YTHDF2 mediates LPS-induced osteoclastogenesis and inflammatory response via the NF-κB and MAPK signaling pathways

Aberrant elevation of osteoclast differentiation and function is responsible for disrupting bone homeostasis in various inflammatory bone diseases. YTH domain family 2 (YTHDF2) is a well-known m⁶A-binding protein that plays an essential role in regulating cell differentiation and inflammatory processes by mediating mRNA degradation. However, the regulatory role of YTHDF2 in inflammatory osteoclast differentiation remains unelucidated. Here, we detected the expression of m⁶A-related genes and found that YTHDF2 was upregulated in RANKL-primed osteoclast precursors stimulated with lipopolysaccharide (LPS). Ythdf2 knockdown in RAW264.7 cells and primary bone marrow-derived macrophages (BMMs) enhanced osteoclast formation and bone resorption, which was assessed by TRAP staining assay and pit formation assay. Ythdf2 depletion upregulated osteoclast-related gene expression and proinflammatory cytokine secretion. In contrast, overexpression of Ythdf2 produced the reverse effect. Furthermore, Ythdf2 knockdown enhanced the phosphorylation of IKKα/β, IκBα, ERK, P38 and JNK. NF-κB and MAPK signaling pathway inhibitors effectively abrogated the enhanced expression of Nfact1, c-Fos, IL-1β and TNF-α caused by Ythdf2 knockdown. Mechanistically, the mRNA stability assay revealed that Ythdf2 depletion led to stabilization of Tnfrsf11a, Traf6, Map4k4, Map2k3, Map2k4 and Nfatc1 mRNA. In summary, our findings demonstrated that YTHDF2 has a negative regulatory role in LPS-induced osteoclast differentiation and the inflammatory response via the NF-κB and MAPK signaling pathways.     

CpG oligonucleotides induce an immune response of odontoblasts through the TLR9, MyD88 and NF-κB pathways

Odontoblasts are the first-line defense cells against invading microorganisms. Toll-like receptors (TLRs) play a crucial role in innate immunity, and TLR9 is involved in the recognition of microbial DNA. This study aimed to investigate whether odontoblasts can respond to CpG DNA and to determine the intracellular signaling pathways triggered by CpG DNA. We found that the mouse odontoblast-like cell line MDPC-23 constitutively expressed TLR9. Exposure to CpG ODN induced a potent proinflammatory response based on an increase of IL-6 and TNF-α expression. Pretreatment with an inhibitory MyD88 peptide or a specific inhibitor for TLR9, NF-κB or IκBα markedly inhibited CpG ODN-induced IL-6 and TNF-α expression. Moreover, the CpG ODN-mediated increase of κB-luciferase activity in MDPC-23 cells was suppressed by the overexpression of dominant negative mutants of TLR9, MyD88 and IκBα, but not by the dominant negative mutant of TLR4. This result suggests a possible role for the CpG DNA-mediated immune response in odontoblasts and indicates that TLR9, MyD88 and NF-κB are involved in this process.     

Down-regulation of PKHD1 induces cell apoptosis through PI3K and NF-κB pathways

Mutations in PKHD1 (polycystic kidney and hepatic disease gene 1) gene cause the autosomal recessive polycystic kidney disease (ARPKD). Fibrocystin/polyductin (FPC), encoded by PKHD1, is a membrane-associated receptor-like protein. Although it is widely accepted that cystogenesis is mostly due to aberrant cell proliferation and apoptosis, it is still unclear how apoptosis is regulated. The aim of this study is to analyze the relationship among apoptosis, phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor κB (NF-κB) in FPC knockdown kidney cells. We show that PKHD1-silenced HEK293 cells demonstrate a higher PI3K/Akt activity. Selective inhibition of PI3K/Akt using LY294002 or wortmannin in these cells increases serum starvation-induced HEK293 cell apoptosis with a concomitant decrease in cell proliferation and higher caspase-3 activity. PI3K/Akt inhibition also leads to increased NF-κB activity in these cells. We conclude that the PI3K/Akt pathway is involved in apoptotic function in PKHD1-silenced cells, and PI3K/Akt inhibition correlates with upregulation of NF-κB activity. These observations provide a potential platform for determining FPC function and therapeutic investigation of ARPKD.     

