Optineurin is required for CYLD-dependent inhibition of TNFα-induced NF-κB activation

The nuclear factor kappa B (NF-κB) regulates genes that function in diverse cellular processes like inflammation, immunity and cell survival. The activation of NF-κB is tightly controlled and the deubiquitinase CYLD has emerged as a key negative regulator of NF-κB signalling. Optineurin, mutated in certain glaucomas and amyotrophic lateral sclerosis, is also a negative regulator of NF-κB activation. It competes with NEMO (NF-κB essential modulator) for binding to ubiquitinated RIP (receptor interacting protein) to prevent NF-κB activation. Recently we identified CYLD as optineurin-interacting protein. Here we have analysed the functional significance of interaction of optineurin with CYLD. Our results show that a glaucoma-associated mutant of optineurin, H486R, is altered in its interaction with CYLD. Unlike wild-type optineurin, the H486R mutant did not inhibit tumour necrosis factor α (TNFα)-induced NF-κB activation. CYLD mediated inhibition of TNFα-induced NF-κB activation was abrogated by expression of the H486R mutant. Upon knockdown of optineurin, CYLD was unable to inhibit TNFα-induced NF-κB activation and showed drastically reduced interaction with ubiquitinated RIP. The level of ubiquitinated RIP was increased in optineurin knockdown cells. Deubiquitination of RIP by over-expressed CYLD was abrogated in optineurin knockdown cells. These results suggest that optineurin regulates NF-κB activation by mediating interaction of CYLD with ubiquitinated RIP thus facilitating deubiquitination of RIP.     

Fas ligation and tumor necrosis factor α activation of murine astrocytes promote heat shock factor-1 activation and heat shock protein expression leading to chemokine induction and cell survival

Death-inducing ligands tumor necrosis factor alpha (TNFα) and Fas ligand (FasL) do not kill cultured astrocytes; instead they induce a variety of chemokines including macrophage-inflammatory protein-1α/CC chemokine ligand 3 (CCL3), monocyte chemoattractant protein-1 (CC CCL-2), macrophage-inflammatory protein-2/CXC chemokine ligand 2 (CXCL2, a murine homologue of interleukin 8), and interferon-induced protein of 10 kDa (CXCL10). Induction is enhanced by protein synthesis inhibition suggesting the existence of endogenous inhibitors. ERK, NF-κB, heat shock factor-1 (HSF-1) and heat shock proteins were examined for their possible roles in signal transduction. Inhibition of ERK activation by PD98059 partially inhibited expression of all but FasL-induced CXCL10. Although inhibition of NF-κB DNA binding inhibited chemokine induction, PD98059 did not inhibit TNFα-induced NF-κB DNA binding suggesting that ERK serves an NF-κB-independent pathway. Heat shock itself induced astrocytic chemokine expression; both TNFα and FasL induced HSF-1 DNA binding and Hsp72 production; and Hsp72-induced chemokine expression. Inhibition of either HSF-1 binding with quercetin or heat shock protein synthesis with KNK437 compromised chemokine induction without compromising cell survival. These data suggest that the induction of heat shock proteins via HSF-1 contribute to the TNFα- and FasL-induced expression of chemokines in astrocytes.     

c-Jun N-terminal kinase attenuates TNFα signaling by reducing Nox1-dependent endosomal ROS production in vascular smooth muscle cells

Tumor necrosis factor-α (TNFα), a proinflammatory cytokine, causes vascular smooth muscle cell (VSMC) proliferation and migration and promotes inflammatory vascular lesions. Nuclear factor-kappa B (NF-κB) activation by TNFα requires endosomal superoxide production by Nox1. In endothelial cells, TNFα stimulates c-Jun N-terminal kinase (JNK), which inhibits NF-κB signaling. The mechanism by which JNK negatively regulates TNFα-induced NF-κB activation has not been defined. We hypothesized that JNK modulates NF-κB activation in VSMC, and does so via a Nox1-dependent mechanism. TNFα-induced NF-κB activation was TNFR1- and endocytosis-dependent. Inhibition of endocytosis with dominant-negative dynamin (DynK44A) potentiated TNFα-induced JNK activation, but decreased ERK activation, while p38 kinase phosphorylation was not altered. DynK44A attenuated intracellular, endosomal superoxide production in wild-type (WT) VSMC, but not in NADPH oxidase 1 (Nox1) knockout (KO) cells. siRNA targeting JNK1 or JNK2 potentiated, while a JNK activator (anisomycin) inhibited, TNFα-induced NF-κB activation in WT, but not in Nox1 KO cells. TNFα-stimulated superoxide generation was enhanced by JNK1 inhibition in WT, but not in Nox1 KO VSMC. These data suggest that JNK suppresses the inflammatory response to TNFα by reducing Nox1-dependent endosomal ROS production. JNK and endosomal superoxide may represent novel targets for pharmacologic modulation of TNFα signaling and vascular inflammation.     

