Murine Colonic Epithelial Cells Research Articles

Differential Secreted Proteome Approach in Murine Model for Candidate Biomarker Discovery in Colon Cancer

The complexity and heterogeneity of the plasma proteome have presented significant challenges in the identification of protein changes associated with tumor development. We used cell culture as a model system and identified differentially expressed, secreted proteins which may constitute serological biomarkers. A stable isotope labeling by amino acids in cell culture (SILAC) approach was used to label the entire secreted proteomes of the CT26 murine colon cancer cell line and normal young adult mouse colon (YAMC) cell line, thereby creating a stable isotope labeled proteome (SILAP) standard. This SILAP standard was added to unlabeled murine CT26 colon cancer cell or normal murine YAMC colon epithelial cell secreted proteome samples. A multidimensional approach combining isoelectric focusing (IEF), strong cation exchange (SCX) followed by reversed phase liquid chromatography was used for extensive protein and peptide separation. A total of 614 and 929 proteins were identified from the YAMC and CT26 cell lines, with 418 proteins common to both cell lines. Twenty highly abundant differentially expressed proteins from these groups were selected for liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS) analysis in sera. Differential secretion into the serum was observed for several proteins when Apc(min) mice were compared with control mice. These findings were then confirmed by Western blot analysis.

Enterococcus faecalis Induces Aneuploidy and Tetraploidy in Colonic Epithelial Cells through a Bystander Effect

Intestinal commensals are potential important contributors to the etiology of sporadic colorectal cancer, but mechanisms by which bacteria can initiate tumors remain uncertain. Herein, we describe mechanisms that link Enterococcus faecalis, a bacterium known to produce extracellular superoxide, to the acute induction of chromosomal instability. Immortalized human and nontransformed murine colonic epithelial cells, along with a mouse colonic ligation model, were used to assess the effect of E. faecalis on genomic DNA stability and damage. We found that this human intestinal commensal generated aneuploidy, tetraploidy, and gammaH2AX foci in HCT116, RKO, and YAMC cells. In addition, direct exposure of E. faecalis to these cells induced a G2 cell cycle arrest. Similar observations were noted by exposing cells to E. faecalis-infected macrophages in a dual-chamber coculture system for detecting bystander effects. Manganese superoxide dismutase, catalase, and tocopherols attenuated, and caffeine and inhibitors of glutathione synthase exacerbated, the aneugenic effects and linked the redox-active phenotype of this intestinal commensal to potentially transforming events. These findings provide novel insights into mechanisms by which E. faecalis and intestinal commensals can contribute to cellular transformation and tumorigenesis.

Genetic Ablation of M3 Muscarinic Receptors Attenuates Murine Colon Epithelial Cell Proliferation and Neoplasia

Colon epithelial cells express and most colon cancers overexpress M(3) muscarinic receptors (M(3)R). In human colon cancer cells, post-M(3)R signaling stimulates proliferation. To explore the importance of M(3)R expression in vivo, we used the azoxymethane-induced colon neoplasia model. Mice treated with weekly i.p. injection of saline [10 wild-type (WT) mice] or azoxymethane (22 WT and 16 M(3)R(-/-) mice) for 6 weeks were euthanized at 20 weeks. At week 20, azoxymethane-treated WT mice weighed approximately 16% more than M(3)R(-/-) mice (33.4 grams +/- 1.0 grams versus 27.9 grams +/- 0.5 grams; mean +/- SE, P < 0.001). In azoxymethane-treated M(3)R(-/-) mice, cell proliferation (BrdUrd staining) was reduced 43% compared with azoxymethane-treated WT mice (P < 0.05). Whereas control mice (both WT and M(3)R(-/-)) had no colon tumors, azoxymethane-treated WT mice had 5.3 +/- 0.5 tumors per animal. Strikingly, azoxymethane-treated M(3)R(-/-) mice had only 3.2 +/- 0.3 tumors per mouse (P < 0.05), a 40% reduction. Tumor volume in azoxymethane-treated M(3)R(-/-) mice was reduced 60% compared with azoxymethane-treated WT mice (8.1 mm(3) +/- 1.5 mm(3) versus 20.3 mm(3) +/- 4.1 mm(3); P < 0.05). Compared with WT, fewer M(3)R(-/-) mice had adenomas (6% versus 36%; P = 0.05), and M(3)R(-/-) mice had fewer adenocarcinomas per mouse (0.6 +/- 0.1 versus 1.7 +/- 0.4; P < 0.05). Eleven of 22 WT but no M(3)R(-/-) mice had multiple adenocarcinomas (P < 0.001). Compared with WT, azoxymethane-treated M(3)R-deficient mice have attenuated epithelial cell proliferation, tumor number, and size. M(3)R and post-M(3)R signaling are novel therapeutic targets for colon cancer.

