Diacylglycerol kinase α enhances protein kinase Cζ-dependent phosphorylation at Ser311 of p65/RelA subunit of nuclear factor-κB

We recently reported that diacylglycerol kinase (DGK) α enhanced tumor necrosis factor-α (TNF-α)-induced activation of nuclear factor-κB (NF-κB). However, the signaling pathway between DGKα and NF-κB remains unclear. Here, we found that small interfering RNA-mediated knockdown of DGKα strongly attenuated protein kinase C (PKC) ζ-dependent phosphorylation of a large subunit of NF-κB, p65/RelA, at Ser311 but not PKCζ-independent phosphorylation at Ser468 or Ser536. Moreover, knockdown and overexpression of PKCζ suppressed and synergistically enhanced DGKα-mediated NF-κB activation, respectively. These results strongly suggest that DGKα positively regulates TNF-α-dependent NF-κB activation via the PKCζ-mediated Ser311 phosphorylation of p65/RelA.     

Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-α

POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-α (TNF-α), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-α-induced down-regulation of POEM gene expression occurred in both time- and dose-dependent manners through the nuclear factor kappa B (NF-κB) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-α in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-α-induced inhibition of osteoblast differentiation. These results suggest that TNF-α inhibits POEM expression through the NF-κB signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-α.     

Inhibition of TNF-α-mediated inflammatory responses by a benzodioxolylacetylamino-linked benzothiazole analog in human fibroblast-like synoviocytes

The pathologic processes of rheumatoid arthritis are mediated by a number of cytokines, chemokines, and matrix metalloproteinases, the expressions of which are controlled by NF-κB. This study was performed to explore the effects of a benzothiazole analog, SPA0537, on the control of the NF-κB activation pathway. We also investigated whether SPA0537 had any anti-inflammatory effects in human rheumatoid fibroblast-like synoviocytes (FLS). SPA0537 inhibited the nuclear translocation and the DNA binding of NF-κB subunits, which correlated with the inhibitory effects on IKK phosphorylation and IκBα degradation in TNF-α-stimulated rheumatoid FLS. These events further suppressed chemokine production, matrix metalloproteinase secretion, and TNF-α-induced cell proliferation. In addition, SPA0537 inhibited the osteoclast differentiation induced by macrophage colony-stimulating factor (MCSF) and receptor activator of the NF-κB ligand (RANKL) in bone marrow macrophages. These findings suggest that SPA0537 exerts anti-inflammatory effects in rheumatoid FLS through the inhibition of the NF-κB pathway. Therefore, it may have therapeutic value for the treatment of rheumatoid arthritis.     

Eicosapentaenoic acid inhibits TNF-alpha-induced matrix metalloproteinase-9 expression in human keratinocytes, HaCaT cells

Eicosapentaenoic acid (EPA) is an omega-3 (ω-3) polyunsaturated fatty acid (PUFA), which has anti-inflammatory and anti-cancer properties. Some reports have demonstrated that EPA inhibits NF-κB activation induced by tumor necrosis factor (TNF)-α or lipopolysaccharide (LPS) in various cells. However, its detailed mode of action is unclear. In this report, we investigated whether EPA inhibits the expression of TNF-α-induced matrix metalloproteinases (MMP)-9 in human immortalized keratinocytes (HaCaT). TNF-α induced MMP-9 expression by NF-κB-dependent pathway. Pretreatment of EPA inhibited TNF-α-induced MMP-9 expression and p65 phosphorylation. However, EPA could not affect IκB-α phosphorylation, nuclear translocation of p65, and DNA binding activity of NF-κB. EPA inhibited TNF-α-induced p65 phosphorylation through p38 and Akt inhibition and this inhibition was IKKα-dependent event. Taken together, we demonstrate that EPA inhibits TNF-α-induced MMP-9 expression through inhibition of p38 and Akt activation.     

