Yoshiaki Toyama

Osteoclast stimulatory transmembrane protein and dendritic cell–specific transmembrane protein cooperatively modulate cell–cell fusion to form osteoclasts and foreign body giant cells

Cell–cell fusion is a dynamic phenomenon promoting cytoskeletal reorganization and phenotypic changes. To characterize factors essential for fusion of macrophage lineage cells, we identified the multitransmembrane protein, osteoclast stimulatory transmembrane protein (OC‐STAMP), and analyzed its function. OC‐STAMP–deficient mice exhibited a complete lack of cell–cell fusion of osteoclasts and foreign body giant cells (FBGCs), both of which are macrophage‐lineage multinuclear cells, although expression of dendritic cell specific transmembrane protein (DC‐STAMP), which is also essential for osteoclast/FBGC fusion, was normal. Crossing OC‐STAMP–overexpressing transgenic mice with OC‐STAMP–deficient mice restored inhibited osteoclast and FBGC cell–cell fusion seen in OC‐STAMP–deficient mice. Thus, fusogenic mechanisms in macrophage‐lineage cells are regulated via OC‐STAMP and DC‐STAMP.

Comparative study of spinopelvic sagittal alignment between patients with and without degenerative spondylolisthesis.

IntroductionTo date, few studies have focused on spinopelvic sagittal alignment as a predisposing factor for the development of degenerative spondylolisthesis (DS). The objectives of this study were to compare differences in spinopelvic sagittal alignment between patients with or without DS and to elucidate factors related to spinopelvic sagittal alignment.Materials and methodsA total of 100 patients with or without DS who underwent surgery for lumbar spinal canal stenosis were assessed in this study. Fifty patients with DS (DS group) and 50 age- and gender-matched patients without DS (non-DS group) were enrolled. Spinopelvic parameters including pelvic incidence (PI), sacral slope (SS), pelvic tilt (PT), L4 slope, L5 slope, thoracic kyphosis (TK), lumbar lordosis (LL) and sagittal balance were compared between the two groups. In the DS group, the percentage of vertebral slip (% slip) was also measured.ResultsSeveral spinopelvic parameters, PI, SS, L4 slope, L5 slope, TK and LL, in the DS group were significantly greater than those in the non-DS group, and PI had positive correlation with % slip (rxa0=xa00.35, pxa0<xa00.05). Degrees of correlations among spinopelvic parameters differed between the two groups. In the DS group, PI was more strongly correlated with SS (rxa0=xa00.82, pxa0<xa00.001) than with PT (rxa0=xa00.41, pxa0<xa00.01). In the non-DS group, PI was more strongly correlated with PT (rxa0=xa00.73, pxa0<xa00.001) than with SS (rxa0=xa00.38, pxa0<xa00.01).ConclusionsGreater PI may lead to the development and the progression of vertebral slip. Different compensatory mechanisms may contribute to the maintenance of spinopelvic sagittal alignment in DS and non-DS patients.

Prolyl Hydroxylase 3 (PHD3) Modulates Catabolic Effects of Tumor Necrosis Factor-α (TNF-α) on Cells of the Nucleus Pulposus through Co-activation of Nuclear Factor κB (NF-κB)/p65 Signaling

Background: The regulation of PHD expression and function under inflammatory conditions in the nucleus pulposus is unknown. Results: Expression of PHD3 is regulated by TNF-α and IL-1β. PHD3 controls TNF-α activity by modulating NF-κB signaling. Conclusion: PHD3 promotes the catabolic effects of TNF-α on nucleus pulposus cells. Significance: PHD3 may play an important role in pathogenesis of disc disease. Recent studies suggest a differential role of prolyl hydroxylase (PHD) isoforms in controlling hypoxia-inducible factor (HIF)-α degradation and activity in nucleus pulposus (NP) cells. However, the regulation and function of PHDs under inflammatory conditions that characterize disc disease are not yet known. Here, we show that in NP cells, TNF-α and IL-1β induce PHD3 expression through NF-κB. Lentiviral delivery of Sh-p65 and Sh-IKKβ confirms that cytokine-mediated PHD3 expression is NF-κB-dependent. It is noteworthy that although both cytokines induce HIF activity, mechanistic studies using Sh-HIF-1α and PHD3 promoter/enhancer constructs harboring well characterized hypoxia response element (HRE) show lack of HIF involvement in cytokine-mediated PHD3 expression. Loss-of-function studies clearly indicate that PHD3 serves as a co-activator of NF-κB signaling activity in NP cells; PHD3 interacts with, and co-localizes with, p65. We observed that when PHD3 is silenced, there is a significant decrease in TNF-α-induced expression of catabolic markers that include ADAMTS5, syndecan4, MMP13, and COX2, and at the same time, there is restoration of aggrecan and collagen type II expression. It is noteworthy that hydroxylase function of PHDs is not required for mediating cytokine-dependent gene expression. These findings show that by enhancing the activity of inflammatory cytokines, PHD3 may serve a critical role in degenerative disc disease.

