Curcumin analogue NL04 inhibits spinal cord central sensitization in rats with bone cancer pain by inhibiting NLRP3 inflammasome activation and reducing IL-1ß production.

The management and therapy of bone cancer pain (BCP) remain formidable clinical challenges. Curcumin and its analogues have been shown to have anti-inflammatory and analgesic properties. In the present study, we investigated the efficacy of curcumin analogue NL04 (NL04) in modulating inflammation in spinal dorsal horn (SDH), thereby exploring its potential to reduce central sensitization of BCP in a rat model. Differing doses of NL04 and curcumin were administered intrathecally either once (on day 12 of BCP) or over seven consecutive days (from day 6-12 of BCP). Results indicated that the ED50 for NL04 and curcumin ameliorating BCP-induced mechanical hyperalgesia is 49.08 µg/kg and 489.6 µg/kg, respectively. The analgesic effects at various doses of NL04 lasted between 4 and 8 h, with sustained administration over a week maintaining pain relief for 1-4 days, while also ameliorating locomotor gait via gait analysis and reducing depressive and anxiety-like behaviors via open-field and light-dark transition tests. The analgesic effects at various doses of curcumin lasted 4 h, with sustained administration over a week maintaining pain relief for 0-2 days. ELISA, Western blotting, qPCR, and immunofluorescence assays substantiated that intrathecal administration of NL04 on days 6-12 of BCP dose-dependently lowered spinal IL-1ß and IL-18 levels and significantly reduced the expression of IKKß genes and proteins, as well as the downstream cleavage of the trans-Golgi network (TGN). Whole-cell patch-clamp results demonstrated that NL04 inhibits potassium ion efflux in rat primary spinal neurons. Thus, NL04 exhibits significant analgesic effects in a BCP rat model by downregulating IKKß expression and inhibiting neuronal potassium ion efflux, which, in turn, suppresses the activation of NLRP3 inflammasomes and reduces IL-1ß production, potentially ameliorating pain management in BCP.

Diosgenin protects against cationic bovine serum albumin-induced membranous glomerulonephritis by attenuating oxidative stress and renal inflammation via the NF-κB pathway.

CONTEXT: Membranous glomerulonephritis (MGN) is a leading cause of nephrotic syndrome in adults. Diosgenin (DG) has been reported to exert antioxidative and anti-inflammatory effects. OBJECTIVE: To investigate the renoprotective activity of DG in a cationic bovine serum albumin-induced rat model of MGN. MATERIALS AND METHODS: Fourty male Sprague-Dawley rats were randomized into four groups. The MGN model was established and treated with a DG dose (10 mg/kg) and a positive control (TPCA1, 10 mg/kg), while normal control and MGN groups received distilled water by gavage for four consecutive weeks. At the end of the experiment, 24 h urinary protein, biochemical indices, oxidation and antioxidant levels, inflammatory parameters, histopathological examination, immunohistochemistry and immunoblotting were evaluated. RESULTS: DG significantly ameliorated kidney dysfunction by decreasing urinary protein (0.56-fold), serum creatinine (SCr) (0.78-fold), BUN (0.71-fold), TC (0.66-fold) and TG (0.73-fold) levels, and increasing ALB (1.44-fold). DG also reduced MDA (0.82-fold) and NO (0.83-fold) levels while increasing the activity of SOD (1.56-fold), CAT (1.25-fold), glutathione peroxidase (GPx) (1.55-fold) and GSH (1.81-fold). Furthermore, DG reduced Keap1 (0.76-fold) expression, Nrf2 nuclear translocation (0.79-fold), and induced NQO1 (1.25-fold) and HO-1 (1.46-fold) expression. Additionally, DG decreased IL-2 (0.55-fold), TNF-α (0.80-fold) and IL-6 (0.75-fold) levels, and reduced protein expression of NF-κB p65 (0.80-fold), IKKß (0.93-fold), p-IKKß (0.89-fold), ICAM-1 (0.88-fold), VCAM-1 (0.91-fold), MCP-1 (0.88-fold) and E-selectin (0.87-fold), and also inhibited the nuclear translocation of NF-κB p65 (0.64-fold). DISCUSSION AND CONCLUSIONS: The results suggest a potential therapeutic benefit of DG against MGN due to the inhibition of the NF-κB pathway, supporting the need for further clinical trials.

Dual regulation of NEMO by Nrf2 and miR-125a inhibits ferroptosis and protects liver from endoplasmic reticulum stress-induced injury.

