CARD4/Nod1 mediates NF-κB and JNK activation by invasive Shigella flexneri

Epithelial cells are refractory to extracellular lipopolysaccharide (LPS), yet when presented inside the cell, it is capable of initiating an inflammatory response. Using invasive Shigella flexneri to deliver LPS into the cytosol, we examined how this factor, once intracellular, activates both NF-κB and c-Jun N-terminal kinase (JNK). Surprisingly, the mode of activation is distinct from that induced by toll-like receptors (TLRs), which mediate LPS responsiveness from the outside-in. Instead, our findings demonstrate that this response is mediated by a cytosolic, plant disease resistance-like protein called CARD4/Nod1. Biochemical studies reveal enhanced oligomerization of CARD4 upon S. flexneri infection, an event necessary for NF-κB induction. Dominant-negative versions of CARD4 block activation of NF-κB and JNK by S. flexneri as well as microinjected LPS. Finally, we showed that invasive S. flexneri triggers the formation of a transient complex involving CARD4, RICK and the IKK complex. This study demonstrates that in addition to the extracellular LPS sensing system mediated by TLRs, mammalian cells also possess a cytoplasmic means of LPS detection via a molecule that is related to plant disease-resistance proteins.     

GEF-H1/RhoA signalling pathway mediates lipopolysaccharide-induced intercellular adhesion molecular-1 expression in endothelial cells via activation of p38 and NF-κB

The purpose of study is to investigate the effects of GEF-H1/RhoA pathway in regulating intercellular adhesion molecule-1 (ICAM-1) expression in lipopolysaccharide (LPS)-activated endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) to LPS induced GEF-H1 and ICAM-1 expression in dose- and time-dependent up-regulating manners. Pretreatment with Clostridium difficile toxin B-10463 (TcdB-10463), an inhibitor of Rho activity, reduced LPS-related phosphorylation of p65 at Ser 536 in a dose-dependent manner. Inhibition of TLR4 expression significantly blocked LPS-induced RhoA activity, NF-κB transactivation, GEF-H1 and ICAM-1 expression. Coimmunoprecipitation assay indicated that LPS-activated TLR4 and GEF-H1 formed a signalling complex, suggesting that LPS, acting through TLR4, stimulates GEF-H1 expression and RhoA activity, and thereby induces NF-κB transactivation and ICAM-1 gene expression. However, GEF-H1/RhoA regulates LPS-induced NF-κB transactivation and ICAM-1 expression in a MyD88-independent pathway because inhibition of MyD88 expression could not block LPS-induced RhoA activity. Furthermore, pretreatment with Y-27632, an inhibitor of ROCK, significantly reduced LPS-induced p38, ERK1/2 and p65 phosphorylation, indicating that ROCK acts as an upstream effector of p38 and ERK1/2 to promote LPS-induced NF-κB transactivation and ICAM-1 expression. What is more, the p38 inhibitor (SB203580) but not ERK1/2 inhibitor (PD98059) blocked LPS-induce NF-κB transactivation and ICAM-1 expression, which demonstrates that RhoA mediates LPS-induced NF-κB transactivation and ICAM-1 expression dominantly through p38 but not ERK1/2 activation. In summary, our data suggest that LPS-induced ICAM-1 synthesis in HUVECs is regulated by GEF-H1/RhoA-dependent signaling pathway via activation of p38 and NF-κB.     

NF-κB activation in myeloid cells mediates ventilator-induced lung injury

Background

Although use of the mechanical ventilator is a life-saving intervention, excessive tidal volumes will activate NF-κB in the lung with subsequent induction of lung edema formation, neutrophil infiltration and proinflammatory cytokine/chemokine release. The roles of NF-κB and IL-6 in ventilator-induced lung injury (VILI) remain widely debated.

Methods

To study the molecular mechanisms of the pathogenesis of VILI, mice with a deletion of IкB kinase in the myeloid cells (IKKβ^△mye), IL-6^-/- to WT chimeric mice, and C57BL/6 mice (WT) were placed on a ventilator for 6 hr.WT mice were also given an IL-6-blocking antibody to examine the role of IL-6 in VILI.

Results

Our results revealed that high tidal volume ventilation induced pulmonary capillary permeability, neutrophil sequestration, macrophage drifting as well as increased protein in bronchoalveolar lavage fluid (BALF). IL-6 production and IL-1β, CXCR2, and MIP2 expression were also increased in WT lungs but not in those pretreated with IL-6-blocking antibodies. Further, ventilator-induced protein concentrations and total cells in BALF, as well as lung permeability, were all significantly decreased in IKKβ^△mye mice as well as in IL6^-/- to WT chimeric mice.

