Young Adult Mouse Colon Cells Research Articles

Abstract 306: Alternatively activated macrophages induce bystander effects via arginase 1

Abstract Colorectal cancer is a leading cause of cancer mortality and arises from genetic and epigenetic changes in colon epithelial cells. One of the mechanisms driving colon epithelial cell transformation is the bystander effect (BSE). BSE primarily arises from polarized macrophages (MØs) in stromal tissue. Herein, we report that alternatively activated (M2) MØs, as generated by interleukin (IL)-4, induce double-strand DNA breaks in target cells through BSE. Unlike M1 MØs that are activated by intestinal microbiota, BSE and DNA damage due to M2-like MØs occur through a distinct mechanism that differs from M1 MØs and does not involve pro-inflammatory mediators (e.g., cyclooxygenase-2 or tumor necrosis factor-α). Murine MØs (RAW264.7 cells) treated with IL-4 were polarized to an M2-like phenotype and found to strongly express arginase 1 (ARG1), a prototypical enzyme that converts L-arginine to L-ornithine and urea. L-Ornithine is a precursor in polyamine synthesis with H2O2 being a key oxidizing byproduct of polyamine catabolism. In a dual-chamber system using Young Adult Mouse Colon (YAMC) cells as targets for BSE generated by M2-like MØs, inhibition of ARG1 by N-hydroxy-nor-L-arginine abolished DNA damage (as measured by γH2AX). Direct treatment of YAMC cells with L-ornithine resulted in similar levels of DNA damage as M2-like MØs. YAMC cells exposed to M2-like MØs had increased intracellular H2O2. MDL-72527, an inhibitor polyamine oxidase and polyamine catabolism, reduced intracellular H2O2 to background levels and decreased DNA damage. YAMC cells sustaining DNA damage due to BSE from M2-like MØs showed specific phosphorylation of p53(ser15) and NF-κB(ser536) with no evidence for active caspase-3. These data along with an increased expression of cyclinD1 in YAMC cells indicated that DNA damage caused by M2-like MØs occurred via polyamine catabolism and was not due to cellular toxicity or apoptosis. Pathway analysis of RNAseq data for YAMC cells exposed to M2-like MØs identified multiple activated pathways including DNA repair, ubiquitination, Nrf2-mediated stress responses, endoplasmic reticulum overload responses, and lipid biosynthesis. These results provide additional evidence for global cellular responses to BSE generated by M2-like MØs. In conclusion, our findings show that M2-like MØs can generate BSE via a novel mechanism involving ARG1. Induction of BSE by M2-like MØs results in acute DNA damage in colon epithelial cells and activates multiple signaling pathways in target epithelial cells. These data imply a role for alternatively activated M2-like MØs in the malignant transformation of epithelial cells. Citation Format: Ram B. Undi, Hunter L. Porter, Jonathan D. Wren, Naushad Ali, Mark M. Huycke. Alternatively activated macrophages induce bystander effects via arginase 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 306.

Wnt signaling promotes IL‐33 expression in the colonic epithelium

BackgroundInterleukin‐33 (IL‐33) released from the colonic epithelium promotes type 2 innate lymphoid cell activity as part of the colonic injury response. We have previously shown that the intracellular signaling regulator, Sprouty2, restrains colonic IL‐33 via a GSK3β‐dependent mechanism. GSK3β can regulate canonical Wnt signaling, but the role of Wnt signaling and the specific components of this pathway involved in driving IL‐33 are unknown. We tested the hypothesis that activation of discrete arms of Wnt signaling induce epithelial IL‐33 expression.MethodsBulk RNA sequencing was performed on colons from mice with an intestinal epithelial‐specific Sprouty2 deletion (Spry2IEKO) or littermate controls to determine the effect of Sprouty2 on Wnt targets. Colonoid and young adult mouse colon (YAMC) epithelial cell cultures were treated with Wnt ligands. Small molecule inhibitors were used to inhibit CBP‐ or p300‐mediated Wnt signaling arms (ICG‐001 or IQ‐1 respectively), in YAMC cells. IL‐33 levels were assayed by qPCR and ELISA.ResultsSpry2IEKO mice have increased colonic transcript levels of the Wnt target genes Mmp7 (p<0.01), Irx3 (p<0.05), Mapk10 (p<0.05), and Jag1 (p<0.05), correlating with the increased expression of IL‐33 that we have previously reported in these mice. Pathway analysis suggests specifically altered CBP/p300‐mediated Wnt signaling in Spry2IEKO colons. Treatment of wild‐type colonoids or YAMC cells with the Wnt ligands Wnt3a and R‐spondin induced IL‐33 expression by 2.1‐fold and 2.3‐fold respectively (p<0.05). Pre‐treatment of cells with ICG‐001 blocked the Wnt‐induced increase in IL‐33 at both the RNA and protein level, whereas IQ‐1 had no effect.ConclusionCBP‐mediated Wnt signaling promotes colonic epithelial expression of IL‐33 in vitro. Elevated Wnt activity in Spry2IEKO mice is associated with elevated IL‐33 levels. Dysregulated Wnt signaling may result in impairment of the injury response in inflammatory disease.

