MEKK3 is required for lysophosphatidic acid-induced NF-κB activation

Lysophosphatidic acid (LPA) is a potent agonist that exerts various cellular functions on many cell types through binding to its cognate G protein-coupled receptors (GPCRs). Although LPA induces NF-κB activation by acting on its GPCR receptor, the molecular mechanism of LPA receptor-mediated NF-κB activation remains to be well defined. In the present study, by using MEKK3-, TAK1-, and IKKβ-deficient murine embryonic fibroblasts (MEFs), we found that MEKK3 but not TAK1 deficiency impairs LPA and protein kinase C (PKC)-induced IκB kinase (IKK)-NF-κB activation, and IKKβ is required for PKC-induced NF-κB activation. In addition, we demonstrate that LPA and PKC-induced IL-6 and MIP-2 production are abolished in the absence of MEKK3 but not TAK1. Together, our results provide the genetic evidence that MEKK3 but not TAK1 is required for LPA receptor-mediated IKK-NF-κB activation.     

Rho GTPases Are Involved in the Regulation of NF-κB by Genotoxic Stress

A common cellular response to genotoxic agents and inflammatory cytokines is the activation of NF-κB. Here, we addressed the question of whether small GTPases of the Rho family are involved in the stimulation of NF-κB signaling by genotoxic agents or TNFα in HeLa cells. Inhibition of isoprenylation of Rho proteins by use of the HMG-CoA reductase inhibitor lovastatin attenuated UV-, doxorubicin-, and TNFα-induced degradation of IκBα as well as drug-stimulated DNA binding activity of NF-κB. Furthermore, NF-κB-regulated gene expression stimulated by either UV irradiation or treatment with TNFα was abrogated by lovastatin pretreatment. This indicates that isoprenylated regulatory proteins participate in the regulation of NF-κB by DNA-damaging agents as well as by TNFα. Specific blockage of Rho signaling by Clostridium difficile toxin B attenuated UV- and doxorubicin-induced activation of NF-κB, but did not affect stimulation of NF-κB by TNFα. Obviously, signaling to NF-κB by genotoxic and nongenotoxic stimuli occurs via different molecular mechanisms, either involving Rho GTPases or not. Based on the data, we suggest Rho GTPases to be essentially required for genotoxic stress-induced signaling to NF-κB.     

EFFECT OF MELATONIN ON NF-κB DNA-BINDING ACTIVITY IN THE RAT SPLEEN

It was recently demonstrated that the pineal neurohormone melatonin is a hydroxyl radical scavenger and antioxidant, and that it plays an important role in the immune system. In studies reported herein, we have investigated the relationship of the melatonin level and the NF-κ B DNA binding activity in the spleen of Sprague—Dawley rats. These in vivo results indicate that NF- κB DNA binding activity in the spleen is lower at night, when endogenous melatonin levels are elevated, than during the day, when endogenous melatonin levels are lower. Furthermore, exogenously administered melatonin (10mg/kg) was shown to cause a significant decrease in NF-κB DNA binding activity in the spleen at 60min after intraperitoneal injection (as compared with vehicle-treated rats). These new findings suggest that the normal night time rise which can be expected for melatonin may be associated with increased NF-κB DNA binding activity in the spleen. The melatonin, therefore, could potentially act to modulate spleen function and/or the immune system by regulating the NF-κB DNA binding activity in the spleen.     

USP1 Inhibits NF-κB/NLRP3 Induced Pyroptosis through TRAF6 in Osteoblastic MC3T3-E1 Cells

Objectives:Deubiquitinase Ubiquitin Specific Protease 1 (USP1) is essential for bone formation, but how USP1 regulates bone formation in response to oxidative stress remains unclear. In this study, we aim to investigate the biological function of USP1 in osteoblastic MC3T3-E1 cells.Methods:Hydrogen peroxide (H₂O₂) as an oxidative reagent was used to trigger osteoblastic MC3T3-E1 cellular damage. Flow cytometry was used to evaluate ROS production, apoptosis, and pyroptosis. Real-time PCR and western bolt assay were used to detect the mRNA and protein levels of USP1. Moreover, coimmunoprecipitation was used to validate the relationship between USP1 and TRAF6.Results:We demonstrated that USP1 was significantly decreased in MC3T3-E1 cells after H₂O₂ treatment. Overexpressing USP1 restored H₂O₂-decreased alkaline phosphatase activity and reactive oxygen species production. USP1 overexpression inhibited cytokine release and NLP3 inflammasome activation, which was mediated by NF-κB. Overexpressing USP1 prevented NF-κB translocation. USP1 formed a complex with TRAF6, inhibiting TRAF6 ubiquitination.Conclusion:USP1 exhibits protective role in MC3T3-E1 cells by suppressing NF-κB-NLRP3 mediated pyroptosis in response to H₂O₂. The involvement of USP1 and TRAF6 in NLRP3 inflammasome signaling suggests a future therapeutic potential to improve clinical symptoms in osteoporosis.     

