Particulate Matter Decreases Intestinal Barrier-Associated Proteins Levels in 3D Human Intestinal Model.

(1) Background: The gastrointestinal tract (GI) tract is one of the main organs exposed to particulate matter (PM) directly through ingestion of contaminated food or indirectly through inhalation. Previous studies have investigated the effects of chronic PM exposure on intestinal epithelia in vitro using Caco-2 cells and in vivo using mice. In this study, we hypothesized that chronic PM exposure would increase epithelial permeability and decrease barrier function due to altered redox homeostasis, which alters levels and/or localization of barrier-associated proteins in human three-dimensional (3D) intestinal tissues. (2) Methods: Transepithelial electrical resistance (TEER) in tissues exposed to 50, 100, 150, 250, and 500 µg/cm2 of PM for 1 week and 2 weeks was analyzed. Levels and localization of tight junction proteins zonula occludens protein 1 (ZO-1) and claudin-1 and desmosome-associated desmocollin were analyzed using immunofluorescence. As a marker of oxidative stress, levels of 4-hydroxy-nonenal (4HNE) adducts were measured. (3) Results: No differences in TEER measurements were observed between exposed and un-exposed tissues. However, increased levels of 4HNE adducts in exposed tissues were observed. Additionally, decreased levels of ZO-1, claudin-1, and desmocollin were demonstrated. (4) Conclusion: These data suggest that chronic PM exposure results in an increase of oxidative stress; modified levels of barrier-associated proteins could possibly link to GI tract inflammatory conditions.

Escherichia Coli Outer Membrane Vesicles Induced DNA Double-Strand Breaks in Intestinal Epithelial Caco-2 Cells.

BACKGROUND Recent studies have shown that Escherichia coli induced digestive tract diseases may be related to outer membrane vesicles (OMVs) induced intestinal double-strand breaks (DSBs) in intestinal epithelial cells. This study aimed to compare the impact of OMVs forces on DSBs in intestinal epithelial Caco-2 cells, and provide a new treatment for digestive diseases caused by E. coli. MATERIAL AND METHODS E.coli OMVs were prepared and co-cultured with Caco-2 cells. The uptake of OMVs by Caco-2 cells was observed by confocal microscopy. The γ-H2AX protein was detected by western-blots. The DSBs caused by OMVs was detected by single cell gel electrophoresis. RESULTS The particle size analyzer showed that the average diameters of OMVs centrifuged at 20 000×g and 50 000×g were 217.5±7.29 nm and 186.3±6.59 nm (P<0.05), respectively. Transmission electron microscopy of the OMVs revealed a lipid bilayer structure with a variety of different sizes. Confocal fluorescence microscopy revealed that OMVs almost completely entered Caco-2 cells after 24 hours. The ratio of γ-H2AX protein band gray value normalized data in the OMVs centrifuged at 20 000×g and 50 000×g, and the control group (without OMVs) were 2.23±0.18, 1.58±0.20, 1±0.30 (P<0.05), respectively, while DNA levels of the comet tail (TailDNA%, TDNA%) were 72.21±14.61%, 23.11±4.98%, and 1.02±1.41% (P<0.05), respectively. The corresponding DNA damage was categorized as high (grade 3), moderate (grade 2), and no damage (grade 0). CONCLUSIONS Different sizes of OMVs induced different degrees of DNA damage in intestinal epithelial Caco-2 cells.

[Effects of short chain fatty acid on barrier disruption of human intestinal epithelial cell induced by endotoxin/lipopolysaccharide and the related mechanism].