NF-κB signaling participates in both RANKL- and IL-4-induced macrophage fusion: receptor cross-talk leads to alterations in NF-κB pathways

NF-κB activation is essential for receptor activator for NF-κB ligand (RANKL)-induced osteoclast formation. IL-4 is known to inhibit the RANKL-induced osteoclast differentiation while at the same time promoting macrophage fusion to form multinucleated giant cells (MNG). Several groups have proposed that IL-4 inhibition of osteoclastogenesis is mediated by suppressing the RANKL-induced activation of NF-κB. However, we found that IL-4 did not block proximal, canonical NF-κB signaling. Instead, we found that IL-4 inhibited alternative NF-κB signaling and induced p105/50 expression. Interestingly, in nfκb1(-/-) bone marrow-derived macrophages (BMM), the formation of both multinucleated osteoclast and MNG induced by RANKL or IL-4, respectively, was impaired. This suggests that NF-κB signaling also plays an important role in IL-4-induced macrophage fusion. Indeed, we found that the RANKL-induced and IL-4-induced macrophage fusion were both inhibited by the NF-κB inhibitors IκB kinase 2 inhibitor and NF-κB essential modulator inhibitory peptide. Furthermore, overexpression of p50, p65, p52, and RelB individually in nfκb1(-/-) or nfκb1(+/+) BMM enhanced both giant osteoclast and MNG formation. Interestingly, knockdown of nfκb2 in wild-type BMM dramatically enhanced both osteoclast and MNG formation. In addition, both RANKL- and IL-4-induced macrophage fusion were impaired in NF-κB-inducing kinase(-/-) BMM. These results suggest IL-4 influences NF-κB pathways by increasing p105/p50 and suppressing RANKL-induced p52 translocation and that NF-κB pathways participate in both RANKL- and IL-4-induced giant cell formation.     

Jolkinolide B inhibits RANKL-induced osteoclastogenesis by suppressing the activation NF-κB and MAPK signaling pathways

Osteoclasts together with osteoblasts play pivotal roles in bone remodeling. The unique function and ability of osteoclasts to resorb bone makes them critical in both normal bone homeostasis and pathologic bone diseases such as osteoporosis and rheumatoid arthritis. Thus, new compounds that may inhibit osteoclastogenesis and osteoclast function may be of great value in the treatment of osteoclast-related diseases. In the present study, we examined the effect of jolkinolide B (JB), isolated from the root of Euphorbia fischeriana Steud on receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation. We found that JB inhibited RANKL-induced osteoclast differentiation from bone marrow macrophages (BMMs) without cytotoxicity. Furthermore, the expression of osteoclastic marker genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (CtsK), and calcitonin receptor (CTR), was significantly inhibited. JB inhibited RANKL-induced activation of NF-κB by suppressing RANKL-mediated IκBα degradation. Moreover, JB inhibited RANKL-induced phosphorylation of mitogen-activated protein kinases (p38, JNK, and ERK). This study thus identifies JB as an inhibitor of osteoclast formation and provides evidence that JB might be an alternative medicine for preventing and treating osteolysis.     

Icariin inhibits RANKL-induced osteoclastogenesis via modulation of the NF-κB and MAPK signaling pathways

The receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-RANK regulatory axis is a major regulator of osteoclast differentiation and activation. Icariin, a flavonol glycoside isolated from the Epimedium herb, has been reported to prevents bone loss in ovariectomized mice and inhibits wear particle-induced osteolysis. However, the molecular mechanism through which icariin inhibits RANKL-induced osteoclastogenesis has not been fully understood. Therefore, we aimed to investigate the effects of icariin on RANKL-induced osteoclastogenesis and to elucidate the mechanism underlying this effect. Our results showed that RANKL-induced osteoclastogenesis was inhibited by icariin in bone marrow macrophages (BMMs) and RAW264.7?cells, and that this effect was due to suppression of NF-κB and mitogen-activated protein kinase (MAPK) activation. In addition, icariin inhibited F-actin ring formation and attenuated the bone resorption ability of mature osteoclasts. Collectively, our results indicate that icariin may be a promising potential candidate for the treatment of osteolytic diseases such as osteoporosis. Moreover, our findings lay the foundation for understanding and intervening in osteoclast-related diseases at the molecular level.     

