Transcriptome analysis reveals the essential role of NK2 homeobox 1/thyroid transcription factor 1 (NKX2-1/TTF-1) in gastric adenocarcinoma of fundic-gland type.

BACKGROUND: Gastric adenocarcinoma of fundic-gland type (GA-FG) is a gastric malignancy with little relation to Helicobacter pylori. Clinical characteristics of GA-FG have been established, but molecular mechanisms leading to tumorigenesis have not yet been elucidated. METHODS: We subjected three GA-FG tumors-normal mucosa pairs to microarray analysis. Network analysis was performed for the top 30 up-regulated gene transcripts, followed by immunohistochemical staining to confirm the gene expression analysis results. AGS and NUGC4 cells were transfected with the gene-encoding NK2 homeobox 1/thyroid transcription factor 1 (NKX2-1/TTF-1) to evaluate transcriptional changes in its target genes. RESULTS: Comprehensive gene expression analysis identified 1410 up-regulated and 1395 down-regulated gene probes with ≥ two-fold difference in expression. Among the top 30 up-regulated genes in GA-FG, we identified transcription factor NKX2-1/TTF-1, a master regulator of lung/thyroid differentiation, together with surfactant protein B (SFTPB), SFTPC, and secretoglobin family 3A member 2(SCGB3A2), which are regulated by NKX2-1/TTF-1. Immunohistochemical analysis of 16 GA-FG specimens demonstrated significantly higher NKX2-1/TTF-1 and SFTPB levels, as compared to that in adjacent normal mucosa (P < 0.05), while SCGB3A2 levels did not differ (P = 0.341). Transduction of NKX2-1/TTF-1 into AGS and NUGC4 cells induced transactivation of SFTPB and SFTPC, indicating that NKX2-1/TTF-1 can function as normally in gastric cells as it can in the lung cells. CONCLUSIONS: Our first transcriptome analysis of GA-FG indicates significant expression of NKX2-1/TTF1 in GA-FG. Immunohistochemistry and cell biology show ectopic expression and normal transactivation ability of NKX2-1/TTF-1, suggesting that it plays an essential role in GA-FG development.

Transduced caudal-type homeobox (CDX) 2/CDX1 can induce growth inhibition on CDX-deficient gastric cancer by rapid intestinal differentiation.

Intestinal metaplasia induced by ectopic expression of caudal-type homeobox (CDX)2 and/or CDX1 (CDX) is frequently observed around gastric cancer (GC). Abnormal expression of CDX is also observed in GC and suggests that inappropriate gastrointestinal differentiation plays essential roles in gastric tumorigenesis, but their roles on tumorigenesis remain unelucidated. Publicly available databases show that GC patients with higher CDX expression have significantly better clinical outcomes. We introduced CDX2 and CDX1 genes separately into GC-originated MKN7 and TMK1 cells deficient in CDX. Marked suppression of cell growth and dramatic morphological change into spindle-shaped flat form were observed along with induction of intestinal marker genes. G0-G1 growth arrest was accompanied by changed expression of cell cycle-related genes but not with apoptosis or senescence. Microarray analyses additionally showed decreased expression of gastric marker genes and increased expression of stemness-associated genes. Hierarchical clustering of 111 GC tissues and 21 non-cancerous gastric tissues by selected 18 signature genes based on our transcriptome analyses clearly categorized the 132 tissues into non-cancer, "CDX signature"-positive GC, and "CDX signature"-negative GC. Gene set enrichment analysis indicated that "CDX signature"-positive GC has lower malignant features. Immunohistochemistry of 89 GC specimens showed that 50.6% were CDX2-deficient, 66.3% were CDX1-deficient, and 44.9% were concomitant CDX2/CDX1-deficient, suggesting that potentially targetable GC cases by induced intestinal differentiation are quite common. In conclusion, exogenous expression of CDX2/CDX1 can lead to efficient growth inhibition of CDX-deficient GC cells. It is based on rapidly induced intestinal differentiation, which may be a future therapeutic strategy.

