Baeckein E suppressed NLRP3 inflammasome activation through inhibiting both the priming and assembly procedure: Implications for gout therapy

BackgroundBaeckein E (BF-2) was isolated from the aerial parts of Baeckea frutescens L., which has a long history of use in traditional medicine in Southeast Asia to treat inflammatory disease.PurposeBF-2 was identified to have inhibitory activity on nucleotide oligomerization domain (NOD)-like receptor protein-3 inflammasome (NLRP3) activation. This study aimed to investigate the related signaling cascade of BF-2 in both lipopolysaccharides (LPS)/ATP induced pyroptosis in J774A.1 macrophages and its application in a mouse model of gout induced by monosodium urate crystal (MSU).MethodsThe effect of BF-2 on NLRP3 inflammasome activation and gouty arthritis was studied in J774A.1 macrophages and male C57BL/6 mice. The J774A.1 macrophages were primed with LPS and stained by propidium iodide (PI) for cell pyroptosis detection. A gout mouse model was established by subcutaneous injection of MSU crystals into the hind paw of C57BL/6 mice. Mice were then randomly divided into different groups. The concentrations of IL-1β and IL-18 in both J774A.1 macrophage and gout mouse model were analyzed by ELISA. The NLRP3 inflammasome related protein expression was detected by western blot analysis. The inhibitory effects of BF-2 on NLRP3 inflammasome assembly were analyzed by immunoprecipitation assay. The roles of BF-2 in mitochondrial damage were imaged by Mito Tracker Green and Mito Tracker Red probes. The inhibitory effects of BF-2 on ROS production were imaged by DCF (2′,7′-dichlorofluorescein diacetate) probe.ResultsThe results demonstrated BF-2 could significantly suppress the cell pyroptosis and IL-1β secretion in macrophages. Furthermore, BF-2 significantly inhibited NLRP3 inflammasome activation and reduced ankle swelling in the gout mouse model. In detail, it alleviated mitochondrial damage mediated oxidative stress and inhibited the assembly of NLRP3 inflammasome by affecting the binding of pro-Caspase 1 and ASC. Moreover, BF-2 blocked NLRP3 activation by inhibiting the MAPK/NF-κB signaling pathways.ConclusionsResults demonstrated BF-2 inhibited NLRP3 inflammasome activation in both LPS primed macrophages and mouse model of gout through blocking MAPK/NF-κB signaling pathway and mitochondrial damage mediated oxidative stress. This study strongly suggests BF-2 could be a promising drug candidate against inflammatory diseases associated with NLRP3 inflammasome activation.     

Peroxidized unsaturated fatty acids stimulate Toll-like receptor 4 signaling in endothelial cells

AimAlthough unsaturated fatty acids are assumed to be protective against inflammatory disorders that include a pathway involving Toll-like receptor 4 (TLR4) activation, they might actually be toxic because of their high susceptibility to lipid peroxidation. Here we studied the effects of peroxidized unsaturated fatty acids on the TLR4–nuclear factor (NF)-κB pathway in endothelial cells.Main methodsConfluent cultured endothelial cells from bovine aorta were incubated for 1 h with fatty acids integrated into phosphatidylcholine vesicles. Lipopolysaccharide (LPS) or phosphatidylcholine vesicles without fatty acids were also applied as a positive control or a control for fatty acid groups, respectively. Activation of TLR4 and downstream signaling was assessed by membrane fractionation and Western blotting or immunofluorescent staining.Key findingsIn the same way as LPS, application of sufficiently peroxidized unsaturated fatty acids like oleic acid or docosahexaenoic acid, acutely caused TLR4 translocation to caveolae/raft membranes, leading to activation of NF-κB signaling in endothelial cells. In contrast, saturated fatty acids did not show such effects. Applying well-peroxidized unsaturated fatty acids, but not saturated fatty acids, acutely activates the TLR4/NF-κB pathway.SignificancePeroxidation of unsaturated fatty acid is essential for the acute activation of TLR4 by the fatty acids that follow the same pathway as the activation by LPS. Unsaturated fatty acids have been assumed to be protective against inflammatory disorders, and drugs containing unsaturated fatty acids are now developed and provided. Our result suggests that, for inflammatory disorders involving TLR4 signaling, using unsaturated fatty acids as anti-inflammatory drugs may cause contrary effects.     