Schisandra extract ameliorates arthritis pathogenesis by suppressing the NF-κB and MAPK signalling pathways

Schisandra chinensis is a medicinal plant used to treat various diseases. Extracts from the leaves or fruits of S. chinensis and their components are used in osteoarthritis (OA). The OA inhibitory effect of schisandrol A, one of its components, has been previously confirmed. We aimed to confirm the OA inhibitory effect of Schisandra (including components like schisandrol A) to identify why the inhibitory effect of the Schisandra extract is better. First, we investigated the effects of the Schisandra extract on OA as a potential therapeutic. Experimental OA was induced in a mouse model via destabilized medial meniscus surgery. The animals were orally administered the Schisandra extract; the inhibition of cartilage destruction was confirmed using histological analysis. In vitro analysis showed that the Schisandra extract attenuated osteoarthritic cartilage destruction by regulating IL-1β-induced MMP3 and COX-2 levels. The Schisandra extract inhibited IL-1β-induced degradation of IκB (NF-κB pathway) and IL-1β-induced phosphorylation of p38 and JNK (mitogen-activated protein kinase (MAPK) pathway). RNA-sequencing analysis showed that the Schisandra extract decreased the expression of IL-1β-induced MAPK and NF-κB signalling pathway-related genes more than schisandrol A alone. Therefore, Schisandra extract may be more effective than schisandrol A in preventing OA progression by regulating MAPK and NF-κB signalling.     

P53 modulates hepatic insulin sensitivity through NF-κB and p38/ERK MAPK pathways

Besides its well-established oncosuppressor activity, the role of p53 in regulating metabolic pathways has been recently identified. Nevertheless, the function of p53 with respect to insulin resistance appears highly controversial. To address this issue, we investigated the expression of p53 in experimental model of insulin resistance. Then we used activator (nutlin-3α) and inhibitor (pifithrin-α, PFT-α) of p53 in HepG2 cell. Here we showed that p53 protein level was decreased in the hepatic tissue of high-fat diet-induced insulin resistance mice, genetically diabetic ob/ob mice and palmitate (PA) treated HepG2 cells. And high expression of phosphor-p38, ERK1/2 and nuclear factor kappa B (NF-κB) p65 accompanied with low expression of p53. But activation of p53 with nutlin-3α prevented PA-induced reduction of glucose consumption and suppression of insulin signaling pathways. At the same time, nutlin-3α downregulated the activation of NF-κB, p38 and ERK1/2 pathways upon stimulation with PA. In contrast, inhibition of p53 with PFT-α decreased glucose consumption and suppressed insulin signaling pathway. Furthermore, PFT-α activated NF-κB, p38 and ERK1/2 pathways in HepG2 cells. Overall, these results suggest that p53 is involved in improving insulin sensitivity of hepatic cells via inhibition of mitogen-activated protein kinases (MAPKs) and NF-κB pathways.     

The plant limonoid 7-oxo-deacetoxygedunin inhibits RANKL-induced osteoclastogenesis by suppressing activation of the NF-κB and MAPK pathways

Osteoclasts together with osteoblasts play pivotal roles in bone remodeling. Aberrations in osteoclast differentiation and activity contribute to osteopenic disease. Osteoclasts differentiate from monocyte/macrophage progenitors, a process that is initiated by the interaction between receptor activator of NF-κB (RANK) and its ligand, RANKL. In this study, we identified 7-oxo-7-deacetoxygedunin (7-OG), a gedunin type limonoid from seeds of the mangrove Xylocarpus moluccensis, as a potent inhibitor of osteoclastogenesis. Additionally, 7-OG showed strong anti-osteoclastogenic activity with low cytotoxicity against the monocyte/macrophage progenitor cell line, RAW264.7. The IC50 for anti-osteoclastogenic activity was 4.14 μM. Treatment with 7-OG completely abolished the appearance of multinucleated giant cells with tartrate-resistant acid phosphatase activity in RAW264.7 cells stimulated with RANKL. When the expression of genes related to osteoclastogenesis was investigated, a complete downregulation of NFATc1 and cathepsin K and a delayed downregulation of irf8 were observed upon 7-OG treatment in the presence of RANKL. Furthermore, treatment with this limonoid suppressed RANKL-induced activation of p38, MAPK and Erk and nuclear localization of NF-κB p65. Taken together, we present evidence indicating a plant limonoid as a novel osteoclastogenic inhibitor that could be used for osteoporosis and related conditions.     

