Surface Mucous Cells Research Articles

Novel gene expression responses in the ovine abomasal mucosa to infection with the gastric nematode Teladorsagia circumcincta

Infection of sheep with the gastric nematode Teladorsagia circumcincta results in distinct Th2-type changes in the mucosa, including mucous neck cell and mast cell hyperplasia, eosinophilia, recruitment of IgA/IgE producing cells and neutrophils, altered T-cell subsets and mucosal hypertrophy. To address the protective mechanisms generated in animals on previous exposure to this parasite, gene expression profiling was carried out using samples of abomasal mucosa collected pre- and post- challenge from animals of differing immune status, using an experimental model of T. circumcincta infection. Recently developed ovine cDNA arrays were used to compare the abomasal responses of sheep immunised by trickle infection with worm-naïve sheep, following a single oral challenge of 50 000 T. circumcincta L3. Key changes were validated using qRT-PCR techniques. Immune animals demonstrated highly significant increases in levels of transcripts normally associated with cytotoxicity such as granulysin and granzymes A, B and H, as well as mucous-cell derived transcripts, predominantly calcium-activated chloride channel 1 (CLCA1). Challenge infection also induced up-regulation of transcripts potentially involved in initiating or modulating the immune response, such as heat shock proteins, complement factors and the chemokine CCL2. In contrast, there was marked infection-associated down-regulation of gene expression of members of the gastric lysozyme family. The changes in gene expression levels described here may reflect roles in direct anti-parasitic effects, immuno-modulation or tissue repair. (Funding; DEFRA/SHEFC (VT0102) and the BBSRC (BB/E01867X/1)).

Requirement of the Epithelium-specific Ets Transcription Factor Spdef for Mucous Gland Cell Function in the Gastric Antrum

Mucus-secreting cells of the stomach epithelium provide a protective barrier against damage that might result from bacterial colonization or other stimuli. Impaired barrier function contributes to chronic inflammation and cancer. Knock-out mice for the epithelium-specific transcription factor Spdef (also called Pdef) have defects in terminal differentiation of intestinal and bronchial secretory cells. We sought to determine the physiologic function of Spdef in the stomach, another site of significant levels of Spdef expression. We used in situ hybridization and immunohistochemistry to localize Spdef-expressing cells in the mouse stomach; targeted gene disruption to generate mice lacking Spdef; and histologic, immunologic, and transcriptional profiling approaches to determine the requirements of Spdef in stomach epithelial homeostasis. In wild-type mice, Spdef RNA and protein are expressed predominantly in mucous gland cells of the antrum and in mucous neck cells of the glandular corpus. Within 1.5 years, nearly half of homozygous mutant mice developed profound mucosal hyperplasia of the gastric antrum. Submucosal infiltration of inflammatory cells preceded antral hyperplasia by several weeks. The absence of Spdef impaired terminal maturation of antral mucous gland cells, as reflected in reduced expression of Muc6 and Tff2 and reduced numbers of secretory granules. Antral gene expression abnormalities overlapped significantly with those in Spdef(-/-) colon, including genes implicated in secretory granule traffic and functions. Spdef is required for terminal maturation of antral mucous gland cells to protect animals from gastric inflammation and resulting hyperplasia. These requirements parallel Spdef functions in secretory intestinal cells and suggest a common molecular mechanism for maturation of gastrointestinal secretory lineages.

Hedgehog signaling regulates E-cadherin expression for the maintenance of the actin cytoskeleton and tight junctions

In the stomach, strictly regulated cell adherens junctions are crucial in determining epithelial cell differentiation. Sonic Hedgehog (Shh) regulates epithelial cell differentiation in the adult stomach. We sought to identify whether Shh plays a role in regulating adherens junction protein E-cadherin as a mechanism for epithelial cell differentiation. Mouse nontumorigenic gastric epithelial (IMGE-5) cells treated with Hedgehog signaling inhibitor cyclopamine and anti-Shh 5E1 antibody or transduced with short hairpin RNA against Skinny Hedgehog (IMGE-5(Ski)) were cultured. A mouse model expressing a parietal cell-specific deletion of Shh (HKCre/Shh(KO)) was used to identify further changes in adherens and tight junctions. Inhibition of Hedgehog signaling in IMGE-5 cells caused loss of E-cadherin expression accompanied by disruption of F-actin cortical expression and relocalization of zonula occludens-1 (ZO-1). Loss of E-cadherin was also associated with increased proliferation in IMGE-5(Ski) cells and increased expression of the mucous neck cell lineage marker MUC6. Compared with membrane-expressed E-cadherin and ZO-1 protein in controls, dissociation of E-cadherin/β-catenin and ZO-1/occludin protein complexes was observed in HKCre/Shh(KO) mice. In conclusion, we demonstrate that Hedgehog signaling regulates E-cadherin expression that is required for the maintenance of F-actin cortical expression and stability of tight junction protein ZO-1.

