A dual role of TGF-ß in human osteoclast differentiation mediated by Smad1 versus Smad3 signaling.

TGF-ß1 is highly expressed in the synovial tissue of patients with rheumatoid arthritis and is known as a cytokine that plays an important role in tissue repair and immune cell regulation. However, the role of TGF-ß1 is still unclear in osteoclastogenesis. In this study, we examined the effect of TGF-ß1 on osteoclast differentiation and the underlying mechanism using healthy human peripheral blood monocytes. TGF-ß1 was found to inhibit osteoclast differentiation and decrease the expression of osteoclast-specific genes such as acid phosphatase 5, tartrate resistant and cathepsin K. Levels of NFAT1, an important transcription factor in osteoclastogenesis, were also reduced. In addition, TGF-ß1 suppressed receptor activator of NF-κB (RANK) ligand-induced NF-κB and p38 MAPK signaling. Inhibition of osteoclast differentiation by TGF-ß1 was reversed by 1 µM SB431542 (an inhibitor of ALK4/5/7), which inhibited TGF-ß1-induced phosphorylation of SMAD1, but not that of SMAD3. TGF-ß1 also restricted RANK expression, and this was partially reversed by 1 µM SB431542. In contrast, the inhibition of SMAD3 by SIS3 (an inhibitor of SMAD3) reduced the osteoclast formation. TGF-ß1 has both inhibitory and stimulatory effects on human osteoclast differentiation, and that these opposing functions are mediated by SMAD1 and SMAD3 signaling, respectively.

Poly-γ-glutamic acid suppresses osteoclastogenesis in human osteoclast precursors and prevents joint damage in a collagen-induced murine arthritis model.

Poly-γ-glutamic acid (γ-PGA), a natural polymer derived from Bacillus subtilis, shows anti-inflammatory activity. However, the effects of γ-PGA on osteoclasts, which are important cells for joint destruction in inflammatory diseases such as rheumatoid arthritis (RA), have not yet been reported. In this study, we show that γ-PGA markedly inhibits osteoclast differentiation in normal PBMC-derived osteoclast precursors and in synovial fluid macrophages of patients with RA. γ-PGA also reduces RANK expression by down-regulating M-CSF receptors. Additionally, oral administration of γ-PGA attenuated bone destruction in a collagen-induced arthritis (CIA) model, demonstrating decreases in inflammation, cartilage damage, and osteoclast formation in histological analyses. Taken together, these data suggest that γ-PGA could be a good candidate for therapeutic prevention of joint destruction in RA.

IL-17A induces osteoblast differentiation by activating JAK2/STAT3 in ankylosing spondylitis.

BACKGROUND: IL-17A has recently emerged as a potential target that regulates the extensive inflammation and abnormal bone formation observed in ankylosing spondylitis (AS). Blocking IL-17A is expected to inhibit bony ankylosis. Here, we investigated the effects of anti IL-17A agents in AS. METHODS: TNFα, IL-17A, and IL-12/23 p40 levels in serum and synovial fluid from patients with ankylosing spondylitis (AS), rheumatoid arthritis (RA), osteoarthritis (OA), or healthy controls (HC) were measured by ELISA. Bone tissue samples were obtained at surgery from the facet joints of ten patients with AS and ten control (Ct) patients with noninflammatory spinal disease. The functional relevance of IL-17A, biological blockades, Janus kinase 2 (JAK2), and non-receptor tyrosine kinase was assessed in vitro with primary bone-derived cells (BdCs) and serum from patients with AS. RESULTS: Basal levels of IL-17A and IL-12/23 p40 in body fluids were elevated in patients with AS. JAK2 was also highly expressed in bone tissue and primary BdCs from patients with AS. Furthermore, addition of exogenous IL-17A to primary Ct-BdCs promoted the osteogenic stimulus-induced increase in ALP activity and mineralization. Intriguingly, blocking IL-17A with serum from patients with AS attenuated ALP activity and mineralization in both Ct and AS-BdCs by inhibiting JAK2 phosphorylation and downregulating osteoblast-involved genes. Moreover, JAK2 inhibitors effectively reduced JAK2-driven ALP activity and JAK2-mediated events. CONCLUSIONS: Our findings indicate that IL-17A regulates osteoblast activity and differentiation via JAK2/STAT3 signaling. They shed light on AS pathogenesis and suggest new rational therapies for clinical AS ankylosis.

