Young Adult Mouse Colonic Cells Research Articles

Exposure to p40 in Early Life Prevents Intestinal Inflammation in Adulthood Through Inducing a Long-Lasting Epigenetic Imprint on TGFβ.

Background & AimsColonization by gut microbiota in early life confers beneficial effects on immunity throughout the host’s lifespan. We sought to elucidate the mechanisms whereby neonatal supplementation with p40, a probiotic functional factor, reprograms intestinal epithelial cells for protection against adult-onset intestinal inflammation.Methodsp40 was used to treat young adult mouse colonic (YAMC) epithelial cells with and without deletion of a methyltransferase, su(var)3-9, enhancer-of-zeste and trithorax domain–containing 1β (Setd1β), and mice in early life or in adulthood. Anti–transforming growth factor β (TGFβ)-neutralizing antibodies were administered to adult mice with and without colitis induced by 2,4,6-trinitrobenzenesulfonic acid or dextran sulfate sodium. We examined Setd1b and Tgfb gene expression, TGFβ production, monomethylation and trimethylation of histone H3 on the lysine 4 residue (H3K4me1/3), H3K4me3 enrichment in Tgfb promoter, differentiation of regulatory T cells (Tregs), and the inflammatory status.Resultsp40 up-regulated expression of Setd1b in YAMC cells. Accordingly, p40 enhanced H3K4me1/3 in YAMC cells in a Setd1β-dependent manner. p40-regulated Setd1β mediated programming the TGFβ locus into a transcriptionally permissive chromatin state and promoting TGFβ production in YAMC. Furthermore, transient exposure to p40 during the neonatal period and in adulthood resulted in the immediate increase in Tgfb gene expression. However, only neonatal p40 supplementation induced the sustained H3K4me1/3 and Tgfb gene expression that persisted into adulthood. Interfering with TGFβ function by neutralizing antibodies diminished the long-lasting effects of neonatal p40 supplementation on differentiation of Tregs and protection against colitis in adult mice.ConclusionsExposure to p40 in early life enables an epigenetic imprint on TGFβ, leading to long-lasting production of TGFβ by intestinal epithelial cells to expand Tregs and protect the gut against inflammation.

Toxoplasma and Eimeria co-opt the host cFos expression for intracellular development in mammalian cells.

Successful asexual reproduction of intracellular pathogens depends on their potential to exploit host resources and subvert antimicrobial defense. In this work, we deployed two prevalent apicomplexan parasites of mammalian cells, namely Toxoplasma gondii and Eimeria falciformis, to identify potential host determinants of infection. Expression analyses of the young adult mouse colonic (YAMC) epithelial cells upon infection by either parasite showed regulation of several distinct transcripts, indicating that these two pathogens program their intracellular niches in a tailored manner. Conversely, parasitized mouse embryonic fibroblasts (MEFs) displayed a divergent transcriptome compared to corresponding YAMC epithelial cells, suggesting that individual host cells mount a fairly discrete response when encountering a particular pathogen. Among several host transcripts similarly altered by T. gondii and E. falciformis, we identified cFos, a master transcription factor, that was consistently induced throughout the infection. Indeed, asexual growth of both parasites was strongly impaired in MEF host cells lacking cFos expression. Last but not the least, our differential transcriptomics of the infected MEFs (parental and cFos-/- mutant) and YAMC epithelial cells disclosed a cFos-centered network, underlying signal cascades, as well as a repertoire of nucleotides- and ion-binding proteins, which presumably act in consort to acclimatize the mammalian cell and thereby facilitate the parasite development.

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.