Objective: To investigate the effects of short chain fatty acid (SCFA) on barrier disruption of human intestinal epithelial cell induced by endotoxin/lipopolysaccharide (LPS) and the related mechanism. Methods: The human intestinal epithelial cell line Caco-2 was used to reproduce monolayer-cells. Cells were divided into control group, LPS group, and SCFA+ LPS group according to the random number table. Cells in control group were only routinely cultured with DMEM medium. Cells in LPS group were cultured with DMEM medium and LPS with final mass concentration of 10 µg/mL. Cells in SCFA+ LPS group were cultured with DMEM medium, LPS and SCFA (consisting of 0.5 mmol/L acetate, 0.01 mmol/L propionate, and 0.01 mmol/L butyrate) with final mass concentration of 10 µg/mL. At post culture hour (PCH) 0, 1, 2, 6, 12, and 24, transepithelial electrical resistance (TER) of cells was determined with an ohmmeter, with sample number of 72. Another portion of cells were divided and treated as above, and then Western blotting was employed to detect the protein expressions of zonula occludens 1 (ZO-1), occludin, and claudin-1 at PCH 24, with sample number of 6. Another portion of cells were divided and treated as above and then immunofluorescence was used to observe cellular morphology and distribution of ZO-1. Data were processed with analysis of variance of factorial design, one-way analysis of variance, least-significant difference test, and Bonferroni correction. Results: (1) Compared with that in control group, TER of cells in LPS group was significantly reduced from PCH 1 to 24 (P<0.01), while TER of cells in SCFA+ LPS group showed no obvious change (P>0.05). TER of cells in SCFA+ LPS group was significantly higher than that in LPS group from PCH 1 to 24 (P<0.01). (2) Compared with the protein expressions of ZO-1, occludin, and claudin-1 of cells in control group (1.25±0.10, 1.17±0.04, and 1.24±0.20), those of cells in LPS group (0.74±0.23, 0.76±0.11, and 0.77±0.11) at PCH 24 were significantly decreased (P<0.05), while those of cells in SCFA+ LPS group (1.23±0.46, 1.05±0.09, and 1.01±0.13) showed no significant differences (P>0.05). Protein expressions of occludin and claudin-1 of cells in SCFA+ LPS group were significantly higher than those in LPS group at PCH 24 (P<0.05). Protein expression of ZO-1 of cells in SCFA+ LPS group was higher than that in LPS group at PCH 24 with no significant difference (P>0.05). (3) At PCH 24, cells in control group were compact in arrangement with pebble-like appearance, and ZO-1 was distributed smoothly and continuously along the cell membrane. In LPS group, cells were sparse in arrangement with change in appearance, and ZO-1 was distributed uncontinuously along the cell membrane with curls and breaks. In SCFA+ LPS group, the appearance of cells and distribution of ZO-1 were remarkably ameliorated compared with those in LPS group. Conclusions: SCFA can alleviate the barrier disruption of human intestinal epithelial cell induced by LPS through interdicting the abnormal distribution of ZO-1 and decrease of TER and tight junction proteins' expressions.

Tumor necrosis factor alpha reduces intestinal vitamin C uptake: a role for NF-κB-mediated signaling.

Sodium-dependent vitamin C transporter-1 (SVCT-1) is the major transporter mediating intestinal vitamin C uptake. Intestinal inflammation and prolonged infection are associated with increased serum and intestinal mucosa levels of tumor necrosis factor-α (TNF-α), which also exerts profound effects on the intestinal absorption process. Elevated levels of TNF-α have been linked to the pathogenesis of inflammatory bowel disease (IBD) and malabsorption of nutrients, and patients with this condition have low levels of vitamin C. To date, little is known about the effect of TNF-α on intestinal absorption of vitamin C. We studied the impact of TNF-α on ascorbic acid (AA) transport using a variety of intestinal preparations. The expression level of human SVCT-1 mRNA is significantly lower in patients with IBD. TNF-α treated Caco-2 cells and mice showed a significant inhibition of intestinal 14C-AA uptake. This inhibition was associated with significant decreases in SVCT-1 protein, mRNA, and heterogeneous nuclear RNA levels in TNF-α treated Caco-2 cells, mouse jejunum, and enteroids. Also, TNF-α caused a significant inhibition in the SLC23A1 promoter activity. Furthermore, treatment of Caco-2 cells with celastrol (NF-κB inhibitor) blocked the inhibitory effect caused by TNF-α on AA uptake, SVCT-1 protein, and mRNA expression, as well as the activity of SLC23A1 promoter. Treatment of TNF-α also led to a significant decrease in the expression of hepatocyte nuclear factor-1-α, which drives the basal activity of SLC23A1 promoter, and this effect was reversed by celastrol. Together, these findings show that TNF-α inhibits intestinal AA uptake, and this effect is mediated, at least in part, at the level of transcription of the SLC23A1 gene via the NF-κB pathway. NEW & NOTEWORTHY Our findings show that tumor necrosis factor-α inhibits intestinal ascorbic acid uptake in both in vitro and in vivo systems, and this inhibitory effect is mediated, at least in part, at the level of transcription of the SLC23A1 (sodium-dependent vitamin C transporter-1) gene via the NF-κB pathway.

Optimization of the Ussing chamber setup with excised rat intestinal segments for dissolution/permeation experiments of poorly soluble drugs.

