To suppress virus multiplication, infected macrophages produce NO. However, it remains unclear how infecting viruses then overcome NO challenge. In the present study, we report the effects of accessory protein C from Sendai virus (SeV), a prototypical paramyxovirus, on NO output. We found that in RAW264.7 murine macrophages, a mutant SeV without C protein (4C(–)) significantly enhanced inducible NO synthase (iNOS) expression and subsequent NO production compared to wild type SeV (wtSeV). SeV 4C(-) infection caused marked production of IFN-β, which is involved in induction of iNOS expression via the JAK-STAT pathway. Addition of anti-IFN-β Ab, however, resulted in only marginal suppression of NO production. In contrast, NF-κB, a primarily important factor for transcription of the iNOS gene, was also activated by 4C(–) infection but not wtSeV infection. Induction of NO production and iNOS expression by 4C(–) was significantly suppressed in cells constitutively expressing influenza virus NS1 protein that can sequester double-stranded (ds)RNA, which triggers activation of signaling pathways leading to activation of NF-κB and IRF3. Therefore, C protein appears to suppress NF-κB activation to inhibit iNOS expression and subsequent NO production, possibly by limiting dsRNA generation in the context of viral infection.

Here we report that LPS induces osteoclast (OC) formation in murine RAW 264.7 macrophage cells in RPMI-1640 medium but not in α-minimum essential medium (α-MEM) as the original culture medium. LPS-induced OC formation in both media was examined to clarify the differential response. Receptor activator of NF-κB ligand induced OC formation in either α-MEM or RPMI-1640 medium. However, LPS-induced OC formation in RAW 264.7 cells maintained in RPMI-1640 medium, but not α-MEM, which was also supported by mouse bone marrow-derived macrophages, although they were less sensitive to LPS than RAW 264.7 cells. LPS augmented the expression of nuclear factor of activated T-cells (NFATc1) as a key transcription factor of osteoclastogenesis in cells maintained in RPMI-1640 medium, but reduced it in cells maintained in α-MEM. A high concentration of LPS was cytotoxic against cells maintained in α-MEM. Glutathione exclusively present in RPMI-1640 medium prevented LPS-induced cell death in α-MEM and augmented LPS-induced NFATc1 expression, followed by enhanced LPS-induced OC formation. LPS induced higher generation of reactive oxygen species in α-MEM than RPMI-1640 medium. An antioxidant enhanced LPS-induced OC formation, whereas a pro-oxidant reduced it. Taken together, redox balance in the culture condition was suggested to regulate in vitro LPS-induced OC formation.

The effect of TGF-β1 on CpG DNA-induced type I IFN production was examined by reconstituting a series of signaling molecules in TLR 3 signaling. TGF-β1 inhibited CpG DNA-induced IFN-α4 productivity in HeLa cells. Transfection of IFN regulatory factor (IRF)7 but not TNF receptor-associated factor (TRAF)6 and TRAF3 into cells triggered IFN-α4 productivity, and TGF-β1 inhibited IRF7-mediated type I IFN production in the presence of TRAF6. TGF-β1 induced ubiquitination of TRAF6, although CpG DNA did not induce it. Moreover, TGF-β1 accelerated the ubiquitination of TRAF6 in the presence of CpG DNA. TGF-β1 ubiquitinated TRAF6 at K63 but not K48. TGF-β1 also induced ubiquitination of IRF7. Further, TGF-β1 did not impair the interaction of IRF7 and TRAF6. CpG DNA induced the phosphorylation of IRF7 in the presence of TRAF6, whereas TGF-β1 inhibited the IRF7 phosphorylation. Blocking of TRAF6 ubiquitination abolished the inhibition of CpG DNA-induced type I IFN production by TGF-β. Taken together, TGF-β was suggested to inhibit CpG DNA-induced type I IFN production transcriptionally via ubiquitination of TRAF6.