Identification and characterization of human bone-derived cells.

This study was designed to identify and characterize primary bone-derived cells (BdCs) and investigate the potential role of osteoblast differentiation. Primary BdCs were isolated from surgical bone for comparative analysis with mesenchymal stem cells (MSCs) and fetal osteoblasts (FOBs) and for potential differentiation to mature osteoblasts. Using three different cells, we successfully cultivated human osteoblast differentiation and activity which were evaluated using microarray and biochemical methods. BdCs are more correlated to MSCs in bioinformatics result and similar with FOBs in gene expression. In particular, Osterix, osteoprogenitor marker, was high expressed in BdCs, while the expression in MSCs and FOBs were very low. Furthermore, BdCs exhibited a marked alkaline phosphatase (ALP) expression, early stage of osteogenic marker, and retained osteogenic properties and physiological changes into maturation as in FOBs. BdCs also showed an increase in bone morphogenic protein 2 (BMP2), osteopontin (OPN), and osteocalcin (OCN) mRNA expressions during differentiation. This study suggests that BdCs may be osteoprogenitor cells or undifferentiated preosteoblasts with strong capacity to differentiate toward mature osteoblasts.

Effects of the predominant bacteria from meju and doenjang on the production of volatile compounds during soybean fermentation.

We inoculated five starter candidates, Enterococcus faecium, Tetragenococcus halophilus, Bacillus licheniformis, Staphylococcus saprophyticus, and Staphylococcus succinus, into sterilized soybeans to predict their effectiveness for flavor production in fermented soybean foods. All of the starter candidates exhibited sufficient growth and acid production on soybean cultures. Twenty-two volatile compounds, such as acids, alcohols, carbonyls, esters, furans, and pyrazines, were detected from the control and starter candidate-inoculated soybean cultures. Principal component analysis of these volatile compounds concluded that E. faecium and T. halophilus produced a similar profile of volatile compounds to soybeans with no dramatic differences in soybean flavor. B. licheniformis and S. succinus produced the crucial volatile compounds that distinguish the flavor profiles of soybean. During soybean fermentation, phenylmethanol and 2,3,5,6-tetramethylpyrazine were determined as odor notes specific to B. licheniformis and 3-methylbutyl acetate as an odor note specific to S. succinus.

A novel role for bone-derived cells in ankylosing spondylitis: Focus on IL-23.

The main aim of this study are to explore the role of bone-derived cells (BdCs) in ankylosing spondylitis (AS) and determine the underlying molecular mechanisms of IL-23 production. Primary BdCs were isolated from diced bone of facet joints obtained during surgery from seven AS patients and seven disease control (Ct) patients. Osteoblastic activity of BdCs was assessed by measuring their alkaline phosphatase activity and by alizarin red staining. Osteoblast and endoplasmic reticulum (ER) stress-related genes were assessed by quantitative PCR, immunoblotting, immunofluorescence, and immunohistochemistry. In addition, expression of IL-23 in response to BIX (selective BIP inducer X)-induced ER stress was evaluated by qPCR and ELISA. Protein interaction and binding to IL-23 promoter were confirmed by Immunoprecipitation and Chromatin immunoprecipitation, respectively. Transcript levels of genes involved in osteoblast function, as well as of the ER stress marker were higher in the AS group than the Ct group, and elevated RUNX2, BiP and IL-23 expression were observed in the BdCs, serum, and bone biopsies from the AS group. BIX-induced ER stress stimulated osteoblastic activity and IL-23 secretion by upregulating RUNX2 expression. Furthermore, in AS BdCs, RUNX2 interacted with C/EBPß to bind to IL-23 promoter and RUNX2 knockdown suppressed IL-23 secretion. These finding may provide a molecular mechanism involved in sustained ER stress in AS BdCs stimulates the activation of RUNX2 and C/EBPß genes, leading to IL-23 production.