Rationale: The surge of severe liver damage underscores the necessity for identifying new targets and therapeutic agents. Endoplasmic reticulum (ER) stress induces ferroptosis with Gα12 overexpression. NF-κB essential modulator (NEMO) is a regulator of inflammation and necroptosis. Nonetheless, the regulatory basis of NEMO de novo synthesis and its impact on hepatocyte ferroptosis need to be established. This study investigated whether Nrf2 transcriptionally induces IKBKG (the NEMO gene) for ferroptosis inhibition and, if so, how NEMO induction protects hepatocytes against ER stress-induced ferroptosis. Methods: Experiments were conducted using human liver tissues, hepatocytes, and injury models, incorporating NEMO overexpression and Gα12 gene modulations. RNA sequencing, immunoblotting, immunohistochemistry, reporter assays, and mutation analyses were done. Results: NEMO downregulation connects closely to ER and oxidative stress, worsening liver damage via hepatocyte ferroptosis. NEMO overexpression protects hepatocytes from ferroptosis by promoting glutathione peroxidase 4 (GPX4) expression. This protective role extends to oxidative and ER stress. Similar shifts occur in nuclear factor erythroid-2-related factor-2 (Nrf2) expression alongside NEMO changes. Nrf2 is newly identified as an IKBKG (NEMO gene) transactivator. Gα12 changes, apart from Nrf2, impact NEMO expression, pointing to post-transcriptional control. Gα12 reduction lowers miR-125a, an inhibitor of NEMO, while overexpression has the opposite effect. NEMO also counters ER stress, which triggers Gα12 overexpression. Gα12's significance in NEMO-dependent hepatocyte survival is confirmed via ROCK1 inhibition, a Gα12 downstream kinase, and miR-125a. The verified alterations or associations within the targeted entities are validated in human liver specimens and datasets originating from livers subjected to exposure to other injurious agents. Conclusions: Hepatic injury prompted by ER stress leads to the suppression of NEMO, thereby facilitating ferroptosis through the inhibition of GPX4. IKBKG is transactivated by Nrf2 against Gα12 overexpression responsible for the increase of miR-125a, an unprecedented NEMO inhibitor, resulting in GPX4 induction. Accordingly, the induction of NEMO mitigates ferroptotic liver injury.

Induction of MTHFD2 in Macrophages Inhibits Reactive Oxygen Species-mediated NF-κB Activation and Protects against Inflammatory Responses.

The one-carbon metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is critical for cancer cell proliferation and immune cell phenotypes, but whether it can contribute to macrophage inflammatory responses remains unclear. In this study, we show that MTHFD2 was upregulated by LPS in murine macrophages upon activation of the TLR4-MyD88-IKKα/ß-NF-κB signaling pathway. MTHFD2 significantly attenuated LPS-induced macrophage proinflammatory cytokine production through its enzymatic activity. Notably, ablation of myeloid MTHFD2 rendered mice more sensitive to septic shock and CCl4-induced acute hepatitis. Mechanistically, MTHFD2 restrained IKKα/ß-NF-κB activation and macrophage inflammatory phenotype by scavenging reactive oxygen species through the generation of NADPH. Our study reveals MTHFD2 as a "self-control" mechanism in macrophage-mediated inflammatory responses.

Oxidative stress-mediated activation of FTO exacerbates impairment of the epithelial barrier by up-regulating IKBKB via N6-methyladenosine-dependent mRNA stability in asthmatic mice exposed to PM2.5.

In order to comprehend the underlying mechanisms contributing to the development and exacerbation of asthma resulting from exposure to fine particulate matter (PM2.5), we established an asthmatic model in fat mass and obesity-associated gene knockdown mice subjected to PM2.5 exposure. Histological analyses using hematoxylin-eosin (HE) and Periodic Acid-Schiff (PAS) staining revealed that the down-regulation of the fat mass and obesity-associated gene (Fto) expression significantly ameliorated the pathophysiological alterations observed in asthmatic mice exposed to PM2.5. Furthermore, the down-regulation of Fto gene expression effectively attenuated damage to the airway epithelial barrier. Additionally, employing in vivo and in vitro models, we elucidated that PM2.5 modulated FTO expression by inducing oxidative stress. Asthmatic mice exposed to PM2.5 exhibited elevated Fto expression, which correlated with increased levels of reactive oxygen species. Similarly, when cells were exposed to PM2.5, FTO expression was up-regulated in a ROS-dependent manner. Notably, the administration of N-acetyl cysteine successfully reversed the PM2.5-induced elevation in FTO expression. Concurrently, we performed transcriptome-wide Methylated RNA immunoprecipitation Sequencing (MeRIP-seq) analysis subsequent to PM2.5 exposure. Through the implementation of Gene Set Enrichment Analysis and m6A-IP-qPCR, we successfully identified inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB) as a target gene regulated by FTO. Interestingly, exposure to PM2.5 led to increased expression of IKBKB, while m6A modification on IKBKB mRNA was reduced. Furthermore, our investigation revealed that PM2.5 also regulated IKBKB through oxidative stress. Significantly, the down-regulation of IKBKB effectively mitigated epithelial barrier damage in cells exposed to PM2.5 by modulating nuclear factor-kappa B (NF-κB) signaling. Importantly, we discovered that decreased m6A modification on IKBKB mRNA facilitated by FTO enhanced its stability, consequently resulting in up-regulation of IKBKB expression. Collectively, our findings propose a novel role for FTO in the regulation of IKBKB through m6A-dependent mRNA stability in the context of PM2.5-induced oxidative stress. Therefore, it is conceivable that the utilization of antioxidants or inhibition of FTO could represent potential therapeutic strategies for the management of asthma exacerbated by PM2.5 exposure.