Aldehyde-stress resulting from Aldh2 mutation promotes osteoporosis due to impaired osteoblastogenesis

Osteoporosis is a complex disease with various causes, such as estrogen loss, genetics, and aging. Here we show that a dominant‐negative form of aldehyde dehydrogenase 2 (ALDH2) protein, ALDH2*2, which is produced by a single nucleotide polymorphism (rs671), promotes osteoporosis due to impaired osteoblastogenesis. Aldh2 plays a role in alcohol‐detoxification by acetaldehyde‐detoxification; however, transgenic mice expressing Aldh2*2 (Aldh2*2 Tg) exhibited severe osteoporosis with increased levels of blood acetaldehyde without alcohol consumption, indicating that Aldh2 regulates physiological bone homeostasis. Wild‐type osteoblast differentiation was severely inhibited by exogenous acetaldehyde, and osteoblastic markers such as osteocalcin, runx2, and osterix expression, or phosphorylation of Smad1,5,8 induced by bone morphogenetic protein 2 (BMP2) was strongly altered by acetaldehyde. Acetaldehyde treatment also inhibits proliferation and induces apoptosis in osteoblasts. The Aldh2*2 transgene or acetaldehyde treatment induced accumulation of the lipid‐oxidant 4‐hydroxy‐2‐nonenal (4HNE) and expression of peroxisome proliferator‐activated receptor gamma (PPARγ), a transcription factor that promotes adipogenesis and inhibits osteoblastogenesis. Antioxidant treatment inhibited acetaldehyde‐induced proliferation‐loss, apoptosis, and PPARγ expression and restored osteoblastogenesis inhibited by acetaldehyde. Treatment with a PPARγ inhibitor also restored acetaldehyde‐mediated osteoblastogenesis inhibition. These results provide new insight into regulation of osteoporosis in a subset of individuals with ALDH2*2 and in alcoholic patients and suggest a novel strategy to promote bone formation in such osteopenic diseases.

Enhanced susceptibility to lipopolysaccharide-induced arthritis and endotoxin shock in interleukin-32 alpha transgenic mice through induction of tumor necrosis factor alpha

IntroductionThe present study assessed the potential functions of interleukin (IL)-32α on inflammatory arthritis and endotoxin shock models using IL-32α transgenic (Tg) mice. The potential signaling pathway for the IL-32-tumor necrosis factor (TNF)α axis was analyzed in vitro.MethodsIL-32α Tg mice were generated under control of a ubiquitous promoter. Two disease models were used to examine in vivo effects of overexpressed IL-32α: Toll-like receptor (TLR) ligand-induced arthritis developed using a single injection of lipopolysaccharide (LPS) or zymosan into the knee joints; and endotoxin shock induced with intraperitoneal injection of LPS and D-galactosamine. TNFα antagonist etanercept was administered simultaneously with LPS in some mice. Using RAW264.7 cells, in vitro effects of exogenous IL-32α on TNFα, IL-6 or macrophage inflammatory protein 2 (MIP-2) production were assessed with or without inhibitors for nuclear factor kappa B (NFκB) or mitogen-activated protein kinase (MAPK).ResultsSingle injection of LPS, but not zymosan, resulted in development of severe synovitis with substantial articular cartilage degradation in knees of the Tg mice. The expression of TNFα mRNA in inflamed synovia was highly upregulated in the LPS-injected Tg mice. Moreover, the Tg mice were more susceptive to endotoxin-induced lethality than the wild-type control mice 48 hours after LPS challenge; but blockade of TNFα by etanercept protected from endotoxin lethality. In cultured bone marrow cells derived from the Tg mice, overexpressed IL-32α accelerated production of TNFα upon stimulation with LPS. Of note, exogenously added IL-32α alone stimulated RAW264.7 cells to express TNFα, IL-6, and MIP-2 mRNAs. Particularly, IL-32α -induced TNFα, but not IL-6 or MIP-2, was inhibited by dehydroxymethylepoxyquinomicin (DHMEQ) and U0126, which are specific inhibitors of nuclear factor kappa B (NFκB) and extracellular signal regulated kinase1/2 (ERK1/2), respectively.ConclusionsThese results show that IL-32α contributed to the development of inflammatory arthritis and endotoxin lethality. Stimulation of TLR signaling with LPS appeared indispensable for activating the IL-32α-TNFα axis in vivo. However, IL-32α alone induced TNFα production in RAW264.7 cells through phosphorylation of inhibitor kappa B (IκB) and ERK1/2 MAPK. Further studies on the potential involvement of IL-32α-TNFα axis will be beneficial in better understanding the pathology of autoimmune-related arthritis and infectious immunity.