YAP1 inhibits the induction of TNF-α-stimulated bone-resorbing mediators by suppressing the NF-κB signaling pathway in MC3T3-E1 cells

Yes-associated protein 1 (YAP1), the core downstream effector of the Hippo signaling cascade, was involved in the regulation of osteoblast and osteoclast differentiation and in bone metabolism. However, the regulatory effects and mechanisms of YAP1 on bone-remodeling molecules in osteoblasts under inflammation remain unknown. In this study, YAP1 expression level was downregulated after treatment with inflammatory cytokine tumor necrosis factor-α (TNF-α) in MC3T3-E1 cells. The key osteoclastogenic molecules induced by TNF-α, namely, interleukin-6 and receptor activator of nuclear factor-κB (NF-κB) ligand, were suppressed after lentivirus-induced YAP1 overexpression, which dramatically increased the expression level of osteoprotegerin. Conversely, the expression levels of the above factors showed opposite trends in the YAP1 small interfering RNA and YAP1 inhibitor (verteporfin) group. Mechanistically, YAP1 attenuated the TNF-α-induced activation of the NF-κB signaling pathway as revealed by the reduced expression of phosphorylated-p65 and NF-κB reporter activity and the nuclear translocation of p65. Moreover, the expression level of YAP1 suppressed by TNF-α was reversed by berberine in concentration-dependent manner. Taken together, our study suggests that YAP1 plays a critical role in the regulation of bone metabolism and is a potential therapeutic target for treating inflammatory bone resorption.     

Astrocyte elevated gene-1 promotes inflammation and invasion of fibroblast-like synoviocytes in rheumatoid arthritis

Astrocyte elevated gene-1 (AEG-1) was initially induced by HIV-1 infection and involved in tumor progression, migration and invasion as a nuclear factor-κB (NF-κB)-dependent gene. The present study we intended to investigate the protein expression of AEG-1 significantly associated with rheumatoid arthritis. Western blot analysis and immunohistochemistry demonstrated that AEG-1 was upregulated in synovial tissue of RA patients compared with the controls. Double immunofluorescent staining suggested that AEG-1 was expressed in fibroblast-like synoviocytes (FLS) of RA patients. Furthermore, the expression of AEG-1 in FLS was increased in time-dependent manner by TNF-α stimulation. Upon TNF-α-treated FLS, AEG-1 transferred from the cytoplasm to nucleus where it interacted with the p65 subunit of NF-κB, as examined by immunoprecipitation and immunofluorescent staining assay. Moreover, the inhibition of AEG-1 by RNA interference significantly suppressed TNF-α-induced IL-6 and MMP-3 expression, leading to attenuation of FLS migration and invasion and markedly decreased the phosphorylation of P65 and IκBα, as well as AKT in FLS. Collectively, Our findings provided evidence that AEG-1 contributed to the production of inflammatory cytokines, migration and invasion of RA FLS, and underscored the importance of AEG-1 in the inflammation process of RA.     

I-κBα depletion by transglutaminase 2 and μ-calpain occurs in parallel with the ubiquitin-proteasome pathway

Transglutaminase 2 (TGase2) is a calcium-dependent, cross-linking enzyme that catalyzes iso-peptide bond formation between peptide-bound lysine and glutamine residues. TGase 2 can activate NF-κB through the polymerization-mediated depletion of I-κBα without IKK activation. This NF-κB activation mechanism is associated with drug resistance in cancer cells. However, the polymers cannot be detected in cells, while TGase 2 over-expression depletes free I-κBα, which raises the question of how the polymerized I-κBα can be metabolized in cells. Among proteasome, lysosome and calpain systems, calpain inhibition was found to effectively increase the accumulation of I-κBα polymers in MCF7 cells transfected with TGase 2, and induced high levels of I-κBα polymers as well in MDA-MB-231 breast cancer cells that naturally express a high level of TGase 2. Inhibition of calpain also boosted the level of I-κBα polymers in HEK-293 cells in case of TGase 2 transfection either with I-κBα or I-κBα mutant (S32A, S36A). Interestingly, the combined inhibition of calpain and the proteasome resulted in an increased accumulation of both I-κBα polymers and I-κBα, concurrent with an inhibition of NF-κB activity in MDA-MB-231 cells. This suggests that μ-calpain proteasome-dependent I-κBα polymer degradation may contribute to cancer progression through constitutive NF-κB activation.     