Value of the NF-κB signalling pathway and the DNA repair gene PARP1 in predicting distant metastasis after breast cancer surgery.

The DNA repair gene PARP1 and NF-κB signalling pathway affect the metastasis of breast cancer by influencing the drug resistance of cancer cells. Therefore, this study focused on the value of the DNA repair gene PARP1 and NF-κB pathway proteins in predicting the postoperative metastasis of breast cancer. A nested case‒control study was performed. Immunohistochemical methods were used to detect the expression of these genes in patients. ROC curves were used to analyse the predictive effect of these factors on distant metastasis. The COX model was used to evaluate the effects of PARP1 and TNF-α on distant metastasis. The results showed that the expression levels of PARP1, IKKß, p50, p65 and TNF-α were significantly increased in the metastasis group (P < 0.001). PARP1 was correlated with IKKß, p50, p65 and TNF-α proteins (P < 0.001). There was a correlation between IKKß, p50, p65 and TNF-α proteins (P < 0.001). ROC curve analysis showed that immunohistochemical scores for PARP1 of > 6, IKKß of > 4, p65 of > 4, p50 of > 2, and TNF-α of > 4 had value in predicting distant metastasis (SePARP1 = 78.35%, SpPARP1 = 79.38%, AUCPARP1 = 0.843; Sep50 = 64.95%, Spp50 = 70.10%, AUCp50 = 0.709; SeTNF-α = 60.82%, SpTNF-α = 69.07%, AUCTNF-α = 0.6884). Cox regression analysis showed that high expression levels of PARP1 and TNF-α were a risk factor for distant metastasis after breast cancer surgery (RRPARP1 = 4.092, 95% CI 2.475-6.766, P < 0.001; RRTNF-α = 1.825, 95% CI 1.189-2.799, P = 0.006). Taken together, PARP1 > 6, p50 > 2, and TNF-α > 4 have a certain value in predicting breast cancer metastasis, and the predictive value is better when they are combined for diagnosis (Secombine = 97.94%, Spcombine = 71.13%).

Amlexanox attenuates LPS-induced neuroinflammatory responses in microglial cells via inhibition of NF-κB and STAT3 signaling pathways.

Amlexanox is an anti-inflammatory and anti-allergic agent used clinically for the treatment of aphthous ulcers, allergic rhinitis, and asthma. Recent studies have demonstrated that amlexanox, a selective inhibitor of IkB kinase epsilon (IKKε) and TANK-binding kinase 1 (TBK1), suppresses a range of diseases or inflammatory conditions, such as obesity-related metabolic dysfunction and type 2 diabetes. However, the effects of amlexanox on neuroinflammatory responses to amlexanox have not yet been comprehensively studied. In this study, we investigated the novel therapeutic effect of amlexanox on LPS-induced neuroinflammation in vivo, and intraperitoneal injection of amlexanox markedly reduced LPS-induced IKKε levels, proinflammatory cytokines, and microglial activation, as evidenced by ionized calcium-binding adapter molecule 1 (Iba1) immunostaining. Furthermore, amlexanox significantly reduced proinflammatory cytokines and chemokines in LPS-induced bone marrow-derived macrophages (BMDM), murine BV2, and human HMC3 microglial cells. This data provided considerable evidence that amlexanox can be used as a preventive and curative therapy for neuroinflammatory and neurodegenerative diseases. In terms of mechanism aspects, our results demonstrated that the anti-inflammatory action of amlexanox in BV2 microglial cells was through the downregulation of NF-κB and STAT3 signaling pathways. In addition, the combination of amlexanox and SPI (a STAT3 selective inhibitor) showed high efficiency in inhibiting the production of neurotoxic and pro-inflammatory mediators. Overall, our data provide rational insights into the mechanisms of amlexanox as a potential therapeutic strategy for neuroinflammation-related diseases.

