Model Of Intestinal Epithelium Research Articles

Primary human polarized small intestinal epithelial barriers respond differently to a hazardous and an innocuous protein

An experimental platform employing human derived intestinal epithelial cell (IEC) line monolayers grown on permeable Transwell® filters was previously investigated to differentiate between hazardous and innocuous proteins. This approach was effective at distinguishing these types of proteins and perturbation of monolayer integrity, particularly transepithelial electrical resistance (TEER), was the most sensitive indicator. In the current report, in vitro indicators of monolayer integrity, cytotoxicity, and inflammation were evaluated using primary (non-transformed) human polarized small intestinal epithelial barriers cultured on Transwell® filters to compare effects of a hazardous protein (Clostridium difficile Toxin A [ToxA]) and an innocuous protein (bovine serum albumin [BSA]). ToxA exerted a reproducible decrease on barrier integrity at doses comparable to those producing effects observed from cell line-derived IEC monolayers, with TEER being the most sensitive indicator. In contrast, BSA, tested at concentrations substantially higher than ToxA, did not cause changes in any of the tested variables. These results demonstrate a similarity in response to certain proteins between cell line-derived polarized IEC models and a primary human polarized small intestinal epithelial barrier model, thereby reinforcing the potential usefulness of cell line-derived polarized IECs as a valid experimental platform to differentiate between hazardous and non-hazardous proteins.

Alpha-ketoglutarate suppresses the NF-κB-mediated inflammatory pathway and enhances the PXR-regulated detoxification pathway.

Alpha-ketoglutarate (AKG) is a critical nutritional factor in the maintenance of intestinal homeostasis. However, the relative mechanism of AKG has not been well understood. It was recently shown that the interaction between nuclear factor kappa B (NF-κB)-mediated inflammatory pathway and pregnane X receptor (PXR)-regulated detoxification pathway is a check and balance mechanism for keeping the homeostatic state of the intestine, preventing the onset of intestinal inflammation which may lead to cancer. In the current study we used lipopolysaccharide (LPS)-challenged piglet and intestinal porcine epithelial cells-J2 models to investigate the effects of dietary AKG supplementation on the intestinal immune system and PXR regulated target expression. We found that LPS induced significant activation of the NF-κB-mediated inflammatory pathway with concomitant impairment of intestinal nutrient absorption. AKG administration increased intracellular AKG and its metabolite concentrations and enhanced the mRNA expression of alpha-ketoglutarate dehydrogenase in vivo and in vitro. Thus dietary AKG supplementation reversed the adverse effects induced by LPS. We also found a strong inhibitory effects on the NF-κB-mediated inflammatory pathway, especially, in the AKG-treated intestinal tissues, LPS-induced NF-κB phosphorylation was inhibited and TNF-α was suppressed. Interestingly, AKG has potent effects in regulating the PXR and its downstream targets such as CYP3As and CYP2Bs in vivo and in vitro, although AKG is not a known PXR ligand. One potential mechanism for the up-regulation of the PXR pathway is through the down-regulation of NF-κB pathway which in turn de-represses the PXR-regulated target expression. Taken together, our results suggest that AKG improves intestinal immune system through modulating the interaction between PXR and NF-κB. Our findings have important implications for the prevention and treatment of intestinal inflammatory diseases in neonates.

Tu1786 - EGCG Reverses LPS-Mediated Changes in Claudin Physiology and Improves Trans-Epithelial Resistance in an Intestinal Epithelium Model

Tu1786 - EGCG Reverses LPS-Mediated Changes in Claudin Physiology and Improves Trans-Epithelial Resistance in an Intestinal Epithelium Model

Lipid nanocapsules maintain full integrity after crossing a human intestinal epithelium model

