TNFα-stimulated Cells Research Articles

Distribution of Signal Peptides in Microvesicles from Activated Macrophage Cells.

Extracellular vesicles, such as microvesicles (LEV) and exosomes (SEV), play an important role in intercellular signaling by encapsulating functional molecules and delivering them to specific cells. Recent studies showed that signal peptides (SPs), which are derived from sequences at the N-terminal of newly synthesized proteins, exhibited biological activity in the extracellular fluid. We previously reported that SPs were secreted into the extracellular fluid via SEV; however, it remains unclear whether the release of SPs occurs via LEV. In the present study, we demonstrated that SP fragments from human placental secreted alkaline phosphatase (SEAP) were present in LEV as well as SEV released from RAW-Blue cells, which stably express an NF-κB-inducible SEAP reporter. When RAW-Blue cells were treated with LPS at 0-10,000 ng/mL, SEAP SP fragments per particle were more abundant in LEV than in SEV, with fragments in LEV and SEV reaching a maximum at 1000 and 100 ng/mL, respectively. The content of SEAP SP fragments in LEV from IFNγ-stimulated RAW-Blue cells was higher than those from TNFα-stimulated cells, whereas that in SEV from TNFα-stimulated RAW-Blue cells was higher than those from IFNγ-stimulated cells. Moreover, the content of SEAP SP fragments in LEV and SEV decreased in the presence of W13, a calmodulin inhibitor. Collectively, these results indicate that the transportation of SP fragments to extracellular vesicles was changed by cellular activation, and calmodulin was involved in their transportation to LEV and SEV.

An adapted in vitro assay to assess Campylobacter jejuni interaction with intestinal epithelial cells: Taking into stimulation with TNFα

Campylobacter jejuni is the most prevalent foodborne bacterial infection agent. This pathogen seems also involved in inflammatory bowel diseases in which pro-inflammatory cytokines, such as tumor necrosis factor α (TNFα), play a major role. C. jejuni pathogenicity has been extensively studied using in vitro cell culture methods, and more precisely “healthy” cells. In fact, no information is available regarding the behavior of C. jejuni in contact with TNFα-stimulated cells. Therefore, this research was designed to investigate the effect of TNFα on C. jejuni interaction with human intestinal epithelial cells (HT29 and HT29-MTX). To ensure IL-8 production induced by TNFα, human rtTNFα was added to HT29 and HT29-MTX before adhesion and invasion assays. About 108 CFU bacteria of C. jejuni strains cells were added to measure their adherence and invasion abilities using TNFα-stimulated cells versus non stimulated cells. Exposure to TNFα results in IL-8 overproduction by intestinal epithelial cells. In addition, the effect of TNFα pre-treatment on C. jejuni adhesion and internalization into eukaryotic cells is strain-dependent. Indeed, the adhesion/invasion process is affected in <50% of the strains tested when TNFα is added to the intestinal cells. Interestingly, TNFα affects more strains in their ability to adhere to and invade the mucus-secreting HT29-MTX cells. Among the 10 strains tested, the aero-tolerant C. jejuni Bf strain is one of the most virulent. These results suggest that the TNFα signalling pathway could participate in the internalization of C. jejuni in human intestinal cells and can help in understanding the pathogenicity of this microorganism in contact with TNFα-stimulated cells.

Cross talk of c-Jun N-terminal kinase and p38 map kinase modulate insulin and TNFα-stimulated VCAM-1 expression in rat aorta endothelial cells

