Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem-cell self-renewal.

The canonical Wnt/ß-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling ß-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5+ intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/ß-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5+ ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5+ ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5+ ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.

An Air-Liquid Interface Culture System for 3D Organoid Culture of Diverse Primary Gastrointestinal Tissues.

Conventional in vitro analysis of gastrointestinal epithelium usually relies on two-dimensional (2D) culture of epithelial cell lines as monolayer on impermeable surfaces. However, the lack of context of differentiation and tissue architecture in 2D culture can hinder the faithful recapitulation of the phenotypic and morphological characteristics of native epithelium. Here, we describe a robust long-term three-dimensional (3D) culture methodology for gastrointestinal culture, which incorporates both epithelial and mesenchymal/stromal components into a collagen-based air-liquid interface 3D culture system. This system allows vigorously expansion of primary gastrointestinal epithelium for over 60 days as organoids with both proliferation and multilineage differentiation, indicating successful long-term intestinal culture within a microenvironment accurately recapitulating the stem cell niche.

Defective goblet cell exocytosis contributes to murine cystic fibrosis-associated intestinal disease.

Cystic fibrosis (CF) intestinal disease is associated with the pathological manifestation mucoviscidosis, which is the secretion of tenacious, viscid mucus that plugs ducts and glands of epithelial-lined organs. Goblet cells are the principal cell type involved in exocytosis of mucin granules; however, little is known about the exocytotic process of goblet cells in the CF intestine. Using intestinal organoids from a CF mouse model, we determined that CF goblet cells have altered exocytotic dynamics, which involved intrathecal granule swelling that was abruptly followed by incomplete release of partially decondensated mucus. Some CF goblet cells exhibited an ectopic granule location and distorted cellular morphology, a phenotype that is consistent with retrograde intracellular granule movement during exocytosis. Increasing the luminal concentration of bicarbonate, which mimics CF transmembrane conductance regulator-mediated anion secretion, increased spontaneous degranulation in WT goblet cells and improved exocytotic dynamics in CF goblet cells; however, there was still an apparent incoordination between granule decondensation and exocytosis in the CF goblet cells. Compared with those within WT goblet cells, mucin granules within CF goblet cells had an alkaline pH, which may adversely affect the polyionic composition of the mucins. Together, these findings indicate that goblet cell dysfunction is an epithelial-autonomous defect in the CF intestine that likely contributes to the pathology of mucoviscidosis and the intestinal manifestations of obstruction and inflammation.

Peculiar histiocytic lesions with massive lanthanum deposition in dialysis patients treated with lanthanum carbonate.

Pathologic lesions caused by lanthanum carbonate (LC), a recently developed phosphate-binding agent, have not been recorded. A peculiar gastroduodenal histiocytic lesion associated with a mucosal lanthanum overload was reported. Our routine gastrointestinal biopsy series included 6 cases with heavy lanthanum burden in the gastroduodenal mucosa. In addition to routine histopathologic examinations, a series of immunohistochemical analysis and electron microscopic examinations associated with x-ray diffraction and elemental analysis were performed. Six cases, 3 of male and 3 of female individuals with ages from 59 to 69 years, were all patients of end-stage renal diseases managed under dialysis and treated with LC for >21 months. Endoscopic examinations demonstrated gastric erosions in 3, gastric polyps in 2, and duodenal ulcer in 1. In the mucosal layer, there were numerous non-Langerhans cell histiocytes, stained with CD68 but not S100 protein, engulfing a large amount of mineral-like materials. An electron microscopic and elemental analysis revealed a similar distribution of lanthanum and phosphorus in the histiocytes. Long-standing LC administration can cause massive mucosal accumulation of lanthanum in the tissue histiocytes associated with several forms of gastroduodenal lesions. A long-standing outcome is not clear at present; hence, careful follow-up studies of these patients may be needed.

Gastric mesenchymal myofibroblasts maintain stem cell activity and proliferation of murine gastric epithelium in vitro.