Dietary Interventions Ameliorate Infectious Colitis by Restoring the Microbiome and Promoting Stem Cell Proliferation in Mice.

Decreases in short-chain-fatty-acids (SCFAs) are linked to inflammatory bowel disease (IBD). Yet, the mechanisms through which SCFAs promote wound healing, orchestrated by intestinal stem cells, are poorly understood. We discovered that, in mice with Citrobacter rodentium (CR)-induced infectious colitis, treatment with Pectin and Tributyrin diets reduced the severity of colitis by restoring Firmicutes and Bacteroidetes and by increasing mucus production. RNA-seq in young adult mouse colon (YAMC) cells identified higher expression of Lgr4, Lgr6, DCLK1, Muc2, and SIGGIR after Butyrate treatment. Lineage tracing in CR-infected Lgr5-EGFP-IRES-CreERT2/ROSA26-LacZ (Lgr5-R) mice also revealed an expansion of LacZ-labeled Lgr5(+) stem cells in the colons of both Pectin and Tributyrin-treated mice compared to control. Interestingly, gut microbiota was required for Pectin but not Tributyrin-induced Lgr5(+) stem cell expansion. YAMC cells treated with sodium butyrate exhibited increased Lgr5 promoter reporter activity due to direct Butyrate binding with Lgr5 at −4.0 Kcal/mol, leading to thermal stabilization. Upon ChIP-seq, H3K4me3 increased near Lgr5 transcription start site that contained the consensus binding motif for a transcriptional activator of Lgr5 (SPIB). Thus, a multitude of effects on gut microbiome, differential gene expression, and/or expansion of Lgr5(+) stem cells seem to underlie amelioration of colitis following dietary intervention.

Mechanism of M2 macrophage-derived extracellular vesicles carrying lncRNA MEG3 in inflammatory responses in ulcerative colitis

ABSTRACT Ulcerative colitis (UC) is a chronic inflammatory disease of the colon. M2 macrophages possess certain anti-inflammation activity. Accordingly, the current study set out to investigate the potential mechanism of M2 macrophage-derived extracellular vesicles (M2-EVs) in UC inflammation. Firstly, mouse peritoneal macrophages were induced to M2 phenotype, and M2-EVs were isolated. , the murine model of UC was established, and the length and weight of the colon, disease activity index (DAI), apoptosis, and inflammatory response of UC mice were measured. Young adult mouse colon (YAMC) cells were induced with the help of lipopolysaccharide. LncRNA maternally expressed 3 (LncRNA MEG3), miR-20b-5p, and cAMP responsive element binding protein 1 (CREB1) expression patterns were detected in UC models. In addition, we analyzed the binding relationship among MEG3, miR-20b-5p, and CREB1. UC mice presented with shortened colon length, lightened weight, increased DAI score, enhanced apoptosis, and significant inflammatory cell infiltration, while M2-EVs reversed these trends. In vitro, M2-EVs increased UC cell viability and reduced inflammation. Mechanistic experimentation revealed that M2-EVs transferred MEG3 into YAMC cells to up-regulate MEG3 expression and promote CREB1 transcription by competitively binding to miR-20b-5p. Moreover, up-regulation of MEG3 in M2-EVs enhanced the protective effect of M2-EVs on UC cells, while over-expression of miR-20b-5p attenuated the aforementioned protective effect of M2-EVs on UC mice and cells. Collectively, our findings revealed that M2-EVs carrying MEG3 enhanced UC cell viability and reduced inflammatory responses via the miR-20b-5p/CREB1 axis, thus alleviating UC inflammation.

Long non-coding RNA SNHG5 regulates ulcerative colitis via microRNA-375 / Janus kinase-2 axis

ABSTRACT Ulcerative colitis (UC) is an intestinal inflammatory disorder. Long non-coding RNAs (lncRNAs) are collectively involved in UC. This study is designed to explore the roles of lncRNA (small nucleolar RNA host gene 5) SNHG5 in UC. Gene or microRNA (miRNA) expression was detected using RT-qPCR and western blot, respectively. Cellular functions were analyzed by cell counting kit 8 (CCK8), 5-ethynyl-2′-deoxyuridine (EdU) assay, flow cytometry, and the terminal deoxyribonucleotidyl transferase (TDT)‐mediated dUTP‐digoxigenin nick end labeling (TUNEL) assays. Lactate dehydrogenase (LDH) content was determined by a cell cytotoxicity assay. The interactions between miR-375 and SNHG5 or Janus kinase-2 (JAK2) were verified by a luciferase reporter assay. SNHG5 was up-regulated in intestinal mucosa tissues of UC patients as well as tumor necrosis factor alpha-treated (TNF-α-treated) young adult mouse colon (YAMC) cells. Down-regulated SNHG5 promoted cell proliferation and inhibited apoptosis of YAMC cells. miR-375 was verified to be a target of SNHG5 and was suppressed by TNF-α treatment in YAMC cells. Over-expression of miR-375 restored YAMC cellular functions. Additionally, miR-375 targeted JAK2, which was up-regulated by TNF-α treated YAMC cells. Up-regulation of JAK2 induced the dysfunction of YAMC cells. Knockdown of SNHG5 promoted the proliferation and suppressed the apoptosis of YAMC cells via regulating miR-375/JAK2 axis. Therefore, knockdown of SNHG5 may be a promising therapy for UC.

