Identification of genes responsive to sodium butyrate in colonic epithelial cells

We identified genes responsive to sodium butyrate (SB) in colonic epithelial cells using cDNA microarrays. Treatment with 2 mM SB of colonic epithelial cells (MCE301), which was derived from transgenic mice harboring a temperature-sensitive simian virus 40 large T-antigen, arrested cell growth and showed a differentiated phenotype accompanying an increase in alkaline phosphatase activity. Of the approximately 900 genes analyzed, SB down-regulated 25 genes and up-regulated 88 genes by a factor of 2.0 or greater. Northern blot or TaqMan and Western blot analyses confirmed that the mRNA and protein levels of cyclin D1 and the level of proliferating cell nuclear antigen decreased, whereas the levels of integrin beta1 and osteopontin increased. The present results regarding the changes in gene expression, arrived at using microarrays, will provide a basis for a further understanding of the molecular mechanisms of cell growth arrest and differentiation in response to SB in colonic epithelial cells.     

Inhibition of transforming growth factor-β signaling potentiates tumor cell invasion into collagen matrix induced by fibroblast-derived hepatocyte growth factor

Interaction between tumor cells and stromal fibroblasts plays essential roles in tumor progression. However, its detailed molecular mechanism remains unclear. To understand the mechanism, we investigated molecules mediating this interaction using the three-dimensional (3D) co-culture system of Panc-1 pancreatic carcinoma cells with normal fibroblasts. When the two kinds of cells were placed on the top of collagen gel, the tumor cells scattered into the fibroblast layer, apparently undergoing epithelial‐mesenchymal transition. When fibroblasts were placed within collagen gel, Panc-1 cells actively invaded into the collagen gel, extending a microtubule-based long protrusion. Although transforming growth factor-β (TGF-β) and hepatocyte growth factor (HGF) individually stimulated the tumor cell invasion into collagen gel without fibroblasts, TGF-β signaling inhibitors (SB431542 and LY2157299) significantly enhanced the Panc-1 cell invasion in the 3D co-culture with fibroblasts. Experiments with HGF/Met signaling inhibitors or with the fibroblast conditioned medium revealed that HGF was a major invasion-promoting factor secreted from fibroblasts and SB431542 increased the HGF secretion by blocking the HGF-suppressing activity of cancer cell-derived TGF-β. These results indicate that HGF and TGF-β are critical regulators for both tumor–stroma interaction and tumor invasion. The results also suggest that TGF-β signaling inhibitors may promote tumor progression under some pathological conditions.     

粪菌移植对小鼠实验性结肠炎TLR4信号通路及肠黏膜屏障的影响

目的探讨粪菌移植(FMT)对溃疡性结肠炎(UC)小鼠肠黏膜屏障的影响及可能机制。方法小鼠饮用2.0%葡聚糖硫酸钠(DSS)溶液构建小鼠UC模型;50只成年雄性C57BL/6J小鼠,随机留取10只取粪便(这10只不参与后续的实验),其余40只称重、编号,随机分为空白对照组(Con组)、DSS模型对照组(Model组)、美沙拉嗪组(Model+5-ASA组)和粪菌液组(Model+FMT组),每组10只,Con组和Model组均给予0.9%NaCl溶液灌肠,给药组分别给予美沙拉嗪、粪便滤液灌肠;评估疾病活动指数(DAI)、各组结肠组织病理情况,用透射电镜检测各组小鼠的结肠黏膜上皮细胞结构的变化情况,ELISA检测各组血清内毒素、炎症因子TNF-α水平变化,免疫组化法检测结肠组织Toll样受体4(TLR4)及核因子-κB(NF-κB)的表达变化,Western blot检测各组ZO-1蛋白表达。结果与Model组相比,粪菌移植明显改善小鼠的DAI指数和结肠组织的病理损伤,结肠上皮细胞间隙增宽程度减轻,腺上皮细胞间连接较紧密,结肠黏膜上皮细胞微绒毛完整,排列整齐,内毒素、TNF-α的含量明显下降,TLR4及NF-κB在结肠组织的表达明显下降,ZO-1蛋白表达明显升高,促进结肠黏膜屏障的修复,差异具有统计学意义(t=7.9543,P<0.0001;t=3.7641,P=0.0010;t=4.5899,P=0.0020;t=13.2886,P<0.0001;t=4.9750,P=0.0010;t=6.9388,P<0.0001;t=8.3744,P<0.0001)。结论FMT可减少内毒素及炎症因子的产生,改善结肠炎症,TLR4-NF-κB信号通路可能是FMT修复结肠黏膜屏障功能的机制之一。     