Licorice-derived dehydroglyasperin C increases MKP-1 expression and suppresses inflammation-mediated neurodegeneration

Recent studies have demonstrated that microglial hyperactivation-mediated neuroinflammation is involved in the pathogenesis of several neurodegenerative diseases. Thus, inhibiting microglial production of the neurotoxic mediator tumor necrosis factor-α (TNF-α) is considered a promising strategy to protect against neurodegeneration. Here, we investigated the inhibitory effect of licorice-derived dehydroglyasperin C (DGC) on lipopolysaccharide (LPS)-induced TNF-α production and inflammation-mediated neurodegeneration. We found that DGC pre-treatment attenuated TNF-α production in response to LPS stimulation of BV-2 microglia. DGC pre-treatment attenuated LPS-induced inhibitor of κB-α (IκB-α) and p65 phosphorylation and decreased the DNA binding activity of nuclear factor-κB (NF-κB). DGC pre-treatment also inhibited LPS-mediated phosphorylation of p38 mitogen-activated protein kinases (MAPKs) and extracellular signal-regulated kinase (ERK). Interestingly, DGC treatment of BV-2 microglia significantly increased MAPK phosphatase 1 (MKP-1) mRNA and protein expression, which is a phosphatase of p38 MAPK and ERK, suggesting that the DGC-mediated increase in MKP-1 expression might inhibit LPS-induced MAPKs and NF-κB activation and further TNF-α production. We also found that LPS-mediated microglial neurotoxicity can be attenuated by DGC. The addition of conditioned media (CM) from DGC- and LPS-treated microglia to neurons helped maintain healthy cell body and neurite morphology and increased the number of microtubule-associated protein 2-positive cells and the level of synaptophysin compared to treatment with CM from LPS-treated microglia. Taken together, these data suggest that DGC isolated from licorice may inhibit microglia hyperactivation by increasing MKP-1 expression and acting as a potent anti-neurodegenerative agent.     

Interleukin-1β and tumor necrosis factor-α augment acidosis-induced rat articular chondrocyte apoptosis via nuclear factor-kappaB-dependent upregulation of ASIC1a channel

The acute-phase proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) demonstrate high-level expression and pleiotropic biological effects, and contribute to the progression and persistence of rheumatoid arthritis (RA). Acid hydrarthrosis is also an important pathological characteristic of RA, and the acid-sensing ion channel 1a (ASIC1a) plays a critical role in acidosis-induced chondrocyte cytotoxicity. However, the roles of IL-1β and TNF-α in acid-induced apoptosis of chondrocytes remain unclear. Rat adjuvant arthritis and primary articular chondrocytes were used as in vivo and in vitro model systems, respectively. ASIC1a expression in articular cartilage was increased and highly colocalized with nuclear factor (NF)-κB expression in vivo. IL-1β and TNF-α could upregulate ASIC1a expression. These cytokines activated mitogen-activated protein kinase and NF-κB pathways in chondrocytes, while the respective inhibitors of these signaling pathways could partially reverse the ASIC1a upregulation induced by IL-1β and TNF-α. Dual luciferase and gel-shift assays and chromatin immunoprecipitation-polymerase chain reaction demonstrated that IL-1β and TNF-α enhanced ASIC1a promoter activity in chondrocytes by increasing NF-κB DNA-binding activities, which was in turn prevented by the NF-κB inhibitor ammonium pyrrolidinedithiocarbamate. IL-1β and TNF-α also decreased cell viability but enhanced LDH release, intracellular Ca²⁺ concentration elevation, loss of mitochondrial membrane potential, cleaved PARP and cleaved caspase-3/9 expression, and apoptosis in acid-stimulated chondrocytes, which effects could be abrogated by the specific ASIC1a inhibitor psalmotoxin-1 (PcTX-1), ASIC1a-short hairpin RNA or calcium chelating agent BAPTA-AM. These results indicate that IL-1β and TNF-α can augment acidosis-induced cytotoxicity through NF-κB-dependent up-regulation of ASIC1a channel expression in primary articular chondrocytes.     

