(2S)-2'-Methoxykurarinone inhibits osteoclastogenesis and bone resorption through down-regulation of RANKL signaling.

(2S)-2'-Methoxykurarinone (MK), a compound isolated from the roots of Sophora flavescens, has various physiological properties, such as anti-inflammatory, antipyretic, antidiabetic, and antineoplastic effects. However, the effect of S. flavescens-derived MK on osteoclastogenesis remains unknown. Therefore, we examined the effect and mechanism of action of MK on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and bone resorption. MK inhibited osteoclast differentiation in bone marrow cell-osteoblast cocultures but did not affect the RANKL-to-osteoprotegerin ratio induced by osteoclastogenic factors in osteoblasts. MK also inhibited RANKL-induced osteoclast differentiation from bone marrow macrophages in a dose-dependent manner, without cytotoxicity. Pretreatment with MK significantly suppressed the Akt, p38, c-Jun N terminal kinase (JNK), c-Fos, and nuclear factor of activated T cells c1 (NFATc1) pathways and inhibited the bone-resorbing activity of mature osteoclasts. These results collectively suggest that MK inhibits osteoclast differentiation and bone resorption through RANKL-induced mitogen-activated protein kinases (MAPKs) and c-Fos-NFATc1 signaling pathways.

Costunolide inhibits osteoclast differentiation by suppressing c-Fos transcriptional activity.

Costunolide, a sesquiterpene lactone, exhibits anti-inflammatory and anti-oxidant properties and mediates apoptosis. However, its effects and mechanism of action in osteoclasts remain unknown. Herein, we found that costunolide significantly inhibited RANKL-induced BMM differentiation into osteoclasts in a dose-dependent manner without affecting cytotoxicity. Costunolide did not regulate the early signaling pathways of RANKL, including the mitogen-activated protein kinase and NF-κB pathways. However, costunolide suppressed nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) expression via inhibition of c-Fos transcriptional activity without affecting RANKL-induced c-Fos expression. The inhibitory effects of costunolide were rescued by overexpression of constitutively active (CA)-NFATc1. Taken together, our results suggest that costunolide inhibited RANKL-induced osteoclast differentiation by suppressing RANKL-mediated c-Fos transcriptional activity.

Oleanolic acid acetate inhibits osteoclast differentiation by downregulating PLCγ2-Ca(2+)-NFATc1 signaling, and suppresses bone loss in mice.

Owing to their potential pharmacological activities in human disease, natural plant-derived compounds have recently become the focus of increased research interest. In this study, we first isolated oleanolic acid acetate (OAA), a triterpenoid compound, from Vigna angularis (azuki bean) to discover anti-bone resorptive agents. Many studies have identified and described the various medicinal effects of V. angularis extract. However, the pharmacological effect of OAA-derived V. angularis extract, particularly the effect on osteoclastogenesis, is not known. Therefore, we investigated the effect and mechanism of OAA in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. OAA inhibited RANKL-induced osteoclast differentiation in bone marrow macrophages (BMMs) without any evidence of cytotoxicity. Interestingly, OAA significantly inhibited Btk phosphorylation, phospholipase Cγ2 (PLCγ2) phosphorylation, calcium ion (Ca(2+)) oscillation, and nuclear factor of activated T cell c1 (NFATc1) expression in RANKL-stimulated BMMs, but did not affect RANKL-induced mitogen-activated protein kinase. OAA also inhibited the bone-resorbing activity of mature osteoclasts. Furthermore, mice treated with OAA demonstrated marked attenuation of lipopolysaccharide-induced bone erosion based on micro-computed tomography and histologic analysis of femurs. Taken together, the results suggested that OAA inhibited RANKL-mediated osteoclastogenesis via PLCγ2-Ca(2+)-NFATc1 signaling in vitro and suppressed inflammatory bone loss in vivo.

Glutaredoxin2 isoform b (Glrx2b) promotes RANKL-induced osteoclastogenesis through activation of the p38-MAPK signaling pathway.

