Dongmei Ye

Mechanism of IL-1β-Induced Increase in Intestinal Epithelial Tight Junction Permeability

The IL-1β-induced increase in intestinal epithelial tight junction (TJ) permeability has been postulated to be an important mechanism contributing to intestinal inflammation of Crohn’s disease and other inflammatory conditions of the gut. The intracellular and molecular mechanisms that mediate the IL-1β-induced increase in intestinal TJ permeability remain unclear. The purpose of this study was to elucidate the mechanisms that mediate the IL-1β-induced increase in intestinal TJ permeability. Specifically, the role of myosin L chain kinase (MLCK) was investigated. IL-1β caused a progressive increase in MLCK protein expression. The time course of IL-1β-induced increase in MLCK level correlated linearly with increase in Caco-2 TJ permeability. Inhibition of the IL-1β-induced increase in MLCK protein expression prevented the increase in Caco-2 TJ permeability. Inhibition of the IL-1β-induced increase in MLCK activity also prevented the increase in Caco-2 TJ permeability. Additionally, knock-down of MLCK protein expression by small interference RNA prevented the IL-1β-induced increase in Caco-2 TJ permeability. The IL-1β-induced increase in MLCK protein expression was preceded by an increase in MLCK mRNA expression. The IL-1β-induced increase in MLCK mRNA transcription and subsequent increase in MLCK protein expression and Caco-2 TJ permeability was mediated by activation of NF-κB. In conclusion, our data indicate that the IL-1β increase in Caco-2 TJ permeability was mediated by an increase in MLCK expression and activity. Our findings also indicate that the IL-1β-induced increase in MLCK protein expression and Caco-2 TJ permeability was mediated by an NF-κB-dependent increase in MLCK gene transcription.

Occludin regulates macromolecule flux across the intestinal epithelial tight junction barrier

Defective intestinal epithelial tight junction (TJ) barrier has been shown to be an important pathogenic factor contributing to the development of intestinal inflammation. The expression of occludin is markedly decreased in intestinal permeability disorders, including in Crohns disease, ulcerative colitis, and celiac disease, suggesting that the decrease in occludin expression may play a role in the increase in intestinal permeability. The purpose of this study was to delineate the involvement of occludin in intestinal epithelial TJ barrier by selective knock down of occludin in in vitro (filter-grown Caco-2 monolayers) and in vivo (recycling perfusion of mouse intestine) intestinal epithelial models. Our results indicated that occludin small-interfering RNA (siRNA) transfection causes an increase in transepithelial flux of various-sized probes, including urea, mannitol, inulin, and dextran, across the Caco-2 monolayers, without affecting the transepithelial resistance. The increase in relative flux rate was progressively greater for larger-sized probes, indicating that occludin depletion has the greatest effect on the flux of large macromolecules. siRNA-induced knock down of occludin in mouse intestine in vivo also caused an increase in intestinal permeability to dextran but did not affect intestinal tissue transepithelial resistance. In conclusion, these results show for the first time that occludin depletion in intestinal epithelial cells in vitro and in vivo leads to a selective or preferential increase in macromolecule flux, suggesting that occludin plays a crucial role in the maintenance of TJ barrier through the large-channel TJ pathway, the pathway responsible for the macromolecule flux.

IL-1β-Induced Increase in Intestinal Epithelial Tight Junction Permeability Is Mediated by MEKK-1 Activation of Canonical NF-κB Pathway

IL-1β is a proinflammatory cytokine that plays a central role in the inflammatory process of the gut. IL-1β causes an increase in intestinal epithelial tight junction (TJ) permeability, but the intracellular pathways that mediate intestinal TJ permeability remain unclear. The major aims of this study were to delineate the protein kinases that regulate the IL-1β modulation of intestinal TJ barrier function and to determine the intracellular mechanisms involved, using filter-grown Caco-2 monolayers as the in vitro model system. Our results showed that IL-1β caused a rapid activation of MEKK-1 and NIK. The knockdown of MEKK-1, but not NIK, inhibited the IL-1β increase in Caco-2 TJ permeability. IL-1β caused an activation of both canonical and noncanonical NF-κB pathways; MEKK-1 regulated the activation of the canonical pathway, while NIK regulated the activation of the noncanonical pathway. Inhibition of MEKK-1 activation of the canonical pathway prevented the IL-1β increase in TJ permeability. Our data also indicated that inhibitory κB kinase was the catalytic subunit primarily involved in canonical pathway activation and TJ barrier opening. MEKK-1 also played an essential role in myosin light chain kinase gene activation. In conclusion, our data show for the first time that MEKK-1 plays an integral role in IL-1β modulation of Caco-2 TJ barrier function by regulating the activation of the canonical NF-κB pathway and the MLCK gene.

TNF-α modulation of intestinal epithelial tight junction barrier is regulated by ERK1/2 activation of Elk-1.

