Gremlin 1 identifies a skeletal stem cell with bone, cartilage, and reticular stromal potential.

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).

Coordinate expression loss of GKN1 and GKN2 in gastric cancer via impairment of a glucocorticoid-responsive enhancer.

Gastrokines (GKNs) are anti-inflammatory proteins secreted by gastric epithelial (surface mucous and pit) cells, with their aberrant loss of expression causally linked to premalignant inflammation and gastric cancer (GC). Transcriptional mechanisms accounting for GKN expression loss have not been elucidated. Using human clinical cohorts, mouse transgenics, bioinformatics, and transfection/reporter assays, we report a novel mechanism of GKN gene transcriptional regulation and its impairment in GC. GKN1/GKN2 loss is highly coordinated, with both genes showing parallel downregulation during human and mouse GC development, suggesting joint transcriptional control. In BAC transgenic studies, we defined a 152-kb genomic region surrounding the human GKN1/GKN2 genes sufficient to direct their tissue- and lineage-restricted expression. A screen of the 152-kb region for candidate regulatory elements identified a DNase I hypersensitive site (CR2) located 4 kb upstream of the GKN1 gene. CR2 showed overlapping enrichment of enhancer-related histone marks (H3K27Ac), a consensus binding site (GRE) for the glucocorticoid receptor (GR), strong GR occupancy in ChIP-seq data sets and, critically, exhibited dexamethasone-sensitive enhancer activity in reporter assays. Strikingly, GR showed progressive expression loss, paralleling that of GKN1/2, in human and mouse GC, suggesting desensitized glucocorticoid signaling as a mechanism underlying GKN loss. Finally, mouse adrenalectomy studies revealed a critical role for endogenous glucocorticoids in sustaining correct expression (and anti-inflammatory restraint) of GKNs in vivo. Together, these data link the coordinate expression of GKNs to a glucocorticoid-responsive and likely shared transcriptional enhancer mechanism, with its compromised activation contributing to dual GKN loss during GC progression.NEW & NOTEWORTHY Gastrokine 2 (GKN2) is an anti-inflammatory protein produced by the gastric epithelium. GKN2 expression is progressively lost during gastric cancer (GC), which is believed to play a casual role in GC development. Here, we use bacterial artificial chromosome transgenic studies to identify a glucocorticoid-responsive enhancer element that likely governs expression of GKN1/GKN2, which, via parallel expression loss of the anti-inflammatory glucocorticoid receptor, reveals a novel mechanism to explain the loss of GKN2 during GC pathogenesis.

Overexpression of IL-11 promotes premalignant gastric epithelial hyperplasia in isolation from germline gp130-JAK-STAT driver mutations.

Expression of the cytokine IL-11 is elevated in human Helicobacter pylori infection and progressively increases with worsening gastric pathology. Additionally, IL-11 is required for tumor development in STAT3-dependent murine models of gastric cancer (GC) and, when administered acutely, causes resolving atrophic gastritis. However, it is unclear whether locally elevated IL-11 ligand expression can, in isolation from oncogenic gp130-JAK-STAT pathway mutations, initiate GC pathogenesis. Here we developed a transgenic mouse model of stomach-specific (keratin 19 promoter) IL-11 ligand overexpression. Keratin 19 promoter-IL-11 transgenic ( K19-IL11Tg) mice showed specific IL-11 overexpression in gastric corpus and antrum but not elsewhere in the gastrointestinal tract or in other tissues. K19-IL11Tg mice developed spontaneous premalignant disease of the gastric epithelium, progressing from atrophic gastritis to TFF2-positive metaplasia and severe epithelial hyperplasia, including adenoma-like lesions in a subset of older (1 yr old) animals. Although locally advanced, the hyperplastic lesions remained noninvasive. H. pylori infection in K19-IL11Tg mice accelerated some aspects of the premalignant phenotype. Finally, K19-IL11Tg mice had splenomegaly in association with elevated serum IL-11, with spleens showing an expanded myeloid compartment. Our results provide direct in vivo functional evidence that stomach-specific overexpression of IL-11, in isolation from germline gp130-JAK-STAT3 genetic drivers, is sufficient for premalignant progression. These findings have important functional implications for human GC, in which frequent IL-11 overexpression occurs in the reported absence of somatic mutations in gp130 signaling components. NEW & NOTEWORTHY We provide direct in vivo functional evidence that stomach-specific overexpression of the cytokine IL-11, in isolation from gp130-JAK-STAT3 pathway mutations, can trigger spontaneous atrophic gastritis progressing to locally advanced epithelial hyperplasia (but not dysplasia or carcinoma), which does not require, but may be accelerated by, concomitant Helicobacter pylori infection.

