Mechanism of glucocorticoid regulation of the intestinal tight junction barrier

A defective intestinal epithelial tight 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.     

TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation

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 down-regulation 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-kappa B-dependent downregulation of ZO-1 protein expression and alteration in junctional localization.     

Cellular and molecular mechanisms that mediate basal and tumour necrosis factor-alpha-induced regulation of myosin light chain kinase gene activity.

The patients with Crohn's disease (CD) have a 'leaky gut' manifested by an increase in intestinal epithelial tight junction (TJ) permeability. Tumour necrosis factor-alpha (TNF-alpha) is a proto-typical pro-inflammatory cytokine that plays a central role in intestinal inflammation of CD. An important pro-inflammatory action of TNF-alpha is to cause a functional opening of intestinal TJ barrier. Previous studies have shown that TNF-alpha 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-alpha-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-alpha-induced increase in MLCK promoter activity was mediated by NF-kappaB activation. There were eight kappaB binding sites on MLCK promoter. The NF-kappaB1 site at +48 to +57 mediated TNF-alpha-induced increase in MLCK promoter activity. The NF-kappaB2 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-kappaB dimer type binding to the kappaB sites. p50/p65 dimer preferentially binds to the NF-kappaB1 site and up-regulates promoter activity; while p50/p50 dimer preferentially binds to the NF-kappaB2 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-alpha-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-alpha-induced modulation of MLCK gene activity.     

Cytochalasin B modulation of Caco-2 tight junction barrier: role of myosin light chain kinase

The intracellular mechanisms that mediate cytochalasin-induced increase in intestinal epithelial tight junction (TJ) permeability are unclear. In this study, we examined the involvement of myosin light chain kinase (MLCK) in this process, using the filter-grown Caco-2 intestinal epithelial monolayers. Cytochalasin B (Cyto B) (5 microg/ml) produced an increase in Caco-2 MLCK activity, which correlated with the increase in Caco-2 TJ permeability. The inhibition of Cyto B-induced MLCK activation prevented the increase in Caco-2 TJ permeability. Additionally, myosin-Mg(2+)-ATPase inhibitor and metabolic inhibitors (which inhibit MLCK induced actin-myosin contraction) also prevented the Cyto B-induced increase in Caco-2 TJ permeability. Cyto B caused a late-phase (15-30 min) aggregation of actin fragments into large actin clumps, which was also inhibited by MLCK inhibitors. Cyto B produced a morphological disturbance of the ZO-1 TJ proteins, visually correlating with the functional increase in Caco-2 TJ permeability. The MLCK and myosin-Mg(2+)-ATPase inhibitors prevented both the functional increase in TJ permeability and disruption of ZO-1 proteins. These findings suggested that Cyto B-induced increase in Caco-2 TJ permeability is regulated by MLCK activation.     

Physiologically relevant increase in temperature causes an increase in intestinal epithelial tight junction permeability

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.     

IL-1beta causes an increase in intestinal epithelial tight junction permeability

IL-1beta is a prototypical proinflammatory cytokine that plays a central role in the intestinal inflammation amplification cascade. Recent studies have indicated that a TNF-alpha- and IFN-gamma-induced increase in intestinal epithelial paracellular permeability may be an important mechanism contributing to intestinal inflammation. Despite its central role in promoting intestinal inflammation, the role of IL-1beta on intestinal epithelial tight junction (TJ) barrier function remains unclear. The major aims of this study were to determine the effect of IL-1beta on intestinal epithelial TJ permeability and to elucidate the mechanisms involved in this process, using a well-established in vitro intestinal epithelial model system consisting of filter-grown Caco-2 intestinal epithelial monolayers. IL-1beta (0-100 ng/ml) produced a concentration- and time-dependent decrease in Caco-2 transepithelial resistance. Conversely, IL-1beta caused a progressive time-dependent increase in transepithelial permeability to paracellular marker inulin. IL-1beta-induced increase in Caco-2 TJ permeability was accompanied by a rapid activation of NF-kappaB. NF-kappaB inhibitors, pyrrolidine dithiocarbamate and curcumin, prevented the IL-1beta-induced increase in Caco-2 TJ permeability. To further confirm the role of NF-kappaB in the IL-1beta-induced increase in Caco-2 TJ permeability, NF-kappaB p65 expression was silenced by small interfering RNA transfection. NF-kappaB p65 depletion completely inhibited the IL-1beta-induced increase in Caco-2 TJ permeability. IL-1beta did not induce apoptosis in the Caco-2 cell. In conclusion, our findings show for the first time that IL-1beta at physiologically relevant concentrations causes an increase in intestinal epithelial TJ permeability. The IL-1beta-induced increase in Caco-2 TJ permeability was mediated in part by the activation of NF-kappaB pathways but not apoptosis.     

