Increased Impact of Serum Uric Acid on Arterial Stiffness and Atherosclerosis in Females.

AIMS: Serum uric acid increases with metabolic disorders; however, whether the effects of uric acid on atherosclerosis are different in females and males has not been sufficiently evaluated. Therefore, this study compared the impact of uric acid on arterial stiffness and atherosclerosis between females and males. METHODS: We enrolled 10196 untreated middle-aged subjects (46±8 years, 3021 females and 7175 males) who underwent periodic health check-ups. Serum uric acid levels were measured and arterial stiffness and atherosclerosis were assessed by the cardio-ankle vascular index (CAVI), carotid intima-media thickness (IMT), and plaque, using ultrasound imaging. RESULTS: Females with increased arterial stiffness (CAVI ≥ 8.0) or carotid plaques had higher uric acid than those without (P＜0.0001), but males did not. In multivariable regression analyses including overall participants, uric acid was significantly associated with the CAVI, where sex interacted with uric acid. In sex-specific analyses, uric acid was significantly associated with the CAVI, but not with carotid IMT, in both sexes. However, logistic regression analyses revealed that serum uric acid was independently associated with the presence of carotid plaques in females. The exclusion of subjects with abdominal obesity or metabolic syndrome from the analysis did not alter the results in females. CONCLUSIONS: Serum uric acid was significantly associated with the CAVI in both sexes, but the interaction of sex was confirmed and associated with a carotid plaque only in females. These findings support the increased impact of serum uric acid on arterial stiffness and atherosclerosis in females.

Examination of Large Artery Atherosclerosis could Reveal Small Artery Retinopathy in Untreated Middle-Aged Individuals.

AIMS: Small arteries can be visualized in the ocular fundus, and findings of retinopathy based on Scheie classification are often applied to evaluate the impact of hypertension and atherosclerosis. However, the relationship between damage in the large and small arteries has not been investigated sufficiently, especially in the early stages. The present study investigated possible associations between large artery atherosclerosis and small artery retinopathy in untreated middle-aged individuals. METHODS: Untreated middle-aged workers undergoing periodic health check-ups (n=7,730, 45±8 years) were enrolled in this study. The absence or presence and extent of retinopathy were characterized by ophthalmologists as hypertensive (H0-4) and atherosclerotic grades (S0-4) based on Scheie classification. Large artery atherosclerosis was examined based on functional assessment of the cardio-ankle vascular index (CAVI) and morphological assessment of the carotid intima-media thickness (IMT) by ultrasound. RESULTS: We found significant differences in CAVI and carotid IMT between individuals with and without hypertensive or atherosclerotic retinopathy. Multivariable regression analysis showed that the presence of hypertensive and atherosclerotic retinopathy was significantly associated with CAVI and carotid IMT. Logistic regression analysis with the endpoint of a hypertensive or atherosclerotic lesion revealed that CAVI and carotid IMT are independent determinants of retinopathy. CONCLUSIONS: CAVI and carotid IMT were significantly associated with the presence of retinopathy based on Scheie classification in untreated middle-aged subjects, implying that atherosclerotic examination in large arteries could reveal early-stage small artery retinopathy.

A body shape index could serve to identify individuals with metabolic syndrome and increased arterial stiffness in the middle-aged population.

BACKGROUND: A body shape index (ABSI) is a novel anthropometric measure calculated using waist circumference (WC), body mass index (BMI), and body height. This study investigated the usefulness of ABSI to identify individuals with metabolic syndrome (MetS) and increased arterial stiffness in the middle-aged population. METHODS: Middle-aged workers who underwent periodic health check-ups and who were without previous cardiovascular events were enrolled (n = 10,182). In addition to ABSI, visceral fat area (VFA) was evaluated using computed tomography. Obesity and MetS were diagnosed on the basis of WC, VFA, and ABSI. Arterial stiffness was examined by measuring the cardio-ankle vascular index (CAVI). RESULTS: ABSI was significantly associated with CAVI in multivariable regression analysis. Logistic regression analysis revealed that ABSI was independently associated with the presence of MetS diagnosed on the basis of WC or VFA after adjustment for potential confounders, including BMI. Subjects with MetS diagnosed on the basis of each obesity index showed higher CAVI values than those without. Among subjects with MetS diagnosed on the basis of WC or VFA, those with MetS who met the definition of ABSI obesity showed significantly higher CAVI than those who did not. The other logistic regression analysis demonstrated that CAVI was independently associated with MetS defined on the basis of ABSI. CONCLUSIONS: ABSI was significantly associated with CAVI and the presence of MetS in the middle-aged population and helped to discriminate individuals with MetS and increased CAVI. ABSI could serve to identify individuals with MetS and increased arterial stiffness.