绝经后骨质疏松症TNF-α通过激活NF-κB促进RANKL诱导的破骨细胞形成

本研究检测了绝经后骨质疏松症妇女的肿瘤坏死因子-α(TNF-α)和雌激素水平,并探讨了TNF-α对破骨前体细胞RAW264.7中破骨细胞标志物核因子κB受体激活因子(nuclear factor kappa-B, RANK)、组织蛋白酶K (Cathepsin K, CTSK)和凝血酶受体激活肽(thrombin receptor activating peptide, TRAP)以及核因子-κB (NF-κB)亚基(p65)和NF-κB抑制蛋白(IκBα)的影响。研究结果表明,绝经后骨质疏松症患者的TNF-α水平显著升高,而雌二醇水平显著降低。核因子κB受体激活因子配体(receptor activator for NF-κBligand, RANKL)处理1周后,破骨前体细胞RAW264.7中破骨细胞标志物RANK、CTSK和TRAP的mRNA和蛋白高度表达。与RANKL对照组相比,TNF-α处理可上调RANK、CTSK和TRAP m RNA的表达。但是,仅TNF-α不能诱导培养的RAW264.7细胞分化为破骨细胞成。TNF-α以剂量依赖性方式诱导NF-κB亚基p65和IκBα磷酸化,而NF-κB抑制剂处理则有效降低了RANK和TRAP的表达。本研究结论表明,绝经后骨质疏松症中TNF-α通过激活NF-κB来促进RANKL诱导的破骨细胞形成。     

Aristolochic acid downregulates monocytic matrix metalloproteinase-9 by inhibiting nuclear factor-κB activation

Aristolochic acid (AA)-associated nephropathy was described as being characterized by a rapid progressive enhancement of interstitial renal fibrosis. Renal tissue fibrosis occurs because of an imbalance of extracellular matrix (ECM) accumulation and matrix metalloproteinase (MMP) activation. Much evidence indicates that inflammatory renal disease including monocyte and mesangial interactions is linked to the development and progression of renal remodeling. In this study, we found that AA showed concentration-dependent inhibition of tumor necrosis factor (TNF)-α-induced MMP-9 activation with an IC₅₀ value of 6.4 ± 0.5 μM in human monocytic THP-1 cells. A similar effect was also noted with different ratios of AAs (types I and II). However, AA had no inhibitory effect on the intact enzymatic activity of MMP-9 at a concentration of 20 μM. On the other hand, the level of tissue inhibitor of metalloproteinase (TIMP)-1 was not induced by AA, but it suppressed TNF-α-induced MMP-9 protein and messenger RNA expressions. AA also significantly inhibited TNF-α-induced IκBα degradation. Furthermore, an electrophoretic mobility shift assay and a reported gene study, respectively, revealed that AA inhibited TNF-α-induced NF-κB translocation and activation. In addition, compared to other NF-κB inhibitors, AA exerted significant inhibition of MMP-9 activation and monocyte chemotactic protein-1-directed invasion. From these results, we concluded that AA, a natural compound, inhibits TNF-α-induced MMP-9 in human monocytic cells possibly through the NF-κB signal pathway. These results also imply that AA may be involved in alteration of matrix homeostasis during renal fibrosis in vivo.     