16 CCL11 EXACERBATES COLITIS VIA MODULATION OF EPITHELIAL WOUND REPAIR

Abstract Background We have shown that CCL11 (eotaxin-1), an eosinophil chemoattractant, is significantly increased in the serum of ulcerative colitis and Crohn’s disease patients vs controls and is also increased in dextran sulfate sodium (DSS)-induced murine colitis. In response to azoxymethane (AOM)-DSS, Ccl11–/– mice have significantly decreased colonic tumor number and burden, histologic injury, and colonic eosinophil infiltration vs wild-type (WT) mice. Ccl11 is expressed by isolated colonic epithelial and lamina propria immune cells. Studies in bone marrow chimera mice revealed that both hematopoietic- and epithelial-cell derived CCL11 were important for protection in the AOM-DSS model. Our aim was to assess the role of CCL11 in an injury and recovery colitis model and in an epithelial wound restitution model. Methods C57BL/6 WT and Ccl11–/– mice were exposed to 4% DSS in the drinking water for 5 days followed by 5 days of water for an injury and recovery model. Body weights were assessed daily. Histologic injury was assessed by a validated scoring system including depth and percent involvement of inflammation and crypt damage. Colonic eosinophil and neutrophil infiltration was determined by immunohistochemistry for major basic protein (MBP) and myeloperoxidase (MPO), respectively. The number of MBP- and MPO-positive cells per high-powered field (HPF) were quantified in a blinded manner. Young Adult Mouse Colon (YAMC) epithelial cells were stained with antibodies to the CCL11 receptors CCR2, CCR3, and CCR5 for assessment by flow cytometry. YAMC cell monolayers were wounded and followed for 24 h in normal media or with the addition of 100ng/mL recombinant CCL11 (rCCL11). The wound area was measured at 0 and 24 h to determine percent restitution. RNA was isolated and assessed for Ccl11 mRNA expression. Results In the injury and recovery model, Ccl11–/– mice exhibited decreased body weight loss (p<0.05), histologic injury (20.6 ± 1.5 vs 12.4 ± 1.6, p<0.001), and fewer MBP-positive cells/HPF (23.5 ± 1.4 vs 2.4 ± 0.2, p<0.001) vs WT mice. While colonic MPO-positive cells were increased with DSS exposure, there was no difference in Ccl11–/– mice. Naïve/unstimulated YAMC cells expressed both CCR3 and CCR5. Wounding the YAMC monolayer alone did not lead to increased Ccl11 mRNA expression. However, in the presence of rCCL11, there was a decrease in wound restitution at 24 h (48.0% ± 1.7 vs 31.3% ± 2.1, p<0.001). Conclusions Loss of CCL11 leads to clinical and histologic improvement in an injury and recovery colitis model. Colonic epithelial cells express both CCR3 and CCR5, and epithelial wound restitution is decreased in the presence of rCCL11. These data suggest that CCL11 has a deleterious effect on epithelial restitution leading to an exacerbation of colitis. Therefore, treatment with anti-CCL11 antibodies may be a therapeutic strategy in IBD patients.

Role of intestinal Hsp70 in barrier maintenance: contribution of milk to the induction of Hsp70.2.

Necrotizing enterocolitis (NEC) is a gastrointestinal disease of complex etiology resulting in devastating systemic inflammation and often death in premature newborns. We previously demonstrated that formula feeding inhibits ileal expression of heat shock protein-70 (Hsp70), a critical stress protein within the intestine. Barrier function for the premature intestine is critical. We sought to determine whether reduced Hsp70 protein expression increases neonatal intestinal permeability. Young adult mouse colon cells (YAMC) were utilized to evaluate barrier function as well as intestine from Hsp70-/- pups (KO). Sections of intestine were analyzed by Western blot, immunohistochemistry, and real time PCR. YAMC cells were sub-lethally heated or treated with expressed milk (EM) to induce Hsp70. Immunostaining demonstrates co-localized Hsp70 and tight junction protein zona occludens-1 (ZO-1), suggesting physical interaction to protect tight junction function. The permeability of YAMC monolayers increases following oxidant injury and is partially blocked by Hsp70 induction either by prior heat stress or EM. RT-PCR analysis demonstrated that the Hsp70 isoforms, 70.1 and 70.3, predominate in WT pup; however, Hsp70.2 predominates in the KO pups. While Hsp70 is present in WT milk, it is not present in KO EM. Hsp70 associates with ZO-1 to maintain epithelial barrier function. Both induction of Hsp70 and exposure to EM prevent stress-induced increased permeability. Hsp70.2 is present in both WT and KO neonatal intestine, suggesting a crucial role in epithelial integrity. Induction of the Hsp70.2 isoform appears to be mediated by mother's milk. These results suggest that mother's milk feeding modulates Hsp70.2 expression and could attenuate injury leading to NEC. Level III.