Lipid nanocapsules (LNCs) have demonstrated great potential for the oral delivery of drugs having very limited oral bioavailability (BCS class II, III and IV molecules). It has been shown previously that orally-administered LNCs can permeate through mucus, increase drug absorption by the epithelial tissue, and finally, increase drug bioavailability. However, even if transport mechanisms through mucus and the intestinal barrier have already been clarified, the preservation of particle integrity is still not known. The aim of the present work is to study in vitro the fate of LNCs after their transportation across an intestinal epithelium model (Caco-2 cell model). For this, two complementary techniques were employed: Förster Resonance Energy Transfer (FRET) and Nanoparticle Tracking Analysis (NTA). Results showed, after 2h, the presence of nanoparticles in the basolateral side of the cell layer and a measurable FRET signal. This provides very good evidence for the transcellular intact crossing of the nanocarriers.

Three-dimensional organotypic co-culture model of intestinal epithelial cells and macrophages to study Salmonella enterica colonization patterns

Three-dimensional models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by two-dimensional monolayers and respond to Salmonella in key ways that reflect in vivo infections. To further enhance the physiological relevance of three-dimensional models to more closely approximate in vivo intestinal microenvironments encountered by Salmonella, we developed and validated a novel three-dimensional co-culture infection model of colonic epithelial cells and macrophages using the NASA Rotating Wall Vessel bioreactor. First, U937 cells were activated upon collagen-coated scaffolds. HT-29 epithelial cells were then added and the three-dimensional model was cultured in the bioreactor until optimal differentiation was reached, as assessed by immunohistochemical profiling and bead uptake assays. The new co-culture model exhibited in vivo-like structural and phenotypic characteristics, including three-dimensional architecture, apical-basolateral polarity, well-formed tight/adherens junctions, mucin, multiple epithelial cell types, and functional macrophages. Phagocytic activity of macrophages was confirmed by uptake of inert, bacteria-sized beads. Contribution of macrophages to infection was assessed by colonization studies of Salmonella pathovars with different host adaptations and disease phenotypes (Typhimurium ST19 strain SL1344 and ST313 strain D23580; Typhi Ty2). In addition, Salmonella were cultured aerobically or microaerobically, recapitulating environments encountered prior to and during intestinal infection, respectively. All Salmonella strains exhibited decreased colonization in co-culture (HT-29-U937) relative to epithelial (HT-29) models, indicating antimicrobial function of macrophages. Interestingly, D23580 exhibited enhanced replication/survival in both models following invasion. Pathovar-specific differences in colonization and intracellular co-localization patterns were observed. These findings emphasize the power of incorporating a series of related three-dimensional models within a study to identify microenvironmental factors important for regulating infection.

Titanium dioxide nanoparticle ingestion alters nutrient absorption in an in vitro model of the small intestine

Ingestion of titanium dioxide (TiO2) nanoparticles from products such as agricultural chemicals, processed food, and nutritional supplements is nearly unavoidable. The gastrointestinal tract serves as a critical interface between the body and the external environment, and is the site of essential nutrient absorption. The goal of this study was to examine the effects of ingesting the 30nm TiO2 nanoparticles with an in vitro cell culture model of the small intestinal epithelium, and to determine how acute or chronic exposure to nano-TiO2 influences intestinal barrier function, reactive oxygen species generation, proinflammatory signaling, nutrient absorption (iron, zinc, fatty acids), and brush border membrane enzyme function (intestinal alkaline phosphatase). A Caco-2/HT29-MTX cell culture model was exposed to physiologically relevant doses of TiO2 nanoparticles for acute (4h) or chronic (five days) time periods. Exposure to TiO2 nanoparticles significantly decreased intestinal barrier function following chronic exposure. Reactive oxygen species (ROS) generation, proinflammatory signaling, and intestinal alkaline phosphatase activity all showed increases in response to nano-TiO2. Iron, zinc, and fatty acid transport were significantly decreased following exposure to TiO2 nanoparticles. This is because nanoparticle exposure induced a decrease in absorptive microvilli in the intestinal epithelial cells. Nutrient transporter protein gene expression was also altered, suggesting that cells are working to regulate the transport mechanisms disturbed by nanoparticle ingestion. Overall, these results show that intestinal epithelial cells are affected at a functional level by physiologically relevant exposure to nanoparticles commonly ingested from food.

