NOTCH1 and NOTCH2 regulate epithelial cell proliferation in mouse and human gastric corpus.

The Notch signaling pathway is known to regulate stem cells and epithelial cell homeostasis in gastrointestinal tissues; however, Notch function in the corpus region of the stomach is poorly understood. In this study we examined the consequences of Notch inhibition and activation on cellular proliferation and differentiation and defined the specific Notch receptors functioning in the mouse and human corpus. Notch pathway activity was observed in the mouse corpus epithelium, and gene expression analysis revealed NOTCH1 and NOTCH2 to be the predominant Notch receptors in both mouse and human. Global Notch inhibition for 5 days reduced progenitor cell proliferation in the mouse corpus, as well as in organoids derived from mouse and human corpus tissue. Proliferation effects were mediated through both NOTCH1 and NOTCH2 receptors, as demonstrated by targeting each receptor alone or in combination with Notch receptor inhibitory antibodies. Analysis of differentiation by marker expression showed no change to the major cell lineages; however, there was a modest increase in the number of transitional cells coexpressing markers of mucous neck and chief cells. In contrast to reduced proliferation after pathway inhibition, Notch activation in the adult stomach resulted in increased proliferation coupled with reduced differentiation. These findings suggest that NOTCH1 and NOTCH2 signaling promotes progenitor cell proliferation in the mouse and human gastric corpus, which is consistent with previously defined roles for Notch in promoting stem and progenitor cell proliferation in the intestine and antral stomach. NEW & NOTEWORTHY: Here we demonstrate that the Notch signaling pathway is essential for proliferation of stem cells in the mouse and human gastric corpus. We identify NOTCH1 and NOTCH2 as the predominant Notch receptors expressed in both mouse and human corpus and show that both receptors are required for corpus stem cell proliferation. We show that chronic Notch activation in corpus stem cells induces hyperproliferation and tissue hypertrophy, suggesting that Notch may drive gastric tumorigenesis.

Caudal type homeobox 2 expression induced by leukocytapheresis might be associated with mucosal healing in ulcerative colitis.

BACKGROUND AND AIM: Ulcerative colitis (UC) is a chronic inflammatory disease of the colon with an intractable, recurrent course. Although the goal of UC therapy has recently been to target mucosal healing, the molecular mechanism of mucosal healing remains unknown. In this study, we aimed to elucidate the molecular dynamics related to the proliferation and differentiation of intestinal epithelial cells during cytapheresis therapy in a short duration. METHODS: Endoscopy was performed in 26 patients with UC in multicentre hospitals, and biopsy specimens were collected from the rectum before and within two weeks after leukocytapheresis (LCAP). The expression of representative proteins in intestinal epithelial cells and pathological findings was compared before and after LCAP. RESULTS: The expression of caudal type homeobox 2 (CDX2) and a hes family bHLH transcription factor 1(HES1) markedly increased after LCAP. Patients with endoscopic improvement after LCAP showed the expression of CDX2 before LCAP. Moreover, the number of goblet cells significantly increased after LCAP. Patients without endoscopic improvement after LCAP did not show the expression of CDX2 before LCAP. However, the expression of CDX2 markedly increased after LCAP. CONCLUSION: This study suggests that cytapheresis might induce CDX2 expression without affecting the cell proliferation, thus resulting in mucosal healing with goblet cell restoration.

Atonal homolog 1 protein stabilized by tumor necrosis factor α induces high malignant potential in colon cancer cell line.

Patients with inflammatory bowel disease (IBD) have an increased risk of developing colitis-associated colorectal cancer (CAC). CAC cells often develop chemoresistance, resulting in a poorer prognosis than that of sporadic colorectal cancer (CRC). The mechanism by which CAC enhances malignant potential remains unknown. We have previously reported that the proteasomal degradation of the transcription factor Atonal homolog 1 (Atoh1) protein results in the non-mucinous form of CRC. It also remains unknown whether Atoh1 protein is expressed in CAC. Therefore, in the present study, we investigated whether Atoh1 protein stabilizes in CAC. Consequently, the treatment with TNF-α stabilized Atoh1 protein through the inactivation of GSK-3ß via Akt, resulting in the mucinous form of CRC cell lines. Atoh1 protein also enriched cancer stem cells with upregulated Lgr5 expression and cells in G0/G1 cell cycle phase, resulting in both the chemoresistance to 5-fluorouracil and oxaliplatin and the promotion of cell migration. Immunofluorescence of the human mucinous CAC specimens showed the accumulation of NF-κB p65 at nuclei with the expression of Atoh1 in mucinous cancer. In conclusion, the inflammation associated with carcinogenesis may preserve the differentiation system of intestinal epithelial cell (IEC), resulting in the acquisition of both the mucinous phenotype and high malignant potential associated with the enrichment of cancer stem cell.

Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt.

BACKGROUND AND AIMS: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. METHODS: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. RESULTS: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. CONCLUSIONS: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.

[Undifferentiated carcinoma of the jejunum producing granulocyte colony-stimulating factor].

An 80-year-old man presented with abdominal fullness and vomiting. Laboratory data revealed severe anemia, an inflammatory response, and elevated white blood cell counts. Abdominal computed tomography indicated ileus caused by a jejunal tumor measuring 8cm in diameter. Although small-bowel endoscopy enabled visualization of the tumor, adequate biopsy specimens could not be obtained for accurate diagnosis. The patient's condition rapidly deteriorated, because of which surgical treatment could not be initiated. The patient died approximately 3 weeks after admission. High serum granulocyte colony-stimulating factor (G-CSF) levels were detected at autopsy. Immunohistochemical staining of the autopsy specimen indicated positive G-CSF levels in the jejunal tumor. On the basis of these findings, a final diagnosis of undifferentiated carcinoma of the jejunum producing G-CSF was made.

The acquisition of malignant potential in colon cancer is regulated by the stabilization of Atonal homolog 1 protein.

The transcription factor Atonal homolog 1 (Atoh1) plays crucial roles in the differentiation of intestinal epithelium cells. Although we have reported that the Atoh1 protein was degraded in colon cancer by aberrant Wnt signaling, a recent study has indicated that the Atoh1 protein is expressed in mucinous colon cancer (MC) and signet ring cell carcinoma (SRCC). However, the roles of the Atoh1 protein in MC are unknown. To mimic MC, a mutated Atoh1 protein was stably expressed in undifferentiated colon cancer cells. Microarray analysis revealed the acquisition of not only the differentiated cell form, but also malignant potential by Atoh1 protein stabilization. In particular, Atoh1 enhanced Wnt signaling, resulting in the induction of Lgr5 as a representative stem cell marker with the enrichment of cancer stem cells. Moreover, the fluorescent ubiquitination-based cell cycle indicator system with time-lapse live imaging demonstrated cell cycle arrest in the G0/G1 phase by Atoh1 protein stabilization. In conclusion, the Atoh1 protein regulates malignant potential rather than the differentiation phenotype of MC, suggesting the mechanism by which MC and SRCC are more malignant than non-mucinous adenocarcinoma.

Delta-like 1-Expressing Cells at the Gland Base Promote Proliferation of Gastric Antral Stem Cells in Mouse.

BACKGROUND & AIMS: Notch pathway signaling maintains gastric epithelial cell homeostasis by regulating stem cell proliferation and differentiation. We previously identified NOTCH1 and NOTCH2 as the key Notch receptors controlling gastric stem cell function. Here, we identify the niche cells and critical Notch ligand responsible for regulating stem cell proliferation in the distal mouse stomach. METHODS: Expression of Notch ligands in the gastric antrum was determined by quantitative reverse-transcriptase polymerase chain reaction and cellular localization was determined by in situ hybridization and immunostaining. The contribution of specific Notch ligands to regulate epithelial cell proliferation in adult mice was determined by inducible gene deletion, or by pharmacologic inhibition using antibodies directed against specific Notch ligands. Mouse gastric organoid cultures were used to confirm that Notch ligand signaling was epithelial specific. RESULTS: Delta-like 1 (DLL1) and Jagged 1 (JAG1) were the most abundantly expressed Notch ligands in the adult mouse stomach, with DLL1 restricted to the antral gland base and JAG1 localized to the upper gland region. Inhibition of DLL1 alone or in combination with other Notch ligands significantly reduced epithelial cell proliferation and the growth of gastric antral organoids, while inhibition of the other Notch ligands, DLL4, JAG1, and JAG2, did not affect proliferation or organoid growth. Similarly, DLL1, and not DLL4, regulated proliferation of LGR5+ antral stem cells, which express the NOTCH1 receptor. CONCLUSIONS: DLL1 is the key Notch ligand regulating epithelial cell proliferation in the gastric antrum. We propose that DLL1-expressing cells at the gland base are Notch niche cells that signal to adjacent LGR5+ antral stem cells to regulate stem cell proliferation and epithelial homeostasis.

Long-term Inflammation Transforms Intestinal Epithelial Cells of Colonic Organoids.