Cathelicidin-encoding Lactococcus lactis promotes mucosal repair in murine experimental colitis.

The preventive effect of intrarectal administration of mouse cathelicidin (mCRAMP) and oral administration of mCRAMP-encoding Lactococcus lactis (N4I) has been shown in murine experimental colitis. It is pivotal to understand the ability of N4I whether it can promote mucosal repair in existing colitis. Mice with dextran sulfate sodium-induced ulcerative colitis (UC) were treated orally with L. lactis or its transformed strain with or without nisin induction. The body weight, clinical symptoms, and histological changes of colonic tissues were determined. Sulfasalazine was used as a reference drug. Young adult mouse colon cells were used to further elucidate the direct action and possible mechanisms of mCRAMP to promote colonic wound repair. Results showed that N4I could improve the clinical symptoms, maintain crypt integrity and preserve mucus-secreting layer in colitis animals. The preparation also could prevent cell death and promote cell proliferation. In contrast, effective dose of sulfasalazine only alleviated clinical symptoms but not the mucosal damage and repair in the colon. In vitro study further showed that mCRAMP could directly promote wound repair by accelerating cell migration but not cell proliferation through the GPCR/MAPK pathway. mCRAMP-encoding L. lactis could be a potential therapeutic preparation better than the traditional anti-inflammatory agent in the treatment of UC.

Constitutively active RAS signaling reduces 1,25 dihydroxyvitamin D-mediated gene transcription in intestinal epithelial cells by reducing vitamin D receptor expression

High vitamin D status is associated with reduced colon cancer risk but these studies ignore the diversity in the molecular etiology of colon cancer. RAS activating mutations are common in colon cancer and they activate pro-proliferative signaling pathways. We examined the impact of RAS activating mutations on 1,25 dihydroxyvitamin D (1,25(OH)2D)-mediated gene expression in cultured colon and intestinal cell lines. Transient transfection of Caco-2 cells with a constitutively active mutant K-RAS (G12 V) significantly reduced 1,25(OH)2D-induced activity of both a human 25-hydroxyvitamin D, 24 hydroxyase (CYP24A1) promoter-luciferase and an artificial 3X vitamin D response element (VDRE) promoter-luciferase reporter gene. Young Adult Mouse Colon (YAMC) and Rat Intestinal Epithelial (RIE) cell lines with stable expression of mutant H-RAS had suppressed 1,25(OH)2D-mediated induction of CYP24A1 mRNA. The RAS effects were associated with lower Vitamin D receptor (VDR) mRNA and protein levels in YAMC and RIE cells and they could be partially reversed by VDR overexpression. RAS-mediated suppression of VDR levels was not due to either reduced VDR mRNA stability or increased VDR gene methylation. However, chromatin accessibility to the VDR gene at the proximal promoter (−300bp), an enhancer region at −6kb, and an enhancer region located in exon 3 was significantly reduced in RAS transformed YAMC cells (YAMC-RAS). These data show that constitutively active RAS signaling suppresses 1,25(OH)2D-mediated gene transcription in colon epithelial cells by reducing VDR gene transcription but the mechanism for this suppression is not yet known. These data suggest that cancers with RAS-activating mutations may be less responsive to vitamin D mediated treatment or chemoprevention.

Inhibitor of Apoptosis Protein-1 Regulates Tumor Necrosis Factor–Mediated Destruction of Intestinal Epithelial Cells

Tumor necrosis factor (TNF) is a cytokine that promotes inflammation and contributes to pathogenesis of inflammatory bowel diseases. Unlike other cells and tissues, intestinal epithelial cells undergo rapid cell death upon exposure to TNF, by unclear mechanisms. We investigated the roles of inhibitor of apoptosis proteins (IAPs) in the regulation of TNF-induced cell death in the intestinal epithelium of mice and intestinal organoids. RNA from cell lines and tissues was analyzed by quantitative polymerase chain reaction, protein levels were analyzed by immunoblot assays. BIRC2 (also called cIAP1) was expressed upon induction from lentiviral vectors in young adult mouse colon (YAMC) cells. YAMC cells, the mouse colon carcinoma cell line MC38, the mouse macrophage cell line RAW 264.7, or mouse and human organoids were incubated with second mitochondrial activator of caspases (Smac)-mimetic compound LCL161 or recombinant TNF-like weak inducer of apoptosis (TNFSF12) along with TNF, and cell death was quantified. C57BL/6 mice with disruption of Xiap, Birc2 (encodes cIAP1), Birc3 (encodes cIAP2), Tnfrsf1a, or Tnfrsf1b (Tnfrsf1a and b encode TNF receptors) were injected with TNF or saline (control); liver and intestinal tissues were collected and analyzed for apoptosis induction by cleaved caspase 3 immunohistochemistry. We also measured levels of TNF and alanine aminotransferase in serum from mice. YAMC cells, and mouse and human intestinal organoids, died rapidly in response to TNF. YAMC and intestinal crypts expressed lower levels of XIAP, cIAP1, cIAP2, and cFLIP than liver tissue. Smac-mimetics reduced levels of cIAP1 and XIAP in MC38 and YAMC cells, and Smac-mimetics and TNF-related weak inducer of apoptosis increased TNF-induced cell death in YAMC cells and organoids-most likely by sequestering and degrading cIAP1. Injection of TNF greatly increased levels of cell death in intestinal tissue of cIAP1-null mice, compared with wild-type C57BL/6 mice, cIAP2-null mice, or XIAP-null mice. Excessive TNF-induced cell death in the intestinal epithelium was mediated TNF receptor1. In a study of mouse and human cell lines, organoids, and tissues, we found cIAP1 to be required for regulation of TNF-induced intestinal epithelial cell death and survival. These findings have important implications for the pathogenesis of TNF-mediated enteropathies and chronic inflammatory diseases of the intestine.