Narciclasine inhibits LPS-induced neuroinflammation by modulating the Akt/IKK/NF-κB and JNK signaling pathways

BackgroundNeuroinflammation is defined as innate immune system activation in the central nervous system, and is a complex response involved in removing pathogens, toxic components, and dead cells by activating microglial cells. However, over-activated microglia have been implicated in the pathogenesis of neurodegenerative diseases, because they release large amounts of neurotoxic factors. Thus, inhibiting microglial activation may represent an attractive approach for preventing neuroinflammatory disorders. The objective of this study was to investigate the effect of narciclasine (NA) on lipopolysaccharide (LPS)-induced neuroinflammation by evaluating related markers and neurotoxic factors.MethodsBV-2 cells were pre-incubated with NA at 0.1, 0.2, and 0.3 µM for 1h, and then co-treated with LPS for 12 h. Cellular medium and lysates were measured using a nitric oxide assay, enzyme-link immunosorbent assay (ELISA), western blotting, kinase activity assay, luciferase assay, and immunofluorescence assay. C57BL/6N mice were orally administered NA and intraperitoneally injected with LPS, and the cerebral cortex was examined using western blotting and immunofluorescence assays.ResultsNA showed novel pharmacological activity, inhibiting pro-inflammatory factors, including TNF-α, IL-6, IL-18, NO, and PGE₂, but increasing the anti-inflammatory cytokines IL-10 and TGF-β1 in LPS-induced microglial cells. Moreover, NA also attenuated the LPS-induced mRNA and proteins of iNOS and COX-2. The mechanistic study indicated that NA attenuates the secretion of pro-inflammatory factor by down-regulating the Akt/IKK/NF-κB and JNK signaling pathways, and directly inhibits the catalytic activity of IKKα/β. Furthermore, we found that NA also reduced the expression of the microglial markers Iba-1, COX-2, and TNF-α in the mouse brain.ConclusionNA inhibits the over-expression of pro-inflammatory factors but it promotes anti-inflammatory cytokines by down-regulating the Akt/IKK/NF-κB and JNK signaling pathways in experimental models. Thus, NA may be a potential candidate for relieving neuroinflammation.     

Mogrol,an aglycone of mogrosides,attenuates ulcerative colitis by promoting AMPK activation

BackgroundUlcerative colitis (UC) is a non-specific chronic inflammatory disease. The incidence of UC in China has been increasing in recent years. Mogrol is an aglycone of mogrosides. Studies have shown that mogrosides have anti-oxygenation, anti-inflammatory, and laxative effects as well as other biological activities.PurposeTo investigate the beneficial effects of mogrol on UC and identify its underlying mechanisms.Study designWe used the dextran sodium sulphate (DSS)-induced UC model in mice, TNF-α-damaged NCM460 colonic epithelial cells, macrophage cells THP-M stimulated with lipopolysaccharide (LPS) / adenosine triphosphate (ATP) and compound C (an AMPK inhibitor) to confirm the key role of AMPK (AMP-activated protein kinase) activation.MethodsHistological evaluation, immunohistochemical staining, Western blot analysis, immunofluorescence assay and quantitative real time-PCR were used in the study.ResultsOral administration of mogrol (5 mg/kg/daily) in vivo significantly attenuated pathological colonic damage, inhibited inflammatory infiltration and improved the abnormal expression of NLRP3 inflammasome in colonic mucosa via the AMPK and NF-κB signaling pathways. In vitro, mogrol protected against intestinal epithelial barrier dysfunction by activating AMPK in TNF-α-treated NCM460 cells and inhibited the production of inflammatory mediator in LPS-stimulated THP-M cells. Furthermore, mogrol's effects were reversed by compound C intervention in DSS-induced UC model.ConclusionMogrol exerts protective effects in experimental UC and inhibits production of inflammatory mediators through activation of AMPK-mediated signaling pathways.     