Induction of Regulatory T Cells as a Novel Mechanism Underlying the Therapeutic Action of Kakkonto, a Traditional Japanese Herbal Medicine, in a Murine Food Allergy Model.

BACKGROUND: The number of patients with food allergy (FA) has dramatically increased. Although satisfactory drug therapies for FA are not available, we have found that kakkonto, a traditional Japanese herbal medicine, suppressed the occurrence of allergic symptoms in an FA mouse model. Thus, we investigated whether kakkonto could regulate the activation and differentiation of T cells in the colon. METHODS: BALB/c mice were systemically sensitized and then orally challenged with ovalbumin. FA mice were orally treated with kakkonto. Lamina propria (LP) cells from their colons were isolated and analyzed. RESULTS: Kakkonto significantly reduced the proportion of CD69+ cells and the elevated helper T cell type 2-specific transcription factor GATA-3 mRNA expression in the LP CD4+ T cells, showing that kakkonto has a suppressive effect on the activation and Th2 differentiation of LP effector CD4+ T cells of the FA mouse colon. Furthermore, kakkonto significantly increased the proportion of Foxp3+CD4+ regulatory T cells in the LP CD4+ T cells of the FA mouse colon. Similarly, the number of Foxp3-positive cells was dramatically increased in the colonic mucosa of kakkonto-administered FA mice. However, the pharmacological effect and Foxp3+CD4+ regulatory T cell-inducing ability of kakkonto were not attenuated by the administration of an anti-CD25 monoclonal antibody in the FA model. CONCLUSIONS: The induction of Foxp3+CD4+CD25- regulatory T cells in the colon as a novel mechanism underlying the therapeutic action of kakkonto could be utilized for the development of a novel anti-FA drug.

Atrophic gastritis and enlarged gastric folds diagnosed by double-contrast upper gastrointestinal barium X-ray radiography are useful to predict future gastric cancer development based on the 3-year prospective observation.

BACKGROUND: Double-contrast upper gastrointestinal barium X-ray radiography (UGI-XR) is the standard gastric cancer screening method in Japan. Atrophic gastritis and enlarged gastric folds are considered the two major features of Helicobacter pylori-induced chronic gastritis, but the clinical meaning of evaluating them by UGI-XR has not been elucidated. METHODS: We analyzed healthy UGI-XR examinees without a history of gastrectomy, previous Helicobacter pylori eradication and usage of gastric acid suppressants. RESULTS AND CONCLUSIONS: Of the 6433 subjects, 1936 (30.1 %) had atrophic gastritis and 1253 (19.5 %) had enlarged gastric folds. During the 3-year prospective observational follow-up, gastric cancer developed in seven subjects, six of whom (85.7 %) had atrophic gastritis with H. pylori infection and five of whom (71.4 %) had enlarged gastric folds with H. pylori infection. The Kaplan-Meier method with log-rank testing revealed that both UGI-XR-based atrophic gastritis (p = 0.0011) and enlarged gastric folds (p = 0.0003) are significant predictors for future gastric cancer incidence.

Marked intestinal trans-differentiation by autoimmune gastritis along with ectopic pancreatic and pulmonary trans-differentiation.