The MUC1 mucin protects against Helicobacter pylori pathogenesis in mice by regulation of the NLRP3 inflammasome.

OBJECTIVES: The mucin MUC1, best known for providing an epithelial barrier, is an important protective host factor in both humans and mice during Helicobacter pylori pathogenesis. This study aimed to identify the long-term consequences of MUC1 deficiency on H. pylori pathogenesis and the mechanism by which MUC1 protects against H. pylori gastritis. DESIGN: Wildtype and Muc1(-/-) mice were infected for up to 9 months, and the gastric pathology, immunological response and epigenetic changes assessed. The effects of MUC1 on the inflammasome, a potent inflammatory pathway, were examined in macrophages and H. pylori-infected mice deficient in both MUC1 and inflammasome components. RESULTS: Muc1(-/-) mice began to die 6 months after challenge, indicating Muc1 deficiency made H. pylori a lethal infection. Surprisingly, chimaeric mouse infections revealed MUC1 expression by haematopoietic-derived immune cells limits H. pylori-induced gastritis. Gastritis in infected Muc1(-/-) mice was associated with elevated interleukin (IL)-1ß and epigenetic changes in their gastric mucosa similar to those in transgenic mice overexpressing gastric IL-1ß, implicating MUC1 regulation of an inflammasome. In support of this, infected Muc1(-/-)Casp1(-/-) mice did not develop severe gastritis. Further, MUC1 regulated Nlrp3 expression via an nuclear factor (NF)-κB-dependent pathway and reduced NF-κB pathway activation via inhibition of IRAK4 phosphorylation. The importance of this regulation was proven using Muc1(-/-)Nlrp3(-/-) mice, which did not develop severe gastritis. CONCLUSIONS: MUC1 is an important, previously unidentified negative regulator of the NLRP3 inflammasome. H. pylori activation of the NLRP3 inflammasome is normally tightly regulated by MUC1, and loss of this critical regulation results in the development of severe pathology.

STAT3: a critical component in the response to Helicobacter pylori infection.

STAT3 imparts a profound influence on both the epithelial and immune components of the gastric mucosa, and through regulation of key intracellular signal transduction events, is well placed to control inflammatory and oncogenic outcomes in the context of Helicobacter (H.) pylori infection. Here we review the roles of STAT3 in the host immune response to H. pylori infection, from both gastric mucosal and systemic perspectives, as well as alluding more specifically to STAT3-dependent mechanisms that might be exploited as drug targets.

TFF2 deficiency exacerbates weight loss and alters immune cell and cytokine profiles in DSS colitis, and this cannot be rescued by wild-type bone marrow.

The trefoil factor TFF2 is a member of a tripartite family of small proteins that is produced by the stomach and the colon. Recombinant TFF2, when applied intrarectally in a rodent model of hapten colitis, hastens mucosal healing and reduces inflammatory indexes. Additionally, TFF2 is expressed in immune organs, supporting a potential immunomodulatory and reparative role in the bowel. In this study we confirm that TFF2 is expressed in the colon and is specifically enriched in epithelial cells relative to colonic leukocytes. TFF2-deficient, but not TFF1-deficient, mice exhibit a more severe response to acute or chronic dextran sulfate (DSS)-induced colitis that correlates with a 50% loss of expression of TFF3, the principal colonic trefoil. In addition, the response to acute colitis is associated with altered expression of IL-6 and IL-33, but not other inflammatory cytokines. While TFF2 can reduce macrophage responsiveness and block inflammatory cell recruitment to the colon, the major role in limiting the susceptibility to acute colitis appears to be maintenance of barrier function. Bone marrow transfer experiments demonstrate that leukocyte expression of TFF2 is not sufficient for prevention of colitis induction but, rather, that the gastrointestinal epithelium is the primary source of TFF2. Together, these findings illustrate that epithelial TFF2 is an important endogenous regulator of gut mucosal homeostasis that can modulate immune and epithelial compartments. Because of its extreme stability, even in the corrosive gut lumen, TFF2 is an attractive candidate as an oral therapeutic scaffold for future drug development in the treatment of inflammatory bowel disease.