Close Association between Subclinical Atherosclerosis and Pulmonary Function in Middle-Aged Male Smokers.

AIMS: Cigarette smoking provokes deleterious influences on cardiovascular and pulmonary systems, although the underlying relationship has not been sufficiently investigated especially in early-stage disease. The present study investigated possible associations between subclinical atherosclerosis and pulmonary function in middle-aged male smokers. METHODS: Male smokers undergoing their periodic health check-up were enrolled in this study (n=3,775, 45±8 years). Pulmonary function was evaluated using spirometry by calculating forced vital capacity (FVC) as a percentage of predicted value (FVC%-predicted), forced expiratory volume in one second (FEV1) as a percentage of predicted value (FEV1%-predicted), and the ratio of FEV1 to FVC (FEV1/FVC). Subclinical atherosclerosis was assessed based on ankle-brachial pressure index (ABI), cardio-ankle vascular index (CAVI), ultrasound examination of the carotid intima-media thickness (IMT), and presence of plaque. RESULTS: Multivariate regression analysis showed that ABI was positively associated with FVC%-predicted and FEV1%-predicted after adjustment for confounders including smoking intensity, while CAVI or carotid IMT was inversely associated with both. Participants with chronic obstructive pulmonary disease (COPD, n=256) showed reduced ABI and increased CAVI or carotid IMT compared with those without COPD, and participants with carotid plaque had lower pulmonary function than those without plaque. Reduced FEV1/FVC was an independent determinant of carotid plaque and decreased ABI was an independent determinant of COPD, as revealed by logistic regression analysis with the endpoint of carotid plaque presence or a diagnosis of COPD revealed. CONCLUSIONS: Middle-aged male smokers showed a close association between subclinical atherosclerosis and pulmonary function, implying that smoking induced-vascular and pulmonary damage are interacting in early-stage disease.

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.

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.

Poor toe flexor strength, but not handgrip strength, is associated with the prevalence of diabetes mellitus in middle-aged males.

Previous studies suggested that reduced muscular strength was one of the potential predictor of prevalence of diabetes mellitus. The purpose of this study was to investigate the association between toe flexor strength (TFS) and handgrip strength (HGS) and the prevalence of diabetes mellitus. Cross-sectional analysis was conducted using data from 1,390 Japanese males (35-59 years). TFS and HGS were measured and medical examinations undertaken. The prevalence of diabetes mellitus was defined as fasting blood glucose ≥126 mg/dL, glycated hemoglobin ≥6.5% (48 mmol/mol), and/or current use of anti-diabetes mellitus drugs. A total of 114 participants had diabetes mellitus. TFS in participants with diabetes mellitus was significantly lower than that in persons not suffering from diabetes mellitus but HGS was not. Odds ratio (OR) and 95% confidence interval (CI) per 1-standard deviation-increase in muscular strength measurements for the prevalence of diabetes mellitus were obtained using a multiple logistic regression model. Prevalence of diabetes mellitus was inversely related to TFS (OR 0.769, 95% CI 0.614-0.963), TFS/body mass (BM) (0.696, 0.545-0.889) and TFS/body mass index (BMI) (0.690, 0.539-0.882) after adjustment of covariates. Such associations were not observed in HGS (OR 0.976, 95% CI 0.773-1.232), HGS/BM (0.868, 0.666-1.133) or HGS/BMI (0.826, 0.642-1.062). These results suggested that poor TFS was associated with an increased prevalence of diabetes mellitus independent of visceral fat accumulation, but HGS was not, in middle-aged males. TFS may be a better marker for the prevalence of diabetes mellitus than HGS.