Hochuekkito, a Kampo (Traditional Japanese Herbal) Medicine, and its Polysaccharide Portion Stimulate G-CSF Secretion from Intestinal Epithelial Cells

Kampo (traditional Japanese herbal) medicines are taken orally due to which the gastric mucosal immune system may act as one of the major targets for the expression of pharmacological activity. The inner surface of the intestinal tract possesses a large area of mucosal membranes, and the intestinal epithelial cells sit at the interface between a lumen and a lymphocyte-rich lamina propria. The cross talk that occurs between these compartments serves to maintain intestinal homeostasis, and the cytokine network plays an important role in the cross talk. In this study, the effect of Hochuekkito (HET), one of Kampo medicines, on cytokine secretion of intestinal epithelial cells was investigated. When murine normal colonic epithelial cell-line MCE301 cells were stimulated with HET, the contents of granulocyte colony-stimulating factor (G-CSF) in the conditioned medium were significantly increased in dose- and time-dependent manners. The enhanced G-CSF gene transcription in MCE301 cells by the stimulation of HET was observed by RT-PCR. The enhanced G-CSF secretion by HET was also observed in C3H/HeJ mice-derived primary cultured colonic epithelial cells. When the HET was fractionated, only the polysaccharide fraction (F-5) enhanced the G-CSF secretion of MCE301 cells, and the activity of F-5 lost after the treatment of periodate that can degrade the carbohydrate moiety. These results suggest that HET enhances secretion of G-CSF from colonic epithelial cells and the polysaccharide is one of the active ingredients of HET. The enhanced G-CSF secretion by HET may partly contribute to the clinically observed various pharmacological activities of HET including immunomodulating activity.

Stimulatory effect of a pectic polysaccharide from a medicinal herb, the roots of Bupleurum falcatum L., on G-CSF secretion from intestinal epithelial cells

The inner surface of the intestinal tract possesses a large area of mucosal membranes, and the intestinal epithelial cells exist at the interface between an antigen-rich lumen and a lymphocyte-rich lamina propria. The crosstalk that occurs between these compartments serves to maintain intestinal homeostasis, and the cytokine network plays an important role in the crosstalk. In this study, the effect of a pectic polysaccharide, bupleuran 2IIc from Bupleurum falcatum L., on cytokine secretion of intestinal epithelial cells was investigated in vitro. When murine normal colonic epithelial cell line MCE301 cells were stimulated with bupleuran 2IIc, the contents of granulocyte colony-stimulating factor (G-CSF) in the conditioned medium were significantly increased in dose- and time-dependent manners. The enhanced G-CSF gene transcription in MCE301 cells by the stimulation of bupleuran 2IIc was observed by RT-PCR. The enhanced G-CSF secretion by bupleuran 2IIc was also observed in C3H/HeJ mice derived primary cultured colonic epithelial cells. Bupleuran 2IIc was digested with endo-(1→4)-α-d-polygalacturonase, and the resulting bupleuran 2IIc/PG-1 (“ramified” region) showed potent G-CSF secretion enhancing activity. The activity of bupleuran 2IIc/PG-1 disappeared after the removal of arabinosyl residues from bupleuran 2IIc/PG-1 by endo-(1→5)-α-l-arabinanase digestion. These results suggest that the “ramified” region (bupleuran 2IIc/PG-1) is the active site for the G-CSF secretion enhancing activity of bupleuran 2IIc, and the arabinan moiety of bupleuran 2IIc/PG-1 plays an important role in expression of the activity.