Vitamin D Regulation of the Uridine Phosphorylase 1 Gene and Uridine-Induced DNA Damage in Colon in African Americans and European Americans

African Americans have the greatest colorectal cancer (CRC) burden in the United States; interethnic differences in protective effects of vitamin D might contribute to disparities. 1α,25(OH)2D3 vitamin D (the active form of vitamin D) induces transcription of the uridine phosphorylase gene (UPP1) in colon tissues of European Americans but to a lesser extent in colon tissues of African Americans. UPP1-knockout mice have increased intestinal concentrations of uridine and Deoxyuridine triphosphate (dUTP), have increased uridine-induced DNA damage, and develop colon tumors. We studied 1α,25(OH)2D3 regulation of UPP1 and uridine-induced DNA damage in the colon and differences in these processes between African and European Americans. We quantified expression and activity of UPP1 in response to 1α,25(OH)2D3 in young adult mouse colonic cells, human CRC cells (LS174T), and organoids (derived from rectosigmoid biopsy samples of healthy individuals undergoing colonoscopies) using quantitative polymerase chain reaction, immunoblot, and immunocytochemistry assays. Binding of the vitamin D receptor to UPP1 was tested by chromatin immunoprecipitation. Uridine-induced DNA damage was measured by fragment-length analysis in repair enzyme assays. Allele-specific 1α,25(OH)2D3 responses were tested using luciferase assays. Vitamin D increased levels of UPP1 mRNA, protein, and enzymatic activity and increased vitamin D receptor binding to the UPP1 promoter in young adult mouse colonic cells, LS174T cells, and organoids. 1α,25(OH)2D3 significantly reduced levels of uridine and uridine-induced DNA damage in these cells, which required UPP1 expression. Organoids derived from colon tissues of African Americans expressed lower levels of UPP1 after exposure to 1α,25(OH)2D3 and had increased uridine-induced DNA damage compared with organoids derived from tissues of European Americans. Luciferase assays with the T allele of single nucleotide polymorphism rs28605337 near UPP1, which is found more frequently in African Americans than European Americans, expressed lower levels of UPP1 after exposure to 1α,25(OH)2D3 than assays without this variant. We found vitamin D to increase expression of UPP1, leading to reduce uridine-induced DNA damage, in colon cells and organoids. A polymorphism in UPP1 found more frequently in African Americans than European Americans reduced UPP1 expression upon cell exposure to 1α,25(OH)2D3. Differences in expression of UPP1 in response to vitamin D could contribute to the increased risk of CRC in African Americans.

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.

Editor's Highlight: Microbial-Derived 1,4-Dihydroxy-2-naphthoic Acid and Related Compounds as Aryl Hydrocarbon Receptor Agonists/Antagonists: Structure-Activity Relationships and Receptor Modeling.

1,4-Dihydroxy-2-naphthoic acid (1,4-DHNA) is a bacterial-derived metabolite that binds the aryl hydrocarbon receptor (AhR) and exhibits anti-inflammatory activity in the gut. The structure-dependent AhR activity of hydroxyl/carboxy-substituted naphthoic acids (NAs) was determined in young adult mouse colonic (YAMC) cells and human Caco2 colon cancer cells using CYP1A1/CYP1B1 mRNAs as Ah-responsive genes. Compounds used in this study include 1,4-, 3,5-, and 3,7-DHNA, 1,4-dimethoxy-2-naphthoic acid (1,4-DMNA), 1- and 4-hydroxy-2-naphthoic acid (1-HNA, 4-HNA), 1- and 2-naphthoic acid (1-NA, 2-NA), and 1- and 2-naphthol (1-NOH, 2-NOH). 1,4-DHNA was the most potent compound among hydroxyl/carboxy naphthalene derivatives, and the fold induction response for CYP1A1 and CYP1B1 was similar to that observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in YAMC and Caco2 cells. 1- and 4-HNA were less potent than 1,4-DHNA but induced maximal (TCDD-like) response for CYP1B1 (both cell lines) and CYP1A1 (Caco2 cells). With the exception of 1- and 2-NA, all compounds significantly induced Cyp1b1 in YAMC cells and these responses were not observed in AhR-deficient YAMC cells generated using CRISPR/Cas9 technology. In addition, we also observed that 1- and 2-NOH (and 1,4-DHNA) were weak AhR agonists, and 1- and 2-NOH also exhibited partial AhR antagonist activity. Structure-activity relationship studies for CYP1A1 but not CYP1B1 were similar in both cell lines, and CYP1A1 induction required one or both 1,4-dihydroxy substituents and activity was significantly enhanced by the 2-carboxyl group. We also used computational analysis to show that 1,4-DHNA and TCDD share similar interactions within the AhR binding pocket and differ primarily due to the negatively charged group of 1,4-DHNA.

