug-free recovery period. To examine the marked hypergastrinemia observed in DMP 777-treated rats, the effects of the drug on acid secretion were studied in the rat pylorus-ligation model, where DMP-777 was shown to be a potent inhibitor of gastric acid secretion when dosed orally. Additional in vitro tests showed that DMP-777 did not block histamine (H2), acetylcholine (M3) or gastrin receptors (novel and CCK-B receptors). In isolated rabbit parietal cells, 3 pM DMP-777 completely inhibited basal, histaminestimulated and forskolin-stimulated aminopyrine accumulation (ECso---0.6 pM). While the drug had no direct effect on the activity of the H/K-ATPase, 1-3 laM DMP-777 could dissipate completely an established stimulated proton gradient in isolated tubulovesicles loaded with acridine orange. These latter results suggest that DMP-777 is a protonophore. Since other regions of the gastrointestinal mucosa are not affected by the drug, unexpected effects of DMP-777 on the gastric mucosa may be due to a specific interaction with parietal cell, perhaps involving a specific partitioning of the drug into the parietal cell secretory canalicolus. Short term DMP-777 dosing of rats provides the first readily reversible model of parietal cell loss and mucous cell hyperplasia. This work was funded by DuPont-Merck Pharmaceuticals.

Activation of genes for rat spasmolytic peptide (rSP) and transforming growth factor alpha (TGFα) during adaptation to aspirin injury

Activation of genes for rat spasmolytic peptide (rSP) and transforming growth factor alpha (TGFα) during adaptation to aspirin injury

Human spasmolytic polypeptide decreases proton permeation through gastric mucus in vivo and in vitro.

Exogenously administered trefoil peptides are gastroprotective in rat injury models. We hypothesized that trefoil-associated gastroprotection occurred by decreasing the rate of proton permeation through mucus. Gastric surface cell intracellular pH and mucus gel thickness were measured by in vivo microscopy. Gastric mucosal blood flow was measured by laser-Doppler flowmetry. The effect of human spasmolytic peptide (hSP) on H+ diffusion through 5% purified porcine mucin was measured using an Ussing chamber. Buffering action of mucin was measured by titration. In vivo, gastric mucosal blood flow and mucus gel thickness were not affected by any of the treatments. Topical hSP, but not intravenous hSP, decreased initial acidification rate and elevated the intracellular pH of gastric surface cells during luminal acid challenge. In in vitro studies, hSP dose dependently decreased the diffusion coefficient of H+ through 5% porcine mucin solution. hSP had no significant effect on the buffering action of mucin solutions. These data support our hypothesis that hSP interacts with gastric mucin in a manner that inhibits proton permeation through the mucus gel layer.

Role of spasmolytic polypeptide in healing of stress-induced gastric lesions in rats

Stress is known to induce gastric ulcerations but the mechanism of their healing has been little studied. This paper describes the studies on mucosal expression and the effect on ulcer healing of spasmolytic peptide (SP), one of the members of the trefoil peptide family. Gastric ulcerations were induced in rats by the exposition to 3.5 h of water immersion and restraint stress. It was found that the number of these lesions gradually declined at 4, 8 and 12 h after stress and this spontaneous healing was significantly accelerated by s.c. infusion of human recombinant SP in a constant dose of 50 μg kg −1 h −1. The healing of the stress-induced ulcerations was accompanied by a gradual restoration of gastric mucosal blood flow and the decrease in gastric acid and pepsin secretion towards the normal values. The expression of SP in rats (rSP) was detected by RT–PCR in the intact mucosa and during all tested time periods reaching a peak at 4 h after the stress. Immunostaining for rSP in the intact mucosa was confined to the mucous neck cells, but following the exposure to stress it was significantly enhanced and occurred also in the cells of the basal region of gastric glands, reaching a peak at 4 h after the stress. We conclude that SP plays an important role in healing of stress-induced gastric lesions possibly by the acceleration of the mucosal repair, the enhancement of mucosal blood flow and the inhibition of gastric secretion.

Cloning of contiguous genomic fragments from human chromosome 21 harbouring three trefoil peptide genes.