6-姜烯酚对溃疡性结肠炎小鼠结肠上皮细胞Notch信号通路的影响

目的:观察6-姜烯酚对溃疡性结肠炎小鼠结肠上皮细胞Notch信号通路的调控作用。方法:清洁级昆明小鼠40只随机分为正常组(10只)和造模组(30只),造模组采用2%葡聚糖硫酸钠(DSS)自由饮用诱导溃疡性结肠炎模型,造模15 d后分为模型组,6-姜烯酚组,阳性对照组,每组10只。正常组、模型组灌胃生理盐水,6-姜烯酚组采用6-姜烯酚100 mg/(kg·d)灌胃,阳性对照组采用柳氮磺吡啶100 mg/(kg·d)灌胃,给药20 d后处死,观察小鼠结肠病理组织学改变,免疫荧光双标法检测结肠上皮细胞Hes-1、Math-1蛋白的表达,RT-PCR法检测结肠上皮组织Notch-1、Hes-1、Math-1 mRNA的表达,Western blot法检测结肠上皮组织Notch-1、Hes-1、Math-1蛋白的表达。结果:与正常组相比,模型组小鼠结肠上皮组织Notch-1、Hes-1蛋白和mRNA相对表达量显著升高( P＜0.01), Math-1蛋白和mRNA相对表达量显著降低( P＜0.01);与模型组比较,6-姜烯酚组、柳氮磺吡啶组小鼠结肠上皮组织Notch-1、Hes-1蛋白和mRNA相对表达量显著降低( P＜0.01), Math-1蛋白和mRNA相对表达量显著升高( P＜0.01) 。结论:6-姜烯酚能够抑制Notch通路的过度活化,调节结肠上皮吸收细胞系和分泌细胞系之间分化的平衡,修复受损结肠粘膜组织。     

Insulin-like growth factor-1 receptor transactivation modulates the inflammatory and proliferative responses of neurotensin in human colonic epithelial cells

Neurotensin (NT) is a gastrointestinal neuropeptide that modulates intestinal inflammation and healing by binding to its high-affinity receptor NTR1. The dual role of NT in inflammation and healing is demonstrated in models of colitis induced by Clostridium difficile toxin A and dextran sulfate sodium, respectively, and involves NF-κB-dependent IL-8 expression and EGF receptor-mediated MAPK activation in human colonocytes. However, the detailed signaling pathways involved in these responses remain to be elucidated. We report here that NT/NTR1 coupling in human colonic epithelial NCM460 cells activates tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) in a time- and dose-dependent manner. NT also rapidly induces Src tyrosine phosphorylation, whereas pretreatment of cells with the Src inhibitor PP2 before NT exposure decreases NT-induced IGF-1R phosphorylation. In addition, inhibition of IGF-1R activation by either its specific antagonist AG1024 or siRNA against IGF-1 significantly reduces NT-induced IL-8 expression and NF-κB-dependent reporter gene expression. Pretreatment with AG1024 also inhibits Akt activation and apoptosis induced by NT. Silencing of Akt expression by siRNA also substantially attenuates NT-induced IL-8 promoter activity and NF-κB-dependent reporter gene expression. This is the first report to indicate that NT transactivates IGF-1R and that this response is linked to Akt phosphorylation and NF-κB activation, contributing to both pro-inflammatory and tissue repair signaling pathways in response to NT in colonic epithelial cells. We propose that IGF-1R activation represents a previously unrecognized key pathway involved in the mechanisms by which NT and NTR1 modulate colonic inflammation and inflammatory bowel disease.     

The effect of selenium on cell proliferation in liver and colon

Epidemiologic and experimental evidence support a chemoprotective role for selenium (Se) in malignancy. Many mechanisms have been proposed to explain this phenomenon. In this study, the effect of Se intake on proliferation of hepatocytes and normal colonic epithelial cells in rats was determined using autoradiographic analysis of thymidine incorporation into DNA. Hepatocyte proliferation was measured 24 h after partial hepatectomy. Selenium-dosed animals demonstrated a significant reduction in hepatocyte labeling compared to the control group (6.1±2.6 vs 29.2±15.6,p=0.003). However, Se dosing did not affect the thymidine-labeling indices or distribution of labeling in colonic epithelium. Selenium may inhibit cell proliferation when it is the result of an unusually intense stimulus. This finding could explain in part the inhibitory effect of Se in some experimental cancer models. Dr. Tempero is a recipient of a Junior Clinical Faculty Fellowship from the American Cancer Society.     