Clostridium difficile Toxins TcdA and TcdB Cause Colonic Tissue Damage by Distinct Mechanisms.

As the major cause of antibiotic-associated diarrhea, Clostridium difficile is a serious problem in health care facilities worldwide. C. difficile produces two large toxins, TcdA and TcdB, which are the primary virulence factors in disease. The respective functions of these toxins have been difficult to discern, in part because the cytotoxicity profiles for these toxins differ with concentration and cell type. The goal of this study was to develop a cell culture model that would allow a side-by-side mechanistic comparison of the toxins. Conditionally immortalized, young adult mouse colonic (YAMC) epithelial cells demonstrate an exquisite sensitivity to both toxins with phenotypes that agree with observations in tissue explants. TcdA intoxication results in an apoptotic cell death that is dependent on the glucosyltransferase activity of the toxin. In contrast, TcdB has a bimodal mechanism; it induces apoptosis in a glucosyltransferase-dependent manner at lower concentrations and glucosyltransferase-independent necrotic death at higher concentrations. The direct comparison of the responses to TcdA and TcdB in cells and colonic explants provides the opportunity to unify a large body of observations made by many independent investigators.

TNFα/IFNγ Mediated Intestinal Epithelial Barrier Dysfunction Is Attenuated by MicroRNA-93 Downregulation of PTK6 in Mouse Colonic Epithelial Cells.

Since inflammatory bowel diseases (IBD) represent significant morbidity and mortality in the US, the need for defining novel drug targets and inflammatory mechanisms would be of considerable benefit. Although protein tyrosine kinase 6 (PTK6, also known as breast tumor kinase BRK) has been primarily studied in an oncogenic context, it was noted that PTK6 null mice exhibited significantly enhanced colonic epithelial barrier function. Considering that the inflammatory functions of PTK6 have not yet been explored, we hypothesized that cytokines responsible for mediating IBD, such as TNFα/IFNγ, may solicit the action of PTK6 to alter barrier function. After first assessing critical mediators of TNFα/IFNγ driven epithelial barrier dysfunction, we further explored the possibility of PTK6 in this inflammatory context. In this report, we showed that PTK6 siRNA and PTK6 null young adult mouse colonic epithelial cells (YAMC) exhibited significant attenuation of TNFα/IFNγ induced barrier dysfunction as measured by electric cell-substrate impedance sensing (ECIS) assay and permeability assays. In addition, PTK6 null cells transfected with PTK6 cDNA displayed restored barrier dysfunction in response to TNFα/IFNγ, while the cells transfected with vector alone showed similar attenuation of barrier dysfunction. Furthermore, using subcellular fractionation and immunocytochemistry experiments, we found that PTK6 plays a role in FoxO1 nuclear accumulation leading to down-regulation of claudin-3, a tight junction protein. Moreover, we searched for relevant miRNA candidates putative for targeting PTK6 in order to identify and assess the impact of microRNA that target PTK6 with respect to TNFα/IFNγ induced barrier dysfunction. Subsequently, we assayed likely targets and determined their effectiveness in attenuating PTK6 expression as well as cytokine induced barrier dysfunction. Results showed that miR-93 reduced PTK6 expression and attenuated TNFα/IFNγ imposed decrease in transepithelial electrical resistance (TER), as well as excluded FoxO1 from the nucleus. Our results indicate that PTK6 may act as a novel mediator of intestinal epithelial permeability during inflammatory injury, and miR-93 may protect intestinal epithelial barrier function, at least in part, by targeting PTK6.

Glutamine‐mediated Dual Regulation of Heat Shock Transcription Factor‐1 Activation and Expression