CONTEXT: Prediction of the in vivo absorption of poorly soluble drugs may require simultaneous dissolution/permeation experiments. In vivo predictive media have been modified for permeation experiments with Caco-2 cells, but not for excised rat intestinal segments. OBJECTIVE: The present study aimed at improving the setup of dissolution/permeation experiments with excised rat intestinal segments by assessing suitable donor and receiver media. METHODS: The regional compatibility of rat intestine in Ussing chambers with modified Fasted and Fed State Simulated Intestinal Fluids (Fa/FeSSIFmod) as donor media was evaluated via several parameters that reflect the viability of the excised intestinal segments. Receiver media that establish sink conditions were investigated for their foaming potential and toxicity. Dissolution/permeation experiments with the optimized conditions were then tested for two particle sizes of the BCS class II drug aprepitant. RESULTS: Fa/FeSSIFmod were toxic for excised rat ileal sheets but not duodenal sheets, the compatibility with jejunal segments depended on the bile salt concentration. A non-foaming receiver medium containing bovine serum albumin (BSA) and Antifoam B was nontoxic. With these conditions, the permeation of nanosized aprepitant was higher than of the unmilled drug formulations. DISCUSSION: The compatibility of Fa/FeSSIFmod depends on the excised intestinal region. The chosen conditions enable dissolution/permeation experiments with excised rat duodenal segments. The experiments correctly predicted the superior permeation of nanosized over unmilled aprepitant that is observed in vivo. CONCLUSION: The optimized setup uses FaSSIFmod as donor medium, excised rat duodenal sheets as permeation membrane and a receiver medium containing BSA and Antifoam B.

[Activation of cofilin and its relation with distribution of tight junction protein zonula occludens 1 in hypoxic human intestinal epithelial cells].

OBJECTIVE: To study the effect of hypoxia on cofilin activation in intestinal epithelial cells and its relation with distribution of tight junction protein zonula occludens 1 (ZO-1). METHODS: The human intestinal epithelial cell line Caco-2 was used to reproduce monolayer cells. The monolayer-cell specimens were divided into control group (no treatment), hypoxic group ( exposed to hypoxia), and normoxic group (exposed to normoxia) according to the random number table. Western blotting was used to detect the protein expressions of cofilin and phosphorylatedl cofilin (p-cofilin) of cells in normoxic group and hypoxic group exposed to normoxia or hypoxia for 1, 2, 6, 12, and 24 h and control group, with 9 samples in control group and 9 samples at each time point in the other two groups. The other monolayer-cell specimens were divided into hypoxic group (exposed to hypoxia) and control group (no treatment) according to the random number table. Cells in hypoxic group exposed to hypoxia for 1, 2, 6, 12, and 24 h and control group were obtained. Morphology and distribution of F-actin was observd with laser scanning confocal microscopy, the ratio of F-actin to G-actin was determined by fluorescence method, and distribution of ZO-l and cellular morphology were observed with laser scanning confocal microscopy. The sample number of last 3 experiments was respectively 3, 6, and 3 in both hypoxic group (at each time point) and control group. Data were processed with paired ttest, analysis of variance of repeated measurement, and LSD-t test. RESULTS: The protein expressions of cofilin and p-cofilin of cells between normoxic group exposed to normoxia for 1 to 24 h and control group showed no significant changes (with values from -0.385 to 1.701, t(p-cofilin)values from 0. 040 to 1.538, P values above 0.05). There were no obvious differences in protein expressions of en filmn of cells between hypoxic group exposed to hypoxia for 1 to 24 h and control group ( with values from 1.032 to 2.390, P values above 0.05). Compared with that in control group, the protein expressions of p-cofilin of cells were greatly reduced in hypoxic group exposed to hypoxia for 1 to 24 h (with values from 4.563 to 22.678, P values below 0.01), especially exposed to hypoxia for 24 h. The protein expressions of cofilin of cells between normoxic group and hypoxic group at each time point were close ( with t values from -0.904 to 1.433, P values above 0.05). In hypoxic group, the protein expressions of p-cofilin of cells exposed to hypoxia for 1, 2, 6, 12, and 24 h were 0.87 +/- 08, 0.780 .05, 0.89 +/- 0.07, 0.68+0. 07, and 0.57 +/- 0.06, respectively, significantly lower than those in normoxic group (0.90 +/- 0.07, 0.97 +/- 0.06, 1.00 +/- 0.06, 1.00 +/- 0.05, and 0.99 +/- 0.05, with t values from 3.193 to 16.434, P values below 0.01). In control group, F-actin in the cytoplasm was abundant, most of it was in bunches. The trend of F-actin was disorderly in hypoxic group from being exposed to hypoxia for 1 h, shortened in length or even dissipated. The ratios of F-actin to G-actin of cells in hypoxic group exposed to hypoxia for 12 and 24 h (0.89 +/- 0.12 and 0.84 +/- 0.19) were obviously decreased as compared with that in control group (1. 00, with t values respectively 3. 622 and 3. 577, P values below 0.01). There were no obvious differences in the ratios of F-actin to G-actin of cells between hypoxic group exposed to hypoxia for 1, 2, and 6 h and control group ( with values from 0.447 to 1.526, P values above 0.05). In control group, cells were compact in arrangement, and ZO-1 was distributed continuously along the cytomnembrane. From being exposed to hypoxia for 2 h, cells became irregular in shape in hypoxic group. ZO-1 was distributed in discontinuous fashion along the cytomembrane with breakage in hypoxic group exposed to hypoxia for 24 h. CONCLUSIONS: Hypoxia may cause the disorder of dynamic balance between F-actin and G-actin by inducing cofilin activation, which in turn leads to the changes in distribution of tight junction protein ZO-1 in intestinal epithelial cells.