XBP1 Controls Maturation of Gastric Zymogenic Cells by Induction of MIST1 and Expansion of the Rough Endoplasmic Reticulum

The transition of gastric epithelial mucous neck cells (NCs) to digestive enzyme-secreting zymogenic cells (ZCs) involves an increase in rough endoplasmic reticulum (ER) and formation of many large secretory vesicles. The transcription factor MIST1 is required for granulogenesis of ZCs. The transcription factor XBP1 binds the Mist1 promoter and induces its expression in vitro and expands the ER in other cell types. We investigated whether XBP1 activates Mist1 to regulate ZC differentiation. Xbp1 was inducibly deleted in mice using a tamoxifen/Cre-loxP system; effects on ZC size and structure (ER and granule formation) and gastric differentiation were studied and quantified for up to 13 months after deletion using morphologic, immunofluorescence, quantitative reverse-transcriptase polymerase chain reaction, and immunoblot analyses. Interactions between XBP1 and the Mist1 promoter were studied by chromatin immunoprecipitation from mouse stomach and in XBP1-transfected gastric cell lines. Tamoxifen-induced deletion of Xbp1 (Xbp1Δ) did not affect survival of ZCs but prevented formation of their structure. Xbp1Δ ZCs shrank 4-fold, compared with those of wild-type mice, with granulogenesis and cell shape abnormalities and disrupted rough ER. XBP1 was required and sufficient for transcriptional activation of MIST1. ZCs that developed in the absence of XBP1 induced ZC markers (intrinsic factor, pepsinogen C) but showed abnormal retention of progenitor NC markers. XBP1 controls the transcriptional regulation of ZC structural development; it expands the lamellar rough ER and induces MIST1 expression to regulate formation of large granules. XBP1 is also required for loss of mucous NC markers as ZCs form.

Cytodifferentiation of the postnatal mouse stomach in normal and Huntingtin-interacting protein 1-related-deficient mice

Huntingtin-interacting protein 1-related (Hip1r) is highly expressed in gastric parietal cells, where it participates in vesicular trafficking associated with acid secretion. Hip1r-deficient mice have a progressive remodeling of the mucosa, including apoptotic loss of parietal cells, glandular hypertrophy, mucous cell metaplasia, and reduced numbers of zymogenic cells. In this study, we characterized gastric gland development in wild-type and Hip1r-deficient mice to define normal development, as well as the timing and sequence of the cellular transformation events in the mutant stomach. Postnatal (newborn to 8-wk-old) stomachs were examined by histological and gene expression analysis. At birth, gastric glands in wild-type and mutant mice were rudimentary and mature gastric epithelial cells were not apparent, although marker expression was detected for most cell lineages. Interestingly, newborns exhibited unusual cell types, including a novel surface cell filled with lipid and cells that coexpressed markers of mature mucous neck and zymogenic cells. Glandular morphogenesis proceeded rapidly in both genotypes, with gastric glands formed by weaning at 3 wk of age. In the Hip1r-deficient stomach, epithelial cell remodeling developed in a progressive manner. Initially, in the perinatal stomach, cellular changes were limited to parietal cell apoptosis. Other epithelial cell changes, including apoptotic loss of zymogenic cells and expansion of metaplastic mucous cells, emerged several weeks later when the glands were morphologically mature. Thus, parietal cell loss appeared to be the initiating event in Hip1r-deficient mice, with secondary remodeling of the other gastric epithelial cells.

