Effect of abatacept treatment on serum osteoclast-related biomarkers in patients with rheumatoid arthritis (RA): A multicenter RA ultrasound prospective cohort in Japan.

ABSTRACT: We evaluated the effect of abatacept treatment on osteoclast-related biomarkers and explored whether the biomarkers are associated with the therapeutic response in rheumatoid arthritis (RA) patients treated with abatacept.We enrolled 44 RA patients treated with abatacept from a multicenter prospective ultrasound cohort study of patients who received biologic or targeted synthetic disease-modifying antirheumatic drug therapy. We evaluated the disease activity score (DAS) 28-CRP (C-reactive protein), musculoskeletal ultrasound scores including the total grayscale score (GS)/power Doppler (PD) score and the serum concentrations of isoform 5b of tartrate-resistant acid phosphate (TRACP-5b) and soluble receptor activator of nuclear factor-κB ligand (sRANKL) at baseline and at 3 and 6 months of treatment. "PD responder" was defined as a patient whose Δtotal PD score over 6 months was greater than the median change of that.Abatacept significantly improved DAS28-CRP as well as the total GS/PD score over 6 months. Serum TRACP-5b was significantly elevated and serum sRANKL was significantly decreased at 6 months (P < .0001 and P < .01, respectively). At 6 months, serum sRANKL was significantly decreased in the patients who achieved DAS28-CRP remission and the PD responders but not in those who did not. However, serum TRACP-5b rose regardless of the therapeutic response.Among RA patients treated with abatacept, serum sRANKL decreased in the patients with a good therapeutic response, but serum TRACP-5b elevated paradoxically regardless of the therapeutic response.

Postoperative expressions of TRACP5b and CA125 in patients with breast cancer and their values for monitoring bone metastasis.

PURPOSE: To explore the diagnostic values of serum tartrate-resistant acid phosphatase 5b (TRACP5b) and serum carbohydrate antigen 125 (CA125) for bone metastasis of breast cancer. METHODS: 118 patients pathologically diagnosed with breast cancer in the second People's Hospital of Lianyungang from September 2014 to June 2017 were selected. Among them, 60 patients who were confirmed with bone metastasis by whole-body bone imaging combined with clinical manifestations and other imaging methods were included in a bone metastasis group, and 58 patients who were confirmed without bone metastasis were included in a non-bone metastasis group. Another 61 patients who were pathologically confirmed with benign breast lesion formed a benign lesion group. Enzyme-linked immunosorbent assay (ELISA) was used to detect TRACP5b level and electrochemiluminescence (ECL) was used to detect CA125 level. RESULTS: The expression levels of TRACP5b and CA125 in the bone metastasis group were significantly higher than those in the non-bone metastasis and benign lesion groups (p<0.05), and the expression levels in the non-bone metastasis group were higher than those in the benign lesion group (p<0.05). In bone metastasis of breast cancer, the expression level of TRACP5b was correlated with the number of tumor nodules, lymph node metastasis, tumor local infiltration and TNM staging (p<0.05), while the expression level of CA125 was correlated with the number of tumor nodules, lymph node metastasis and TNM staging (p<0.05). Logistic regression analysis showed that TNM staging, estrogen receptor (ER), TRACP5b, and CA125 were risk factors for bone metastasis of breast cancer patients. CONCLUSION: In conclusion, TRACP5b and CA125 may be involved in the occurrence and progression of bone metastasis of breast cancer. Detection of TRACP5b and CA125 has good sensitivity and specificity in diagnosing bone metastasis of breast cancer, so TRACP5b and CA125 may become new biomarkers for diagnosing the disease.

MicroRNA-21 affects mechanical force-induced midpalatal suture remodelling.