Galangin suppresses RANKL-induced osteoclastogenesis via inhibiting MAPK and NF-κB signalling pathways

Osteoclasts play a critical role in osteoporosis; thus, inhibiting osteoclastogenesis is a therapeutic strategy for osteoporosis. Galangin, a natural bioflavonoid extracted from a traditional Chinese herb, possesses a variety of biological activities, including anti-inflammation and anti-oxidation. However, its effects on osteoporosis have not been elucidated. In this study, we found that galangin treatment dose-dependently decreased osteoclastogenesis in bone marrow–derived macrophages (BMMs). Moreover, during osteoclastogenesis, osteoclast-specific genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (CtsK), ATPase, H + transporting, lysosomal V0 subunit D2 (V-ATPase d2) and dendritic cell–specific transmembrane protein (DC-STAMP), were down-regulated by galangin treatment. Furthermore, the results of the pit formation assay and F-actin ring staining revealed impaired osteoclastic bone resorption in the galangin-treated group compared with that in the control group. Additionally, galangin treatment also inhibited the phosphorylation of p38 and ERK of MAPK signalling pathway, as well as downstream factors of NFATc1, C-Jun and C-Fos. Consistent with our in vitro results, galangin suppressed lipopolysaccharide (LPS)-induced bone resorption via inhibition of osteoclastogenesis. Taken together, our findings provide evidence that galangin is a promising natural compound for the treatment of osteoporosis and may be associated with the inhibition of MAPK and NF-κB signalling pathways.     

TRIM59 interacts with ECSIT and negatively regulates NF-κB and IRF-3/7-mediated signal pathways

A series of inhibitors of d-amino acid oxidase (DAAO) are specific in blocking chronic pain, including formalin-induced tonic pain, neuropathic pain and bone cancer pain. This study used RNA interference technology to further validate the notion that spinal DAAO mediates formalin-induced pain. To target DAAO, a siRNA/DAAO formulated in polyetherimide (PEI) complexation and a shRNA/DAAO (shDAAO, with the same sequence as siRNA/DAAO after intracellular processing) expressed in recombinant adenoviral vectors were designed. The siRNA/DAAO was effective in blocking DAAO expression in NRK-52E rat kidney tubule epithelial cells, compared to the nonspecific oligonucleotides. Furthermore, multiple-daily intrathecal injections of both siRNA/DAAO and Ad-shDAAO for 7 days significantly inhibited spinal DAAO expression by 50-80% as measured by real-time quantitative PCR and Western blot, and blocked spinal DAAO enzymatic activity by approximately 60%. Meanwhile, both siRNA/DAAO and Ad-shDAAO prevented formalin-induced tonic phase pain by approximately 60%. Multiple-daily intrathecal injections of siRNA/DAAO and Ad-shDAAO also blocked more than 30% spinal expression of GFAP, a biomarker for the activation of astrocytes. These results further suggest that down-regulation of spinal DAAO expression and enzymatic activity leads to analgesia with its mechanism potentially related to activation of astrocytes in the spinal cord.     

Selenium suppressed the LPS-induced inflammation of bovine endometrial epithelial cells through NF-κB and MAPK pathways under high cortisol background

The bovine uterus is susceptible to infection, and the elevated cortisol level due to stress are common in cows after delivery. The essential trace element selenium plays a pivotal role in the antioxidant and anti-inflammatory defence system of body. This study investigated whether selenium supplementation protected endometrial cells from inflammation in the presence of high-level cortisol. The primary bovine endometrial epithelial cells were subjected to Escherichia coli lipopolysaccharide to establish cellular inflammation model. The gene expression of inflammatory mediators and proinflammatory cytokines was measured by quantitative PCR. The key proteins of NF-κB and MAPK signalling pathways were detected by Western blot and immunofluorescence. The result showed that pre-treatment of Na₂SeO₃ (1, 2 and 4 μΜ) decreased the mRNA expression of proinflammatory genes, inhibited the activation of NF-κB and suppressed the phosphorylation of extracellular signal-regulated kinase, P38MAPK and c-Jun N-terminal kinase. This inhibition of inflammation was more apparent in the presence of high-level cortisol (30 ng/mL). These results indicated that selenium has an anti-inflammatory effect, which is mediated via NF-κB and MAPK signalling pathways and is augmented by cortisol in bovine endometrial epithelial cells.     