Differential effects of selenium and knock-down of glutathione peroxidases on TNFα and flagellin inflammatory responses in gut epithelial cells

Selenium (Se) is essential for human health. Despite evidence that Se intake affects inflammatory responses, the mechanisms by which Se and the selenoproteins modulate inflammatory signalling, especially in the gut, are not yet defined. The aim of this work was to assess effects of altered Se supply and knock-down of individual selenoproteins on NF-κB activation in gut epithelial cells. Caco-2 cells were stably transfected with gene constructs expressing luciferase linked either to three upstream NF-κB response elements and a TATA box or only a TATA box. TNFα and flagellin activated NF-κB-dependent luciferase activity and increased IL-8 expression. Se depletion decreased expression of glutathione peroxidase1 (GPX1) and selenoproteins H and W and increased TNFα-stimulated luciferase activity, endogenous IL-8 expression and reactive oxygen species (ROS) production. These effects were not mimicked by independent knock-down of either GPX1, selenoprotein H or W; indeed, GPX1 knock-down lowered TNFα-induced NF-κB activation and did not affect ROS levels. GPX4 knock-down decreased NF-κB activation by flagellin but not by TNFα. We hypothesise that Se depletion alters the pattern of expression of multiple selenoproteins that in turn increases ROS and modulates NF-κB activation in epithelial cells, but that the effect of GPX1 knock-down is ROS-independent.     

Procyanidins protect Caco-2 cells from bile acid- and oxidant-induced damage

Procyanidins can exert cytoprotective, anti-inflammatory, and anticarcinogenic actions in the gastrointestinal tract. Previous evidence has shown that procyanidins can interact with synthetic membranes and protect them from oxidation and disruption. Thus, in this study we investigated the capacity of a hexameric procyanidin fraction (Hex) isolated from cocoa to protect Caco-2 cells from deoxycholic (DOC)-induced cytotoxicity, cell oxidant increase, and loss of monolayer integrity. Hex interacted with the cell membranes without affecting their integrity, as evidenced by a Hex-mediated increase in the transepithelial electrical resistance, and inhibition of DOC-induced cytotoxicity. DOC induced an increase in cell oxidants, alterations in the paracellular transport, and redistribution of the protein ZO-1 from cell-cell contacts into the cytoplasm. Hex partially inhibited all these events at concentrations ranging from 2.5 to 20 microM. Similarly, Hex (5-10 microM) inhibited the increase in cell oxidants, and the loss of integrity of polarized Caco-2 cell monolayers induced by a lipophilic oxidant (2,2'-azobis (2,4-dimethylvaleronitrile). Results show that the assayed procyanidin fraction can interact with cell membranes and protect Caco-2 cells from DOC-induced cytotoxicity, oxidant generation, and loss of monolayer integrity. At the gastrointestinal tract, large procyanidins may exert beneficial effects in pathologies such us inflammatory diseases, alterations in intestinal barrier permeability, and cancer.     

Eicosapentaenoic acid inhibits TNF-alpha-induced matrix metalloproteinase-9 expression in human keratinocytes, HaCaT cells

Eicosapentaenoic acid (EPA) is an omega-3 (ω-3) polyunsaturated fatty acid (PUFA), which has anti-inflammatory and anti-cancer properties. Some reports have demonstrated that EPA inhibits NF-κB activation induced by tumor necrosis factor (TNF)-α or lipopolysaccharide (LPS) in various cells. However, its detailed mode of action is unclear. In this report, we investigated whether EPA inhibits the expression of TNF-α-induced matrix metalloproteinases (MMP)-9 in human immortalized keratinocytes (HaCaT). TNF-α induced MMP-9 expression by NF-κB-dependent pathway. Pretreatment of EPA inhibited TNF-α-induced MMP-9 expression and p65 phosphorylation. However, EPA could not affect IκB-α phosphorylation, nuclear translocation of p65, and DNA binding activity of NF-κB. EPA inhibited TNF-α-induced p65 phosphorylation through p38 and Akt inhibition and this inhibition was IKKα-dependent event. Taken together, we demonstrate that EPA inhibits TNF-α-induced MMP-9 expression through inhibition of p38 and Akt activation.     

