Abstract: Exposure of macrophages to bacterial products such as lipopolysaccharide (LPS) results in activation of the NF-κB transcription factor, which orchestrates a gene expression programme that underpins the macrophage-dependent immune response. These changes include the induction or repression of a wide range of genes that regulate inflammation, cell proliferation, migration and cell survival. This process is tightly regulated and loss of control is associated with conditions such as septic shock, inflammatory diseases and cancer. To study this response, it is important to have in vitro model systems that reflect the behaviour of cells in vivo. In addition, it is necessary to understand the natural differences that can occur between individuals. In this report, we have investigated and compared the LPS response in macrophage derived cell lines and peripheral blood mononuclear cell (PBMC) derived macrophages. Gene expression profiles were determined following LPS treatment of THP-1 cells for 1 and 4 hours. LPS significantly induced or repressed 72 out of 465 genes selected as being known or putative NF-κB target genes, which exhibited 4 temporal patterns of expression. Results for 34 of these genes, including several genes not previously identified as LPS target genes, were validated using real time PCR. A high correlation between microarray and real time PCR data was found. Significantly, the LPS induced expression profile of THP-1 cells, as determined using real time PCR, was found to be very similar to that of human PBMC derived macrophages. Interestingly, some differences were observed in the LPS response between the two donor PBMC macrophage populations. Surprisingly, we found that the LPS response in U937 cells was dramatically different to both THP-1 and PBMC derived macrophages. This study revealed a dynamic and diverse transcriptional response to LPS in macrophages, involving both the induction and repression of gene expression in a time dependent manner. Moreover, we demonstrated that the LPS induced transcriptional response in the THP-1 cell line is very similar to primary PBMC derived macrophages. Therefore, THP-1 cells represent a good model system for studying the mechanisms of LPS and NF-κB dependent gene expression.

Abstract: LPS stimulates monocytes/macrophages through the activation of signaling events that modulate the production of inflammatory cytokines. Apigenin, a flavonoid abundantly found in fruits and vegetables, exhibits anti-proliferative and anti-inflammatory activities through poorly defined mechanisms. In this study, we demonstrate that apigenin inhibits the production of proinflammatory cytokines IL-1beta, IL-8, and TNF in LPS-stimulated human monocytes and mouse macrophages. The inhibitory effect on proinflammatory cytokine production persists even when apigenin is administered after LPS stimulation. Transient transfection experiments using NF-kappaB reporter constructs indicated that apigenin inhibits the transcriptional activity of NF-kappaB in LPS-stimulated mouse macrophages. The classical proteasome-dependent degradation of the NF-kappaB inhibitor IkappaBalpha was observed in apigenin LPS-stimulated human monocytes. Using EMSA, we found that apigenin does not alter NF-kappaB-DNA binding activity in human monocytes. Instead we show that apigenin, as part of a non-canonical pathway, regulates NF-kappaB activity through hypophosphorylation of Ser536 in the p65 subunit and the inactivation of the IKK complex stimulated by LPS. The decreased phosphorylation on Ser536 observed in LPS-stimulated mouse macrophages treated with apigenin was overcome by the over-expression of IKKbeta. In addition, our studies indicate that apigenin inhibits in vivo LPS-induced TNF and the mortality induced by lethal doses of LPS. Collectively, these findings suggest a molecular mechanism by which apigenin suppresses inflammation and modulates the immune response in vivo.