Establishment of 3D Intestinal Organoid Cultures from Intestinal Stem Cells.

The intestinal epithelium is the most rapidly renewed tissue in adult mammals, and its renewal is strictly controlled by intestinal stem cells. Extensive studies using genetic models of intestinal epithelium have revealed the mechanisms underlying the self-renewal of intestinal stem cells. Exploiting this knowledge, we developed a novel 3D culture system that enables the outgrowth of intestinal Lgr5+ stem cells derived from mouse and human tissues into ever-expanding crypt-villus mini-guts, known as intestinal epithelial organoids. These organoids are maintained by the self-renewal of stem cells and give rise to all differentiated cell types of the intestinal epithelium. Once established, organoids can be cryopreserved and thawed when needed. This culture system has been widely used for studying stem cell behavior and gene function and for disease modeling.

In vitro investigation of intestinal transport mechanism of silicon, supplied as orthosilicic acid-vanillin complex.

Silicon (Si) is one of the most abundant trace elements in the body. Although pharmacokinetics data described its absorption from the diet and its body excretion, the mechanisms involved in the uptake and transport of Si across the gut wall have not been established. Caco-2 cells were used as a well-accepted in vitro model of the human intestinal epithelium to investigate the transport, across the intestinal barrier in both the absorption and excretion directions, of Si supplied as orthosilicic acid stabilized by vanillin complex (OSA-VC). The transport of this species was found proportional to the initial concentration and to the duration of incubation, with absorption and excretion mean rates similar to those of Lucifer yellow, a marker of paracellular diffusion, and increasing in the presence of EGTA, a chelator of divalents cations including calcium. A cellular accumulation of Si, polarized from the apical side of cells, was furthermore detected. These results provide evidence that Si, ingested as a food supplement containing OSA-VC, crosses the intestinal mucosa by passive diffusion via the paracellular pathway through the intercellular tight junctions and accumulates intracellularly, probably by an uptake mechanism of facilitated diffusion. This study can help to further understand the kinetic of absorption of Si.

Oral Toxicity and Intestinal Transport Mechanism of Colloidal Gold Nanoparticle-Treated Red Ginseng.

(1) Background: Application of nanotechnology or nanomaterials in agricultural food crops has attracted increasing attention with regard to improving crop production, quality, and nutrient utilization. Gold nanoparticles (Au-NPs) have been reported to enhance seed yield, germination rate, and anti-oxidant potential in food crops, raising concerns about their toxicity potential. In this study, we evaluated the oral toxicity of red ginseng exposed to colloidal Au-NPs during cultivation (G-red ginseng) in rats and their intestinal transport mechanism. (2) Methods: 14-day repeated oral administration of G-red ginseng extract to rats was performed, and body weight, hematological, serum biochemical, and histopathological values were analyzed. An in vitro model of human intestinal follicle-associated epithelium (FAE) and an intestinal epithelial monolayer system were used for intestinal transport mechanistic study. (3) Results: No remarkable oral toxicity of G-red ginseng extract in rats was found, and Au-NPs did not accumulate in any organ, although Au-NP transfer to G-red ginseng and some increased saponin levels were confirmed. Au-NPs were transcytozed by microfold (M) cells, but not by a paracellular pathway in the intestinal epithelium. (4) Conclusion: These findings suggest great potential of Au-NPs for agricultural food crops at safe levels. Further study is required to elucidate the functional effects of Au-NPs on ginseng and long-term toxicity.

