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.     

IL-6 induces NF-kappa B activation in the intestinal epithelia

IL-6 is a potent proinflammatory cytokine that has been shown to play an important role in the pathogenesis of inflammatory bowel disease (IBD). It is classically known to activate gene expression via the STAT-3 pathway. Given the crucial role of IL-6 in the pathogenesis of chronic intestinal inflammation, it is not known whether IL-6 activates NF-kappaB, a central mediator of intestinal inflammation. The model intestinal epithelial cell line, Caco2-BBE, was used to study IL-6 signaling and to analyze whether suppressor of cytokine signaling 3 (SOCS-3) proteins play a role in the negative regulation of IL-6 signaling. We show that IL-6 receptors are present in intestinal epithelia in a polarized fashion. Basolateral IL-6 and, to a lesser extent, apical IL-6 induces the activation of the NF-kappaB pathway. Basolateral IL-6 stimulation results in a maximal induction of NF-kappaB activation and NF-kappaB nuclear translocation at 2 h. IL-6 induces polarized expression of ICAM-1, an adhesion molecule shown to be important in the neutrophil-epithelial interactions in IBD. Using various deletion constructs of ICAM-1 promoter, we show that ICAM-1 induction by IL-6 requires the activation of NF-kappaB. We also demonstrate that overexpression of SOCS-3, a protein known to inhibit STAT activation in response to IL-6, down-regulates IL-6-induced NF-kappaB activation and ICAM-1 expression. In summary, we demonstrate the activation of NF-kappaB by IL-6 in intestinal epithelia and the down-regulation of NF-kappaB induction by SOCS-3. These data may have mechanistic and therapeutic implications in diseases such as IBD and rheumatoid arthritis in which IL-6 plays an important role in the pathogenesis.     

Cholecystokinin induction of mob-1 chemokine expression in pancreatic acinar cells requires NF-kappa B activation

Inflammatory mediators are involved in the early phase of acutepancreatitis, but the cellular mechanisms responsible for theirgeneration within pancreatic cells are unknown. We examined the role ofnuclear factor-B (NF-B) in cholecystokinin octapeptide (CCK-8)-induced mob-1 chemokineexpression in pancreatic acinar cells in vitro. Supraphysiological, butnot physiological, concentrations of CCK-8 increased inhibitory B(IB-) degradation, NF-B activation, andmob-1 gene expression in isolatedpancreatic acinar cells. CCK-8-induced IB- degradation wasmaximal within 1 h. Expression ofmob-1 was maximal within 2 h. Neitherbombesin nor carbachol significantly increasedmob-1 mRNA or induced IB-degradation. Thus the concentration, time, and secretagogue dependenceof mob-1 gene expression and IB-degradation were similar. Inhibition of NF-B with pharmacologicalagents or by adenovirus-mediated expression of the inhibitory proteinIB- also inhibited mob-1 geneexpression. These data indicate that the NF-B signaling pathway isrequired for CCK-8-mediated induction ofmob-1 chemokine expression inpancreatic acinar cells. This supports the hypothesis that NF-Bsignaling is of central importance in the initiation of acute pancreatitis.

     

Oncogenic and anti-apoptotic activity of NF-kappa B in human thyroid carcinomas

Thyroid cancer includes three types of carcinomas classified as differentiated thyroid carcinomas (DTC), medullary thyroid carcinomas, and undifferentiated carcinomas (UTC). DTC and medullary thyroid carcinomas generally have a good prognosis, but UTC are usually fatal. Consequently, there is a need for new effective therapeutic modalities to improve the survival of UTC patients. Here we show that NF-kappa B is activated in human thyroid neoplasms, particularly in undifferentiated carcinomas. Thyroid cell lines, reproducing in vitro the different thyroid neoplasias, also show basal NF-kappa B activity and resistance to drug-induced apoptosis, which correlates with the level of NF-kappa B activation. Activation of NF-kappa B in the DTC cell line NPA renders these cells resistant to drug-induced apoptosis. Stable expression of a super-repressor form of I kappa B alpha (I kappa B alpha M) in the UTC cell line FRO results in enhanced sensitivity to drug-induced apoptosis, to the loss of the ability of these cells to form colonies in soft agar, and to induce tumor growth in nude mice. In addition, we show that FRO cells display a very low JNK activity that is restored in FRO-I kappa B alpha M clones. Moreover, inhibition of JNK activity renders FRO-I kappa B alpha M clones resistant to apoptosis induced by chemotherapeutic agents. Our results indicate that NF-kappa B plays a pivotal role in thyroid carcinogenesis, being required for tumor growth and for resistance to drug-induced apoptosis, the latter function very likely through the inhibition of JNK activity. Furthermore, the strong constitutive NF-kappa B activity in human anaplastic thyroid carcinomas, besides representing a novel diagnostic tool, makes NF-kappa B a target for the development of novel therapeutic strategies.     