Effects of hangeshashinto on butyrate-induced cell death in murine colonic epithelial cell

Effects of hangeshashinto on butyrate-induced cell death in murine colonic epithelial cell

Human epithelial-specific response to pathogenicCampylobacter jejuni

The gastrointestinal epithelia of mammals are tolerant of their resident gut microbiota but are usually highly responsive to entero-pathogens; the host-specific responses have not been well characterized. To this end, the transcriptional responses of cultured human (Caco-2) and murine (CT-26) colonic epithelial cells were compared after exposure with the microfloral bacterium Lactobacillus reuteri or the human gastrointestinal pathogen Campylobacter jejuni. When in bacterial broth, both species elicit a stronger differential gene expression response in human colonic cells compared with mouse colonic cells. However, when these data are adjusted to remove bacterial broth effects, only human colonic epithelia exposed to C. jejuni show altered gene expression, suggesting that the human pathogen C. jejuni induces a host-specific response. The genes with altered expression are involved in growth, transcription, and steroid biosynthesis. Interestingly, human genes involved in cell polarity and water transport were significantly changed in response to C. jejuni exposure, linking infection with gastrointestinal disease. This study demonstrates that mouse and human colonic epithelia remain relatively unresponsive to commensal bacterial challenge, while the human pathogen C. jejuni elicits a host-specific response.

Deletion of the genes encoding the type III secretion system and cytotoxic enterotoxin alters host responses to Aeromonas hydrophila infection

In our previous study, we deleted the gene encoding Aeromonas outer membrane protein B (AopB), a structural component of the type III secretion system (T3SS) from a cytotoxic enterotoxin gene ( act)-minus diarrheal isolate SSU of Aeromonas hydrophila. Our laboratory also molecularly characterized the cytotoxic enterotoxin (Act), which is secreted by the bacterium utilizing the type II secretion system (T2SS). The act/aopB mutant exhibited significantly reduced cytotoxicity to cultured cells (e.g. RAW 264.7 murine macrophages and HT-29 human colonic epithelial cells) and was avirulent in mice. In this study, we developed additional A. hydrophila mutants in which T3SS-associated ascV and acrV genes were deleted, either individually or in combination with that of the act gene, to examine host–pathogen interactions. A significant reduction in the induction of inflammatory cytokines and chemokines was noted in the sera of mice infected with these mutants when compared to animals infected with wild-type (WT) A. hydrophila. After infection with the WT and act/aopB mutant, we performed microarray analyses on RNA from the above-mentioned murine macrophages and human colonic epithelial cells to examine global cellular transcriptional responses. Based on three independent experiments, WT A. hydrophila altered the expression of 434 genes in RAW 264.7 cells and 80 genes in HT-29 cells. Alteration in the expression of 209 macrophage and 32 epithelial cell genes was reduced when the act/ aopB mutant was used, compared to when cells were infected with the WT bacterium, indicating the involvement of Act and/or AopB in transcriptional regulation of these genes. We verified up-regulation of 15 genes by real-time reverse transcriptase-polymerase chain reaction and confirmed A. hydrophila WT–versus mutant -induced production of cytokines/chemokines in supernatants from RAW 264.7 and HT-29 cells. This is the first description of host cell transcriptional responses to A. hydrophila infection.

Butyrate Induces Necrotic Cell Death in Murine Colonic Epithelial Cell MCE301

Recent findings have suggested that organic acids produced by anaerobic intestinal bacteria might contribute to the pathogenesis of colonic ulcers. In this study, it was shown that butyrate caused potent cytotoxicity in the murine normal colonic epithelial cells MCE301 at physiological concentrations. Several markers of apoptosis, such as phosphatidyl serine externalization, cytochrome c release, DNA fragmentation, and chromatin condensation were negative after butyrate exposure. Inhibitor of caspases failed to protect against butyrate cytotoxicity. By transmission electron microscopy, marked swollen mitochondria and vacuolization within the cytoplasm was observed by treatment of butyrate. Collective, these data indicated that butyrate-induced cell death caused through a necrosis-like process. Butyrate induced cell death was reduced partially by treatment with prednisolone or 5-aminosalicylates in a concentration dependent manner. These results suggest that (1) butyrate induces necrotic cell death but not apoptotic cell death, and (2) the necrotic cell death induced by butyrate may be useful as a novel in vitro model of ulcerative colitis to screen useful drugs for the treatment of the disease.