Human ß-Defensin 3 Inhibition of P. gingivalis LPS-Induced IL-1ß Production by BV-2 Microglia through Suppression of Cathepsins B and L.

Cathepsin B (CatB) is thought to be essential for the induction of Porphyromonas gingivalis lipopolysaccharide (Pg LPS)-induced Alzheimer's disease-like pathologies in mice, including interleukin-1ß (IL-1ß) production and cognitive decline. However, little is known about the role of CatB in Pg virulence factor-induced IL-1ß production by microglia. We first subjected IL-1ß-luciferase reporter BV-2 microglia to inhibitors of Toll-like receptors (TLRs), IκB kinase, and the NLRP3 inflammasome following stimulation with Pg LPS and outer membrane vesicles (OMVs). To clarify the involvement of CatB, we used several known CatB inhibitors, including CA-074Me, ZRLR, and human ß-defensin 3 (hBD3). IL-1ß production in BV-2 microglia induced by Pg LPS and OMVs was significantly inhibited by the TLR2 inhibitor C29 and the IκB kinase inhibitor wedelolactonne, but not by the NLRPs inhibitor MCC950. Both hBD3 and CA-074Me significantly inhibited Pg LPS-induced IL-1ß production in BV-2 microglia. Although CA-074Me also suppressed OMV-induced IL-1ß production, hBD3 did not inhibit it. Furthermore, both hBD3 and CA-074Me significantly blocked Pg LPS-induced nuclear NF-κB p65 translocation and IκBα degradation. In contrast, hBD3 and CA-074Me did not block OMV-induced nuclear NF-κB p65 translocation or IκBα degradation. Furthermore, neither ZRLR, a specific CatB inhibitor, nor shRNA-mediated knockdown of CatB expression had any effect on Pg virulence factor-induced IL-1ß production. Interestingly, phagocytosis of OMVs by BV-2 microglia induced IL-1ß production. Finally, the structural models generated by AlphaFold indicated that hBD3 can bind to the substrate-binding pocket of CatB, and possibly CatL as well. These results suggest that Pg LPS induces CatB/CatL-dependent synthesis and processing of pro-IL-1ß without activation of the NLRP3 inflammasome. In contrast, OMVs promote the synthesis and processing of pro-IL-1ß through CatB/CatL-independent phagocytic mechanisms. Thus, hBD3 can improve the IL-1ß-associated vicious inflammatory cycle induced by microglia through inhibition of CatB/CatL.

IKKß stabilizes Mitofusin 2 and suppresses doxorubicin cardiomyopathy.

AIMS: The mitochondrial dynamics protein Mitofusin 2 (MFN2) coordinates critical cellular processes including mitochondrial bioenergetics, quality control, and cell viability. The NF-κB kinase IKKß suppresses mitochondrial injury in doxorubicin cardiomyopathy, but the underlying mechanism is undefined. METHODS AND RESULTS: Herein, we identify a novel signalling axis that functionally connects IKKß and doxorubicin cardiomyopathy to a mechanism that impinges upon the proteasomal stabilization of MFN2. In contrast to vehicle-treated cells, MFN2 was highly ubiquitinated and rapidly degraded by the proteasomal-regulated pathway in cardiac myocytes treated with doxorubicin. The loss of MFN2 activity resulted in mitochondrial perturbations, including increased reactive oxygen species (ROS) production, impaired respiration, and necrotic cell death. Interestingly, doxorubicin-induced degradation of MFN2 and mitochondrial-regulated cell death were contingent upon IKKß kinase activity. Notably, immunoprecipitation and proximity ligation assays revealed that IKKß interacted with MFN2 suggesting that MFN2 may be a phosphorylation target of IKKß. To explore this possibility, mass spectrometry analysis identified a novel MFN2 phospho-acceptor site at serine 53 that was phosphorylated by wild-type IKKß but not by a kinase-inactive mutant IKKßK-M. Based on these findings, we reasoned that IKKß-mediated phosphorylation of serine 53 may influence MFN2 protein stability. Consistent with this view, an IKKß-phosphomimetic MFN2 (MFN2S53D) was resistant to proteasomal degradation induced by doxorubicin whereas wild-type MFN2 and IKKß-phosphorylation defective MFN2 mutant (MFNS53A) were readily degraded in cardiac myocytes treated with doxorubicin. Concordantly, gain of function of IKKß or MFN2S53D suppressed doxorubicin-induced mitochondrial injury and cell death. CONCLUSIONS: The findings of this study reveal a novel survival pathway for IKKß that is mutually dependent upon and obligatory linked to the phosphorylation and stabilization of the mitochondrial dynamics protein MFN2.