Serum-reduced and serum-free media for differentiation of Caco-2 cells.

Human intestinal Caco-2 cells were differentiated using serum-reduced medium with fetal bovine serum (FBS) added only to the basolateral (BL) medium, and four serum-free media, containing insulin, transferrin, selenium (ITS), or MITO+™ serum extender (ITS plus growth factors), with or without addition of a lipid mixture, respectively. Differentiation was assessed by monitoring monolayer permeability, alkaline phosphatase and sucrase activities, and the transport of digoxin and cephalexin. Notably, the serum-reduced protocol produced results that were comparable to cells differentiated in the control medium and should be recommended as an alternative to the use of 10% FBS in both apical (AP) and BL media. ITS serum-free medium elicited permeability values and cephalexin transport similar to control cells. MITO+™ medium was the most efficient in promoting the two transport activities investigated, and it should be further evaluated with a larger set of substances, although its undisclosed composition represents a limit that may override these advantages.

In vitro assessment of epithelial electrical resistance in human esophageal and jejunal mucosae and in Caco-2 cell layers.

OBJECTIVE: There is a need for a technique allowing studies of human mucosal specimens collected during different clinical conditions. This study elucidates if square wave pulse analysis discriminates between epithelial and transmural electrical resistance and if there is an association with transepithelial permeability of molecular probes. METHODS: Mucosae from esophagus (surgical resections: n = 14; endoscopic biopsies: n = 15) and jejunum (n = 12) and Caco-2 cell monolayers were investigated in Ussing chambers. Transmural and epithelial electrical resistance were recorded by the use of standardized current pulses. Permeability was assessed using two fluorescein-labeled probes (weight 376 and 4000 Da). RESULTS: Baseline epithelial electrical resistance was higher in esophageal mucosa (~280 Ω*cm(2)), than in jejunal (~10 Ω*cm(2)) and Caco-2 cells (~140 Ω*cm(2)). The subepithelial contribution to the transmural resistance was higher in jejunal preparations (+88%) and Caco-2 cells (+75%), than in esophageal (+30%). During hypoxia the subepithelial resistance was unchanged, whereas the epithelial resistance decreased significantly in jejunal mucosa and Caco-2 cells. These findings coincided with increased transepithelial probe permeability and signs of disturbed morphology. Esophageal epithelia were resistant to hypoxia. However, exposure to deoxycholic acid and trypsin abolished the esophageal epithelial resistance and increased probe permeability. Endoscopic esophageal biopsies from patients with erosive reflux disease exhibited significantly lower epithelial resistance and higher current than healthy subjects. CONCLUSION: Square wave pulse analysis in Ussing chambers is suitable for assessment of epithelial electrical resistance that can reflect transepithelial permeability of molecular probes with known size. Moreover, the technique discriminated between healthy and reflux-diseased esophageal mucosal biopsies.

Lactobacillus amylophilus D14 protects tight junction from enteropathogenic bacteria damage in Caco-2 cells.

Enterotoxigenic Escherichia coli and Salmonella Typhimurium could adhere to epithelial tissue and destroy cell junctions, leading to intestinal inflammation and diarrhea. Lactobacillus could prevent the adhesion of pathogens to host cells and protect the mucosal barrier. The objective of this study was to investigate the protective effects of Lactobacillus amylophilus D14 on Caco-2 cells against the invasion of enterotoxigenic Escherichia coli K88 and Salmonella Typhimurium SL1344. We found that with a reduction in dextran permeability and an increase in transepithelial electrical resistance, L. amylophilus D14 could ameliorate the damage to cell integrity caused by pathogens. Furthermore, L. amylophilus D14 reduced the expression of phosphorylated extracellular signal-regulated protein kinase and phospho-c-jun N-terminal kinase, and it decreased the secretion of IL-8. The abilities of the Lactobacillus to protect the cell junctions were then evaluated on Caco-2 cells. Increased expression and amelioration distribution of tight junction proteins (zonula occludens-1, claudin-1, and E-cadherin) were observed when the cells were cocultured with pathogens and Lactobacillus simultaneously. Lactobacillus amylophilus D14 may influence the mitogen-activated protein kinase pathway to regulate the correct assembly of the tight junction and adherens junction, protecting the cell junctions and mucosal barrier damaged by enterotoxigenic E. coli K88 or Salmonella Typhimurium SL1344 infection.