The Type III Secretion System and Cytotoxic Enterotoxin Alter the Virulence of Aeromonas hydrophila

Many gram-negative bacteria use a type III secretion system (TTSS) to deliver effector proteins into host cells. Here we report the characterization of a TTSS chromosomal operon from the diarrheal isolate SSU of Aeromonas hydrophila. We deleted the gene encoding Aeromonas outer membrane protein B (AopB), which is predicted to be involved in the formation of the TTSS translocon, from wild-type (WT) A. hydrophila as well as from a previously characterized cytotoxic enterotoxin gene (act)-minus strain of A. hydrophila, thus generating aopB and act/aopB isogenic mutants. The act gene encodes a type II-secreted cytotoxic enterotoxin (Act) that has hemolytic, cytotoxic, and enterotoxic activities and induces lethality in a mouse model. These isogenic mutants (aopB, act, and act/aopB) were highly attenuated in their ability to induce cytotoxicity in RAW 264.7 murine macrophages and HT-29 human colonic epithelial cells. The act/aopB mutant demonstrated the greatest reduction in cytotoxicity to cultured cells after 4 h of infection, as measured by the release of lactate dehydrogenase enzyme, and was avirulent in mice, with a 90% survival rate compared to that of animals infected with Act and AopB mutants, which caused 50 to 60% of the animals to die at a dose of three 50% lethal doses. In contrast, WT A. hydrophila killed 100% of the mice within 48 h. The effects of these mutations on cytotoxicity could be complemented with the native genes. Our studies further revealed that the production of lactones, which are involved in quorum sensing (QS), was decreased in the act (32%) and aopB (64%) mutants and was minimal (only 8%) in the act/aopB mutant, compared to that of WT A. hydrophila SSU. The effects of act and aopB gene deletions on lactone production could also be complemented with the native genes, indicating specific effects of Act and the TTSS on lactone production. Although recent studies with other bacteria have indicated TTSS regulation by QS, this is the first report describing a correlation between the TTSS and Act of A. hydrophila and the production of lactones.

Topical antisense oligonucleotide therapy against LIX, an enterocyte-expressed CXC chemokine, reduces murine colitis

Epithelial neutrophil-activating peptide-78 (ENA-78), a member of the CXC chemokine subfamily, is induced by inflammatory cytokines in human colonic enterocyte cell lines and increased in the colon of patients with inflammatory bowel disease (IBD). Lipopolysaccharide-induced CXC-chemokine (LIX) was recently identified as the murine homolog of ENA-78. Here we show that, similar to ENA-78, inflammatory cytokine stimulation of a murine colonic epithelial cell line, MODE-K, results in increased LIX expression. Consistent with the expression pattern of ENA-78 in IBD, LIX expression is significantly increased in mice with colitis induced by the ingestion of dextran sodium sulfate (DSS). Treating mice with antisense oligonucleotides to LIX via rectal enema delivery before DSS treatment results in colonic enterocyte uptake and a significant reduction in neutrophil infiltration and severity of colitis. These findings indicate that LIX plays an integral role in the pathogenesis of DSS-induced colitis. Similarly, enterocyte-derived CXC chemokines may play a key role in regulating neutrophil recruitment and intestinal injury in IBD. The intracolonic administration of ENA-78 antisense oligonucleotides may be effective in treating distal ulcerative colitis in humans.