TFF2 mRNA Transcript Expression Marks a Gland Progenitor Cell of the Gastric Oxyntic Mucosa

Gastric stem cells are located in the isthmus of the gastric glands and give rise to epithelial progenitors that undergo bipolar migration and differentiation into pit and oxyntic lineages. Although gastric mucus neck cells located below the isthmus express trefoil factor family 2 (TFF2) protein, TFF2 messenger RNA transcripts are concentrated in cells above the neck region in normal corpus mucosa, suggesting that TFF2 transcription is a marker of gastric progenitor cells. Using a BAC strategy, we generated a transgenic mouse with a tamoxifen-inducible Cre under the control of the TFF2 promoter (TFF2-BAC-Cre(ERT2)) and analyzed the lineage derivation from TFF2 mRNA transcript-expressing (TTE) cells. TTE cells were localized to the isthmus, above and distinct from TFF2 protein-expressing mucus neck cells. Lineage tracing revealed that these cells migrated toward the bottom of the gland within 20 days, giving rise to parietal, mucous neck, and chief cells, but not to enterochromaffin-like-cell. Surface mucus cells were not derived from TTE cells and the progeny of the TTE lineage did not survive beyond 200 days. TTE cells were localized in the isthmus adjacent to doublecortin CaM kinase-like-1(+) putative progenitor cells. Induction of spasmolytic polypeptide-expressing metaplasia with DMP-777-induced acute parietal cell loss revealed that this metaplastic phenotype might arise in part through transdifferentiation of chief cells as opposed to expansion of mucus neck or progenitor cells. TFF2 transcript-expressing cells are progenitors for mucus neck, parietal and zymogenic, but not for pit or enterochromaffin-like cell lineages in the oxyntic gastric mucosa.

Regeneration profiling of human gastric glands: differences between fundic and antral surface mucous cells

Introduction: Maintenance of the surface integrity of mucous epithelia is vital for physiological functions of many organs such as the stomach. The human gastric mucosa can be divided into three different functional regions, i.e., the cardia, the corpus, and the antrum. Classically, the cellular constituents are defined as surface mucous cells (SMCs)/gastric pit cells, oxyntic cells, mucous neck and antral gland cells, zymogenic cells and endocrine cells. Here, a first expression profile of human gastric glands is presented using RT-PCR analysis after laser microdissection.

Calpain 8/nCL-2 and Calpain 9/nCL-4 Constitute an Active Protease Complex, G-Calpain, Involved in Gastric Mucosal Defense

Calpains constitute a superfamily of Ca2+-dependent cysteine proteases, indispensable for various cellular processes. Among the 15 mammalian calpains, calpain 8/nCL-2 and calpain 9/nCL-4 are predominantly expressed in the gastrointestinal tract and are restricted to the gastric surface mucus (pit) cells in the stomach. Possible functions reported for calpain 8 are in vesicle trafficking between ER and Golgi, and calpain 9 are implicated in suppressing tumorigenesis. These highlight that calpains 8 and 9 are regulated differently from each other and from conventional calpains and, thus, have potentially important, specific functions in the gastrointestinal tract. However, there is no direct evidence implicating calpain 8 or 9 in human disease, and their properties and physiological functions are currently unknown. To address their physiological roles, we analyzed mice with mutations in the genes for these calpains, Capn8 and Capn9. Capn8−/− and Capn9−/− mice were fertile, and their gastric mucosae appeared normal. However, both mice were susceptible to gastric mucosal injury induced by ethanol administration. Moreover, the Capn8−/− stomach showed significant decreases in both calpains 9 and 8, and the same was true for Capn9−/−. Consistent with this finding, in the wild-type stomach, calpains 8 and 9 formed a complex we termed “G-calpain,” in which both were essential for activity. This is the first example of a “hybrid” calpain complex. To address the physiological relevance of the calpain 8 proteolytic activity, we generated calpain 8:C105S “knock-in” (Capn8CS/CS) mice, which expressed a proteolytically inactive, but structurally intact, calpain 8. Although, unlike the Capn8−/− stomach, that of the Capn8CS/CS mice expressed a stable and active calpain 9, the mice were susceptible to ethanol-induced gastric injury. These results provide the first evidence that both of the gastrointestinal-tract-specific calpains are essential for gastric mucosal defense, and they point to G-calpain as a potential target for gastropathies caused by external stresses.