Stem cells are influenced by a microenvironmental niche that includes mesenchymal cells. We established a novel long-term method for primary mouse glandular stomach culture with mesenchymal myofibroblasts to investigate gastric epithelial-mesenchymal interactions. A gastric mesenchymal myofibroblast (GMF) cell line was established from mouse glandular stomach. Glandular stomach cells from neonatal mice and GMF cells were co-cultured in a collagen gel. Cultured stomach cells yielded expanding sphere-like structures. In the GMF co-culture system, the number and size of gastrospheres were increased compared with control cultures (P = 0.009 and 0.008, respectively). Immunohistochemistry showed cells positive for human gastric mucin, HIK1083, and chromogranin A, indicating differentiation into surface mucous cells, mucous neck cells, and enteroendocrine cells, respectively. RNA in situ hybridization for Lgr5 showed Lgr5(+) stem cells in the cultured gastrospheres. Lgr5(+) cells were observed persistently in the epithelium of gastrospheres in the GMF co-culture system for 2 months. GMFs allowed the cultured gastric epithelium to maintain active proliferation similar to that seen in vivo. Real-time quantitative RT-PCR showed that Gas1 expression was higher in GMFs (P = 0.0445), and Hoxc8, Notch1, and Sox10 expressions were higher in intestinal mesenchymal myofibroblasts (P = 0.0003, 0.0143, and 0.0488, respectively). We show the potential role of GMFs in sustaining Lgr5(+) stem cell activity and affecting normal gastric epithelial differentiation and proliferation.

Metastatic tumor evolution and organoid modeling implicate TGFBR2 as a cancer driver in diffuse gastric cancer.

BACKGROUND: Gastric cancer is the second-leading cause of global cancer deaths, with metastatic disease representing the primary cause of mortality. To identify candidate drivers involved in oncogenesis and tumor evolution, we conduct an extensive genome sequencing analysis of metastatic progression in a diffuse gastric cancer. This involves a comparison between a primary tumor from a hereditary diffuse gastric cancer syndrome proband and its recurrence as an ovarian metastasis. RESULTS: Both the primary tumor and ovarian metastasis have common biallelic loss-of-function of both the CDH1 and TP53 tumor suppressors, indicating a common genetic origin. While the primary tumor exhibits amplification of the Fibroblast growth factor receptor 2 (FGFR2) gene, the metastasis notably lacks FGFR2 amplification but rather possesses unique biallelic alterations of Transforming growth factor-beta receptor 2 (TGFBR2), indicating the divergent in vivo evolution of a TGFBR2-mutant metastatic clonal population in this patient. As TGFBR2 mutations have not previously been functionally validated in gastric cancer, we modeled the metastatic potential of TGFBR2 loss in a murine three-dimensional primary gastric organoid culture. The Tgfbr2 shRNA knockdown within Cdh1-/-; Tp53-/- organoids generates invasion in vitro and robust metastatic tumorigenicity in vivo, confirming Tgfbr2 metastasis suppressor activity. CONCLUSIONS: We document the metastatic differentiation and genetic heterogeneity of diffuse gastric cancer and reveal the potential metastatic role of TGFBR2 loss-of-function. In support of this study, we apply a murine primary organoid culture method capable of recapitulating in vivo metastatic gastric cancer. Overall, we describe an integrated approach to identify and functionally validate putative cancer drivers involved in metastasis.

Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture.

The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here we used a single air-liquid interface culture method without modification to engineer oncogenic mutations into primary epithelial and mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia as a result of expression of Kras carrying the G12D mutation (Kras(G12D)), p53 loss or both and readily generated adenocarcinoma after in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, Kras(G12D) and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), as compared to the more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the IGF2 (insulin-like growth factor-2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues.

Establishment of a long-term three-dimensional primary culture of mouse glandular stomach epithelial cells within the stem cell niche.