Abstract: MAPK-p38 plays an important role in inflammation. Several studies have shown that blocking p38 activity attenuates the transcriptional activity of the proinflammatory transcription factor NF-κB without altering its DNA-binding activity. We have also observed that blocking p38 in human primary astrocytes suppresses the transcriptional but not the DNA-binding activity of NF-κB and down-regulates the expression of an NF-κB-dependent gene, inducible NO synthase. However, the molecular mechanism of p38-mediated regulation of NF-κB remains largely unknown. In this study, we delineate that p38 controls the transcriptional activity of NF-κB by regulating acetylation of p65, but not its phosphorylation. The combination of IL-1β and IFN-γ, previously shown to strongly induce inducible NO synthase in human primary astrocytes, induced p38-dependent phosphorylation of acetyltransferase coactivator p300, but not p65, and subsequent association of p300 with p65. Furthermore, immunocomplex-histone acetyltransferase assays demonstrated that cytokine-induced association of p65 with biologically active immunocomplex-histone acetyltransferase assay was dependent on p38. It has been previously reported that acetylation of p65 at K310 residue is important for transcriptional activity of NF-κB. Accordingly, we found that cytokine-induced association of p65 with p300 led to acetylation of p65 at K310. Because p38 regulated the association between p65 and p300, blocking p38 activity also led to attenuation of p65-K310 acetylation in cytokine-stimulated astrocytes. Taken together, this study illuminates a novel regulatory role of p38 during neuroinflammation where this MAP kinase controls acetylation of NF-κB p65 by regulating acetyltransferase activity of coactivator p300.

Abstract: Triggering of antigen receptors on lymphocytes is critical for initiating adaptive immune response against pathogens. T-cell receptor (TCR) engagement induces the formation of the Carma1-Bcl10-Malt1 (CBM) complex that is essential for activation of the IkappaB kinase (IKK)/NF-kappaB pathway. However, the molecular mechanisms that link CBM complex formation to IKK activation remain unclear. Here we report that Malt1 is polyubiquitinated upon T-cell activation. Ubiquitin chains on Malt1 provide a docking surface for the recruitment of the IKK regulatory subunit NEMO/IKKgamma. TRAF6 associates with Malt1 in response to T-cell activation and can function as an E3 ligase for Malt1 in vitro and in vivo, mediating lysine 63-linked ubiquitination of Malt1. Multiple lysine residues in the C-terminus of Malt1 serve as acceptor sites for the assembly of polyubiquitin chains. Malt1 mutants that lack C-terminal ubiquitin acceptor lysines are impaired in rescuing NF-kappaB signaling and IL-2 production in Malt1-/- T cells. Thus, our data demonstrate that induced Malt1 ubiquitination is critical for the engagement of CBM and IKK complexes, thereby directing TCR signals to the canonical NF-kappaB pathway.

Abstract: The NF-κB transcription factor is normally transiently activated by proinflammatory cytokines and bacterial lipopolysaccharide (LPS); however, persistent NF-κB activation is commonly observed in inflammatory disease and malignancy. The ubiquitin editing enzyme A20 serves an essential role in the termination of TNF-α- and LPS-mediated NF-κB signaling by inactivating key signaling molecules. However, little is known about how A20 is regulated and if other molecules play a role in the termination of NF-κB signaling. Here we demonstrate that Tax1-binding protein 1 (TAX1BP1) is essential for the termination of NF-κB and JNK activation in response to TNF-α, IL-1 and LPS stimulation. In TAX1BP1-deficient mouse fibroblasts, TNF-α-, IL-1- and LPS-mediated IKK and JNK activation is elevated and persistent owing to enhanced ubiquitination of RIP1 and TRAF6. Furthermore, in the absence of TAX1BP1, A20 is impaired in RIP1 binding, deubiquitination of TRAF6 and inhibition of NF-κB activation. Thus, TAX1BP1 is pivotal for the termination of NF-κB and JNK signaling by functioning as an essential regulator of A20.

Abstract: Prolonged and repeated exposure of the skin to ultraviolet light (UV) leads not only to aging of the skin but also increases the incidence of non-melanoma skin cancer (NMSC). Damage of cells induced by ultraviolet B (UVB) light both at the DNA level and molecular level initiates the activation of transcription factor pathways, which in turn regulate the expression of a number of genes termed the “UV response genes”. Two such transcription factor families that are activated in this way are those of the nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) families. These two transcription factor families have been identified to be involved in the processes of cell proliferation, cell differentiation and cell survival and therefore play important roles in tumorigenesis. The study of these two transcription factor pathways and the cross-talk between them in response to UVB exposure may help with the development of new chemopreventive strategies for the prevention of UVB-induced skin carcinogenesis.