BACKGROUND AND AIMS: Patients with ulcerative colitis [UC] are at an increased risk of developing colitis-associated cancer [CAC], suggesting that continuous inflammation in the colon promotes the transformation of colonic epithelial cells. However, the mechanisms underlying cell transformation in UC remain unknown. We therefore aimed to investigate the effect of long-term inflammation on intestinal epithelial cells [IECs] using organoid culture. METHODS: IECs were isolated from mouse colon, and were cultured according to a method for a three-dimensional [3D] organoid culture. To mimic chronic inflammation, a mixture of cytokines and bacterial components were added to the medium for over a year. Cell signal intensity was assessed by 3D immunofluorescence. Cell transformation was assessed by microarray with gene set enrichment analysis. RESULTS: Stimulation with cytokines resulted in a significant induction of target genes for the nuclear factor [NF]-κB pathway in colonic organoids. Following 60 weeks of continuous stimulation, cell differentiation was suppressed. Continuous stimulation also resulted in significant amplification of NF-κB signalling. Amplified NF-κB signalling by long-term stimulation remained in colonic organoids even 11 weeks after the removal of all cytokines. Some genes were specifically upregulated only in colonic organoids after the removal all cytokines following long-term stimulation. CONCLUSIONS: Colonic organoids stimulated with cytokines for a prolonged period were established as in vitro model to assess long-term epithelial responses to inflammatory cytokines. Chronic inflammation led to sustained NF-κB signalling activation in colonic organoids, resulting in cell transformation that might be related to the carcinogenesis of CAC in UC.

BRAFV600E cooperates with CDX2 inactivation to promote serrated colorectal tumorigenesis.

While 20-30% of colorectal cancers (CRCs) may arise from precursors with serrated glands, only 8-10% of CRCs manifest serrated morphology at diagnosis. Markers for distinguishing CRCs arising from 'serrated' versus 'conventional adenoma' precursors are lacking. We studied 36 human serrated CRCs and found CDX2 loss or BRAF mutations in ~60% of cases and often together (p=0.04). CDX2Null/BRAFV600E expression in adult mouse intestinal epithelium led to serrated morphology tumors (including carcinomas) and BRAFV600E potently interacted with CDX2 silencing to alter gene expression. Like human serrated lesions, CDX2Null/BRAFV600E-mutant epithelium expressed gastric markers. Organoids from CDX2Null/BRAFV600E-mutant colon epithelium showed serrated features, and partially recapitulated the gene expression pattern in mouse colon tissues. We present a novel mouse tumor model based on signature defects seen in many human serrated CRCs - CDX2 loss and BRAFV600E. The mouse intestinal tumors show significant phenotypic similarities to human serrated CRCs and inform about serrated CRC pathogenesis.

Reduced Human α-defensin 6 in Noninflamed Jejunal Tissue of Patients with Crohn's Disease.

BACKGROUND: Mucosal barrier dysfunction is considered a critical component of Crohn's disease (CD) pathogenesis after the identification of susceptibility genes. However, the precise mechanism underlying mucosal barrier dysfunction has not yet been elucidated. We therefore aimed to elucidate the molecular mechanism underlying the expression of human α-defensin 6 (HD6) in patients with CD. METHODS: HD6 expression was induced by the transfection of an atonal homolog 1 (Atoh1) transgene and was assessed by reverse transcription polymerase chain reaction. The HD6 promoter region targeted by Atoh1 and ß-catenin was determined by reporter analysis and chromatin immunoprecipitation assay. HD5/HD6/Atoh1/ß-catenin expression in noninflamed jejunal samples collected by balloon endoscopy from 15 patients with CD and 9 non-inflammatory bowel disease patients were assessed by immunofluorescence. RESULTS: Both promoter activity and gene expression of HD6 was significantly upregulated by the Atoh1 transgene in human colonic cancer cell line. We identified a TCF4 binding site and an E-box site, critical for the regulation of HD6 transcriptional activity by directly binding of Atoh1 in the 200-bp HD6 promoter region. The treatment with ß-catenin inhibitor also decreases HD6 promoter activity and gene expression. Moreover, HD6 expression, but not HD5 expression, was found to be decreased in noninflamed jejunal regions from patients with CD. In HD6-negative crypts, nuclear accumulation of ß-catenin was impaired. CONCLUSIONS: HD6 expression was found to be regulated by cooperation between Atoh1 and ß-catenin within the HD6 promoter region. Downregulation of HD6 in noninflamed mucosa may contribute to mucosal barrier dysfunction of patients with CD.