Conclusion

Given that IKKβ^△mye mice demonstrated a significant decrease in ventilator-induced IL-6 production, we conclude that NF-κB–IL-6 signaling pathways induce inflammation, contributing to VILI, and IкB kinase in the myeloid cells mediates ventilator-induced IL-6 production, inflammation, and lung injury.     

Silencing of TNF-α receptors coordinately suppresses TNF-α expression through NF-κB activation blockade in THP-1 macrophage

Persistently elevated level of TNF-α has been implicated in several inflammatory disorders, however, its autocrine production through TNF-α receptors signaling is poorly understood. Here we report that simultaneous silencing of TNF-receptors, R1 and R2 by DNAzyme or siRNA suppressed TNF-α expression more efficiently than silencing them individually in lipopolysaccharides (LPS) stimulated THP-1 macrophages. Co-silencing of TNF-receptors also inhibited TNF-α induced NF-κB activation to a higher extent. It was further observed that NF-κB inhibitor but not c-Jun N-terminal kinase inhibitor (SP600125) suppressed TNF-α expression. All these results suggest that TNF-α expression is regulated by synergistic signaling of TNF receptors through downstream NF-κB activation.     

ARD1 binding to RIP1 mediates doxorubicin-induced NF-κB activation

NF-κB is activated by several cellular stresses. Of these, the TNFα-induced activation pathway has been examined in detail. It was recently reported that receptor-interacting protein 1 (RIP1) is involved in DNA damage-induced NF-κB activation by forming a complex with the p53 interacting death domain protein (PIDD) and NF-κB essential modulator (NEMO) in the nucleus, although the underlying mechanism of this interaction has yet to be clarified. This study shows that siRNA knock-down of arrest-defective 1 protein (ARD1) abrogated doxorubicin- but not TNFα-induced activation. Conversely, the over-expression of ARD1 greatly enhanced NF-κB activation induced by doxorubicin. Immunoprecipitation experiments revealed that ARD1 interacted with RIP1 via the acetyltransferase domain. Furthermore, the over-expression of several domain-deleted ARD1 constructs demonstrated that the N-terminal and acetyltransferase domains of ARD1 were required for doxorubicin-induced NF-κB activation. Treatment of deacetylase inhibitor, trichostatin A, significantly increased doxorubicin-induced NF-κB activation in the presence of ARD1 but not acetyltransferase-defective ARD1 mutant. Moreover, N-terminal domain-deleted ARD1 could not be localized in the nucleus in response to doxorubicin treatment. These data indicate that the interaction between ARD1 and RIP1 plays an important role in the DNA damage-induced NF-κB activation, and that the acetyltransferase activity of ARD1 and its localization in to the nucleus are involved in such stress response.     

Possible participation of intracellular platelet-activating factor in NF-κB activation in rat peritoneal macrophages

As we had found previously that thapsigargin, an endomembrane Ca²⁺-ATPase inhibitor, induces production of intracellular platelet-activating factor (PAF) [Br. J. Pharmacol. 116 (1995) 2141], we decided to investigate the possible roles of intracellular PAF in nuclear factor (NF)-κB activation of thapsigargin-stimulated rat peritoneal macrophages. When rat peritoneal macrophages were stimulated with thapsigargin, the level of inhibitory protein of NF-κB-α (IκB-α) was decreased and the nuclear translocation of NF-κB was increased. The thapsigargin-induced activation of NF-κB was inhibited by the PAF synthesis inhibitor SK&F 98625 and the PAF antagonist E6123. Structurally unrelated PAF antagonists such as E5880 and L-652,731 also inhibited the thapsigargin-induced activation of NF-κB. Lipopolysaccharide (LPS)-induced activation of NF-κB was also suppressed by these drugs. In a culture of rat peritoneal macrophages, exogenously added PAF did not induce degradation of IκB-α. These findings suggest that the intracellular PAF produced by the stimulation with thapsigargin or LPS is involved in activation of the NF-κB pathway.     