Foveolar cell hyperplasia at the pyloric canal: an unusual cause of gastric outlet obstruction in 3 young children

Foveolar cell hyperplasia (FCH) in the pylorus is rare in children. Between January 1993 and December 2008, 3 children with pyloric canal FCH presented with gastric outlet obstruction and/or anemia and underwent operation because a clear diagnosis could not be established with conservative management. The median age of the children was 18 months. The onset of FCH at the pyloric canal is problematic, and its diagnosis is difficult to make in pediatric patients. Exploratory laparotomy is a reasonable method of establishing the diagnosis and managing the condition.

Cell cycle regulators p16INK4a in metaplasia, dysplasia, and adenocarcinoma of Barrett's esophagus.

e14656 Background: p16INK4a is a cyclin-dependent kinase inhibitor, together with p21, p53 and p63 playing an important role in the regulation of the cell cycle and histomorphology in embryology, regeneration and in tumours. The variety of neoplastic and non-neoplastic types of epithelia and their architecture in Barrett esophagus may be associated with structural and functional proteins such as cell-cycle regulators. Loss of heterozygosity in p16INK4a/CDKN2A occurs in 75% of Barrett-carcinomas and is a amrker of better prognosis. Methods: Twenty formalin-fixed, paraffine embeded biopsies of Barrett esophagus with epithelia of specialised (colonic) type, twenty cases with metaplastic gastric foveolar type/cardiac type and ten cases of Barrett-associated adenocarcinoma (diffuse, intestinal and exophytic papillary type) were analysed for the expression and distribution of p16INK4a (Antibodies: mtm Heidelberg).All cases histologically and endopscopically confirmed (Munic Type C patients). Results: Nonneoplastic Barrett: Strong,expression of p16INK4a in the stratum spinosum cells of regenerative hyperplastic squamous epithelium: mostly coherent cells in columnar or reticulated arrangement. No expression in the foveolar cells of esophageal gastric heterotopia. Crypts and intestinal- metaplastic cardia-glands show slight to moderate nuclear and cytoplasmic expreession. Dysplasia and neoplasia in Barrett: Low p16INK4a positivity in colon-type specialized epithelium of low grade dysplasia. Moderarte positivity in high grade dysplasia. Most cases indeterminate for dysplasia are negative. Two different patterns of p16INK4a expression in invasive adenocarcinomas of Barrett epithelia: well-differentiated papillary and tubuloglandular cancers highly positive for p16INK4a; carcinomas of macroglandular or diffuse pattern or signet-cell-type no or low expression. Conclusions: Inactivation of CDKN2A /p16 and is an early event in Barrett

Effects of weaning and infection with Teladorsagia circumcincta on mucin carbohydrate profiles of early weaned lambs

Differences in mucin glycosylation in milk-fed and early weaned lambs may influence susceptibility to parasitism, particularly the greater cellular content and higher sulphation of mucins in young and unweaned lambs. Weaning also reduced the percentage of Gal (p<0.05) in fundic mucin and galactosamine (GalN) (p<0.01) in duodenal mucin, but had no noticeable effect on fucosylation or sialylation. Four experimental groups of lambs were studied (n=3): (1) 3 days old; (2) 9 weeks old milk-fed; (3) 9 weeks old weaned at 3 weeks-of-age on to lucerne chaff and cereal-based pellets (4) 9 weeks old weaned and infected with 1000 Teladorsagia circumcincta L3 twice weekly for 5 weeks. Fundic and duodenal mucin monosaccharides were analysed chemically and fundic, antral and duodenal tissues were stained with lectins, periodic acid Schiff (PAS), Alcian blue/PAS (AB/PAS) and high iron diamine. Age-related maturation of mucin glycosylation was prominent in young lambs: reduced total fundic mucins and increasing fucosylation and decreasing sialylation and sulphation of all mucins, as well as changes in the types of linkages of Fuc and sialic acids. By 9 weeks-of-age, there were no longer sialylated mucins in fundic surface mucus cells, only neutral mucins, while in Brunner's glands, there was reduced sialylation and large amounts of neutral mucins. In the neonates, both fundic and duodenal tissues contained only small amounts of mucins terminating with alpha-1,2-linked Fuc, which became the principal linkage in 9 weeks old lambs. Duodenal mucins in 3 days old lambs contained both alpha-2,6- and alpha-2,3-linked sialic acids, whereas the alpha-2,3 linkage was not present in older lambs. Parasitism increased the percentage of galactose, but reduced total and neutral fundic mucins, as well as sulphation and sialylation. There was both decreased sialylation and sulphation in duodenal mucins. Although no change in fucosylation was apparent from chemical analysis, infection reduced lectin staining for alpha-1,2-linked fucose in antral and duodenal tissues and alpha-1,6- and alpha-1,3-linked fucose in the duodenum. These changes in fundic and duodenal mucins were similar to those previously seen on Day 28 p.i. after a single infection of 4-9 months old sheep with T. circumcincta larvae.

