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Background: Defective intestinal epithelial tight junction (TJ), characterized by an increase in intestinal TJ permeability, has been shown to play a critical role in the pathogenesis of inflammatory bowel disease (IBD). Tumor necrosis factor-a (TNF-α) is a key pro-inflammatory cytokine involved in the immunopathology of IBD and has been shown to cause an increase in intestinal epithelial TJ permeability. Although TNF-α antibodies and other biologics have been advanced for use in IBD treatment, these therapies are associated with severe side effects and have limited efficacy, and there is an urgent need for therapieswith benign profiles and high therapeutic efficacy. Probiotic bacteria have beneficial effects and are generally safe and represent an important class of potential therapeutic agents in IBD. Lactobacillus acidophilus (LA) is one of the most used probiotics for wide-ranging health benefits, including in gastrointestinal, metabolic, and inflammatory disorders. A specific strain of LA, LA1, was recently demonstrated to have protective and therapeutic effects on the intestinal epithelial TJ barrier. However, the mechanisms of actions of LA1 remain largely unknown. Methods: The primary aim of this study was to investigate microbial-epithelial interactions and novel signaling pathways that regulate the effect of LA1 on TNFa- induced increase in intestinal epithelial TJ permeability, using cell culture and animal model systems. Results and Conclusion: Pre-treatment of filter-grown Caco-2 monolayers with LA1 prevented the TNF-α-induced increase in intestinal epithelial TJ permeability by inhibiting TNF-α-induced activation of NF-kB p50/p65 and myosin light chain kinase (MLCK) gene and kinase activity in a TLR-2-dependent manner. LA1 produced a TLR-2- and MyD88-dependent activation of NF-kB p50/p65 in immune cells; however, LA1, in intestinal cells, inhibited the NF-kB p50/p65 activation in a TLR-2-dependent but MyD88-independent manner. In addition, LA1 inhibition of NF-kB p50/p65 and MLCK gene was mediated by TLR-2 pathway activation of phosphatidylinositol 3-kinase (PI3K) and IKK-a phosphorylation. Our results demonstrated novel intracellular signaling pathways by which LA1/TLR-2 suppresses the TNF-α pathway activation of NF-kB p50/p65 in intestinal epithelial cells and protects against the TNF-α-induced increase in intestinal epithelial TJ permeability. [ABSTRACT FROM AUTHOR]

Interleukin-1β (IL-1β) is a prototypical multifunctional cytokine that plays an important role in intestinal inflammation of Crohn's disease and other inflammatory conditions of the gut. Previous studies have shown that IL-1β causes an increase in intestinal epithelial tight junction (TJ) permeability both in in vivo animal and in vitro cell culture model systems. The IL-1β-induced increase in intestinal epithelial TJ permeability has been postulated to be an important pathogenic mechanism contributing to intestinal inflammation. However, the signalling pathways and the molecular processes that mediate the IL-1β modulation of intestinal epithelial TJ barrier remain unclear. Here, we show that the IL-1β-induced increase in Caco-2 monolayer TJ permeability was mediated by activation of extracellular signal-regulated kinases 1/2 (ERK1/2) signalling pathway and that inhibition of ERK1/2 activity inhibits the IL-1β-induced increase in Caco-2 TJ permeability. The activation of ERK1/2 pathway caused a downstream activation of nuclear transcription factor Elk-1. The activated Elk-1 translocated to the nucleus and binds to the cis-binding motif on myosin light chain kinase (MLCK) promoter region, triggering MLCK gene activation, MLCK mRNA transcription and MLCK protein synthesis and MLCK catalysed opening of the intestinal epithelial TJ barrier. These studies provide novel insight into the cellular and molecular processes that mediate the IL-1β-induced increase in intestinal epithelial TJ permeability.

The reduced folate carrier (RFC) is a major folate transport system in mammalian cells. RFC is highly expressed in the intestine and believed to play a role in folate absorption. Studies from our laboratory and others have characterized different aspects of the intestinal folate absorption process, but little is known about possible existence of accessory protein(s) that interacts with RFC and influences its physiology and/or cell biology. We investigated this issue by employing a bacterial two-hybrid system to screen a BacterioMatch II human intestinal cDNA library using the large intracellular loop between transmembrane domains 6 and 7 of the human RFC (hRFC) as bait. Our screening has resulted in the identification of dynein light chain road block-1 (DYNLRB1) as an interacting partner with hRFC. Existence of a direct protein-protein interaction between hRFC and DYNLRB1 was confirmed by in vitro pull-down assay and in vivo mammalian two-hybrid luciferase assay and coimmunoprecipitation analysis. Furthermore, confocal imaging of live human intestinal epithelial HuTu-80 cells demonstrated colocalization of DYNLRB1 with hRFC. Coexpression of DYNLRB1 with hRFC led to a significant (P < 0.05) increase in folate uptake. On the other hand, inhibiting the endogenous DYNLRB1 with gene-specific small interfering RNA or pharmacologically with a specific inhibitor (vanadate) led to a significant (P < 0.05) decrease in folate uptake. This study demonstrates for the first time the identification of DYNLRB1 as an interacting protein partner with hRFC. Furthermore, DYNLRB1 appears to influence the function and cell biology of hRFC. folate transport; protein-protein interactions; two-hybrid analysis; accessory proteins