BACKGROUND: Autoimmune gastritis (AIG) is a prevalent chronic inflammatory disease with oncogenic potential that causes destruction of parietal cells and severe mucosal atrophy. We aimed to explore the distinctive gene expression profiles, activated signaling pathways, and their underlying mechanisms. METHODS: A comprehensive gene expression analysis was conducted using biopsy specimens from AIG, Helicobacter pylori-associated gastritis (HPG), and non-inflammatory normal stomachs. Gastric cancer cell lines were cultured under acidic (pH 6.5) conditions to evaluate changes in gene expression. RESULTS: Gastric mucosa with AIG had a unique gene expression profile compared with that with HPG and normal mucosa, such as extensively low expression of ATP4A and high expression of GAST and PAPPA2, which are involved in neuroendocrine tumorigenesis. Additionally, the mucosa with AIG and HPG showed the downregulation of stomach-specific genes and upregulation of small intestine-specific genes; however, intestinal trans-differentiation was much more prominent in AIG samples, likely in a CDX-dependent manner. Furthermore, AIG induced ectopic expression of pancreatic digestion-related genes, PNLIP, CEL, CTRB1, and CTRC; and a master regulator gene of the lung, NKX2-1/TTF1 with alveolar fluid secretion-related genes, SFTPB and SFTPC. Mechanistically, acidic conditions led to the downregulation of master regulator and stemness control genes of small intestine, suggesting that increased environmental pH may cause abnormal intestinal differentiation in the stomach. CONCLUSIONS: AIG induces diverse trans-differentiation in the gastric mucosa, characterized by the transactivation of genes specific to the small intestine, pancreas, and lung. Increased environmental pH owing to AIG may cause abnormal differentiation of the gastric mucosa.

Rebamipide induces dendritic cell recruitment to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-exposed rat gastric mucosa based on IL-1ß upregulation.

Rebamipide is usually used for mucosal protection, healing of gastric ulcers, treatment of gastritis, etc., but its effects on gastric malignancy have not been elucidated. Using Lewis and Buffalo rat strains treated with peroral administration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), we evaluated the effect of rebamipide on the induction of tumor-suppressive dendritic cells, which are known to be heterogeneous antigen-presenting cells of bone marrow origin and are critical for the initiation of primary T-cell responses. Using CD68 as a marker for dendritic cells, the stomach pyloric mucosae of Lewis and Buffalo rats were immunohistochemically analyzed in the presence or absence of rebamipide and MNNG. After a 14-day treatment of rebamipide alone, no significant change in number of CD68-expressing cells was detected in either rat strain. However, after concurrent exposure to MNNG for 14 days, treatment with rebamipide slightly increased CD68-positive cells in the Lewis strain, and significantly increased them in the Buffalo strain. Analysis of two chemotactic factors of dendritic cells, IL-1ß and TNF-α, in the gastric cancer cells showed that expression of IL-1ß, but not TNF-α, was induced by rebamipide in a dose-dependent manner. A luciferase promoter assay using gastric SH-10-TC cells demonstrated that an element mediating rebamipide action exists in the IL-1ß gene promoter region. In conclusion, rebamipide has potential tumor-suppressive effects on gastric tumorigenesis via the recruitment of dendritic cells, based on the upregulation of the IL-1ß gene in gastric epithelial cells.

Oral tolerance induced by transfer of food antigens via breast milk of allergic mothers prevents offspring from developing allergic symptoms in a mouse food allergy model.

We examined whether maternal exposure to food antigens during lactation and maternal allergic status would affect the development of food allergy in offspring. OVA-sensitized or OVA-nonsensitized BALB/c female mice were exposed or unexposed to OVA during lactation. After weaning, their offspring were systemically sensitized twice with OVA and repeatedly given OVA by oral intubation. While 97.1% of the mice breastfed by OVA-nonsensitized and OVA-unexposed mothers developed allergic diarrhea, 59.7% of the mice breastfed by OVA-exposed nonallergic mothers during lactation and 24.6% of the mice breastfed by OVA-exposed allergic mothers during lactation developed food allergy. Furthermore, OVA was detected in breast-milk from OVA-exposed nonallergic mothers during lactation (4.6 ± 0.5 µg/mL). In addition, OVA-specific IgG1 titers were markedly increased in breast milk from allergic mothers (OVA-sensitized and OVA-unexposed mother: 11.0 ± 0.5, OVA-sensitized and OVA-exposed mother: 12.3 ± 0.3). Our results suggest that oral tolerance induced by breast milk-mediated transfer of dietary antigens along with their specific immunoglobulins to offspring leads to antigen-specific protection from food allergy.