Expression of TNFAIP3 in intestinal epithelial cells protects from DSS- but not TNBS-induced colitis

Intestinal epithelial cells (IEC) maintain gastrointestinal homeostasis by providing a physical and functional barrier between the intestinal lumen and underlying mucosal immune system. The activation of NF-κB and prevention of apoptosis in IEC are required to maintain the intestinal barrier and prevent colitis. How NF-κB activation in IEC prevents colitis is not fully understood. TNFα-induced protein 3 (TNFAIP3) is a NF-κB-induced gene that acts in a negative-feedback loop to inhibit NF-κB activation and also to inhibit apoptosis; therefore, we investigated whether TNFAIP3 expression in the intestinal epithelium impacts susceptibility of mice to colitis. Transgenic mice expressing TNFAIP3 in IEC (villin-TNFAIP3 Tg mice) were exposed to dextran sodium sulfate (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS), and the severity and characteristics of mucosal inflammation and barrier function were compared with wild-type mice. Villin-TNFAIP3 Tg mice were protected from DSS-induced colitis and displayed reduced production of NF-κB-dependent inflammatory cytokines. Villin-TNFAIP3 Tg mice were also protected from DSS-induced increases in intestinal permeability and induction of IEC death. Villin-TNFAIP3 Tg mice were not protected from colitis induced by TNBS. These results indicate that TNFAIP3 expression in IEC prevents colitis involving DSS-induced IEC death, but not colitis driven by T cell-mediated inflammation. As TNFAIP3 inhibits NF-κB activation and IEC death, expression of TNFAIP3 in IEC may provide an avenue to inhibit IEC NF-κB activation without inducing IEC death and inflammation.     

Tumor necrosis factor α-mediated induction of interleukin 17C in human keratinocytes is controlled by nuclear factor κB

IL-17C is a member of the IL-17 family of cytokines. The expression of IL-17C has been demonstrated to be strongly induced by TNFα in human keratinocytes, and recently the level of IL-17C was found to be increased in the inflammatory skin disease psoriasis. However, little is known about the molecular mechanisms involved in the regulation of IL-17C. Here, we show that pretreatment of cultured human keratinocytes with the inhibitor of κB kinase 2 inhibitor, SC-514, resulted in a significant reduction in both IL-17C mRNA and protein expression, indicating the significance of this pathway in the regulation of IL-17C. NF-κB binding sites were identified upstream from the IL-17C gene, and by electrophoretic mobility shift assay NF-κB was shown to bind to all three identified binding sites. Moreover, NF-κB binding to these sites was inducible by TNFα. Supershift analysis revealed binding of the NF-κB subunits p65 and p50 to all three NF-κB binding sites. To determine the contribution of NF-κB in IL-17C expression, we conducted luciferase gene reporter experiments and demonstrated that a 3204-bp promoter fragment of IL-17C containing three putative NF-κB binding sites was strongly activated by TNFα. Interestingly, mutations of the three NF-κB binding sites revealed that one specific NF-κB binding site was crucial for the TNFα-mediated IL-17C induction because mutation of this specific site completely abolished TNFα-induced IL-17C promoter activation. We conclude that the activation of NF-κB (p65/p50) is crucial for the TNFα-induced stimulation of IL-17C expression in human keratinocytes.     

TRAF2 Suppresses Basal IKK Activity in Resting Cells and TNFα Can Activate IKK in TRAF2 and TRAF5 Double Knockout Cells

Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) and TRAF5 are adapter proteins involved in TNFα-induced activation of the c-Jun N-terminal kinase and nuclear factor κB (NF-κB) pathways. Currently, TNFα-induced NF-κB activation is believed to be impaired in TRAF2 and TRAF5 double knockout (T2/5 DKO) cells. Here, we report instead that T2/5 DKO cells exhibit high basal IκB kinase (IKK) activity and elevated expression of NF-κB-dependent genes in unstimulated conditions. Although TNFα-induced receptor-interacting protein 1 ubiquitination is indeed impaired in T2/5 DKO cells, TNFα stimulation further increases IKK activity in these cells, resulting in significantly elevated expression of NF-κB target genes to a level higher than that in wild-type cells. Inhibition of NIK in T2/5 DKO cells attenuates basal IKK activity and restores robust TNFα-induced IKK activation to a level comparable with that seen in wild-type cells. This suggests that TNFα can activate IKK in the absence of TRAF2 and TRAF5 expression and receptor-interacting protein 1 ubiquitination. In addition, both the basal and TNFα-induced expression of anti-apoptotic proteins are normal in T2/5 DKO cells, yet these DKO cells remain sensitive to TNFα-induced cell death, due to the impaired recruitment of anti-apoptotic proteins to the TNFR1 complex in the absence of TRAF2. Thus, our data demonstrate that TRAF2 negatively regulates basal IKK activity in resting cells and inhibits TNFα-induced cell death by recruiting anti-apoptotic proteins to the TNFR1 complex rather than by activating the NF-κB pathway.     