Tumor necrosis factor (TNF-α) is a proinflammatory cytokine that plays a critical role in the pathogenesis of inflammatory bowel disease. TNF-α causes an increase in intestinal permeability; however, the signaling pathways and the molecular mechanisms involved remain unclear. The major purpose of this study was to investigate the role of MAP kinase pathways (ERK1/2 and p38 kinase) and the molecular processes involved. An in vitro intestinal epithelial model system consisting of Caco-2 monolayers and an in vivo mouse model system were used to delineate the cellular and molecular mechanisms involved in TNF-α effects on tight junction barrier. The TNF-α-induced increase in Caco-2 tight junction permeability was mediated by activation of the ERK1/2 signaling pathway, but not the p38 kinase pathway. Activation of the ERK1/2 pathway led to phosphorylation and activation of the ETS domain-containing transcription factor Elk-1. The activated Elk-1 translocated to the nucleus, where it bound to its binding motif on the myosin light chain kinase (MLCK) promoter region, leading to the activation of MLCK promoter activity and gene transcription. In addition, in vivo intestinal perfusion studies also indicated that the TNF-α-induced increase in mouse intestinal permeability requires ERK1/2-dependent activation of Elk-1. These studies provide novel insight into the cellular and molecular processes that regulate the TNF-α-induced increase in intestinal epithelial tight junction permeability.

Interleukin-6 modulation of intestinal epithelial tight junction permeability is mediated by JNK pathway activation of claudin-2 gene.

Defective intestinal epithelial tight junction (TJ) barrier has been shown to be a pathogenic factor in the development of intestinal inflammation. Interleukin-6 (IL-6) is a pleiotropic, pro-inflammatory cytokine which plays an important role in promoting inflammatory response in the gut and in the systemic circulation. Despite its key role in mediating variety inflammatory response, the effect of IL-6 on intestinal epithelial barrier remains unclear. The purpose of this study was to investigate the effect of IL-6 on intestinal epithelial TJ barrier and to delineate the intracellular mechanisms involved using in-vitro (filter-grown Caco-2 monolayers) and in-vivo model (mouse intestinal perfusion) systems. Our results indicated that IL-6 causes a site-selective increase in Caco-2 intestinal epithelia TJ permeability, causing an increase in flux of small-sized molecules having molecular radius <4 Å. The size-selective increase in Caco-2 TJ permeability was regulated by protein-specific increase in claudin-2 expression. The IL-6 increase in TJ permeability required activation of JNK signaling cascade. The JNK pathway activation of AP-1 resulted in AP-1 binding to its binding sequence on the claudin-2 promoter region, leading to promoter activation and subsequent increase in claudin-2 gene transcription and protein synthesis and TJ permeability. Our in-vivo mouse perfusion showed that IL-6 modulation of mouse intestinal permeability was also mediated by AP-1 dependent increase in claudin-2 expression. In conclusion, our studies show for the first time that the IL-6 modulation of intestinal TJ permeability was regulated by JNK activation of AP-1 and AP-1 activation of claudin-2 gene.

Mechanism of IL-1β Modulation of Intestinal Epithelial Barrier Involves p38 Kinase and Activating Transcription Factor-2 Activation

The defective intestinal epithelial tight junction (TJ) barrier has been postulated to be an important pathogenic factor contributing to intestinal inflammation. It has been shown that the proinflammatory cytokine IL-1β causes an increase in intestinal permeability; however, the signaling pathways and the molecular mechanisms involved remain unclear. The major purpose of this study was to investigate the role of the p38 kinase pathway and the molecular processes involved. In these studies, the in vitro intestinal epithelial model system (Caco-2 monolayers) was used to delineate the cellular and molecular mechanisms, and a complementary in vivo mouse model system (intestinal perfusion) was used to assess the in vivo relevance of the in vitro findings. Our data indicated that the IL-1β increase in Caco-2 TJ permeability correlated with an activation of p38 kinase. The activation of p38 kinase caused phosphorylation and activation of p38 kinase substrate, activating transcription factor (ATF)-2. The activated ATF-2 translocated to the nucleus where it attached to its binding motif on the myosin L chain kinase (MLCK) promoter region, leading to the activation of MLCK promoter activity and gene transcription. Small interfering RNA induced silencing of ATF-2, or mutation of the ATF-2 binding motif prevented the activation of MLCK promoter and MLCK mRNA transcription. Additionally, in vivo intestinal perfusion studies also indicated that the IL-1β increase in mouse intestinal permeability required p38 kinase–dependent activation of ATF-2. In conclusion, these studies show that the IL-1β–induced increase in intestinal TJ permeability in vitro and in vivo was regulated by p38 kinase activation of ATF-2 and by ATF-2 regulation of MLCK gene activity.