Sphingosine kinase-1-dependent and -independent inhibitory effects of zanthoxyli fructus to attenuate the activation of mucosal mast cells and ameliorate food allergies in mice.

Food allergy (FA) is relatively a common disease in infants, but effective drug therapies are not yet available. Notably, mucosal mast cells, but not connective-tissue mast cells, play important roles in food allergic reactions via the release of inflammatory mediators. Therefore, we screened medicinal herb extracts for in vitro and in vivo antiallergic activity through inhibiting mucosal mast cell activation. As a result, both antigen-induced and calcium ionophore-induced degranulation was significantly inhibited by Zanthoxyli Fructus water extract (ZF) in mucosal-type murine bone marrow-derived mast cells (mBMMCs). ZF suppressed the antigen-induced [Ca(2+)](i) elevation and the antigen-enhanced mRNA expression of TNF-α, IL-4, and IL-13. The transcriptome and real-time PCR analyses revealed that ZF greatly decreased the antigen-enhanced expression level of sphingosine kinase 1 (Sphk1), which plays a key role in the FcεRI-mediated immune responses in mast cells. Furthermore, ZF inhibited allergic symptoms in an ovalbumin-caused murine FA model and decreased the number of infiltrating mucosal mast cells and the enhanced mRNA expression levels of IL-4 and Sphk1 in the FA mice colons. These results indicate that ZF suppresses mucosal mast cell activities mainly through Sphk1-dependent mechanism, and ZF is utilized for the development of a novel, potent anti-FA agent.

Transcriptome of sessile serrated adenoma/polyps is associated with MSI-high colorectal cancer and decreased expression of CDX2.

The objective of this study was to elucidate the molecular background of sessile serrated adenoma/polyp (SSA/P) endoscopically resected with comprehensive gene expression analysis. Gene expression profiling was performed for 10 tumor-normal pairs of SSA/P. Cluster analysis, gene set enrichment analysis (GSEA), and consensus molecular subtype (CMS) classification of colorectal cancer (CRC) were applied to our transcriptome analysis. Unsupervised cluster analysis showed that the gene expression profile of SSA/Ps is different from that of adjacent normal epithelial cells, even in the very early stage of tumorigenesis. According to the CMS classification, our microarray data indicated that SSA/Ps were classified as CMS1. GSEA demonstrated a strong association between SSA/P and microsatellite instability-high (MSI-H) CRC (p < 10-5 ). Transcriptome analysis of five MSI-related genes (MSH2, MSH6, MLH1, PMS1, and PMS2) and five CRC-related genes (BRAF, KRAS, APC, TP53, and CDX2) showed that CDX2 expression was most severely decreased in SSA/P. Immunohistochemical staining confirmed that CDX2 protein was reduced compared with the surrounding mucosa. Direct sequencing of the BRAF gene showed that the BRAF V600E mutation was detected in only nine of 36 cases. In a mouse model, BRAF, APC, or CDX2 deficiency indicated that the gene expression pattern with loss of CDX2 is more similar to our SSA/Ps compared with those induced by BRAF or APC mutation. Transcriptome analysis of SSA/Ps showed characteristic gene expression with a strong resemblance to MSI-H CRC. Downregulation of CDX2 expression is an essential molecular mechanism involved in the initial stage of SSA/P tumorigenesis. (UMIN000027365).

Suppression of phospholipase Cγ1 phosphorylation by cinnamaldehyde inhibits antigen-induced extracellular calcium influx and degranulation in mucosal mast cells.