Biokinetics of food additive silica nanoparticles and their interactions with food components

Nanomaterials have been widely utilized in the food industry in production, packaging, sensors, nutrient delivery systems, and food additives. However, research on the interactions between food-grade nanoparticles and biomolecules as well as their potential toxicity is limited. In the present study, the in vivo solubility, oral absorption, tissue distribution, and excretion kinetics of one of the most extensively used food additives, silica (SiO2) were evaluated with respect to particle size (nano vs bulk) following single-dose oral administration to rats. Intestinal transport mechanism was investigated using a 3D culture system, in vitro model of human intestinal follicle-associated epithelium (FAE). The effect of the presence of food components, such as sugar and protein, on the oral absorption of nanoparticles was also evaluated with focus on their interactions. The results obtained demonstrated that the oral absorption of nanoparticles (3.94±0.38%) was greater than that of bulk materials (2.95±0.37%), possibly due to intestinal transport by microfold (M) cells. On the other hand, particle size was found to have no significant effect on in vivo dissolution property, biodistribution, or excretion kinetics. Oral absorption profile of silica nanoparticles was highly dependent on the presence of sugar or protein, showing rapid absorption rate in glucose, presumably due to their surface interaction on nanoparticles. These findings will be useful for predicting the potential toxicity of food-grade nanoparticles and for understanding biological interactions.

A Facile Method for Separating and Enriching Nano and Submicron Particles from Titanium Dioxide Found in Food and Pharmaceutical Products.

Recent studies indicate the presence of nano-scale titanium dioxide (TiO2) as an additive in human foodstuffs, but a practical protocol to isolate and separate nano-fractions from soluble foodstuffs as a source of material remains elusive. As such, we developed a method for separating the nano and submicron fractions found in commercial-grade TiO2 (E171) and E171 extracted from soluble foodstuffs and pharmaceutical products (e.g., chewing gum, pain reliever, and allergy medicine). Primary particle analysis of commercial-grade E171 indicated that 54% of particles were nano-sized (i.e., < 100 nm). Isolation and primary particle analysis of five consumer goods intended to be ingested revealed differences in the percent of nano-sized particles from 32%‒58%. Separation and enrichment of nano- and submicron-sized particles from commercial-grade E171 and E171 isolated from foodstuffs and pharmaceuticals was accomplished using rate-zonal centrifugation. Commercial-grade E171 was separated into nano- and submicron-enriched fractions consisting of a nano:submicron fraction of approximately 0.45:1 and 3.2:1, respectively. E171 extracted from gum had nano:submicron fractions of 1.4:1 and 0.19:1 for nano- and submicron-enriched, respectively. We show a difference in particle adhesion to the cell surface, which was found to be dependent on particle size and epithelial orientation. Finally, we provide evidence that E171 particles are not immediately cytotoxic to the Caco-2 human intestinal epithelium model. These data suggest that this separation method is appropriate for studies interested in isolating the nano-sized particle fraction taken directly from consumer products, in order to study separately the effects of nano and submicron particles.

Gastrointestinal digestates of Grana Padano and Trentingrana cheeses promote intestinal calcium uptake and extracellular bone matrix formation in vitro

In the present work, Grana Padano (GP) and Trentingrana (TN) cheeses at different ripening time were in vitro digested. To study calcium uptake and utilization, the intact digestates (selected doses that do not alter cell viability and Transepithelial Electrical Resistance) were administered to Caco2/HT-29 70/30 cells, cultured on a semipermeable membrane in transwells, as a model of human intestinal epithelium. Intact digestates as well as the whole basolateral solutions (mimicking the passage of digestates through intestinal cells before reaching the blood flow and bone) in parallel were further administered to human osteoblast-like cells SaOS-2 to study the extracellular bone matrix formation. In vitro digestates deriving from GP and TN promoted calcium uptake and extracellular bone matrix formation independently of both the cheese type and its ripening period (13, 19 or 26months). The present study reports the ability of whole digestates of GP and TN cheeses to improve intestinal calcium absorption and bone matrix formation in vitro. Once fully explored at bone level, this finding could better support the role of cheese in ameliorating calcium deficiencies and associated diseases in vivo.