Abstract: Mutations in the parkin gene are a major cause of autosomal recessive Parkinson's disease. Here we show that the E3 ubiquitin ligase parkin activates signaling through the IκB kinase (IKK)/nuclear factor κB (NF-κB) pathway. Our analysis revealed that activation of this signaling cascade is causally linked to the neuroprotective potential of parkin. Inhibition of NF-κB activation by an IκB super-repressor or a kinase-inactive IKKβ interferes with the neuroprotective activity of parkin. Furthermore, pathogenic parkin mutants with an impaired neuroprotective capacity show a reduced ability to stimulate NF-κB-dependent transcription. Finally, we present evidence that parkin interacts with and promotes degradation-independent ubiquitylation of IKKγ/NEMO (NF-κB essential modifier) and TRAF2 [TNF (tumor necrosis factor) receptor-associated factor 2], two critical components of the NF-κB pathway. Thus, our results support a direct link between the neuroprotective activity of parkin and ubiquitin signaling in the IKK/NF-κB pathway.

Abstract: NF-κB signaling drives much of the initial acute inflammatory response to innate immune stimulation. Cells of the innate immune system express membrane-bound Toll-like receptors (TLRs) and intracellular pattern recognition receptors, such as the NOD proteins and RIG-I, all of which are responsible for recognizing specific components of bacterial, viral, or fungal organisms (21, 30, 31). Upon activation of these receptors, cells of the innate immune system activate divergent signal transduction pathways that ultimately converge to activate the IκB kinase (IKK) complex (11, 30). This complex consists, in its simplest form, of the kinases IKKα and IKKβ bound together by NEMO (IKKγ). Upon activation, this kinase complex phosphorylates IκBα, the inhibitor of NF-κB. This phosphorylation event leads to IκBα's ubiquitination and proteasomal degradation, allowing NF-κB transcription factors to stimulate the transcription of inflammatory genes (11). While these initial mechanisms of NF-κB activation have been well studied, the mechanisms by which NF-κB is downregulated are less well understood. The numerous signaling pathways causing NF-κB activation must be tightly regulated. Failure to mount a sufficient immune response results in susceptibility to infectious disease, and failure to properly downregulate NF-κB results in autoimmune or inflammatory disorders (11). Numerous studies have demonstrated that diverse inflammatory pathways are regulated in large part by coordinated phosphorylation and ubiquitination events (6, 16, 24, 28). The phosphorylation- and ubiquitin-mediated degradation of IκBα is the most well-known example of this coordination. In addition, TLR and RIP2/NOD2 activation results in the K63-linked ubiquitination of TRAF6 and NEMO (1, 2, 7, 30). Unlike K48-linked polyubiquitin chains, which target a protein for proteasomal degradation, the nondegradative K63-linked polyubiquitinated chains are thought to nucleate a signaling complex that activates the kinases TAK1 and IKKα/β (6, 16). In a similar way, K63-linked ubiquitination of TRAF2 and RIP1 is responsible for activating the IKK complex following activation of the tumor necrosis factor (TNF) receptor by TNF-α (6). Ubiquitination is a rapidly reversible modification, as deubiquitinating enzymes such as A20 (TNFAIP3) decrease the amplitude of ubiquitin-mediated NF-κB signaling (9, 29, 35). A20 contains a deubiquitinase domain at its N terminus and a series of seven zinc fingers at its C terminus (5, 9, 35). These C-terminal zinc fingers mediate K48-linked polyubiquitination. A20 deubiquitinates K63-polyubiquitinated proteins and then helps to conjugate K48-linked polyubiquitin chains such that the protein is targeted for degradation (5, 35). K63-linked polyubiquitination of many NF-κB regulators, such as RIP, TRAF6, TRAF2, and NEMO, positively regulates NF-κB activity (6, 16), and A20 has been shown to interact with and/or deubiquitinate the same proteins to ultimately inhibit the NF-κB signaling pathway (12, 29, 35, 37). The importance of A20 in the downregulation of inflammatory signaling pathways is further illustrated by A20−/− mice, which show uncontrolled whole-body inflammation and hypersensitivity to TNF-α and lipopolysaccharide (LPS) (5, 18). A20 is known to be transcriptionally upregulated following activation of NF-κB, but mechanisms regulating the intrinsic enzymatic activity of A20 have not been reported (17, 23). IKKβ is a serine/threonine kinase that is essential for the activation of the canonical NF-κB signaling pathway. Its activation of NF-κB is important for cell survival pathways, and a role in insulin signaling pathways was suggested following a report that IKKβ can phosphorylate IRS-1 (10, 25). IKKβ has also been shown to have a role in the development of a number of inflammation-related cancers (reviewed in references 3 and 15). While several IKKβ substrates have been recognized, both the importance of IKKβ and the breadth of signaling pathways it affects suggest that the complete repertoire of intracellular substrates has yet to be identified. Given this possibility, we developed and utilized an unbiased method to identify novel substrates of IKKβ. The optimal phosphorylation motif for IKKβ was identified using a positional scanning peptide library technique. A combination of several bioinformatic approaches was then used to identify the most likely candidate IKKβ substrates, and the deubiquitinating enzyme A20 was identified as a putative substrate of IKKβ. Serine 381 (S381) on A20 was identified as a target phosphorylation site for IKKβ and was verified as an in vitro and in vivo IKKβ phosphorylation site. We further found that phosphorylation of this site increases the ability of A20 to downregulate NF-κB in response to multiple stimuli. These findings show that phosphorylation of A20 by IKKβ modulates NF-κB signaling and represents a novel mechanism of regulation within innate immune signaling pathways.