IKBKE promotes the ZEB2-mediated EMT process by phosphorylating HMGA1a in glioblastoma.

IKBKE (Inhibitor of Nuclear Factor Kappa-B Kinase Subunit Epsilon) is an important oncogenic protein in a variety of tumors, which can promote tumor growth, proliferation, invasion and drug resistance, and plays a critical regulatory role in the occurrence and progression of malignant tumors. HMGA1a (High Mobility Group AT-hook 1a) functions as a cofactor for proper transcriptional regulation and is highly expressed in multiple types of tumors. ZEB2 (Zinc finger E-box Binding homeobox 2) exerts active functions in epithelial mesenchymal transformation (EMT). In our current study, we confirmed that IKBKE can increase the proliferation, invasion and migration of glioblastoma cells. We then found that IKBKE can phosphorylate HMGA1a at Ser 36 and/or Ser 44 sites and inhibit the degradation process of HMGA1a, and regulate the nuclear translocation of HMGA1a. Crucially, we observed that HMGA1a can regulate ZEB2 gene expression by interacting with ZEB2 promoter region. Hence, HMGA1a was found to promote the ZEB2-related metastasis. Consequently, we demonstrated that IKBKE can exert its oncogenic functions via the IKBKE/HMGA1a/ZEB2 signalling axis, and IKBKE may be a prominent biomarker for the treatment of glioblastoma in the future.

IκB kinase thwarts aggregation: Phosphorylating TDP-43 for degradation.

TDP-43 aggregation is a hallmark of neurodegeneration. In this issue, Iguchi et al. (https://doi.org/10.1083/jcb.202302048) report that IκB kinase (IKK), an important mediator of inflammation, phosphorylates cytoplasmic TDP-43 to promote proteasomal degradation, revealing an unexpected link between inflammation and TDP-43 homeostasis.

6-Gingerol ameliorates alveolar hypercoagulation and fibrinolytic inhibition in LPS-provoked ARDS via RUNX1/NF-κB signaling pathway.

BACKGROUND: Alveolar hypercoagulation and fibrinolytic inhibition play a central role in refractory hypoxemia in acute respiratory distress syndrome (ARDS), but it lacks effective drugs for prevention and treatment of this pathophysiology. Our previous experiment confirmed that RUNX1 promoted alveolar hypercoagulation and fibrinolytic inhibition through NF-κB pathway. Other studies demonstrated that 6-gingerol regulated inflammation and metabolism by inhibiting the NF-κB signaling pathway. We assume that 6-gingerol would ameliorate alveolar hypercoagulation and fibrinolytic inhibition via RUNX1/ NF-κB pathway in LPS-induced ARDS. METHODS: Rat ARDS model was replicated through LPS inhalation. Before LPS inhalation, the rats were intraperitoneally treated with different doses of 6-gingerol or the same volume of normal saline (NS) for 12 h, and then intratracheal inhalation of LPS for 24 h. In cell experiment, alveolar epithelial cell type II (AECII) was treated with 6-gingerol for 6 h and then with LPS for another 24 h. RUNX1 gene was down-regulated both in pulmonary tissue and in cells. Tissue factor (TF), plasminogen Activator Inhibitor 1(PAI-1) and thrombin were determined by Wester-blot (WB), qPCR or by enzyme-linked immunosorbent (ELISA). Lung injury score, pulmonary edema and pulmonary collagen III in rat were assessed. NF-κB pathway were also observed in vivo and in vitro. The direct binding capability of 6-gingerol to RUNX1 was confirmed by using Drug Affinity Responsive Target Stability test (DARTS). RESULTS: 6-gingerol dose-dependently attenuated LPS-induced lung injury and pulmonary edema. LPS administration caused excessive TF and PAI-1 expression both in pulmonary tissue and in AECII cell and a large amount of TF, PAI-1 and thrombin in bronchial alveolar lavage fluid (BALF), which all were effectively decreased by 6-gingerol treatment in a dose-dependent manner. The high collagen Ⅲ level in lung tissue provoked by LPS was significantly abated by 6-gingerol. 6-gingerol was seen to dramatically inhibit the LPS-stimulated activation of NF-κB pathway, indicated by decreases of p-p65/total p65, p-IKKß/total IKKß, and also to suppress the RUNX1 expression. RUNX1 gene knock down or RUNX1 inhibitor Ro5-3335 significantly enhanced the efficacies of 6-gingerol in vivo and in vitro, but RUNX1 over expression remarkably impaired the effects of 6-gingerol on TF, PAI-1 and on NF-κB pathway. DARTS result showed that 6-gingerol directly bond to RUNX1 molecules. CONCLUSIONS: Our experimental data demonstrated that 6-gingerol ameliorates alveolar hypercoagulation and fibrinolytic inhibition via RUNX1/NF-κB pathway in LPS-induced ARDS. 6-gingerol is expected to be an effective drug in ARDS.