Mechanistic insights into the contribution of epithelial damage to airway remodeling. Novel therapeutic targets for asthma.

It has been suggested that an inherent airway epithelial repair defect is the root cause of airway remodeling in asthma. However, the relationship between airway epithelial injury and repair, airway remodeling, and airway hyperresponsiveness (AHR) has not been directly examined. We investigated the contribution of epithelial damage and repair to the development of airway remodeling and AHR using a validated naphthalene (NA)-induced murine model of airway injury. In addition, we examined the endogenous versus exogenous role of the epithelial repair peptide trefoil factor 2 (TFF2) in disease pathogenesis. A single dose of NA (200 mg/kg in 10 ml/kg body weight corn oil [CO] vehicle, intraperitoneally) was administered to mice. Control mice were treated with CO (10 ml/kg body weight, intraperitoneally). At 12, 24, 48, and 72 hours after NA or CO injection, AHR and various measures of airway remodeling were examined by invasive plethysmography and morphometric analyses, respectively. TFF2-deficient mice and intranasal treatment were used to examine the role of the epithelial repair peptide. NA treatment induced denudation and apoptosis of airway epithelial cells, goblet cell metaplasia, elevated AHR, and increased levels of endogenous TFF2. Airway epithelial changes peaked at 12 hours after NA treatment, whereas airway remodeling changes were observed from 48 hours. TFF2 was protective against epithelial damage and induced remodeling and was found to mediate organ protection via a platelet-derived growth factor-associated mechanism. Our findings directly demonstrate the contribution of epithelial damage to airway remodeling and AHR and suggest that preventing airway epithelial damage and promoting epithelial repair may have therapeutic implications for asthma treatment.

Increased immunoexpression of trefoil factors in salivary gland tumors.

OBJECTIVE: Very little is known about the role of trefoil factors (TFFs) in salivary gland tumors, and TFF immunoexpression has never been investigated in such tumors. The aim of this study was to evaluate TFF immunoexpression in benign and malignant salivary gland tumors. MATERIALS AND METHODS: Benign (n = 25) and malignant (n = 25) salivary gland tumor specimens were included in this study, using mucocele (n = 25) specimens as a control group. Immunohistochemical staining was performed to evaluate the expression of TFFs (TFF1, TFF2, and TFF3) by semiquantitative means. RESULTS: Expression of TFF1, TFF2, and TFF3 was significantly increased in benign (p = 0.001, p = 0.005, p < 0.001, respectively) and malignant (p < 0.001, p < 0.001, p < 0.001, respectively) groups as compared with the control group. Patterns of co-expression between TFF1/TFF2, TFF2/TFF3, and TFF1/TFF3 were different among the three groups. CONCLUSIONS: The present study provided new information showing that all TFFs were significantly increased in benign and malignant salivary gland tumors, and overexpression of TFFs could be associated with neoplastic transformation in salivary gland tissues. CLINICAL RELEVANCE: Overexpression of TFFs may be useful as biomarkers in terms of differential diagnosis between salivary gland tumors and other oral neoplasms for which clinical manifestations are indistinguishable.

Trefoil factor-2 reverses airway remodeling changes in allergic airways disease.

Trefoil factor 2 (TFF2) is a small peptide with an important role in mucosal repair. TFF2 is up-regulated in asthma, suggesting a role in asthma pathogenesis. Given its known biological role in promoting epithelial repair, TFF2 might be expected to exert a protective function in limiting the progression of airway remodeling in asthma. The contribution of TFF2 to airway remodeling in asthma was investigated by examining the expression of TFF2 in the airway and lung, and evaluating the effects of recombinant TFF2 treatment on established airway remodeling in a murine model of chronic allergic airways disease (AAD). BALB/c mice were sensitized and challenged with ovalbumin (OVA) or saline for 9 weeks, whereas mice with established OVA-induced AAD were treated with TFF2 or vehicle control (intranasally for 14 d). Effects on airway remodeling, airway inflammation, and airway hyperresponsiveness were then assessed, whereas TFF2 expression was determined by immunohistochemistry. TFF2 expression was significantly increased in the airways of mice with AAD, compared with expression levels in control mice. TFF2 treatment resulted in reduced epithelial thickening, subepithelial collagen deposition, goblet-cell metaplasia, bronchial epithelium apoptosis, and airway hyperresponsiveness (all P < 0.05, versus vehicle control), but TFF2 treatment did not influence airway inflammation. The increased expression of endogenous TFF2 in response to chronic allergic inflammation is insufficient to prevent the progression of airway inflammation and remodeling in a murine model of chronic AAD. However, exogenous TFF2 treatment is effective in reversing aspects of established airway remodeling. TFF2 has potential as a novel treatment for airway remodeling in asthma.