The L-Arginine Transporter Solute Carrier Family 7 Member 2 Mediates the Immunopathogenesis of Attaching and Effacing Bacteria.

Solute carrier family 7 member 2 (SLC7A2) is an inducible transporter of the semi-essential amino acid L-arginine (L-Arg), which has been implicated in immune responses to pathogens. We assessed the role of SLC7A2 in murine infection with Citrobacter rodentium, an attaching and effacing enteric pathogen that causes colitis. Induction of SLC7A2 was upregulated in colitis tissues, and localized predominantly to colonic epithelial cells. Compared to wild-type mice, Slc7a2 –/–mice infected with C. rodentium had improved survival and decreased weight loss, colon weight, and histologic injury; this was associated with decreased colonic macrophages, dendritic cells, granulocytes, and Th1 and Th17 cells. In infected Slc7a2 –/–mice, there were decreased levels of the proinflammatory cytokines G-CSF, TNF-α, IL-1α, IL-1β, and the chemokines CXCL1, CCL2, CCL3, CCL4, CXCL2, and CCL5. In bone marrow chimeras, the recipient genotype drove the colitis phenotype, indicative of the importance of epithelial, rather than myeloid SLC7A2. Mice lacking Slc7a2 exhibited reduced adherence of C. rodentium to the colonic epithelium and decreased expression of Talin-1, a focal adhesion protein involved in the attachment of the bacterium. The importance of SLC7A2 and Talin-1 in the intimate attachment of C. rodentium and induction of inflammatory response was confirmed in vitro, using conditionally-immortalized young adult mouse colon (YAMC) cells with shRNA knockdown of Slc7a2 or Tln1. Inhibition of L-Arg uptake with the competitive inhibitor, L-lysine (L-Lys), also prevented attachment of C. rodentium and chemokine expression. L-Lys and siRNA knockdown confirmed the role of L-Arg and SLC7A2 in human Caco-2 cells co-cultured with enteropathogenic Escherichia coli. Overexpression of SLC7A2 in human embryonic kidney cells increased bacterial adherence and chemokine expression. Taken together, our data indicate that C. rodentium enhances its own pathogenicity by inducing the expression of SLC7A2 to favor its attachment to the epithelium and thus create its ecological niche.

Sa1746 Cationic Amino Acid Transporter 2 and L-Arginine Availability Are Required for Talin-1-Dependent Adherence of the Colonic Pathogen Citrobacter Rodentium and Subsequent Pro-Inflammatory Responses

Background: L-arginine (L-Arg) uptake in cells is mediated by cationic amino acid transporter (CAT) proteins, and CAT2 is the inducible form. We have found that CAT2-/mice exhibit less colitis in the IBD model induced by infection with Citrobacter rodentium, a rodent pathogen similar to EPEC that acts by forming attaching and effacing lesions on epithelial cells. The translocated intimin receptor is injected into host cells by C. rodentium and forms a complex with talin-1 that mediates bacterial binding and downstream signaling. Our aim was to investigate the role of talin-1 in C. rodentium infection. Methods: Male C57BL/6 wild-type (WT) or CAT2-/mice were infected with C. rodentium for 14 days. Talin-1 and the C. rodentium protein espB were assesed by confocal microscopy and Western blotting. mRNA levels of talin-1 and the proinflammatory CXC chemokines, KC and MIP-2, were determined by qPCR. Young adult mouse colon (YAMC) cells were transduced with control or talin-1 shRNA, and activated with C. rodentium at an MOI of 200. Bacterial adherence was assessed by culture after saponin treatment of cells. Levels of the NF-κB subunit p65 were assessed by immunofluorescence at 30 min, and of talin-1, KC and MIP-2 by qPCR at 4 h. Talin-1 mRNA stability was assessed in YAMC cells using the transcriptional inhibitor actinomycin D followed by serial assessment of mRNA levels. Results: C. rodentium infection in vivo induced a 4-fold increase in talin-1 mRNA and protein expression in whole tissues and a 236-fold increase in mRNA levels in colonic epithelial cells (CECs). Talin-1 and espB protein levels were markedly decreased in colon tissues from infected CAT2-/vs. WT mice; this was confirmed by confocal microscopy and there was also less colocalization of these proteins. Talin-1, KC and MIP-2 mRNA levels were each decreased by >92% in CAT2-/vs. WT mice (p<0.01). L-lysine, a competitive inhibitor of L-Arg uptake, reduced induction of talin-1, KC, and MIP-2 mRNA expression by 60.4 ± 18.3%, 91.7 ± 2.6%, and 68.9 ± 12.2%, respectively, in YAMC cells (p<0.01). In talin-1 knockdown cells, levels of adherent C. rodentium decreased from 4.9 ± 0.9 to 0.8 ± 0.2 bacteria/cell (p<0.05), and there was less nuclear translocation of p65. Similarly, KC and MIP-2 levels were decreased by 90.6 ± 0.3% and 92.5 ± 3.1%, respectively, in knockdown cells (p<0.01). There was a 4.2 ± 0.5-fold increase in talin-1 mRNA levels when cells starved of L-Arg were repleted with L-Arg for 4 h. Talin-1 mRNA was less stable in the absence of L-Arg; the mRNA half-life was 5.2fold (p<0.01) greater in cells repleted with L-Arg. Conclusions: Talin-1 is essential for C. rodentium adherence to colonic epithelial cells and induction of host inflammatory responses. L-Arg availability is needed for talin-1 mRNA stability, and thus mediates the immunopathogenesis of C. rodentium infection.