Study of the effect of keap1 on oxidative stress in human umbilical cord mesenchymal stem cells.

BACKGROUND: HucMSCs had shown promising efficacy in treating childhood diseases, but oxidative stress induced by the poor microenvironment at the site of damage resulted in low cell survival after transplantation, thus preventing the cells from maximizing therapeutic efficacy. Therefore, this study aimed to investigate the role and mechanism of keap1 in oxidative stress injury of human umbilical cord mesenchymal stem cells (hucMSCs), and to provide theoretical support for improving the efficacy of stem cell therapy. METHODS: The hucMSCs were treated with hypoxic low-sugar-free serum (GSDH) to mimic the damaged site microenvironment after implantation. Adenoviral overexpression of keap1 gene of hucMSCs was performed in vitro, and cell proliferation ability was detected by CCK8 assay, crystal violet staining assay, and cell cycle assay. Cellular redox level was assessed by Amplex Red, MDA, and GSH/GSSG kit. Mitochondrial morphology was evaluated by mitotracker Red staining. ATP production was estimated by ATP detection kit. The mRNA and protein expression levels were tested by western blotting and RT-qPCR. RESULTS: GSDH treatment substantially upregulated keap1 expression. Subsequently, we found that overexpression of keap1 notably inhibited cell proliferation and caused cells to stagnate in G1 phase. At the same time, overexpression of keap1 induced the production of large amounts of H2O2 and the accumulation of MDA, but suppressed the GSH/GSSG ratio and the expression of antioxidant proteins NQO1 and SOD1, which caused oxidative stress damage. Overexpression of keap1 induced cells to produce a large number of dysfunctional mitochondria resulting in reduced ATP production. Moreover, Overexpression of keap1 significantly decreased the IKKß protein level, while upregulating IkB mRNA levels and downregulating P50 mRNA levels. CONCLUSIONS: Overexpression of keap1 may induce oxidative stress injury in hucMSCs by down-regulating IKKß expression and inhibiting NF-κB pathway activation. This implies the importance of keap1 in hucMSCs and it may be a potential gene for genetic modification of hucMSCs.

Role of Influenza A virus protein NS1 in regulating host nuclear body ND10 complex formation and its involvement in establishment of viral pathogenesis.

Influenza viral infection is a seasonal infection which causes widespread acute respiratory issues among humans globally. This virus changes its surface receptor composition to escape the recognition process by the host's immune cells. Therefore, the present study focussed to identify some other important viral proteins which have a significant role in establishment of infection and having apparent conserved structural composition. This could facilitate the permanent vaccine development process or help in designing a drug against IAV (influenza A virus) infection which will eliminate the seasonal flu shot vaccination process. The NS1 (Non-structural protein 1) protein of IAV maintains a conserved structural motif. Earlier studies have shown its significant role in infection establishment. However, the mechanism by which viruses escape the host's ND10 antiviral action remains elusive. The present study clearly showed that IAV infection and NS1 transfection in A549 cells degraded the main component of the ND10 anti-viral complex, PML and therefore, inhibited the formation of Daxx-sp100-p53-PML complex (ND10) at the mid phase of infection/transfection. PML degradation activated the stress axis which increased cellular ROS (reactive oxygen species) levels as well as mitochondrial dysfunction. Additionally, IAV/NS1 increased cellular stress and p53 accumulation at the late phase of infection. These collectively activated apoptotic pathway in the host cells. Along with the inactivation of several interferon proteins, IAV was found to decrease p-IKKε. A549 cells transfected with pcDNA3.1-NS1 showed a similar effect in the interferon axis and IKKε. Moreover, NS1 induced the disintegration of the host's ND10 complex through the changes in the SUMOylation pattern of the PML nuclear body. These findings suggest the possible mechanism of how NS1 helps IAV to establish infection in the host cells. However, it demands further detailed study before targeting NS1 to develop permanent vaccines or novel drugs against IAV in future.

IKKε and TBK1 prevent RIPK1 dependent and independent inflammation.