Genetic Diversity and Antibiotic Resistance of Enterococcus faecalis Isolates from Traditional Korean Fermented Soybean Foods.

Eighty-five Enterococcus faecalis isolates collected from animals (40 isolates), meju (a Korean fermented soybean product; 27 isolates), humans (10 isolates), and various environmental samples (8 isolates) were subjected to multilocus sequence typing (MLST) to identify genetic differences between samples of different origins. MLST analysis resulted in 44 sequence types (STs), and the eBURST algorithm clustered the STs into 21 clonal complexes (CCs) and 17 singletons. The predominant STs, ST695 (21.1%, 18/85) and ST694 (9.4%, 8/85), were singletons, and only contained isolates originating from meju. None of the STs in the current study belonged to CC2 or CC9, which comprise clinical isolates with high levels of antibiotic resistance. The E. faecalis isolates showed the highest rates of resistance to tetracycline (32.9%), followed by erythromycin (9.4%) and vancomycin (2.4%). All isolates from meju were sensitive to these three antibiotics. Hence, MLST uncovered genetic diversity within E. faecalis, and clustering of the STs using eBURST revealed a correlation between the genotypes and origins of the isolates.

Safety and technological characterization of coagulase-negative staphylococci isolates from traditional Korean fermented soybean foods for starter development.

To select starters for the production of meju and doenjang, traditional Korean fermented soybean foods, we assessed the safety and technological properties of their predominant isolates, Staphylococcus saprophyticus, Staphylococcus succinus and Staphylococcus xylosus. Phenotypic antibiotic resistance, hemolysis and biofilm formation were strain-specific. None of the S. succinus isolates exhibited antibiotic resistance or hemolytic activities. Thirty-three selected strains, identified through safety assessments of 81 coagulase-negative staphylococci (CNS) isolates, produced cadaverine, putrescine, and tyramine, but not histamine in the laboratory setting. The production of these three biogenic amines may, however, be insignificant considering the high levels of tyramine produced by the control, Enterococcus faecalis. The 33 CNS strains could grow on tryptic soy agar containing 21% NaCl (w/v), exhibited acid producing activity at 15% NaCl, and expressed strain-specific protease and lipase activities. S. succinus 14BME1, the selected starter candidate, produced significant amounts of benzeneacetic acid, 2,3-butanediol, trimethylpyrazine, and tetramethylpyrazine through soybean fermentation.

TREM-1, a negative regulator of human osteoclastogenesis.

Triggering receptor expressed on myeloid cells (TREMs) are a family of cell surface receptors that play important roles in innate and adaptive immunity. Among them, TREM-2 has been extensively studied for its role in osteoclast differentiation and its essential role in human osteoclastogenesis has been well established. However, much less has been discovered about the role of TREM-1 in human osteoclast differentiation. In this study, we investigate the role of TREM-1 in human osteoclast differentiation. Consistent with previous reports, TREM-2 expression was strongly increased during the generation of human osteoclast precursors. In contrast, TREM-1 expression was decreased during the generation of human osteoclast precursors. Stimulation of TREM-1 using agonistic anti-TREM-1 antibody resulted in suppression of RANKL-induced osteoclastogenesis, as evidenced by diminished formation of TRAP+ multinucleated cells. In addition, TREM-1 stimulation strongly suppressed RANKL-induced expression of osteoclast-related genes such as cathepsin K and NFATc1. TREM-1 stimulation also down-regulated gene expression and cell surface expression of M-CSF receptor that is essential for osteoclast differentiation and survival. In synovial fluid macrophages of rheumatoid arthritis (RA) patients, TREM-1 stimulation suppressed osteoclastogenesis. In conclusion, we demonstrate that TREM-1 acts as a negative regulator of human osteoclast differentiation and identify a novel mechanism of negative regulation of osteoclastogenesis that plays a role in inflammation.

