Effect of Stem Cell Factor,Interleukin–6, Nitric Oxide and Transforming Growth Factor–β on the Osteoclast Differentiation Induced by 1α,25–(OH)2D3 in Primary Murine Bone Marrow Cultures

Abstract: Osteotropic hormones and cytokines are involved in the differentiation of osteoclast progenitors from haematopoietic stem cells to multinucleated osteoclasts which mediate bone resorption. Stem cell factor, interleukin–6, nitric oxide, and transforming growth factor–β are implicated in the regulation of bone resorption by osteoclast. We test whether stem cell factor, interleukin–6, nitric oxide, and transforming growth factor–β affect the generation of osteoclast–like multinucleated cells induced by 1α,25–(OH)₂D₃. 1α,25–(OH)₂D₃ increases the generation of osteoclast–like cells retaining osteoclast characteristics including multinuclearity and positive staining for tartrate–resistant acid phosphatase. Combined treatment of stem cell factor with interleukin–6 synergistically potentiates the ability of 1α,25–(OH)₂D₃ to generate tartrate–resistant acid phosphatase–positive multinucleated cells. However, either stem cell factor or interleukin–6 alone does not induce the generation of tartrate–resistant acid phosphatase–positive multinucleated cells. Transforming growth factor–β produces a biphasic effect on osteoclast generation induced by 1α,25–(OH)₂D₃. Transforming growth factor–β stimulates osteoclast generation at low concentration (0.1 ng/ml) whereas it suppresses the formation of osteoclast–like cell at higher concentration (1 ng/ml). Sodium nitroprusside, a donor of nitric oxide, almost completely inhibits the generation of 1α,25–(OH)₂D₃–induced osteoclast at high concentration (100 μM), but it significantly enhances the osteoclast generation at low concentrations (3 μM). These results suggest that stem cell factor, interleukin–6, transforming growth factor–β, and nitric oxide interact with 1α,25–(OH)₂D₃ to modulate the differentiation of hematopoietic precursors toward committed osteoclast precursors.     

Hyperosmotic Stimulus Down-regulates 1��, 25-dihydroxyvitamin D3-induced Osteoclastogenesis by Suppressing the RANKL Expression in a Co-culture System

The hyperosmotic stimulus is regarded as a mechanical factor for bone remodeling. However, whether the hyperosmotic stimulus affects 1α, 25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃)-induced osteoclastogenesis is not clear. In the present study, the effect of the hyperosmotic stimulus on 1α,25(OH)₂D₃-induced osteoclastogenesis was investigated in an osteoblast-preosteoclast co-culture system. Serial doses of sucrose were applied as a mechanical force. These hyperosmotic stimuli significantly evoked a reduced number of 1α,25(OH)₂D₃-induced tartrate-resistant acid phosphatase-positive multinucleated cells and 1α,25(OH)₂D₃-induced bone-resorbing pit area in a co-culture system. In osteoblastic cells, receptor activator of nuclear factor κB ligand (RANKL) and Runx2 expressions were down-regulated in response to 1α,25(OH)₂D₃. Knockdown of Runx2 inhibited 1α,25(OH)₂D₃-induced RANKL expression in osteoblastic cells. Finally, the hyperosmotic stimulus induced the overexpression of TonEBP in osteoblastic cells. These results suggest that hyperosmolarity leads to the down-regulation of 1α,25(OH)₂D₃-induced osteoclastogenesis, suppressing Runx2 and RANKL expression due to the TonEBP overexpression in osteoblastic cells.     

Zoledronic acid inhibits RANK expression and migration of osteoclast precursors during osteoclastogenesis

Bisphosphonates have been known to directly inhibit bone resorption and promote apoptosis in mature osteoclasts. Although bisphosphonates have been recognized as the most effective drugs to treat osteoporosis and bone cancer metastasis, the exact effects and mechanism(s) of bisphosphonates on osteoclastogenesis are unclear. The aim of this study was to clarify whether nitrogen-containing bisphosphonates affect recruitment and differentiation in osteoclasts. We examined the effects of zoledronic acid on receptor activator of NF-κB (RANK) expression and cell migration during osteoclastogenesis in two types of osteoclast precursors, RAW264.7 cells and Bone marrow cells (BMCs). Tumor necrosis factor-α (TNF-α) and RANK ligand (RANKL) upregulated RANK expression in RAW264.7 and BMCs in the presence of macrophage colony stimulating factor in a time-dependent manner. Zoledronic acid (30 and 50 μM) had no effect on cell viability in osteoclast precursors after 36 h of cultivation. Zoledronic acid (10 and 30 μM) strongly inhibited TNF-α- and RANKL-induced upregulation of RANK in a dose-dependent manner. The inhibitory effects on RANK expression were likely to be associated with the suppression of the NF-κB pathway, but not other downstream signaling pathways. Zoledronic acid (30 μM) also suppressed the TNF-α- and RANKL-induced migration of precursors by inhibiting the mevalonic acid pathway. Our results suggest that nitrogen-containing bisphosphonates not only inhibit mature osteoclasts but also prevent osteoclast precursors from differentiating and migrating towards inflammatory osteolysis lesions.     

