rhHMGB1 drives osteoblast migration in a TLR2/TLR4- and NF-κB-dependent manner

Osteoblast migration is significant in skeletal development. Recently, high mobility group box 1 protein (HMGB1) has been shown to highly expressed in cartilage to regulate endochondral ossification. Nevertheless, whether HMGB1 can modulate osteoblast proliferation and migration is poorly understood, as well as the intracellular signalling pathways that are involved in this process. Herein, we examined the effects of recombinant human HMGB1 (rhHMGB1) on the proliferation and migration of rat osteoblasts and investigated whether Toll-like receptor 2 (TLR2)- and TLR4-dependent signalling pathways are involved in the regulation of intracellular signalling. A transwell chamber assay was used to evaluate the migration of osteoblasts and the MTT assay was used to assess osteoblast proliferation. rhHMGB1 could significantly promote the migration of osteoblasts without inhibiting their proliferation. Meanwhile, rhHMGB1 can increase the nuclear translocation of nuclear factor-kappa B (NF-κB) p65. Specific siRNA constructs that target TLR2 or TLR4 could markedly inhibit HMGB1-induced migration of osteoblasts and HMGB1-enhanced activation of NF-κB. Collectively, HMGB1 could significantly enhance the migration of osteoblasts in vitro, and TLR2/TLR4-dependent NF-κB pathways are involved in HMGB1-induced osteoblast migration.     

TRP14 Inhibits Osteoclast Differentiation via Its Catalytic Activity

We previously reported the inhibitory role of thioredoxin-related protein of 14 kDa (TRP14), a novel disulfide reductase, in nuclear factor-κB (NF-κB) activation, but its biological function has remained to be explored. Here, we evaluated the role of TRP14 in the differentiation and function of osteoclasts (OCs), for which NF-κB and cellular redox regulation have been known to be crucial, using RAW 264.7 macrophage cells expressing wild-type TRP14 or a catalytically inactive mutant, as well as its small interfering RNA. TRP14 depletion enhanced OC differentiation, actin ring formation, and bone resorption, as well as the accumulation of reactive oxygen species (ROS). TRP14 depletion promoted the activation of NF-κB, c-Jun NH₂-terminal kinase, and p38, the expression of c-Fos, and the consequent induction of nuclear factor of activated T cell, cytoplasmic 1 (NFATc1), a key determinant of osteoclastogenesis. However, pretreatment with N-acetylcysteine or diphenylene iodonium significantly reduced the OC differentiation, as well as the ROS accumulation and NF-κB activation, that were enhanced by TRP14 depletion. Furthermore, receptor activator of NF-κB ligand (RANKL)-induced ROS accumulation, NF-κB activation, and OC differentiation were inhibited by the ectopic expression of wild-type TRP14 but not by its catalytically inactive mutant. These results suggest that TRP14 regulates OC differentiation and bone resorption through its catalytic activity and that enhancing TRP14 may present a new strategy for preventing bone resorption diseases.     

Novel Changes in NF-κB Activity during Progression and Regression Phases of Hyperplasia: ROLE OF MEK,ERK, AND p38*