PER1 modulates SGLT1 transcription in vitro independent of E-box status.

BACKGROUND AND AIMS: The intestine demonstrates profound circadian rhythmicity in glucose absorption in rodents, mediated entirely by rhythmicity in the transcription, translation, and function of the sodium glucose co-transporter SGLT1 (Slc5a1). Clock genes are rhythmic in the intestine and have been implicated in the regulation of rhythmicity of other intestinal genes; however, their role in the regulation of SGLT1 is unknown. We investigated the effects of one clock gene, PER1, on SGLT1 transcription in vitro. METHODS: Caco-2 cells were stably transfected with knockdown vectors for PER1 and mRNA expression of clock genes and SGLT1 determined using quantitative polymerase chain reaction (qPCR). Chinese hamster ovary (CHO) cells were transiently cotransfected with combinations of the PER1 expression vectors and the wild-type SGLT1-luciferase promoter construct or the promoter with mutated E-box sequences. RESULTS: Knockdown of PER1 increased native SGLT1 expression in Caco-2 enterocytes, while promoter studies confirmed that the inhibitory activity of PER1 on SGLT1 occurs via the proximal 1 kb of the SGLT1 promoter. E-box sites exerted a suppressive effect on the SGLT1 promoter; however, mutation of E-boxes had little effect on the inhibitory activity of PER1 on the SGLT1 promoter suggesting that the actions of PER1 on SGLT1 are independent of E-boxes. CONCLUSIONS: Our findings suggest that PER1 exerts an indirect suppressive effect on SGLT1, possibly acting via other clock-controlled genes binding to non-E-box sites on the SGLT1 promoter. Understanding the regulation of rhythmicity of SGLT1 may lead to new treatments for the modulation of SGLT1 expression in conditions such as malabsorption, diabetes, and obesity.

Particulate matter air pollution causes oxidant-mediated increase in gut permeability in mice.

BACKGROUND: Exposure to particulate matter (PM) air pollution may be an important environmental factor leading to exacerbations of inflammatory illnesses in the GI tract. PM can gain access to the gastrointestinal (GI) tract via swallowing of air or secretions from the upper airways or mucociliary clearance of inhaled particles. METHODS: We measured PM-induced cell death and mitochondrial ROS generation in Caco-2 cells stably expressing oxidant sensitive GFP localized to mitochondria in the absence or presence of an antioxidant. C57BL/6 mice were exposed to a very high dose of urban PM from Washington, DC (200 µg/mouse) or saline via gastric gavage and small bowel and colonic tissue were harvested for histologic evaluation, and RNA isolation up to 48 hours. Permeability to 4 kD dextran was measured at 48 hours. RESULTS: PM induced mitochondrial ROS generation and cell death in Caco-2 cells. PM also caused oxidant-dependent NF-κB activation, disruption of tight junctions and increased permeability of Caco-2 monolayers. Mice exposed to PM had increased intestinal permeability compared with PBS treated mice. In the small bowel, colocalization of the tight junction protein, ZO-1 was lower in the PM treated animals. In the small bowel and colon, PM exposed mice had higher levels of IL-6 mRNA and reduced levels of ZO-1 mRNA. Increased apoptosis was observed in the colon of PM exposed mice. CONCLUSIONS: Exposure to high doses of urban PM causes oxidant dependent GI epithelial cell death, disruption of tight junction proteins, inflammation and increased permeability in the gut in vitro and in vivo. These PM-induced changes may contribute to exacerbations of inflammatory disorders of the gut.

Epithelial inducible nitric oxide synthase causes bacterial translocation by impairment of enterocytic tight junctions via intracellular signals of Rho-associated kinase and protein kinase C zeta.