Heat shock factor‐1 (HSF1) is the master regulator for cytoprotective heat shock protein (Hsp) expression. It is widely thought that HSF1 expression is non‐inducible and thus the key control point of HSP expression is regulation of HSF1's transactivation activity. How HSF1 expression is regulated remains unknown. Herein, we demonstrate that glutamine (Gln) enhanced Hsp expression both in rat colonic epithelium in vivo and in cultured non‐transformed colonic YAMC cells. This was associated with upregulated HSF1's transactivation activity via increased HSF1 trimerization, nuclear localization and DNA binding. Intriguingly, Gln enhanced HSF1 protein and mRNA expression and Hsf1 gene promoter activity. Within the −281/−200 region of the Hsf1 promoter, deletion of the putative CCAAT enhancer binding protein (C/EBP) binding site abolished Gln's HSF1 response. Gln availability strikingly altered the ratio of C/EBPβ inhibitory and active isoforms, i.e., liver‐enriched inhibitory protein (LIP) and liver‐enriched activating protein (LAP). LIP and LAP were further shown to be independent repressor and activator respectively for Hsf1 gene transcription and the relative abundance of these two C/EBPβ isoforms was demonstrated to determine Hsf1 transcription. We show for the first time that Gln not only enhances HSF1's transactivation, but also induces Hsf1 gene transcription in a C/EBPβ‐dependent manner.

Glutamine-mediated Dual Regulation of Heat Shock Transcription Factor-1 Activation and Expression

Regulation of transcriptional activity of heat shock factor-1 (HSF1) is widely thought to be the main point of control for heat shock protein (Hsp) expression. Glutamine increases Hsf1 gene transcription in a C/EBPβ-dependent manner and up-regulates HSF1 activity. Glutamine is an activator for both HSF1 expression and transactivation. Glutamine-induced HSF1 expression provides a novel mechanistic frame for HSF1-Hsp axis regulation. Heat shock transcription factor-1 (HSF1) is the master regulator for cytoprotective heat shock protein (Hsp) expression. It is widely thought that HSF1 expression is non-inducible, and thus the key control point of Hsp expression is regulation of the transactivation activity of HSF1. How HSF1 expression is regulated remains unknown. Herein we demonstrate that glutamine (Gln), a preferred fuel substrate for the gut, enhanced Hsp expression both in rat colonic epithelium in vivo and in cultured non-transformed young adult mouse colonic epithelial cells. This was associated with up-regulation of the transactivation activity of HSF1 via increased HSF1 trimerization, nuclear localization, DNA binding, and relative abundance of activating phosphorylation at Ser-230 of HSF1. More intriguingly, Gln enhanced HSF1 protein and mRNA expression and Hsf1 gene promoter activity. Within the -281/-200 region of the Hsf1 promoter, deletion of the putative CCAAT enhancer-binding protein (C/EBP) binding site abolished the HSF1 response to Gln. C/EBPβ was further shown to bind to this 82-bp sequence both in vitro and in vivo. Gln availability strikingly altered the ratio of C/EBPβ inhibitory and active isoforms, i.e. liver-enriched inhibitory protein and liver-enriched activating protein. Liver-enriched inhibitory protein and liver-enriched activating protein were further shown to be an independent repressor and activator, respectively, for Hsf1 gene transcription, and the relative abundance of these two C/EBPβ isoforms was demonstrated to determine Hsf1 transcription. We show for the first time that Gln not only enhances the transactivation of HSF1 but also induces Hsf1 expression by activating its transcription in a C/EBPβ-dependent manner.

The Australian fruit Illawarra plum (Podocarpus elatusEndl., Podocarpaceae) inhibits telomerase, increases histone deacetylase activity and decreases proliferation of colon cancer cells

Fruit antioxidants have many health benefits including prevention of cancer development. The native Australian bush fruit Illawarra plum (Podocarpus elatus Endl., Podocarpaceae) has a high content of anthocyanin-rich phenolics, with an antioxidant capacity at levels higher than most fruits. In the present study the molecular mechanisms of the anti-proliferative activity of Illawarra plum on colorectal cancer cells were investigated. Non-tumorigenic young adult mouse colonic (YAMC) cells and tumorigenic human colonic (HT-29) cells were treated with a polyphenolic-rich Illawarra plum extract (0-1000 microg/ml). Illawarra plum had anti-proliferative properties in only the cancer cells, with growth suppressed in a dose- and time-dependent manner. Treatment of HT-29 cells with Illawarra plum extract (500 mg/ml; 24 h) was also associated with a 2-fold increase in apoptosis, and a cell cycle delay in the S phase (P < 0.01). Assessment of biomarkers for DNA damage revealed that plum treatment caused a 93% down-regulation of telomerase activity (P < 0.001) and a decrease in telomere length (up to 75%; P < 0.01). Treatment with Illawarra plum extract also induced morphological alterations to HT-29 cells that were suggestive of induction of autophagy, as the formation of cytoplasmic vacuoles was observed in many cells. This could be induced by the increased (6-fold) histone deacetylase (HDAC) activity (P < 0.001) and the trend for increased expression of the class III HDAC sirtuin 1. The present study has shown that Illawarra plum extract is able to reduce the proliferation of colon cancer cells by altering the cell cycle, increasing apoptosis and possibly inducing autophagy. The active ingredients in Illawarra plum may provide an alternative chemoprevention strategy to conventional chemotherapy.