Adherence to HEp-2 cells and replication in macrophages of Salmonella derby of human origin.

Adherence to a HEp-2 cell monolayer was tested for in four strains of Salmonella derby which were isolated from patients with diarrhea. One strain, SB1, was highly adherent and another strain, SB4, was nonadherent. The other two strains exhibited moderate adherence. Further in vitro study of invasion of HEp-2 cells by S. derby and its replication in murine peritoneal macrophages was carried out using SB1 and SB4. Thin section electron micrographs revealed that SB1 invaded HEp-2 cells but SB4 did not. The number of viable bacteria within macrophages was determined at intervals after inoculation of bacteria. The result indicates that SB1 can replicate in the macrophages but SB4 cannot. Flagella and fimbriae were compared by electron microscopy between SB1 and SB4, and their lipopolysaccharides and outer membrane proteins were also compared with each other by SDS-polyacrylamide gel electrophoresis. The presence of a 41 kDa protein in the outer membranes of SB1 was only the difference detected, suggesting that this protein could be a factor required for adherence of this serovar to epithelial cells.     

Hepatocyte growth factor promotes colonic epithelial regeneration via Akt signaling

Hepatocyte growth factor (HGF) can promote the regeneration of injured organs, including HGF gene therapy by electroporation (EP) for liver injury. In this study, we investigated the effect of HGF on dextran sulfate sodium-induced colitis and tried to clarify the regenerative mechanisms of colonic epithelial cells and the signaling pathway involved. Colitis was induced by dextran sulfate sodium in mice, together with HGF gene transfer by EP. On day 10, the colitis was evaluated histologically and by Western blot analysis. The colonic epithelial cell line MCE301 was exposed to HGF protein, and its proliferation and activated signaling pathway were analyzed. In vivo, the histological score improved and the number of Ki-67-positive epithelial cells increased in the HGF-treated mice compared with the controls. Western blot analysis showed enhanced expression of phospho-Akt in the HGF-treated mice compared with the controls. In vitro, HGF stimulated the proliferation of MCE301 cells. There was enhanced phospho-Akt expression for more than 48 h after HGF stimulation, although phospho-ERK1/2 was enhanced for only 10 min. LY-294002 or Akt small interfering RNA suppressed cell proliferation induced by HGF. Thus HGF induces the proliferation of colonic epithelial cells via the phosphatidylinositol 3-kinase/Akt signaling pathway. HGF gene therapy can attenuate acute colitis via epithelial cell proliferation through the PI3K/Akt pathway. These data suggested that HGF gene therapy by EP may be effective for the regeneration and repair of injured epithelial cells in inflammatory bowel disease.     

Altered SK3/KCa2.3-mediated migration in adenomatous polyposis coli (Apc) mutated mouse colon epithelial cells

Lost of adenomatous polyposis coli gene (Apc) disturbs the migration of intestinal epithelial cells but the mechanisms have not been fully characterized. Since we have demonstrated that SK3/KCa2.3 channel promotes cancer cell migration, we hypothesized that Apc mutation may affect SK3/KCa2.3 channel-mediated colon epithelial cell motility. We report evidence that SK3/KCa2.3 channel promotes colon epithelial cells motility. Following Apc mutation SK3/KCa2.3 expression is largely reduced leading to a suppression of the SK3/KCa2.3 channel mediated-cell migration. Our findings reveal a previously unknown function of the SK3/KCa2.3 channel in epithelial colonic cells, and suggest that Apc is a powerful regulator SK3/KCa2.3 channel.     

Delta-like 1 expression promotes goblet cell differentiation in Notch-inactivated human colonic epithelial cells

Notch signaling has previously been implicated in the regulation of the cell fate of intestinal epithelial cells. However, the expression and function of Notch ligands in the human intestine remain largely unknown. In the present study, we showed that Notch ligands Delta-like 1 (Dll1) and Delta-like 4 (Dll4) are expressed in a goblet cell-specific manner in human colonic tissue. Additionally, we found that Dll1 and Dll4 expression was regulated in-parallel with Atoh1 and MUC2, which are both under the control of the Notch-Hes1 signaling pathway. Because knockdown of Dll1 expression completely abrogated the acquisition of the goblet cell phenotype in Notch-inactivated colonic epithelial cells, we postulate that Dll1 might function as a cis-acting regulatory element that induces undifferentiated cells to become goblet cells. Our results suggest a link between Dll1 expression and human goblet cell differentiation that might be mediated by a function that is distinct from its role as a Notch receptor ligand.     