Celastrol is a novel selective agonist of cannabinoid receptor 2 with anti-inflammatory and anti-fibrotic activity in a mouse model of systemic sclerosis

BackgroundIncreasing evidence indicated that the cannabinoid receptors were involved in the pathogenesis of organ fibrogenesis.PurposeThe purpose of this study was to discover novel cannabinoid receptor 2 (CB2) agonist and assess the potential of CB2 activation in treating systemic sclerosis.MethodsA gaussia princeps luciferase–based split luciferase complementation assay (SLCA) was developed for detection of the interaction between CB2 and β-arrestin2. A library of 366 natural products was then screened as potential CB2 agonist using SLCA approach. Several GPCR functional assays, including HTRF-based cAMP assay and calcium mobilization were also utilized to evaluated CB2 activation. Bleomycin-induced experimental systemic sclerosis was used to assess the in vivo anti-fibrotic effects. Dermal thickness and collagen content were evaluated via H&E and sirius red staining.ResultsCelastrol was identified as a new agonist of CB2 by using SLCA. Furthermore, celastrol triggers several CB2-mediated downstream signaling pathways, including calcium mobilization, inhibition of cAMP accumulation, and receptor desensitization in a dose-dependent manner, and it has a moderate selectivity on CB1. In addition, celastrol exhibited the anti-inflammatory properties on lipopolysaccharide (LPS) treated murine Raw 264.7 macrophages and primary macrophages. Finally, we found that celastrol exerts anti-fibrotic effects in the bleomycin-induced systemic sclerosis mouse model accompanied by reduced inflammatory conditions.ConclusionTaken together, celastrol is identified a novel selective CB2 agonist using a new developed arrestin-based SLCA, and CB2 activation by celastrol reduces the inflammatory response, and prevents the development of dermal fibrosis in bleomycin-induced systemic sclerosis mouse model.     

Dehydrozingerone ameliorates Lipopolysaccharide induced acute respiratory distress syndrome by inhibiting cytokine storm,oxidative stress via modulating the MAPK/NF-κB pathway

BackgroundInflammation-mediated lung injury is a major cause of health problems in many countries and has been the leading cause of morbidity/mortality in intensive care units. In the current COVID-19 pandemic, the majority of the patients experienced serious pneumonia resulting from inflammation (Acute respiratory distress syndrome/ARDS). Pathogenic infections cause cytokine release syndrome (CRS) by hyperactivation of immune cells, which in turn release excessive cytokines causing ARDS. Currently, there are no standard therapies for viral, bacterial or pathogen-mediated CRS.PurposeThis study aimed to investigate and validate the protective effects of Dehydrozingerone (DHZ) against LPS induced lung cell injury by in-vitro and in-vivo models and to gain insights into the molecular mechanisms that mediate these therapeutic effects.MethodsThe therapeutic activity of DHZ was determined in in-vitro models by pre-treating the cells with DHZ and exposed to LPS to stimulate the inflammatory cascade of events. We analysed the effect of DHZ on LPS induced inflammatory cytokines, chemokines and cell damage markers expression/levels using various cell lines. We performed gene expression, ELISA, and western blot analysis to elucidate the effect of DHZ on inflammation and its modulation of MAPK and NF-κB pathways. Further, the prophylactic and therapeutic effect of DHZ was evaluated against the LPS induced ARDS model in rats.ResultsDHZ significantly (p < 0.01) attenuated the LPS induced ROS, inflammatory cytokine, chemokine gene expression and protein release in macrophages. Similarly, DHZ treatment protected the lung epithelial and endothelial cells by mitigating the LPS induced inflammatory events in a dose-dependent manner. In vivo analysis showed that DHZ treatment significantly (p < 0.001) mitigated the LPS induced ARDS pathophysiology of increase in the inflammatory cells in BALF, inflammatory cytokine and chemokines in lung tissues. LPS stimulated neutrophil-mediated events, apoptosis, alveolar wall thickening and alveolar inflammation were profoundly reduced by DHZ treatment in a rat model.ConclusionThis study demonstrates for the first time that DHZ has the potential to ameliorate LPS induced ARDS by inhibiting cytokine storm and oxidative through modulating the MAPK and NF-κB pathways. This data provides pre-clinical support to develop DHZ as a potential therapeutic agent against ARDS.     