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 Crohn's 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.     

Interleukin-6 (IL-6) regulates claudin-2 expression and tight junction permeability in intestinal epithelium

In inflammatory bowel diseases (IBD), intestinal barrier function is impaired as a result of deteriorations in epithelial tight junction (TJ) structure. IL-6, a pleiotropic cytokine, is elevated in IBD patients, although the role of IL-6 in barrier function remains unknown. We present evidence that IL-6 increases TJ permeability by stimulating the expression of channel-forming claudin-2, which is required for increased caudal-related homeobox (Cdx) 2 through the MEK/ERK and PI3K pathways in intestinal epithelial cells. IL-6 increases the cation-selective TJ permeability without any changes to uncharged dextran flux or cell viability in Caco-2 cells. IL-6 markedly induces claudin-2 expression, which is associated with increased TJ permeability. The colonic mucosa of mice injected with IL-6 also exhibits an increase in claudin-2 expression. The claudin-2 expression and TJ permeability induced by IL-6 are sensitive to the inhibition of gp130, MEK, and PI3K. Furthermore, expression of WT-MEK1 induces claudin-2 expression in Caco-2 cells. Claudin-2 promoter activity is increased by IL-6 in a MEK/ERK and PI3K-dependent manner, and deletion of Cdx binding sites in the promoter sequence results in a loss of IL-6-induced promoter activity. IL-6 increases Cdx2 protein expression, which is suppressed by the inhibition of MEK and PI3K. These observations may reveal an important mechanism by which IL-6 can undermine the integrity of the intestinal barrier.     

Effect of neuronal PC12 cells on the functional properties of intestinal epithelial Caco-2 cells

The effect of neuronal cells on the functional properties of intestinal epithelial cells was examined by using an in vitro coculture system. Two cell lines, Caco-2 and PC12, were respectively used as intestinal epithelial and enteric neuronal cell models. Coculture of differentiated Caco-2 cells with PC12 caused a significant decrease in the transepithelial electrical resistance (TER) value of the Caco-2 monolayer. The permeability to lucifer yellow (LY) was also significantly increased, suggesting that the tight junction (TJ) of the Caco-2 monolayers was modulated by coculturing with PC12. To identify the TJ-modulating factor presumably secreted from PC12, the effects of the major neurotransmitters on the TER value and LY transport were examined, but no influence was apparent. The TJ-modulating effect of PC12 was prevented by exposing PC12 to cycloheximide, suggesting that new protein synthesis in PC12 was necessary for this regulation.     

Protein kinase C-alpha mediates endothelial barrier dysfunction induced by TNF-alpha

We tested the hypothesis that protein kinase C-alpha (PKC-alpha) mediates tumor necrosis factor-alpha (TNF-alpha)-induced alterations in permeability of pulmonary microvessel endothelial monolayers (PEM). The permeability of PEM was assessed by the clearance rate of Evans blue-labeled albumin. PEM lysates were analyzed for PKC-alpha mRNA (Northern cDNA blot), protein (Western immunoblot), and activity (translocation and phosphorylation of myristoylated arginine-rich C kinase substrate). Incubation of PEM with TNF-alpha (1,000 U/ml) for 4 h resulted in increases in 1) PKC-alpha protein, 2) cytoskeletal-associated PKC-alpha, 3) PKC-alpha activity, and 4) permeability to albumin. The TNF-alpha-induced increase in PKC-alpha protein, PKC-alpha activity, and permeability was prevented by a 4-h pretreatment with PKC-alpha antisense oligonucleotide but not by the scrambled nonsense oligonucleotide. The TNF-alpha-induced increase in permeability to albumin was prevented by myristoylated protein kinase C inhibitor (an inhibitor of PKC-alpha/beta, 100 microM) and calphostin (an inhibitor of the classic and novel PKC isotypes, 200 nM). The treatment with calphostin from 0.5 to 3.0 h after TNF-alpha still prevented barrier dysfunction induced by 4 h of TNF-alpha treatment. The data indicate that prolonged activation of PKC-alpha, maintained by a translation-dependent pool of PKC-alpha protein, mediates TNF-alpha-induced increases in endothelial permeability in PEM.     