Abstract: Nuclear factor kappa B (NF-kappaB) is a redox-associated transcription factor that is involved in the activation of survival pathways. We have previously shown that deoxycholate (DOC) activates NF-kappaB in hepatocytes and colon epithelial cells and that persistent exposure of HCT-116 cells to increasing concentrations of DOC results in the constitutive activation of NF-kappaB, which is associated with the development of apoptosis resistance. The mechanisms by which DOC activates NF-kappaB in colon epithelial cells, and whether natural antioxidants can reduce DOC-induced NF-kappaB activation, however, are not known. Also, it is not known if DOC can generate reactive oxygen species within mitochondria as a possible pathway of stress-related NF-kappaB activation. Since we have previously shown that DOC activates the NF-kappaB stress-response pathway in HCT-116 cells, we used this cell line to further explore the mechanisms of NF-kappaB activation. We found that DOC induces mitochondrial oxidative stress and activates NF-kappaB in HCT-116 cells through multiple mechanisms involving NAD(P)H oxidase, Na+/K+-ATPase, cytochrome P450, Ca++ and the terminal mitochondrial respiratory complex IV. DOC-induced NF-kappaB activation was significantly (P < 0.05) inhibited by pre-treatment of cells with CAPE, EGCG, TMS, DPI, NaN3, EGTA, Ouabain and RuR. The NF-kappaB-activating pathways, induced by the dietary-related endogenous detergent DOC, provide mechanisms for promotion of colon cancer and identify possible new targets for chemoprevention.