The human sodium-dependent multivitamin transporter (hSMVT) mediates sodium-dependent uptake of biotin in renal and intestinal epithelia. To date, however, there is nothing known about the structure-function relationship or targeting sequences in the hSMVT polypeptide that control its polarized expression within epithelia. Here, we focused on the role of the COOH-terminal tail of hSMVT in the targeting and functionality of this transporter. A full-length hSMVT-green fluorescent protein (GFP) fusion protein was functional and expressed at the apical membrane in renal and intestinal cell lines. Microtubule disrupting agents disrupted the mobility of trafficking vesicles and impaired cell surface delivery of hSMVT, which was also prevented in cells treated with dynamitin (p50), brefeldin, or monensin. Progressive truncation of the COOH-terminal tail impaired the functionality and targeting of the transporter. First, biotin transport decreased by approximately 20-30% on deletion of up to 15 COOH-terminal amino acids of hSMVT, a decrease mimicked solely by deletion of the terminal PDZ motif (TSL). Second, deletions into the COOH-terminal tail (between residues 584-612, containing a region of predicted high surface accessibility) resulted in a further drop in hSMVT transport (to ~40% of wild-type). Third, apical targeting was lost on deletion of a helical-prone region between amino acids 570-584. We conclude that the COOH tail of hSMVT contains several determinants important for polarized targeting and biotin transport. biotin; epithelial polarity; vitamin; microtubule

Occludin, a transmembrane (TM)-spanning protein, is an integral component of the tight junctional (TJ) complexes that regulate epithelial integrity and paracellular barrier function. However, the molecular determinants that dictate occludin targeting and delivery to the TJs remain unclear. Here, using live cell imaging of yellow fluorescent protein-labeled occludin fragments, we resolved the intracellular trafficking of occludin-fusion proteins in polarized Madin-Darby canine kidney and Caco-2 cells to delineate the regions within the occludin polypeptide that are important for occludin targeting to the TJs. Live cell confocal imaging showed that complete or partial truncation of the COOH-terminal tail of the occludin polypeptide did not prevent occludin targeting to the TJs in epithelial cell lines. Progressive truncations into the COOH-terminal tail decreased the efficiency of occludin expression; after the removal of the regions proximal to the fourth transmembrane domain (TM4), the efficiency of expression increased. However, further deletions into the TM4 abolished TJ targeting, which resulted in constructs that were retained intracellularly within the endoplasmic reticulum. The full-length occludin polypeptide trafficked to the cell surface within a heterogenous population of intracellular vesicles that delivered occludin to the plasma membrane in a microtubule- and temperature-dependent manner. In contrast, the steady-state localization of occludin at the cell surface was dependent on intact microfilaments but not microtubules. epithelium