Compared to the small intestine and colon, little is known about stem cells in the stomach because of a lack of specific stem cell markers and an in vitro system that allows long-term culture. Here we describe a long-term three-dimensional (3D) primary gastric culture system within the stem cell niche. Glandular stomach cells from neonatal mice cultured in collagen gel yielded expanding sphere-like structures for 3months. The wall of the gastrospheres consisted of a highly polarized epithelial monolayer with an outer lining of myofibroblasts. The epithelial cells showed a tall columnar cell shape, basal round nuclei, and mucus-filled cytoplasm as well as expression of MUC5AC, indicating differentiation into gastric surface mucous cells. These cells demonstrated the features of fully differentiated gastric surface mucous cells such as microvilli, junctional complexes, and glycogen and secretory granules. Fewer than 1% of cultured epithelial cells differentiated into enteroendocrine cells. Active proliferation of the epithelial cells and many apoptotic cells in the inner lumen revealed the rapid cell turnover in gastrospheres in vitro. This method enables us to investigate the role of signaling between cell-cell and epithelial-mesenchymal interactions in an environment that is extremely similar to the in vivo environment.

Functional Cftr in crypt epithelium of organotypic enteroid cultures from murine small intestine.

Physiological studies of intact crypt epithelium have been limited by problems of accessibility in vivo and dedifferentiation in standard primary culture. Investigations of murine intestinal stem cells have recently yielded a primary intestinal culture in three-dimensional gel suspension that recapitulates crypt structure and epithelial differentiation (Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, Van Es JH, Abo A, Kujala P, Peters PJ, Clevers H. Nature 459: 262-265, 2009). We investigated the utility of murine intestinal crypt cultures (termed "enteroids") for physiological studies of crypt epithelium by focusing on the transport activity of the cystic fibrosis transmembrane conductance regulator Cftr. Enteroids had multiple crypts with well-differentiated goblet and Paneth cells that degranulated on exposure to the muscarinic agonist carbachol. Modified growth medium provided a crypt proliferation rate, as measured by 5-ethynyl-2'-deoxyuridine labeling, which was similar to proliferation in vivo. Immunoblots demonstrated equivalent Cftr expression in comparisons of freshly isolated crypts with primary and passage 1 enteroids. Apparent enteroid differences in mRNA expression of other transporters were primarily associated with villous epithelial contamination of freshly isolated crypts. Microelectrode analysis revealed cAMP-stimulated membrane depolarization in enteroid epithelium from wild-type (WT) but not Cftr knockout (KO) mice. Morphological and microfluorimetric studies, respectively, demonstrated Cftr-dependent cell shrinkage and lower intracellular pH in WT enteroid epithelium in contrast to Cftr KO epithelium or WT epithelium treated with Cftr inhibitor 172. We conclude that crypt epithelium of murine enteroids exhibit Cftr expression and activity that recapitulates crypt epithelium in vivo. Enteroids provide a primary culture model that is suitable for physiological studies of regenerating crypt epithelium.

The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations.

The small intestine epithelium undergoes rapid and continuous regeneration supported by crypt intestinal stem cells (ISCs). Bmi1 and Lgr5 have been independently identified to mark long-lived multipotent ISCs by lineage tracing in mice; however, the functional distinctions between these two populations remain undefined. Here, we demonstrate that Bmi1 and Lgr5 mark two functionally distinct ISCs in vivo. Lgr5 marks mitotically active ISCs that exhibit exquisite sensitivity to canonical Wnt modulation, contribute robustly to homeostatic regeneration, and are quantitatively ablated by irradiation. In contrast, Bmi1 marks quiescent ISCs that are insensitive to Wnt perturbations, contribute weakly to homeostatic regeneration, and are resistant to high-dose radiation injury. After irradiation, however, the normally quiescent Bmi1(+) ISCs dramatically proliferate to clonally repopulate multiple contiguous crypts and villi. Clonogenic culture of isolated single Bmi1(+) ISCs yields long-lived self-renewing spheroids of intestinal epithelium that produce Lgr5-expressing cells, thereby establishing a lineage relationship between these two populations in vitro. Taken together, these data provide direct evidence that Bmi1 marks quiescent, injury-inducible reserve ISCs that exhibit striking functional distinctions from Lgr5(+) ISCs and support a model whereby distinct ISC populations facilitate homeostatic vs. injury-induced regeneration.