Vitamin D receptor is a novel transcriptional regulator for Axin1

BackgroundAxin1 is a scaffold protein in the β-catenin destruction complex, which, if disrupted, contributes to pathogenesis of various human diseases, including colorectal carcinogenesis and inflammatory bowel diseases (IBD). We have previously demonstrated that Salmonella infection promotes the degradation and plasma sequestration of Axin1, leading to bacterial invasiveness and inflammatory responses. Vitamin D and the vitamin D receptor (VDR) appear to be important regulators of IBD and colon cancer. Although VDR and Axin1 are all involved in intestinal inflammation, it remains unclear whether these processes are related or function independently. In the current study, we hypothesize that VDR is an important regulator for the maintenance of physiological level of Axin1. MethodsUsing the intestinal epithelial conditional VDR knockout mouse model (VDRΔIEC) and cultured cell lines, influences of VDR status on the expression of Axin1 was evaluated by Western blots and real-time PCR. Loss- and gain-of-function assays were used to investigate the regulation of VDR on Axin1 at the transcriptional and translational levels. Cells were treated with cycloheximide or actinomycin for molecular mechanistic studies. Candidate genomic VDR binding sites for Axin1 were tested by chromatin immunoprecipitation (ChIP) assay. Physical interactions among VDR, Axin1, and β-catenin were tested by immunoprecipitation. Cellular localization of Axin1 with different VDR status was determined by fractionation and immunohistochemistry. ResultsWe found that VDR deletion led to lower protein and mRNA levels of Axin1, whereas knockdown of Axin1 did not change the expression level of VDR protein. Immunoprecipitation data did not support physical interaction between VDR and Axin1. The VDR regulation of Axin1 was through a VDR genomic binding site for Axin1 gene on the regulatory region. Fractionation data showed that cytosolic Axin1 was significantly reduced due to VDR deletion, leaving the nuclear fraction unchanged. In ileum, Axin1 was distributed in the cytosol of apical epithelium and crypts. ConclusionVDR is important for the maintenance of physiological level of Axin1. The discovery of Axin1 as a VDR target gene provides novel and fundamental insights into the interactions between the VDR and β-catenin signaling pathways.

Nutraceutical Improvement Increases the Protective Activity of Broccoli Sprout Juice in a Human Intestinal Cell Model of Gut Inflammation.

Benefits to health from a high consumption of fruits and vegetables are well established and have been attributed to bioactive secondary metabolites present in edible plants. However, the effects of specific health-related phytochemicals within a complex food matrix are difficult to assess. In an attempt to address this problem, we have used elicitation to improve the nutraceutical content of seedlings of Brassica oleracea grown under controlled conditions. Analysis, by LC-MS, of the glucosinolate, isothiocyanate and phenolic compound content of juices obtained from sprouts indicated that elicitation induces an enrichment of several phenolics, particularly of the anthocyanin fraction. To test the biological activity of basal and enriched juices we took advantage of a recently developed in vitro model of inflamed human intestinal epithelium. Both sprouts’ juices protected intestinal barrier integrity in Caco-2 cells exposed to tumor necrosis factor α under marginal zinc deprivation, with the enriched juice showing higher protection. Multivariate regression analysis indicated that the extent of rescue from stress-induced epithelial dysfunction correlated with the composition in bioactive molecules of the juices and, in particular, with a group of phenolic compounds, including several anthocyanins, quercetin-3-Glc, cryptochlorogenic, neochlorogenic and cinnamic acids.

Influence of sweet whey protein concentrate and its hydrolysates on host-pathogen interactions in the emerging foodborne pathogen Cronobacter sakazakii.