Abstract: Cystic fibrosis (CF) is characterized by prolonged and excessive inflammatory responses in the lung and increased activation of NF-κB. Parthenolide is a sesquiterpene lactone derived from the plant feverfew, which has been used in folk medicine for anti-inflammatory activity. Several studies suggest that this compound inhibits the NF-κB pathway, but the exact site is controversial. We hypothesized that parthenolide might ameliorate the excessive inflammatory response in CF models by inhibiting activation of NF-κB. This was tested in vitro, using two pairs of cell lines with defective versus normal CF transmembrane conductance regulator (CFTR) (antisense/sense transfected 16 HBE and IB-3/S9), and in vivo, using CFTR-knockout (KO) mice. All cell lines were pretreated with parthenolide and then stimulated with IL-1β and/or TNF. Parthenolide significantly inhibited IL-8 secretion induced by these cytokines and prevented NF-κB activation, IκBα degradation, and IκB Kinase complex activity. CFTR-KO and wild-type...

Abstract: Identifying the active chemical ingredients of ancient medicines and the molecular targets of those ingredients is an attractive therapeutic objective. Embelin, identified primarily from the Embelia ribes plant, is one such compound shown to exhibit chemopreventive, anti-inflammatory, and apoptotic activities through an unknown mechanism. Because nuclear factor-kappaB (NF-kappaB) regulates several genes associated with inflammation, proliferation, carcinogenesis, and apoptosis, we postulated that embelin might mediate its activity through modulation of NF-kappaB activation. We found that embelin inhibited tumor necrosis factor (TNF) alpha-induced NF-kappaB activation. Both inducible and constitutive NF-kappaB activation were abrogated by embelin. In addition, NF-kappaB activated by diverse stimuli such as interleukin-1beta, lipopolysaccharide, phorbol myristate acetate, okadaic acid, hydrogen peroxide, and cigarette smoke condensate also was suppressed. We found that embelin inhibited sequentially the TNFalpha-induced activation of the inhibitory subunit of NF-kappaBalpha (IkappaBalpha) kinase, IkappaBalpha phosphorylation, IkappaBalpha degradation, and p65 phosphorylation and nuclear translocation. Embelin also suppressed NF-kappaB-dependent reporter gene transcription induced by TNFalpha, TNF receptor-1 (TNFR1), TNFR1-associated death domain protein, TNFR-associated factor-2, NF-kappaB-inducing kinase, and IkappaBalpha kinase but not by p65. Furthermore, we found that embelin down-regulated gene products involved in cell survival, proliferation, invasion, and metastasis of the tumor. This down-regulation was associated with enhanced apoptosis by cytokine and chemotherapeutic agents. Together, our results indicate that embelin is a novel NF-kappaB blocker and potential suppressor of tumorigenesis.

Abstract: Background Human mast cells are multifunctional cells capable of a wide variety of inflammatory responses. Baicalein (BAI), isolated from the traditional Chinese herbal medicine Huangqin (Scutellaria baicalensis Georgi), has been shown to have anti-inflammatory effects. We examined its effects and mechanisms on the expression of inflammatory cytokines in an IL-1β- and TNF-α-activated human mast cell line, HMC-1.

Abstract: Sustained hepatic inflammation induced by various causes can lead to liver fibrosis. Transcription factor NF-κB is important in regulating inflammatory responses, especially in macrophages. We pres...