Secretory phospholipase A2-potentiated inducible nitric oxide synthase expression by macrophages requires NF-kappa B activation

The effect of secretory group II phospholipase A2 (sPLA2) on the expression of the inducible NO synthase (iNOS) and the production of NO by macrophages was investigated. sPLA2 by itself barely stimulated nitrite production and iNOS expression in Raw264.7 cells. However, in combination with LPS, the effects were synergistic. This potentiation was shown for sPLA2 enzymes from sPLA2-transfected stable cells or for purified sPLA2 from human synovial fluid. The effect of PLA2 on iNOS induction appears to be specific for the secretory type of PLA2. LPS-stimulated activation of iNOS was inhibited by the well-known selective inhibitors of sPLA2 such as 12-epi-scalaradial and p-bromophenacyl bromide. In contrast, the cytosolic PLA2-specific inhibitors methyl arachidonyl fluorophosphate and arachidonyltrifluoromethyl ketone did not affect LPS-induced nitrite production and iNOS expression. Moreover, when we transfected cDNA-encoding type II sPLA2, we observed that the sPLA2-transfected cells produced two times more nitrites than the empty vector or cytosolic PLA2-transfected cells. The sPLA2-potentiated iNOS expression was associated with the activation of NF-kappa B. We found that the NF-kappa B inhibitor pyrrolidinedithiocarbamate prevented nitrite production, iNOS induction, and mRNA accumulation by sPLA2 plus LPS in Raw264.7 cells. Furthermore, EMSA analysis of the activation of the NF-kappa B involved in iNOS induction demonstrated that pyrrolidinedithiocarbamate prevented the NF-kappa B binding by sPLA2 plus LPS. Our findings indicated that sPLA2, in the presence of LPS, is a potent activator of macrophages. It stimulates iNOS expression and nitrite production by a mechanism that requires the activation of NF-kappa B.     

IL-2 gene expression and NF-kappa B activation through CD28 requires reactive oxygen production by 5-lipoxygenase.

Activation of the CD28 surface receptor provides a major costimulatory signal for T cell activation resulting in enhanced production of interleukin-2 (IL-2) and cell proliferation. In primary T lymphocytes we show that CD28 ligation leads to the rapid intracellular formation of reactive oxygen intermediates (ROIs) which are required for CD28-mediated activation of the NF-kappa B/CD28-responsive complex and IL-2 expression. Delineation of the CD28 signaling cascade was found to involve protein tyrosine kinase activity, followed by the activation of phospholipase A2 and 5-lipoxygenase. Our data suggest that lipoxygenase metabolites activate ROI formation which then induce IL-2 expression via NF-kappa B activation. These findings should be useful for therapeutic strategies and the development of immunosuppressants targeting the CD28 costimulatory pathway.     

A novel NF-kappa B-inducing kinase-MAPK signaling pathway up-regulates NF-kappa B activity in melanoma cells.