OBJECTIVES: miR-21 can promote osteoblast differentiation of periodontal ligament stem cells. However, the effect of miR-21 on bone remodelling in the midpalatal suture is unclear. This study aimed to elucidate the effects of miR-21 on the midpalatal suture bone remodelling by expanding the palatal sutures. MATERIALS AND METHODS: miR-21 deficient (miR-21-/- ) and wild-type (WT) mice were used to establish animal models by expanding the palatal sutures. Micro-CT, haematoxylin-eosin (HE) staining, tartrate-resistant acid phosphatase (TRAP) staining, fluorescence labelling and immunohistochemistry were used to investigate the function of miR-21 in midpalatal suture bone remodelling. Besides, bone mesenchymal stem cells (BMSCs) derived from both miR-21-/- and WT mice were cultured. The MTT, CCK8, EdU analysis, transwell and wound healing test were used to assess the effects of miR-21 on the characteristics of cells. RESULTS: The expression of ALP was suppressed in miR-21-/- mice after expansion except 28 days. The expression of Ocn in WT mice was much higher than that of miR-21-/-  mice. Besides, with mechanical force, miR-21 deficiency downregulated the expression of Opg, upregulated the expression of Rankl, and induced more osteoclasts as TRAP staining showed. After injecting agomir-21  to miR-21-/- mice, the expression of Alp, Ocn and Opg/Rankl were rescued. In vitro, the experiments suggested that miR-21 deficiency reduced proliferation and migration ability of BMSCs. CONCLUSIONS: The results showed that miR-21 deficiency reduced the rate of bone formation and prolonged the process of bone formation. miR-21 regulated the bone resorption and osteoclastogenesis by affecting the cell abilities of proliferation and migration.

Constant hypoxia inhibits osteoclast differentiation and bone resorption by regulating phosphorylation of JNK and IκBα.

BACKGROUND: Osteoclasts are responsible for the bone loss in rheumatoid arthritis (RA). Hypoxia has been suggested to play key roles in pathological bone loss. However, the current understanding of the effects of hypoxia on osteoclastogenesis is controversial. Effects of hypoxia on both the formation and function of osteoclasts requires examination. In the current study, we aimed to explore the effect of hypoxia on osteoclast differentiation and the underlying mechanisms. METHODS: RAW264.7 cells and murine bone-marrow-derived monocytes were used to induce osteoclastogenesis in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL). Hypoxic conditions were maintained in a hypoxic chamber at 5% CO2 and 1% O2, balanced with N2. Osteoclasts were detected by tartrate-resistant acid phosphatase (TRAP) staining. A bone resorption assay was carried out in vitro using bone slices. RT-PCR was conducted to detect osteoclast markers and transcription factors. The phosphorylation of nuclear factor-κBα (IκBα), c-Jun N-terminal kinase (JNK), extracellular regulated protein kinase (ERK), and p38 was detected by western blotting. Mann-Whitney U test or Student's t test was used to compare differences between the two groups. RESULTS: TRAP staining and the bone resorption assay revealed that hypoxia-restrained osteoclast differentiation and bone resorption. Expression of osteoclast markers including cathepsin K, RANK, and TRAP decreased during osteoclast differentiation under hypoxic conditions (all P < 0.05). Hypoxia at 1% O2 did not affect cell viability, whereas it dramatically abated RANKL-dependent phosphorylation of the JNK-mitogen-activated protein kinases (MAPK) and IκBα pathways. Moreover, the expression of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) was inhibited under hypoxic conditions (all P < 0.05). CONCLUSIONS: These results suggest that constant hypoxia at 1% O2 significantly restrains osteoclast formation and resorbing function without affecting cell viability. Constant hypoxia might inhibit RANKL-induced osteoclastogenesis by regulating NFATc1 expression via interfering the phosphorylation of JNK and IκBα.

Manipulation of osteoclastogenesis: Bioactive multiphasic silica/collagen composites and their effects of surface and degradation products.