Leptin, Insulin-Like Growth Factor-1, and Insulin-Like Growth Factor-2 Are Mitogens in ApcMin/+ but not Apc+/+ Colonic Epithelial Cell Lines

The obese state is associated with elevated circulating levels of insulin, insulin-like growth factors (IGF), and leptin. Research is contradictory regarding the role of these elevated growth factors in colon cancer risk. We hypothesized that colonic epithelial cells that were Apc deficient (ApcMin/+) but not those expressing wild-type Apc (Apc+/+) would experience a hyperproliferative and antiapoptotic phenotype when exposed to these growth factors. This hypothesis was addressed using two nontumorigenic murine colonic epithelial cell lines with distinct Apc genotypes: Apc+/+ YAMC cells and ApcMin/+ IMCE cells. Cells were treated for 48 hours with various concentrations of leptin (0.001-50 ng/mL), IGF-1 (0.1-200 ng/mL), or IGF-2 (0.1-600 ng/mL). In YAMC cells, leptin caused a significant decrease in cell proliferation (P < 0.01) compared with controls due to induction of caspase activity and apoptosis. In contrast, in the IMCE cells, leptin induced a 75% increase in cell proliferation compared with controls (P < 0.0001). IGF-1 and IGF-2 also induced 50% greater proliferation in the IMCE cells (P < 0.001) compared with controls. Cotreatment of IMCE cells with leptin and either IGF-1 or IGF-2 induced greater proliferation than either growth factor alone (P < 0.0001). IMCE cell proliferation caused by leptin only treatment was associated with activation of p42/44 mitogen-activated protein kinase (MAPK), p38 MAPK, and nuclear factor-kappaB nuclear translocation but not with MAPK kinase or Janus-activated kinase/signal transducers and activators of transcription activation. These data provide the first evidence that leptin may interact with IGFs to promote survival and expansion of colonic epithelial cells that were Apc deficient (ApcMin/+) but not those expressing wild-type Apc (Apc+/+).

Vascular endothelial growth factor and enhanced angiogenesis do not promote metastatic conversion of a newly established azoxymethane‐induced colon cancer cell line

The organo-specific carcinogen, azoxymethane (AOM), produces colon tumors in mice that share many pathological features with sporadic human colorectal cancer (CRC). An important distinction between AOM-induced CRC and human CRC is lack of mucosal invasion in the murine model. To assess the role of the microenvironment in preventing the invasive phenotype, multiple benign in situ adenocarcinomas were harvested from AOM-treated mice and cultured in vitro. However, tumor cell growth was extremely limiting under standard culturing conditions. Thus, we injected tumor cells directly into nude mice and performed two serial transplants, and successfully explanted a rapidly growing epithelial tumor cell line (AJ02nm(0)). When injected subcutaneously (sc) into nude mice, AJ02nm(0) cells formed well-differentiated adenocarcinomas with minimal tumor invasive capacity. To define whether metastatic and invasive potential were related to lack of angiogenic stimuli, the AJ02nm(0) cells were transfected to overexpress murine vascular endothelial growth factor-164 (VEGF(164)). AJ02nm-VEGF cells produced rapidly growing tumors in nude mice that exhibited extensive pseudo-epithelial ductal architecture and supporting vasculature, but without increased invasive potential compared to controls. The established murine colon epithelial cell line provides a useful experimental model to further elaborate genetic and epigenetic factors that may promote or inhibit colon tumorigenesis and metastasis.

Different cytokine response of primary colonic epithelial cells to commensal bacteria

