Sendai virus C protein affects macrophage function, which plays a critical role in modulating disease severity during Sendai virus infection in mice.

Sendai virus (SeV) accessory protein C limits the generation of double-stranded RNAs, defective interfering RNAs, or both, during viral transcription and replication, thereby limiting interferon-ß production. Our recent in vitro analyses on murine macrophage cell lines demonstrated that this protein also contributes to restricting macrophage function, including the production of nitric oxide (NO) and inflammatory cytokines in addition to interferon-ß, in infected macrophages. This study showed that depletion of airway macrophages by clodronate-loaded liposomes led to the development of severe viral pneumonia in recombinant C gene-knockout SeV (SeV∆C)-infected mice, but did not modulate disease severity in wild-type SeV-infected mice. Furthermore, the severe disease observed in macrophage-depleted, SeV∆C-infected mice was associated with exacerbated virus replication in the lungs, leading to severe airway inflammation and pulmonary edema, indicating lung injury. These results suggested that the antimacrophage activity of SeV C protein might play a critical role in modulating lung injury and associated diseases caused by SeV.

Cellular Anti-Inflammatory and Antioxidant Activities of Bamboo Sasa albomarginata Leaf Extract and Its Constituent Coumaric Acid Methyl Ester.

Background: Hot water extract of Sasa albomarginata (Kumazasa) leaves is commercially available and used as a dietary supplement or skincare cream. The extract possesses anti-inflammatory activity on the mouse atopic dermatitis model. To elucidate the mechanism of in vivo activity, we have studied the cellular anti-inflammatory and antioxidant activities of the extract and its constituents. Methods: Secretion of mouse and human IL-6 was measured by ELISA. ROS production was measured by a fluorescent reagent. Ultrahigh performance liquid chromatography (UHPLC)/MS was used for the ingredient analysis. Results: The Sasa albomarginata extract inhibited inflammatory mediators such as LPS-induced NO, IL-6, and ROS production in mouse monocyte leukemia RAW264.7 cells. It also inhibited iNOS, IL-6, and IL-1ß expressions. Moreover, it inhibited LPS-induced IL-6 expression and production in human monocyte leukemia THP-1 cells differentiated into macrophages. The HPLC analysis of the extract revealed the existence of coumaric acid, ferulic acid, and coumaric acid methyl ester. Coumaric acid methyl ester but not coumaric acid or ferulic acid inhibited LPS-induced NO, IL-6, and ROS production in RAW264.7 cells and IL-6 production in differentiated THP-1 cells. Conclusion: The hot water extract of Sasa albomarginata leaves and one of its constituents possess cellular anti-inflammatory and antioxidant activities.

Sendai Virus V Protein Inhibits the Secretion of Interleukin-1ß by Preventing NLRP3 Inflammasome Assembly.

Inflammasomes play a key role in host innate immune responses to viral infection by caspase-1 (Casp-1) activation to facilitate interleukin-1ß (IL-1ß) secretion, which contributes to the host antiviral defense. The NLRP3 inflammasome consists of the cytoplasmic sensor molecule NLRP3, adaptor protein ASC, and effector protein pro-caspase-1 (pro-Casp-1). NLRP3 and ASC promote pro-Casp-1 cleavage, leading to IL-1ß maturation and secretion. However, as a countermeasure, viral pathogens have evolved virulence factors to antagonize inflammasome pathways. Here we report that V gene knockout Sendai virus [SeV V(-)] induced markedly greater amounts of IL-1ß than wild-type SeV in infected THP1 macrophages. Deficiency of NLRP3 in cells inhibited SeV V(-)-induced IL-1ß secretion, indicating an essential role for NLRP3 in SeV V(-)-induced IL-1ß activation. Moreover, SeV V protein inhibited the assembly of NLRP3 inflammasomes, including NLRP3-dependent ASC oligomerization, NLRP3-ASC association, NLRP3 self-oligomerization, and intermolecular interactions between NLRP3 molecules. Furthermore, a high correlation between the NLRP3-binding capacity of V protein and the ability to block inflammasome complex assembly was observed. Therefore, SeV V protein likely inhibits NLRP3 self-oligomerization by interacting with NLRP3 and inhibiting subsequent recruitment of ASC to block NLRP3-dependent ASC oligomerization, in turn blocking full activation of the NLRP3 inflammasome and thus blocking IL-1ß secretion. Notably, the inhibitory action of SeV V protein on NLRP3 inflammasome activation is shared by other paramyxovirus V proteins, such as Nipah virus and human parainfluenza virus type 2. We thus reveal a mechanism by which paramyxovirus inhibits inflammatory responses by inhibiting NLRP3 inflammasome complex assembly and IL-1ß activation.IMPORTANCE The present study demonstrates that the V protein of SeV, Nipah virus, and human parainfluenza virus type 2 interacts with NLRP3 to inhibit NLRP3 inflammasome activation, potentially suggesting a novel strategy by which viruses evade the host innate immune response. As all members of the Paramyxovirinae subfamily carry similar V genes, this new finding may also lead to identification of novel therapeutic targets for paramyxovirus infection and related diseases.