Antigen-IgE-mediated mucosal mast-cell activation is critical in the development of food allergies. Cinnamaldehyde, a major constituent of Cinnamomi cortex, dose-dependently inhibited the antigen-IgE-induced degranulation of mucosal-type bone-marrow derived mast cells (mBMMCs) and RBL-2H3 cells. Cinnamaldehyde also suppressed the elevation of the intracellular Ca(2+) level that is induced by the extracellular Ca(2+) influx in antigen-IgE-stimulated mBMMCs. Furthermore, tyrosine phosphorylation of phospholipase C (PLC) γ1, which is a crucial activation switch for the intracellular Ca(2+) mobilization in mast cells, was attenuated by cinnamaldehyde. Together, our results demonstrated that cinnamaldehyde suppressed the intracellular Ca(2+) mobilization and the degranulation of mucosal mast cells by inhibiting the activity of the IgE receptor-PLCγ-Ca(2+) influx pathway. These findings suggest that cinnamaldehyde may have therapeutic potential in mucosal mast cell-related allergic diseases, such as food allergies.

High glucose-induced apoptosis in human coronary artery endothelial cells involves up-regulation of death receptors.

BACKGROUND: High glucose can induce apoptosis in vascular endothelial cells, which may contribute to the development of vascular complications in diabetes. We evaluated the role of the death receptor pathway of apoptotic signaling in high glucose-induced apoptosis in human coronary artery endothelial cells (HCAECs). METHODS: HCAECs were treated with media containing 5.6, 11.1, and 16.7 mM of glucose for 24 h in the presence or absence of tumor necrosis factor (TNF)-α. For detection of apoptosis, DNA fragmentation assay was used. HCAEC expression of death receptors were analyzed by the PCR and flow cytometry methods. Also, using immunohistochemical techniques, coronary expression of death receptors was assessed in streptozotocin-nicotinamide-induced type 2 diabetic mice. RESULTS: Exposure of HCAECs to high glucose resulted in a significant increase in TNF-R1 and Fas expression, compared with normal glucose. High glucose increased TNF-α production by HCAECs and exogenous TNF-α up-regulated TNF-R1 and Fas expression in HCAECs. High glucose-induced up-regulation of TNF-R1 and Fas expression was undetectable in the presence of TNF-α. Treatment with TNF-R1 neutralizing peptides significantly inhibited high glucose-induced endothelial cell apoptosis. Type 2 diabetic mice displayed appreciable expression of TNF-R1 and Fas in coronary vessels. CONCLUSIONS: In association with increased TNF-α levels, the death receptors, TNF-R1 and Fas, are up-regulated in HCAECs under high glucose conditions, which could in turn play a role in high glucose-induced endothelial cell apoptosis.

Rab5a regulates surface expression of FcεRI and functional activation in mast cells.

Surface expression levels of high-affinity immunoglobulin E (IgE) receptors (FcεRI) on mast cells are regulated by constitutive internalization from the plasma membrane, which is thought to be an important determinant of FcεRI-mediated signaling potential. However, molecular mechanism of FcεRI trafficking has remained poorly understood. Rab proteins are small guanosine 5'-triphosphatases (GTPases) involved in the regulation of membrane traffic. In particular, Rab5 has been shown to regulate transport in the early endocytic pathway, whereas it is not known whether the FcεRI surface expression levels are regulated by Rab5. In this study, we investigated the role of individual Rab5 isoforms in mast cells by small interfering RNA knockdown method. Our results demonstrate that Rab5a knockdown enhanced FcεRI-dependent mast cell activation and upregulated FcεRI surface expression in its steady state. In contrast, Rab5c knockdown caused suppression of the activation. These findings revealed modulatory and individual roles of Rab5 isoforms in mast cell functions.

The traditional herbal medicine saireito exerts its inhibitory effect on murine oxazolone-induced colitis via the induction of Th1-polarized immune responses in the mucosal immune system of the colon.