1α,25-Dihydroxyvitamin D3 upregulates HIF-1 and TREM-1 via mTOR signaling.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is induced by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) in human monocytes/macrophages and epithelial cells. However, little information is available regarding the mechanism of 1,25(OH)2D3-induced TREM-1 expression in human monocytes/macrophages. In this study, 1,25(OH)2D3 was shown to strongly upregulate hypoxia-inducible transcription factor (HIF) in PMA-differentiated U937 cells. However, HIF was not mainly involved in 1,25(OH)2D3-induced TREM-1 expression. Instead, 1,25(OH)2D3-induced expression of TREM-1 was inhibited by rapamycin, a specific inhibitor of the mammalian target of rapamycin (mTOR) signaling pathway, indicating the involvement of mTOR. Induction of HIF proteins by 1,25(OH)2D3 was also inhibited by rapamycin. In addition, 1,25(OH)2D3 induced the phosphorylation of p70S6 kinase, a target of mTOR complex 1 (mTORC1). Our results suggest that 1,25(OH)2D3 induces the expression of TREM-1 through the mTOR signaling pathway in human macrophages.

Regulation of TREM-1 expression by 1,25-dihydroxyvitamin D3 in human monocytes/macrophages.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a recently identified cell surface receptor that is expressed mainly on monocytes and neutrophils, and acts as an amplifier of immune responses. In this study, 1,25(OH)2D3 strongly upregulated the expression of TREM-1 in human monocytes and macrophages. 1,25(OH)2D3 stimulated TREM-1 mRNA expression by augmenting transcription, and not by inhibiting mRNA degradation. The upregulated expression of TREM-1 by 1,25(OH)2D3 was dependent on the NF-κB signaling pathway and required new protein synthesis in differentiated U937 macrophages. Our results show that 1,25(OH)2D3 can affect the innate and inflammatory responses by upregulating TREM-1 expression, and suggest that 1,25(OH)2D3 may function as an enhancer of the innate immune response by upregulating TREM-1 expression, in addition to inducing the antimicrobial peptide cathelicidin.

1,25-Dihydroxyvitamin D3 inhibits directly human osteoclastogenesis by down-regulation of the c-Fms and RANK expression.

OBJECTIVE: 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is a key molecule to maintain calcium homeostasis and bone metabolism. It was recently reported that 1,25(OH)2D3 directly inhibited osteoclast differentiation in mouse bone marrow cells and human bone marrow-derived colony-forming unit granulocyte macrophage (CFU-GM) cells. However, the direct effects of 1,25(OH)2D3 and its affecting mechanisms on the osteoclast differentiation of human osteoclast precursors remain largely unknown. In this study, we examined the direct effects of 1,25(OH)2D3 on the osteoclastogenesis of human peripheral blood (PB) osteoclast precursors. METHODS: In vitro osteoclastogenesis assays were performed using osteoclast precursors from normal PB. The gene expressions were analyzed using real-time PCR. The cell surface proteins, including c-Fms and RANK, were measured by flow cytometry. RESULTS: 1,25(OH)2D3 strongly inhibited osteoclast differentiation and it suppressed the expression of RANK in the human PB osteoclast precursors. One mechanism of RANK inhibition by 1,25(OH)2D3 is down-regulation of the M-CSF receptor c-Fms, which is required for the expression of RANK. In contrast to the previous reports on mouse osteoclast precursors, 1,25(OH)2D3 did not affect the expression of c-Fos. Parallel to the inhibition of osteoclastogenesis, 1,25(OH)2D3 increased the expression and phosphorylation of CCAAT enhancer-binding protein ß (C/EBPß), which is a recently discovered inhibitor of osteoclastogenesis. CONCLUSIONS: Our results show that 1,25(OH)2D3 inhibits human osteoclastogenesis by decreasing the RANK+ osteoclast precursors, and we suggest that 1,25(OH)2D3 may be a powerful therapeutic agent for treating inflammation-induced bone disease that shows excessive osteoclast activation.