To determine if primary murine colonic epithelial cells (CEC) respond to commensal bacteria and discriminate between different types of bacteria. A novel CEC: bacteria co-culture system was used to compare the ability of the colonic commensal bacteria, Bacteroides ovatus, E. coli (SLF) and Lactobacillus rhamnosus (LGG) to modulate production of different cytokines (n = 15) by primary CEC. Antibody staining and flow cytometry were used to investigate Toll-like receptor (TLR) expression by CEC directly ex vivo and TLR responsiveness was determined by examining the ability of TLR ligands to influence CEC cytokine production. Primary CEC constitutively expressed functional TLR2 and TLR4. Cultured in complete medium alone, CEC secreted IL-6, MCP-1 and IP-10 the levels of which were significantly increased upon addition of the TLR ligands peptidoglycan (PGN) and lipopolysaccharide (LPS). Exposure to the commensal bacteria induced or up-regulated different patterns of cytokine production and secretion. E. coli induced production of MIP-1alpha/beta and betadefensin3 whereas B. ovatus and L. rhamnosus exclusively induced MCP-1 and MIP-2alpha expression, respectively. TNFalpha, RANTES and MEC were induced or up-regulated in response to some but not all of the bacteria whereas ENA78 and IP-10 were up-regulated in response to all bacteria. Evidence of bacterial interference and suppression of cytokine production was obtained from mixed bacterial: CEC co-cultures. Probiotic LGG suppressed E. coli- and B. ovatus-induced cytokine mRNA accumulation and protein secretion. These observations demonstrate the ability of primary CEC to respond to and discriminate between different strains of commensal bacteria and identify a mechanism by which probiotic bacteria (LGG) may exert anti-inflammatory effects in vivo.

Fucoidan derived from Cladosiphon okamuranus Tokida ameliorates murine chronic colitis through the down-regulation of interleukin-6 production on colonic epithelial cells.

Our previous study indicated that the interleukin (IL)-6/STAT-3 signal was up-regulated in inflammatory bowel disease (IBD) in both humans and animal models. We also discovered phosphorylated STAT-3 in the nucleus of the colonic epithelial cells in IBD mice. Intestinal epithelial cells (IEC) have been shown to secrete IL-6. Therefore, the secretion of IL-6 from IEC may be one of the mechanisms of STAT-3 phosphorylation in IEC during the pathogenesis of IBD, and inhibition of IL-6 production by IEC may be beneficial in preventing IBD. We examined the preventative effect of various types of fucoidans on IL-6 production in a lipopolysaccharide (LPS)-stimulated murine colonic epithelial cells line, CMT-93, in vitro. We also determined in vivo the effect of fucoidans on murine chronic colitis induced with dextran sodium sulphate. Among fucoidans, those from Cladosiphon okamuranus Tokida and Kjellmaniella crassifolia inhibited IL-6 production in CMT-93 cells with the down-regulation of NF-kappaB nuclear translocation. Analysis of the effect of fucoidan on murine colitis in vivo showed that the disease activity index and myeloperoxidase activity decreased in mice fed Cladosiphon fucoidan, but not Fucus fucoidan. Cytokine profiles in colonic lamina propria indicated that the synthesis of interferon (IFN)-gamma and IL-6 decreased and that of IL-10 and transforming growth factor (TGF)-beta increased in mice fed Cladosiphon fucoidan, compared with mice fed a standard diet or Fucus fucoidan. The levels of IL-6 mRNA in colonic epithelial cells was lower in colitis-induced Balb/c mice fed Cladosiphon fucoidan than those fed a standard diet. Fucoidan improves murine chronic colitis by down-regulating the synthesis of IL-6 in the colonic epithelial cells. Fucoidan derived from C. o. Tokida may be useful as a dietary substance for the patients with inflammatory bowel disease.

Flavonoids Promote Cell Migration in Nontumorigenic Colon Epithelial Cells Differing in Apc Genotype: Implications of Matrix Metalloproteinase Activity