Microbial transformation of isosteviol oxime and the inhibitory effects on NF-κB and AP-1 activation in LPS-stimulated macrophages

Microbial transformation of isosteviol oxime (ent-16-E-hydroxyiminobeyeran-19-oic acid) (2) with Aspergillus niger BCRC 32720 and Absidia pseudocylindrospora ATCC 24169 yielded several compounds. In addition to bioconverting the d-ring to lactone and lactam moieties, 4α-carboxy-13α-hydroxy-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactone (7) and 4α-carboxy-13α-amino-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactam (10), one known compound, ent-1β,7α-dihydroxy-16-oxo-beyeran-19-oic acid (6), and five new compounds, ent-7α-hydroxy-16-E-hydroxyiminobeyeran-19-oic acid (3), ent-1β,7α-dihydroxy-16-E-hydroxyiminobeyeran-19-oic acid (4), ent-1β-hydroxy-16-E-hydroxyiminobeyeran-19-oic acid (5), ent-8β-cyanomethyl-13-methyl-12-podocarpen-19-oic acid (8), and ent-8β-cyanomethyl-13-methyl-13-podocarpen-19-oic acid (9), were isolated from the microbial transformation of 2. Elucidation of the structures of these isolated compounds was primarily based on 1D and 2D NMR, and HRESIMS data, and 3–5 were further confirmed by X-ray crystallographic analyses. Additionally, the inhibitory effects of all of these compounds were evaluated on NF-κB and AP-1 activation in LPS-stimulated RAW 264.7 macrophages. Among the compounds tested, 5 and 10 significantly inhibited NF-κB activation, with 5 showing equal potency to dexamethasone; 3 and 6–9 significantly inhibited AP-1 activation, particularly 8, which showed more inhibitory activity than dexamethasone.     

IRF2 enhances RANKL-induced osteoclast differentiation via regulating NF-κB/NFATc1 signaling

Interferon regulatory factors (IRFs) play roles in various biological processes including cytokine signaling, cell growth regulation and hematopoietic development. Although it has been reported that several IRFs are involved in bone metabolism, the role of IRF2 in bone cells has not been elucidated. Here, we investigated the involvement of IRF2 in RANKL-induced osteoclast differentiation. IRF2 overexpression in osteoclast pre-cursor cells enhanced osteoclast differentiation by regulating the expression of NFATc1, a master regulator of osteoclasto-genesis. Conversely, IRF2 knockdown inhibited osteoclast differentiation and decreased the NFATc1 expression. Moreover, IRF2 increased the translocation of NF-κB subunit p65 to the nucleus in response to RANKL and subsequently induced the expression of NFATc1. IRF2 plays an important role in RANKL-induced osteoclast differentiation by regulating NF-κB/NFATc1 signaling pathway. Taken together, we demonstrated the molecular mechanism of IRF2 in osteoclast differentiation, and provide a molecular basis for potential therapeutic targets for the treatment of bone diseases characterized by excessive bone resorption.     

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.     

Alizarin,a nature compound,inhibits the growth of pancreatic cancer cells by abrogating NF-κB activation

The current performance of nature compounds in antitumor field is gradually attracted more and more attention, we discovered a nature active ingredient alizarin possess potent natural reductive NF-κB activity to against pancreatic cancer. However, the preclinical pharmacology and therapeutic effect, and the underlying mechanisms of alizarin in inhibiting pancreatic cancer are still unclear. After high-throughput screening, this is the first report that alizarin can induce a potent inhibitory effect against pancreatic cancer cells. Alizarin induced cell cycle arrest and promoted cell apoptosis by inhibiting TNF-α-stimulated NF-κB activity and nuclear translocation, and inactivated its related TNF-α-TAK1-NF-κB signaling cascade followed by downregulation of NF-κB target genes involved in cell apoptosis (Bcl-2, Bcl-xL, XIAP) and in the cell cycle and growth (cyclin D, c-myc). Due to the abrogation of NF-κB activity, combination of alizarin and gemcitabine exerted a better inhibitory effect on pancreatic cancer. In summary, natural component alizarin, inhibited cell proliferation and induced apoptosis in vitro and in vivo through targeting of the NF-κB signaling cascade with minimal toxicity, which combine with gemcitabine, can significantly enhance the antitumor capability, playing a synergistic effect. Therefore, alizarin may play a role in reversing gemcitabine resistance caused by overactivated NF-κB in clinical application in the future.