OBJECTIVE: Gut barrier dysfunction and bacterial translocation occur in various disorders, including intestinal obstruction. Overexpression of inducible nitric oxide synthase is implicated in the pathogenesis of bacterial translocation, of which the molecular mechanism remains unclear. Epithelial permeability is regulated by tight junction reorganization and myosin light chain phosphorylation. Our aim was to investigate the roles of Rho-associated kinase and protein kinase C ζ in epithelial nitric oxide synthase-mediated barrier damage. DESIGN: Animal study and cell cultures. SETTING: Research laboratory. SUBJECTS: BALB/c mice. INTERVENTIONS: : Mouse distal small intestine was obstructed in vivo by a 10-cm loop ligation in which vehicle, L-Nil (a nitric oxide synthase inhibitor), or Y27632 (a Rho-associated kinase inhibitor) was luminally administered. After obstruction for 24 hrs, intestinal tissues were mounted on Ussing chambers for macromolecular flux. Liver and spleen tissues were assessed for bacterial counts. Caco-2 cells were exposed to 1 mM S-nitroso-N-acetylpenicillamine (a nitric oxide donor) for 24 hrs, and transepithelial resistance and permeability were evaluated. MEASUREMENTS AND MAIN RESULTS: Mice with intestinal obstruction displayed epithelial barrier dysfunctions, such as permeability rise and bacterial translocation, associated with tight junction disruption and myosin light chain phosphorylation. Increased inducible nitric oxide synthase and phosphorylated protein kinase C ζ were observed in villus epithelium. Enteric instillation of L-Nil and Y27632 attenuated the functional and structural barrier damage caused by intestinal obstruction. L-Nil decreased intestinal obstruction-induced myosin light chain, myosin phosphatase target subunit 1, and protein kinase C ζ phosphorylation, suggesting that inducible nitric oxide synthase is upstream of Rho-associated kinase and protein kinase C ζ signaling. The intestinal phosphorylated myosin light chain level did not increase in inducible nitric oxide synthase(-/-) mice following intestinal obstruction. In vitro studies showed that S-nitroso-N-acetylpenicillamine-induced transepithelial resistance drop and permeability rise was independent of cell apoptosis. Y27632 inhibited S-nitroso-N-acetylpenicillamine-induced myosin light chain phosphorylation and permeability rise. S-nitroso-N-acetylpenicillamine also triggered phosphorylation and membrane translocation of protein kinase C ζ. Inhibitory protein kinase C ζ pseudosubstrate blocked S-nitroso-N-acetylpenicillamine-induced tight junction reorganization, but not myosin light chain phosphorylation. CONCLUSIONS: Epithelial inducible nitric oxide synthase activates two distinct signals, protein kinase C ζ and Rho-associated kinase, to disrupt tight junctions leading to bacterial influx.

Macrophages increase microparticle uptake by enterocyte-like Caco-2 cell monolayers.

Caco-2 cells form an enterocyte-like monolayer that has been used to explore small intestinal microparticle uptake. They are a useful functional model for the investigation of in vivo drug delivery systems and the uptake of particulate environmental pollutants. The aim of this paper was to determine if the previously reported decrease in Caco-2 transepithelial resistance following exposure to macrophages was matched by increased microparticle uptake, especially as macrophage phagocytosis simulates removal of particles from the subepithelial compartment. Caco-2 cells were grown as a monoculture for 21 days on insert membranes. A compartmentalised model involved Caco-2 cells in the upper compartment, with THP-1-derived macrophages adhering to the base of the underlying well, the two cell populations communicating only through the shared culture medium. Caco-2 cells were also cultured in macrophage-conditioned medium and all groups were exposed apically to 2 µm latex particles for 5 or 60 min. Parameters measured were: transepithelial resistance; cytokine levels; cell dimensions and the distribution of nuclei, actin and junctional proteins. Subepithelial particle numbers, defined as those located below the insert membrane, were also counted and were significantly increased in the Caco-2/macrophage model, with over 90% associated with the macrophages. Other changes induced by the presence of macrophages included decreased transepithelial resistance levels, diffuse localisation of some junctional proteins, higher proinflammatory cytokine levels, disorganisation of cell shape and decreased cell height associated with actin reorganisation. Macrophage-conditioned medium produced a smaller transepithelial resistance decrease than the Caco-2/macrophage model and there were few other changes. In conclusion, culture of Caco-2 cells with underlying macrophages produced a lower, less organised epithelium and greater microparticle uptake.

Transcriptional profiling of Clostridium difficile and Caco-2 cells during infection.

Clostridium difficile is well recognized as the most common infectious cause of nosocomial diarrhea. The incidence and severity of C. difficile infection (CDI) is increasing worldwide. Here, we evaluated simultaneously the transcriptional changes in the human colorectal epithelial Caco-2 cells and in C. difficile after infection. A total of 271 transcripts in Caco-2 cells and 207 transcripts in C. difficile were significantly differentially expressed at 1 time point during CDI. We used the gene ontology annotations and protein-protein network interactions to underline a framework of target molecules that could potentially play a key role during CDI. These genes included those associated with cellular metabolism, transcription, transport, cell communication, and signal transduction. Our data identified certain key factors that have previously been reported to be involved in CDI, as well as novel determinants that may participate in a complex mechanism underlying the host response to infection, bacterial adaptation, and pathogenesis.