Tu1892 Decreased SHIP1 Activity in Crohn's Disease

Background: While the precise pathogenesis of ulcerative colitis (UC) remains unknown, the increased oxidative stress and the depressed antioxidant defense system have been involved in the pathogenesis of UC. In previous study, we identified the decreased expression of peroxiredoxin-6 (Prx-6) in murine colonic inflamed mucosa using differential proteomic analysis (J Gastroenterol Hepatol 2010). Although Prx-6 is involved in cell defense against oxidative stress and in redox regulation of intracellular signaling, the functional role of Prx6 in colonic mucosa remains to be determined. In this study, we investigated the role of Prx-6 in inflamed colonic mucosa, and the expression of Prx-6 before and after the treatment with granulocyte and monocyte adsorption (GMA). Materials and methods: The colonic expression and localization of Prx-6 were investigated using Realtime-PCR, western blotting and immunohistochemistry. At colonoscopy, biopsies were taken from normal colon mucosa in healthy volunteers and inflamed mucosa obtained from active UC patients. Furthermore, we investigated the impact of GMA treatment on the mucosal level of Prx-6 in active UC patients. In additional In Vitro study, the cytotoxicity by hydrogen peroxide and cell migration were determined in YAMC (Young Adult Mouse colonic epithelial cells) cells (as normal colonic epithelial cells) with down-regulation of Prx-6 expression by siRNA transfection. Results: The colonic expression of Prx-6 mRNA and protein was down-regulated in inflamed mucosa compared to normal colon mucosa. Immunohistochemical study showed that Prx6 expression was mainly localized in the epithelial cells. Therefore, we investigated the role of Prx-6 in the intestinal epithelial cells (YAMC cells), and hydrogen peroxide-induced cell injury was aggravated in Prx-6-downregulated YAMC cells. The wound assay revealed the delayed restitution of Prx-6-downregulated YAMC cells. In clinical study, 25 patients with active UC (14 male, 11female; mean age 47.7 years) were included and treated with GMA. The mean clinical activity index (CAI) was 12.6, and 11 of 25 patients were considered responders (a decrease of more than 5 points in CAI) after 10 GMA sessions. In responders, the colonic expression levels of Prx-6 after GMA treatment were significantly increased compared to those before GMA treatment. Interestingly, the colonic expression levels of Prx-6 before GMA treatment in responders were significantly higher than in non-responders. Conclusion: These findings indicate that down-regulated expression of Prx-6 plays a crucial role in the pathogenesis of the intestinal mucosal inflammation, and Prx-6 may be a therapeutic molecule and biomarker for the intestinal inflammation.

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.

Linoleic acid and butyrate synergize to increase Bcl-2 levels in colonocytes

The biological properties of polyunsaturated fatty acid (PUFA) classes have been the source of much contention. For example, n-3 PUFA are chemoprotective, whereas n-6 PUFA may promote tumor development. Since dietary components can have combinatorial effects, we further examined the apoptotic properties of n-3 or n-6 fatty acids when combined with different fiber sources. Mice were fed diets supplemented with either fish oil (FO; enriched in n-3 PUFA) or corn oil (CO; enriched in n-6 PUFA) and nonfermentable (cellulose) or fermentable (pectin) fiber sources. In complementary experiments, immortalized young adult mouse colonic (YAMC) cells were treated with docosahexaenoic acid (DHA; 22:6n-3) or linoleic acid (LA; 18:2n-6) with or without butyrate. Mice fed a FO and pectin diet had significantly (p < 0.05) increased levels of apoptosis in colonocytes compared to all other diets. Similarly, apoptosis was highly induced in DHA and butyrate cotreated YAMC cells. In contrast, in both YAMC and mouse models, LA/CO with butyrate/pectin treatment reduced apoptosis and enhanced expression of bcl-2. The LA and butyrate induced antiapoptotic phenotype was reversed by knocking down bcl-2 using targeted siRNA. In comparison, overexpression of bcl-2 blocked the proapoptotic effect of DHA and butyrate. These data provide new mechanistic insights into the regulation of apoptosis by dietary PUFA and fiber.