A Novel Foregut Mucin Characterized by a Murine Monoclonal Autoantibody

Autoantibodies to gastric cellular antigens and glycoproteins including mucins and Lewis X and Y antigens have been implicated in the induction of autoimmune gastritis. Monoclonal antibody D10 (D10 MAb) recognizes a highly conserved mucin expressed in the foregut of mammals and other vertebrates. The objective of this study was to biochemically characterize the autoantigen identified by D10 MAb and examine its autoimmunogenicity in the mouse. Characterization of the mucin autoantigen was undertaken following purification, by amino acid and carbohydrate analyses, deglycosylation, SDS-PAGE, and immunoblotting using D10 MAb. Autoimmune reactivity and specificity of D10 MAb were validated by immunohistochemistry and ELISA using mouse tissue. Induction of autoimmune gastritis was investigated following immunization of mice with D10 MAb-reactive heterologous mucin. D10 MAb was shown to be a murine anti-mucin autoantibody with a unique pattern of immunohistochemical staining of Brunner's glands of the duodenum and the cardiac glands, mucous neck cells, and pyloric glands of the stomach from inbred Balb/c mice in patterns identical to that previously reported in human tissue. Amino acid and carbohydrate analysis of purified D10 mucin reflected a compositional profile of a typical mucin molecule. Confirmation that D10 MAb recognizes a mucin was also provided by demonstration that the carbohydrate epitope resides on a high molecular weight (>1x10(6)Da), high-density (>1.40 g/mL) molecule comprised of greater than 60% carbohydrate. Mice immunized with D10 MAb-reactive, purified, heterologous mucin produced autoantibodies of identical specificity to the original D10 MAb. These data demonstrate the autoimmunogenic properties of a novel foregut mucin and raise the potential of anti-mucin autoantibodies in the induction of autoimmune gastritis.

Loss of Sonic Hedgehog in the adult stomach results in foveolar hyperplasia and delayed differentiation of the zymogenic cell lineage

ObjectiveParietal cell‐expressed Sonic Hedgehog (Shh) regulates function and differentiation of the gastric epithelium. Correlative evidence shows that loss of Shh expression associated with Helicobacter pylori infection results in the disruption of the normal gastric epithelium. We sought to identify whether Shh regulates gastric epithelial cell differentiation in the absence of inflammation.MethodsA parietal cell‐specific, tamoxifen‐inducible deletion of Shh mouse model was developed (HKCreInd/ShhKO). Epithelial cell proliferation and migration was studied using BrdU and Ki67 co‐immunostaining. Cell lineage changes were studied using markers specific for surface mucous (Ulex europaeus, UEAI), mucous neck (Griffonia simplicifolia, GSII) and zymogen (intrinsic factor, IF) cells.ResultsCompared to Controls, HKCreInd/ShhKO mice developed foveolar hyperplasia reflected by an expansion of BrdU/UEAI positive cells. HKCreInd/ShhKO mice also showed an expansion of GSII/IF co‐expressing cells at the base of the gastric gland. Co‐localization of BrdU and Ki67 revealed loss of normal migration of the epithelial cells within HKCreInd/ShhKO mouse stomachs.ConclusionLoss of Shh in the adult stomach results in the development of foveolar hyperplasia and delayed differentiation of the zymogenic cell lineage independent of inflammation.(DHC Pilot and Feasibility Project Award, Zavros)

Protocol for the Examination of Specimens From Patients With Carcinoma of the Distal Extrahepatic Bile Ducts