Gastrokines: stomach-specific proteins with putative homeostatic and tumor suppressor roles.

During the past decade, a new family of stomach-specific proteins has been recognized. Known as "gastrokines" (GKNs), these secreted proteins are products of gastric mucus-producing cell lineages. GKNs are highly conserved in physical structure, and emerging data point to convergent functions in the modulation of gastric mucosal homeostasis and inflammation. While GKNs are highly prevalent in the normal stomach, frequent loss of GKN expression in gastric cancers, coupled with established antiproliferative activity, suggests putative tumor suppressor roles. Conversely, ectopic expression of GKNs in reparative lesions of Crohn's disease alludes to additional activity in epithelial wound healing and/or repair. Modes of action remain unsolved, but the recent demonstration of a GKN2-trefoil factor 1 heterodimer implicates functional interplay with trefoil factors. This review aims to provide a historical account of GKN biology and encapsulate the rapidly accumulating evidence supporting roles in gastric epithelial homeostasis and tumor suppression.

IL-11 is a parietal cell cytokine that induces atrophic gastritis.

BACKGROUND AND AIMS: IL-is important in gastric damage, mucosal repair and gastric cancer progression. We analysed IL-11 expression in H.pylori infected mouse stomach, the site of gastric IL-11 expression in mice and humans, and the effect of exogenous IL-11 on gastric mucosal homeostasis. METHODS: IL-11 protein was localised in mouse and human stomach. The impact of chronic, exogenous IL-11 on normal mouse stomach was examined histologically and transcriptionally by microarray, confirmed by mRNA and protein analysis. Functional impact of IL-11 on gastric acid secretion was determined. RESULTS: In mice infected with H.pylori, IL-11 was increased in fundic mucosa with temporal expression similar to IL-1b. IL-11 protein was localised predominantly to parietal cells in mouse and human stomach. Application of exogenous IL-11 to resulted in fundic parietal and chief cell loss, hyperplasia, mucous cell metaplasia and inflammation. Coincident with cellular changes were an increased gastric pH, altered parietal cell ultrastructure and altered gene expression, particularly genes involved in immune response and ion transport which could result in compromised acid secretion. We confirmed that a single dose of IL-11 effectively ablated the gastric response to histamine. CONCLUSIONS: IL-11 is a parietal cell cytokine that blocks gastric acid secretion, likely via reducing expression of parietal cell ion transport genes, CCKb and histamine H2 receptors. IL-11 expression is increased in H. pylori infected mouse stomach and treatment of wild type mice with IL-11 induced changes in the gastric fundic mucosa reminiscent of chronic atrophic gastritis, a precursor to gastric cancer.

Inhibition of gastric carcinogenesis by the hormone gastrin is mediated by suppression of TFF1 epigenetic silencing.

BACKGROUND & AIMS: Epigenetic alterations have been correlated with field cancerization in human patients, but evidence from experimental models that specific epigenetic changes can initiate cancer has been lacking. Although hormones have been associated with cancer risk, the mechanisms have not been determined. The peptide hormone gastrin exerts a suppressive effect on antral gastric carcinogenesis. METHODS: N-methyl-N-nitrosourea (MNU)-dependent gastric cancer was investigated in hypergastrinemic (INS-GAS), gastrin-deficient (GAS(-/-)), Tff1-deficient (Tff1(+/-)), and wild-type (WT) mice. Epigenetic alterations of the trefoil factor 1 (TFF1) tumor suppressor gene were evaluated in vitro and in vivo. RESULTS: Human intestinal-type gastric cancers in the antrum exhibited progressive TFF1 repression and promoter hypermethylation. Mice treated with MNU exhibited a field defect characterized by widespread Tff1 repression associated with histone H3 lysine 9 methylation and H3 deacetylation at the Tff1 promoter in epithelial cells. In MNU-induced advanced cancers, DNA methylation at the Tff1 promoter was observed. Tumor induction and Tff1 repression were increased in MNU-treated mice by Helicobacter infection. Hypergastrinemia suppressed MNU-dependent tumor initiation and progression in a manner that correlated with gene silencing and epigenetic alterations of Tff1. In contrast, homozygous gastrin-deficient and heterozygous Tff1-deficient mice showed enhanced MNU-dependent field defects and cancer initiation compared with WT mice. In gastric cancer cells, gastrin stimulation partially reversed the epigenetic silencing in the TFF1 promoter. CONCLUSIONS: Initiation of antral gastric cancer is associated with progressive epigenetic silencing of TFF1, which can be suppressed by the hormone gastrin.