Stress granule formation mediates the inhibition of colonic Hsp70 translation by interferon-γ and tumor necrosis factor-α

Mucosal inflammation, through cytokines such as interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), has many effects on the intestinal epithelium, including selective translational inhibition of the cytoprotective protein heat shock protein 70 (Hsp70). To further elucidate the mechanisms underlying this effect, we examined the role of stress granules in mediating the actions of these proinflammatory cytokines. Using conditionally immortalized young adult mouse colonic epithelial cells, we demonstrate that IFN-gamma and TNF-alpha, which upregulate eukaryotic initiation factor-alpha (eIF-2alpha) phosphorylation and reduce Hsp70 translation, significantly enhance stress granule formation in heat-shocked intestinal epithelial cells. The IFN-gamma and TNF-alpha effects in upregulation of stress granule formation and downregulation of Hsp70 were eIF-2alpha dependent, and the effect could be negated by blocking eIF-2alpha phosphorylation with use of an RNA-dependent protein kinase inhibitor. Correspondingly, IFN-gamma and TNF-alpha increased binding of cytoplasmic proteins to the 3'-untranslated region of Hsp70 mRNA, suggesting specific recruitment of Hsp70 to stress granules as the mechanism of IFN-gamma and TNF-alpha inhibition of Hsp70 translation. We thus report a novel linkage between inflammatory cytokine production, stress granule formation, and Hsp70 translation inhibition, providing additional insights into the response of intestinal epithelial cells to inflammatory stress.

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.

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.

Translational Inhibition of Colonic Epithelial Heat Shock Proteins by IFN-γ and TNF-α in Intestinal Inflammation

Inducible heat shock proteins (iHsp), Hsp25/27 and Hsp70, play essential roles in protecting cells against stress and, in intestinal mucosal inflammation, potentially lessening the extent and severity of injury. We examined the expression and regulation of iHsp in human and experimental inflammatory bowel diseases (IBD) and in vitro. iHsp expression and regulation were assessed in normal and IBD colonic biopsy specimens, IL-10(-/-) mice, and young adult mouse colonic epithelial cells by immunohistochemistry, Western blot, and real-time polymerase chain reaction (PCR). Phosphorylation of double-stranded RNA-dependent protein kinase (PKR) and eukaryotic initiation factor-2alpha (eIF-2alpha) was determined by Western blot. Hsp25/27 and Hsp70 levels were selectively reduced in areas of active mucosal inflammation associated with human IBD and IL-10(-/-) mice with colitis. Wild-type mice treated in vivo with interferon (IFN)-gamma + tumor necrosis factor (TNF)-alpha also demonstrated reduced colonic Hsp25/27 and Hsp70. In young adult mouse colonic epithelial cells, IFN-gamma+TNF-alpha inhibited heat induction of Hsp25/27 and Hsp70, an effect not associated with changes in iHsp messenger RNA or protein half-lives but caused by suppressed de novo iHsp synthesis. IFN-gamma+TNF-alpha cotreatment activated PKR, resulting in phosphorylation and inactivation of eIF-2alpha, an essential factor in protein translation. These effects were not due to induced apoptosis and could be negated by PKR-inhibitor and short interfering RNA to PKR. Increased phosphorylation of PKR and eIF-2alpha were also observed in active IBD tissues. Mucosal inflammation is associated with iHsp down-regulation, an effect that appears mediated by translational down-regulation by proinflammatory cytokines. In the context of IBD, we propose that this mechanism contributes to the severity, extent, and persistence of inflammation-induced mucosal injury.