Effects of Japanese mistletoe lectin on cytokine gene expression in human colonic carcinoma cells and in the mouse intestine.

Mistletoe lectins have various biological activities including anti-cancer and immunomodulatory effects. We previously isolated a lectin (ML-J) from Japanese mistletoe. In the present study, we examined the effects of ML-J on cytokine gene expression in human colon adenocarcinoma Caco-2 cells and in the mouse intestine. The results of reverse transcription-polymerase chain reaction and quantitative real-time polymerase chain reaction indicated that ML-J caused an upregulation of the gene expression of the proinflammatory cytokines interleukin (IL)-8, tumor necrosis factor-alpha (TNF-alpha) and IL-6 in Caco-2 cells and TNF-alpha and IL-6 in the duodenum. This study provides the first example to show that a perorally administered plant lectin affects gene expression in the duodenum.

Substance P accelerates intestinal tissue regeneration after gamma-irradiation-induced damage.

Radiation therapy causes varying degrees of damage to biological systems. Many groups are investigating the mechanism underlying radiation-induced cellular damage but there are limited therapeutic solutions for affected patients. Recent studies show that substance P (SP) participates in cell proliferation. In the present study, we characterized the mechanism underlying SP-induced cellular signaling in radiation-induced damage of the intestine. Exposure of Caco-2 cells to SP increases cell proliferation and Erk phosphorylation in a time- and dose-dependent manner. The proliferation of cells exposed to gamma-irradiation is also stimulated by exposure to SP, a phenomenon that may result from inhibition of apoptosis because SP activates Akt and inhibits the cleavage of caspase-3. The effect of SP on cell proliferation and protection was confirmed by investigations in mice. Proliferating cell nuclear antigen staining shows that cell proliferation in radiation-damaged mouse intestine increases significantly upon exposure to SP. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end labeling assay reveals fewer cells stained in SP-treated mice compared with untreated controls. These findings show the potential for SP-induced acceleration of intestinal wound healing and reveal that the mechanism underlying this process involves activation of Erk and Akt and inhibition of caspase-3 cleavage.

Thermal killing of human colon cancer cells is associated with the loss of eukaryotic initiation factor 5A.

Heat-induced cell death appears to be a cell-specific event. Chronic heat stress was lethal to human colon cancer cells (Caco-2, HT29, and HCT116), but not to normal diploid fibroblasts and other cancer cells (BJ-T, WI38, HeLa, ovarian 2008, WI38VA). Acute heat stress (45-51 degrees C, 30 min) caused cell death of colon cancer cells during recovery at physiological temperature. Thermal killing of Caco-2 cells was not mediated via oxidative stress since Caco-2 cells were much more resistant than HeLa and other cancer cells to H(2)O(2)-induced cell death. Acute heat stress caused a striking loss of eukaryotic initiation factor 5A (eIF5A) in colon cancer cells, but not in HeLa and other normal or transformed human fibroblasts. The heat-induced loss of eIF5A is likely to be due to changes in the protein stability. The half-life of eIF5A was changed from >20 h to less than 30 min during the acute heat stress. Sequence analysis of the eIF5A gene from Caco-2 and HeLa cells did not reveal any difference, suggesting that the change in stability in Caco-2 cells was not due to any eIF5A mutation. Pretreatment of cells with protease inhibitors such as phenylmethyl sulfonyl fluoride (PMSF) partially blocked the heat-induced loss of eIF5A and prevented heat-induced cell death. In light of the essential role of eIF5A in cell survival and proliferation, our results suggest that the stability of eIF5A may have an important role in determining the fate of the particular cell type after severe heat stress.

Polyethylene glycol influences microbial interactions with intestinal epithelium.

