Bitnara Lee

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 × 10−7) and rs30187 (p=7.16 × 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=4.71×10−7, CCCC: p=8.56×10−6). 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.

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-1β

IL-1β is a key mediator of bone resorption in inflammatory settings, such as rheumatoid arthritis (RA). IL-1β 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-1β also has suppressive properties on osteoclastogenesis in primary human peripheral blood monocytes and RA synovial macrophages. Early addition of IL-1β, prior to or together with RANKL, strongly inhibited human osteoclastogenesis as assessed by generation of TRAP+ multinucleated cells. IL-1β 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-1β rendered early-stage human OCPs refractory to RANK stimulation. Similar inhibitory effects of IL-1β were observed using RA synovial macrophages. One mechanism of RANK inhibition was IL-1β–induced proteolytic shedding of the M-CSF receptor c-Fms that is required for RANK expression. These results identify a homeostatic function of IL-1β 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.

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 β (C/EBPβ), 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.

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.

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.

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.

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/EBPβ 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/EBPβ genes, leading to IL-23 production.