Constitutive activation of NF-kappa B is an emerging hallmark of various types of tumors including breast, colon, pancreatic, ovarian, and melanoma. In melanoma cells, the basal expression of the CXC chemokine, CXCL1, is constitutively up-regulated. This up-regulation can be attributed in part to constitutive activation of NF-kappa B. Previous studies have shown an elevated basal I kappa B kinase (IKK) activity in Hs294T melanoma cells, which leads to an increased rate of I kappa B phosphorylation and degradation. This increase in I kappa B-alpha phosphorylation and degradation leads to an approximately 19-fold higher nuclear localization of NF-kappa B. However, the upstream IKK kinase activity is up-regulated by only about 2-fold and cannot account for the observed increase in NF-kappa B activity. We now demonstrate that NF-kappa B-inducing kinase (NIK) is highly expressed in melanoma cells, and IKK-associated NIK activity is enhanced in these cells compared with the normal cells. Kinase-dead NIK blocked constitutive NF-kappa B or CXCL1 promoter activity in Hs294T melanoma cells, but not in control normal human epidermal melanocytes. Transient overexpression of wild type NIK results in increased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), which is inhibited in a concentration-dependent manner by PD98059, an inhibitor of p42/44 MAPK. Moreover, the NF-kappa B promoter activity decreased with overexpression of dominant negative ERK expression constructs, and EMSA analyses further support the hypothesis that ERK acts upstream of NF-kappa B and regulates the NF-kappa B DNA binding activity. Taken together, our data implicate involvement of I kappa B kinase and MAPK signaling cascades in NIK-induced constitutive activation of NF-kappa B.     

Lipopolysaccharide-unresponsive mutant pre-B-cell lines blocked in NF-kappa B activation.

NF-kappa B activation is a crucial late step in the induction of immunoglobulin kappa light-chain gene expression in pre-B cells by lipopolysaccharide (LPS). We have analyzed NF-kappa B activation in three independent mutant lines of 70Z/3 pre-B cells which are unresponsive to LPS. All three variant cell lines failed to activate NF-kappa B when induced with LPS or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. However, all three cell lines contained functional NF-kappa B, as revealed by detergent treatment of cytoplasmic extracts. Moreover, cycloheximide induced limited activation of NF-kappa B comparable to that in wild-type 70Z/3 pre-B cells in two of the three variant lines. These results indicate that the mutations blocking kappa gene induction in these variant 70Z/3 pre-B-cell lines affect NF-kappa B activation.     

Lymphotoxin beta receptor induces sequential activation of distinct NF-kappa B factors via separate signaling pathways

Lymphotoxin beta receptor (LTbetaR)-induced activation of NF-kappaB in mouse embryo fibroblasts was mediated by the classical pathway and by an alternative or second pathway. The classical pathway involved the IkappaB kinase (IKK)beta- and IKKgamma-dependent degradation of IkappaBalpha and resulted in the rapid but transient activation of primarily RelA-containing NF-kappaB dimers. The alternative or second pathway proceeded via NF-kappaB-inducing kinase (NIK)-, IKKalpha-, and protein synthesis-dependent processing of the inhibitory NF-kappaB2 p100 precursor protein to the p52 form and resulted in a delayed but sustained activation of primarily RelB-containing NF-kappaB dimers. This second pathway was independent of the classical IKK complex, which is governed by its central IKKgamma regulatory subunit. The sequential engagement of two distinct pathways, coupled with the negative feedback inhibition of RelA complexes by NF-kappaB-induced resynthesis of IkappaBalpha, resulted in a pronounced temporal change in the nature of the NF-kappaB activity during the course of stimulation. Initially dominant RelA complexes were replaced with time by RelB complexes. Therefore, the alternative activation path mediated by processing of p100 was necessary for sustained NF-kappaB activity in mouse embryo fibroblasts in response to LTbetaR stimulation. Based on the phenotype of mice deficient in various components of the LTbetaR-induced activation of p100 processing, we conclude that this pathway is critically involved in the function of stromal cells during the generation of secondary lymphoid organ microarchitectures.     

CRH inhibits NF-kappa B signaling in human melanocytes

Corticotropin releasing hormone (CRH), a messenger of stress at the central level, is expressed in the epidermis where it operates within local equivalent of hypothalamo-pituitary axis. CRH inhibits NF-κB activity in human immortalized epidermal (PIG1) melanocytes. In melanocytes CRH stimulates pro-opiomelanocortin (POMC) mRNA and adrenocorticotropin (ACTH) peptide production. Knockdown of POMC levels by transfecting cells with antisense oligonucleotides blocks the effect of CRH on NF-κB signaling indicating that the above inhibition is indirect, e.g. through activation of POMC. We suggest that induction of POMC by CRH serves as a feedback mechanism to self-restrict inflammatory response in the skin.     