A Lactobacillus rhamnosus GG-derived Soluble Protein, p40, Stimulates Ligand Release from Intestinal Epithelial Cells to Transactivate Epidermal Growth Factor Receptor

p40, a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, ameliorates intestinal injury and colitis, reduces apoptosis, and preserves barrier function by transactivation of the EGF receptor (EGFR) in intestinal epithelial cells. The aim of this study is to determine the mechanisms by which p40 transactivates the EGFR in intestinal epithelial cells. Here we show that p40-conditioned medium activates EGFR in young adult mouse colon epithelial cells and human colonic epithelial cell line, T84 cells. p40 up-regulates a disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) catalytic activity, and broad spectrum metalloproteinase inhibitors block EGFR transactivation by p40 in these two cell lines. In ADAM17-deficient mouse colonic epithelial (ADAM17(-/-) MCE) cells, p40 transactivation of EGFR is blocked, but can be rescued by re-expression with WT ADAM17. Furthermore, p40 stimulates release of heparin binding (HB)-EGF, but not transforming growth factor (TGF)α or amphiregulin, in young adult mouse colon cells and ADAM17(-/-) MCE cells overexpressing WT ADAM17. Knockdown of HB-EGF expression by siRNA suppresses p40 effects on transactivating EGFR and Akt, preventing apoptosis, and preserving tight junction function. The effects of p40 on HB-EGF release and ADAM17 activation in vivo are examined after administration of p40-containing pectin/zein hydrogel beads to mice. p40 stimulates ADAM17 activity and EGFR activation in colonic epithelial cells and increases HB-EGF levels in blood from WT mice, but not from mice with intestinal epithelial cell-specific ADAM17 deletion. Thus, these data define a mechanism of a probiotic-derived soluble protein in modulating intestinal epithelial cell homeostasis through ADAM17-mediated HB-EGF release, leading to transactivation of EGFR.

Chapter Five - Selenoprotein W as Biomarker for the Efficacy of Selenium Compounds to Act as Source for Selenoprotein Biosynthesis

Selenium is an essential trace element and, like all elements, present in many different compounds with unequivocal functions. This fact is only sporadically mentioned when recommended intake or supplementation is indicated just as "selenium." In mammals, selenium is an integral part of selenoproteins as selenocysteine. Selenocysteine is formed from serine at the respective tRNA((ser)sec), a reaction that requires selenophosphate formed from selenide and ATP. Thus, only compounds that can be metabolized into selenide can serve as sources for selenoprotein biosynthesis. We therefore tested the ability of selenium compounds such as sodium selenite, methylseleninic acid (MeSeA), Se-methyl selenocysteine, and selenomethionine to increase the activity, protein, or mRNA levels of commonly used biomarkers of the selenium status, glutathione peroxidase-1 (GPx1) and thioredoxin reductase, and of putatively new biomarkers, selenoprotein W1 (SepW1), selenoprotein H, and selenoprotein 15 in three different cell lines. Selenite and MeSeA were most efficient in increasing all markers tested, whereas the other compounds had only marginal effects. Effects were higher in the noncancerous young adult mouse colon cells than in the cancer cell lines HepG2 and HT-29. At the protein level, SepW1 responded as well as GPx1 and at the mRNA level, even better. Thus, the outcome of selenium treatment strongly depends on the chemical form, the cell type, and the biomarker used for testing efficacy.