A defective intestinal epithelial fight junction (TJ) barrier has been proposed as an important pathogenic factor contributing to the intestinal inflammation of Crohn's disease. Glucocorticoids are first-line therapeutic agents for the treatment of moderate to severe Crohn's disease. Glucocorticoid treatment has been shown to induce retightening of the intestinal TJ barrier defect in Crohn's disease patients. However, the mechanisms that mediate the glucocorticoid therapeutic action on intestinal TJ barrier function remain unknown. The aim of this study was to elucidate the mechanism of glucocorticoid modulation of the intestinal epithelial TJ barrier using an in vitro model system. Filter-grown Caco-2 intestinal epithelial cells were used as an in vitro model to examine the effects of glucocorticoids on basal intestinal epithelial TJ barrier function and on TNF-[alpha]-induced disruption of the TJ barrier. Glucocorticoids (prednisolone and dexamethasone) did not have a significant effect on baseline Caco-2 TJ barrier function but prevented the TNF-[alpha]-induced increase in Caco-2 TJ permeability. The glucocorticoid protective effect against the TNF-[alpha]-induced increase in Caco-2 TJ permeability required activation of the glucocorticoid receptor (GR) complex. The activation of the GR complex resulted in GR complex binding to the glucocorticoid response element (GRE) site on DNA and activation of a GR-responsive promoter. Glucocorticoids inhibited the TNF-[alpha]-induced increase in myosin light chain kinase (MLCK) protein expression, a key process mediating the TNF-[alpha] increase in intestinal TJ permeability. The glucocorticoid inhibition of the TNF-[alpha]-induced increase in MLCK protein expression was due to the binding of the GR complex to a GRE binding site on the MLCK promoter region suppressing the TNF-[alpha]-induced activation. Glucocorticoids inhibit the TNF-[alpha]-induced increase in Caco-2 TJ permeability. The prednisolone protective action was mediated by binding of activated GR complex to the GRE site on the MLCK promoter, suppressing the TNF-[alpha]-induced increase in MLCK gene activity, protein expression, and subsequent opening of the intestinal TJ barrier. prednisolone; inflammation; tumor necrosis factor-a; glucocorticoid receptor; glucocorticoid response element; Crohn's disease

A TNF-[alpha]-induced increase in intestinal epithelial tight junction (TJ) permeability has been proposed to be an important proinflammatory mechanism contributing to intestinal inflammation in Crohn's disease and other inflammatory conditions. Previous studies from our laboratory suggested that the TNF-[alpha]-induced increase in intestinal TJ permeability was mediated by an increase in myosin light chain kinase (MLCK) protein expression. However, the molecular mechanisms that mediate the TNF-[alpha] increase in intestinal TJ permeability and MLCK protein expression remain unknown. The purpose of this study was to delineate the intracellular and molecular mechanisms that mediate the TNF-[alpha]-induced increase in intestinal TJ permeability; using an in vitro intestinal epithelial model system consisting of filter-grown Caco-2 intestinal epithelial monolayers. To examine the molecular mechanisms involved in the TNF-[alpha] regulation of intestinal TJ barrier, we identified and cloned for the first time a functionally active MLCK promoter region. TNF-[alpha] treatment of filter-grown Caco-2 monolayers transfected with plasmid vector containing the MLCK promoter region produced an increase in MLCK promoter activity and MLCK transcription. The TNF-[alpha]-induced increase in MLCK transcription corresponded to a sequential increase in MLCK protein expression, MLCK activity, and Caco-2 TJ permeability. The TNF-[alpha]-induced increase in MLCK promoter activity was mediated by NF-[kappa]B activation, and the inhibition of NF-[kappa]B activation prevented the TNF-[alpha]-induced increase in promoter activity and the subsequent increase in MLCK protein expression and Caco-2 TJ permeability. The TNF-[alpha]-induced activation of MLCK promoter was mediated by binding of the activated NF-[kappa]B p50/p65 dimer to the downstream [kappa]B binding site (-84 to -75) on the MLCK promoter region; deletion of the [kappa]B binding site prevented the TNF-[alpha] increase in promoter activity. Additionally, siRNA silencing of NF-[kappa]B p65 also prevented the TNF-[alpha] increase in MLCK promoter activity. In conclusion, our findings indicated that the TNF-[alpha]-induced increase in intestinal epithelial TJ permeability was mediated by NF-[kappa]B p50/p65 binding and activation of the MLCK promoter. NF-[kappa]B p50/p65 activation of the MLCK promoter then leads to a stepwise increase in MLCK transcription, expression and activity, and MLCK-mediated opening of the intestinal TJ barrier. Crohn's disease: intestinal permeability; myosin light chain kinase

The effects of physiologically relevant increase in temperature (37-41 [degrees]C) on intestinal epithelial tight junction (TJ) barrier have not been previously studied. Additionally, the role of heat-shock proteins (HSPs) in the regulation of intestinal TJ barrier during heat stress remains unknown. Because heat-induced disturbance of intestinal TJ barrier could lead to endotoxemia and bacterial translocation during physiological thermal stress, the purpose of this study was to investigate the effects of modest, physiologically relevant increases in temperature (37-41[degrees]C) on intestinal epithelial TJ barrier and to examine the protective role of HSPs on intestinal TJ barrier. Filter-grown Caco-2 intestinal epithelial cells were used as an in vitro intestinal epithelial model system to assess the effects of heat exposure on intestinal TJ barrier. Exposure of filter-grown Caco-2 monolayers to modest increases in temperatures (37-41[degrees]C) resulted in a significant time- and temperature-dependent increases in Caco-2 TJ permeability. Exposure to modest heat (39 or 41[degrees]C) resulted in rapid and sustained increases in HSP expression; and inhibition of HSP expression produced a marked increase in heat-induced increase in Caco-2 TJ permeability (P < 0.001). Heat exposure (41[degrees]C) resulted in a compensatory increase in Caco-2 occludin protein expression and an increase in junctional localization. Inhibition of HSP expression prevented the compensatory upregulation of occludin protein expression and produced a marked disruption in junctional localization of occludin protein during heat stress. In conclusion, our findings demonstrate for the first time that a modest, physiologically relevant increase in temperature causes an increase in intestinal epithelial TJ permeability. Our data also show that HSPs play an important protective role in preventing the heat-induced disruption of intestinal TJ barrier and suggest that HSP mediated upregulation of occludin expression may be an important mechanism involved in the maintenance of intestinal epithelial TJ barrier function during heat stress. Caco-2; heat-shock protein; tight junction protein