WSS25, a sulfated polysaccharide,inhibits RANKL-induced mouse osteoclast formation by blocking SMAD/ID1 signaling

Aim:

WSS25 is a sulfated polysaccharide extracted from the rhizome of Gastrodia elata BI, which has been found to bind to bone morphogenetic protein 2 (BMP-2) in hepatocellular cancer cells. Since BMP-2 may regulate both osteoclasts and osteoblasts, here we investigated the effects of WSS25 on osteoclastogenesis in vitro and bone loss in ovariectomized mice.

Methods:

RAW264.7 cells or mouse bone marrow macrophages (BMMs) were treated with RANKL to induce osteoclastogenesis, which was assessed using TRAP staining, actin ring formation and pit formation assays, as well as bone resorption assay. Cell viability was detected with MTT assay. The mRNA levels of osteoclastogenesis-related genetic markers (TRAP, NFATc1, MMP-9 and cathepsin K) were detected using RT-PCR, while the protein levels of p-Smad1/5/8 and Id1 were measure with Western blotting. WSS25 was administered to ovariectomized mice (100 mg·kg⁻¹·d⁻¹, po) for 3 months. After the mice were euthanized, total bone mineral density and cortical bone density were measured.

Results:

In RAW264.7 cells and BMMs, WSS25 (2.5, 5, 10 μg/mL) did not affect the cell viability, but dose-dependently inhibited RANKL-induced osteoclastogenesis. Furthermore, WSS25 potently suppressed RANKL-induced expression of TRAP, NFATc1, MMP-9 and cathepsin K in RAW264.7 cells. Treatment of RAW264.7 cells with RANKL increased BMP-2 expression, Smad1/5/8 phosphorylation and Id1 expression, which triggered osteoclast differentiation, whereas co-treatment with WSS25 or the endogenous BMP-2 antagonist noggin suppressed the BMP-2/Smad/Id1 signaling pathway. In RAW264.7 cells, knockdown of Id1 attenuated RANKL-induced osteoclast differentiation, which was partially rescued by Id1 overexpression. In conformity to the in vitro experiments, chronic administration of WSS25 significantly reduced the bone loss in ovariectomized mice.

Conclusion:

WSS25 inhibits RANKL-induced osteoclast formation in RAW264.7 cells and BMMs by blocking the BMP-2/Smad/Id1 signaling pathway. WSS25 administration reduces bone loss in ovariectomized mice, suggesting that it may be a promising therapeutic agent for osteoporosis.     

Ellagic acid inhibits RANKL-induced osteoclast differentiation by suppressing the p38 MAP kinase pathway

Bone undergoes continuous remodeling by a coupled action between osteoblasts and osteoclasts. During osteoporosis, osteoclast activity is often elevated leading to increased bone destruction. Hence, osteoclasts are deemed as potential therapeutic targets to alleviate bone loss. Ellagic acid (EA) is a polyphenol reported to possess anticancer, antioxidant and anti-inflammatory properties. However, its effects on osteoclast formation and function have not yet been examined. Here, we explored the effects of EA on RANKL-induced osteoclast differentiation in RAW264.7 murine macrophages (in vitro) and human CD14⁺monocytes (ex vivo). EA dose-dependently attenuated RANKL-induced TRAP⁺ osteoclast formation in osteoclast progenitors with maximal inhibition seen at 1 µM concentration without cytotoxicity. Moreover, owing to perturbed osteoclastogenesis, EA disrupted actin ring formation and bone resorptive function of osteoclasts. Analysis of the underlying molecular mechanisms revealed that EA suppressed the phosphorylation and activation of the p38 MAP kinase pathway which subsequently impaired the RANKL-induced differentiation of osteoclast progenitors. Taken together, these novel results indicate that EA alleviates osteoclastogenesis by suppressing the p38 signaling pathway downstream of RANKL and exerts inhibitory effects on bone resorption and actin ring formation.     