An Essential Role for STAT6-STAT1 Protein Signaling in Promoting Macrophage Cell-Cell Fusion

Background: The signaling leading to macrophage fusion remains largely unknown. Results: STAT6 deficiency completely inhibited macrophage fusion, although STAT1 deficiency or OC-STAMP/DC-STAMP co-expression was sufficient to promote macrophage fusion. Conclusion: The STAT6-STAT1-OC-STAMP/DC-STAMP axis is required for macrophage fusion. Significance: The STAT6-STAT1-OC-STAMP/DC-STAMP axis is a novel pathway leading to macrophage fusion. Macrophage lineage cells such as osteoclasts and foreign body giant cells (FBGCs) form multinuclear cells by cell-cell fusion of mononuclear cells. Recently, we reported that two seven-transmembrane molecules, osteoclast stimulatory transmembrane protein (OC-STAMP) and dendritic cell-specific transmembrane protein (DC-STAMP), were essential for osteoclast and FBGC cell-cell fusion in vivo and in vitro. However, signaling required to regulate FBGC fusion remained largely unknown. Here, we show that signal transducer and activator of transcription 1 (STAT1) deficiency in macrophages enhanced cell-cell fusion and elevated DC-STAMP expression in FBGCs. By contrast, lack of STAT6 increased STAT1 activation, significantly inhibiting cell-cell fusion and decreasing OC-STAMP and DC-STAMP expression in IL-4-induced FBGCs. Furthermore, either STAT1 loss or co-expression of OC-STAMP/DC-STAMP was sufficient to induce cell-cell fusion of FBGCs without IL-4. We conclude that the STAT6-STAT1 axis regulates OC-STAMP and DC-STAMP expression and governs fusogenic mechanisms in FBGCs.

Clinical significance of cartilage biomarkers for monitoring structural joint damage in rheumatoid arthritis patients treated with anti-TNF therapy.

Purpose With the current use of biologics in rheumatoid arthritis (RA), there is a need to monitor ongoing structural joint damage due to the dissociation of articular cartilage damage from disease activity of RA. This study longitudinally analyzed levels of serum cartilage biomarkers during 54 weeks of infliximab therapy, to evaluate the feasibility of biomarkers for monitoring structural joint damage. Methods Subjects comprised 33 patients with early RA and 33 patients with established RA. All patients received 3 mg/kg of infliximab and methotrexate for 54 weeks. Levels of the following serum cartilage markers were measured at baseline and at weeks 14, 22, and 54: hyaluronan (HA); cartilage oligometric matrix protein (COMP); type II collagen (CII)-related neoepitope (C2C); type II procollagen carboxy-propeptide (CPII); and keratin sulfate (KS). Time courses for each biomarker were assessed, and relationships between these biomarkers and clinical or radiographic parameters generally used for RA were investigated. Results Levels of CRP, MMP-3, DAS28-CRP, and annual progression of TSS were improved to similar degrees in both groups at week 54. HA and C2C/CPII were significantly decreased compared to baseline in the early RA group (p<0.001), whereas HA and COMP, but not C2C/CPII, were decreased in the established RA group. Strikingly, serum C2C/CPII levels were universally improved in early RA, regardless of EULAR response grade. Both ΔHA and ΔC2C/CPII from baseline to week 54 correlated significantly with not only ΔCRP, but also ΔDAS28 in early RA. Interestingly, when partial correlation coefficients were calculated by standardizing CRP levels, the significant correlation of ΔHA to ΔDAS28 disappeared, whereas correlations of ΔC2C/CPII to ΔDAS28, ΔJNS, and ΔHAQ remained significant. These results suggest a role of ΔC2C/CPII as a marker of ongoing structural joint damage with the least association with CRP, and that irreversible cartilage damage in established RA limits restoration of the C2C/CPII level, even with tight control of joint inflammation. Conclusion The temporal course of C2C/CPII level during anti-TNF therapy indicates that CII turnover shifts toward CII synthesis in early RA, but not in established RA, potentially due to irreversible cartilage damage. ΔC2C/CPII appears to offer a useful marker reflecting ongoing structural joint damage, dissociated from inflammatory indices such as CRP and MMP-3.