Upregulation of interleukin-1ß production by 1,25-dihydroxyvitamin D(3) in activated human macrophages.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) plays important roles in the immune system. In contrast to its well known function in the adaptive immune system, much less is known about the immunoregulatory effects of 1,25(OH)(2)D(3) in the innate immune system, especially on activated human macrophages. Here we found that 1,25(OH)(2)D(3) strongly stimulated the production of interleukin-1ß (IL-1ß) in PMA-differentiated U937 cells and human monocyte-derived macrophages treated with lipopolysaccharide (LPS) or PMA. In this study, Erk1/2 appeared to mediate 1,25(OH)(2)D(3)-induced expression of IL-1ß. Parallel to the increased production of IL-1ß, 1,25(OH)(2)D(3) increased the expression and phosphorylation of the CCAAT enhancer-binding protein ß (C/EBPß), which is one of the key transcriptional regulatory factors for IL-1ß transcription. These results suggest that 1,25(OH)(2)D(3) may function as a proinflammatory molecule in inflammatory macrophages.

T-cell responses to versican in ankylosing spondylitis.

The objective of our study was to undertake a systematic analysis of the T-cell response to the proteoglycan versican G1-globular domain (VG1) in ankylosing spondylitis (AS) as immunity to VG1 in mice can induce a pathology closely resembling AS. Peripheral blood lymphocytes from 36 AS patients and 33 healthy controls were incubated with recombinant human VG1 in culture for 6 h. T-cell responses were assessed by FACS analyses using mAb against surface expression of the activation marker CD69 and against the intracellular cytokines interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha. T cells activated by exposure to versican were determined by assessing the percentage of CD4+ or CD8+ T cells that were CD69/cytokine double-positive cells as compared to isotype control staining. In the AS patients, exposure to VG1 resulted in increased expression by CD4+ T cells of IFN-gamma in 55.6% of patients and of TNF-alpha in 52.8% of patients. In the controls, only 36.4% of subjects demonstrated an IFN-gamma response and 36.4% demonstrated a TNF-alpha response (P value 0.148, 0.227, respectively). With respect to CD8+ T-cell responses, versican stimulation enhanced IFN-gamma expression in 44.4% of AS patients and 39.4% of controls, and enhanced TNF-alpha response in 50.0% of AS patients and 39.4% of controls (P value 0.620, 0.327, respectively). Although, there was no statistically significant difference in the magnitude of the IFN-γ or TNF secretion by CD4+ T cells and CD8+ T cells between AS and controls, our results demonstrate an enhanced T-cell response to VG1 in AS.

Genetic studies of ankylosing spondylitis in Koreans confirm associations with ERAP1 and 2p15 reported in white patients.

OBJECTIVE: Investigators from the Australo-Anglo-American Spondyloarthritis Consortium have reported additional genes associated with ankylosing spondylitis (AS) susceptibility including IL1R2, ANTXR2, and gene deserts at 2p15 and 21q22. We evaluated these new candidate genes in a large cohort of Korean patients with AS. METHODS: A group of 1164 patients with AS and 752 healthy controls were enrolled for our study. Eight single-nucleotide polymorphisms (SNP) were analyzed to define genetic association with AS by MassARRAY system. RESULTS: Significant positive associations of AS with endoplasmic reticulum aminopeptidase 1 SNP, rs27037 (p = 1.31 × 10(-4)), and rs27434 (p = 4.59 × 10(-6)), were observed. The rs10865331 of gene desert at 2p15 also showed a significant association with AS (p = 4.63 × 10(-5)). CONCLUSION: This is the first confirmation in a nonwhite population that genetic polymorphisms of rs27037, rs27434, and rs10865331 are associated with AS, implicating common pathogenetic mechanisms in Korean and white patients with AS.