Inhibition of Late and Early Phases of Cancer Metastasis by the NF-κB Inhibitor DHMEQ Derived from Microbial Bioactive Metabolite Epoxyquinomicin: A Review.

We previously designed and synthesized dehydroxyepoxyquinomicin (DHMEQ) as an inhibitor of NF-κB based on the structure of microbial secondary metabolite epoxyquinomicin C. DHMEQ showed anti-inflammatory and anticancer activity in various in vivo disease models without toxicity. On the other hand, the process of cancer metastasis consists of cell detachment from the primary tumor, invasion, transportation by blood or lymphatic vessels, invasion, attachment, and formation of secondary tumor. Cell detachment from the primary tumor and subsequent invasion are considered to be early phases of metastasis, while tumor cell attachment to the tissue and secondary tumor formation the late phases. The assay system for the latter phase was set up with intra-portal-vein injection of pancreatic cancer cells. Intraperitoneal administration of DHMEQ was found to inhibit liver metastasis possibly by decreasing the expression of MMP-9 and IL-8. Also, when the pancreatic cancer cells treated with DHMEQ were inoculated into the peritoneal cavity of mice, the metastatic foci formation was inhibited. These results indicate that DHMEQ is likely to inhibit the late phase of metastasis. Meanwhile, we have recently employed three-dimensional (3D) culture of breast cancer cells for the model of early phase metastasis, since the 3D invasion just includes cell detachment and invasion into the matrix. DHMEQ inhibited the 3D invasion of breast cancer cells at 3D-nontoxic concentrations. In this way, DHMEQ was shown to inhibit the late and early phases of metastasis. Thus, DHMEQ is likely to be useful for the suppression of cancer metastasis.

Rational design, synthesis and in vitro evaluation of novel exo-methylene butyrolactone salicyloylamide as NF-κB inhibitor.

(-)-Dehydroxymethylepoxyquinomicin ((-)-DHMEQ, 1) is a specific inhibitor of NF-κB. It binds to SH group in the specific cysteine residue of NF-κB components with its epoxide moiety to inhibit DNA binding. In the present research, we have designed and synthesized an epoxide-free analog called (S)-ß-salicyloylamino-α-exo-methylene-Æ´-butyrolactone (SEMBL, 3). SEMBL inhibited DNA binding of NF-κB component p65 in vitro. It inhibited LPS-induced NF-κB activation, iNOS expression, and inflammatory cytokine secretions. It also inhibited NF-κB and cellular invasion in ovarian carcinoma ES-2 cells. Moreover, its stability in aqueous solution was greatly enhanced compared with (-)-DHMEQ. Thus, SEMBL has a potential to be a candidate for a new anti-inflammatory and anticancer agent.

GSK2656157, a PERK inhibitor, reduced LPS-induced IL-1ß production through inhibiting Caspase 1 activation in macrophage-like J774.1 cells.

IL-1ß is one of the inflammatory cytokines and is cleaved from pro-IL-1ß proteolytically by activated Caspase 1. For the activation of Caspase 1, inflammasome was formed by two signals, what is called, priming and triggering signals. In this study, it was found that mouse macrophage J774.1 cells, when treated by single large amount of lipopolysaccharide (LPS), produced a significant amount of IL-1ß. On the other hand, IL-1ß production was not detected when treated by a single, small amount of LPS. Then, focusing on endoplasmic reticulum (ER) stress response among stress responses induced by a large amount of LPS, when GSK2656157, a PERK inhibitor, was used for inhibition of ER stress, GSK2656157 reduced IL-1ß production dose-dependently. Next, when Thapsigargin, an ER stress reagent, was added with LPS, IL-1ß production increased more than by LPS alone. Thus, these results suggested that ER stress was involved in LPS-induced IL-1ß production. When the activation of Caspase 1 was examined by fluorescence activated cell sorter analysis, it was found that GSK2656157 inhibited LPS-induced Caspase 1 activation. Further, it was confirmed that GSK2656157 did not affect LPS-induced TNF-α production and activation of NF-κB and specifically inhibited the PERK/eIF-2α pathway. Therefore, it was found that GSK2656157 specifically inhibited ER stress induced by large amount of LPS and reduced LPS-induced IL-1ß production through inhibition of Caspase 1 activation.

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.

Spironolactone inhibits production of proinflammatory mediators in response to lipopolysaccharide via inactivation of nuclear factor-κB.