Interactions among oscillatory pathways in NF-kappa B signaling

Background  

Sustained stimulation with tumour necrosis factor alpha (TNF-alpha) induces substantial oscillations—observed at both the single cell and population levels—in the nuclear factor kappa B (NF-kappa B) system. Although the mechanism has not yet been elucidated fully, a core system has been identified consisting of a negative feedback loop involving NF-kappa B (RelA:p50 hetero-dimer) and its inhibitor I-kappa B-alpha. Many authors have suggested that this core oscillator should couple to other oscillatory pathways.     

Protein kinase C activation modulates pro- and anti-apoptotic signaling pathways

Activation of protein kinase C (PKC) by TPA in human U937 myeloid leukemia cells is associated with induction of adherence, differentiation, and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these differentiating cells about 25% of U937 cells accumulated in the subG1 phase after TPA treatment. This effect proved to be phorbol ester-specific, since other compounds such as retinoic acid or vitamin D3 failed to induce apoptosis in conjunction with differentiation. Only a specific inhibitor of PKC, GF109203X, but not the broad-spectrum kinase inhibitor staurosporine or a tyrosine kinase inhibitor genistein could reverse the induction of apoptosis. Bryostatin-1, another specific PKC activator with distinct biochemical activity failed to induce apoptosis. Moreover, bryostatin-1 completely abolished the induction of apoptosis in U937 cells even if added 8 hours after TPA treatment. Apart from apoptosis induced by various chemotherapeutic drugs, TPA-related cell death is not mediated by an autocrine Fas-FasL loop and could not be prevented by a blocking antibody to the Fas receptor. However, a 75% reduction in the number of apoptotic cells after TPA stimulation was achieved by preincubation with a blocking antibody to the TNFalpha receptor. Tetrapeptide cleavage assays revealed a four-fold increase in the DEVD-cleavage activity in U937 cells compared to a three-fold increase in TUR cells. Immunoblotting demonstrated that TUR cells did not activate significant levels of caspase-3 or -7, whereas in U937 cells a 20-kDa cleavage product corresponding to activated caspase-3 was detectable after 3 d TPA exposure. Moreover, immunoblots revealed a strongly reduced expression of the adaptor molecule APAF-1, which is required for cytochrome c-dependent activation of caspase-9 and subsequently caspase-3. APAF-1 proved to be inducible after PKC activation with phorbol ester in U937, but not in TUR cells. Thus, APAF-1 expression may, at least in part, be regulated by PKC activity and reduced APAF-1 levels are associated with resistance to various inducers of apoptosis. Furthermore, TPA exposure of U937 cells is associated with increased levels of the pro-apoptotic proteins Bak and Bcl-xs, whereas simultaneously a decline in the Bcl-2 expression was noticable.     

IL-1beta stimulates IL-6 production in cultured skeletal muscle cells through activation of MAP kinase signaling pathway and NF-kappa B

Recent studies suggest that the skeletal muscle may be a significant site of IL-6 production in various conditions, including exercise, inflammation, hypoperfusion, denervation, and local muscle injury. The mediators and molecular mechanisms regulating muscle IL-6 production are poorly understood. We tested the hypothesis that IL-6 production in muscle cells is regulated by IL-1beta and that mitogen-activated protein (MAP) kinase signaling and NF-kappaB activation are involved in IL-1beta-induced IL-6 production. Cultured C2C12 cells, a mouse skeletal muscle cell line, were treated with different concentrations (0.1-2 ng/ml) of IL-1beta in the absence or presence of the p38 MAP kinase inhibitor SB-208350 or the p42/44 inhibitor PD-98059. Protein and gene expression of IL-6 were determined by ELISA and real-time PCR, respectively. NF-kappaB DNA binding activity was determined by electrophoretic mobility shift assay and by transfecting myocytes with a luciferase reporter plasmid containing a promoter construct with multiple repeats of NF-kappaB binding site. Treatment of myotubes with IL-1beta resulted in a dose- and time-dependent increase of IL-6 production accompanied by an approximately 25-fold increase in IL-6 mRNA levels. IL-1beta stimulated NF-kappaB DNA binding activity and gene activation. SB-208350 and PD-98059 inhibited the increase in IL-6 production induced by IL-1beta. The present results support the concept that skeletal muscle is an important site of IL-6 production. In addition, the results suggest the IL-1beta regulates muscle IL-6 production at least in part by activating the MAP kinase pathway and NF-kappaB.