Association of body flexibility and carotid atherosclerosis in Japanese middle-aged men: a cross-sectional study.

OBJECTIVE: This study examined the associations of body flexibility with carotid arterial remodelling, including intima-media thickness (IMT) and plaque formation in middle-aged men. METHODS: The subjects of this cross-sectional study included 1354 Japanese men aged 35-59 years without histories of stroke or cardiac diseases. The arm extensibility test, which can estimate flexibility of the upper extremity (composed of shoulder external rotation and forearm supination), and the sit-and-reach test were performed. Common carotid IMT and plaque formation (≥1.1 mm) were estimated by ultrasound. RESULTS: The proportion of subjects who fully completed the arm extensibility test was 55.0%, and who had plaques in the common carotid artery was 37.8%. IMT was associated with poor arm extensibility (ß=-0.073, 95% CI -0.02224 to -0.00041, P=0.004), while plaque formation was associated with poor sit-and-reach (OR 0.98579, 95% CI 0.97257 to 0.99919, P=0.038) after adjustment by all covariates. CONCLUSIONS: This study demonstrated that poor upper extremity and trunk flexibility were associated with characteristics of early onset of atherosclerosis. Furthermore, these associations were independent of covariates such as age, blood pressure, blood lipids glucose levels and abdominal fat accumulation, handgrip strength and lifestyle, including sleeping, drinking, exercise and smoking habits. Poor flexibility may reflect subclinical atherosclerosis in middle-aged men.

Age-related reduction and independent predictors of toe flexor strength in middle-aged men.

BACKGROUND: Toe flexor muscles play an important role in posture and locomotion, and poor toe flexor strength is a risk factor for falls. In this cross-sectional study, we estimated the age-related change in toe flexor strength and compared it with that of handgrip strength. Independent factors predicting toe flexor and handgrip strength were also determined. METHODS: A total of 1401 male (aged 35-59 years) study participants were divided into five groups according to their chronological age; 35-39, 40-44, 45-49, 50-54, and 55-59 years. Toe flexor and handgrip strength, anthropometry, and resting blood pressure were measured. Fasting blood samples were collected to measure blood glucose, triglycerides, high- and low-density lipoprotein-cholesterols, and albumin. A self-administered lifestyle questionnaire was conducted. RESULTS: Decline in absolute toe flexor and handgrip strength began in the age groups 50-55 and 55-59 years, respectively. In comparison to the mean values of the youngest group, relative toe flexor strength (87.0 ± 26.6%) was significantly lower than handgrip strength (94.4 ± 13.1%) for the oldest group. Multiple regression analyses showed that independent factors predicting both toe flexor and handgrip strength were lean body mass, age, serum albumin, drinking habit, and fat mass. Additionally, fasting blood glucose, diastolic blood pressure, sleeping time and exercise habit were predicting factors of toe flexor strength but not of handgrip strength. CONCLUSIONS: Age-related reduction in toe flexor strength was earlier and greater than handgrip strength, and toe flexor strength reflects body composition and metabolic status.

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.

Pulmonary platelet accumulation induced by catecholamines: Its involvement in lipopolysaccharide-induced anaphylaxis-like shock.

Intravenously injected lipopolysaccharides (LPS) rapidly induce pulmonary platelet accumulation (PPA) and anaphylaxis-like shock (ALS) in mice. Macrophages reportedly release catecholamines rapidly upon stimulation with LPS. Here, we examined the involvement of macrophage-derived catecholamines in LPS-induced PPA and ALS. A catecholamine or Klebsiella O3 (KO3) LPS was intravenously injected into mice, with 5-hydroxytryptamine in the lung being measured as a platelet marker. The tested catecholamines induced PPA, leading to shock. Their minimum shock-inducing doses were at the nmol/kg level. The effects of epinephrine and norepinephrine were inhibited by prazosin (α1 antagonist) and by yohimbine (α2 antagonist), while dopamine's were inhibited only by prazosin. Use of synthetic adrenergic α1- and/or α2-agonists, platelet- or macrophage-depleted mice, a complement C5 inhibitor and C5-deficient mice revealed that (a) α2-receptor-mediated PPA and shock depend on both macrophages and complements, while α1-receptor-mediated PPA and shock depend on neither macrophages nor complements, (b) the PPA and ALS induced by KO3-LPS depend on α1- and α2-receptors, macrophages, and complements, and (c) KO3-LPS-induced PPA is preceded by catecholamines decreasing in serum. Together, these results suggest the following. (i) Catecholamines may stimulate macrophages and release complement C5 via α2-receptors. (ii) Macrophage-derived catecholamines may mediate LPS-induced PPA and ALS. (iii) Moderate PPA may serve as a defense mechanism to remove excess catecholamines from the circulation by promoting their rapid uptake, thus preventing excessive systemic effects. (iv) The present findings might provide an insight into possible future pharmacological strategies against such diseases as shock and acute respiratory distress syndrome.