Receptor activator of NF-κB ligand (RANKL) triggers the differentiation of bone marrow-derived monocyte/macrophage precursor cells (BMMs) of hematopoietic origin into osteoclasts through the activation of mitogen-activated protein (MAP) kinases and transcription factors. Recently, reactive oxygen species (ROS) and antioxidant enzymes were shown to be closely associated with RANKL-mediated osteoclast differentiation. Although glutaredoxin2 (Glrx2) plays a role in cellular redox homeostasis, its role in RANKL-mediated osteoclastogenesis is unclear. We found that Glrx2 isoform b (Glrx2b) expression is induced during RANKLmediated osteoclastogenesis. Over-expression of Glrx2b strongly enhanced RANKL- mediated osteoclastogenesis. In addition, Glrx2b-transduced BMMs enhanced the expression of key transcription factors c-Fos and NFATc1, but pre-treatment with SB203580, a p38-specific inhibitor, completely blocked this enhancement. Conversely, down-regulation of Glrx2b decreased RANKL- mediated osteoclastogenesis and the expression of c-Fos and NFATc1 proteins. Also, Glrx2b down-regulation attenuated the RANKL-induced activation of p38. Taken together, these results suggest that Glrx2b enhances RANKL-induced osteoclastogenesis via p38 activation.

Effect of Cornus Officinalis on Receptor Activator of Nuclear Factor-kappaB Ligand (RANKL)-induced Osteoclast Differentiation.

OBJECTIVES: Osteoporosis is a disease of bones that is thought to result from an imbalance between bone resorption and bone formation. Although osteoporosis itself has no symptoms, osteoporosis caused by osteoclasts leads to an increased risk of fracture. Here we examined the effects of cornus officinalis on receptor activator of nuclear factor-kappaB ligand (RANKL)-mediated osteoclast differentiation. METHODS: We evaluated the effects of cornus officinalis on RANKL-induced osteoclast differentiation from bone marrow-derived macrophages (BMMs) and performed a cytotoxicity assay, reverse transcriptase-polymerase chain reaction (RT-PCR), and Western blot analysis. RESULTS: Cornus officinalis significantly inhibits RANKL-mediated osteoclast differentiation in a dose-dependent manner, but without cytotoxicity against BMMs. The mRNA expression of tartrate-resistant acid phosphatase (TRAP), osteoclast-associated receptor (OSCAR), c-Fos, and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) in BMMs treated with RANKL was considerably inhibited by cornus officinalis treatment. Also, cornus officinalis inhibits the protein expression of c-Fos and NFATc1. Cornus officinalis greatly inhibits RANKL-induced phosphorylation of p38 and c-JUN N-terminal kinase (JNK). Also, cornus officinalis significantly suppresses RANKL-induced degradation of I-κB. CONCLUSIONS: Taken together, our results suggest that cornus officinalis may be a useful the treatment of osteoporosis.

Amorphigenin inhibits Osteoclast differentiation by suppressing c-Fos and nuclear factor of activated T cells.

Among the several rotenoids, amorphigenin is isolated from the leaves of Amopha Fruticosa and it is known that has anti-proliferative effects and anti-cnacer effects in many cell types. The main aim of this study was to investigate the effects of amorphigenin on osteoclast differentiation in vitro and on LPS treated inflammatory bone loss model in vivo. We show here that amorphigenin inhibited RANKL-induced osteoclast differentiation from bone marrow macrophages in a dose dependent manner without cellular toxicity. Anti-osteoclastogenic properties of amorphigenin were based on a down-regulation of c-fos and NFATc1. Amorphigenin markedly inhibited RANKL-induced p38 and NF-κB pathways, but other pathways were not affected. Micro-CT analysis of the femurs showed that amorphigenin protected the LPS-induced bone loss. We concluded that amorphigenin can prevent inflammation-induced bone loss. Thus we expect that amorphigenin could be a treatment option for bone erosion caused by inflammation.

Inhibition of osteoclast differentiation and bone resorption by rotenone, through down-regulation of RANKL-induced c-Fos and NFATc1 expression.