Colonic epithelial cell migration is required for movement up to the apex of the crypt and, hence, normal differentiated cell function. This migratory phenotype is dependent upon wild-type adenomatous polyposis coli (Apc) expression. The purpose of this study is to determine whether specific flavonoids induce cell migration in colon epithelial cells either wild type or heterozygous for Apc genotype. Nontumorigenic murine colon epithelial cell lines with distinct Apc genotypes, young adult mouse colon (YAMC; Apc+/+) cells, and Immortomouse/Min colon epithelial (IMCE; ApcMin/+) cells were used to assess the ability of specific flavonoids to induce cell migration relative to migration induced by hepatocyte growth factor (HGF). The citrus flavanones naringenin and hesperetin did not induce cell migration comparable with HGF in either cell type. However, the glycosylated forms of these flavanones, naringin and hesperidin, induced migration differentially in YAMC and IMCE cells. Specifically, naringin and hesperidin induced the greatest migratory response in IMCE cells at 1 μM (P < 0.01) and induced migration greater than untreated control cells (P < 0.05) but equal to HGF-treated cells. In YAMC cells, hesperidin did not induce migration except at the 100-mM concentration. Apigenin induced migration in IMCE cells at 1 (P < 0.05) and 50 μM (P < 0.01) and did not induce migration in the YAMC cells. Catechin induced migration at the highest concentration (300 μM) only in the IMCE cells, whereas epicatechin induced migration at the lowest concentration only (1 mM) in IMCE cells. Overall, the glycosylated citrus flavanones induced the greatest migratory response at the lowest concentration in IMCE cells. Co-treatment of IMCE cells with the global matrix metalloproteinase (MMP) inhibitor Ilomastat® in the presence of naringin, hesperidin, or apigenin demonstrated that flavonoid-induced migration was dependent on MMP activity. Induction of the migratory phenotype by flavonoids in these models of preneoplastic epithelial cells suggests a novel mechanism for colon cancer prevention.

A20 is critical for protection of the intestinal epithelium from LPS or TNF induced apoptosis

Backgromtd/Aims: A20 is an important regulator of TNF induced NF-kB activity, and programmed cell death. Mice deficient for A20 develop colitis The aim of this stud}, was to determine whether A20 plays a role in the regulation of TNF induced NF*kB acti~ W and apoptosis in the intestinal epithelnim Methods: Young adult mouse colonocytes (YAMC), from a conditionally immortalized murine colonic epithelial cell line, were treated with TNF. A20 mRNA and protein expression induced TNF in YAMC cells were assessed by' Northern and Western analysis, respectivdy. TNF-induced NF-kB in YAMC cells was assessed dectrophoretic mobility shiR assay. Wildwpe (wt) and A20-/mice (generated gene targeting) were m/ected with lipopolysaccharide (LPS) or TNF intraperitoneally. The developmere of enteritis and intestinal epithelial cell apoptos~s in these mice were evaluated histology and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphae rock end-labeling (2LINED method. Resuhs: A20 mRNA and protein expression in YAMC ceils were induced by TNF. YAMC ceils responded to TNF by' activating and terminating NF-kB activity. These results suggest that A20 is expressed in mouse intestinal epithelkal cells (IEC) and regulates TNF niduced NF-kB activity in YAMC cells. A20G mice died within 2 hours of injection of either EPS or TNF, where as wt mice survived. A204mice that were treated with LPS or TNF developed severe enteritis with marked intestinal epithehal cell exfoliations. There was a tremendous increase in the number of apoptotic IEC demonstrated in both histology and TUNEr. staining, lntraepithehal lymphocytes may' have an ~mplication on the 1EC apoptosis. However, double mutant A20-/recombinase-activating gene-1 deficient (RAG-i-/-) mice lacking all matm'e lymphocytes also developed severe emerins with marked 1EC apoptosts and died within 2 hours after LPS injection. Conclusions: A20 is expressed in IEC and is cntical for protecting the intestinal epithelium trom LPS or TNF induced enteritis. The protection of the intestinal epithelium A20 may be importam for the prevention of IBD

Membrane-type matrix metalloproteinases mediate curcumin-induced cell migration in non-tumorigenic colon epithelial cells differing in Apc genotype.