Taenia taeniaeformis: colonic hyperplasia in heavily infected rats

Only one study previously mentioned the involvement of colon during Taenia taeniaeformis larvae infection in rats with inconsistent occurrence of lesions. Present study aimed to determine the consistency of histopathologic changes in colonic epithelia, and the proliferation of mucosal cells through BrdU and PCNA immunohistochemistry. Results demonstrated that crypt hyperplasia of the colon was found in all infected rats, although variable in degree even in a single tissue section. Cystic cavities were frequently seen in severely hyperplastic mucosa. Proliferative zone lengths were significantly increased and PCNA positive cells were observed throughout the colonic crypt lengths at 9 but not at 6 weeks post infection. Cell proliferation involving the major types of cells in the epithelial colon was also increased in infected rats at 9 weeks post infection, with labeling indices significantly greater than the control rats throughout the BrdU time course labeling. Findings suggested that massive increases in epithelial cells and depth of colonic crypts were due to a remarkable increase in cell proliferation. The study concluded that enteropathy in the colon during T. taeniaeformis infection could be consistently observed in heavily infected rats.     

Calpains are involved in Entamoeba histolytica-induced death of HT-29 colonic epithelial cells

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that can cause amebic colitis and liver abscess in humans. E. histolytica has the capability to kill colon epithelial cells in vitro; however, information regarding the role of calpain in colon cell death induced by ameba is limited. In this study, we investigated whether calpains are involved in the E. histolytica-induced cell death of HT-29 colonic epithelial cells. When HT-29 cells were co-incubated with E. histolytica, the propidium iodide stained dead cells markedly increased compared to that in HT-29 cells incubated with medium alone. This pro-death effect induced by ameba was effectively blocked by pretreatment of HT-29 cells with the calpain inhibitor, calpeptin. Moreover, knockdown of m- and μ-calpain by siRNA significantly reduced E. histolytica-induced HT-29 cell death. These results suggest that m- and μ-calpain may be involved in colon epithelial cell death induced by E. histolytica.     

Role of Janus Kinase 3 in Mucosal Differentiation and Predisposition to Colitis

Janus kinase 3 (Jak3) is a nonreceptor tyrosine kinase expressed in both hematopoietic and nonhematopoietic cells. Previously, we characterized the functions of Jak3 in cytoskeletal remodeling, epithelial wound healing, and mucosal homeostasis. However, the role of Jak3 in mucosal differentiation and inflammatory bowel disease was not known. In this report, we characterize the role of Jak3 in mucosal differentiation, basal colonic inflammation, and predisposition toward colitis. Using the Jak3 knock-out (KO) mouse model, we show that Jak3 is expressed in colonic mucosa of mice, and the loss of mucosal expression of Jak3 resulted in reduced expression of differentiation markers for the cells of both enterocytic and secretory lineages. Jak3 KO mice showed reduced expression of colonic villin, carbonic anhydrase, secretory mucin muc2, and increased basal colonic inflammation reflected by increased levels of pro-inflammatory cytokines IL-6 and IL-17A in colon along with increased colonic myeloperoxidase activity. The inflammations in KO mice were associated with shortening of colon length, reduced cecum length, decreased crypt heights, and increased severity toward dextran sulfate sodium-induced colitis. In differentiated human colonic epithelial cells, Jak3 redistributed to basolateral surfaces and interacted with adherens junction (AJ) protein β-catenin. Jak3 expression in these cells was essential for AJ localization of β-catenin and maintenance of epithelial barrier functions. Collectively, these results demonstrate the essential role of Jak3 in the colon where it facilitated mucosal differentiation by promoting the expression of differentiation markers and enhanced colonic barrier functions through AJ localization of β-catenin.     