TBK1 and IKKε regulate multiple cellular processes including anti-viral type-I interferon responses, metabolism and TNF receptor signaling. However, the relative contributions and potentially redundant functions of IKKε and TBK1 in cell death, inflammation and tissue homeostasis remain poorly understood. Here we show that IKKε compensates for the loss of TBK1 kinase activity to prevent RIPK1-dependent and -independent inflammation in mice. Combined inhibition of IKKε and TBK1 kinase activities caused embryonic lethality that was rescued by heterozygous expression of kinase-inactive RIPK1. Adult mice expressing kinase-inactive versions of IKKε and TBK1 developed systemic inflammation that was induced by both RIPK1-dependent and -independent mechanisms. Combined inhibition of IKKε and TBK1 kinase activities in myeloid cells induced RIPK1-dependent cell death and systemic inflammation mediated by IL-1 family cytokines. Tissue-specific studies showed that IKKε and TBK1 were required to prevent cell death and inflammation in the intestine but were dispensable for liver and skin homeostasis. Together, these findings revealed that IKKε and TBK1 exhibit tissue-specific functions that are important to prevent cell death and inflammation and maintain tissue homeostasis.

MEK-mediated CHPF2 phosphorylation promotes colorectal cancer cell proliferation and metastasis by activating NF-κB signaling.

The cytokine tumor necrosis factor (TNF) plays a crucial role in the proliferation and metastasis of colorectal cancer (CRC) cells, but the underlying mechanisms remain poorly understood. Here, we report that chondroitin polymerizing factor 2 (CHPF2) promotes CRC cell proliferation and metastasis mediated by TNF, independently of its enzymatic activity. CHPF2 is highly expressed in CRC, and its elevated expression is associated with poor prognosis of CRC patients. Mechanistically, upon TNF stimulation, CHPF2 is phosphorylated at the T588 residue by MEK, enabling CHPF2 to interact with both TAK1 and IKKα. This interaction enhances the binding of TAK1 and IKKα, leading to increased phosphorylation of the IKK complex and activation of NF-κB signaling. As a result, the expression of early growth factors (EGR1) is upregulated to promote CRC cell proliferation and metastasis. In contrast, introduction of a phospho-deficient T588A mutation in CHPF2 weakened the interaction between CHPF2 and TAK1, thus impairing NF-κB signaling. CHPF2 T588A mutation reduced the ability of CHPF2 to promote the proliferation and metastasis of CRC in vitro and in vivo. Furthermore, the NF-κB RELA subunit promotes CHPF2 expression, further amplifying TNF-induced NF-κB signaling activation. These findings identify a moonlighting function of CHPF2 in promoting tumor cell proliferation and metastasis and provide insights into the mechanism by which CHPF2 amplifies TNF-mediated NF-κB signaling activation. Our study provides a molecular basic for the development of therapeutic strategies for CRC treatment.

Brazilin-Ce nanoparticles attenuate inflammation by de/anti-phosphorylation of IKKß.

Inflammation is associated with a series of diseases like cancer, cardiovascular disease and infection, and phosphorylation/dephosphorylation modification of proteins are important in inflammation regulation. Here we designed and synthesized a novel Brazilin-Ce nanoparticle (BX-Ce NPs) using Brazilin, which has been used for anti-inflammation in cardiovascular diseases but with narrow therapeutic window, and Cerium (IV), a lanthanide which has the general activity in catalyzing the hydrolysis of phosphoester bonds, to conferring de/anti-phosphorylation of IKKß. We found that BX-Ce NPs specifically bound to Asn225 and Lys428 of IKKß and inhibited its phosphorylation at Ser181, contributing to appreciably anti-inflammatory effect in cellulo (IC50 = 2.5 µM). In vivo mouse models of myocardial infarction and sepsis also showed that the BX-Ce NPs significantly ameliorated myocardial injury and improved survival in mice with experimental sepsis through downregulating phosphorylation of IKKß. These findings provided insights for developing metal nanoparticles for guided ion interfere therapy, particularly synergistically target de/anti-phosphorylation as promising therapeutic agents for inflammation and related diseases.

IKKß deletion from CNS macrophages increases neuronal excitability and accelerates the onset of EAE, while from peripheral macrophages reduces disease severity.