PDGFBB promotes PDGFRα-positive cell migration into artificial bone in vivo

Bone defects caused by traumatic bone loss or tumor dissection are now treated with auto- or allo-bone graft, and also occasionally by artificial bone transplantation, particularly in the case of large bone defects. However, artificial bones often exhibit poor affinity to host bones followed by bony union failure. Thus therapies combining artificial bones with growth factors have been sought. Here we report that platelet derived growth factor bb (PDGFBB) promotes a significant increase in migration of PDGF receptor α (PDGFRα)-positive mesenchymal stem cells/pre-osteoblastic cells into artificial bone in vivo. Growth factors such as transforming growth factor beta (TGFβ) and hepatocyte growth factor (HGF) reportedly inhibit osteoblast differentiation; however, PDGFBB did not exhibit such inhibitory effects and in fact stimulated osteoblast differentiation in vitro, suggesting that combining artificial bones with PDGFBB treatment could promote host cell migration into artificial bones without inhibiting osteoblastogenesis.

Matrix metalloproteinase 13 in the ligamentum flavum from lumbar spinal canal stenosis patients with and without diabetes mellitus

BackgroundLumbar spinal canal stenosis (LSCS) is one of the most common spinal disorders in the elderly, and ligamentum flavum (LF) hypertrophy is an important cause of LSCS. Matrix metalloproteinase 13 (MMP13) can degrade fibrillar collagens and elastic microfibrils, and is involved in inflammation and fibrosis. The purpose of this study was to compare the expression of MMP13 in the LF from LSCS patients with diabetes mellitus [DM (+)] with that in the LF from patients without DM [DM (−)] and to analyze the relationship among DM, MMP13 expression, and LF hypertrophy.MethodsLFs from 11 DM (+) and 24 DM (−) LSCS patients were analyzed in this study. Histology analysis using hematoxylin and eosin and Masson’s trichrome stain was performed for each LF. The expression of MMP13 was analyzed by quantitative real-time PCR. The thickness of LF was measured by CT.ResultsIn the LF from DM (+) LSCS patients, the elastic fibers were more disorganized and had lower volumes than in the LF from DM (−) LSCS patients, while more fibrotic tissue was observed in the LF from DM (+) than from DM (−) LSCS patients. MMP13 expression was significantly higher in the LF from DM (+) LSCS patients (0.46xa0±xa00.61 vs. 0.05xa0±xa00.09, Pxa0=xa00.002). The LF from the DM (+) LSCS patients was significantly thicker than that from the DM (−) LSCS patients (5.0xa0±xa00.9 vs. 3.1xa0±xa00.8xa0mm, Pxa0<xa00.01), and the thickness was correlated with the expression of MMP13 (correlation coefficientxa0=xa00.43, Pxa0=xa00.01, Pearsons correlation test).ConclusionDM-related MMP13 expression can be one of the factors contributing to fibrosis and hypertrophy of the LF. Further research on the mechanism of this process may lead to new therapies for LF hypertrophy.

GRIP1 enhances estrogen receptor α-dependent extracellular matrix gene expression in chondrogenic cells

OBJECTIVEnThe role of postmenopause on the pathogenesis of cartilage degeneration has been an open question. We assessed cartilage degeneration in estrogen receptor (ER)alpha null mice and examined the role of glucocorticoid receptor-interacting protein 1 (GRIP1) in the ERalpha-dependent transcription of a type II collagen gene (col2a1) with special reference to a crosstalk with the transforming growth factor (TGF)-beta signaling pathway.nnnMETHODSnThe vertebral cartilaginous endplate from female ERalpha null mice was subjected to histological analyses. Col2a1 expression of primary chondrocytes (PCs) obtained from ERalpha null mice after 17beta-estradiol (E(2)) and TGF-beta1 stimulation was examined by reverse transcription polymerase chain reaction (RT-PCR). Estrogen response element (ERE) or col2a1 promoter-enhancer luciferase reporter system was used to investigate the crosstalk among ERalpha, GRIP1, and MKK6. Col2a1 expression and glycosaminoglycan (GAG) content were measured in ATDC5 cells treated with GRIP1 small interfering RNA (siRNA).nnnRESULTSnERalpha deficiency clearly accelerated impairment of the vertebral cartilaginous endplate. E(2) and TGF-beta1 stimulation increased col2a1 expression in PC from wild-type mice, but not that from ERalpha null mice. The same stimulation increased the col2a1 promoter-enhancer reporter activity, and the elevated activity was decreased by dominant-negative ERalpha and p38 mitogen-activated protein kinase (MAPK) inhibitor. GRIP1 increased the E(2)-dependent ERE activation in the presence of ERalpha and constitutive-active MKK6. GRIP1 siRNA repressed col2a1 expression and GAG production in ATDC5 cells.nnnCONCLUSIONSnCrosstalks between ERalpha/GRIP1 and TGF-beta/MKK6/p38 MAPK pathway have protective roles on cartilage metabolism via regulating the extracellular matrices expression. The finding may lead to the development of a novel therapeutic approach for cartilage degeneration.