Probiotics inhibit nuclear factor-κB and induce heat shock proteins in colonic epithelial cells through proteasome inhibition

The extent and severity of mucosal injury in inflammatory bowel diseases are determined by the disequilibrium between 2 opposing processes: reparative and cytoprotective mechanisms vs. inflammation-induced injury. Probiotics may provide clinical benefit by ameliorating colitis; however, their mechanisms of action remain largely unknown. Our objective was to investigate microbial-epithelial interactions that could explain the beneficial therapeutic effects of probiotics. The effect of VSL#3-conditioned media on the nuclear factor-kappaB pathway in young adult mouse colonic epithelial cells was assessed by using monocyte chemoattractant protein-1 enzyme-linked immunosorbent assays; IkappaBalpha, IkappaBbeta, and p105 immunoblot analysis; and nuclear factor-kappaB luciferase reporter gene and proteasome assays. Effects on heat shock proteins were determined by electrophoretic mobility shift assay and immunoblot for heat shock proteins 25 and 72 in young adult mouse colonic cells. Cytoprotection against oxidant injury was determined by chromium 51 release and filamentous and globular actin assays. VSL#3 produces soluble factors that inhibit the chymotrypsin-like activity of the proteasome in gut epithelial cells. Proteasome inhibition is an early event that begins almost immediately after exposure of the epithelial cells to the probiotic-conditioned media. In addition, these bacteria inhibit the proinflammatory nuclear factor-kappaB pathway through a mechanism different from the type III secretory mechanisms described for other nonpathogenic enteric flora. They also induce the expression of cytoprotective heat shock proteins in intestinal epithelial cells. The resulting inhibition of nuclear factor-kappaB and increased expression of heat shock proteins may account for the anti-inflammatory and cytoprotective effects reported for probiotics and may be a novel mechanism of microbial-epithelial interaction. These effects seem to be mediated through the common unifying mechanism of proteasome inhibition.

Chemopreventive n-3 fatty acids activate RXRalpha in colonocytes.

The underlying mechanisms by which n-3 polyunsaturated fatty acids (PUFA) exert a chemopreventive effect in the colon have not been elucidated. Retinoid X receptors (RXR) are a family of nuclear receptors implicated in cancer chemoprevention. Since docosahexaenoic acid (DHA), an n-3 PUFA enriched in fish oil, reduces colonocyte proliferation and enhances apoptosis relative to n-6 PUFA-treated cells, we determined whether DHA can serve as a specific ligand for RXRalpha activation relative to n-6 PUFA in colonocytes. In a mammalian one-hybrid assay, immortalized young adult mouse colonic (YAMC) cells were co-transfected with a yeast galactose upstream activating sequence (UAS)4-tk-Luciferase (Luc) reporter plasmid, plus either GAL4 DNA-binding domain fused to RXRalpha, retinoic acid receptor alpha or GAL4 alone, followed by an n-3, n-6 or n-9 fatty acid incubation. Luc activity levels were dose-dependently elevated only in n-3 PUFA (DHA)-treated RXRalpha. Since RXR homodimers and RXR/peroxisome proliferator-activated receptor (PPAR) heterodimers bind consensus direct repeat (DR1) motifs, YAMC and NCM460 (a normal human colonic cell line), were respectively, co-transfected with RXRalpha and DR1-Luc, followed by different PUFA treatment. Luc activity levels were increased (P < 0.05) only in DHA groups. The DHA-dependent induction of DR-1-Luc was reduced to basal levels upon RXRalpha antagonist-treatment, with no effect on PPARgamma antagonist-treatment. A role for select RXR isoforms in colonocyte biology was also determined by examining nuclear receptor mRNA levels in rat colon following dietary lipid and carcinogen exposure over time. RXRalpha, RXRbeta and RXRgamma were detected in rat colonic mucosa, and the levels of RXRalpha and RXRgamma were elevated in fish oil (n-3 PUFA) versus corn oil (n-6 PUFA) fed animals after 16 weeks. These data indicate that, RXRalpha, an obligatory component of various nuclear receptors, preferentially binds n-3 PUFA in colonocytes, and that the nuclear receptor targets for PUFA in the colon are modulated by dietary lipid exposure.