Adiponectin induces dendritic cell activation via PLCγ/JNK/NF-κB pathways, leading to Th1 and Th17 polarization

Adiponectin (APN) is a crucial regulator for many inflammatory processes, but its effect on Th cell-mediated responses has not been fully understood. Thus, we investigated the immune-modulatory effects of APN on dendritic cells (DCs) controlling Th cell polarization. APN induced maturation and activation of DCs, as demonstrated by the increased expression of MHC class II, costimulatory molecules in both mouse and human DCs, and it significantly enhanced production of proinflammatory cytokines. APN triggered degradation of IκB proteins, nuclear translocation of NF-κB p65 subunit, and phosphorylation of MAPKs in DCs. Pretreatment with a phospholipase C (PLC)γ inhibitor and a JNK inhibitor suppressed IL-12 production and NF-κB binding activity. Additionally, PLCγ inhibitor downregulated phosphorylation of JNK, indicating that PLCγ and JNK may be upstream molecules of NF-κB. Importantly, APN-treated DCs significantly induced both Th1 and Th17 responses in allogeneic CD4(+) T cells. The addition of a neutralizing anti-IL-12 mAb to the cocultures abolished the secretion of IFN-γ, whereas the blockage of IL-23 and IL-1β suppressed APN-induced IL-17 production. Immunization of mice with OVA-pulsed, APN-treated DCs efficiently led to Ag-specific Th1 and Th17 cell responses. Taken together, these results demonstrated that APN effectively induced activation of DCs through PLCγ/JNK/NF-κB-signaling pathways, leading to enhanced Th1 and Th17 responses.     

HMGB-1 induces IL-6 production in human synovial fibroblasts through c-Src, Akt and NF-κB pathways

High mobility group box chromosomal protein 1 (HMGB-1) is a widely studied, ubiquitous nuclear protein that is present in eukaryotic cells, and plays a crucial role in inflammatory response. However, the effects of HMGB-1 on human synovial fibroblasts are largely unknown. In this study, we investigated the intracellular signaling pathway involved in HMGB-1-induced IL-6 production in human synovial fibroblast cells. HMGB-1 caused concentration- and time-dependent increases in IL-6 production. HMGB-1-mediated IL-6 production was attenuated by receptor for advanced glycation end products (RAGE) monoclonal antibody (Ab) or siRNA. Pretreatment with c-Src inhibitor (PP2), Akt inhibitor and NF-κB inhibitor (pyrrolidine dithiocarbamate and L-1-tosylamido-2-phenylenylethyl chloromethyl ketone) also inhibited the potentiating action of HMGB-1. Stimulation of cells with HMGB-1 increased the c-Src and Akt phosphorylation. HMGB-1 increased the accumulation of p-p65 in the nucleus, as well as NF-κB luciferase activity. HMGB-1-mediated increase of NF-κB luciferase activity was inhibited by RAGE Ab, PP2 and Akt inhibitor or RAGE siRNA, or c-Src and Akt mutant. Our results suggest that HMGB-1-increased IL-6 production in human synovial fibroblasts via the RAGE receptor, c-Src, Akt, p65, and NF-κB signaling pathways.     