The intent of the present study was to demonstrate that multiphasic silica/collagen xerogels are able to manipulate cellular processes. These xerogels were prepared by a sol-gel approach allowing the incorporation of mineral phases. The resulting nanocomposites are designed as biomaterial for bone regeneration. Human osteoclasts derived from peripheral blood mononuclear cells were cultured both indirectly and directly, either in presence of different xerogel types or on their surface, to investigate the factor with the main influence on osteoclastogenesis. To this end, the incorporation of a third phase to silica/collagen xerogels was used to affect osteoclastogenesis. In cell culture, ambient ion conditions controlled by both the degradation products of the xerogel and the bioactivity-dependent ion release and reprecipitation were shown to have the main effect on osteoclast specific enzyme tartrate-resistant acid phosphatase (TRAP) 5b. Late stage of osteoclastogenesis characterized by resorption was strongly dependent on the xerogels composition. Surface chemistry of the xerogels was displayed to play an important role in osteoclast resorption. Biphasic silica/collagen xerogels and triphasic xerogels with calcium carbonate offered widespread resorbed areas, whereas hydroxyapatite containing xerogels showed distinctly reduced resorption. The incorporation of strontium carbonate and phosphate, respectively, as third phase changed TRAP 5b activity dose-dependently and inhibited resorption within 21 days. Quantitative evaluation on osteoclast differentiation was carried out using biochemical methods (TRAP 5b, cathepsin K) and was supported by confocal laser scanning microscopy and scanning electron microscopy (SEM). Qualitative estimation of resorption was carried out by SEM.

Continuous application of compressive force induces fusion of osteoclast-like RAW264.7 cells via upregulation of RANK and downregulation of LGR4.

AIMS: During orthodontic treatment, facilitating osteoclastic bone resorption in the alveolar bone exposed to the compressive force (CF) is an important factor for tooth movement. The present study investigated the effect of CF stimulation on the differentiation of RAW264.7 cells from precursors to mature osteoclasts. MAIN METHODS: The cells were continuously stimulated with 0.3, 0.6, or 1.1 g/cm2 CF-which was generated by increasing the volume of culture medium in the wells of a 96-well plate-in the presence or absence of receptor activator of nuclear factor κB (RANK) ligand (RANKL) for 4 days. KEY FINDINGS: In the presence of RANKL, the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and the mRNA levels of dendritic cell-specific transmembrane protein (DC-STAMP) and osteoclast-stimulatory transmembrane protein (OC-STAMP) were increased by application of 0.6 and 1.1 g/cm2 CF as compared to 0.3 g/cm2 CF. The mRNA level of RANK was upregulated whereas that of leucine-rich repeat-containing G-protein-coupled receptor (LGR)4-another RANKL receptor was downregulated by 0.6 and 1.1 g/cm2 CF as compared to 0.3 g/cm2 CF in the absence of RANKL. The proportion of cells with nuclear translocation of the nuclear translocation of nuclear factor of activated T cells (NFAT)c1 was increased by 0.6 and 1.1 g/cm2 CF in the presence of RANKL. SIGNIFICANCE: Continuous application of CF induced the differentiation of RAW264.7 cells into TRAP-positive multinuclear cells by enhancing the expression of DC- and OC-STAMP and the nuclear translocation of NFATc1. This may result from the CF-induced increase in RANK and decrease in LGR4 expression.

Prognostic significance of ACP5 expression in patients with lung adenocarcinoma.

INTRODUCTION: Tartrate-resistant acid phosphatase 5 (ACP5), which is essential for bone resorption and osteoclast differentiation, promotes cell motility through the modulation of focal adhesion kinase phosphorylation. This study seeks to elucidate the association of ACP5 expression and the clinicopathologic characteristics of patients with lung adenocarcinoma (AD). METHODS: The expression of ACP5 was measured by Immunohistochemistry and Western blot analysis in lung AD and matched tumor-adjacent tissues, and the χ2 test was applied to analyze the correlation between ACP5 expression and clinicopathologic features. Using the Kaplan-Meier method, univariate and multivariate regression analysis was to explore the correlation between ACP5 expression and overall survival (OS). RESULTS: We found that ACP5 was frequently upregulated in lung AD tissues. The high expression of ACP5 was significantly related to lymph node status, tumor-node-metastasis (TNM) stage, and differentiation. From the results of univariate survival analysis, it indicated that the patients with high expression of ACP5 expression had a significantly lower OS than the patients with low expression of ACP5 expression. As it showed in Multivariate Cox regression analysis, the high expression of ACP5 expression was an independent prognostic factor for OS. CONCLUSIONS: Our results suggest that high expression of ACP5 correlates with tumor progression and may serve as a potential prognostic biomarker in lung AD.