L-arginine uptake by cationic amino acid transporter 2 is essential for colonic epithelial cell restitution

Restoration of the colonic epithelial barrier is an important response during colitis. L-arginine (L-Arg) is a semiessential amino acid that reduces murine colitis induced by Citrobacter rodentium. Cationic amino acid transporter (CAT) proteins increase L-Arg uptake into cells. L-Arg is utilized to produce nitric oxide (NO), by inducible NO synthase (iNOS), or L-ornithine (L-Orn) by arginase (Arg) enzymes. The latter is followed by generation of polyamines by ornithine decarboxylase (ODC) and L-proline (L-Pro) by ornithine aminotransferase (OAT). We show that L-Arg enhanced epithelial restitution in conditionally immortalized young adult mouse colon (YAMC) cells in a wound repair model, and in isolated mouse colonic epithelial cells (CECs), using a cell migration assay. Restitution was impaired by C. rodentium. Wounding induced CAT2, and inhibition of L-Arg uptake by the competitive inhibitor L-lysine (L-Lys) or by CAT2 shRNA, but not CAT1 shRNA, decreased restitution. Migration was impaired in CECs treated with L-Lys or from CAT2(-/-) mice. Wounding increased Arg1 expression, and inhibition of arginase with S-(2-boronoethyl)-L-cysteine (BEC) or Arg1 shRNA inhibited restitution in YAMC cells; cell migration in CECs was also impaired by BEC. Inhibition of ODC or iNOS did not alter restitution. L-Orn or L-Pro restored restitution in cells treated with BEC or Arg1 shRNA, whereas the polyamine putrescine had no benefit. Wounding increased OAT levels, OAT shRNA inhibited restitution, and L-Pro restored restitution in cells with OAT knockdown. Uptake of L-Arg, and its metabolism by Arg1 to L-Orn and conversion to L-Pro by OAT is essential for colonic epithelial wound repair.

Berberine induces caspase-independent cell death in colon tumor cells through activation of apoptosis-inducing factor

AbstractBerberine, an isoquinoline alkaloid derived from plants, is a traditional medicine for treating bacterial diarrhea and intestinal parasite infections. Although berberine has recently been shown to suppress growth of several tumor cell lines, information regarding the effect of berberine on colon tumor growth is limited. Here, we investigated the mechanisms underlying the effects of berberine on regulating the fate of colon tumor cells, specifically the immorto Min mouse colonic epithelial (IMCE) cells carrying the Apcmin mutation, and of normal colon epithelial cells, namely young adult mouse colon (YAMC) epithelial cells. Berberine decreased colon tumor colony formation in agar, and induced cell death and LDH release in a time- and concentration-dependent manner in IMCE cells. In contrast, YAMC cells were not sensitive to berberine-induced cell death. Berberine did not stimulate caspase activation, and PARP cleavage and berberine-induced cell death were not affected by a caspase inhibitor in IMCE cells. Rather, berberine stimulated a caspase-independent cell death mediator, apoptosis-inducing factor (AIF) release from mitochondria and nuclear translocation in a ROS production-dependent manner. Amelioration of berberine-stimulated ROS production or suppression of AIF expression blocked berberine-induced cell death and LDH release in IMCE cells. Furthermore, two targets of ROS production in cells, cathepsin B release from lysosomes and PARP activation were induced by berberine. Blockage of either of these pathways decreased berberine-induced AIF activation and cell death in IMCE cells. Thus, berberine-stimulated ROS production leads to cathepsin B release and PARP activation-dependent AIF activation, resulting in caspase-independent cell death in colon tumor cells. Notably, normal colon epithelial cells are less susceptible to berberine-induced cell death, which suggests the specific inhibitory effects of berberine on colon tumor cell growth.

Disruption of the Mouse Protein Tyrosine Kinase 6 Gene Prevents STAT3 Activation and Confers Resistance to Azoxymethane

Protein tyrosine kinase 6 (PTK6) is expressed throughout the gastrointestinal tract and is a negative regulator of proliferation that promotes differentiation and DNA-damage-induced apoptosis in the small intestine. PTK6 is not expressed in normal mammary gland, but is induced in most human breast tumors. Signal transducer and activator of transcription 3 (STAT3) mediates pathogenesis of colon cancer and is a substrate of PTK6. We investigated the role of PTK6 in colon tumorigenesis. Ptk6+/+ and Ptk6-/- mice were injected with azoxymethane alone or in combination with dextran sodium sulfate; formation of aberrant crypt foci and colon tumors was examined. Effects of disruption of Ptk6 on proliferation, apoptosis, and STAT3 activation were examined by immunoblot and immunohistochemical analyses. Regulation of STAT3 activation was examined in the HCT116 colon cancer cell line and young adult mouse colon cells. Ptk6-/- mice developed fewer azoxymethane-induced aberrant crypt foci and tumors. Induction of PTK6 increased apoptosis, proliferation, and STAT3 activation in Ptk6+/+ mice injected with azoxymethane. Disruption of Ptk6 impaired STAT3 activation following azoxymethane injection, and reduced active STAT3 levels in Ptk6-/- tumors. Stable knockdown of PTK6 reduced basal levels of active STAT3, as well as activation of STAT3 by epidermal growth factor in HCT116 cells. Disruption of Ptk6 reduced activity of STAT3 in young adult mouse colon cells. PTK6 promotes STAT3 activation in the colon following injection of the carcinogen azoxymethane and regulates STAT3 activity in mouse colon tumors and in the HCT116 and young adult mouse colon cell lines. Disruption of Ptk6 decreases azoxymethane-induced colon tumorigenesis in mice.

TNF transactivation of EGFR stimulates cytoprotective COX-2 expression in gastrointestinal epithelial cells.