The effect of spironolactone (SPIR) on lipopolysaccharide (LPS)-induced production of proinflammatory mediators was examined using RAW 264.7 macrophage-like cells and mouse peritoneal macrophages. SPIR significantly inhibited LPS-induced production of nitric oxide (NO), tumor necrosis factor-α and prostaglandin E2. The inhibition was not mediated by cell death. SPIR reduced the expression of an inducible NO synthase mRNA in response to LPS. SPIR significantly inhibited phosphorylation of p65 nuclear factor (NF)-κB in response to LPS. Furthermore, SPIR inhibited phosphorylation of IκB kinase (IKK) as an upstream molecule of NF-κB in response to LPS. LPS did not induce the production of aldosterone in RAW 264.7 cells. Taken together, SPIR is suggested to inhibit LPS-induced proinflammatory mediators via inactivation of IKK/NF-κB in LPS signaling.

Lipopolysaccharide impairs insulin sensitivity via activation of phosphoinositide 3-kinase in adipocytes.

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.

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.

Phonon mode of TiO2 coupled with the electron transfer from N3 dye.

Low frequency vibrational spectra of submonolayer N3 dye (Ru(4,4(')-dicarboxy-2,2(')-bipyridine)2(NCS)2) adsorbed on TiO2 (110) were reported by using fourth-order coherent Raman spectroscopy, which is interface-sensitive vibrational spectroscopy. Most of the peaks observed in the experiment were at the same frequency as that of Raman and infrared spectra of the dye and TiO2. Two interfacial modes at 640 and 100 cm(-1) and one resonantly enhanced phonon at 146 cm(-1) appeared in addition to the pure TiO2 and N3 spectra. Adsorption of N3 dye on TiO2 contributed to the enhancement of 100 and 146 cm(-1) mode. The results not only reported interfacial low-frequency vibrations of TiO2 (110) with N3 dye adsorption but also suggested the coupling between the surface vibrations of TiO2 and charge transfer between N3 dye and TiO2 on the surface.

SeV C Protein Plays a Role in Restricting Macrophage Phagocytosis by Limiting the Generation of Intracellular Double-Stranded RNA.

Macrophages play a central role in the innate immune response to respiratory viral infections through pro-inflammatory factor secretion and phagocytosis. However, as a countermeasure, viral pathogens have evolved virulence factors to antagonize macrophage function. In our recent in vitro analyses of murine macrophage cell lines, Sendai virus (SeV) accessory protein C inhibited the secretion of pro-inflammatory factors, and C gene-knockout SeV (SeVΔC) caused drastic morphological changes in RAW264.7 macrophages, similar to those observed after stimulation with Lipid A, a well-known activator of actin-rich membrane ruffle formation and phagocytosis. Hence, we sought to determine whether the C protein limits phagocytosis in SeV-infected macrophages through the suppression of membrane ruffling. Phagocytosis assays indicated an upregulation of phagocytosis in both SeVΔC-infected and Lipid A-stimulated macrophages, but not in SeV WT-infected cells. Further, the observed membrane ruffling was associated with phagocytosis. RIG-I is essential for Lipid A-induced phagocytosis; its deficiency inhibited SeVΔC-stimulated phagocytosis and ruffling, confirming the essential role of RIG-I. Moreover, treatment with interferon (IFN)-ß stimulation and neutralizing antibodies against IFN-ß suggested that SeVΔC-induced phagocytosis and ruffling occurred in an IFN-ß-independent manner. A newly isolated SeVΔC strain that does not generate dsRNA further highlighted the importance of dsRNA in the induction of phagocytosis and ruffling. Taken together, the current results suggest that SeV C protein might limit phagocytosis-associated membrane ruffling in an RIG-I-mediated but IFN-independent manner via limiting the generation of intracellular dsRNA.

Calpain regulates thymidylate synthase-5-fluoro-dUMP complex levels associated with response to 5-fluorouracil in gastric cancer cells.