&lt;p&gt;VCAM-1 cell surface expression was determined by flow cytometry in rat aorta endothelial cells stimulated with insulin and/or Tumor Necrosis Factor-a in the absence or presence of short-hairpin RNA inhibitors of c-Jun N-terminal Kinase (JNK) and p38 MAP Kinase. Cells transfected with insulin alone exhibited moderate increases in cell surface VCAM-1 expression, whereas cells stimulated with TNFα alone or in combination with insulin, exhibited a significant (P, 0.05) increase in VCAM-1 surface expression. Cells transfected with shJNK alone showed increased VCAM-1 expression at the cell surface as compared to mock-transfected positive controls. In contrast, cells transfected with shp38 exhibited significant decreased insulin-stimulated VCAM-1 expression at the cell surface, but only moderate decreased VCAM-1 expression in cells stimulated by TNFα alone in combination with insulin. Interestingly, in cells first tranfected with shJNK and then shp38, VCAM-1 expression appeared to increase in an additive fashion. In contrast, cells transfected with shp38 first then shJNK, VCAM-1 expression exhibited increased VCAM-1 expression even in the presence of shp38. One can conclude that JNK is a potent negative regulator of insulin- and TNFα-stimulated cell surface expression of VCAM-1, whereas p38 is mild positive regulator of insulin-, but not TNFα-stimulated VCAM-1 expression. JNK appears to be a more potent mediator of insulin- and TNFα-stimulated cells surface VCAM-1 expression than p38 MAP Kinase. Thus, it could be a therapeutic target for amelioration of inflammation-associated VCAM-1 expression and its sequelae of events that lead to atherosclerosis.&lt;/p&gt;

Cross talk of c-Jun N-terminal kinase and p38 map kinase modulate insulin and TNFα-stimulated VCAM-1 expression in rat aorta endothelial cells

VCAM-1 cell surface expression was determined by flow cytometry in rat aorta endothelial cells stimulated with insulin and/or Tumor Necrosis Factor-a in the absence or presence of short-hairpin RNA inhibitors of c-Jun N-terminal Kinase (JNK) and p38 MAP Kinase. Cells transfected with insulin alone exhibited moderate increases in cell surface VCAM-1 expression, whereas cells stimulated with TNFα alone or in combination with insulin, exhibited a significant (P, 0.05) increase in VCAM-1 surface expression. Cells transfected with shJNK alone showed increased VCAM-1 expression at the cell surface as compared to mock-transfected positive controls. In contrast, cells transfected with shp38 exhibited significant decreased insulin-stimulated VCAM-1 expression at the cell surface, but only moderate decreased VCAM-1 expression in cells stimulated by TNFα alone in combination with insulin. Interestingly, in cells first tranfected with shJNK and then shp38, VCAM-1 expression appeared to increase in an additive fashion. In contrast, cells transfected with shp38 first then shJNK, VCAM-1 expression exhibited increased VCAM-1 expression even in the presence of shp38. One can conclude that JNK is a potent negative regulator of insulin- and TNFα-stimulated cell surface expression of VCAM-1, whereas p38 is mild positive regulator of insulin-, but not TNFα-stimulated VCAM-1 expression. JNK appears to be a more potent mediator of insulin- and TNFα-stimulated cells surface VCAM-1 expression than p38 MAP Kinase. Thus, it could be a therapeutic target for amelioration of inflammation-associated VCAM-1 expression and its sequelae of events that lead to atherosclerosis.

The low molecular weight fraction of human serum albumin upregulates COX2, prostaglandin E2, and prostaglandin D2 under inflammatory conditions in osteoarthritic knee synovial fibroblasts

BackgroundThe ability to decrease inflammation and promote healing is important in the intervention and management of a variety of disease states, including osteoarthritis of the knee (OAK). Even though cyclooxygenase 2 (COX2) has an established pro-inflammatory role, evidence suggests it is also critical to the resolution that occurs after the initial activation phase of the immune response. In this study, we investigated the effects of the low molecular weight fraction of 5% human serum albumin (LMWF-5A), an agent that has proven to decrease pain and improve function in OAK patients after intra-articular injection, on the expression of COX2 and its downstream products, prostaglandins (PGs). MethodsFibroblast-like synoviocytes from the synovial membrane of OAK patients were treated with LMWF-5A or saline as a control with or without the addition of interleukin-1β (IL-1β) or tumor necrosis factor α (TNFα) to elicit an inflammatory response. Cells were harvested for RNA and protein at 2, 4, 8, 12, and 24h, and media was collected at 24h for analysis of secreted products. COX2 mRNA expression was determined by qPCR, and COX2 protein expression was determined by western blot analysis. Levels of prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2) in the media were quantified by competitive ELISA. ResultsIn the presence of either IL-1β or TNFα, LMWF-5A increased the expression of both COX2 mRNA and protein, and this increase was significant compared to that observed with IL-1β- or TNFα-stimulated, saline-treated cells. Downstream of COX2, the levels of PGE2 were increased only in TNFα-stimulated, LMWF-5A-treated cells; however, in both IL-1β- and TNFα-stimulated cells, LMWF-5A increased the release of the anti-inflammatory prostaglandin PGD2. ConclusionLMWF-5A appears to trigger increased anti-inflammatory PG signaling, and this may be a primary component of its therapeutic mode of action in the treatment of OAK.