BACKGROUND: Ulcerative colitis is an intractable inflammatory colonic disease, and its etiology remains unclear. Saireito, a traditional herbal medicine, is widely used for treating ulcerative colitis in Japan. We analyzed the immunological characteristics of an oxazolone (OXZ)-induced colitis (OC) model and examined the effects of sareito on this model. METHODS: OXZ was injected into the colon of BALB/c mice. Saireito was orally administered once a day for 3 consecutive days. Colitis was assessed by scoring the symptoms and macroscopic findings. The transcription patterns in the middle colon and spleen were analyzed with global transcriptome analysis and real-time polymerase chain reaction (PCR). RESULTS: The above-mentioned scores were increased in the OC mice. The transcription levels of Th2 cytokines were significantly upregulated in the spleen and middle colon of the OC mice, whereas those of the Th1 cytokine interferon (IFN)-gamma decreased in the spleen and increased in the middle colon. Saireito significantly ameliorated OC. In the middle colon of the saireito-treated mice, enhanced expression of Th2 cytokine mRNAs was markedly downregulated, while that of IFN-gamma mRNA was further upregulated. In contrast, in the spleen, saireito had no effect on the transcription of either type of cytokine. After global transcriptome analysis, real-time PCR analysis revealed that saireito greatly downregulated the enhanced expression of the suppressor of cytokine signaling (SOCS)-3 mRNA in the middle colon of OC mice. CONCLUSIONS: Saireito exhibits inhibitory effects on OC by the induction of Th1-polarized immune responses in the mucosal immune system of the colon.

The inhibitory effect of ergosterol, a bioactive constituent of a traditional Japanese herbal medicine saireito on the activity of mucosal-type mast cells.

Mucosal inflammation in ulcerative colitis (UC) is presumed to be regulated primarily by type 2 T helper cell immune responses and mucosal mast cells in the colon are thought to play an important role in the pathogenesis of the mucosal inflammation. Saireito, a Japanese herbal medicine of standardized quality, originating from traditional Chinese medicine (Kampo medicine), is composed of two different Kampo medicines (shosaikoto and goreisan) and is often used for UC in Japan. In this study, we examined the direct effects of these Kampo medicines and their constituents on the antigen-induced degranulation of mucosal-type mast cells. Mucosal-type murine bone marrow-derived mast cells (mBMMCs) were pretreated by these drugs for 24 h, and immunoglobulin E (IgE) receptor-triggered degranulation of mBMMCs was assessed by beta-hexosaminidase release. Goreisan showed inhibitory effects on degranulation of mBMMCs in a dose-dependent manner. Among the five constituent medicinal herbs of goreisan, Poria and Polyporus had the inhibitory effects on mBMMCs. Ergosterol, a principal and common component of Poria and Polyporus, also suppressed the degranulation of mBMMCs. Our results provide a molecular basis to explain a portion of the beneficial therapeutic properties of saireito on UC.

Therapeutic effect of kakkonto in a mouse model of food allergy with gastrointestinal symptoms.

BACKGROUND: The number of patients with food allergy has increased dramatically over the last several decades. However, there is no effective drug for food allergies. In the present study, we evaluated the effects of kakkonto, a traditional Japanese herbal medicine, in a mouse model of food allergy with gastrointestinal symptoms. METHODS: BALB/c mice were systemically sensitized twice with ovalbumin (OVA) and then were repeatedly given OVA by oral intubation (OVA mice). Kakkonto was administered orally before the OVA challenges. RESULTS: The OVA mice developed allergic diarrhea (91.8 +/- 3.8% after 6 OVA challenges), and myeloperoxidase (MPO) activity was dramatically elevated in the colons of the OVA mice. Kakkonto significantly suppressed the occurrence of allergic diarrhea and MPO activity in the OVA mice. Furthermore, the number of mucosal mast cells was greatly increased in the proximal colons of the OVA mice, and this was also suppressed by kakkonto. Interestingly, mRNA expression of helper T cell type 1 (Th1) cytokines (IFN-gamma) and Th2 cytokines (IL-4, IL-5 and IL-10) were significantly upregulated in the proximal colons of the OVA mice, an effect which was also reduced by kakkonto. Transcriptome analysis detected increased mRNA expression of suppressor of cytokine signaling-3 in the proximal colons of OVA mice, which was decreased by kakkonto administration. CONCLUSION: Kakkonto has immunosuppressive effects and interferes with the infiltration of mucosal mast cells in the colons of mice with induced food allergy, leading to improvement of allergic symptoms. Kakkonto has potential as a therapeutic drug for treatment of allergic symptoms induced by the disruption of intestinal mucosal immunity.