Abstract: Nuclear factor kappa B (NF-κB) has been shown to play an important role in regulating the expression of many genes involved in cell survival, immunity and in the inflammatory processes. NF-κB activation upregulates inducible nitric oxide synthase leading to enhanced nitric oxide production during an inflammatory response. NF-κB activation is regulated by distinct kinase pathways independent of inhibitor of κB kinase (IKK). Here, we examine the role of protein kinase C isoforms and janus activated kinase 2 (JAK2) activation in NF-κB activation and LPS-stimulated NO production. Murine RAW 264.7 macrophages were treated with lipopolysaccharide (LPS), Phorbol 12-myristate 13-acetate (PMA) and a combination of LPS and PMA in the presence or absence of various inhibitors of PKC isoforms and JAK2. Nuclear translocation of the NF-κB p65 subunit, was assessed by Western blot analysis whilst NO levels were assessed by Greiss assay. LPS-stimulated NO production was attenuated by PMA whilst PMA alone did not affect NO release. These effects were associated with changes in p65 nuclear translocation. The PKCα, β, γ, δ and ζ inhibitor Go 6983 (Go) had no effect on LPS-induced NO release. In contrast, Bisindolymalemide I (Bis), a PKC α, βI, βII, γ, δ and e isoform inhibitors completely inhibited LPS-stimulated NO production without affecting p65 nuclear translocation. Furthermore, a partial inhibitory effect on LPS-induced NO release was seen with the JAK2 inhibitor AG-490 and the p38 MAPK inhibitor SB 203850. The results further define the role of NF-κB in LPS stimulated NO production in RAW macrophages. The data support a function for PKCe, JAK2 and p38 MAPK in NF-κB activation following p65 nuclear import.

Abstract: Numerous recent reports suggest that statins (hydroxy-3-methylglutaryl-CoA reductase inhibitors) exhibit potential to suppress tumorigenesis through a mechanism that is not fully understood. Therefore, in this article, we investigated the effects of simvastatin on TNF-α-induced cell signaling. We found that simvastatin potentiated the apoptosis induced by TNF-α as indicated by intracellular esterase activity, caspase activation, TUNEL, and annexin V staining. This effect of simvastatin correlated with down-regulation of various gene products that mediate cell proliferation (cyclin D1 and cyclooxygenase-2), cell survival (Bcl-2, Bcl-x L , cellular FLIP, inhibitor of apoptosis protein 1, inhibitor of apoptosis protein 2, and survivin), invasion (matrix mellatoproteinase-9 and ICAM-1), and angiogenesis (vascular endothelial growth factor); all known to be regulated by the NF-κB. We found that simvastatin inhibited TNF-α-induced NF-κB activation, and l-mevalonate reversed the suppressive effect, indicating the role of hydroxy-3-methylglutaryl-CoA reductase. Simvastatin suppressed not only the inducible but also the constitutive NF-κB activation. Simvastatin inhibited TNF-α-induced IκBα kinase activation, which led to inhibition of IκBα phosphorylation and degradation, suppression of p65 phosphorylation, and translocation to the nucleus. NF-κB-dependent reporter gene expression induced by TNF-α, TNFR1, TNFR-associated death domain protein, TNFR-associated factor 2, TGF-β-activated kinase 1, receptor-interacting protein, NF-κB-inducing kinase, and IκB kinase β was abolished by simvastatin. Overall, our results provide novel insight into the role of simvastatin in potentially preventing and treating cancer through modulation of IκB kinase and NF-κB-regulated gene products.

Abstract: Redox sensitive, pro-inflammatory nuclear transcription factor NF-κB plays a key role in both inflammation and aging processes In a redox state disrupted by oxidative stress, pro-inflammatory genes are upregulated by the activation of NF-κB through diverse kinases Thus, the search and characterization of new substances that modulate NF-κB are of recent research interest Cinnamaldehyde (CNA) is the major component of cinnamon bark oil, which has been widely used as a flavoring agent in foodstuffs such as beverages and ice cream In the present study, CNA was examined for its molecular modulation of inflammatory NF-κB activation via the redox-related NIK/IKK and MAPK pathways through the reduction of oxidative stress Results show that age-related NF-κB activation upregulated NF-κB targeting genes, inflammatory iNOS, and COX-2, all of which were inhibited effectively by CNA Our study further shows that CNA inhibited the activation of NF-κB via three signal transduction pathways, NIK/IKK, ERK, and p38 MAPK Our results indicate that CNA’s antioxidative effect and the restoration of redox balance were responsible for its anti-inflammatory action Thus, the significance of the current study is the new information revealing the anti-inflammatory properties of CNA and the role it plays in the regulation of age-related alterations in signal transduction pathways