After the liver, the pancreas contains the second highest level of folate among human tissues, and folate deficiency adversely affects its physiological function. Despite that, nothing is currently known about the cellular mechanisms involved in folate uptake by cells of this important exocrine organ or about folate uptake regulation. We have begun to address these issues, and in this report we present the results of our findings on the mechanism of folate uptake by the human-derived pancreatic MIA PaCa-2 cells. Our results show folic acid uptake to be 1) temperature and energy dependent; 2) pH dependent, with a markedly higher uptake at acidic pH compared with neutral or alkaline pH; 3) [Na.sup.+] independent; 4) saturable as a function of substrate concentration (apparent [K.sub.m] = 0.762 [+ or -] 0.10 [micro]M); 5) inhibited (with similar affinity) by reduced, substituted, and oxidized folate derivatives; and 6) sensitive to the inhibitory effect of anion transport inhibitors. RT-PCR and Western blot analysis showed expression of the human reduced folate carrier (hRFC) at the RNA and protein levels, respectively. The functional contribution of hRFC in carrier-mediated folate uptake was confirmed by gene silencing using gene-specific small interfering RNA. Evidence also was found suggesting that the folate uptake process by MIA PaCa-2 cells is regulated by cAMP- and protein tyrosine kinase (PTK)-mediated pathways. These studies demonstrate for the first time the involvement of a specialized, acidic pH-dependent, carrier-mediated mechanism for folate uptake by human pancreatic MIA PaCa-2 cells. The results also show the involvement of hRFC in the uptake process and suggest the possible involvement of intracellular cAMP- and PTK-mediated pathways in the regulation of folate uptake. human reduced folate carrier; small interfering RNA; transport regulation

Crohn's disease (CD) patients have an abnormal increase in intestinal epithelial permeability. The defect in intestinal tight junction (TJ) barrier has been proposed as an important etiologic factor of CD. TNF-[alpha] increases intestinal TJ permeability. Because TNF-[alpha] levels are markedly increased in CD, TNF-[alpha] increase in intestinal TJ permeability could be a contributing factor of intestinal permeability defect in CD. Our purpose was to determine some of the intracellular mechanisms involved in TNF-[alpha] modulation of intestinal epithelial TJ permeability by using an in vitro intestinal epithelial system consisting of filter-grown Caco-2 monolayers. TNF-[alpha] produced a concentration- and time-dependent increase in Caco-2 TJ permeability. TNF-[alpha]-induced increase in Caco-2 TJ permeability correlated with Caco-2 NF-[kappa]B activation. Inhibition of TNF-[alpha]-induced NF-[kappa]B activation by selected NF-[kappa]B inhibitors, curcumin and triptolide, prevented the increase in Caco-2 TJ permeability, indicating that NF-[kappa]B activation was required for the TNF-[alpha]-induced increase in Caco-2 TJ permeability. This increase in Caco-2 TJ permeability was accompanied by downregulation of zonula occludens (ZO)-1 proteins and alteration in junctional localization of ZO-1 proteins. TNF-[alpha] modulation of ZO-1 protein expression and junctional localization were also prevented by NF-[kappa]B inhibitors. TNF-[alpha] did not induce apoptosis in Caco-2 cells, suggesting that apoptosis was not the mechanism involved in TNF[alpha]-induced increase in Caco-2 TJ permeability. These results demonstrate for the first time that TNF-[alpha]-induced increase in Caco-2 TJ permeability was mediated by NF-[kappa]B activation. The increase in permeability was associated with NF-KB-dependent downregulation of ZO-1 protein expression and alteration in junctional localization. intestinal permeability; tumor necrosis factor-[alpha]