The College of American Pathologists offers these protocols to assist pathologists in providing clinically useful and relevant information when reporting results of surgical specimen examinations. The College regards the reporting elements in the ‘‘Surgical Pathology Cancer Case Summary (Checklist)’’ portion of the protocols as essential elements of the pathology report. However, the manner in which these elements are reported is at the discretion of each specific pathologist, taking into account clinician preferences, institutional policies, and individual practice. The College developed these protocols as an educational tool to assist pathologists in the useful reporting of relevant information. It did not issue the protocols for use in litigation, reimbursement, or other contexts. Nevertheless, the College recognizes that the protocols might be used by hospitals, attorneys, payers, and others. Indeed, effective January 1, 2004, the Commission on Cancer of the American College of Surgeons mandated the use of the checklist elements of the protocols as part of its Cancer Program Standards for Approved Cancer Programs. Therefore, it becomes even more important for pathologists to familiarize themselves with these documents. At the same time, the College cautions that use of the protocols other than for their intended educational purpose may involve additional considerations that are beyond the scope of these documents.

Effect of ezrin knockdown on the architecture of gastric epithelia

Ezrin, an adaptor protein that cross‐links plasma membrane‐associated proteins with actin cytoskeleton, is highly concentrated on the apical surface of many epithelial cell types especially in microvilli in small intestines and stomachs. In stomachs, ezrin is predominantly expressed on apical canalicular membrane of parietal cells. The ezrin knockdown (Vil2kd/kd) mice showed achlorhydria due to the impairment of membrane fusion of tubulovesicles with apical membrane. Here, we newly showed the involvement of ezrin in the architecture of gastric glands using the Vil2kd/kd mice. The Vil2kd/kd mice showed hypergastrinemia and hyperplasia of gastric surface epithelial cells. Atrophy was observed in the gastric glands with the number of parietal cells and chief cells being decreased, and the number of mucus neck cells being increased. The expression level of gastric proton pump was significantly decreased whereas the expression level of chloride channel CFTR was increased. These results demonstrate that ezrin is involved in the development of gastric epithelial cells and expression of transport proteins involved in gastric acid secretion.

The characterization of the first anti-mouse Muc6 antibody shows an increased expression of the mucin in pancreatic tissue of Cftr-knockout mice

Gel-forming mucins are large high-molecular weight secreted O-glycoproteins responsible for the gel-properties of the mucus blanket. Five orthologous gel-forming mucins have been cloned in human and mouse. Among them, the mucin MUC6 has been less studied, particularly in rodents and no anti rodent-Muc6 antibody has been reported yet. In order to further study Muc6 in mice, our aims were to obtain a specific Muc6 antibody, to validate it and to test it in Cftr deficient mice. A polyclonal serum named CP4 was isolated from a rabbit immunized by a mouse Muc6 peptide. In Western blot experiments, the antibody detected a high-molecular weight molecule secreted by the gastric tissue. Using immunohistochemistry, we showed that the antibody reacted strongly with deep glands of duodenum and ileum and mucous neck cells of gastric body. CP4 also recognized Muc6 protein secreted at the surface of the stomach and renal collecting tubules. The centroacinar cells of pancreatic tissue also reacted with the antibody. Cftr-/- mice showed a higher expression of Muc6 at both protein and RNA levels compared with their control Cftr+/+ littermates suggesting that as in the human disease, Muc6 may contribute to the formation of materials that block pancreatic acini and ducts in mouse models of cystic fibrosis. The rabbit anti-mouse Muc6 polyclonal antibody seems highly specific to the mouse mucin and will be useful to study pancreatic pathology in cystic fibrosis.