Hyperactivation of Stat3 in gp130 mutant mice promotes gastric hyperproliferation and desensitizes TGF-beta signaling.

The latent transcription factor Stat3 is activated by gp130, the common receptor for the interleukin (IL)-6 cytokine family and other growth factor and cytokine receptors. Ligand-induced dimerization of gp130 leads to activation of the Stat1, Stat3 and Shp2-Ras-Erk signaling pathways. Here we assess genetically the contribution of exaggerated Stat3 activation to the phenotype of gp130 (Y757F/Y757F) mice, in which a knock-in mutation disrupts the negative feedback mechanism on gp130-dependent Stat signaling. Compared to gp130 (Y757F/Y757F) mice, reduced Stat3 activation in gp130 (Y757F/Y757F) Stat3(+/-) mice increased their lifespan, prevented splenomegaly, normalized exaggerated hepatic acute-phase response and lymphocyte trafficking, and suppressed the growth of spontaneously arising gastric adenomas in young mice. These lesions share histological features of gastric polyps in aging mice with monoallelic null mutations in Smad4, which encodes the common transducer for transforming growth factor (TGF)-beta signaling. Indeed, hyperactivation of Stat3 desensitizes gp130 (Y757F/Y757F) cells to the cytostatic effect of TGF-beta through transcriptional induction of inhibitory Smad7, thereby providing a novel link for cross-talk between Stat and Smad signaling in gastric homeostasis.

Investigation of trefoil factor expression in saliva and oral mucosal tissues of patients with oral squamous cell carcinoma.

OBJECTIVES: The aims of our study were to determine levels of trefoil factor (TFF) peptides in saliva and oral mucosal tissues from patients with oral squamous cell carcinoma (OSCC), and to evaluate whether individual members of TFFs (TFF1, TFF2, and TFF3) might act as biomarkers of disease. MATERIALS AND METHODS: Saliva samples were from 23 healthy subjects and 23 OSCC patients. Tissue samples were collected from 32 normal oral mucosa (NOM) and 32 OSCC biopsy specimens. ELISA and immunohistochemical methods were used to evaluate the expression of TFF1, TFF2, and TFF3 in saliva and oral mucosal tissues, respectively. RESULTS: Expression of TFF2 and TFF3 in oral mucosal tissues of OSCC patients was strongly downregulated when compared to healthy subjects (p < 0.001 and p = 0.002, respectively). However, there were no differences in levels of salivary TFF concentrations between OSCC patients and healthy subjects. CONCLUSIONS: The present study extends previous observations, demonstrating the reduction of TFF2 and TFF3 expression in oral mucosal tissues of OSCC patients. CLINICAL RELEVANCE: These findings suggest the clinical significance of TFF2 and TFF3 molecules as negative markers of tumor progression in OSCC. Quantification of TFF levels in saliva may not be optimal in terms of diagnostic or predictive value for OSCC derived from oral mucosa.

IL-33 promotes gastric tumour growth in concert with activation and recruitment of inflammatory myeloid cells.