Glucocorticoids and the non-steroidal selective glucocorticoid receptor modulator, compound A, differentially affect colon cancer-derived myofibroblasts

The glucocorticoid receptor functions as a ligand-dependent transcription factor that positively or negatively regulates the transcription of various specific target genes. Not only steroidal glucocorticoids can bind and activate the glucocorticoid receptor, but also the intensively examined non-steroidal selective glucocorticoid receptor modulators can do so, albeit with a select effector profile skewed to glucocorticoid receptor transrepression. Glucocorticoids are widely used to treat inflammatory afflictions, but also as anti-cancer therapies or adjuvants thereof. As the impact of glucocorticoids and selective glucocorticoid receptor modulators has scarcely been researched in this setting, we focused on colon cancer and its stromal environment, in particular the stromal myofibroblasts, which are known to influence cancer cells via paracrine signaling.In these myofibroblasts, the glucocorticoid dexamethasone is able to drive the glucocorticoid receptor into the nucleus and thus negatively regulates the expression of particular pro-inflammatory genes in TNFα-stimulated cells. The selective glucocorticoid receptor modulator compound A has an impaired ability to translocate GR, presumably underpinning its modest anti-inflammatory properties in these cells. Only dexamethasone, and not compound A, can upregulate the glucocorticoid receptor transactivation-dependent GILZ expression. Neither dexamethasone, nor compound A affects myofibroblast cell viability. However, compound A retards the growth of this myofibroblast cell line. Additionally, dexamethasone can inhibit the expression of Tenascin C, hepatocyte growth factor, and TGFβ, which are all factors known for their impact on colon cancer cell invasion, in a glucocorticoid receptor-dependent manner. In contrast, compound A can only slightly diminish the expression of just hepatocyte growth factor, and not tenascin C or TGFβ. Combined, our results expose new tumor microenvironment-modulating effects of glucocorticoids and the selective GR modulator compound A.

Proliferation of mouse liver stem/progenitor cells induced by plasma from patients with acute liver failure is modulated by P2Y2 receptor-mediated JNK activation

We recently reported that acute liver failure plasma (ALF-P) promotes the proliferation of mouse liver oval cells (OCs) through c-jun N-terminal kinase (JNK) activation. The aim of this study was to investigate the mechanism by which ALF-P induces JNK activation and OC proliferation. OCs and primary hepatocytes were exposed to ALF-P or normal control plasma (NC-P). Cell proliferation and activation of JNK and other JNK signaling molecules were detected subsequently. Next, we determined the effects of extracellular adenosine triphosphate (ATP) and ATP receptors on ALF-P-stimulated cell growth. Finally, the relationship between the tumor necrosis factor alpha (TNFα) and ATP receptor pathways was investigated. Cell proliferation accompanied by JNK activation was only observed in ALF-P-stimulated OCs. ALF-P stimulated the activation of SEK1/MKK4 and ATF2, but not c-Jun. Both PPADS (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid) treatment and P2Y2 (G-protein-coupled) small interfering RNA (siRNA) transfection blocked the effects of ALF-P on cell proliferation and JNK activation. However, ATP levels in ALF-P were significantly lower than that in NC-P, and ATP did not stimulate the proliferation of OCs. On the other hand, TNFα stimulated JNK activation and proliferation of OCs. TNFα receptor antagonist partly inhibited the ALF-P-stimulated proliferation of OCs. Moreover, PPADS significantly inhibited TNFα-stimulated cell proliferation, induced apoptosis, and inhibited the activation of JNK. However, our data showed no significant difference in plasma TNFα levels between the NC-P and ALF-P samples. JNK activation induced by P2Y2 receptor crosstalk with the TNFα signaling pathway is important in mediating the effects of ALF-P on the proliferation and survival of OCs.