Early reduction in toe flexor strength is associated with physical activity in elderly men.

[Purpose] To compare the toe flexor, hand grip and knee extensor strengths of young and elderly men, and to examine the association between toe flexor strength and physical activity or inactivity levels. [Subjects and Methods] Young (n=155, 18-23 years) and elderly (n=60, 65-88 years) men participated in this study. Toe flexor, hand grip, and knee extensor strength were measured. Physical activity (time spent standing/walking per day) and inactivity (time spent sitting per day) were assessed using a self-administered questionnaire. [Results] Toe flexor, hand grip, and knee extensor strength of the elderly men were significantly lower than those of the young men. Standing/walking and sitting times of the elderly men were lower than those of the young men. Toe flexor strength correlated with hand grip and knee extensor strength in both groups. In elderly men, toe flexor strength correlated with standing/walking time. In comparison to the young men's mean values, toe flexor strength was significantly lower than knee extensor and hand grip strength in the elderly group. [Conclusion] The results suggest that age-related reduction in toe flexor strength is greater than those of hand grip and knee extensor strengths. An early loss of toe flexor strength is likely associated with reduced physical activity in elderly men.

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.

Pretreatment of LPS inhibits IFN-ß-induced STAT1 phosphorylation through SOCS3 induced by LPS.

It has been known that LPS activates macrophages and induces IFN-ß production from macrophages. The endogenous IFN-ß produced by LPS stimulates the cells, which plays a role in innate immune. However, it was not elucidated yet if the signaling by exogenous IFN-ß was influenced by LPS stimulation. In this study, it was found pretreatment of LPS interrupted IFN-ß-induced JAK1/STAT1 phosphorylation. LPS pretreatment also reduced IFN-ß-induced ISG54, one of IFN-ß-inducible genes. Pretreatment with LPS for more than 2h shows inhibitory effect on IFN-ß-induced STAT1 phosphorylation but simultaneous treatment or post-treatment of LPS with IFN-ß did not show the inhibitory effect. The study using a neutralizing antibody to IFN-ß indicated that IFN-ß produced by LPS does not take part in the inhibitory effect of LPS. Furthermore, LPS did not affect the expression of IFN αß receptor. A previous report has shown that LPS-induced SOCS3 inhibited IFN-γ-induced STAT1 phosphorylation, likewise, it was also shown in this study that LPS induced SOCS3 expression and its expression inhibited IFN-ß-induced STAT1 phosphorylation which was confirmed by the knockdown study by the siRNA of SOCS3. The real-time PCR and immune-blot studies of SOCS3 indicated that LPS induced SOCS3 is independent of IL-6, IL-10, TNF-α and STAT3, and might depend on p38 activation by LPS. It was suggested that bacterial LPS rather interfere with IFN-ß actions, dependent on the timing of LPS stimulation.

TGF-ß1 inhibits the production of IFN in response to CpG DNA via ubiquitination of TNF receptor-associated factor (TRAF) 6.