RAB26 and RAB3D Are Direct Transcriptional Targets of MIST1 That Regulate Exocrine Granule Maturation

Little is known about how differentiating cells reorganize their cellular structure to perform specialized physiological functions. MIST1, an evolutionarily conserved transcription factor, is required for the formation of large, specialized secretory vesicles in gastric zymogenic (chief) cells (ZCs) as they differentiate from their mucous neck cell progenitors. Here, we show that MIST1 binds to highly conserved CATATG E-boxes to directly activate transcription of 6 genes, including those encoding the small GTPases RAB26 and RAB3D. We next show that RAB26 and RAB3D expression is significantly downregulated in Mist1(-)(/)(-) ZCs, suggesting that MIST1 establishes large secretory granules by inducing RAB transcription. To test this hypothesis, we transfected human gastric cancer cell lines stably expressing MIST1 with red fluorescent protein (RFP)-tagged pepsinogen C, a key secretory product of ZCs. Those cells upregulate expression of RAB26 and RAB3D to form large secretory granules, whereas control, non-MIST1-expressing cells do not. Moreover, granule formation in MIST1-expressing cells requires RAB activity because treatment with a RAB prenylation inhibitor and transfection of dominant negative RAB26 abrogate granule formation. Together, our data establish the molecular process by which a transcription factor can directly induce fundamental cellular architecture changes by increasing transcription of specific cellular effectors that act to organize a unique subcellular compartment.

A Novel Gastrokine, Gkn3, Marks Gastric Atrophy and Shows Evidence of Adaptive Gene Loss in Humans

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. 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. 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. 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.

New Frontiers in Gut Nutrient Sensor Research: Luminal Glutamate–Sensing Cells in Rat Gastric Mucosa

Dietary free glutamate is known to elicit umami, one of the five basic tastes percepted via the specific taste sensor cells on the tongue. Recent studies suggest the specific glutamate sensors exist in the gastric mucosa and contribute to the regulation of gastrointestinal functions, yet the precise mechanism remains still unknown. We established the method to enrich various cell fractions from the isolated rat gastric mucosa and characterized the expression of putative glutamate sensors using such cell fractions. The gastric mucosal cell fractions such as surface mucous, parietal, chief, and endocrine cells were successfully prepared by mucosal protease digestion, elutriation, and gradient centrifugation. The characteristics of these cells were confirmed by real-time RT-PCR using the respective cell-specific markers. Parietal cell fraction exclusively expressed putative umami receptor molecules such as T1R1 and mGluR1 compared to other fractions, although the degree of expression was low. In contrast, the representative taste cell specific markers such as PLCβ2 and TRPM5 were specifically expressed in the smaller endocrine cell fraction. Both parietal and smaller endocrine cell fractions also positively expressed some mGluR subtypes. The chief-cell fraction less expressed T1R1 and mGluR1. These results suggest that multiple glutamate sensors, probably different mechanisms from taste buds, contribute to the glutamate sensing in the gastric mucosa.

Loss of Parietal Cell Expression of Sonic Hedgehog Induces Hypergastrinemia and Hyperproliferation of Surface Mucous Cells

Sonic Hedgehog (Shh) is expressed in the adult stomach, but its role as a gastric morphogen is unclear. We sought to identify mechanisms by which Shh might regulate gastric epithelial cell function and differentiation. Mice with a parietal cell-specific deletion of Shh (HKCre/Shh(KO)) were created. Gastric morphology and function were studied in control and HKCre/Shh(KO) mice between 1 and 8 months of age. In contrast to control mice, HKCre/Shh(KO) mice developed gastric hypochlorhydria, hypergastrinemia, and a phenotype that resembled foveolar hyperplasia. The fundic mucosa of HKCre/Shh(KO) mice had an expanded surface pit cell lineage that was documented by increased incorporation of bromodeoxyuridine and was attributed to the hypergastrinemia. Compared with controls, numbers of total mucous neck and zymogen cells were significantly decreased in stomachs of HKCre/Shh(KO) mice. In addition, zymogen and neck cell markers were coexpressed in the same cell populations, indicating disrupted differentiation of the zymogen cell lineage from the mucous neck cells in the stomachs of HKCre/Shh(KO) mice. Laser capture microdissection of the surface epithelium, followed by quantitative reverse-transcription polymerase chain reaction, revealed a significant increase in expression of Indian Hedgehog, glioma-associated oncogene homolog 1, Wnt, and cyclin D1. Laser capture microdissection analysis also showed a significant increase in Snail with a concomitant decrease in E-cadherin. In the stomachs of adult mice, loss of Shh from parietal cells results in hypochlorhydria and hypergastrinemia. Hypergastrinemia might subsequently induce increased Hedgehog and Wnt signaling in the surface pit epithelium, resulting in hyperproliferation.