Interaction between adipose tissue stromal cells and gastric cancer cells in vitro.

Adipose tissue exists in the gastric submucosa and subserosa. Thus, adipose tissue stromal cells (ATSCs), which include mesenchymal stem cells (MSCs), seem critical for the progression of gastric cancer but their interaction with the cancer cells is unknown. We demonstrated an interaction between these cells, using immunohistochemistry, Western blot and the collagen gel invasion assay system, in which the adenocarcinoma cells (well and poorly differentiated types, MKN28 and MKN45, respectively) were cultured on a ATSC-embedded or ATSC-non-embedded gel. ATSCs promoted the expression of the growth marker, proliferation cell nuclear antigen but inhibited that of the apoptosis marker, single-stranded DNA, in the cancer cell types. ATSCs accelerated the invasion of only MKN28 into the gel and promoted the expression of mitogen-activated protein kinase (MAPK, pERK-1/2) but decreased that of the molecularly targeted protein, HER2, in the cancer cells. ATSCs did not affect the expression of the prostaglandin biosynthetic enzyme cyclooxgenase-2 (COX-2) in the cancer cells. The COX-2 inhibitor celecoxib did not affect the morphology or invasion of the cancer cells. The cancer cell types in turn promoted the display of the myofibroblast marker, α-smooth muscle actin, whereas they decreased that of some MSC markers, e.g., CD44 and CD105, in ATSCs. The data suggest that (1) ATSCs influence the progression of gastric cancer by increasing their growth/invasion and decreasing their apoptosis through MAPK activation in a COX-2-independent way; (2) ATSCs adversely affect HER2-targeted therapy; (3) the cancer cells induce the cancer-associated myofibroblast phenotype in ATSCs.

A promising culture model for analyzing the interaction between adipose tissue and cardiomyocytes.

The heart has epicardial adipose tissue that produces adipokines and mesenchymal stem cells. Systemic adipose tissue is involved in the pathophysiology of obesity-related heart diseases. However, the method for analyzing the direct interaction between adipose tissue and cardiomyocytes has not been established. Here we show the novel model, using collagen gel coculture of adipose tissue fragments (ATFs) and HL-1 cardiomyocytes, and electron microscopy, immunohistochemistry, real-time RT-PCR, and ELISA. HL-1 cells formed a stratified layer on ATF-nonembedded gel, whereas they formed almost a monolayer on ATF-embedded gel. ATFs promoted the apoptosis, lipid accumulation, and fatty acid transport protein (FATP) expression of FATP4 and CD36 in HL-1 cells, whereas ATFs inhibited the growth and mRNA expression of myosin, troponin T, and atrial natriuretic peptide. Treatment of leptin (100 ng/ml) and adiponectin (10 µg/ml) neither replicated nor abolished the ATF-induced morphology of HL-1 cells, whereas that of FATP4 and CD36 antibodies (25 µg/ml) never abolished it. HL-1 cells prohibited the development of CD44+/CD105+ mesenchymal stem cell-like cells and lipid-laden preadipocytes from ATFs. HL-1 cells increased the production of adiponectin in ATFs, whereas they decreased that of leptin. The data indicate that our model actively creates adipose tissue-HL-1 cardiomyocyte interaction, suggesting first that ATFs may be related to the lipotoxiciy of HL-1 cells via unknown factors plus FATP4 and CD36 and second that HL-1 cells may help to retain the static state of ATFs, affecting adipokine secretion. Our model will serve to study adipose tissue-cardiomyocyte interaction and mechanisms of obesity-related lipotoxicity and heart diseases.

Culture models for studying thyroid biology and disorders.