Suppression of homodimerization of toll-like receptor 4 by isoliquiritigenin

Toll-like receptors (TLRs) play important inductive roles in innate immune responses for host defense against invading microbial pathogens. Activation of TLR4 by lipopolysaccharide (LPS) induces dimerization of TLR4 and, subsequently, activation of downstream signaling pathways including nuclear factor-kappa B and interferon regulatory factor 3. TLR4 dimerization may be an early regulatory event in activating signaling pathways induced by LPS. Here, biochemical evidence is reported that isoliquiritigenin, one of the major ingredients derived from licorice root, inhibits LPS-induced TLR4 dimerization resulting in inhibition of nuclear factor-kappa B and interferon regulatory factor 3 activation, and cyclooxygenase-2 and inducible nitric oxide synthase expression. These results suggest that isoliquiritigenin modulates TLR-mediated signaling pathways at the receptor level. Furthermore, these results suggest that TLRs themselves may be important targets for the prevention of chronic inflammatory diseases.     

NOD1配体抑制人早孕期滋养细胞的侵袭功能

目的:明确固有免疫受体NOD1对人早孕期滋养细胞侵袭功能的调控及对侵袭相关因子分泌的影响。方法:采用免疫细胞化学法鉴定原代滋养细胞NOD1的表达,用transwell侵袭实验检测激活NOD1后滋养细胞侵袭功能的改变,ELISA检测配体刺激后滋养细胞MMP2和MMP9的分泌情况。结果:免疫细胞化学结果显示滋养细胞分离鉴定成功,且原代滋养细胞可以表达固有免疫受体NOD1。使用NOD1的特异性配体及非特异性配体,发现激活NOD1可以抑制滋养细胞的侵袭,且非特异性配体LPS可以下调侵袭相关金属基质蛋白酶分子MMP2和MMP9的分泌,特异性配体i E-DAP仅下调MMP9的分泌而对MMP2的分泌无影响。结论:固有免疫模式识别受体NOD1可以在早孕期滋养细胞表达,可调控滋养细胞的侵袭功能,其激活会导致侵袭相关分子MMP2和MMP9的分泌下降。     

Activation of the bile acid receptor TGR5 enhances LPS-induced inflammatory responses in a human monocytic cell line

Abstract

Introduction: Bile acids are recognized as signaling molecules, mediating their effects both through the cell surface receptor TGR5 and the nuclear receptor FXR. After a meal, approximately 95% of the bile acids are transported from terminal ileum and back to the liver via the portal vein, resulting in postprandial elevations of bile acids in blood. During the digestion of fat, components from the microbiota, including LPS, are thought to reach the circulation where it may lead to inflammatory responses after binding TLR4 immune cells. Both LPS and bile acids are present in blood after a high-fat meal; we therefore wanted to study consequences of a possible interplay between TGR5 and TLR4 in human monocytes. Methods: The monocytic cell line U937 stably transfected with the NF-κB reporter plasmid 3x-κB-luc was used as a model system to study the effects of TGR5 and TLR4. Activation of MAP kinases was studied to reveal functional consequences of triggering TGR5 in U937 cells. Effects of TGR5 and TLR4 activation were monitored using NF-κB luciferase assay and by quantification of the pro-inflammatory cytokines IL-6 and IL-8 using ELISA. Results: In this study, results show that triggering TGR5 with the specific agonist betulinic acid (BA), and the bile acids CDCA or DCA, activated both the main MAP kinases ERK1/2, p38 and JNK, and the NF-κB signaling pathway. We further demonstrated that co-triggering of TLR4 and TGR5 enhanced the activation of NF-κB and the release of inflammatory cytokines in a synergistic manner compared to triggering of TLR4 alone. Conclusions: Thus, two different and simultaneous events associated with the digestive process coordinately affect the function of human monocytes and contribute to enhanced inflammation. Because elevated levels of circulatory LPS may contribute to the development of insulin resistance, the results from this study suggest that bile acids through the activation of TGR5 may have a role in the development of insulin resistance as well.     