Porcine reproductive and respiratory syndrome virus induces interleukin-15 through the NF-κB signaling pathway

Porcine reproductive and respiratory syndrome virus (PRRSV) mainly infects macrophages/dendritic cells and modulates cytokine expression in these cells. Interleukin-15 (IL-15) is a pleiotropic cytokine involved in wide range of biological activities. It has been shown to be essential for the generation, activation, and proliferation of NK and NKT cells and for the survival and activation of CD8(+) effector and memory T cells. In this study, we discovered that PRRSV infection upregulated IL-15 production at both the mRNA and protein levels in porcine alveolar macrophages (PAMs), blood monocyte-derived macrophages (BMo), and monocyte-derived dendritic cells (DCs). We subsequently demonstrated that the NF-κB signaling pathway was essential for PRRSV infection-induced IL-15 production. First, addition of an NF-κB inhibitor drastically reduced PRRSV infection-induced IL-15 production. We then found that NF-κB was indeed activated upon PRRSV infection, as evidenced by IκB phosphorylation and degradation. Moreover, we revealed an NF-κB binding motif in the cloned porcine IL-15 (pIL-15) promoter, deletion of which abrogated the pIL-15 promoter activity in PRRSV-infected alveolar macrophages. In addition, we demonstrated that PRRSV nucleocapsid (N) protein had the ability to induce IL-15 production in porcine alveolar macrophage cell line CRL2843 by transient transfection, which was mediated by its multiple motifs, and it also activated NF-κB. These data indicated that PRRSV infection-induced IL-15 production was likely through PRRSV N protein-mediated NF-κB activation. Our findings provide new insights into the molecular mechanisms underling the IL-15 production induced by PRRSV infection.     

Magnesium isoglycyrrhizinate suppresses LPS-induced inflammation and oxidative stress through inhibiting NF-κB and MAPK pathways in RAW264.7 cells

Magnesium Isoglycyrrhizinate (MgIG), a novel molecular compound extracted from licorice root, has exhibited greater anti-inflammatory activity and hepatic protection than glycyrrhizin and β-glycyrrhizic acid. In this study, we investigated the anti-inflammatory effect and the potential mechanism of MgIG on Lipopolysaccharide (LPS)-treated RAW264.7 cells. MgIG down-regulated LPS-induced pro-inflammatory mediators and enzymes in LPS-treated RAW264.7 cells, including TNF-α, IL-6, IL-1β, IL-8, NO and iNOS. The generation of reactive oxygen species (ROS) in LPS-treated RAW264.7 cells was also reduced. MgIG attenuated NF-κB translocation by inhibiting IKK phosphorylation and IκB-α degradation. Simultaneously, MgIG also inhibited LPS-induced activation of MAPKs, including p38, JNK and ERK1/2. Taken together, these results suggest that MgIG suppresses inflammation by blocking NF-κB and MAPK signaling pathways, and down-regulates ROS generation and inflammatory mediators.     

Unfractionated heparin inhibits lipopolysaccharide-induced inflammatory response through blocking p38 MAPK and NF-κB activation on endothelial cell

Heparins, including unfractionated heparin (UFH) and low-molecular-weight heparins (LMWH), are glycosaminoglycans that are largely used as anti-thrombotic drugs. While the mechanisms of their anticoagulant actions in blood have been extensively studied, their effects on the inflammation of the endothelium are still under investigation since the endothelium plays a central role in sepsis. Furthermore, UFH is much cheaper than LMWH. The aim of this study was to determine how UFH regulates lipopolysaccharide (LPS)-induced inflammatory response on endothelial cells in vitro, and define the role of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) in mediating this effect. Human pulmonary microvascular endothelial cells (HPMECs) were pretreated with UFH (0.01U/ml-10U/ml), prior to stimulation with LPS (10μg/ml). Markers of systemic inflammation and endothelial activation were assessed. Interleukin (IL)-1β, IL-6, E-selectin, intercellular adhesion molecule (ICAM)-1 release were subsequently measured at 2h, 6h and 12h. Phosphorylation of p38 MAPK at 2h, 6h and nuclear translocation of the proinflammatory NF-κB at 2h were assessed. In HPMEC, UFH significantly attenuated LPS-induced production of IL-1β, IL-6, E-selectin and ICAM-1, as well as phosphorylation of p38 MAPK and NF-κB translocation, especially in 10U/ml. In conclusion, UFH at high dose significantly protects against endothelial-cell-mediated immune response. The inhibition of p38 MAPK and NF-κB activation certainly represents one of the mechanisms by which UFH exerts its anti-inflammatory effect.