Utilizing the Citrobacter rodentium-induced transmissible murine colonic hyperplasia (TMCH) model, we measured hyperplasia and NF-κB activation during progression (days 6 and 12 post-infection) and regression (days 20–34 post-infection) phases of TMCH. NF-κB activity increased at progression in conjunction with bacterial attachment and translocation to the colonic crypts and decreased 40% by day 20. NF-κB activity at days 27 and 34, however, remained 2–3-fold higher than uninfected control. Expression of the downstream target gene CXCL-1/KC in the crypts correlated with NF-κB activation kinetics. Phosphorylation of cellular IκBα kinase (IKK)α/β (Ser^176/180) was elevated during progression and regression of TMCH. Phosphorylation (Ser^32/36) and degradation of IκBα, however, contributed to NF-κB activation only from days 6 to 20 but not at later time points. Phosphorylation of MEK1/2 (Ser^217/221), ERK1/2 (Thr²⁰²/Tyr²⁰⁴), and p38 (Thr¹⁸⁰/Tyr¹⁸²) paralleled IKKα/β kinetics at days 6 and 12 without declining with regressing hyperplasia. siRNAs to MEK, ERK, and p38 significantly blocked NF-κB activity in vitro, whereas MEK1/2-inhibitor (PD98059) also blocked increases in MEK1/2, ERK1/2, and IKKα/β thereby inhibiting NF-κB activity in vivo. Cellular and nuclear levels of Ser⁵³⁶-phosphorylated (p65⁵³⁶) and Lys³¹⁰-acetylated p65 subunit accompanied functional NF-κB activation during TMCH. RSK-1 phosphorylation at Thr³⁵⁹/Ser³⁶³ in cellular/nuclear extracts and co-immunoprecipitation with cellular p65-NF-κB overlapped with p65⁵³⁶ kinetics. Dietary pectin (6%) blocked NF-κB activity by blocking increases in p65 abundance and nuclear translocation thereby down-regulating CXCL-1/KC expression in the crypts. Thus, NF-κB activation persisted despite the lack of bacterial attachment to colonic mucosa beyond peak hyperplasia. The MEK/ERK/p38 pathway therefore seems to modulate sustained activation of NF-κB in colonic crypts in response to C. rodentium infection.     

CARD9 Mediates Dectin-2-induced IκBα Kinase Ubiquitination Leading to Activation of NF-κB in Response to Stimulation by the Hyphal Form of Candida albicans

The scaffold protein CARD9 plays an essential role in anti-fungus immunity and is implicated in mediating Dectin-1/Syk-induced NF-κB activation in response to Candida albicans infection. However, the molecular mechanism by which CARD9 mediates C. albicans-induced NF-κB activation is not fully characterized. Here we demonstrate that CARD9 is involved in mediating NF-κB activation induced by the hyphal form of C. albicans hyphae (Hyphae) but not by its heat-inactivated unicellular form. Our data show that inhibiting Dectin-2 expression selectively blocked Hyphae-induced NF-κB, whereas inhibiting Dectin-1 mainly suppressed zymosan-induced NF-κB, indicating that Hyphae-induced NF-κB activation is mainly through Dectin-2 and not Dectin-1. Consistently, we find that the hyphae stimulation induces CARD9 association with Bcl10, an adaptor protein that functions downstream of CARD9 and is also involved in C. albicans-induced NF-κB activation. This association is dependent on Dectin-2 but not Dectin-1 following the hyphae stimulation. Finally, we find that although both CARD9 and Syk are required for Hyphae-induced NF-κB activation, they regulate different signaling events in which CARD9 mediates IκBα kinase ubiquitination, whereas Syk regulates IκBα kinase phosphorylation. Together, our data demonstrated that CARD9 is selectively involved in Dectin-2-induced NF-κB activation in response to C. albicans hyphae challenging.     

Interleukin-20 Promotes Migration of Bladder Cancer Cells through Extracellular Signal-regulated Kinase (ERK)-mediated MMP-9 Protein Expression Leading to Nuclear Factor (NF-κB) Activation by Inducing the Up-regulation of p21WAF1 Protein Expression

The role of inflammatory cytokine interleukin-20 (IL-20) has not yet been studied in cancer biology. Here, we demonstrated up-regulation of both IL-20 and IL-20R1 in muscle-invasive bladder cancer patients. The expressions of IL-20 and IL-20R1 were observed in bladder cancer 5637 and T-24 cells. We found that IL-20 significantly increased the expression of matrix metalloproteinase (MMP)-9 via binding activity of NF-κB and AP-1 in bladder cancer cells and stimulated the activation of ERK1/2, JNK, p38 MAPK, and JAK-STAT signaling. Among the pathways examined, only ERK1/2 inhibitor U0126 significantly inhibited IL-20-induced migration and invasion. Moreover, siRNA knockdown of IL-20R1 suppressed migration, invasion, ERK1/2 activation, and NF-κB-mediated MMP-9 expression induced by IL-20. Unexpectedly, the cell cycle inhibitor p21^WAF1 was induced by IL-20 treatment without altering cell cycle progression. Blockade of p21^WAF1 function by siRNA reversed migration, invasion, activation of ERK signaling, MMP-9 expression, and activation of NF-κB in IL-20-treated cells. In addition, IL-20 induced the activation of IκB kinase, the degradation and phosphorylation of IκBα, and NF-κB p65 nuclear translocation, which was regulated by ERK1/2. IL-20 stimulated the recruitment of p65 to the MMP-9 promoter region. Finally, the IL-20-induced migration and invasion of cells was confirmed by IL-20 gene transfection and by addition of anti-IL-20 antibody. This is the first report that p21^WAF1 is involved in ERK1/2-mediated MMP-9 expression via increased binding activity of NF-κB, which resulted in the induction of migration in IL-20/IL-20R1 dyad-induced bladder cancer cells. These unexpected results might provide a critical new target for the treatment of bladder cancer.     

