The TRIF-dependent signaling pathway is not required for acute cerebral ischemia/reperfusion injury in mice

TIR domain-containing adaptor protein (TRIF) is an adaptor protein in Toll-like receptor (TLR) signaling pathways. Activation of TRIF leads to the activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB). While studies have shown that TLRs are implicated in cerebral ischemia/reperfusion (I/R) injury and in neuroprotection against ischemia afforded by preconditioning, little is known about TRIF’s role in the pathological process following cerebral I/R. The present study investigated the role that TRIF may play in acute cerebral I/R injury. In a mouse model of cerebral I/R induced by transient middle cerebral artery occlusion, we examined the activation of NF-κB and IRF3 signaling in ischemic cerebral tissue using ELISA and Western blots. Neurological function and cerebral infarct size were also evaluated 24 h after cerebral I/R. NF-κB activity and phosphorylation of the inhibitor of kappa B (IκBα) increased in ischemic brains, but IRF3, inhibitor of κB kinase complex-ε (IKKε), and TANK-binding kinase1 (TBK1) were not activated after cerebral I/R in wild-type (WT) mice. Interestingly, TRIF deficit did not inhibit NF-κB activity or p-IκBα induced by cerebral I/R. Moreover, although cerebral I/R induced neurological and functional impairments and brain infarction in WT mice, the deficits were not improved and brain infarct size was not reduced in TRIF knockout mice compared to WT mice. Our results demonstrate that the TRIF-dependent signaling pathway is not required for the activation of NF-κB signaling and brain injury after acute cerebral I/R.     

ABIN1 protein cooperates with TAX1BP1 and A20 proteins to inhibit antiviral signaling

Upon virus infection, the innate immune response provides the first line of protection and rapidly induces type I interferons (IFNα/β), which mediate potent antiviral effects. To maintain homeostasis and prevent autoimmunity, IFN production is tightly regulated; however, the mechanisms of negative regulation are poorly understood. Herein, we demonstrate that the A20 binding inhibitor of NF-κB 1 (ABIN1) is a novel negative regulator of antiviral signaling. Overexpression of ABIN1 inhibited IFN-β promoter activation in response to virus infection or poly(I:C) transfection, whereas siRNA-mediated knockdown of ABIN1 enhanced IFN-β production upon virus infection. ABIN1 interacted with the A20 regulatory molecule TAX1BP1 and was essential for the recruitment of TAX1BP1 and A20 to the noncanonical IκB kinases TBK1 and IKKi in response to poly(I:C) transfection. ABIN1 and TAX1BP1 together disrupted the interactions between the E3 ubiquitin ligase TRAF3 and TBK1/IKKi to attenuate lysine 63-linked polyubiquitination of TBK1/IKKi. Finally, an intact ubiquitin binding domain of ABIN1 was essential for ABIN1 to interact with TBK1/IKKi and inhibit IFN-β production upon poly(I:C) transfection or virus infection. Together, these results suggest that ABIN1 requires its ubiquitin binding domain and cooperates with TAX1BP1 and A20 to restrict antiviral signaling.     

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.     

Regulation of NF-κB signaling by caspases and MALT1 paracaspase

Caspases are intracellular proteases that are best known for their function in apoptosis signaling. It has become evident that many caspases also function in other signaling pathways that propagate cell proliferation and inflammation, but studies on the inflammatory function of caspases have mainly been limited to caspase-1-mediated cytokine processing. Emerging evidence, however, indicates an important contribution of caspases as mediators or regulators of nuclear factor-κB (NF-κB) signaling, which plays a key role in inflammation and immunity. Much still needs to be learned about the mechanisms that govern the activation and regulation of NF-κB by caspases, and this review provides an update of this area. Whereas apoptosis signaling is dependent on the catalytic activity of caspases, they mainly act as scaffolding platforms for other signaling proteins in the case of NF-κB signaling. Caspase proteolytic activity, however, counteracts the pro-survival function of NF-κB by cleaving specific signaling molecules. A striking exception is the paracaspase mucosa-associated lymphoid tissue 1 (MALT1), whose adaptor and proteolytic activity are both needed to initiate a full blown NF-κB response in antigen-stimulated lymphocytes. Understanding the role of caspases and MALT1 in the regulation of NF-κB signaling is of high interest for therapeutic immunomodulation.     