TRAF6-mediated ubiquitination of NEMO requires p62/sequestosome-1

The atypical protein kinase C-interacting protein p62/sequestosome-1 (p62) has emerged as a crucial molecule in a variety of cellular functions due to its involvement in various signaling mechanisms. p62 has been implicated in the activation of NF-κB in TNFα-stimulated cells and has been shown to be activated in response to interleukin-1β (IL-1β). Here we demonstrate that p62 interacts with NEMO, the regulatory subunit of the complex responsible for activation of NF-κB transcription factor. Depletion of p62 obtained through a short interfering RNA targeting p62 mRNA abrogated TRAF6 capacity to promote NEMO ubiquitination and severely impairs NF-κB activation following IL-1β stimulation.Together, these results indicate that p62 is an important intermediary in the NF-κB activation pathways implemented through non-degradative ubiquitination events.

Human Metabolic Transformation of Quercetin Blocks Its Capacity To Decrease Endothelial Nitric Oxide Synthase (eNOS) Expression and Endothelin-1 Secretion by Human Endothelial Cells

The major dietary flavonol quercetin, which has been shown to improve endothelial function and decrease blood pressure, is extensively metabolized during absorption. This study examined the relative abilities of quercetin and its human metabolites to modulate the expression of eNOS and ET-1, which are involved in regulating endothelial homeostasis. Quercetin aglycone significantly reduced both eNOS protein and gene expression in HUVEC, mirroring the effects of the pro-inflammatory cytokine TNFα. In the presence of TNFα the aglycone caused further reductions in eNOS, whereas the metabolites were without effect in either TNFα-stimulated or unstimulated cells. ET-1 expression was significantly reduced by quercetin in both TNFα-stimulated or unstimulated HUVECs. The metabolites had no effect on ET-1 expression with the exception of quercetin-3'-sulfate, which caused a moderate increase in TNFα-stimulated cells. These results suggest that metabolic transformation of quercetin prevents it from causing a potentially deleterious decrease in eNOS in endothelial cells.

Analysis of Nuclear Factor-κB (NF-κB) Essential Modulator (NEMO) Binding to Linear and Lysine-linked Ubiquitin Chains and Its Role in the Activation of NF-κB

Nuclear factor-κB (NF-κB) essential modulator (NEMO), a component of the inhibitor of κB kinase (IKK) complex, controls NF-κB signaling by binding to ubiquitin chains. Structural studies of NEMO provided a rationale for the specific binding between the UBAN (ubiquitin binding in ABIN and NEMO) domain of NEMO and linear (Met-1-linked) di-ubiquitin chains. Full-length NEMO can also interact with Lys-11-, Lys-48-, and Lys-63-linked ubiquitin chains of varying length in cells. Here, we show that purified full-length NEMO binds preferentially to linear ubiquitin chains in competition with lysine-linked ubiquitin chains of defined length, including long Lys-63-linked deca-ubiquitins. Linear di-ubiquitins were sufficient to activate both the IKK complex in vitro and to trigger maximal NF-κB activation in cells. In TNFα-stimulated cells, NEMO chimeras engineered to bind exclusively to Lys-63-linked ubiquitin chains mediated partial NF-κB activation compared with cells expressing NEMO that binds to linear ubiquitin chains. We propose that NEMO functions as a high affinity receptor for linear ubiquitin chains and a low affinity receptor for long lysine-linked ubiquitin chains. This phenomenon could explain quantitatively distinct NF-κB activation patterns in response to numerous cell stimuli.