A group of small peptides with a typical cysteine-rich domain (termed trefoil motif or P-domain) is abundantly expressed at mucosal surfaces of specific normal and neoplastic tissues. Their association with the maintenance of surface integrity was suggested. The first known human trefoil peptide (pS2) was isolated from breast cancer cells (MCF7). Its oestrogen-inducible gene, and the human homologue to the porcine spasmolytic peptide gene (hSP/SML1) appear synchronously expressed in healthy stomach mucosa and several carcinomas of the gastrointestinal tract. Both genes were shown to be localised at 21q22.3. A new trefoil peptide from human intestinal mucosa (hITF/hP1.B) and its gene were described recently. By using suitable oligonucleotide primers and PCR and isolating large (110-250 kb) genomic recombinants cloned in the bacterial artificial chromosome (BAC) system, we present a genomic region from chromosome band 21q22.3 cloned in contiguous sequences and encoding all three members of human P-domain/trefoil peptides proving a physical linkage of all three trefoil peptide genes. Such genomic sequences will provide useful experimental material for analysis of gene regulation, for gene modification experiments and for establishing transgenic cells or animals.

Expression of trefoil peptides in the gastric mucosa of transgenic mice overexpressing transforming growth factor-alpha.

Overexpression of transforming growth factor-alpha (TGF-alpha) in the gastric mucosa of metallothionein-TGF alpha (MT-TGF alpha) transgenic mice leads to a marked alteration in the ontogeny of the fundic cellular lineages. Induction of the transgene leads to the over-production of mucous cells with a concomitant diminution in the development of parietal cell and chief cell lineages. We have sought to define more precisely the mucous cell lineages involved in the mucous cell hyperplasia in MT-TGF alpha mice by investigating the expression of trefoil peptides in MT-TGF alpha mice. MT-TGF alpha mice and their non-transgenic littermates were treated with cadmium sulfate beginning at 13 days of age. Animals were then sacrificed at intervals over the following 2 weeks and gastric mucosa was examined for expression of trefoil peptides and TGF alpha by immunohistochemistry and in situ hybridization. No TGF alpha mRNA expression could be demonstrated by in situ hybridization in non-transgenic mice. In MT-TGF alpha mice, in situ grains for TGF alpha mRNA were detected at the base of fundic glands in 13 day old animals, whereas the expression was observed more widely in the mucosa of older animals (28 days). TGF alpha immunoreactivity was observed in foveolar mucous cells and residual parietal cells in MT-TGF alpha mice at all ages. By in situ hybridization, pS2 mRNA was detected in the surface mucous cells in normal gastric mucosa. In MT-TGF alpha mice, pS2 mRNA was found throughout the expanded foveolar region. By in situ hybridization, spasmolytic peptide (SP) expression was observed in the region of the progenitor zone in both groups of mice. By immunohistochemistry, SP expression was noted in a broad band of mucous neck cells deep to the progenitor zone. No gastric expression of intestinal trefoil factor (ITF) was noted in either group of mice. The results demonstrate that the expansion of the foveolar mucous cell compartment in MT-TGF alpha mice is due to the hyperplasia of normal surface cells expressing their particular mucin-associated trefoil peptide, pS2.

A third P-domain peptide gene (TFF3), human intestinal trefoil factor, maps to 21 q22.3

Small peptides displaying a cysteine-rich module (termed P-domain or trefoil motif) form a recently increasing group of peptides abundantly expressed at mucosal surfaces of specific tissues and are associated with the maintenance of surface integrity. The estrogen-inducible pS2 gene (BCEI) and the human homolog to the porcine spasmolytic peptide (hsP) gene (SML1) appear synchronously expressed in healthy stomach mucosa and several carcinomas of the gastrointestinal tract. Both genes were shown to be located at 21q22.3. A new trefoil peptide from human intestinal mucosa (hITF/hP1.B) and its gene (TFF3) were described recently. By PCR analysis of a somatic cell hybrid panel and FISH using two large genomic recombinants (110 kb, 210 kb) cloned in the Bacterial Artificial Chromosome (BAC) system, we show that this gene coding for the new member of human P-domain/trefoil peptides also maps to chromosome region 21q22.3 suggesting a physical linkage of all three trefoil peptide genes.