The effect of TGF-ß1 on CpG DNA-induced type I IFN production was examined by reconstituting a series of signaling molecules in TLR 3 signaling. TGF-ß1 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-ß1 inhibited IRF7-mediated type I IFN production in the presence of TRAF6. TGF-ß1 induced ubiquitination of TRAF6, although CpG DNA did not induce it. Moreover, TGF-ß1 accelerated the ubiquitination of TRAF6 in the presence of CpG DNA. TGF-ß1 ubiquitinated TRAF6 at K63 but not K48. TGF-ß1 also induced ubiquitination of IRF7. Further, TGF-ß1 did not impair the interaction of IRF7 and TRAF6. CpG DNA induced the phosphorylation of IRF7 in the presence of TRAF6, whereas TGF-ß1 inhibited the IRF7 phosphorylation. Blocking of TRAF6 ubiquitination abolished the inhibition of CpG DNA-induced type I IFN production by TGF-ß. Taken together, TGF-ß was suggested to inhibit CpG DNA-induced type I IFN production transcriptionally via ubiquitination of TRAF6.

Irbesartan attenuates production of high-mobility group box 1 in response to lipopolysaccharide via downregulation of interferon-ß production.

High-mobility group box 1 (HMGB1) is suggested to participate in development of local and systemic inflammatory disorders. Irbesartan (IRB), an angiotensin II type1 receptor blocker, is widely used for treatment of hypertension, especially in patients with diabetic nephropathy. The effect of IRB on lipopolysaccharide (LPS)-induced HMGB1 and nitric oxide (NO) production was examined using RAW 264.7 macrophage-like cells. IRB inhibited LPS-induced HMGB1 production. IRB also reduced LPS-induced expression of an inducible NO synthase, and inhibited LPS-induced NO production. The expression levels of IFN-ß protein and mRNA, which is a key molecule in MyD88-independent pathway of LPS signaling, were exclusively inhibited by IRB. Peroxisome proliferator-activated receptor-γ and angiotensin II type 1 receptor were not involved in the inhibitory action of IRB on LPS-induced HMGB1 and NO production. Collectively, IRB was suggested to inhibit LPS-induced HMGB1 production via downregulation of IFN-ß production in the MyD88-independent pathway.

Inhibition of RANKL- and LPS-induced osteoclast differentiations by novel NF-κB inhibitor DTCM-glutarimide.

We have isolated 9-methylstreptimidone from microorganism as a new NF-κB inhibitor. Later, we designed 3-[(dodecylthiocarbonyl) methyl]-glutarimide (DTCM-glutarimide) as an analog of this compound, which shows anti-inflammatory activity in vivo. In the present research, we found that DTCM-glutarimide inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation of mouse bone marrow-derived macrophages and RANKL- or lipopolysaccharide (LPS)-induced osteoclast differentiation of RAW 264.7 cells without any toxicity. It also inhibited the RANKL-induced NFATc1 expression. Upstream signaling involving phosphorylation of Akt and GSK-3ß was induced by RANKL, of which the signaling was inhibited by DTCM-glutarimide. Then DTCM-glutarimide was confirmed to inhibit RANKL-induced NF-κB activity, possibly by inhibiting the Akt-mediated activation of IKK. Thus, DTCM-glutarimide inhibited osteoclastogenesis by blocking both the Akt-GSK3ß-NFATc1 and NF-κB-NFATc1 pathways. DTCM-glutarimide may be a candidate as a chemotherapeutic agent for severe bone resorption diseases.

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.

Lipopolysaccharide downregulates the expression of p53 through activation of MDM2 and enhances activation of nuclear factor-kappa B.

The effect of lipopolysaccharide (LPS) on the expression of p53 protein in RAW 264.7 macrophage cells was examined. LPS downregulated the expression of p53 protein 4-24 h after the stimulation. LPS-induced p53 inhibition was restored with pharmacological inhibitors of c-jun N-terminal kinase (JNK) and phosphatidylinositol 3-kinase (PI3K). It was also restored by inhibitors of MDM2 activation and proteasome. LPS-induced p53 inhibition corresponded to activation of MDM2. LPS-induced MDM2 activation was prevented by inhibitors of JNK and PI3K. The expression of p65 NF-κB at a late stage after LPS stimulation was downregulated in the presence of a MDM2 inhibitor. Nutlin-3 as a MDM2 inhibitor reduced LPS-induced production of nitric oxide but not tumor necrosis factor-α. Administration of LPS into mice downregulated the in vivo expression of p53 in the livers. Taken together, LPS was suggested to downregulate the expression of p53 via activation of MDM2 and enhance the activation of NF-κB at a late stage.

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.