USP4 targets TAK1 to downregulate TNFα-induced NF-κB activation

Lys63-linked polyubiquitination of transforming growth factor-β-activated kinase 1 (TAK1) has an important role in tumor necrosis factor-α (TNFα)-induced NF-κB activation. Using a functional genomic approach, we have identified ubiquitin-specific peptidase 4 (USP4) as a deubiquitinase for TAK1. USP4 deubiquitinates TAK1 in vitro and in vivo. TNFα induces association of USP4 with TAK1 to deubiquitinate TAK1 and downregulate TAK1-mediated NF-κB activation. Overexpression of USP4 wild type, but not deuibiquitinase-deficient C311A mutant, inhibits both TNFα- and TAK1/TAB1 co-overexpression-induced TAK1 polyubiquitination and NF-κB activation. Notably, knockdown of USP4 in HeLa cells enhances TNFα-induced TAK1 polyubiquitination, IκB kinase phosphorylation, IκBα phosphorylation and ubiquitination, as well as NF-κB-dependent gene expression. Moreover, USP4 negatively regulates IL-1β-, LPS- and TGFβ-induced NF-κB activation. Together, our results demonstrate that USP4 serves as a critical control to downregulate TNFα-induced NF-κB activation through deubiquitinating TAK1.     

Omentin inhibits TNF-α-induced expression of adhesion molecules in endothelial cells via ERK/NF-κB pathway

In the present study, we investigated whether omentin affected the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor-α (TNF-α) induced human umbilical vein endothelial cells (HUVECs). Our data showed that omentin decreased TNF-α-induced expression of ICAM-1 and VCAM-1 in HUVECs. In addition, omentin inhibited TNF-α-induced adhesion of THP-1 cells to HUVECs. Further, we found that omentin inhibited TNF-α-activated signal pathway of nuclear factor-κB (NF-κB) by preventing NF-κB inhibitory protein (IκBα) degradation and NF-κB/DNA binding activity. Omentin pretreatment significantly inhibited TNF-α-induced ERK activity and ERK phosphorylation in HUVECs. Pretreatment with PD98059 suppressed TNF-α-induced NF-κB activity. Omentin, NF-kB inhibitor (BAY11-7082) and ERK inhibitor (PD98059) reduced the up-regulation of ICAM-1 and VCAM-1 induced by TNF-α. These results suggest that omentin may inhibit TNF-α-induced expression of adhesion molecules in endothelial cells via blocking ERK/NF-κB pathway.     

Exogenous regucalcin stimulates osteoclastogenesis and suppresses osteoblastogenesis through NF-κB activation