Antimicrobial resistance poses a significant global healthcare predicament. An attractive approach to the dilemma of drug-resistant bacteria is the development and use of agents that interfere with the ability of pathogens to adhere to human tissue. The influence of sweet whey protein concentrate (SWPC), and selected hydrolysates of this material, on host-pathogen interactions of Cronobacter sakazakii (ATCC 29544) was investigated. CaCo-2 cell line was selected as a suitable model for the human intestinal epithelium. Cronobacter sakazakiiATCC 29544 was identified as the strain with the highest adhesion efficiency. SWPC reduced its association by 80% (P<0·01), invasion 35% (P<0·01), and translocation >95% (P<0·001). SWPC enzymatically modified with lipase, trypsin and pepsin had variable effects on these behaviours with the most significant effect exhibited with the lipase treatment. SWPC produced an almost total inhibition of translocation of C.sakazakii across a CaCo-2 cell monolayer. Lipase and pepsin treated SWPC also reduced translocation by 75% and 90% respectively. However, trypsin treatment nullified the effect SWPC had on translocation. The presence of viable bacterial cells and SWPC both increased expression of IL-8 following Cronobacter invasion into CaCo-2 cells. Factors governing adherence, invasion and translocation of Cronobacter spp. to human intestinal cells are multi-factorial and digested milk products exhibit varying effects dependant on their enzyme modification and protein lipid content. These findings contribute to our, as yet, incomplete understanding of Cronobacter pathogenesis, and suggest that SWPC in whole and enzymatically hydrolysed forms, may provide a cost-effective source of bioactive materials with inhibitory effects on bacterial virulence.

The Ferroxidase Hephaestin (HEPH) is Not Required for Iron Efflux Under Conditions of Enhanced Iron Transport in an Established Model of the Human Intestinal Epithelium (Caco‐2 Cells)

The Ferroxidase Hephaestin (HEPH) is Not Required for Iron Efflux Under Conditions of Enhanced Iron Transport in an Established Model of the Human Intestinal Epithelium (Caco‐2 Cells)

Anti‐inflammatory properties of ginger extract in an in vitro model of inflamed human intestinal epithelium

Ginger is rich in phenolic compounds; the beneficial effect of ginger might be attributed not only to the antioxidant properties of the phenolic compounds but also to the modulation of signaling cascades in physiological and pathophysiological conditions such as inflammatory processes. This study assessed the potential anti‐inflammatory activity of a polyphenol‐enriched ginger extract in a cellular model of intestinal inflammation and the effects of ginger extract on tight junction (TJ) molecules by using cytokine‐stimulated Caco‐2 epithelial cells. To develop an in vitro model of the acute phase of intestinal inflammation, Caco‐2 cells were exposed to a mixture of inflammatory mediators such as interleukin (IL)‐1β, tumor necrosis factor (TNF)‐α, interferon (IFN)‐γ, and lipopolysaccharide (LPS). Responses to the mixture of inflammatory mediators were assessed by measuring the secretion level of IL‐6 and IL‐8 and mRNA expression of cyclooxygenase (Cox)‐2 and inducible nitric oxide synthase (iNOS). In addition, the level of nuclear factor (NF)‐κB activation and phosphorylation of mitogen‐activated protein kinase (MAPK) were examined. Transepithelial electrical resistance (TEER) and expression of TJ proteins, zonula occludens (ZO)‐1, ZO‐2, occludin, junctional adhesion molecule‐1 (JAM), claudin‐1, claudin‐3, and claudin‐4 were measured. Ginseng extract and its constituents such as 6‐shogaol and 6‐gingerol improved the inflammatory responses by decreasing NF‐κB activation and MAPK phosphorylation. It also significantly ameliorated iNOS and Cox‐2 mRNA expression levels. Ginger extract increased the TEER, indicating increased TJ integrity, and upregulated the mRNA expression levels of TJ proteins. These results show that ginger extract may be developed as a functional supplement to maintain gastrointestinal health.Support or Funding Information

Transport of six tyrosine kinase inhibitors: active or passive?