Thymidylate synthase (TS) plays a major role in the response to 5-fluorouracil (5-FU) by binding directly to the 5-FU metabolite, 5-fluoro-dUMP (FdUMP). The change in the TS expression levels after 5-FU administration was examined in parallel to 5-FU responsiveness in six human gastric adenocarcinoma cell lines to elucidate the source of variability of 5-FU sensitivity. MKN-1, SH-10-TC and MKN-74 cells were more resistant to 5-FU than MKN-28, KATO III and MKN-45 cells. Western blotting analysis revealed that the 5-FU sensitivity of these cells did not correlate with the basal TS expression levels but did correlate with rapid detection of the TS-FdUMP complex after exposure to 5-FU. In 5-FU-resistant cells, very low levels of the TS-FdUMP complex early after 5-FU exposure were elevated by pretreatment with calpain inhibitors such as benzyloxycarbonyl-leucyl-leucinal (ZLLH), benzyloxycarbonyl-leucyl-leucyl-leucinal (ZLLLH) and ALLN, but not by other protease inhibitors. In contrast, ONO-3403, which causes calpain activation, stimulated downregulation of the TS-FdUMP complex in 5-FU-sensitive cells. The expression levels of calpastatin, an endogenous calpain inhibitor, were higher in 5-FU-sensitive cells than in 5-FU-resistant cells. ZLLH increased the 5-FU sensitivity of 5-FU-resistant cells, whereas ONO-3403 decreased the sensitivity of 5-FU-sensitive cells. In addition, knockdown of m-calpain by siRNA increased the 5-FU sensitivity in 5-FU-resistant cells, while knockdown of calpastatin reduced the sensitivity in 5-FU-sensitive cells. These results suggest that calpain might reduce the chemosensitivity of human gastric cancer cells to 5-FU possibly by rapid degradation of the TS-FdUMP complex, a finding that is considered to have novel therapeutic implications.

An ADP ribosylation factor-GTPase activating protein negatively regulates the production of proinflammatory mediators in response to lipopolysaccharide.

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-ß, 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.

Thalidomide inhibits interferon-γ-mediated nitric oxide production in mouse vascular endothelial cells.

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 inhibits lipopolysaccharide-induced TNF-α production through inactivation of nuclear factor-κB and mitogen-activated protein kinases in the MyD88-dependent pathway.

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 designed NF-κB inhibitor, DHMEQ, inhibits KISS1R-mediated invasion and increases drug-sensitivity in mouse plasmacytoma SP2/0 cells.

Plasmacytoma is one of the most difficult types of leukemia to treat, and it often invades the bone down to the marrow resulting in the development of multiple myeloma. NF-κB is often constitutively activated, and promotes metastasis and drug resistance in neoplastic cells. The present study assessed the cellular anticancer activity of an NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on mouse plasmacytoma SP2/0 cells. Cellular invasion was measured by Matrigel chamber assay, and apoptosis was assessed by detecting caspase-3 cleavage and by flow cytometric analysis with Annexin V. DHMEQ inhibited constitutively activated NF-κB at nontoxic concentrations. DHMEQ was also shown to inhibit cellular invasion of SP2/0 cells, as well as human myeloma KMS-11 and RPMI-8226 cells. The metastasis PCR array indicated that DHMEQ induced a decrease in KISS1 receptor (KISS1R) expression in SP2/0 cells. Knockdown of KISS1R by small interfering RNA suppressed cellular invasion, suggesting that KISS1R may serve an essential role in the invasion of SP2/0 cells. Furthermore, DHMEQ enhanced cytotoxicity of the anticancer agent melphalan in SP2/0 cells. Notably, DHMEQ inhibited the expression of NF-κB-dependent anti-apoptotic proteins, such as Bcl-XL, FLIP, and Bfl-1. In conclusion, inhibition of constitutively activated NF-κB by DHMEQ may be useful for future anti-metastatic and anticancer strategies for the treatment of plasmacytoma.

Tumor necrosis factor-a augments lipopolysaccharide-induced suppressor of cytokine signalling 3 (SOCS-3) protein expression by preventing the degradation.

The regulatory role of tumour necrosis factor-a (TNF-a) on the expression of suppressor of cytokine signalling 3 (SOCS-3) in response to lipopolysaccharide (LPS) was examined using peritoneal macrophages from TNF-a-deficient mice. The LPS-induced SOCS-3 expression was markedly augmented in macrophages from wild-type mice whereas such augmentation was not seen in the cells from TNF-a-deficient mice. However, there was no significant difference in the level of SOCS-3 messenger RNA expression between macrophages from wild-type mice and those from TNF-a-deficient mice. The addition of exogenous TNF-a augmented the LPS-induced SOCS-3 expression in macrophages from TNF-a-deficient mice. The pulse chase analysis suggested augmented degradation of LPS-induced SOCS-3 protein in macrophages from TNF-a-deficient mice. Moreover, MG 132, a 26S proteasome inhibitor, sustained the LPS-induced SOCS-3 expression in those cells. The tyrosine phosphorylation of SOCS-3 was definitely induced in LPS-stimulated macrophages from TNF-a-deficient mice but not wild-type mice. A tyrosine phosphatase inhibitor enhanced the tyrosine phosphorylation of SOCS-3 in wild-type mice and accelerated the degradation. Therefore, it was suggested that TNF-a prevented the degradation of SOCS-3 protein via inhibition of the tyrosine phosphorylation in LPS-stimulated macrophages.

Seladin-1 is a novel lipopolysaccharide (LPS)-responsive gene and inhibits the tumour necrosis factor-alpha production and osteoclast formation in response to LPS.

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.