Regucalcin plays a pivotal role in regulating intracellular calcium homeostasis and consequently has a profound effect on multiple intracellular signal transduction pathways. The regucalcin transgenic rat displays pronounced bone loss, and bone marrow from these animals exhibits significantly elevated osteoclast formation. Consistent with these effects exogenous regucalcin promotes osteoclastogenesis in mouse bone marrow cultures, but interestingly regucalcin suppresses the differentiation and mineralization of MC3T3 osteoblast precursors. However, the molecular mechanisms involved are presently unclear. As the nuclear factor-kappa B (NF-κB) signal transduction pathway is critical to osteoclastogenesis but inhibitory of osteoblastogenesis, we hypothesized that regucalcin may promote osteoclastogenesis and suppress osteoblastogenesis upregulating NF-κB signal transduction. In this study, we examined the effect of regucalcin on receptor activator of NF-κB (RANK) ligand (RANKL) -induced osteoclast formation using the RAW264.7 monocytic cell line and osteoblast formation using the pre-osteoblastic cell line MC3T3. As expected, culture with exogenous regucalcin was found to enhance RANKL-induced osteoclastogenesis. Consistent with this effect regucalcin increased basal and RANKL-induced NF-κB activation as assessed by NF-κB luciferase assay. The capacity of regucalcin to augment RANKL-induced NF-κB activity was inhibited by menaquinone-7, a potent NF-κB antagonist, while the Erk inhibitor PD98059 and staurosporine had no effect, demonstrating a specific effect on NF-κB signaling. By contrast, regucalcin inhibited mineralization of MC3T3 cells and enhanced tumor necrosis factor-α (TNFα)-induced NF-κB activation. As with NF-κB induction in osteoclasts, NF-κB activation was abolished by addition of the NF-κB antagonist menaquinone-7, but not by PD98059 and staurosporine. Transforming growth factor-β (TGFβ) and bone morphogenic protein-2 (BMP2) are potent early commitment and late osteoblast differentiation factors, respectively, and both mediate their actions through the Smad-signal transduction pathway, a system that is extremely sensitive to and inhibited by TNFα-induced NF-κB. We consequently examined the effect of regucalcin on TGFβ and BMP2-induced Smad activation in the presence and absence of TNFα. While regucalcin had no effect on basal Smad activation by TGFβ and BMP2, it enhanced the suppressive effect of TNFα on both TGFβ- and BMP2-induced Smad activations. Taken together, present data suggest that regucalcin may induce bone loss in vivo by promoting osteoclasts and simultaneously suppressing osteoblasts through amplification of basal and/or cytokine-induced NF-κB activation. Regucalcin may have a role as a modulator in NF-κB activation.     

The role of viral hemorrhagic septicemia virus (VHSV) NV gene in TNF-α- and VHSV infection-mediated NF-κB activation

The role of viral hemorrhagic septicemia virus (VHSV) NV gene in nuclear factor-κB (NF-κB) activation was investigated. Epithelioma papulosum cyprini (EPC) cells pre-treated with tumor necrosis factor (TNF)-α showed a strong resistance against VHSV infection, but cells treated with TNF-α after VHSV infection showed no resistance, suggesting that immediate early TNF-α-mediated responses inhibit VHSV replication. Activation of NF-κB is a key step in TNF-α-mediated immunomodulatory pathways. In this study, activation of NF-κB by TNF-α exposure was inhibited in EPC cells harboring NV gene expressing vectors, indicating that the NV gene of VHSV can suppress TNF-α-mediated NF-κB activation. Furthermore, the NV gene knock-out recombinant VHSV (rVHSV-ΔNV-EGFP) induced significantly higher NF-κB activity in EPC cells than wild-type VHSV, suggesting that VHSV adopted a strategy to suppress early activation of NF-κB in host cells through and NV gene.     

Aristolochic acid downregulates monocytic matrix metalloproteinase-9 by inhibiting nuclear factor-κB activation

Aristolochic acid (AA)-associated nephropathy was described as being characterized by a rapid progressive enhancement of interstitial renal fibrosis. Renal tissue fibrosis occurs because of an imbalance of extracellular matrix (ECM) accumulation and matrix metalloproteinase (MMP) activation. Much evidence indicates that inflammatory renal disease including monocyte and mesangial interactions is linked to the development and progression of renal remodeling. In this study, we found that AA showed concentration-dependent inhibition of tumor necrosis factor (TNF)-α-induced MMP-9 activation with an IC₅₀ value of 6.4 ± 0.5 μM in human monocytic THP-1 cells. A similar effect was also noted with different ratios of AAs (types I and II). However, AA had no inhibitory effect on the intact enzymatic activity of MMP-9 at a concentration of 20 μM. On the other hand, the level of tissue inhibitor of metalloproteinase (TIMP)-1 was not induced by AA, but it suppressed TNF-α-induced MMP-9 protein and messenger RNA expressions. AA also significantly inhibited TNF-α-induced IκBα degradation. Furthermore, an electrophoretic mobility shift assay and a reported gene study, respectively, revealed that AA inhibited TNF-α-induced NF-κB translocation and activation. In addition, compared to other NF-κB inhibitors, AA exerted significant inhibition of MMP-9 activation and monocyte chemotactic protein-1-directed invasion. From these results, we concluded that AA, a natural compound, inhibits TNF-α-induced MMP-9 in human monocytic cells possibly through the NF-κB signal pathway. These results also imply that AA may be involved in alteration of matrix homeostasis during renal fibrosis in vivo.