Interleukin-33 (IL-33) is an IL-1 family cytokine known to promote T-helper (Th) type 2 immune responses that are often deregulated in gastric cancer (GC). IL-33 is overexpressed in human gastric tumours suggesting a role in driving GC progression although a causal link has not been proven. Here, we investigated the impact of IL-33 genetic deficiency in the well-characterized gp130 F/F mouse model of GC. Expression of IL-33 (and it's cognate receptor, ST2) was increased in human and mouse GC progression. IL-33 deficient gp130 F/F /Il33 -/- mice had reduced gastric tumour growth and reduced recruitment of pro-tumorigenic myeloid cells including key mast cell subsets and type-2 (M2) macrophages. Cell sorting of gastric tumours revealed that IL-33 chiefly localized to gastric (tumour) epithelial cells and was absent from tumour-infiltrating immune cells (except modest IL-33 enrichment within CD11b+ CX3CR1+CD64+MHCII+ macrophages). By contrast, ST2 was absent from gastric epithelial cells and localized exclusively within the (non-macrophage) immune cell fraction together with mast cell markers, Mcpt1 and Mcpt2. Collectively, we show that IL-33 is required for gastric tumour growth and provide evidence of a likely mechanism by which gastric epithelial-derived IL-33 drives mobilization of tumour-promoting inflammatory myeloid cells.

Random mutagenesis of the mouse genome: a strategy for discovering gene function and the molecular basis of disease.

Mutagenesis of mice with N-ethyl-N-nitrosourea (ENU) is a phenotype-driven approach to unravel gene function and discover new biological pathways. Phenotype-driven approaches have the advantage of making no assumptions about the function of genes and their products and have been successfully applied to the discovery of novel gene-phenotype relationships in many physiological systems. ENU mutagenesis of mice is used in many large-scale and more focused projects to generate and identify novel mouse models for the study of gene functions and human disease. This review examines the strategies and tools used in ENU mutagenesis screens to efficiently generate and identify functional mutations.

Helicobacter pylori infection promotes methylation and silencing of trefoil factor 2, leading to gastric tumor development in mice and humans.

BACKGROUND & AIMS: Trefoil factors (TFFs) regulate mucosal repair and suppress tumor formation in the stomach. Tff1 deficiency results in gastric cancer, whereas Tff2 deficiency increases gastric inflammation. TFF2 expression is frequently lost in gastric neoplasms, but the nature of the silencing mechanism and associated impact on tumorigenesis have not been determined. METHODS: We investigated the epigenetic silencing of TFF2 in gastric biopsy specimens from individuals with Helicobacter pylori-positive gastritis, intestinal metaplasia, gastric cancer, and disease-free controls. TFF2 function and methylation were manipulated in gastric cancer cell lines. The effects of Tff2 deficiency on tumor growth were investigated in the gp130(F/F) mouse model of gastric cancer. RESULTS: In human tissue samples, DNA methylation at the TFF2 promoter began at the time of H pylori infection and increased throughout gastric tumor progression. TFF2 methylation levels were inversely correlated with TFF2 messenger RNA levels and could be used to discriminate between disease-free controls, H pylori-infected, and tumor tissues. Genome demethylation restored TFF2 expression in gastric cancer cell lines, so TFF2 silencing requires methylation. In Tff2-deficient gp130(F/F)/Tff2(-/-) mice, proliferation of mucosal cells and release of T helper cell type-1 (Th-1) 1 cytokines increased, whereas expression of gastric tumor suppressor genes and Th-2 cytokines were reduced, compared with gp130(F/F)controls. The fundus of gp130(F/F)/Tff2(-/-) mice displayed glandular atrophy and metaplasia, indicating accelerated preneoplasia. Experimental H pylori infection in wild-type mice reduced antral expression of Tff2 by increased promoter methylation. CONCLUSIONS: TFF2 negatively regulates preneoplastic progression and subsequent tumor development in the stomach, a role that is subverted by promoter methylation during H pylori infection.

A novel gastrokine, Gkn3, marks gastric atrophy and shows evidence of adaptive gene loss in humans.