(+)-Nootkatone inhibits tumor necrosis factor α/interferon γ-induced production of chemokines in HaCaT cells

Chemokines are important mediators of cell migration, and thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22) are well-known typical inflammatory chemokines involved in atopic dermatitis (AD). (+)-Nootkatone is the major component of Cyperus rotundus. (+)-Nootkatone has antiallergic, anti-inflammatory, and antiplatelet activities. The purpose of this study was to investigate the effect of (+)-nootkatone on tumor necrosis factor α (TNF-α)/interferon γ (IFN-γ)-induced expression of Th2 chemokines in HaCaT cells. We found that (+)-nootkatone inhibited the TNF-α/IFN-γ-induced expression of TARC/CCL17 and MDC/CCL22 mRNA in HaCaT cells. It also significantly inhibited TNF-α/IFN-γ-induced activation of nuclear factor kappa B (NF-κB), p38 mitogen-activated protein kinase (MAPK), and protein kinase Cζ (PKCζ). Furthermore, we showed that PKCζ and p38 MAPK contributed to the inhibition of TNF-α/IFN-γ-induced TARC/CCL17 and MDC/CCL22 expression by blocking IκBα degradation in HaCaT cells. Taken together, these results suggest that (+)-nootkatone may suppress TNF-α/IFN-γ-induced TARC/CCL17 and MDC/CCL22 expression in HaCaT cells by inhibiting of PKCζ and p38 MAPK signaling pathways that lead to activation of NF-κB. We propose that (+)-nootkatone may be a useful therapeutic candidate for inflammatory skin diseases such as AD.     

JAK2 is an important signal transducer in IL-33-induced NF-κB activation

IL-33, a member of the IL-1 family of cytokines, has been shown to activate NF-κB and MAP kinase family through the IL-1 receptor-related protein, ST2L. In this study, we found that IL-33 rapidly activated a tyrosine kinase, JAK2. Interestingly, we demonstrated the functional involvement of JAK2 in IL-33-induced IκBα degradation and NF-κB activation, since a JAK2 inhibitor, AG490, effectively inhibited this signaling pathway. Furthermore, IL-33 failed to induce IκBα degradation and NF-κB activation in JAK2-deficient MEFs expressing ST2L, compared with wild-type MEFs expressing ST2L. In addition, the introduction of wild-type JAK2 but not kinase dead JAK2 mutant (K882R) restored the IL-33-induced efficient activation of NF-κB in JAK2-deficient MEFs expressing ST2L, resulting in the induction of IL-6, CCL2/MCP-1 and CXCL1/KC expression. On the other hand, the activation of ERK, JNK and p38 was unaffected by JAK2 inhibition and JAK2 deficiency. Thus, these data demonstrate that JAK2 plays an important role in regulating IL-33-induced NF-κB activation.     

TNF-α刺激大鼠骨髓间充质干细胞的机制研究

目的：研究肿瘤坏死因子-α（Tumor necrosis factor-α,TNF-α）刺激大鼠骨髓间充质干细胞（marrow-derived mesenchymalstem cells,MSCs）的作用机制。方法：采取大鼠骨髓,以密度梯度离心分离出单个核细胞（MNCs）,于体外培养并由牛垂体提取物（PEX）诱导扩增传代培养出骨髓间充质干细胞（MSCs）。经形态学和流式细胞仪检测MSCs表面标志物鉴定后,用TNF-α刺激骨髓间充质干细胞（MSCs）,通过酶联免疫吸附剂测定法（enzyme linked immunosorbent assay,ELISA）观察比较不同组别细胞的生长因子分泌和蛋白印迹法（western blot）来观察细胞中蛋白的变化。结果：①经形态学观察和流式细胞仪检测MSCs表面标志物鉴定,提示骨髓间充质干细胞的培养成功。②无TNF-α刺激组与TNF-α刺激组比较,TNF-α刺激组的生长因子分泌显著性增加,而通过磷酸化IκB的表达量显著性增加提示NF-κB被激活（P〈0.05）;同时TNF-α刺激组与TNF-α＋NF-κB抑制剂组比较,TNF-α＋NF-κB抑制剂组的生长因子分泌显著降低,而通过磷酸化IκB的表达量显著减少提示NF-κB的活性被抑制（P〈0.05）。结论：NF-κB对TNF-α刺激下的骨髓间充质干细胞分泌生长因子有关键性作用。