Trolox inhibits osteolytic bone metastasis of breast cancer through both PGE2-dependent and independent mechanisms

Bone is a preferred site of metastasis from breast cancer, and increased osteoclast activity is implicated in breast cancer outgrowth in the bone microenvironment. Our previous observation of an anti-osteoclastic activity of Trolox, a vitamin E analog, led us to investigate whether Trolox inhibits bone metastasis and osteolysis caused by breast cancer. Administration of Trolox markedly inhibited osteolytic bone metastasis in an experimental metastasis model by intracardiac injection of 4T1 breast cancer cells. Trolox inhibited proliferation of 4T1 cells in the bone marrow but not in the mammary fat pad. In addition, Trolox could reduce tumor burden, osteolysis, and prostaglandin E₂ (PGE₂) production induced by direct inoculation of 4T1 cells into the marrow cavity of the tibia. Trolox decreased the migratory and invasive activities of 4T1 cells via PGE₂-dependent and independent mechanisms. It also inhibited the ability of 4T1 cells to stimulate the expression of receptor activator of nuclear factor-κB ligand (RANKL), a key cytokine for osteoclast differentiation factor, in osteoblasts. In addition, Trolox suppressed RANKL expression in osteoblasts induced by soluble factors from 4T1 cells. Furthermore, Trolox suppressed 4T1 cell-induced osteoclast differentiation in the co-culture of bone marrow cells and osteoblasts via both PGE₂-dependent and independent mechanisms. Taken together, these results suggest that Trolox inhibits breast cancer cell-induced osteoclast differentiation and the invasive behavior of cancer cells through PGE₂-dependent and independent mechanisms, thereby suppressing osteolytic bone metastasis of breast cancer.     

Advances in the treatment of osteoporosis

Drugs used to treat osteoporosis act either by inhibiting bone resorption or stimulating bone formation. Osteoclast formation and bone resorption require cell-to-cell contact between osteoblasts and osteoclast precursors and osteoclasts in the marrow. Interaction between the receptor for activation of nuclear factor kappa B (RANK) on the surface of preosteoclasts and osteoclasts and RANK ligand on the surface of osteoblasts is required to stimulate osteoclast formation and activation. Binding of the RANK ligand to its receptor and osteoclastogenesis are prevented by osteoprotegerin (OPG), a decoy receptor produced by osteoblasts and marrow stromal cells. Thus, interference in binding of the RANK ligand to RANK by OPG determines the rate of bone resorption. Antiresorptive drugs such as estrogen, raloxifene, bisphosphonates, salmon calcitonin, and osteoprotegerin increase bone mass by inhibiting osteoclast function and bone resorption. Osteoprotegerin is more potent since it also inhibits osteoclast formation. Raloxifene, a selective estrogen receptor modulator (SERM), is a member of a class of compounds that act through estrogen receptors and are agonists for bone, antagonists for breast and uterine tissue and may be cardioprotective. The drug was shown to prevent vertebral fractures. Alendronate and bisphosphonates are the only antiresorptive drugs that have been shown to decrease fracture rates for the hip in addition to spine and other sites. Bone morphogenetic proteins stimulate bone formation at local sites and are being developed to stimulate fracture healing. Parathyroid hormone (1-34) stimulates osteoblastic bone formation, markedly increases bone mass, prevents vertebral fractures and is under development to treat osteoporosis.     

Rolofylline,an adenosine A1 receptor antagonist,inhibits osteoclast differentiation as an inverse agonist

BACKGROUND AND PURPOSE

Adenosine may be generated by hydrolysis of extracellular nucleotides by ectonucleotidases, including ectonucleoside triphosphate diphosphohydrolase 1 (CD39), ecto-5′-nucleotidase (CD73), nucleotide pyrophosphatase phosphodiesterase 1 (NPP-1) and tissue non-specific alkaline phosphatase (TNAP). Previous work from our laboratory has uncovered a critical role for adenosine A₁ receptors (A₁R) in osteoclastogenesis; blockade or deletion of these receptors diminishes osteoclast differentiation. Interestingly, selective A₁R agonists neither affect basal osteoclastogenesis nor do they reverse A₁R antagonist-mediated inhibition of osteoclastogenesis. In this study, we determined whether ectonucleotidase-mediated adenosine production was required for A₁R antagonist-mediated inhibition, and, when we saw no effect, determined whether A₁R was constitutively activated and the antagonist was acting as an inverse agonist to diminish osteoclast differentiation.

EXPERIMENTAL APPROACH

Osteoclast formation derived from wild-type, CD39 knockout (KO), CD73 KO, NPP-1 KO and TNAP KO mice was examined by tartrate-resistant acid phosphatase staining of receptor activator of NF-κB ligand–macrophage colony-stimulating factor-stimulated osteoclasts and osteoclast gene expression (Ctsk, Acp5, MMP-9 and NFATc1). Intracellular cAMP concentration was determined by elisa.

KEY RESULTS

Rolofylline inhibited osteoclast formation in a dose-dependent manner (IC₅₀ = 20–70 nM) in mice lacking all four of these phosphatases, although baseline osteoclast formation was significantly less in precursors from CD73 KO mice. Rolofylline (1 μM) stimulates cAMP production in bone marrow macrophages by 10.23 ± 0.89-fold.