NIMA-related kinase 7 amplifies NLRP3 inflammasome pro-inflammatory signaling in microglia/macrophages and mice models of spinal cord injury

BackgroundNIMA-related kinase-7 (NEK7) is a serine/threonine kinase that drives cell-cycle dynamics by modulating mitotic spindle formation and cytokinesis. It is also a crucial modulator of the pro-inflammatory effects of NOD-like receptor 3 (NLRP3) inflammasome. However, the role of NEK7 in microglia/macrophages post-spinal cord injury (SCI) is not well defined.MethodsIn this study, we performed both in vivo and in vitro experiments. Using an in vivo mouse SCI model, NEK7 siRNAs were administered intraspinally. For in vitro analysis, BV-2 microglia cells with NEK7-siRNA were stimulated with 1 μg/ml lipopolysaccharide (LPS) and 2 mM Adenosine triphosphate (ATP).ResultsHere, we found that the mRNA and protein levels of NEK7 and NLRP3 inflammasomes were upregulated in spinal cord tissues of injured mice and BV-2 microglia cells exposed to Lipopolysaccharide (LPS) and Adenosine triphosphate (ATP). Further experiments established that NEK7 and NLRP3 interacted in BV-2 microglia cells, an effect that was eliminated following NEK7 ablation. Moreover, NEK7 ablation suppressed the activation of NLRP3 inflammasomes. Although NEK7 inhibition did not significantly improve motor function post-SCI in mice, it was found to attenuate local inflammatory response and inhibit the activation of NLRP3 inflammasome in microglia/macrophages of the injured spinal cord.ConclusionNEK7 amplifies NLRP3 inflammasome pro-inflammatory signaling in BV-2 microglia cells and mice models of SCI. Therefore, agents targeting the NEK7/NLRP3 signaling offers great promise in the treatment of inflammatory response post-SCI.     

The Role of p38 and CK2 Protein Kinases in the Response of RAW 264.7 Macrophages to Lipopolysaccharide

The role of protein kinases p38 and CK2 (casein kinase II) in the response of RAW 264.7 macrophages to the lipopolysaccharide (LPS) from gram-negative bacteria was studied. Using specific p38 and CK2 inhibitors (p38 MAP kinase Inhibitor XI and casein kinase II Inhibitor III, respectively), we investigated the effects of these protein kinases on (i) LPS-induced activation of signaling pathways involving nuclear factor κB (NF-κB), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38, and interferon regulatory factor 3 (IRF3); (ii) expression of Toll-like receptor 4 (TLR4) and inducible heat-shock proteins HSP72 and HSP90; and (iii) production of interleukins IL-1α, IL-1β, IL-6, tumor necrosis factor α, and IL-10. Activation of the proapoptotic signaling in the macrophages was evaluated from the ratio between the active and inactive caspase-3 forms and p53 phosphorylation. Six hours after LPS addition (2.5 μg/ml) to RAW 264.7 cells, activation of the TLR4 signaling pathways was observed that was accompanied by a significant increase in phosphorylation of IκB kinase α/β, NF-κB (at both Ser536 and Ser276), p38, JNK, and IRF3. Other effects of macrophage incubation with LPS were an increase in the contents of TLR4, inducible heat-shock proteins (HSPs), and pro- and anti-inflammatory cytokines, as well as slight activation of the pro-apoptotic signaling in the cells. Using inhibitor analysis, we found that during the early response of macrophages to the LPS, both CK2 and p38 modulate activation of MAP kinase and NF-κB signaling pathways and p65 phosphorylation at Ser276/Ser536 and cause accumulation of HSP72, HSP90 and the LPS-recognizing receptor TLR4. Suppression of the p38 MAP kinase and CK2 activities by specific inhibitors (Inhibitor XI and Inhibitor III, respectively) resulted in the impairment of the macrophage effector function manifested as a decrease in the production of the early-response proinflammatory cytokines and disbalance between the pro- and anti-apoptotic signaling pathways leading presumably to apoptosis development. Taken together, our data indicate the inefficiency of therapeutic application of p38 and CK2 inhibitors during the early stages of inflammatory response.     