The thyroid is composed of thyroid follicles supported by extracellular matrix, capillary network, and stromal cell types such as fibroblasts. The follicles consist of thyrocytes and C cells. In this microenvironment, thyrocytes are highly integrated in their specific structural and functional polarization, but monolayer and floating cultures cannot allow thyrocytes to organize the follicles with such polarity. In contrast, three-dimensional (3-D) collagen gel culture enables thyrocytes to form 3-D follicles with normal polarity. However, these systems never reconstruct the follicles consisting of both thyrocytes and C cells. Thyroid tissue-organotypic culture retains 3-D follicles with both thyrocytes and C cells. To create more appropriate experimental models, we here characterize four culture systems above and then introduce the models for studying thyroid biology and disorders. Finally, we propose a new approach to the cell type-specific culture systems on the basis of in vivo microenvironments of various cell types.

Adipose tissue explants and MDCK cells reciprocally regulate their morphogenesis in coculture.

Adipokine-producing fatty tissues, composed of preadipocytes, adipocytes, and mesenchymal stem cells, surround the kidney. To study the interaction between renal tubular cells and adipose tissue, we cocultured adipose tissue fragments and MDCK cells. MDCK cells in the coculture showed a taller columnar shape with improved organization of their microvilli and basal lamina than that seen in MDCK cell monoculture. The adipose tissue-induced change in morphology was replicated when we added leptin to MDCK cells cultured alone. Adiponectin abolished the leptin effect. Adipose tissue fragments inhibited MDCK cell division and also the formation of single-stranded DNA, an indicator of apoptosis. The fragments promoted the expression of polarity-associated proteins, including the tight junction molecules, ZO-1, atypical protein kinase C, and Cdc42. Further, the fragments also accelerated the expression of pendrin, the chloride/iodide transporter in the MDCK cells. In turn, MDCK cells decreased the number of preadipocytes and CD44+/CD105+ mesenchymal stem cells in the fragments, and promoted adiponectin production from the fragments. Thus, our study shows that adipose tissue fragments promote the hypertrophy, polarization, and differentiation of MDCK cells by attenuating their growth and apoptosis through opposing endocrine or paracrine effects of leptin and adiponectin. Further, MDCK cells inhibit the regeneration of preadipocytes and mesenchymal stem cells in adipose tissue.

Adipose tissue-organotypic culture system as a promising model for studying adipose tissue biology and regeneration.

Adipose tissue consists of mature adipocytes, preadipocytes and mesenchymal stem cells (MSCs), but a culture system for analyzing their cell types within the tissue has not been established. We have recently developed "adipose tissue-organotypic culture system" that maintains unilocular structure, proliferative ability and functions of mature adipocytes for a long term, using three-dimensional collagen gel culture of the tissue fragments. In this system, both preadipocytes and MSCs regenerate actively at the peripheral zone of the fragments. Our method will open up a new way for studying both multiple cell types within adipose tissue and the cell-based mechanisms of obesity and metabolic syndrome. Thus, it seems to be a promising model for investigating adipose tissue biology and regeneration. In this article, we introduce adipose tissue-organotypic culture, and propose two theories regarding the mechanism of tissue regeneration that occurs specifically at peripheral zone of tissue fragments in vitro.

Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche.

The in vitro analysis of intestinal epithelium has been hampered by a lack of suitable culture systems. Here we describe robust long-term methodology for small and large intestinal culture, incorporating an air-liquid interface and underlying stromal elements. These cultures showed prolonged intestinal epithelial expansion as sphere-like organoids with proliferation and multilineage differentiation. The Wnt growth factor family positively regulates proliferation of the intestinal epithelium in vivo. Accordingly, culture growth was inhibited by the Wnt antagonist Dickkopf-1 (Dkk1) and markedly stimulated by a fusion protein between the Wnt agonist R-spondin-1 and immunoglobulin Fc (RSpo1-Fc). Furthermore, treatment with the gamma-secretase inhibitor dibenzazepine and neurogenin-3 overexpression induced goblet cell and enteroendocrine cell differentiation, respectively, consistent with endogenous Notch signaling and lineage plasticity. Epithelial cells derived from both leucine-rich repeat-containing G protein-coupled receptor-5-positive (Lgr5(+)) and B lymphoma moloney murine leukemia virus insertion region homolog-1-positive (Bmi1(+)) lineages, representing putative intestinal stem cell (ISC) populations, were present in vitro and were expanded by treatment with RSpo1-Fc; this increased number of Lgr5(+) cells upon RSpo1-Fc treatment was subsequently confirmed in vivo. Our results indicate successful long-term intestinal culture within a microenvironment accurately recapitulating the Wnt- and Notch-dependent ISC niche.