Tandem repeats of the 5' flanking region of human MUC5AC have a role as a novel enhancer in MUC5AC gene expression.

MUC5AC is a well-known gastric differentiation marker, which has been frequently used for the classification of stomach cancer. However, the molecular mechanism of regulation of MUC5AC expression remains to be elucidated. In previous studies, we have shown that Gli regulated MUC5AC transcription through the Gli-binding motif in the 5' region of MUC5AC. Gli played important roles, but independently was not sufficient for MUC5AC expression. In this study, we analyzed a 4010 bp fragment of the 5'-flanking promoter region of the human MUC5AC gene by luciferase assay, and found a novel distal enhancer region located between -1434 bp to -3000 bp upstream from the first ATG initiation codon. This region is composed of repetitive DNA sequences 5'-TCACTCAC-3'. The strength of enhancer activities depended on the length of the repetitive region. The tandem repeats are conserved among primates, but not in other mammals. The tandem repeat regions enhanced promoter activities not only of MUC5AC but also of other genes. The enhancer effect of the tandem repeat regions was maintained even when inverted. ChIP analysis revealed that H3K9me3 binds to the tandem repeat regions. Together, our results suggest that the tandem repeat region in the MUC5AC promoter has the potential to act as a strong enhancer, and H3K9me3 may contribute to histone modifications of this region.

Identification of marker genes and pathways specific to precancerous duodenal adenomas and early stage adenocarcinomas.

BACKGROUND: The mechanism behind the pathogenesis and carcinogenesis of these neoplasms is not fully understood. The objective of this study was to identify genetic markers and pathways specific to precancerous duodenal adenomas and early stage adenocarcinomas through gene expression analysis. METHODS: Gene expression profiling was performed in 4 pairs of duodenal adenoma/adenocarcinomas and corresponding matched normal tissue. Genes with consistent expression differences were identified and confirmed in 7 independent pairs. Gene set enrichment analysis (GSEA) was performed to characterize gene expression profiles of duodenal adenoma/adenocarcinomas, together with immunohistochemical staining of candidate oncogenic genes. RESULTS: 626 probes consistently demonstrated over a twofold expression difference between tumor-normal pairs. Reverse transcriptase polymerase chain reaction of genes with the most prominent difference in expression between tumors and normal mucosa (KLK7, KLK6, CEMIP, MMP7, KRT17, LGR5, G6PC, S100G, APOA1) validated the results of gene expression analysis. GSEA demonstrated a strong association between duodenal adenoma/adenocarcinomas with colorectal adenomas (p < 10-5) and gene expression patterns seen after APC gene knockout (p < 10-5), suggesting that the Wnt/ß-catenin pathway plays a crucial role in the carcinogenesis of these neoplasms. Immunohistochemical staining of an independent group of duodenal adenomas confirmed over-accumulation of ß-catenin in 80.0% (16/20). CONCLUSIONS: Precancerous duodenal adenomas and early stage adenocarcinomas demonstrate gene expression characteristics with a strong resemblance to colorectal adenomas. The results of this study strongly suggest that upregulation of the Wnt/ß-catenin pathway is the major factor involved in the initial stages of the carcinogenesis of duodenal adenocarcinomas.

IgE-induced degranulation of mucosal mast cells is negatively regulated via nicotinic acetylcholine receptors.