NMR-based structural studies of the pNR-2/pS2 single domain trefoil peptide. Similarities to porcine spasmolytic peptide and evidence for a monomeric structure.

NMR spectroscopy measurements have been used to obtain structural information about the pNR-2/pS2 single-domain trefoil peptide. NMR data from 2D (two dimensional) double-quantum-filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), NOE spectroscopy (NOESY), rotating frame NOE spectroscopy (ROESY) and 2D 13C-1H heteronuclear single-quantum coherence (HSQC) and 13C-1H HSQC-TOCSY spectra have been analysed to provide essentially complete 1H and 13C sequence-specific assignments for the pNR-2/pS2 protein. From a consideration of the NOE intensities, 3J(NH-alpha CH) coupling constants, 1H and 13C chemical shifts of backbone atoms and amide-proton exchange rates, the pNR-2/pS2 was found to contain two short antiparallel beta-strands (32-35 and 43-46), a short helix (25-30) and a type I beta-turn (11-15). These elements of secondary structure are very similar to those found in the two trefoil domains of pSP for which detailed structural information is already available. Similar 1H chemical shifts were noted for several conserved residues in pNR-2/pS2 and pSP and a characteristic Phe residue with a slowly flipping ring was found in the pNR-2/pS2 variant and in both domains of pSP. The tertiary structures of the domains therefore appear to be very similar in the two proteins and it is likely that the pNR-2/pS2 has the same pattern of disulphide bonds (1-5, 2-4, 3-6) as pSP. Correlation time measurements derived from 1H-1H NOE measurements indicate that the Cys58-->Ser form of the pNR-2/pS2 protein used in this study is monomeric in solution at approximately 2 mM.

Subcellular distribution of peptides associated with gastric mucosal healing and neoplasia

The trefoil peptides pS2 and human spasmolytic peptide are putative growth factors, particularly associated with mucus-producing cells of the gastrointestinal tract including those of the stomach. The receptor for transforming growth factor alpha (TGF alpha) takes its name from one of its alternative ligands, epidermal growth factor, and is called the epidermal growth factor receptor. Although there is immunoreactive epidermal growth factor in the stomach, it is TGF alpha and the epidermal growth factor receptor that are abundant. Immunolabelling at electron microscope level allows for subcellular localisation of antigens; pS2 and human spasmolytic peptide co-localise to cytomembranes, including the Golgi apparatus, and thecae of surface/pit mucous cells. TGF alpha is abundant on the membranes of tubulovesicles of parietal cells and is also present in chief cells: in mucous producing cells it can be detected but not in association with mucous. The distribution of the epidermal growth factor receptor mimics that of TGF alpha but with preferential clustering on the basolateral membranes of gastric cells. The trefoil peptides are associated with healing and probably act, together with mucus, to protect the gastric mucosa and maintain a viable environment. TGF alpha, transduced via the epidermal growth factor receptor, inhibits gastric acid secretion, thus aids the trefoils in the maintenance of a gastric microenvironment conducive to healing after damage. TGF alpha, however, is also a potent mitogen; while this property plays a vital part in repairing mucosal defects, if this peptide or indeed its receptor are overexpressed, the result can be neoplasia.

Expression of the breast cancer associated gene pS2 and the pancreatic spasmolytic polypeptide gene (hSP) in diffuse type of stomach carcinoma

Expression of the pancreatic spasmolytic peptide (hSP) gene and pS2 (a gene isolated from oestrogen-induced breast carcinoma cells) were analysed in 36 samples of human stomach carcinoma. 17 tumours were investigated at the RNA level (by northern blots) as well as at the gene product level (by immunochemistry). Since pS2 had been shown to be expressed in normal stomach mucosa its activity in carcinoma samples was expected. Surprisingly, strong pS2 immunoreactivity was noted in the diffuse carcinoma type, whereas the intestinal type displayed weak reactivity. The tumour samples showing strong immunostaining expressed the regular 0.6 kb pS2 RNA band and weak staining was paralleled by aberrant transcripts. Additionally, only in tumour samples with regular pS2 transcription was the typical 0.7 kb hSP RNA band seen; samples with aberrant pS2 bands did not express hSP at all. This is the first demonstration of hSP gene activity in a human tumour.