Cellular and molecular mechanisms that mediate basal and tumour necrosis factor‐α‐induced regulation of myosin light chain kinase gene activity

The patients with Crohns disease (CD) have a ‘leaky gut’ manifested by an increase in intestinal epithelial tight junction (TJ) permeability. Tumour necrosis factor‐α (TNF‐α) is a proto‐typical pro‐inflammatory cytokine that plays a central role in intestinal inflammation of CD. An important pro‐inflammatory action of TNF‐α is to cause a functional opening of intestinal TJ barrier. Previous studies have shown that TNF‐α increase in TJ permeability was regulated by an increase in myosin light chain kinase (MLCK) gene activity and protein expression. The major aim of this study was to elucidate the cellular and molecular mechanisms that mediate basal and TNF‐α‐induced increase in MLCK gene activity. By progressive 5′ deletion, minimal MLCK promoter was localized between −313 to +118 on MLCK promoter. A p53 binding site located within minimal promoter region was identified as an essential determinant for basal promoter activity. A 4 bp start site and a 5 bp downstream promoter element were required for MLCK gene activity. TNF‐α‐induced increase in MLCK promoter activity was mediated by NF‐κB activation. There were eight κB binding sites on MLCK promoter. The NF‐κB1 site at +48 to +57 mediated TNF‐α‐induced increase in MLCK promoter activity. The NF‐κB2 site at −325 to −316 had a repressive role on promoter activity. The opposite effects on promoter activity were due to differences in the NF‐κB dimer type binding to the κB sites. p50/p65 dimer preferentially binds to the NF‐κB1 site and up‐regulates promoter activity; while p50/p50 dimer preferentially binds to the NF‐κB2 site and down‐regulates promoter activity. In conclusion, we have identified the minimal MLCK promoter region, essential molecular determinants and molecular mechanisms that mediate basal and TNF‐α‐induced modulation of MLCK promoter activity in Caco‐2 intestinal epithelial cells. These studies provide novel insight into the cellular and molecular mechanisms that regulate basal and TNF‐α‐induced modulation of MLCK gene activity.

Cellular and molecular mechanisms of heat stress-induced up-regulation of occludin protein expression: regulatory role of heat shock factor-1.

The heat stress (HS)-induced increase in occludin protein expression has been postulated to be a protective response against HS-induced disruption of the intestinal epithelial tight junction barrier. The aim of this study was to elucidate the cellular and molecular processes that mediate the HS-induced up-regulation of occludin expression in Caco-2 cells. Exposure to HS (39 degrees C or 41 degrees C) resulted in increased expression of occludin protein; this was preceded by an increase in occludin mRNA transcription and promoter activity. HS-induced activation of heat shock factor-1 (HSF-1) resulted in cytoplasmic-to-nuclear translocation of HSF-1 and binding to its binding motif in the occludin promoter region. HSF-1 activation was associated with an increase in occludin promoter activity, mRNA transcription, and protein expression; which were abolished by the HSF-1 inhibitor quercetin. Targeted HSF-1 knock-down by siRNA transfection inhibited the HSF-1-induced increase in occulin expression and junctional localization of occulin protein. Site-directed mutagenesis of the HSF-1 binding motif in the occludin promoter region inhibited HS-induced binding of HSF-1 to the occludin promoter region and subsequent promoter activity. In conclusion, our data show for the first time that the HS-induced increase in occludin protein expression is mediated by HSF-1 activation and subsequent binding of HSF-1 to the occludin promoter, which initiates a series of molecular and cellular events culminating in increased junctional localization of occludin protein.

Cellular and molecular mechanism of interleukin‐1β modulation of Caco‐2 intestinal epithelial tight junction barrier

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.

Mechanism of interleukin-1β induced-increase in mouse intestinal permeability in vivo.

UNLABELLED Interleukin-1β (IL-1β) has been shown to play an essential role in mediating intestinal inflammation of Crohns disease and other inflammatory conditions of the gut. Previous studies from our laboratory have shown that IL-1β causes an increase in intestinal tight-junction permeability in Caco-2 monolayers in vitro. However, the IL-1β effect on the intestinal epithelial barrier in vivo remains unclear. AIMS the major aims of this study were to examine the effect of IL-1β on mouse intestinal epithelial barrier in vivo and to delineate the mechanisms involved using an in vivo model system consisting of a recycling perfusion of mouse small intestine. Intraperitonial injection of IL-1β at varying doses (0-10 μg) caused a concentration-dependent increase in mouse intestinal permeability to the paracellular marker dextran (10 KD), and the maximal increase in dextran flux occurred at IL-1β dose of 5 μg. IL-1β treatment caused an increase in myosin light-chain kinase (MLCK) mRNA and protein expression in the small intestinal tissue starting at 24 h, which continued up to 72 h. Additionally, IL-1β did not cause an increase in intestinal permeability in MLCK-deficient mice (C57BL/6 MLCK(-/-)). MLCK inhibitor ML-7 (2 mg/kg body weight) also inhibited the IL-1β-induced increase in small intestinal permeability. The IL-1β-induced increase in mouse intestinal permeability was associated with an increase in NF-κB activation. The intestinal tissue-specific silencing of NF-κB p65 inhibited the IL-1β-induced increase in intestinal permeability and increase in MLCK expression. These data show for the first time that IL-1β causes an increase in mouse intestinal permeability in vivo. These data suggested that the mechanism of IL-1β-induced increase in mouse intestinal permeability in vivo involved NF-κB p65-induced activation of the mouse enterocyte MLCK gene.