The autonomic nervous system is known to mediate mast cell activation. We investigated expression of nicotinic acetylcholine receptors (nAChRs) in mucosal-type mast cells and their contribution to the regulation of mast cell activation. Expression of mRNA of nAChR alpha4, alpha7, and beta2 subunits were detected in specially differentiated mucosal-type murine bone marrow-derived mast cells (mBMMCs). Pretreatment with non-specific nAChRs agonists, acetylcholine, nicotine and epibatidine and a specific alpha7 subunit agonist GTS-21 significantly inhibited antigen-induced degranulation of mBMMCs in a dose-dependent manner and GTS-21-induced inhibition was significantly blocked by alpha7 subunit antagonist, alpha-bungarotoxin. Furthermore, confocal microscopy also demonstrated surface binding of alpha-bungarotoxin on mBMMCs. Our findings indicate that mucosal mast cell activation may be negatively regulated mainly through nAChR alpha7 subunit, suggesting that nAChRs are involved in neuronal-mucosal mast cell interactions.

Transmembrane topology of ceramide synthase in yeast.

Ceramide plays a crucial role as a basic building block of sphingolipids, but also as a signalling molecule mediating cell-fate decisions. Three genes, LAG1, LAC1 and LIP1, have been shown to be required for ceramide synthase activity in Saccharomyces cerevisiae [Guillas, Kirchman, Chuard, Pfefferli, Jiang, Jazwinski and Conzelman (2001) EMBO J. 20, 2655-2665; Schorling, Vallee, Barz, Reizman and Oesterhelt (2001) Mol. Biol. Cell 12, 3417-3427; Vallee and Riezman (2005) EMBO J. 24, 730-741]. In the present study, the topology of the Lag1p and Lac1p subunits was investigated. The N- and C-termini of the proteins are in the cytoplasm and eight putative membrane-spanning domains were identified in Lag1p and Lac1p by insertion of glycosylation and factor Xa cleavage sites at various positions. The conserved Lag motif, potentially containing the active site, is most likely embedded in the membrane. We also present evidence that histidine and aspartic acid residues in the Lag motif are essential for the function of Lag1p in vivo.

Shikonin, a constituent of Lithospermum erythrorhizon exhibits anti-allergic effects by suppressing orphan nuclear receptor Nr4a family gene expression as a new prototype of calcineurin inhibitors in mast cells.

Over the last few decades, food allergy (FA) has become a common disease in infants in advanced countries. However, anti-allergic medicines available in the market have no effect on FA, and consequently effective drug therapies for FA are not yet available. We have already demonstrated that mucosal mast cells play an essential role in the development of FA in a murine model. Thus, we screened many constituents from medicinal herbs for the ability to inhibit rat basophilic leukemia-2H3 mast-like cell degranulation, and found that shikonin, a naphthoquinone dye from Lithospermum erythrorhizon, exhibited the most potent inhibitory effect among them. Furthermore, shikonin extremely inhibited the IgE/antigen-induced and calcium ionophore-induced upregulation of tumor necrosis factor (TNF)-α mRNA expression in mucosal-type bone marrow-derived mast cells (mBMMCs). Global gene expression analysis confirmed by real-time PCR revealed that shikonin drastically inhibited the IgE/antigen-induced and calcium ionophore-induced upregulation of mRNA expression of the nuclear orphan receptor 4a family (Nr4a1, Nr4a2 and Nr4a3) in mBMMCs, and knockdown of Nr4a1 or Nr4a2 suppressed the IgE/antigen-induced upregulation of TNF-α mRNA expression. Computational docking simulation of a small molecule for a target protein is a useful technique to elucidate the molecular mechanisms underlying the effects of drugs. Therefore, the simulation revealed that the predicted binding sites of shikonin to immunophilins (cyclophilin A and FK506 binding protein (FKBP) 12) were almost the same as the binding sites of immunosuppressants (cyclosporin A and FK506) to immunophilins. Indeed, shikonin inhibited the calcineurin activity to a similar extent as cyclosporin A that markedly suppressed the IgE/antigen-enhanced mRNA expression of TNF-α and the Nr4a family in mBMMCs. These findings suggest that shikonin suppresses mucosal mast cell activation by reducing Nr4a family gene expression through the inhibition of calcineurin activity. Therefore, shikonin has therapeutic potential for the treatment of allergic diseases as a new calcineurin inhibitor.