Osteoclasts are responsible for bone erosion in diseases as diverse as osteoporosis, periodontitis, and rheumatoid arthritis. Natural plant-derived products have received recent attention as potential therapeutic and preventative drugs in human disease. The effect of rotenone in RANKL-induced osteoclast differentiation was examined in this study. Rotenone inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages (BMMs) in a dose-dependent manner without any evidence of cytotoxicity. The mRNA expression of c-Fos, NFATc1, TRAP, and OSCAR in RANKL-treated BMMs was inhibited by rotenone treatment. Rotenone strongly inhibited p38 and ERK phosphorylation and I-kappaB degradation in RANKL-stimulated BMMs, and did not inhibit JNK phosphorylation. Further, RANKL-induced c-Fos and NFATc1 protein expression was suppressed by rotenone. Rotenone additionally inhibited the bone resorptive activity of differentiated osteoclasts. A lipopolysaccharide (LPS)-induced bone erosion study was also performed to assess the effects of rotenone in vivo. Mice treated with rotenone demonstrated marked attenuation of bone erosion based on Micro CT and histologic analysis of femurs. These results collectively suggested that rotenone demonstrated inhibitory effects on osteoclast differentiation in vitro and suppressed inflammatory bone loss in vivo. Rotenone may therefore serve as a useful drug in the prevention of bone loss.

Risedronate directly inhibits osteoclast differentiation and inflammatory bone loss.

Risedronate, a nitrogen-containing bisphosphonate, is widely used in the clinical field for the treatment of osteoporosis. Risedronate is known to exert its effects through binding to hydroxyapatite in bone tissue, inhibiting osteoclastic activity, and inducing apoptosis of osteoclasts. The purpose of this study was to determine the effects of risedronate on osteoclast differentiation in vitro and on an inflammatory bone loss model in vivo. Risedronate inhibited osteoclast differentiation in co-culture of bone marrow cells (BMCs) and osteoblasts, and suppressed receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-mediated osteoclast differentiation from bone marrow-derived macrophages (BMMs) in a dose-dependent manner without toxicity. Risedronate significantly inhibited expression of c-Fos and nuclear factor of activated T cells (NFAT) c1 induced by RANKL. To examine the effect of risedronate on bone loss in vivo, we used a mouse model of lipopolysaccharide (LPS)-mediated bone loss. Micro-CT analysis of the femurs showed that LPS treatment caused bone loss. However, bone loss was significantly attenuated in mice administered with risedronate. Taken together, we conclude that risedronate exerts beneficial effects on osteoporosis by inhibiting osteoclast differentiation both directly and indirectly. In infectious conditions, the inhibitory effect of risedronate on bone erosion was excellent. Thus risedronate could be a treatment option for osteoporosis caused by inflammatory and infectious conditions.

Pyridone 6, a pan-Janus-activated kinase inhibitor, suppresses osteoclast formation and bone resorption through down-regulation of receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL)-induced c-Fos and nuclear factor of activated T cells (NFAT) c1 expression.

It has been reported that Janus tyrosine kinase (JAK)-dependent signaling pathways play a critical role in the pathogenesis of numerous malignancies and immune reactions, and inhibition of JAK has been implicated in cell growth inhibition. The role which JAK has on osteoclast differentiation and anti-bone resorptive activity is not well understood. In this study, we investigated the effects of a pan-JAK inhibitor, pyridone 6, on osteoclast differentiation and bone-resorption in vitro and ex vivo. Pyridone 6 inhibited osteoclast differentiation in mouse bone marrow macrophage (BMM) cultures stimulated by the receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) and co-cultures of bone marrow cells and osteoblasts. Pyridone 6 suppressed the expression of c-Fos and nuclear factor of activated T cells (NFAT) c1 in BMMs. It also inhibited the bone resorptive activity of mature osteoclasts that was accompanied by disruption of actin rings. Pyridone 6 also suppressed I-kappaB degradation and extracellular signal-regulated kinase (ERK) in mature osteoclasts, suggesting that these are the key molecules that pyridone 6 targets in the inhibition of osteoclast function. These results demonstrate inhibition of JAK may be useful for the treatment of bone-resorptive diseases, such as osteoporosis.

Polymorphisms in the IL-13 and IL-4 receptor alpha genes and allergic rhinitis.