Oxidatively Truncated Phospholipids Are Required Agents of Tumor Necrosis Factor α (TNFα)-induced Apoptosis

TNFα generates reactive oxygen species (ROS) at the cell surface that induce cell death, but how ROS communicate to mitochondria and their specific apoptotic action(s) are both undefined. ROS oxidize phospholipids to hydroperoxides that are friable and fragment adjacent to the (hydro)peroxide function, forming truncated phospholipids, such as azelaoyl phosphatidylcholine (Az-PC). Az-PC is relatively soluble, and exogenous Az-PC rapidly enters cells to damage mitochondrial integrity and initiate intrinsic apoptosis. We determined whether this toxic phospholipid is formed within cells during TNFα stimulation in sufficient quantities to induce apoptosis and if they are essential in TNFα-induced cytotoxicity. We found that TNFα induced ROS formation and phospholipid peroxidation in Jurkat cells, and either chemical interference with NADPH oxidase activity or siRNA suppression of the NADPH oxidase-4 subunit blocked ROS accumulation and phospholipid peroxidation. Mass spectrometry showed that phospholipid peroxides and then Az-PC increased after TNFα exposure, whereas ROS inhibition abolished Az-PC accumulation and TNFα-induced cell death. Glutathione peroxidase-4 (GPx4), which specifically metabolizes lipid hydroperoxides, fell in TNFα-stimulated cells prior to death. Ectopic GPx4 overcame this, reduced peroxidized phospholipid accumulation, blocked Az-PC accumulation, and prevented death. Conversely, GPx4 siRNA knockdown enhanced phospholipid peroxidation, increasing TNFα-stimulated Az-PC formation and apoptosis. Truncated phospholipids were essential elements of TNFα-induced apoptosis because overexpression of PAFAH2 (a phospholipase A(2) that selectively hydrolyzes truncated phospholipids) blocked TNFα-induced Az-PC accumulation without affecting phospholipid peroxidation. PAFAH2 also abolished apoptosis. Thus, phospholipid oxidation and truncation to apoptotic phospholipids comprise an essential element connecting TNFα receptor signaling to mitochondrial damage and apoptotic death.

Helicobacter pylori-induced tyrosine phosphorylation of IKKβ contributes to NF-κB activation

Helicobacter pylori, the etiological agent of several human gastric diseases, induces the transcription factor nuclear factor-κB (NF-κB) in colonized epithelial cells leading to the release of proinflammatory mediators. Activation of NF-κB involves the IκB kinase (IKK)-complex composed of two catalytic subunits, IKKα and IKKβ, and a regulatory scaffold protein, IKKγ. IKKβ was shown to be essential for NF-κB activation in response to a variety of stimuli including H. pylori. In addition to the phosphorylation of serine residues, tyrosine phosphorylation could be crucial for IKKβ activation. Here we provide evidence that IKKβ phosphorylation is induced in lipid rafts (DRM fractions) of H. pylori-infected cells, but not TNFα-stimulated cells. Furthermore, H. pylori transiently induces binding of IKKβ to c-Src kinase. Inhibition of c-Src by specific inhibitors as well as knockdown of c-Src by small interfering RNA reduced phosphorylation of IκBα as well as of p65. Thus, tyrosine-phosphorylated IKKβ contributes at least in part to NF-κB activation in response to H. pylori infection.