BACKGROUND: Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease characterized by motor deficits and cognitive decline. Many immune aspects of the disease are understood through studies in the experimental autoimmune encephalomyelitis (EAE) model, including the contribution of the NF-κB transcription factor to neuroinflammation. However, the cell-specific roles of NF-κB to EAE and its cognitive comorbidities still needs further investigation. We have previously shown that the myeloid cell NF-κB plays a role in the healthy brain by exerting homeostatic regulation of neuronal excitability and synaptic plasticity and here we investigated its role in EAE. METHODS: We used constitutive MφIKKßΚΟ mice, in which depletion of IKKß, the main activating kinase of NF-κB, was global to CNS and peripheral macrophages, and ΜgΙΚΚßKO mice, in which depletion was inducible and specific to CNS macrophages by 28 days after tamoxifen administration. We subjected these mice to MOG35-55 induced EAE and cuprizone-induced demyelination. We measured pathology by immunohistochemistry, investigated molecular mechanisms by RNA sequencing analysis and studied neuronal functions by in vivo electrophysiology in awake animals. RESULTS: Global depletion of IKKß from myeloid cells in MφIKKßΚΟ mice accelerated the onset and significantly supressed chronic EAE. Knocking out IKKß only from CNS resident macrophages accelerated the onset and exacerbated chronic EAE, accompanied by earlier demyelination and immune cell infiltration but had no effect in cuprizone-induced demyelination. Peripheral T cell effector functions were not affected by myeloid cell deletion of IKKß, but CNS resident mechanisms, such as microglial activation and neuronal hyperexcitability were altered from early in EAE. Lastly, depletion of myeloid cell IKKß resulted in enhanced late long-term potentiation in EAE. CONCLUSIONS: IKKß-mediated activation of NF-κΒ in myeloid cells has opposing roles in EAE depending on the cell type and the disease stage. In CNS macrophages it is protective while in peripheral macrophages it is disease-promoting and acts mainly during chronic disease. Although clinically protective, CNS myeloid cell IKKß deletion dysregulates neuronal excitability and synaptic plasticity in EAE. These effects of IKKß on brain cognitive abilities deserve special consideration when therapeutic interventions that inhibit NF-κB are used in MS.

Molecular cloning and functional characterization of pigeon IKKε.

Inhibitor of nuclear factor kappa-B kinase ε (IKKε), a member of the non-canonical IκB kinase family, plays a critical role in connecting various signaling pathways associated with the initiation of type I interferon (IFN) production. Although the importance of IKKε in innate immunity has been well established in mammals and fish, its characterization and function in pigeons have remained largely unexplored. In this study, we successfully cloned pigeon IKKε (piIKKε) from pigeon embryo fibroblasts (PEFs) for the first time. This gene encodes 722 amino acids and shares high amino acid similarity with its duck and goose counterparts. piIKKε showed a diffuse cytoplasmic distribution and broad expression in all tissues examined. Overexpression of piIKKε in PEFs significantly activated the IFN-ß promoter, with both the kinase and CC domains of piIKKε playing key roles in initiating IFN-ß expression. Knockdown of piIKKε using small interfering RNA significantly reduced the levels of IFN-ß induced by NDV, AIV, poly (I:C), or SeV. Furthermore, the presence of piIKKε resulted in a remarkable reduction in the replication of both avian influenza virus (AIV) H9N2 and Newcastle disease virus (NDV) in PEFs. Our results demonstrate that piIKKε plays a critical role in mediating antiviral innate immunity in pigeons.

A molluscan IRF interacts with IKKα/ß family protein and modulates NF-κB and MAPK activity.

Interferon regulatory factor (IRF) family proteins are key transcription factors involved in vital physiological processes such as immune defense. However, the function of IRF in invertebrates, especially in marine shellfish is not clear. In this study, a new IRF gene (CfIRF2) was identified in the Zhikong scallop, Chlamys farreri, and its immune function was analyzed. CfIRF2 has an open reading frame of 1107 bp encoding 368 amino acids. The N-terminus of CfIRF2 consists of a typical IRF domain, with conserved amino acid sequences. Phylogenetic analysis suggested close evolutionary relationship with shellfish IRF1 subfamily proteins. Expression pattern analysis showed that CfIRF2 mRNA was expressed in all tissues, with the highest expression in the hepatopancreas and gills. CfIRF2 gene expression was substantially enhanced by a pathogenic virus (such as acute viral necrosis virus) and poly(I:C) challenge. Co-immunoprecipitation assay identified CfIRF2 interaction with the IKKα/ß family protein CfIKK1 of C. farreri, demonstrating a unique signal transduction mechanism in marine mollusks. Moreover, CfIRF2 interacted with itself to form homologous dimers. Overexpression of CfIRF2 in HEK293T cells activated reporter genes containing interferon stimulated response elements and NF-κB genes in a dose-dependent manner and promoted the phosphorylation of protein kinases (JNK, Erk1/2, and P38). Our results provide insights into the functions of IRF in mollusks innate immunity and also provide valuable information for enriching comparative immunological theory for the prevention of diseases in scallop farming.