Direct inhibition of human RANK+ osteoclast precursors identifies a homeostatic function of IL-1beta.

IL-1ß is a key mediator of bone resorption in inflammatory settings, such as rheumatoid arthritis (RA). IL-1ß promotes osteoclastogenesis by inducing RANKL expression on stromal cells and synergizing with RANKL to promote later stages of osteoclast differentiation. Because IL-1Rs share a cytosolic Toll-IL-1R domain and common intracellular signaling molecules with TLRs that can directly inhibit early steps of human osteoclast differentiation, we tested whether IL-1ß also has suppressive properties on osteoclastogenesis in primary human peripheral blood monocytes and RA synovial macrophages. Early addition of IL-1ß, prior to or together with RANKL, strongly inhibited human osteoclastogenesis as assessed by generation of TRAP(+) multinucleated cells. IL-1ß acted directly on human osteoclast precursors (OCPs) to strongly suppress expression of RANK, of the costimulatory triggering receptor expressed on myeloid cells 2 receptor, and of the B cell linker adaptor important for transmitting RANK-induced signals. Thus, IL-1ß rendered early-stage human OCPs refractory to RANK stimulation. Similar inhibitory effects of IL-1ß were observed using RA synovial macrophages. One mechanism of RANK inhibition was IL-1ß-induced proteolytic shedding of the M-CSF receptor c-Fms that is required for RANK expression. These results identify a homeostatic function of IL-1ß in suppressing early OCPs that contrasts with its well-established role in promoting later stages of osteoclast differentiation. Thus, the rate of IL-1-driven bone destruction in inflammatory diseases, such as RA, can be restrained by its direct inhibitory effects on early OCPs to limit the extent of inflammatory osteolysis.

ARTS1 polymorphisms are associated with ankylosing spondylitis in Koreans.

OBJECTIVE: To test the association between ARTS1 polymorphisms and Koreans with ankylosing spondylitis (AS). METHODS: All patients and controls were Korean. 872 patients with AS fulfilling the modified New York criteria and 403 healthy controls were genotyped for five single nucleotide polymorphisms (SNPs), rs27044, rs17482078, rs10050860, rs30107 and rs2287987, known to be associated with AS in Caucasians. RESULTS: SNPs rs27044 (p=9.37 x 10(-7)) and rs30187 (p=7.16 x 10(-6)) of ARTS1 were significantly associated with AS in Koreans. There was no significant association for rs17482078, rs10050860 and rs2287987. Two four-marker haplotypes were found to be associated with AS (GCCT: p=4.71 x 10(-7), CCCC: p=8.56 x 10(-6)). CONCLUSIONS: This is first confirmation in a non-Caucasian population that genetic polymorphisms in ARTS1 are associated with AS, implicating common pathogenetic mechanisms in Korean and Caucasian patients with AS.

IL-23R polymorphisms in patients with ankylosing spondylitis in Korea.

OBJECTIVE: IL23R polymorphisms have been shown to have a significant association with ankylosing spondylitis (AS). To date, these studies have been restricted to Caucasian patients with AS. Our study addresses this relationship in Korean patients with AS. METHODS: A total of 451 patients with AS and 392 ethnically matched healthy controls were enrolled. All patients were native Koreans with AS satisfying the modified New York criteria. In total, 10 single nucleotide polymorphisms (SNP) within the IL-23R gene cluster were genotyped. RESULTS: No IL-23R SNP were found to be associated with AS in Koreans. CONCLUSION: The association of IL23R and AS that is seen in Caucasian patients with AS is not present in Korean patients with AS.