The inhibitory effect of valproic acid (VPA) on lipopolysaccharide (LPS)-induced inflammatory response was studied by using mouse RAW 264.7 macrophage-like cells. VPA pretreatment attenuated LPS-induced phosphorylation of phosphatidylinositol 3-kinase (PI3K) and Akt, but not nuclear factor (NF)-κB and mitogen-activated protein kinases. VPA reduced phosphorylation of MDM2, an ubiquitin ligase and then prevented LPS-induced p53 degradation, followed by enhanced p53 expression. Moreover, p53 small interfering RNA (siRNA) abolished the inhibitory action of VPA on LPS-induced NF-κB p65 transcriptional activation and further LPS-induced tumor necrosis factor (TNF)-α and interleukin (IL)-6 production. VPA prevented LPS-induced degradation of phosphatase and tensin homologue deleted on chromosome ten (PTEN) and up-regulated the PTEN expression. Taken together, VPA was suggested to down-regulate LPS-induced NF-κB-dependent transcriptional activity via impaired PI3K/Akt/MDM2 activation and enhanced p53 expression. A detailed mechanism for inhibition of LPS-induced inflammatory response by VPA is discussed.

The effect of lipopolysaccharide (LPS) on insulin sensitivity in adipocytes were examined by using differentiated 3T3-L1 adipocytes. Insulin-mediated activation of insulin receptor substrate (IRS) 1/2 was inhibited in LPS-pretreated adipocytes and IRS1/2-mediated Akt activation was also attenuated in those cells. LPS inhibited activation of glycogen synthase kinase 3 as a negative regulator of glycogenesis and impaired the glycogen synthesis in response to insulin. LPS-induced activation of phosphoinositide 3-kinase (PI3K) in adipocytes. Involvement of suppressor of cytokine signaling 3 (SOCS3) in LPS-induced IRS1/2 inhibition was excluded. Considering that both insulin and LPS were able to activate the PI3K/Akt signaling pathway, LPS was suggested to impair insulin sensitivity of adipocytes through down-regulating insulin-mediated PI3K/Akt activation.

The effect of LPS on the production of vascular endothelial growth factor (VEGF) was examined using RAW 264.7 macrophage cells. LPS induced VEGF production in RAW 264.7 cells and mouse peritoneal cells. LPS induced VEGF production via the expression of hypoxia inducible factor-1α and LPS-induced VEGF production was dependent on the activation of p38 MAPK and NF-κB activation· Transforming growth factor (TGF)-β1 augmented LPS-induced VEGF production, although TGF-β1 alone did not induce VEGF production. The augmentation of LPS-induced VEGF production by TGF-β1 was inhibited by a p38 MAPK inhibitor and was correlated with the phosphorylation of Smad3. The enhancing effect of TGF-β1 on LPS-induced VEGF production was observed in vivo in the skin lesions of mice receiving a subcutaneous injection of LPS. Taken together, it is suggested that LPS induced the VEGF production in macrophages and that it was augmented by TGF-β1 in vitro and in vivo.

The effect of lipopolysaccharide (LPS) on valproic acid (VPA)-induced cell death was examined by using mouse RAW 264.7 macrophage cells. LPS inhibited the activation of caspase 3 and poly (ADP-ribose) polymerase and prevented VPA-induced apoptosis. LPS inhibited VPA-induced p53 activation and pifithrin-α as a p53 inhibitor as well as LPS prevented VPA-induced apoptosis. LPS abolished the increase of Bax/Bcl-2 ratio, which is a critical indicator of p53-mediated mitochondrial damage, in response to VPA. The nuclear factor (NF)-κB inhibitors, Bay 11-7082 and parthenolide, abolished the preventive action of LPS on VPA-induced apoptosis. A series of toll-like receptor ligands, Pam3CSK4, poly I:C, and CpG DNA as well as LPS prevented VPA-induced apoptosis. Taken together, LPS was suggested to prevent VPA-induced apoptosis via activation of anti-apoptotic NF-κB and inhibition of pro-apoptotic p53 activation. The detailed inhibitory mechanism of VPA-induced apoptosis by LPS is discussed.