MNF, an ankyrin repeat protein of myxoma virus, is part of a native cellular SCF complex during viral infection

Myxoma virus (MYXV), a member of the Poxviridae family, is the agent responsible for myxomatosis, a fatal disease in the European rabbit (Oryctolagus cuniculus). Like all poxviruses, MYXV is known for encoding multiple proteins that regulate cellular signaling pathways. Among them, four proteins share the same ANK/PRANC structure: M148R, M149R, MNF (Myxoma Nuclear factor) and M-T5, all of them described as virulence factors. This family of poxvirus proteins, recently identified, has drawn considerable attention for its potential role in modulating the host ubiquitin-proteasome system during viral infection. To date, many members of this novel protein family have been shown to interact with SCF components, in vitro. Here, we focus on MNF gene, which has been shown to express a nuclear protein presenting nine ANK repeats, one of which has been identified as a nuclear localization signal. In transfection, MNF has been shown to colocalise with the transcription factor NF-κB in the nucleus of TNFα-stimulated cells. Functionally, MNF is a critical virulence factor since its deletion generates an almost apathogenic virus. In this study, to pursue the investigation of proteins interacting with MNF and of its mechanism of action, we engineered a recombinant MYXV expressing a GFP-linked MNF under the control of MNF native promoter. Infection of rabbits with MYXV-GFPMNF recombinant virus provided the evidence that the GFP fusion does not disturb the main function of MNF. Hence, cells were infected with MYXV-GFPMNF and immunoprecipitation of the GFPMNF fusion protein was performed to identify MNF's partners. For the first time, endogenous components of SCF (Cullin-1 and Skp1) were co-precipitated with an ANK myxoma virus protein, expressed in an infectious context, and without over-expression of any protein.

Denbinobin, a naturally occurring 1,4-phenanthrenequinone, inhibits HIV-1 replication through an NF-κB-dependent pathway

Anthraquinones and structurally related compounds have been recently shown to exert antiviral activities and thus exhibit a therapeutic potential. In this study we report the isolation of the 1,4-phenanthrenequinone, denbinobin, from a variety of Cannabis sativa. Denbinobin does not affect the reverse transcription and integration steps of the viral cycle but prevents HIV-1 reactivation in Jurkat T cells activated by TNFα, mAbs anti-CD3/CD28 or PMA. In addition, denbinobin inhibits HIV-1-LTR activity at the level of transcription elongation and also TNFα-induced HIV-1-LTR transcriptional activity. We found that denbinobin prevents the binding of NF-κB to DNA and the phosphorylation and degradation of NF-κB inhibitory protein, IκBα, and inhibits the phosphorylation of the NF-κB p65 subunit in TNFα-stimulated cells. These results highlight the potential of the NF-κB transcription factor as a target for natural anti-HIV-1 compounds such as 1,4-phenanthrenequinones, which could serve as lead compounds for the development of an alternative therapeutic approach against AIDS.

Dynein Light Chain LC8 Negatively Regulates NF-κB through the Redox-dependent Interaction with IκBα

Redox regulation of nuclear factor kappaB (NF-kappaB) has been described, but the molecular mechanism underlying such regulation has remained unclear. We recently showed that a novel disulfide reductase, TRP14, inhibits tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation, and we identified the dynein light chain LC8, which interacts with the NF-kappaB inhibitor IkappaBalpha, as a potential substrate of TRP14. We now show the molecular mechanism by which NF-kappaB activation is redox-dependently regulated through LC8. LC8 inhibited TNFalpha-induced NF-kappaB activation in HeLa cells by interacting with IkappaBalpha and thereby preventing its phosphorylation by IkappaB kinase (IKK), without affecting the activity of IKK itself. TNFalpha induced the production of reactive oxygen species, which oxidized LC8 to a homodimer linked by the reversible formation of a disulfide bond between the Cys(2) residues of each subunit and thereby resulted in its dissociation from IkappaBalpha. Butylated hydroxyanisol, an antioxidant, and diphenyleneiodonium, an inhibitor of NADPH oxidase, attenuated the phosphorylation and degradation of IkappaBalpha by TNFalpha stimulation. In addition LC8 inhibited NF-kappaB activation by other stimuli including interleukin-1beta and lipopolysaccharide, both of which generated reactive oxygen species. Furthermore, TRP14 catalyzed reduction of oxidized LC8. Together, our results indicate that LC8 binds IkappaBalpha in a redox-dependent manner and thereby prevents its phosphorylation by IKK. TRP14 contributes to this inhibitory activity by maintaining LC8 in a reduced state.