BACKGROUND & AIMS: Gastrokines are stomach mucus cell-secreted proteins; 2 gastrokines are known, GKN1 and GKN2. Gastrokine expression is lost in gastric cancer, indicating a possible function in tumor suppression. We have identified a third gastrokine gene in mammals. METHODS: Gkn3 was characterized by studies of molecular structure, evolutionary conservation, and tissue expression as well as transcriptional/translational outcome in mouse genetic models of gastric pathology. The functional consequences of Gkn3 overexpression were evaluated in transfected cell lines. RESULTS: Gkn3 encodes a secreted (approximately 19 kilodalton) protein that is co-expressed with trefoil factor (Tff)2 in the distal stomach and discriminates a Griffinia simplicifolia lectin (GS)-II-positive mucus neck cell (MNC) subpopulation in the proximal stomach. In humans, widespread homozygosity for a premature stop codon polymorphism, W59X, has likely rendered GKN3 non-functional. Population genetic analysis revealed an ancestral GKN3 read-through allele that predominates in Africans and indicates the rapid expansion of W59X among non-Africans during recent evolution. Mouse Gkn3 expression is strongly up-regulated in (Tff2-deficient) gastric atrophy, a pre-cancerous state that is typically associated with Helicobacter pylori and marks a non-proliferative, GS-II positive lineage with features of spasmolytic polypeptide-expressing metaplasia (SPEM). Gkn3 overexpression inhibits proliferation in gastric epithelial cell lines, independently of incubation with recombinant human TFF2 or apoptosis. CONCLUSIONS: Gkn3 encodes a novel, functionally distinct gastrokine that is overexpressed and might restrain epithelial cell proliferation in gastric atrophy. Spread of the human GKN3 stop allele W59X might have been selected for among non-Africans because of its effects on pre-neoplastic outcomes in the stomach.

Reciprocal regulation of gastrointestinal homeostasis by SHP2 and STAT-mediated trefoil gene activation in gp130 mutant mice.

The intracellular signaling mechanisms that specify tissue-specific responses to the interleukin-6 (IL-6) family of cytokines are not well understood. Here, we evaluated the functions of the two major signaling pathways, the signal transducers and activators of transcription 1 and 3 (STAT1/3) and the Src-homology tyrosine phosphatase 2 (SHP2)-Ras-ERK, emanating from the common signal transducer, gp130, in the gastrointestinal tract. Gp130(757F) mice, with a 'knock-in' mutation abrogating SHP2-Ras-ERK signaling, developed gastric adenomas by three months of age. In contrast, mice harboring the reciprocal mutation ablating STAT1/3 signaling (gp130(Delta STAT)), or deficient in IL-6-mediated gp130 signaling (IL-6(-/-) mice), showed impaired colonic mucosal wound healing. These gastrointestinal phenotypes are highly similar to the phenotypes exhibited by mice deficient in trefoil factor 1 (pS2/TFF1) and intestinal trefoil factor (ITF)/TFF3, respectively, and corresponded closely with the capacity of the two pathways to stimulate transcription of the genes encoding pS2/TFF1 and ITF/TFF3. We propose a model whereby mucosal wound healing depends solely on activation of STAT1/3, whereas gastric hyperplasia ensues when the coordinated activation of the STAT1/3 and SHP2-Ras-ERK pathways is disrupted.

The interleukin-6 family cytokine interleukin-11 regulates homeostatic epithelial cell turnover and promotes gastric tumor development.

BACKGROUND & AIMS: Gastric cancer is the second most common cause of cancer-related mortality worldwide, mainly as a result of late-stage detection. Interleukin (IL)-11 is a multifunctional cytokine reported to be up-regulated in human gastric cancer. METHODS: We investigated the importance of IL-11 in gastric cancer progression by examining its role in a variety of mouse gastric tumor models, as well as in nonneoplastic and tumor tissues taken from gastric cancer patients. We then determined the transcriptional and translational outcomes of IL-11 overexpression in normal gastric mucosa and identified a novel gene signature important early in the progression toward gastric tumorigenesis. RESULTS: IL-11 was up-regulated significantly in 4 diverse mouse models of gastric pathology as well as in human biopsy specimens adjacent to and within gastric cancer. Removal of IL-11 co-receptor alpha significantly reduced HKbeta-/- mouse fundic hyperplasia and ablated gp130(757F/F) mouse tumorigenesis. Exogenous IL-11 but not IL-6 activated oncogenic signal transducer and activator of transcription-3, and altered expression of novel proliferative and cytoprotective genes RegIII-beta, RegIII-gamma, gremlin-1, clusterin, and growth arrest specific-1 in wild-type gastric mucosa, a gene signature common in gp130(757F/F) and HKbeta-/- tumors as well as nonneoplastic mucosa of gastric cancer patients. One week of chronic IL-11 administration in wild-type mice sustained the gene signature, causing pretumorigenic changes in both antrum and fundus. CONCLUSIONS: Increased gastric IL-11 alters expression of proliferative and cytoprotective genes and promotes pretumorigenic cellular changes.