Colonic epithelial cell migration is required for normal differentiated cell function. This migratory phenotype is dependent upon wild-type adenomatous polyposis coli (Apc) expression. Non-tumorigenic murine colon epithelial cell lines with distinct Apc genotypes, i.e. young adult mouse colon (YAMC; Apc(+/+)) and immortomouse/Min colon epithelial (IMCE; Apc(Min/+) cells) were used to assess the association between the Apc genotype, cell motility and matrix metalloproteinase (MMP) activity. Cells were treated with epidermal growth factor (EGF; 1, 10 and 25 ng/ml), hepatocyte growth factor (HGF; 1, 10 and 25 ng/ml) and/or curcumin (0.1-100 microM). EGF (25 ng/ml) and HGF (25 ng/ml) induced a greater migratory response in YAMC compared with IMCE cells after 24 h (P < 0.05). Treatment with curcumin induced a greater or equivalent migratory response in IMCE than YAMC cells. When migrating cells were treated with Ilomastat (MMP inhibitor), migration was inhibited in both cell types. High concentrations of Ilomastat (25 and 50 microM) inhibited migration in both cell types, while low concentrations (10 microM) inhibited HGF-induced IMCE migration. Curcumin-induced migration was inhibited in both cell types at the highest concentration of Ilomastat (50 microM). Immuno-localization analysis of membrane type-1 (MT1)-MMP indicated that migration is associated with the redistribution of this protein from the endoplasmic reticulum to the plasma membrane. Addition of neutralizing polyclonal antibodies against MT1-MMP or a mixture of MT1, 2- and 3-MMPs demonstrated partial or complete inhibition of cell migration in both cell types, respectively. The data provide the first evidence that migration in non-tumorigenic murine colon epithelial cells is: (i) inducible by EGF and HGF in an Apc genotype-dependent manner, (ii) dependent on MT-MMP activity and (iii) inducible by curcumin in an Apc genotype-independent manner. The data suggest a potential mechanism by which curcumin may induce cells heterozygous for Apc to overcome defective cell migration, a phenotype associated with cell differentiation and apoptosis.

Expression of prostaglandin endoperoxide H synthase-2 induced by nitric oxide in conditionally immortalized murine colonic epithelial cells.

Increased expression of prostaglandin endoperoxide H synthase-2 (PGHS-2) has been implicated in pathological conditions such as inflammatory bowel diseases and colon cancer. Recently, it has been demonstrated that inducible nitric oxide synthase (NOS II) expression and nitric oxide (NO) production are up-regulated in these diseases as well. However, the apparent link between PGHS-2 and NOS II has not been thoroughly investigated in nontransformed and nontumorigenic colonic epithelial cells. In the present study, we examined the concomitant expression of PGHS-2 and NOS II as well as the production of prostaglandin E2 (PGE2) and NO in conditionally immortalized mouse colonic epithelial cells, namely YAMC (Apc(+/+)). We found that the induction of PGHS-2 and generation of PGE2 in these cells by IFN-gamma and lipopolysaccharide (LPS) were greatly reduced by two selective NOS II inhibitors, L-NIL and SMT. To ascertain the effect of NO on PGHS-2 overexpression, we tested NO-releasing compounds, NOR-1 and SNAP, and found that they caused PGHS-2 expression and PGE2 production. This effect was abolished by hemoglobin, a NO scavenger. Using electrophoretic mobility shift assays, we found that both NOR-1 and SNAP caused beta-catenin/LEF-1 DNA complex formation. Super-shift by anti-beta-catenin antibody confirmed the presence of beta-catenin in the complex. Cell fractionation studies indicated that NO donors caused an increase in free soluble cytoplasmic beta-catenin. This is further corroborated by the immunocytochemistry data showing the redistribution of beta-catenin from the predominantly membrane localization into the cytoplasm and nucleus after treatment with NO donors. To further explore the possible connection between PGHS-2 expression and beta-catenin/LEF-1 DNA complex formation, we studied IMCE (Apc(Min/+)) cells, a sister cell line of YAMC with similar genetic background but differing in Apc genotype and, consequently, their beta-catenin levels. We found that IMCE cells, in comparison with YAMC cells, had markedly higher beta-catenin/LEF-1 DNA complex formation under both resting conditions as well as after induction with NO. In parallel fashion, IMCE cells expressed significantly higher levels of PGHS-2 mRNA and protein, and generated more PGE2. Overall, this study suggests that NO may be involved in PGHS-2 overexpression in conditionally immortalized mouse colonic epithelial cells. Although the molecular mechanism of the link is still under investigation, this effect of NO appears directly or indirectly to be a result of the increase in free soluble beta-catenin and the formation of nuclear beta-catenin/LEF-1 DNA complex.

Antigen presentation capabilities of primary murine colonic epithelial cells

Antigen presentation capabilities of primary murine colonic epithelial cells