Novel phosphorylation-dependent ubiquitination of tristetraprolin by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1) and tumor necrosis factor receptor-associated factor 2 (TRAF2)

Acute versus chronic inflammation is controlled by the accurate activation and regulation of interdependent signaling cascades. TNF-receptor 1 engagement concomitantly activates NF-κB and JNK signaling. The correctly timed activation of these pathways is the key to account for the balance between NF-κB-mediated cell survival and cell death, the latter fostered by prolonged JNK activation. Tristetraprolin (TTP), initially described as an mRNA destabilizing protein, acts as negative feedback regulator of the inflammatory response: it destabilizes cytokine-mRNAs but also acts as an NF-κB inhibitor by interfering with the p65/RelA nuclear import pathway. Our biochemical studies provide evidence that TTP contributes to the NF-κB/JNK balance. We find that the MAP 3-kinase MEKK1 acts as a novel TTP kinase that, together with the TNF receptor-associated factor 2 (TRAF2), constitutes not only a main determinate of the NF-κB-JNK cross-talk but also facilitates "TTP hypermodification": MEKK1 triggers TTP phosphorylation as prerequisite for its Lys-63-linked, TRAF2-mediated ubiquitination. Consequently, TTP no longer affects NF-κB activity but promotes the activation of JNK. Based on our data, we suggest a model where upon TNFα induction, TTP transits a hypo- to hypermodified state, thereby contributing to the molecular regulation of NF-κB versus JNK signaling cascades.     

Salidroside promotes rat spinal cord injury recovery by inhibiting inflammatory cytokine expression and NF-κB and MAPK signaling pathways

Spinal cord injury (SCI) is a public health problem in the world. The SCI usually triggers an excessive inflammatory response that brings about a secondary tissue wreck leading to further cellular and organ dysfunction. Hence, there is great potential of reducing inflammation for therapeutic strategies of SCI. In this study, we aim to investigate if Salidroside (SAD) exerts an anti-inflammatory effect and promotes recovery of motor function on SCI through suppressing nuclear factor-κB (NF-κB) and the mitogen-activated protein kinase (MAPK) pathways. In vitro, real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) were used to examine the inhibitory effect of SAD on the expression and release of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) activated by lipopolysaccharide (LPS) in astrocytes. In addition, SAD was found to inhibit NF-κB, p38 and extracellular-regulated protein kinases (ERK) signaling pathways by western blot analysis. Further, in vivo study showed that SAD was able to improve hind limb motor function and reduce tissue damage accompanied by the suppressed expression of inflammatory cytokines IL-1β, IL-6, and TNF-α. Overall, SAD could reduce the inflammatory response and promote motor function recovery in rats after SCI by inhibiting NF-κB, p38, and ERK signaling pathways.     

Phospholipase Cγ-2 and intracellular calcium are required for lipopolysaccharide-induced Toll-like receptor 4 (TLR4) endocytosis and interferon regulatory factor 3 (IRF3) activation

Toll-like receptor 4 (TLR4) is unique among the TLRs in its use of multiple adaptor proteins leading to activation of both the interferon regulatory factor 3 (IRF3) and nuclear factor κB (NF-κB) pathways. Previous work has demonstrated that TLR4 initiates NF-κB activation from the plasma membrane, but that subsequent TLR4 translocation to the endosomes is required for IRF3 activation. Here we have characterized several components of the signaling pathway that governs TLR4 translocation and subsequent IRF3 activation. We find that phospholipase C γ2 (PLCγ2) accounts for LPS-induced inositol 1,4,5-trisphosphate (IP(3)) production and subsequent calcium (Ca(2+)) release. Blockage of PLCγ2 function by inhibitors or knockdown of PLCγ2 expression by siRNAs in RAW 264.7 macrophages lead to reduced IRF3, but enhanced NF-κB activation. In addition, bone marrow-derived macrophages from PLCγ2-deficient mice showed impaired IRF3 phosphorylation and expression of IRF3-regulated genes after LPS stimulation. Using cell fractionation, we show that PLCγ2-IP(3)-Ca(2+) signaling cascade is required for TLR4 endocytosis following LPS stimulation. In conclusion, our results describe a novel role of the PLCγ2-IP(3)-Ca(2+) cascade in the LPS-induced innate immune response pathway where release of intracellular Ca(2+) mediates TLR4 trafficking and subsequent activation of IRF3.     

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.     

Bronchial epithelial cells release IL-6, CXCL1 and CXCL8 upon mast cell interaction

Although mast cells have been found in increased numbers in bronchial epithelium in asthma patients, the pathogenic role of the interaction of mast cells with bronchial epithelial cells in the development of local inflammation in asthma is not well understood. In this study, primary human bronchial epithelial cells and a human mast cell line (HMC-1) were cultured either together or separately in the presence or absence of various signaling molecule inhibitors or dexamethasone. Cytokine IL-6, and chemokines including CXCL1 and CXCL8 in cell culture supernatant were assayed by enzyme-linked immunosorbent assay (ELISA), and the activity of mitogen-activated protein kinases (MAPKs), or nuclear factor-κB (NF-κB) in co-culture system was analyzed by ELISA. Co-culture of bronchial epithelial cells and mast cells induced a significant elevation of IL-6, CXCL1 and CXCL8 in bronchial epithelial cells, and both IL-17A and IL-17F could further enhance the release of these inflammatory mediators from co-culture. The induction of IL-6, CXCL1 and CXCL8 upon the interaction of bronchial epithelial cells with mast cells was mediated by MAPKs and NF-κB signaling pathways. These data indicate that the interaction of mast cells with bronchial epithelial cells may represent a crucial mechanism of regulating local inflammatory response in allergic asthma.     

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.