G-protein coupled receptor kinase 5 mediates lipopolysaccharide-induced NFκB activation in primary macrophages and modulates inflammation in vivo in mice

G-protein coupled receptor kinase-5 (GRK5) is a serine/threonine kinase discovered for its role in the regulation of G-protein coupled receptor signaling. Recent studies have shown that GRK5 is also an important regulator of signaling pathways stimulated by non-GPCRs. This study was undertaken to determine the physiological role of GRK5 in Toll-like receptor-4-induced inflammatory signaling pathways in vivo and in vitro. Using mice genetically deficient in GRK5 (GRK5(-/-) ) we demonstrate here that GRK5 is an important positive regulator of lipopolysaccharide (LPS, a TLR4 agonist)-induced inflammatory cytokine and chemokine production in vivo. Consistent with this role, LPS-induced neutrophil infiltration in the lungs (assessed by myeloperoxidase activity) was markedly attenuated in the GRK5(-/-) mice compared to the GRK5(+/+) mice. Similar to the in vivo studies, primary macrophages from GRK5(-/-) mice showed attenuated cytokine production in response to LPS. Our results also identify TLR4-induced NFκB pathway in macrophages to be selectively regulated by GRK5. LPS-induced IκBα phosphorylation, NFκB p65 nuclear translocation, and NFκB binding were markedly attenuated in GRK5(-/-) macrophages. Together, our findings demonstrate that GRK5 is a positive regulator of TLR4-induced IκBα-NFκB pathway as well as a key modulator of LPS-induced inflammatory response.     

Thiol modulation of TNFα and IL-1 induced MnSOD gene expression and activation of NF-κB

TNF and IL-1 each can activate NF-B and induce gene expression of manganese superoxide dismutase (MnSOD), a mitochondrial matrix enzyme which can provide critical protection against hyperoxic lung injury. The regulation of MnSOD gene expression is not well understood. Since redox status can modulate NF-B and potential B site(s) exist in the MnSOD promoter, the effect of thiols (including NAC, DTT and 2-ME) on TNF and IL-1 induced activation of NF-B and MnSOD gene expression was investigated. Activation of NF-kB and increased MnSOD expression were potentiated by thiol reducing agents. In contrast, thiol oxidizing or alkylating agents inhibited both NF-B activation and elevated MnSOD expression in response to TNF or IL-1. Since protease inhibitors TPCK and TLCK can inhibit NF-B activation, we also investigated the effect of these compounds on MnSOD expression and NF-B activation. TPCK and TLCK each inhibited MnSOD gene expression and NF-B activation. Since the MnSOD promoter also contains anAP-1 binding site, the effect of thiols and thiol modifying agents on AP-1 activation was investigated. Thiols had no consistent effect onAP-1 activation. Likewise, some of the thiol modifying compounds inhibited AP-1 activation by TNF or IL-1, whereas others did not. Since diverse agents had similar effects on activation of NF-B and MnSOD gene expression, we have demonstrated that activation of NF-B and MnSOD gene expression are closely associated and that reduced sulfhydryl groups are required for cytokine mediation of both processes.Abbreviations O₂ Superoxide radical - H₂O₂ Hydrogen peroxide - NAC N-acetyl L-cysteine - DTT Dithiothreitol - 2-ME 2-Mercaptoethanol - MnSOD Manganese superoxide dismutase - NF-B Nuclear factor kappa B - AP-1 Activator protein-1 - NBT Nitroblue tetrazolium - CAT Chloramphenicol acetyltransferase - TPCK N-tosyl-L-phenylalanine chloromethyl ketone - TLCK Na-p-tosyl-L-lysine chloromethyl ketone - TAME N--p-tosyl-L-arginine methyl ester - NEM N-ethyl maleimide - DEM Diethyl maleate - CDNB 1-chloro-2,4-dinitrobenzene - DTT_OX Oxidized dithiothreitol     

Blockade of CD38 diminishes lipopolysaccharide-induced macrophage classical activation and acute kidney injury involving NF-κB signaling suppression

The CD38, possessing ADP-ribosyl cyclase (ADPR-cyclase) and cyclic ADP-ribose hydrolase (cADPR-hydrolase), is able to regulate a variety of cellular activities. However, the role and mechanisms for CD38 in macrophage activation and sepsis-induced acute kidney injury (AKI) remain to be determined. Here we report that in cultured macrophages, Lipopolysaccharide (LPS) could upregulate CD38 expression in time and dose dependent manner. Knocking down or blockade of CD38 in macrophages could inhibit LPS-induced macrophage M1 polarization accompanied by diminished NF-κB signaling activation. In mouse model with LPS-induced acute kidney injury, blocking CD38 with quercetin could significantly relieve kidney dysfunction, kidney pathological changes as well as inflammatory cell accumulation. Similar to those in the cultured cells, quercetin could inhibit macrophage M1 polarization and NF-κB signaling activation in macrophages from kidneys and spleens in mice after LPS injection. Together, these results demonstrate that CD38 mediates LPS-induced macrophage activation and AKI, which may be treated as a therapeutic target for sepsis-induced AKI in patients.     