The IL-4 receptor (IL-4R) and IL-13 genes are candidate genes in atopic diseases. The IL-4Ralpha chain and IL-13 promoter polymorphisms are gain-of-function mutations associated with atopy. We tested whether polymorphisms in the IL-4Ralpha chain and the coding region of the IL-13 gene are associated with allergic rhinitis in a Korean population. Polymerase chain reaction-based assays for IL-4Ralpha Gln551Arg and IL-13 exon 4 G2044A were used for genotyping. There were no differences in the frequencies of the genotypes and alleles of IL-4Ralpha between the controls and patients. The frequency of the IL-13 exon 4 2044A allele was statistically different between the controls and patients. Our results suggest that the IL-13 exon 4 G2044A polymorphism confers susceptibility to the development of allergic rhinitis in Koreans, whereas the IL-4Ralpha Gln551Arg polymorphism is not related to allergic rhinitis.

Molecular variations in Th1-specific cell surface gene Tim-3.

The family of T-cell immunoglobulin domain and mucin domain (TIM) proteins is identified to be expressed on T cells. A member of Tim family, Tim-3 (T cell immunoglobulin mucin 3) is selectively expressed on the surface of differentiated Th1 cells. Tim-3 might have an important role in the induction of autoimmune diseases by regulating macrophage activation and interacts with Tim-3 ligand to regulate Th1 responses. To determine the variation sites in the coding and promoter region of human Tim-3 gene, we performed variation scanning by direct sequencing using the genomic DNA isolated from the patients with asthma or allergic rhinitis and healthy controls without asthma and allergic rhinitis. We identified four single nucleotide polymorphisms (SNPs) including one novel SNPs (-1541C>T) and two variation sites (-1292_-1289delTAAA and -1282_-1278dupTAAAA) in the coding and promoter region of human Tim-3 gene in both the patients and healthy groups.

Association between polymorphisms of the angiotensin-converting enzyme and angiotensinogen genes and allergic rhinitis in a Korean population.

Angiotensin-converting enzyme (ACE) inactivates bradykinin, substance P, and neurokinin A, which are thought to play important roles in the pathogenesis of inflammatory diseases. Expression of angiotensinogen, a precursor of angiotensin, is enhanced by augmented secretion of proinflammatory cytokines (eg, interleukin-1) in the site of inflammation. Insertion or deletion (I/D) ACE and M235T angiotensinogen gene polymorphisms were reported to be associated with atopy in a Czech population. Using polymerase chain reaction restriction fragment length polymorphism and SNaPshot typing analysis, we investigated the frequencies of the genotypes and alleles of the ACE gene in 137 patients with allergic rhinitis, of the M235T angiotensinogen gene in 186 patients with allergic rhinitis, and of both in 219 healthy control subjects. There was no difference in the frequency of the DD genotype of the ACE gene in the controls and patients (odds ratio, 1.32 [0.66-2.60]; p > .05). The D allele was more frequent in patients, but the difference was not statistically significant (odds ratio, 1.21 [0.89-1.64]; p > .05). There was no difference in the frequency of the TT genotype of the angiotensinogen gene in the controls and patients (odds ratio, 1.01 [0.38-2.69]; p > .05). The T allele was more frequent in patients, but the difference was not statistically significant (odds ratio, 1.10 [0.78-1.55]; p > .05). Our results indicate that polymorphisms in the genes for ACE and angiotensinogen may not be related to the development of allergic rhinitis in the Korean population.

Chemokine RANTES promoter polymorphisms in allergic rhinitis.

OBJECTIVES/HYPOTHESIS: RANTES is one of the most widely studied of the chemokines linked to allergic diseases. Two polymorphisms of the RANTES promoter region (-403 G/A and -28 C/G) have been found. The authors investigated whether these RANTES promoter polymorphisms were associated with allergic rhinitis. STUDY DESIGN: Case-control study. METHODS: Blood samples for genetic analysis were obtained from 151 individuals with allergic rhinitis and from 278 healthy individuals without atopic disease. Polymerase chain reaction-based assays for detection of the -403 G/A and -28 C/G polymorphisms of the RANTES gene were used for genotyping. RESULTS: The frequencies of both the RANTES -403A and -28G alleles were significantly higher in patients with allergic rhinitis than in control subjects (P <.05 for both). CONCLUSION: The study results indicated that the -403 and -28 alleles in the RANTES promoter region belong to the predictor gene set for allergic rhinitis and could be used in genomic analysis.

Association analysis of novel TBX21 variants with asthma phenotypes.