Indigestible material attenuated changes in apoptosis in the fasted rat jejunal mucosa.

We have previously demonstrated that fasting induced apoptosis and decreased cell proliferation in the rat intestinal mucosa. The aim was to investigate the effect of expanded polystyrene as indigestible material on apoptosis and cell proliferation in rat small intestinal mucosa during fasting. Male SD rats were divided into 3 groups. The first group was fed with chow and water ad libitum. The second group fasted for 72 hrs. The third group was fasted for 24 hrs and was fed expanded polystyrene. Intestinal apoptosis was evaluated by percent fragmented DNA assay, terminal deoxynucleotidyl transferase-mediated dUDP-biotin nick end-labeling (TUNEL) staining, and caspase-3 assay. Cell proliferation was analyzed by 5-bromo-2'-deoxyuridine (5-BrdU) uptake. Truncal vagotomy was performed to evaluate a role of the central nervous system. In the 72-hr fasted rat, mucosal height of the rat jejunum was decreased to 73% of that in rats fed ad libitum, and this decrease was partly restored to 90% in rats fed expanded polystyrene. The fragmented DNA was increased in fasted rats (28.0%) when compared with that in rats fed ad libitum (2.6%). The increase in fragmented DNA in fasted rats was recovered by feeding them expanded polystyrene (8.3%). TUNEL staining confirmed this result. The effect of polystyrene on apoptosis was decreased by truncal vagotomy. Expression of cleaved caspase-3 was increased in fasted rats, which was then decreased by feeding of expanded polystyrene. In contrast to apoptosis, feeding of expanded polystyrene had no reconstructive effect on 5-BrdU uptake in the intestinal epithelium, which was decreased by fasting to 60% of that in rats fed ad libitum. In conclusion, feeding of indigestible material partly restored the decrease in intestinal mucosal length in the fasted rats through the apoptotic pathway without any influence on BrdU uptake. Further exploration focused on the mechanism of this effect of indigestible material is required.

Differentiation of gastric surface mucous cells (GSM06) induced by air-liquid interface is regulated partly through mitogen-activated protein kinase pathway.

BACKGROUND AND AIM: The aim of the present study was to examine the role of mitogen-activated protein (MAP) kinase pathway on gastric surface epithelium using an established cell culture model in which differentiation is promoted in GSM06 cells by air-liquid interface. METHODS: A double-dish culture system of mouse gastric surface mucous cell line GSM06 in Ham's F12 medium supplemented with 10% fetal calf serum and 50 microg/mL gentamicin at 37 degrees C in a humidified atmosphere of 5% CO(2) in air was used for an air-liquid interface. Culture cells were examined on histology, cell proliferation was evaluated by bromodeoxy-uridine (BrdU) uptake, and western blot analysis of extracellular signal-regulated kinase (ERK)1/2 and phosphate ERK1/2. On day 3, U0126, an inhibitor of MAP kinase kinase (MEK), was added to medium of incubated cells. RESULTS: GSM06 cells were differentiated with an air-liquid interface for 3 weeks. Compared to immersion control culture, phosphorylated ERK 1/2 expression increased significantly. This increase was completely suppressed with U0126, and tall columnar cells developed by air-liquid interface in GSM06 were not observed in U0126-treated cells. Increase in BrdU uptake with air-liquid interface was suppressed by U0126. CONCLUSION: These results suggested that MAP kinase signaling, activated by air-liquid interface, was, at least in part, related to cell differentiation in GSM06 cells induced by air-liquid interface.