Koumine modulates spinal microglial M1 polarization and the inflammatory response through the Notch-RBP-Jκ signaling pathway,ameliorating diabetic neuropathic pain in rats

BackgroundDiabetic neuropathic pain (DNP), a complication of diabetes, has serious impacts on human health. As the pathogenesis of DNP is very complex, clinical treatments for DNP is limited. Koumine (KM) is an active ingredient extracted from Gelsemium elegans Benth. that exerts an inhibitory effect on neuropathic pain (NP) in several animal models.PurposeTo clarify the anti-NP effect of KM on rats with DNP and the molecular mechanisms involving the Notch- Jκ recombination signal binding protein (RBP-Jκ) signaling pathway.MethodsMale Sprague-Dawley rats were administered streptozocin (STZ) by intraperitoneal injection to induce DNP. The effect of KM on mechanical hyperalgesia in rats with DNP was evaluated using the Von Frey test. Microglial polarization in the spinal cord was examined using western blotting and quantitative real-time PCR. The Notch-RBP-Jκ signaling pathway was analysed using western blotting.ResultsKM attenuated DNP during the observation period. In addition, KM alleviated M1 microglial polarization in STZ-induced rats. Subsequent experiments revealed that Notch-RBP-Jκ signaling pathway was activated in the spinal cord of rats with DNP, and the activation of this pathways was decreased by KM. Additionally, KM-mediated analgesia and deactivation of the Notch-RBP-Jκ signaling pathway were inhibited by the Notch signaling agonist jagged 1, indicating that the anti-DNP effect of KM may be regulated by the Notch-RBP-Jκ signaling pathway.ConclusionsKM is a potentially desirable candidate treatment for DNP that may inhibit microglial M1 polarization through the Notch-RBP-Jκ signaling pathway.     

Resveratrol improves oxidative stress and prevents the progression of periodontitis via the activation of the Sirt1/AMPK and the Nrf2/antioxidant defense pathways in a rat periodontitis model

Oxidative stress is a key factor regulating the systemic pathophysiological effects associated with periodontitis. Resveratrol is a phytochemical with antioxidant and anti-inflammatory properties that can reduce oxidative stress and inflammation. We hypothesized that resveratrol may prevent the progression of periodontitis and reduce systemic oxidative stress through the activation of the sirtuin 1 (Sirt1)/AMP-activated protein kinase (AMPK) and the nuclear factor E2-related factor 2 (Nrf2)/antioxidant defense pathways. Three groups of male Wistar rats (periodontitis treated with melinjo resveratrol, periodontitis without resveratrol, and control rats with no periodontitis or resveratrol treatment) were examined. A ligature was placed around the maxillary molars for 3 weeks to induce periodontitis, and the rats were then given drinking water with or without melinjo resveratrol. In rats with periodontitis, ligature placement induced alveolar bone resorption, quantified using three-dimensional images taken by micro-CT, and increased proinflammatory cytokine levels in gingival tissue. Melinjo resveratrol intake relieved alveolar bone resorption and activated the Sirt1/AMPK and the Nrf2/antioxidant defense pathways in inflamed gingival tissues. Further, melinjo resveratrol improved the systemic levels of 8-hydroxydeoxyguanosine, dityrosine, nitric oxide metabolism, nitrotyrosine, and proinflammatory cytokines. We conclude that oral administration of melinjo resveratrol may prevent the progression of ligature-induced periodontitis and improve systemic oxidative and nitrosative stress.     

Involvement of the MAPK and PI3K pathways in chitinase 3-like 1-regulated hyperoxia-induced airway epithelial cell death