The pyrin and HIN-domain (PYHIN) family member1 (pyhin1) is a member of PYHIN proteins and involved in transcriptional regulation of genes important for cell cycle control, differentiation and apoptosis. The regulatory action of mouse pyhin1 on LPS-induced inflammatory response was examined. LPS augmented the pyhin1 mRNA expression in murine RAW 264.7 macrophage cells and peritoneal macrophages. The augmentation of pyhin1 mRNA expression was abolished by parthenolide, a NF-κB inhibitor. Silencing of pyhin1 with small interfering RNA reduced the production of IFN-β and NO. However, pyhin1 silencing did not affect the production of TNF-α, IL-6, IL-10 and prostaglandin E2. Reduced IFN-β production by pyhin1 silencing caused inactivation of STAT1 and reduced expression of IRF1. Pyhin1 silencing inhibited the expression of TRAF6, TBK1 and TRIF, which trigger IFN-β production in the MyD88-independent pathway. However, pyhin1 silencing did not affect the expression of MyD88, IRAK4 and several mitogen-activated protein kinases in the MyD88-dependent pathway. Taken together, mouse pyhin1 was suggested to be a NF-κB-responsible gene in response to LPS and positively regulate LPS-induced IFN-β and NO production through up-regulating the MyD88-independent signaling pathway.

SummaryThe effect of Pam3CSK4, a Toll‐like receptor 2 (TLR2) ligand, on interferon‐γ (IFN‐γ) ‐induced nitric oxide (NO) production in mouse vascular endothelial END‐D cells was studied. Pre‐treatment or post‐treatment with Pam3CSK4 augmented IFN‐γ‐induced NO production via enhanced expression of an inducible NO synthase (iNOS) protein and mRNA. Pam3CSK4 augmented phosphorylation of Janus kinase 1 and 2, followed by enhanced phosphorylation of signal transducer and activator of transcription 1 (STAT1) at tyrosine 701. Subsequently, the enhanced STAT1 activation augmented IFN‐γ‐induced IFN‐regulatory factor 1 expression leading to the iNOS expression. Pam3CSK4 also induced the activation of p38 and subsequent phosphorylation of STAT1 at serine 727. A pharmacological p38 inhibitor abolished the augmentation of IFN‐γ‐induced NO production by Pam3CSK4. Surprisingly, Pam3CSK4 enhanced a physical association of MyD88 and IFN‐γ receptor. Together, these findings suggest that Pam3CSK4 up‐regulates IFN‐γ signalling in vascular endothelial cells via the physical association between MyD88 and IFN‐γ receptor α, and p38‐dependent serine 727 STAT1 phosphorylation.

A toll-like receptor 4 (TLR-4) ligand, lipopolysaccharide (LPS) not only activates expression and secretion of inflammatory cytokines, but it also often shows toxicity in monocytes. Whether an oncogenic protein, β-catenin, is positively involved in LPS-induced cytotoxicity in a mouse leukemic monocyte cell line, RAW 264.7, was examined. TWS119, a GSK-3β inhibitor, increased LPS-induced β-catenin accumulation in the nucleus and augmented LPS-induced cytotoxicity. Cardamonin, a β-catenin inhibitor, inhibited LPS-induced β-catenin accumulation in the nucleus and reduced LPS-induced cytotoxicity. To confirm that β-catenin is involved in LPS-induced cytotoxicity, silencing of β-catenin expression by siRNA was carried out. The results were that knockdown of β-catenin reduced LPS-induced cytotoxicity. Interestingly, Cardamonin treatment or β-catenin silencing reduced LPS-induced endoplasmic reticulum (ER) stress responses such as PERK and e1F-2α phosphorylation and CHOP expression. Moreover, TWS119 increased LPS-induced ER stress responses. On the basis of these results, the oncogenic protein β-catenin is considered to be positively involved in LPS-induced cytotoxicity, possibly by downregulating ER stress responses.

An ADP ribosylation factor-GTPase activating protein (ASAP1) is highly expressed in a variety of tumor cells and is involved in the cell motility, invasion, and metastasis. In order to elucidate the involvement of ASAP1 in lipopolysaccharide (LPS)-mediated inflammatory response, the effect of ASAP1 silencing on LPS-induced proinflammatory mediators production was examined by using RAW 264.7 macrophage-like cells. ASAP1 was constitutively expressed in the cells and the expression was augmented by LPS stimulation. Silencing of ASAP1 with small interfering RNA enhanced the production of tumor necrosis factor-α, interleukin 6, interferon-β, and nitric oxide in response to LPS. ASAP1 silencing augmented the activation of nuclear factor (NF)-κB and several mitogen-activated protein kinases (MAPKs). On the other hand, ASAP1 silencing did not affect the expression of IRAK4, TRAF6, and Akt as the upstream molecules of NF-κB signaling. A series of toll-like receptor ligands as well as LPS augmented the ASAP1 expression. Taken together, ASAP1 was suggested to negatively regulate LPS-induced proinflammatory mediators production through down-regulating LPS signaling. The feedback function of ASAP1 in LPS-mediated inflammatory response is discussed.