There is emerging evidence that polyethylene glycol (PEG), widely used as a bowel preparation before surgery, may protect the intestinal epithelium from microbial invasion. Experiments were designed to study the effects of both low-molecular-weight (LMW; 3.35 kd) and high-molecular-weight (HMW; 15-20 kd) PEG on interactions of Escherichia coli, Candida albicans, and Candida glabrata with intestinal epithelium (these three intestinal microbes are frequently involved in systemic infection in shock and trauma patients.) In vitro experiments studied the effects of PEG on mature Caco-2 enterocytes. Using the gentamicin protection assay, both HMW and LMW PEG inhibited E. coli internalization by Caco-2 enterocytes. Using an immunosorbent assay, both HMW and LMW PEG inhibited C. albicans and C. glabrata adherence to Caco-2 enterocytes. Scanning electron micrographs of Caco-2 cells incubated in HMW or LMW PEG showed globular material distributed randomly over the epithelial surface, and apical microvilli seemed distorted. As an in vivo correlate to these experiments, separate groups of antibiotic-treated mice were orally associated with either E. coli, C. albicans, or C. glabrata, and cohort groups were given drinking water containing 5% HMW or 5% LMW PEG. Cecal colonization of E. coli was decreased in mice given HMW but not LMW PEG. Cecal colonization with C. albicans or C. glabrata was decreased in mice given either HMW or LMW PEG. These data provide further evidence that PEG may decrease microbial colonization and microbial interactions with intestinal epithelium.

Effects of MAPK signaling on 1,25-dihydroxyvitamin D-mediated CYP24 gene expression in the enterocyte-like cell line, Caco-2.

We examined the role of the extracellular signal regulated kinases (ERK) in 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3))-induced gene expression in the differentiated Caco-2 cells. 1,25(OH)(2)D(3)-regulated expression of the 25-hydroxyvitamin D, 24-hydroxylase (CYP24) gene (both natural gene and promoter construct) was strongly modulated by altering ERK activity (i.e., reduced by MEK inhibitors and dominant negative (dn) ERK1 and ERK2, activated by epidermal growth factor) but ERK inhibition had no effect on 1,25(OH)(2)D(3)-regulated expression of the transient receptor potential cation channel, subfamily V, member 6 (TRPV6). ERK5-mediated phosphorylation of the transcription factor Ets-1 enhanced 1,25(OH)(2)D(3)-mediated CYP24 gene transcription in proliferating but not differentiated Caco-2 cells due to reduced levels of ERK5 and Ets-1 (total and phosphoprotein levels) in differentiated cells. MEK inhibition reduced 1,25(OH)(2)D(3)-induced 3X-VDRE promoter activity but had no impact on the association of vitamin D receptor (VDR) with chromatin suggesting a role for co-activator recruitment in ERK-modulation of vitamin D-regulated CYP24 gene activation. Chromatin immunoprecipitation assays revealed that the ERK1/2 target, mediator 1 (MED1), is recruited to the CYP24, but not the TRPV6, promoter following 1,25(OH)(2)D(3) treatment. MED1 phosphorylation was sensitive to activators and inhibitors of the ERK1/2 signaling and MED1 siRNA reduced 1,25(OH)(2)D(3)-regulated human CYP24 promoter activity. This suggests ERK1/2 signaling enhances 1,25(OH)(2)D(3) effects on the CYP24 promoter by MED1-mediated events. Our data show that there are both promoter-specific and cell stage-specific roles for the ERK signaling pathway on 1,25(OH)(2)D(3)-mediated gene induction in enterocyte-like Caco-2 cells.

Potential molecular mechanism for c-Src kinase-mediated regulation of intestinal cell migration.

The ubiquitously expressed Src tyrosine kinases (c-Src, c-Yes, and c-Fyn) regulate intestinal cell growth and differentiation. Src activity is also elevated in the majority of malignant and premalignant tumors of the colon. The development of fibroblasts with the three ubiquitously expressed kinases deleted (SYF cells) has identified the role of Src proteins in the regulation of actin dynamics associated with increased cell migration and invasion. Despite this, unexpectedly nothing is known about the role of the individual Src kinases on intestinal cell cytoskeleton and/or cell migration. We have previously reported that villin, an epithelial cell-specific actin-modifying protein that regulates actin reorganization, cell morphology, cell migration, cell invasion, and apoptosis, is tyrosine-phosphorylated. In this report using the SYF cells reconstituted individually with c-Src, c-Yes, c-Fyn, and wild type or phosphorylation site mutants of villin, we demonstrate for the first time the absolute requirement for c-Src in villin-induced regulation of cell migration. The other major finding of our study is that contrary to previous reports, the nonreceptor tyrosine kinase, Jak3 (Janus kinase 3), does not regulate phosphorylation of villin or villin-induced cell migration and is, in fact, not expressed in intestinal epithelial cells. Further, we identify SHP-2 and PTP-PEST (protein-tyrosine phosphatase proline-, glutamate-, serine-, and threonine-rich sequence) as negative regulators of c-Src kinase and demonstrate a new function for these phosphatases in intestinal cell migration. Together, these data suggest that in colorectal carcinogenesis, elevation of c-Src or down-regulation of SHP-2 and/or PTP-PEST may promote cancer metastases and invasion by regulating villin-induced cell migration and cell invasion.