Diarylheptanoid Hirsutenone Attenuates Osteoclastogenesis by Suppressing IFNγ and NF-κB Signaling in Th1 and Preosteoclastic Cells.

The aim of the present study was to examine the inhibitory roles and mechanisms of hirsutenone (HTN) in the regulation of osteoclastogenesis. Gene levels were compared to assure the effects of HTN on osteoclastogenesis in mouse splenocytes/CD4+ T cells, mouse macrophage-like cell line RAW264.7 (preosteoclast), MG63 (osteoblast), and RPMI1788 (B cell) cells. The mechanism by which HTN regulates the degradation of tumor necrosis factor receptor-associated factor 6 (TRAF6) and inhibits inhibitor of kappaB (IκB) and nuclear factor-kappaB (NF-κB) signaling was examined by Western blotting and luciferase reporter assays. Our results demonstrated that HTN effectively downregulated the expression of interferon γ (IFNγ), interleukin-22 (IL-22), IL-1ß, and tartrate-resistant acid phosphatase (TRAP) in splenocyte-/CD4+-RAW264.7 co-culture system. Moreover, receptor activator of nuclear factor-κB ligand (RANKL) and CD25 expression were also significantly inhibited in MG63 and CD4+ single culture system, suggesting an additional independent effect of HTN on osteoclastogenesis. Notably, TRAF6 was markedly degraded along with a decrease in nuclear factor of activated T-cells (NFATc) and NF-κB activities in RAW264.7 cells. Finally, we concluded that HTN directly or indirectly inhibits osteoclastogenesis via the inhibition of NF-κB signaling by promoting TRAF6 degradation, and plays a crucial role in suppressing the expression of RANKL and cytokines expressed in IFNγ-producing T-helper 1 (Th1) cells. These findings suggest that HTN may be a promising therapeutic candidate for diseases resulting from bone loss.

Nitidine chloride prevents OVX-induced bone loss via suppressing NFATc1-mediated osteoclast differentiation.

Nitidine chloride (NC), a bioactive alkaloid isolated from Zanthoxylum nitidum, has been used as a herbal ingredient in toothpaste that prevents cavities for decades. It also displays potential antitumor and anti-inflammation properties. However, its anticatabolic effect on bone is not known. We investigated the effect of NC on osteoclastogenesis, bone resorption and RANKL-induced NF-κB and NFATc1 signalling. In mouse-derived bone marrow monocytes (BMMs), NC suppressed RANKL-induced multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclast formation and bone resorption in a dose dependent manner. NC attenuated the expression of osteoclast marker genes including cathepsin K, D2, calcitonin receptor, NFATc1, and TRAP. Further, NC inhibited RANKL-activated NF-κB and NFATc1 signalling pathways. In vivo study revealed that NC abrogated oestrogen deficiency-induced bone loss in ovariectomized mice. Histological analysis showed that the number of osteoclasts was significantly lower in NC-treated groups. Collectively, our data demonstrate that NC suppressed osteoclastogenesis and prevented OVX-induced bone loss by inhibiting RANKL-induced NF-κB and NFATc1 signalling pathways. NC may be a natural and novel treatment for osteoclast-related bone lytic diseases.

Effects of 10-hydroxycamptothecin on differentiation of RAW264.7 cells into osteoclasts.