Anti-Inflammatory Effects of Progesterone in Lipopolysaccharide-Stimulated BV-2 Microglia

Female sex is associated with improved outcome in experimental brain injury models, such as traumatic brain injury, ischemic stroke, and intracerebral hemorrhage. This implies female gonadal steroids may be neuroprotective. A mechanism for this may involve modulation of post-injury neuroinflammation. As the resident immunomodulatory cells in central nervous system, microglia are activated during acute brain injury and produce inflammatory mediators which contribute to secondary injury including proinflammatory cytokines, and nitric oxide (NO) and prostaglandin E₂ (PGE₂), mediated by inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively. We hypothesized that female gonadal steroids reduce microglia mediated neuroinflammation. In this study, the progesterone’s effects on tumor necrosis factor alpha (TNF-α), iNOS, and COX-2 expression were investigated in lipopolysaccharide (LPS)-stimulated BV-2 microglia. Further, investigation included nuclear factor kappa B (NF-κB) and mitogen activated protein kinase (MAPK) pathways. LPS (30 ng/ml) upregulated TNF-α, iNOS, and COX-2 protein expression in BV-2 cells. Progesterone pretreatment attenuated LPS-stimulated TNF-α, iNOS, and COX-2 expression in a dose-dependent fashion. Progesterone suppressed LPS-induced NF-κB activation by decreasing inhibitory κBα and NF-κB p65 phosphorylation and p65 nuclear translocation. Progesterone decreased LPS-mediated phosphorylation of p38, c-Jun N-terminal kinase and extracellular regulated kinase MAPKs. These progesterone effects were inhibited by its antagonist mifepristone. In conclusion, progesterone exhibits pleiotropic anti-inflammatory effects in LPS-stimulated BV-2 microglia by down-regulating proinflammatory mediators corresponding to suppression of NF-κB and MAPK activation. This suggests progesterone may be used as a potential neurotherapeutic to treat inflammatory components of acute brain injury.     

Epstein-Barr Virus-Encoded Latent Membrane Protein 1 Activates the JNK Pathway through Its Extreme C Terminus via a Mechanism Involving TRADD and TRAF2

The transforming Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) activates signalling on the NF-κB axis through two distinct domains in its cytoplasmic C terminus, namely, CTAR1 (amino acids [aa] 187 to 231) and CTAR2 (aa 351 to 386). The ability of CTAR1 to activate NF-κB appears to be attributable to the direct interaction of tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), while recent work indicates that CTAR2-induced NF-κB is mediated through its association with TNF receptor-associated death domain (TRADD). LMP1 expression also results in activation of the c-Jun N-terminal kinase (JNK) (also known as stress-activated protein kinase) cascade, an effect which is mediated exclusively through CTAR2 and can be dissociated from NF-κB induction. The organization and signalling components involved in LMP1-induced JNK activation are not known. In this study we have dissected the extreme C terminus of LMP1 and have identified the last 8 aa of the protein (aa 378 to 386) as being important for JNK signalling. Using a series of fine mutants in which single amino acids between codons 379 and 386 were changed to glycine, we have found that mutations of Pro³⁷⁹, Glu³⁸¹, Ser³⁸³, or Tyr³⁸⁴ diminish the ability of LMP1 CTAR2 to engage JNK signalling. Interestingly, this region was also found to be essential for CTAR2-mediated NF-κB induction and coincides with the LMP1 amino acid sequences shown to bind TRADD. Furthermore, we have found that LMP1-mediated JNK activation is synergistically augmented by low levels of TRADD expression, suggesting that this adapter protein is critical for LMP1 signalling. TRAF2 is known to associate with TRADD, and expression of a dominant-negative N-terminal deletion TRAF2 mutant was found to partially inhibit LMP1-induced JNK activation in 293 cells. In addition, the TRAF2-interacting protein A20 blocked both LMP1-induced JNK and NF-κB activation, further implicating TRAF2 in these phenomena. While expression of a kinase-inactive mutated NF-κB-inducing kinase (NIK), a mitogen-activated protein kinase kinase kinase which also associates with TRAF2, impaired LMP1 signalling on the NF-κB axis, it did not inhibit LMP1-induced JNK activation, suggesting that these two pathways may bifurcate at the level of TRAF2. These data further define a role for TRADD and TRAF2 in JNK activation and confirm that LMP1 utilizes signalling mechanisms used by the TNF receptor/CD40 family to elicit its pleiotropic activities.     