BackgroundExposure to 100% oxygen causes hyperoxic acute lung injury characterized by cell death and injury of alveolar epithelial cells. Recently, the role of chitinase 3-like 1 (CHI3L1), a member of the glycosyl hydrolase 18 family that lacks chitinase activity, in oxidative stress was demonstrated in murine models. High levels of serum CHI3L1 have been associated with various diseases of the lung, such as asthma, chronic obstructive pulmonary disease, and cancer. However, the role of CHI3L1 in human airway epithelial cells undergoing oxidative stress remains unknown. In addition, the signaling pathways associated with CHI3L1 in this process are poorly understood.PurposeIn this study, we demonstrate the role of CHI3L1, along with the MAPK and PI3K signaling pathways, in hyperoxia-exposed airway epithelial cells.MethodThe human airway epithelial cell line, BEAS-2B, was exposed to >95% oxygen (hyperoxia) for up to 72 h. Hyperoxia-induced cell death was determined by assessing cell viability, Annexin-V FITC staining, caspase-3 and -7 expression, and electron microscopy. CHI3L1 knockdown and overexpression studies were conducted in BEAS-2B cells to examine the role of CHI3L1 in hyperoxia-induced apoptosis. Activation of the MAPK and PI3K pathways was also investigated to determine the role of these signaling cascades in this process.ResultsHyperoxia exposure increased CHI3L1 expression and apoptosis in a time-dependent manner. CHI3L1 knockdown protected cells from hyperoxia-induced apoptosis. In contrast, CHI3L1 overexpression promoted cell death after hyperoxia exposure. Finally, phosphorylation of ERK1/2, p38, and Akt were affected by CHI3L1 knockdown.ConclusionThis study indicates that CHI3L1 is involved in hyperoxia-induced cell death, suggesting that CHI3L1 may be one of several cell death regulators influencing the MAPK and PI3K pathways during oxidative stress in human airway epithelial cells.     

Sodium current abnormalities and deregulation of Wnt/β-catenin signaling in iPSC-derived cardiomyocytes generated from patient with arrhythmogenic cardiomyopathy harboring compound genetic variants in plakophilin 2 gene

BackgroundMutations in desmosomal genes linked to arrhythmogenic cardiomyopathy are commonly associated with Wnt/β-catenin signaling abnormalities and reduction of the sodium current density. Inhibitors of GSK3B were reported to restore sodium current and improve heart function in various arrhythmogenic cardiomyopathy models, but mechanisms underlying this effect remain unclear. We hypothesized that there is a crosstalk between desmosomal proteins, signaling pathways, and cardiac sodium channels.Methods and resultsTo reveal molecular mechanisms of arrhythmogenic cardiomyopathy, we established human iPSC-based model of this pathology. iPSC-derived cardiomyocytes from patient carrying two genetic variants in PKP2 gene demonstrated that PKP2 haploinsufficiency due to frameshift variant, in combination with the missense variant expressed from the second allele, was associated with decreased Wnt/β-catenin activity and reduced sodium current. Different approaches were tested to restore impaired cardiomyocytes functions, including wild type PKP2 transduction, GSK3B inhibition and Wnt/β-catenin signaling modulation. Inhibition of GSK3B led to the restoration of both Wnt/β-catenin signaling activity and sodium current density in patient-specific cardiomyocytes while GSK3B activation led to the reduction of sodium current density. Moreover, we found that upon inhibition GSK3B sodium current was restored through Wnt/β-catenin-independent mechanism.ConclusionWe propose that alterations in GSK3B-Wnt/β-catenin signaling pathways lead to regulation of sodium current implying its role in molecular pathogenesis of arrhythmogenic cardiomyopathy.     

Loss of the Polarity Protein PAR3 Activates STAT3 Signaling via an Atypical Protein Kinase C (aPKC)/NF-κB/Interleukin-6 (IL-6) Axis in Mouse Mammary Cells

PAR3 suppresses tumor growth and metastasis in vivo and cell invasion through matrix in vitro. We propose that PAR3 organizes and limits multiple signaling pathways and that inappropriate activation of these pathways occurs without PAR3. Silencing Pard3 in conjunction with oncogenic activation promotes invasion and metastasis via constitutive STAT3 activity in mouse models, but the mechanism for this is unknown. We now show that loss of PAR3 triggers increased production of interleukin-6, which induces STAT3 signaling in an autocrine manner. Activation of atypical protein kinase C ι/λ (aPKCι/λ) mediates this effect by stimulating NF-κB signaling and IL-6 expression. Our results suggest that PAR3 restrains aPKCι/λ activity and thus prevents aPKCι/λ from activating an oncogenic signaling network.