Mitochondrial Extrusion through the Cytoplasmic Vacuoles during Cell Death

Under various conditions, noxious stimuli damage mitochondria, resulting in mitochondrial fragmentation; however, the mechanisms by which fragmented mitochondria are eliminated from the cells remain largely unknown. Here we show that cytoplasmic vacuoles originating from the plasma membrane engulfed fragmented mitochondria and subsequently extruded them into the extracellular spaces in undergoing acute tumor necrosis factor alpha-induced cell death in a caspase-dependent fashion. Notably, upon fusion of the membrane encapsulating mitochondria to the plasma membrane, naked mitochondria were released into the extracellular spaces in an exocytotic manner. Mitochondrial extrusion was specific to tumor necrosis factor alpha-induced cell death, because a genotoxic stress-inducing agent such as cisplatin did not elicit mitochondrial extrusion. Moreover, intact actin and tubulin cytoskeletons were required for mitochondrial extrusion as well as membrane blebbing. Furthermore, fragmented mitochondria were engulfed by cytoplasmic vacuoles and extruded from hepatocytes of mice injected with anti-Fas antibody, suggesting that mitochondrial extrusion can be observed in vivo under pathological conditions. Mitochondria are eliminated during erythrocyte maturation under physiological conditions, and anti-mitochondrial antibody is detected in some autoimmune diseases. Thus, elucidating the mechanism underlying mitochondrial extrusion will open a novel avenue leading to better understanding of various diseases caused by mitochondrial malfunction as well as mitochondrial biology.

Optineurin Negatively Regulates TNFα- Induced NF-κB Activation by Competing with NEMO for Ubiquitinated RIP

NF-kappaB essential modulator (NEMO), the regulatory subunit of the IkappaB kinase (IKK) that activates NF-kappaB, is essential for NF-kappaB activation. NEMO was recently found to contain a region that preferentially binds Lys (K)63-linked but not K48-linked polyubiquitin (polyUb) chains, and the ability of NEMO to bind to K63-linked polyUb RIP (receptor-interacting protein) is necessary for efficient tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation. Optineurin is a homolog of NEMO, and mutations in the optineurin gene are found in a subset of patients with glaucoma, a neurodegenerative disease involving the loss of retinal ganglion cells. Although optineurin shares considerable homology with NEMO, in resting cells, it is not present in the high-molecular-weight complex containing IKKalpha and IKKbeta, and optineurin cannot substitute for NEMO in lipopolysaccharide (LPS)-induced NF-kappaB activation. On the other hand, the overexpression of optineurin blocks the protective effect of E3-14.7K on cell death caused by the overexpression of TNFalpha receptor 1 (TNFR1). Here we show that optineurin has a K63-linked polyUb-binding region similar to that of NEMO, and like NEMO, it bound K63- but not K48-linked polyUb. Optineurin competitively antagonized NEMO's binding to polyUb RIP, and its overexpression inhibited TNFalpha-induced NF-kappaB activation. This competition occurs at physiologic protein levels because microRNA silencing of optineurin resulted in markedly enhanced TNFalpha-induced NF-kappaB activity. These results reveal a physiologic role for optineurin in dampening TNFalpha signaling, and this role might provide an explanation for its association with glaucoma.

The Molecular Mechanisms Underlying the Proinflammatory Actions of Thiazolidinediones in Human Macrophages