Glucagon-like peptide 2 improves intestinal wound healing through induction of epithelial cell migration in vitro-evidence for a TGF--beta-mediated effect

BACKGROUND/AIMS: In vitro studies suggest that glucagon-like peptide 2 (GLP-2), secreted from enteroendocrine cells in the gastrointestinal tract after food intake, is able to ameliorate mucosal injury in settings of human disease characterized by injury and dysfunction of the intestinal mucosal epithelium. We evaluated this potential of GLP-2 after epithelial trauma by using two in vitro models measuring intestinal epithelial cell proliferation and cell migration. MATERIALS AND METHODS: Injuries were induced in confluent monolayers of the small intestinal cells lines IEC-6 and IEC-18, as well as in the colonic cell lines Caco-2 and Colo 320. GLP-2 (50-500 nM) or other peptides were added to the media. Wound healing was investigated after 24 h by quantification of the number of cells migrating across the wound edge. Proliferation of cells was assessed by using photometric mitochondrial incorporation measurement of MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide). Monoclonal TGF-beta antibodies were added to wounded monolayers to examine whether the GLP-2-induced wound healing was TGF-beta-mediated. RESULTS: Migration assessments revealed a significant stimulation of GLP-2-induced migration in IEC-6 and IEC-18 monolayers compared to the placebo group. No effect was observed in the colon cancer cell lines Caco-2 and Colo 320. Results of the proliferation assays show a significant inhibition of proliferation by GLP-2 in small intestinal cell lines whereas a dose-dependent stimulation of proliferation in colonic epithelial cells was observed. Addition of neutralizing TGF-beta1 antibodies to wounded IEC-6 and IEC-18 monolayers incubated with GLP-2 significantly reduced the number of migrating cells to the level of the placebo group. CONCLUSIONS: In our in vitro model, it was shown that the GLP-2-induced improvement of intestinal wound healing is TGF-beta-mediated. These effects were predominant in the epithelium of the small intestine compared to colonic epithelium. Our findings provide further insight into mechanisms leading to GLP-2-induced mucosal wound healing. These results suggest that GLP-2 or analogues of this peptide may potentially be useful for the treatment of intestinal disorders characterized by injury and ineffective repair of the intestinal mucosa.     

Epithelial cell cultures from the colon of the suckling rat

Summary Epithelial cells from the colon of suckling rats have been propagated in vitro. The colons were excised and cut longitudinally. The epithelial sheets were peeled off and dissociated in 0.1% trypsin solution at 25°C for 10 min. The first cell suspension was discarded and the remaining fragments trypsinized again for an additional 20 min. The dissociated cells were washed and cultured. Forty-eight hours later, several epithelial colonies consisting of closely packed polygonal cells were formed. Transmission and scanning electron microscope examination of the colonies showed numerous regularly spaced microvilli on the surface and tight junctions and desmosomes between adjacent cells. Immunocytochemical studies with antiserum prepared against the brush-border membrane of the colonic epithelium showed specific staining of the epithelial colonies. Epithelial colonies were subcultured by the penicylinder method. Although the subcultured cells retained their epithelial characteristics, the proliferative activity of the cells gradually decreased. Currently, efforts are being made to determine the optimum nutritional requirements of the primary and low-passage cultures.     

Endothelin-3 stimulates survival of goblet cells in organotypic cultures of fetal human colonic epithelium

Cells within the normal human colonic epithelium undergo a dynamic cycle of growth, differentiation, and death. The organotypic culture system of human fetal colonic epithelial cells seeded on top of collagen gels with embedded colonic fibroblasts allowed prolonged culture of the colonic epithelial cells (Kalabis J, Patterson MJ, Enders GM, Marian B, Iozzo RV, Rogler G, Gimotty PA, Herlyn M. FASEB J 17: 1115-1117, 2003). Herein, we have evaluated the role of endothelin-3 (ET3) and both cognate endothelin receptors (ETRA, ETRB) for human colonic epithelial cell growth and survival. ET3 was produced continuously by the fibroblasts as a result of adenovirus-mediated gene transfer. The presence and function of the endothelin receptors (ETRs) in epithelial cells was evaluated by [(3)H]thymidine incorporation using primary epithelial cells in monoculture and by immunohistochemistry on human fetal and adult paraffin-embedded tissues. In organotypic culture, ET3 increased the number of goblet cells but not of enteroendocrine cells. The increase in goblet cells was caused by prolonged cell survival and differentiation. The inhibition of both ETRA and ETRB significantly decreased the number of goblet cells and proliferation in epithelial cells, whereas the number of enteroendocrine cells remained unchanged. ET3 induced activation of IkappaB and MAPK in the epithelial cells, suggesting that these signaling pathways mediate its proproliferation and prosurvival activities. Our results demonstrate that ET3 is involved in regulating human colonic epithelial cell proliferation and survival, particularly for goblet cells, and may be an important component of colonic homeostasis.