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.     

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.     

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.     

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.     

Efficient synthesis of (±)-parasitenone, a novel inhibitor of NF-κB

Dehydroxymethylepoxyquinomicin (DHMEQ, 1) is a novel nuclear factor-κB (NF-κB) inhibitor that inhibits DNA binding of NF-κB components including p65. To inspect its biological activity of 1, we synthesized parasitenone (3), possessing the common epoxycyclohexenone moiety of 1. Assessment of the inhibitory activity against NF-κB indicated that the epoxycyclohexenone moiety is the most essential element for the NF-κB inhibitory activity and the salicylic acid moiety may contribute the binding efficiency and specificity.     

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.     

Molecular dynamics simulation and docking analysis of NF-κB protein binding with sulindac acid

It is of interest to document the Molecular Dynamics Simulation and docking analysis of NF-κB target with sulindac sodium in combating COVID-19 for further consideration. Sulindac is a nonsteroidal anti-inflammatory drug (NSAID) of the arylalkanoic acid class that is marketed by Merck under the brand name Clinoril. We show the binding features of sulindac sodium with NF-κB that can be useful in drug repurposing in COVID-19 therapy.     

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.     

S‐nitrosylated protein disulfide isomerase contributes to mutant SOD1 aggregates in amyotrophic lateral sclerosis

A major hallmark of mutant superoxide dismutase (SOD1)‐linked familial amyotrophic lateral sclerosis is SOD1‐immunopositive inclusions found within motor neurons. The mechanism by which SOD1 becomes aggregated, however, remains unclear. In this study, we aimed to investigate the role of nitrosative stress and S‐nitrosylation of protein disulfide isomerase (PDI) in the formation of SOD1 aggregates. Our data show that with disease progression inducible nitric oxide synthase (iNOS) was up‐regulated, which generated high levels of nitric oxide (NO) and subsequently induced S‐nitrosylation of PDI in the spinal cord of mutant SOD1 transgenic mice. This was further confirmed by in vitro observation that treating SH‐SY5Y cells with NO donor S‐nitrosocysteine triggered a dose‐dependent formation of S‐nitrosylated PDI. When mutant SOD1 was over‐expressed in SH‐SY5Y cells, the iNOS expression was up‐regulated, and NO generation was consequently increased. Furthermore, both S‐nitrosylation of PDI and the formation of mutant SOD1 aggregates were detected in the cells expressing mutant SOD1^G93A. Blocking NO generation with the NOS inhibitor N‐nitro‐l ‐arginine attenuated the S‐nitrosylation of PDI and inhibited the formation of mutant SOD1 aggregates. We conclude that NO‐mediated S‐nitrosylation of PDI is a contributing factor to the accumulation of mutant SOD1 aggregates in amyotrophic lateral sclerosis.     

AKR1B10 confers resistance to radiotherapy via FFA/TLR4/NF-κB axis in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is one kind of human head and neck cancers with high incidence in Southern China, Southeast Asia and North Africa. In spite of great innovations in radiation and chemotherapy treatments, the 5-year survival rate is not satisfactory. One of the main reasons is resistance to radiotherapy which leads to therapy failure and recurrence of NPC. The mechanism underlying remains to be fully elucidated. Aldo-keto reductase B10 (AKR1B10) plays a role in the formation and development of carcinomas. However, its role in resistance to radiotherapy of NPC is not clear. In this research, the relationships between AKR1B10 expression and the treatment effect of NPC patients, NPC cell survival, cell apoptosis, and DNA damage repair, as well as the effect and mechanism of AKR1B10 expression on NPC radioresistance were explored. A total of 58 paraffin tissues of NPC patients received radiotherapy were collected including 30 patients with radiosensitivity and 28 patients with radioresistance. The relationships between AKR1B10 expression and the treatment effect as well as clinical characteristics were analyzed by immuno-histochemical experiments, and the roles of AKR1B10 in cell survival, apoptosis and DNA damage repair were detected using the AKR1B10 overexpressed cell models. Furthermore the mechanism of AKR1B10 in NPC radioresistance was explored. Finally, the radioresistance effect of AKR1B10 expression was evaluated by the tumor xenograft model of nude mice and the method of radiotherapy. The results showed AKR1B10 expression level was correlated with radiotherapy resistance, and AKR1B10 overexpression promoted proliferation of NPC cells, reduced apoptosis and decreased cellular DNA damage after radiotherapy. The probable molecular mechanism is that AKR1B10 expression activated FFA/TLR4/NF-κB axis in NPC cells. This was validated by using the TLR4 inhibitor TAK242 to treat NPC cells with AKR1B10 expression, which reduced the phosphorylation of NF-κB. This study suggests that AKR1B10 can induce radiotherapy resistance and promote cell survival via FFA/TLR4/NF-κB axis in NPC, which may provide a novel target to fight against radiotherapy resistance of NPC.