The effect of pyrroloquinoline quinine (PQQ) on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation was examined using RAW 264.7 macrophage-like cells. RANKL led to the formation of osteoclasts identified as tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in the culture of RAW 264.7 cells. However, PQQ inhibited the appearance of osteoclasts and prevented the decrease of F4/80 macrophage maturation marker on RANKL-stimulated cells, suggesting a preventive action of PQQ on RANKL-induced osteoclast differentiation. PQQ inhibited the activation of nuclear factor of activated T cells (NFATc1), a key transcription factor of osteoclastogenesis, in RANKL-stimulated cells. On the other hand, PQQ did not inhibit the signaling pathway from RANK/RANKL binding to NFATc1 activation, including NF-κB and mitogen-activated protein kinases (MAPKs). PQQ augmented the expression of type I interferon receptor (IFNAR) and enhanced the IFN-β-mediated janus kinase (JAK1) and signal transducer and activator of transcription (STAT1) expression. Moreover, PQQ reduced the expression level of c-Fos leading to the activation of NFATc1. Taken together, PQQ was suggested to prevent RANKL-induced osteoclast formation via the inactivation of NFATc1 by reduced c-Fos expression. The reduced c-Fos expression might be mediated by the enhanced IFN-β signaling due to augmented IFNAR expression.

Thalidomide is known as an anti-angiogenic, anti-tumor, and anti-proliferative agent, widely used in the treatment of some immunological disorders and cancers. The effect of thalidomide on interferon (IFN)-γ induced nitric oxide (NO) production in mouse vascular endothelial cells was examined in order to elucidate the anti-angiogenic or anti-inflammatory action. Thalidomide inhibited IFN-γ-induced NO production in mouse END-D cells via reduced expression of an inducible type of NO synthase (iNOS) protein and mRNA. Since thalidomide did not alter the cell surface expression of IFN-γ receptor, the NO inhibition was suggested to be due to the impairment of IFN-γ-induced intracellular event by thalidomide. Thalidomide inhibited the phosphorylation of IRF1, which was required for the iNOS expression. Moreover, it inhibited the phosphorylation of STAT1, an upstream molecule of IRF1, in IFN-γ signaling. Thalidomide did not inhibit the JAK activation in response to IFN-γ. A phosphatase inhibitor, sodium orthovanadate, abolished the inhibitory action of thalidomide. Therefore, thalidomide was suggested to inhibit IFN-γ-induced NO production via impaired STAT1 phosphorylation.

Flavopiridol is a cyclin-dependent kinase inhibitor and inhibits the growth of various cancer cells. The effect of flavopiridol on lipopolysaccharide (LPS)-induced proinflammatory mediator production was examined in RAW 264.7 macrophage-like cells. Flavopiridol significantly reduced the production of tumor necrosis factor-α and, to a lesser extent, nitric oxide in LPS-stimulated cells. Flavopiridol inhibited the activation of nuclear factor-κB and IκB kinase in response to LPS. Flavopiridol also inhibited the activation of a series of mitogen-activated protein kinases, such as p38, stress-activated protein kinase/c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in response to LPS. However, flavopiridol did not alter the expression of tumor necrosis factor receptor-associated factor 6, myeloid differentiation factor 88 (MyD88) or CD14/toll-like receptor (TLR) 4. Flavopiridol inhibited nitric oxide production induced by a MyD88-dependent TLR2 ligand, but not a MyD88-independent TLR3 ligand. Further, flavopiridol did not alter the phosphorylation of interferon regulatory factor 3 in the MyD88-independent pathway. Therefore, it was suggested that flavopiridol exclusively inhibited the activation of nuclear factor-κB and mitogen-activated protein kinases in the MyD88-dependent pathway. Flavopiridol might be useful for the prevention of LPS-induced inflammatory response.