Aquaporin 5 increases keratinocyte-derived chemokine expression and NF-κB activity through ERK activation

Aquaporin-5 (AQP5) is a water-selective channel protein that is expressed in submucosal glands and alveolar epithelial cells in the lungs. Recent studies have revealed that AQPs regulate not only water metabolism, but also some cellular functions such as cell growth and migration. Here, we report the role of AQP5 in inflammatory responses. In MLE-12 cells, knockdown of AQP5 using siRNA (10–50 nM) attenuated TNF-α-induced expression of keratinocyte chemoattractant (KC) mRNA and protein. Conversely, in NIH-3T3 cells, overexpression of AQP5 increased KC expression, NF-κB activation, and ERK phosphorylation. The AQP5-induced increase of KC expression was diminished by treatment with ERK inhibitors. Taken together, we propose a new function of AQP5 as an inflammatory signal potentiator, which may be mediated by increased activation of ERK and NF-κB.     

Salicortin suppresses lipopolysaccharide-stimulated inflammatory responses via blockade of NF-κB and JNK activation in RAW 264.7 macrophages

We isolated the phenolic glucoside salicortin from a Populus euramericana bark extract, and examined its ability to suppress inflammatory responses as well as the molecular mechanisms underlying these abilities, using lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Salicortin inhibited iNOS expression and the subsequent production of NO in a dose-dependent manner in the LPS-stimulated RAW 264.7 cells. Salicortin significantly suppressed LPS-induced signal cascades of NF-κB activation, such as IKK activation, IκBα phosphorylation and p65 phosphorylation in RAW 264.7 cells. In addition, salicortin inhibited the LPS-induced activation of JNK, but not ERK or p38 MAPK. Furthermore, salicortin significantly inhibited production of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6 in the LPS-stimulated RAW 264.7 cells. These findings suggest that salicortin may show its anti-inflammatory activity by suppressing the LPS-induced expression of pro-inflammatory mediators through inhibition of NF-κB and JNK MAPK signaling cascades in macrophages. [BMB Reports 2014; 47(6): 318-323]     

Role of post-translational modifications of HTLV-1 Tax in NF-κB activation

Human T-cell leukemia virus type 1 (HTLV-1), the first human retrovirus discovered, is the etiological agent of adult-T-cell leukemia/lymphoma. The HTLV-1 encoded Tax protein is a potent oncoprotein that deregulates gene expression by constitutively activating nuclear factor-κB (NF-κB). Tax activation of NF-κB is critical for the immortalization and survival of HTLV-1-infected T cells. In this review, we summarize the present knowledge on mechanisms underlying Tax-mediated NF-κB activation, with an emphasis on post-translational modifications of Tax.     

MiRNA-891a-5p mediates HIV-1 Tat and KSHV Orf-K1 synergistic induction of angiogenesis by activating NF-κB signaling

Co-infection with HIV-1 and Kaposi''s sarcoma-associated herpesvirus (KSHV) is the cause of aggressive AIDS-related Kaposi''s sarcoma (AIDS-KS) characterized by abnormal angiogenesis. The impact of HIV-1 and KSHV interaction on the pathogenesis and extensive angiogenesis of AIDS-KS remains unclear. Here, we explored the synergistic effect of HIV-1 Tat and KSHV oncogene Orf-K1 on angiogenesis. Our results showed that soluble Tat or ectopic expression of Tat enhanced K1-induced cell proliferation, microtubule formation and angiogenesis in chorioallantoic membrane and nude mice models. Mechanistic studies revealed that Tat promoted K1-induced angiogenesis by enhancing NF-κB signaling. Mechanistically, we showed that Tat synergized with K1 to induce the expression of miR-891a-5p, which directly targeted IκBα 3′ untranslated region, leading to NF-κB activation. Consequently, inhibition of miR-891a-5p increased IκBα level, prevented nuclear translocation of NF-κB p65 and ultimately suppressed the synergistic effect of Tat- and K1-induced angiogenesis. Our results illustrate that, by targeting IκBα to activate the NF-κB pathway, miR-891a-5p mediates Tat and K1 synergistic induction of angiogenesis. Therefore, the miR-891a-5p/NF-κB pathway is important in the pathogenesis of AIDS-KS, which could be an attractive therapeutic target for AIDS-KS.     

BRCA-1 depletion impairs pro-inflammatory polarization and activation of RAW 264.7 macrophages in a NF-κB-dependent mechanism

Molecular and Cellular Biochemistry - Inadequate migration and invasion of the trophoblast cells during embryo implantation is one of the reasons for pregnancy-related complications such as...