Ubiquitin-associated Domain-containing Ubiquitin Regulatory X (UBX) Protein UBXN1 Is a Negative Regulator of Nuclear Factor κB (NF-κB) Signaling

Excessive nuclear factor κB (NF-κB) activation should be precisely controlled as it contributes to multiple immune and inflammatory diseases. However, the negative regulatory mechanisms of NF-κB activation still need to be elucidated. Various types of polyubiquitin chains have proved to be involved in the process of NF-κB activation. Many negative regulators linked to ubiquitination, such as A20 and CYLD, inhibit IκB kinase activation in the NF-κB signaling pathway. To find new NF-κB signaling regulators linked to ubiquitination, we used a small scale siRNA library against 51 ubiquitin-associated domain-containing proteins and screened out UBXN1, which contained both ubiquitin-associated and ubiquitin regulatory X (UBX) domains as a negative regulator of TNFα-triggered NF-κB activation. Overexpression of UBXN1 inhibited TNFα-triggered NF-κB activation, although knockdown of UBXN1 had the opposite effect. UBX domain-containing proteins usually act as valosin-containing protein (VCP)/p97 cofactors. However, knockdown of VCP/p97 barely affected UBXN1-mediated NF-κB inhibition. At the same time, we found that UBXN1 interacted with cellular inhibitors of apoptosis proteins (cIAPs), E3 ubiquitin ligases of RIP1 in the TNFα receptor complex. UBXN1 competitively bound to cIAP1, blocked cIAP1 recruitment to TNFR1, and sequentially inhibited RIP1 polyubiquitination in response to TNFα. Therefore, our findings demonstrate that UBXN1 is an important negative regulator of the TNFα-triggered NF-κB signaling pathway by mediating cIAP recruitment independent of VCP/p97.     

Co-Regulation of NF-κB and Inflammasome-Mediated Inflammatory Responses by Myxoma Virus Pyrin Domain-Containing Protein M013

NF-κB and inflammasomes both play central roles in orchestrating anti-pathogen responses by rapidly inducing a variety of early-response cytokines and chemokines following infection. Myxoma virus (MYXV), a pathogenic poxvirus of rabbits, encodes a member of the cellular pyrin domain (PYD) superfamily, called M013. The viral M013 protein was previously shown to bind host ASC-1 protein and inhibit the cellular inflammasome complex that regulates the activation and secretion of caspase 1-regulated cytokines such as IL-1β and IL-18. Here, we report that human THP-1 monocytic cells infected with a MYXV construct deleted for the M013L gene (vMyxM013-KO), in stark contrast to the parental MYXV, rapidly induce high levels of secreted pro-inflammatory cytokines like TNF, IL-6, and MCP-1, all of which are regulated by NF-κB. The induction of these NF-κB regulated cytokines following infection with vMyxM013-KO was also confirmed in vivo using THP-1 derived xenografts in NOD-SCID mice. vMyxM013-KO virus infection specifically induced the rapid phosphorylation of IKK and degradation of IκBα, which was followed by nuclear translocation of NF-κB/p65. Even in the absence of virus infection, transiently expressed M013 protein alone inhibited cellular NF-κB-mediated reporter gene expression and nuclear translocation of NF-κB/p65. Using protein/protein interaction analysis, we show that M013 protein also binds directly with cellular NF-κB1, suggesting a direct physical and functional linkage between NF-κB1 and ASC-1. We further demonstrate that inhibition of the inflammasome with a caspase-1 inhibitor did not prevent the induction of NF-κB regulated cytokines following infection with vMyxM013-KO virus, but did block the activation of IL-1β. Thus, the poxviral M013 inhibitor exerts a dual immuno-subversive role in the simultaneous co-regulation of both the cellular inflammasome complex and NF-κB-mediated pro-inflammatory responses.     