The involvement of retinoblastoma protein-interacting zinc finger 1 (RIZ1), a tumor suppressor, in lipopolysaccharide (LPS)-induced inflammatory responses was investigated by using RAW 264.7 macrophage-like cells. LPS significantly augmented the expression of RIZ1 and the augmentation was mediated by the activation of nuclear factor (NF)-κB and Akt. The silencing of RIZ1 with the siRNA led to the inactivation of NF-κB in response to LPS. Moreover, the RIZ1 silencing caused the down-regulation of p53 activation and a p53 pharmacological inhibitor attenuated the RIZ1 expression. LPS-induced tumor necrosis factor-α and interleukin-6 production was prevented by RIZ1 siRNA or a p53 pharmacological inhibitor. Therefore, RIZ1 was suggested to augment LPS-induced NF-κB activation in collaboration with p53 and enhance the production of proinflammatory cytokines in response to LPS.

The role of retinoblastoma protein-interacting zinc finger 1 (RIZ1) on the cell growth of mouse and human monocytic leukemia cells was examined. RIZ1 expression was induced in response to tumor necrosis factor (TNF)-α. The expression was dependent on the nuclear factor-κB and AKT signaling. Further, RIZ1 expression led to the augmentation of p53 expression and the silencing of RIZ1 prevented it. On the other hand, a p53 inhibitor enhanced the TNF-α-induced RIZ1 expression. Silencing of RIZ1 augmented the proliferative activity of TNF-α-treated cells. Therefore, it is suggested that RIZ1 negatively regulated the cell proliferation of monocytic leukemia cells via activation of p53.

Selective Alzheimer disease indicator-1 (seladin-1) is a broadly expressed oxidoreductase and is related to Alzheimer disease, cholesterol metabolism and carcinogenesis. The effect of lipopolysaccharide (LPS) on the expression of seladin-1 was examined using RAW 264.7 macrophage-like cells and murine peritoneal macrophages. Lipopolysaccharide induced the expression of seladin-1 protein and messenger RNA in those macrophages. The seladin-1 expression was also augmented by a series of Toll-like receptor ligands. The LPS augmented the expression of seladin-1 via reactive oxygen species generation and p38 activation. Seladin-1 inhibited LPS-induced activation of p38 but not nuclear factor-kappaB and inhibited the production of tumour necrosis factor-alpha in response to LPS. Moreover, seladin-1 inhibited LPS-induced osteoclast formation and enhanced LPS-induced alkaline phosphatase activity. Therefore, it was suggested that seladin-1 might be an LPS-responsible gene product and regulate the LPS-induced inflammatory response negatively.

The LPS-mediated lethality of NC/Nga mice, having fewer NKT cells, was examined by using d-galactosamine (d-GalN)-sensitization. The NC/Nga mice were not killed by a simultaneous administration of d-GalN and LPS whereas all C57BL/6 (B6) control mice were killed. The injection of d-GalN and LPS failed to elevate the levels of serum alanine aminotransferase and caspase 3 in the liver tissues of NC/Nga mice. Further, the nitric oxide (NO) level of the d-GalN- and LPS-injected NC/Nga mice was much lower than those of the B6 mice. The expression of an inducible NO synthase (iNOS) was significantly reduced in the livers of NC/Nga mice. However, there was no significant difference in LPS-induced TNF-α production between B6 mice and NC/Nga mice. The NC/Nga mice had an impaired expression of IFN-γ protein and mRNA in response to d-GalN and LPS. The pretreatment with α-galactosylceramide (α-GalCer), which activates Vα14+ NKT cells and induces the production of IFN-γ, rendered NC/Nga mice more susceptible to the LPS-mediated lethality. The livers of NC/Nga mice had fewer NKT cells compared to B6 mice. Taken together, it is suggested that the resistance of NC/Nga mice to the LPS-mediated lethality with d-GalN sensitization depended on the impaired IFN-γ production caused by fewer NKT cells and reduced NO production that followed.