It is hypothesized that the antiinflammatory actions of peroxisome proliferator-activated receptors (PPARs) may explain the protective effect of these receptors in diabetes, atherosclerosis, cancer, and other inflammatory diseases. However, emerging evidence for proinflammatory activities of activated PPARs is concerning in light of new studies that associate PPAR modulators with an increased incidence of both cardiovascular events in humans and the sporadic formation of tumors in rodents. In an attempt to define the role of each PPAR subtype in inflammation, we made the unexpected observation that human PPARdelta is a positive regulator of inflammatory responses in both monocytes and macrophages. Notably, TNFalpha-stimulated cells administered PPARdelta agonists express and secrete elevated levels of inflammatory cytokines. Most surprising, however, was the finding that thiazolidinediones (TZDs) and other known PPARgamma ligands display different degrees of proinflammatory activities in a PPARgamma- and PPARalpha-independent manner via their ability to augment PPARdelta signaling. A series of mechanistic studies revealed that TZDs, at clinically relevant concentrations, bind and activate the transcriptional activity of PPARdelta. Collectively, these studies suggest that the observed proinflammatory and potentially deleterious effects of PPARgamma ligands may be mediated through an off-target effect on PPARdelta. These studies highlight the need for PPAR modulators with increased receptor subtype specificity. Furthermore, they suggest that differences in systemic exposure and consequently in the activation of PPARgamma and PPARdelta may explain why TZDs can exhibit both inflammatory and antiinflammatory activities in humans.

CSN controls NF-κB by deubiquitinylation of IκBα

The COP9 signalosome (CSN) is a conserved protein complex that regulates assembly and activity of cullin-RING ubiquitin ligases (CRLs). Ubiquitin-dependent degradation of the NF-kappaB inhibitor IkappaBalpha preceeds nuclear translocation of NF-kappaB. For the first time, we show here an inducible interaction of the CSN with IkappaBalpha and that the CSN controls IkappaBalpha and NF-kappaB activity. Strikingly, disruption of the CSN by a small interfering RNA-mediated knockdown of single CSN subunits results in a reduced re-accumulation of IkappaBalpha and prolonged nuclear translocation of NF-kappaB in TNFalpha-stimulated cells. The control of IkappaBalpha by the CSN is regulated by deubiquitinylation of IkappaBalpha conferred by the CSN-associated deubiquitinylase USP15. Protein expression levels of cullin1 and the CRL substrate adapter beta-TrCP are reduced in nonstimulated cells with a disrupted function of the CSN, which might account for an impaired basal turnover of IkappaBalpha. We propose that the CSN controls both CRL activity and stability of the CRL substrate IkappaBalpha. In consequence, basal and signal-induced CRL-dependent turnover of IkappaBalpha is precisely adapted to specific cellular needs.

TNFα-induced cytoprotection requires the production of free radicals within mitochondria in C 2C 12 myotubes

We previously reported that tumour necrosis factor alpha (TNFα) can mimic classic ischemic preconditioning (IPC) in both cells and heart. However, the signalling pathways involved remain incompletely understood. One potential protective pathway could be TNFα-induced reactive oxygen species (ROS). We hypothesized that TNFα cytoprotection occurs through the generation of ROS which originate within the mitochondria. C 2C 12 myotubes were preconditioned with either a short period of hypoxia (IPC) or a low concentration of TNFα (0.5 ng/ml) prior to a simulated ischemic insult. ROS generation was evaluated on cells stained with dichlorofluorescin diacetate (DCFH-DA) by flow cytometry. The source of TNFα-induced ROS was examined with Mitotracker Red CM-H 2XRos. The bioenergetics of the mitochondria were evaluated by investigation of the respiratory parameters and the inner mitochondrial membrane potential. Pretreatment with TNFα improved cell viability compared with the simulated ischemic control (TNFα: 75 ± 1% versus 34 ± 1% for the control: p < 0.001). The ROS scavenger, N-2-mercaptopropionyl-glycine (MPG), reduced the viability of TNFα-stimulated cells to 15 ± 1% ( p < 0.001 versus TNFα). Similar results were obtained with IPC. TNFα stimulation increased ROS production mainly in the mitochondria, and this increase was abolished in the presence of MPG. Addition of TNFα to the cells increased State 2 respiration and modestly depolarised the membrane potential prior to the ischemic insult. In conclusion, TNFα-induced ROS generation can occur within the mitochondria, resulting in temporal mitochondrial perturbations which may initiate the cytoprotective effect of TNFα.