Human TBX21 expressed in T Cells (T-BOX21) is a Th1-specific T-box transcription factor that controls the expression of the hallmark Th1 cytokine, IFNG. As a potent candidate gene for asthma genetic study, we have sequenced the full gene of human TBX21, including the -1,500bp promoter region to identify its gene polymorphisms. Twenty-three single nucleotide polymorphisms (SNPs) were identified; one in promoter region (c.-1514T>C), one in 5'UTR (c.-138C>A), two in exon 1 (c.99C>G (p.His33Gln), c.390A>G), sixteen in introns (c.492+806T>C, c.492+1170C>A, c.492+1514G>A, c.492+1907A>C, c.492+2116G>A, c.492+2516A>G, c.492+2953C>T, c.492+4207A>T, c.492+4211A>T, c.492+4985T>A, c.492+4207G>A, c.492+5533C>T, c.492+7889T>A, c.492+8270G>C, c.768+417T>C and c.989+183C>T), one in exon 4 (c.831C>T), one in exon 6 (c.1455G>A), and one in 3'UTR (c.2103A>C). Among twenty-three identified variants, seven were selected for larger scale genotyping (n=721) for asthma association study based on frequencies and location. Haplotypes, their frequencies and linkage disequilibrium coefficients (mid R:D'mid R:) between SNP pairs were estimated. The associations with risk of asthma, skin-test reactivity and total serum IgE levels were analyzed. Using statistical analyses for association of TBX21 polymorphisms with these three asthma phenotypes, no significant signals were detected. In conclusion, we identified twenty-three genetic polymorphisms in the important TBX21 gene, but no significant associations of TBX21 variants with asthma phenotypes were detected. TBX21 variation/haplotype information identified in this study will provide valuable information for future association studies of other immunological diseases.

Prognostic potential of glutathione S-transferase M1 and T1 null genotypes for gastric cancer progression.

To improve understanding of glutathione S-transferase (GST) behavior in terms of a development and prognostic factor for gastric adenocarcinoma, we investigated the association between the GSTM1 and GSTT1 null genotypes and gastric cancer risk or the prognostic value of the GSTM1 and GSTT1 null genotypes was evaluated. Using a polymerase chain reaction-based method, the frequencies of GSTM1 and GSTT1 genotypes and prognostic factors, such as staging, differentiation, and histologic type (intestinal vs. diffuse), were evaluated in 80 patients with curatively resected primary gastric adenocarcinoma. The frequencies of GSTM1 and GSTT1 null individuals were higher in the gastric cancer group, but the differences were not statistically significant (for GSTM1 null odds ratio (OR)=0.86; 95% confidence interval (CI)=0.49-1.51 and for GSTT1, OR=0.97; 95% CI=0.55-1.71). Since the GSTM1 and GSTT1 null genotypes are potential indicators of gastric adenocarcinoma, we examined the relationship between the GSTM1 and GSTT1 genotypes and prognostic factors. In terms of the histologically diffuse type of cancer, GSTM1 indicated an approximately 3.24-fold increase (OR=3.24; 95% CI=1.05-10.17). With respect to gastric cancer differentiation, the frequency of the GSTM1 null genotype was linked with a statistically significant increase in risk (3.42-fold) for the high-grade type (OR=3.42; CI=1.02-13.24). Our results indicate that there is no obvious relationship between GSTM1 and GSTT1 polymorphisms and the development of gastric cancer. However, in Korean gastric adenocarcinoma patients the GSTM1 null genotype appears to be associated with a poorer prognosis.

Inhibitory effect of Artemisia capillaris on ethanol-induced cytokines (TNF-alpha, IL-1alpha) secretion in Hep G2 cells.

A human hepatoma cell line, Hep G2 cell, is reliable for the study of alcohol-induced hepatotoxicity. In this study, we investigated the effect of an aqueous extract of Artemisia capillaris Thunb (Compositae) plant (AC) on ethanol (EtOH)-induced cytotoxicity in Hep G2 cells. AC (0.5-5 microg/mL) inhibited the secretion of EtOH-induced interluekin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha). AC also inhibited the EtOH-, IL-1alpha-, and TNF-alpha-induced cytotoxicity. Furthermore, we found that AC inhibited the EtOH-induced apoptosis of Hep G2 cells. These results suggest that AC may prevent the EtOH-induced cytotoxicity through inhibition of the apoptosis of Hep G2 cells.