Poly I:C enhances production of nitric oxide in response to interferon-γ via upregulation of interferon regulatory factor 7 in vascular endothelial cells.

The effect of poly I:C on interferon (IFN)-γ-induced nitric oxide (NO) production in vascular endothelial cells was examined using murine aortic endothelial END-D cells. Poly I:C augmented IFN-γ-induced NO production although it alone did not induce the NO production. Poly I:C augmented the NO production via enhanced expression of an inducible NO synthase protein. Poly I:C did not affect the activation of Janus kinase (JAK) 1/2, and signal transducer and activator of transcription (STAT) 1 in IFN-γ signaling. Moreover, there was no significant difference in the IFN-γ-induced interferon regulatory factor (IRF) 1 expression between the presence and absence of poly I:C. Poly I:C led to the activation of IRF7 in END-D cells. Inhibition of poly I:C signaling by amlexanox, an inhibitor of TANK-binding kinase (TBK) 1 and IκB kinase (IKK) ε, abolished the augmentation of IFN-γ-induced NO production. Therefore, poly I:C was suggested to augment IFN-γ-induced NO production at the transcriptional level via enhanced IRF7 activation.

A Toll-like receptor 2 ligand, Pam3CSK4, augments interferon-γ-induced nitric oxide production via a physical association between MyD88 and interferon-γ receptor in vascular endothelial cells.

The 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.

Lipopolysaccharide prevents valproic acid-induced apoptosis via activation of nuclear factor-κB and inhibition of p53 activation.

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.

Sendai virus C protein limits NO production in infected RAW264.7 macrophages.

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-ß, which is involved in induction of iNOS expression via the JAK-STAT pathway. Addition of anti-IFN-ß 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.

Involvement of redox balance in in vitro osteoclast formation of RAW 264.7 macrophage cells in response to LPS.

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.

A novel mechanism for inhibition of lipopolysaccharide-induced proinflammatory cytokine production by valproic acid.

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.

Augmentation of LPS-induced vascular endothelial cell growth factor production in macrophages by transforming growth factor-ß1.

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)-ß1 augmented LPS-induced VEGF production, although TGF-ß1 alone did not induce VEGF production. The augmentation of LPS-induced VEGF production by TGF-ß1 was inhibited by a p38 MAPK inhibitor and was correlated with the phosphorylation of Smad3. The enhancing effect of TGF-ß1 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-ß1 in vitro and in vivo.

Mouse pyrin and HIN domain family member 1 (pyhin1) protein positively regulates LPS-induced IFN-ß and NO production in macrophages.

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-ß and NO. However, pyhin1 silencing did not affect the production of TNF-α, IL-6, IL-10 and prostaglandin E2. Reduced IFN-ß 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-ß 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-ß and NO production through up-regulating the MyD88-independent signaling pathway.

Involvement of oncogenic protein ß-catenin in LPS-induced cytotoxicity in mouse mononuclear leukemia RAW 264.7 cells.

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, ß-catenin, is positively involved in LPS-induced cytotoxicity in a mouse leukemic monocyte cell line, RAW 264.7, was examined. TWS119, a GSK-3ß inhibitor, increased LPS-induced ß-catenin accumulation in the nucleus and augmented LPS-induced cytotoxicity. Cardamonin, a ß-catenin inhibitor, inhibited LPS-induced ß-catenin accumulation in the nucleus and reduced LPS-induced cytotoxicity. To confirm that ß-catenin is involved in LPS-induced cytotoxicity, silencing of ß-catenin expression by siRNA was carried out. The results were that knockdown of ß-catenin reduced LPS-induced cytotoxicity. Interestingly, Cardamonin treatment or ß-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 ß-catenin is considered to be positively involved in LPS-induced cytotoxicity, possibly by downregulating ER stress responses.

Inhibition of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation by pyrroloquinoline quinine (PQQ).

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-ß-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-ß signaling due to augmented IFNAR expression.