&lt;p class="ADMETabstracttext"&gt;Transport of erlotinib, gefitinib, sorafenib, sunitinib, dasatinib and crizotinib can be active or passive, which was studied by measuring uptake at low (4 °C; passive) and normal temperature (37 °C; active and passive) and by the use of specific organic cation transporter (OCT) inhibitors. Intracellular accumulation was determined using Caco-2 as monolayers, while for gut permeation we used differentiated Caco-2 as model for intestinal epithelium in the Transwell system. Sorafenib and crizotinib uptake are likely to be dependent on passive transport. Gefitinib, dasatinib and sunitinib uptake seem to be active. Erlotinib’s transport also seems to be active. This study suggests that hOCTs might be involved in the apical to basolateral transport of gefitinib and crizotinib. Overall it can be concluded that the accumulation and transport of these six TKIs are very different, despite the fact that they are all tyrosine kinase inhibitors.&lt;/p&gt;

Establishment of Caco-2 cell monolayer model and barrier permeability

Objective To establish and evaluate intestinal epithelial barrier model using Caco-2 cell so as to play a foundation for next study of barrier permeability. Methods Caco-2 cells were cultured in vitro then seeded into Transwell cell culture inserts.The permeability of the intestinal epithelial barrier was detected by transepithelial electrical resistance(TEER)and lucifer yellow flux, and verified by transmission electron microscope.Different concentrations of PAF(0, 50, 100, and 200 nmol/L)were exposed for 24 hours to Caco-2 monolayer when cultured 21 days.The tight junction was observed under transmission electron microscope.Assessment of ZO-1 protein localization and expression were detected by immunofluorescence and Western blot analysis. Results Cultured Caco-2 cell confluencd as monolayer with time passed.From 5th day, TEER increased, then reached 600Ω·cm2 at 15th day and lasted to 21st day, there was little flux of lucifer yellow, transmission electron microscopy also found cells differentiated better, had well-arranged villi and polarity alined as monolayer, forming completed tight junction which was the marker of intestinal epithelial barrier model in vitro.TEER decreased and lucifer yellow flux increased in cells exposed to PAF.The permeability reached the peak when exposed to 100 nmol/L PAF(P< 0.01), tight junction disrupted, ZO-1 protein expression downregulated, abnormal localization and distribution was assessed by immunofluorescence staining. Conclusion Cultured Caco-2 cells for 2-3w can be used to study intestinal epithelial barrier as a model in vitro.PAF increased intestinal epithelial permeability, which would correlate to the decreased protein expression and abnormal distribution of ZO-1. Key words: Human colon carcinoma cell line(Caco-2); Permeability; Transepithelial electrical resistance; Platelet-activating factor(PAF); ZO-1

TNF-α Modulation of Intestinal Tight Junction Permeability Is Mediated by NIK/IKK-α Axis Activation of the Canonical NF-κB Pathway

Tumor necrosis factor (TNF)-α, a key mediator of intestinal inflammation, causes an increase in intestinal epithelial tight junction (TJ) permeability by activating myosin light chain kinase (MLCK; official name MYLK3) gene. However, the precise signaling cascades that mediate the TNF-α-induced activation of MLCK gene and increase in TJ permeability remain unclear. Our aims were to delineate the upstream signaling mechanisms that regulate the TNF-α modulation of intestinal TJ barrier function with the use of invitro and invivo intestinal epithelial model systems. TNF-α caused a rapid activation of both canonical and noncanonical NF-κB pathway. NF-κB-inducing kinase (NIK) and mitogen-activated protein kinase kinase-1 (MEKK-1) were activated in response to TNF-α. NIK mediated the TNF-α activation of inhibitory κB kinase (IKK)-α, and MEKK1 mediated the activation of IKK complex, including IKK-β. NIK/IKK-α axis regulated the activation of both NF-κB p50/p65 and RelB/p52 pathways. Surprisingly, the siRNA induced knockdown of NIK, but not MEKK-1, prevented the TNF-α activation of both NF-κB p50/p65 and RelB/p52 and the increase in intestinal TJ permeability. Moreover, NIK/IKK-α/NF-κB p50/p65 axis mediated the TNF-α-induced MLCK gene activation and the subsequent MLCK increase in intestinal TJ permeability. In conclusion, our data show that NIK/IKK-α/regulates the activation of NF-κB p50/p65 and plays an integral role in the TNF-α-induced activation of MLCK gene and increase in intestinal TJ permeability.