TNF and epidermal growth factor (EGF) are well-known stimuli of cyclooxygenase (COX)-2 expression, and TNF stimulates transactivation of EGF receptor (EGFR) signaling to promote survival in colon epithelial cells. We hypothesized that COX-2 induction and cell survival signaling downstream of TNF are mediated by EGFR transactivation. TNF treatment was more cytotoxic to COX-2(-/-) mouse colon epithelial (MCE) cells than wild-type (WT) young adult mouse colon (YAMC) epithelial cells or COX-1(-/-) cells. TNF also induced COX-2 protein and mRNA expression in YAMC cells, but blockade of EGFR kinase activity or expression inhibited COX-2 upregulation. TNF-induced COX-2 expression was reduced and absent in EGFR(-/-) and TNF receptor-1 (TNFR1) knockout MCE cells, respectively, but was restored upon expression of the WT receptors. Inhibition of mediators of EGFR transactivation, Src family kinases and p38 MAPK, blocked TNF-induced COX-2 protein and mRNA expression. Finally, TNF injection increased COX-2 expression in colon epithelium of WT, but not kinase-defective EGFR(wa2) and EGFR(wa5), mice. These data indicate that TNFR1-dependent transactivation of EGFR through a p38- and/or an Src-dependent mechanism stimulates COX-2 expression to promote cell survival. This highlights an EGFR-dependent cell signaling pathway and response that may be significant in colitis-associated carcinoma.

Novel mechanism for obesity-induced colon cancer progression

Adipose tissue secretes factors linked to colon cancer risk including leptin. A hallmark of cancer is sustained angiogenesis. While leptin promotes angiogenesis in adipose tissue, it is unknown whether leptin can induce epithelial cells to produce factors that may drive angiogenesis, vascular development and therefore cancer progression. The purpose of this study was to compare the effects of leptin-stimulated colon epithelial cells differing in adenomatous polyposis coli (Apc) genotype (gatekeeper tumor suppressor gene for colon cancer) on angiogenesis. We employed novel colonic epithelial cell lines derived from the Immorto mouse [young adult mouse colon (YAMC)] and the Immorto-Min mouse [Immorto-Min colonic epithelial cell (IMCE)], which carries the Apc Min mutation, to study the effects of leptin-stimulated colon epithelial cells on angiogenesis. We utilized ex vivo rat mesenteric capillary bioassay and human umbilical vein endothelial cell (HUVEC) models to study angiogenesis. IMCE cells stimulated with leptin produced significantly more vascular endothelial growth factor (VEGF) than YAMC (268 +/- 18 versus 124 +/- 8 pg/ml; P < 0.01) cells. Leptin treatment induced dose-dependent increases in VEGF only in IMCE cells. Conditioned media from leptin (50 ng/ml)-treated IMCE cells induced significant capillary formation compared with control, which was blocked by the addition of a neutralizing antibody against VEGF. Conditioned media from leptin-treated IMCE cells also induced HUVEC cell proliferation, chemotaxis, upregulation of adhesion proteins and cell-signaling activation resulting in nuclear factor kappa B nuclear translocation and DNA binding due to VEGF. This is the first study demonstrating that leptin can induce preneoplastic colon epithelial cells to orchestrate VEGF-driven angiogenesis and vascular development, thus providing a specific mechanism and potential target for obesity-associated cancer.

The ErbB4 Growth Factor Receptor Is Required for Colon Epithelial Cell Survival in the Presence of TNF

The ErbB4 receptor tyrosine kinase regulates cell growth, survival, and differentiation in several tissues, but its role in the gastrointestinal tract has not been reported. We tested the hypothesis that ErbB4 promotes intestinal cell survival and restitution following injury or inflammation. ErbB4 expression in human inflammatory bowel disease was determined by immunohistochemistry. Mice were subjected to dextran sulfate sodium (DSS, 3%) colitis or injected with tumor necrosis factor (TNF), and ErbB4 expression was quantified by immunohistochemistry and Western blot. Cultured young adult mouse colon (YAMC) cells were exposed to TNF, and ErbB4 messenger RNA, protein, and phosphorylation levels were measured. Cells transfected with ErbB4 small interfering RNA (siRNA), or over expressing ErbB4, were subjected to wound healing and apoptosis assays. ErbB4 levels increased in Crohn's colitis and the colon epithelium of mice with DSS colitis or injected with TNF. In YAMC cells, TNF induced ErbB4 messenger RNA, protein, and phosphorylation; nuclear factor kappaB activation also stimulated ErbB4 accumulation. ErbB4 siRNA sensitized cells to TNF-stimulated apoptosis, while over expression blocked apoptosis induced by TNF plus cycloheximide. Additionally, ErbB4 siRNA decreased YAMC cell wound healing. ErbB4 knockdown attenuated, while over expression elevated, phosphorylation of Akt in response to TNF. Inhibition of the phosphatidylinositol 3-kinase/Akt signaling cascade reversed the ability of ErbB4 over expression to protect from cytokine-induced apoptosis. ErbB4 expression and signaling are key elements for TNF responses in vivo and in cell culture, protecting intestinal epithelial cells from apoptosis in the inflammatory environment, possibly through Akt activation.