Cofilin mediates tight-junction opening by redistributing actin and tight-junction proteins

We previously found that capsaicin induces tight-junction (TJ) opening accompanied with cofilin dephosphorylation/activation in intestinal Caco-2 cells. Here, we examined the role of cofilin in TJ regulation, and analyzed the structural events that lead to TJ opening. We transfected Caco-2 cells with wild-type cofilin [cofilin(wt)] or its constitutively active mutant cofilin(S3A). We found that the decreases in transepithelial electrical resistance (TER) was slower in cofilin(wt) transfectants and faster in cofilin(S3A) mutants than in vector controls. Moreover, cofilin dephosphorylation corresponded to the rate of TER decrease. Capsaicin treatment changed the localization of TJ proteins and altered the F-actin structure, but in a manner different from those depend on myosin light chain kinase (MLCK). These results strongly support the importance of cofilin in TJ opening, suggesting cofilin as a target for TJ permeability regulation in epithelial cells.     

Numb modulates the paracellular permeability of intestinal epithelial cells through regulating apical junctional complex assembly and myosin light chain phosphorylation

Numb is highly expressed throughout the crypt-villus axis of intestinal mucosa and functions as cell fate determinant and integrator of cell-to-cell adhesion. Increased paracellular permeability of intestinal epithelial cells is associated with the epithelial barrier dysfunction of inflammatory bowel diseases (IBDs). The apical junctional complex (AJC) assembly and myosin light chain (MLC) phosphorylation regulate adherens junctions (AJ) and tight junctions (TJ). We determined whether and how Numb modulate the paracellular permeability of intestinal epithelial cells. Caco-2 intestinal epithelial cells and their Numb-interfered counterparts were used in the study for physiological, morphological and biological analyses. Numb, expressed in intestinal epithelial cells and located at the plasma membrane of Caco-2 cells in a basolateral to apical distribution, increased in the intestinal epithelial cells with the formation of the intestinal epithelial barrier. Numb expression decreased and accumulated in the cytoplasm of intestinal epithelial cells in a DSS-induced colitis mouse model. Numb co-localized with E-cadherin, ZO-1 and Par3 at the plasma membrane and interacted with E-cadherin and Par3. Knockdown of Numb in Caco-2 cells altered the F-actin structure during the Ca²⁺ switch assay, enhanced TNFα-/INF-γ-induced intestinal epithelial barrier dysfunction and TJ destruction, and increased the Claudin-2 protein level. Immunofluorescence experiments revealed that NMIIA and F-actin co-localized at the cell surface of Caco-2 cells. Numb knockdown in Caco-2 cells increased F-actin contraction and the abundance of phosphorylated MLC. Numb modulated the intestinal epithelial barrier in a Notch signaling-independent manner. These findings suggest that Numb modulates the paracellular permeability by affecting AJC assembly and MLC phosphorylation.     

Analysis of the mechanism of the tight-junctional permeability increase by capsaicin treatment on the intestinal Caco-2 cells

In a previous experiment (Isoda et al., 2001), we showed that the tight-junctional (TJ) permeability increase in Caco-2 cells during capsaicin exposure was through binding of the capsaicin molecule to a capsaicin receptor-like protein. In the present study, we examined how actin, which modulates TJ permeability, is influenced by capsaicin. We showed that after treatment of the Caco-2 cells with capsaicin, the volume of F-actin decreased. Moreover, we also examined protein kinase C (PKC) and heat shock protein 47 (HSP47), which act as probable second messengers in causing TJ permeability increase. We showed that after capsaicin treatment, HSP47 was activated. However, PKC activity was the same in both control and treatment setups. These results suggest that, while PKC is not involved, it is highly possible that HSP47plays a role in TJ permeability increase in intestinal Caco-2 cells exposed to capsaicin. This revised version was published online in August 2006 with corrections to the Cover Date.