This study was designed to investigate the effect of 10-hydroxycamptothecin (10-HCPT) on osteoclast formation. RAW264.7 cells were cultured in vitro with 100 ng/ml receptor activator for nuclear factor-κ B ligand (RANKL) and 30 ng/ml recombinant macrophage colony stimulating factor (M-CSF), and 10-HCPT with different solubilities were added. After five-day cultivation, tartrate-resistant acid phosphatase (TRAP) staining was used to observe the number of osteoclasts. mRNA expression of osteoclast-specific genes, such as TRAP, cathepsin K (CTSK) and matrix metalloproteinase protease 9 (MMP-9), was detected by real-time Polymerase Chain Reaction (PCR). The effect of 10-HCPT on the proliferation activity of RAW264.7 cells was detected using Cell Counting Kit-8 (CCK-8). CCK-8 detection showed that 10-HCPT with a certain concentration (1 ng/ml to 5 ng/ml) had no effect on cell proliferation (P>0.05); 10-HCPT could inhibit the generation of osteoclasts. With the increase of the concentration of 10-HCPT, the number of osteoclasts generated from cells cultured with 10-HCPT [1 ng/ml (86±11.14), 2 ng/ml (66.67±7.51), 5ng/ml (27.67±6.51)] was much lower than that of the control group (145±8.19), and the difference was statistically significant (all P=0, P less than 0.05); mRNA expression of osteoclast-specific gene TRAP [1 ng/ml (24.38±0.68), 2 ng/ml (20.09±1.86), 5 ng/ml (6.23±0.53)], CTSK [1 ng/ml (10.08±0.81), 2 ng/ml (7.30±0.30), 5 ng/ml (3.20±0.56)] and MMP-9 [1 ng/ml (43.54±6.96), 2 ng/ml (28.28±5.83), 5 ng/ml (11.07±2.53)] was much lower than that of the groups added with RANKL and M-CSF only (all P=0, P less than 0.05), and with the increase of concentration of 10-HCPT, the expression of osteoclast-specific genes showed a decreasing tendency. All the findings suggest that 10-HCPT can inhibit the formation of osteoclasts by reducing the expression of osteoclast-specific genes such as TRAP, CTSK and MMP-9.

Effects of alendronate on osteoclast formation and activity in vitro.

INTRODUCTION: Root resorption is a common complication after replantation following traumatic dental avulsion. Endodontic therapy combined with local and intracanal medications aims to avoid osteoclastic activity. In such cases, the application of alendronate (ALN), a bisphosphonate widely used for the treatment of bone disorders, could be of clinical relevance. This study evaluated alendronate biocompatibility on periodontal ligament cells as well as its effects on an in vitro osteoclastogenesis model. METHODS: Alendronate cytotoxicity (10(-3) to 10(-9) mol/L) in human periodontal ligament fibroblasts, human osteogenic sarcoma cells, and murine osteoclastic precursors (RAW 264.7) was analyzed using cell number determination, cell viability, and proliferation assays. ALN (10(-6) to 10(-12) mol/L) effects on RANKL-induced osteoclastogenesis of RAW cells were assessed by tartrate-resistant acid phosphatase (TRAP) staining and activity and real-time polymerase chain reaction. RESULTS: ALN at higher concentrations was cytotoxic for all cell types, inhibiting significantly the proliferation of human osteogenic sarcoma cells and human periodontal ligament fibroblasts (≥10(-5) mol/L). TRAP activity and expression of the osteoclast markers TRAP and cathepsin K by RAW-derived osteoclasts decreased significantly with ALN at low concentrations, reaching the maximum effect at 10(-10) mol/L. CONCLUSIONS: We showed that ALN at very low concentrations is an effective inhibitor of RANKL-generated osteoclasts, without causing cytotoxic effects on their precursors or periapical cells. ALN at such concentrations might be useful to prevent replacement resorption in avulsed teeth.