Brap2 Regulates Temporal Control of NF-κB Localization Mediated by Inflammatory Response

Nuclear factor-kappaB (NF-κB) is critical for the expression of multiple genes involved in inflammatory responses and cellular survival. NF-κB is normally sequestered in the cytoplasm through interaction with an inhibitor of NF-κB (IκB), but inflammatory stimulation induces proteasomal degradation of IκB, followed by NF-κB nuclear translocation. The degradation of IκB is mediated by a SCF (Skp1-Cullin1-F-box protein)-type ubiquitin ligase complex that is post-translationaly modified by a ubiquitin-like molecule Nedd8. In this study, we report that BRCA1-associated protein 2 (Brap2) is a novel Nedd8-binding protein that interacts with SCF complex, and is involved in NF-κB translocation following TNF-α stimulation. We also found a putative neddylation site in Brap2 associated with NF-κB activity. Our findings suggest that Brap2 is a novel modulator that associates with SCF complex and controls TNF-α-induced NF-κB nuclear translocation.     

Anisomycin protects against sepsis by attenuating IκB kinase-dependent NF-κB activation and inflammatory gene expression

Anisomycin is known to inhibit eukaryotic protein synthesis and has been established as an antibiotic and anticancer drug. However, the molecular targets of anisomycin and its mechanism of action have not been explained in macrophages. Here, we demonstrated the anti-inflammatory effects of anisomycin both in vivo and in vitro. We found that anisomycin decreased the mortality rate of macrophages in cecal ligation and puncture (CLP)- and lipopolysaccharide (LPS)-induced acute sepsis. It also declined the gene expression of proinflammatory mediators such as inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin-1β as well as the nitric oxide and proinflammatory cytokines production in macrophages subjected to LPS-induced acute sepsis. Furthermore, anisomycin attenuated nuclear factor (NF)-κB activation in LPS-induced macrophages, which correlated with the inhibition of phosphorylation of NF-κB-inducing kinase and IκB kinase, phosphorylation and IκBα proteolytic degradation, and NF-κB p65 subunit nuclear translocation. These results suggest that anisomycin prevented acute inflammation by inhibiting NF-κB-related inflammatory gene expression and could be a potential therapeutic candidate for sepsis.     

RIP4 (DIK/PKK), a novel member of the RIP kinase family,activates NF-kappa B and is processed during apoptosis

RIP1 and its homologs, RIP2 and RIP3, form part of a family of Ser/Thr kinases that regulate signal transduction processes leading to NF-κB activation. Here, we identify RIP4 (DIK/PKK) as a novel member of the RIP kinase family. RIP4 contains an N-terminal RIP-like kinase domain and a C-terminal region characterized by the presence of 11 ankyrin repeats. Overexpression of RIP4 leads to activation of NF-κB and JNK. Kinase inactive RIP4 or a truncated version containing the ankyrin repeats have a dominant negative (DN) effect on NF-κB induction by multiple stimuli. RIP4 binds to several members of the TRAF protein family, and DN versions of TRAF1, TRAF3 and TRAF6 inhibit RIP4-induced NF-κB activation. Moreover, RIP4 is cleaved after Asp340 and Asp378 during Fas-induced apoptosis. These data suggest that RIP4 is involved in NF-κB and JNK signaling and that caspase-dependent processing of RIP4 may negatively regulate NF-κB-dependent pro-survival or pro-inflammatory signals.