CONCLUSIONS AND IMPLICATIONS

Based on these findings, we hypothesize that the A₁R is constitutively activated in osteoclast precursors, thereby diminishing basal AC activity, and that A₁R antagonists act as inverse agonists to release A₁R-mediated inhibition of basal AC activity and permit osteoclast differentiation. The constitutive activity of A₁R promotes osteoclast formation and down-regulation of this activity blocks osteoclast formation.     

Nitric Oxide is a Regulator of Bone Remodelling

Nitric oxide (NO) is known to be implicated in the metabolism of bone, especially as a mediator of cytokine effects on the remodelling of bone tissue. In this study we examine whether NO affects the osteoblast activation or the osteoclast differentiation of primary mouse osteoblast-like and osteosarcoma ROS 17/2.8 cell lines. Primary osteoblast and ROS 17/2.8 cells released NO upon stimulation of interleukin-1β, tumour necrosis factor-α, and interferon-γ. Sodium nitroprusside, a donor of nitric oxide, increased the activity of alkaline phosphatase in ROS 17/2.8 cells as well as the number of calcified nodule formations in primary mouse osteoblast-like cells. Sodium nitroprusside also completely inhibited 1α,25-(OH)₂D₃-induced osteoclast generation in a high concentration (100 μm ). However, a low concentration of sodium nitroprusside (3–30 μm ) significantly increased the generation of osteoclasts. These results indicated that NO appears to be an important regulatory molecule in the processes of bone formation and resorption. Hence, NO may be involved in the pathogenesis of bone loss in diseases associated with cytokine activation, such as periodontal disease and rheumatoid arthritis.     

Potencies of vitamin D analogs, 1α‐hydroxyvitamin D3, 1α‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3, in lowering cholesterol in hypercholesterolemic mice in vivo

Vitamin D₃ and the synthetic vitamin D analogs, 1α‐hydroxyvitamin D₃ [1α(OH)D₃], 1α‐hydroxyvitamin D₂ [1α(OH)D₂] and 25‐hydroxyvitamin D₃ [25(OH)D₃] were appraised for their vitamin D receptor (VDR) associated‐potencies as cholesterol lowering agents in mice in vivo. These precursors are activated in vivo: 1α(OH)D₃ and 1α(OH)D₂ are transformed by liver CYP2R1 and CYP27A1 to active VDR ligands, 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] and 1α,25‐dihydroxyvitamin D₂ [1,25(OH)₂D₂]_, respectively. 1α(OH)D₂ may also be activated by CYP24A1 to 1α,24‐dihydroxyvitamin D₂ [1,24(OH)₂D₂], another active VDR ligand. 25(OH)D₃, the metabolite formed via CYP2R1 and or CYP27A1 in liver from vitamin D₃, is activated by CYP27B1 in the kidney to 1,25(OH)₂D₃. In C57BL/6 mice fed the high fat/high cholesterol Western diet for 3 weeks, vitamin D analogs were administered every other day intraperitoneally during the last week of the diet. The rank order for cholesterol lowering, achieved via mouse liver small heterodimer partner (Shp) inhibition and increased cholesterol 7α‐hydroxylase (Cyp7a1) expression, was: 1.75 nmol/kg 1α(OH)D₃ > 1248 nmol/kg 25(OH)D₃ (dose ratio of 0.0014) > > 1625 nmol/kg vitamin D₃. Except for 1.21 nmol/kg 1α(OH)D₂ that failed to lower liver and plasma cholesterol contents, a significant negative correlation was observed between the liver concentration of 1,25(OH)₂D₃ formed from the precursors and liver cholesterol levels. The composite results show that vitamin D analogs 1α(OH)D₃ and 25(OH)D₃ exhibit cholesterol lowering properties upon activation to 1,25(OH)₂D₃: 1α(OH)D₃ is rapidly activated by liver enzymes and 25(OH)D₃ is slowly activated by renal Cyp27b1 in mouse.     

Current topics in pharmacological research on bone metabolism: osteoclast differentiation regulated by glycosphingolipids

Glycosphingolipids are thought to play important roles in the development and function of several tissues, although the function of glycolipids in osteoclastogenesis has not been clearly demonstrated. In the present study, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), a glucosylceramide synthase inhibitor, completely inhibited osteoclastogenesis induced by macrophage-colony stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). Following treatment with D-PDMP, nearly all glycosphingolipid expression was dramatically reduced on the surface of bone marrow cells, which suggests that glycosphingolipids are necessary for osteoclastogenesis. To determine which kinds of glycolipids are important for osteoclastogenesis, we added several types of purified glycolipids to D-PDMP treated bone marrow cells, as the precursor of osteoclasts is known to express glucosylceramide (GlcCer) and lactosylceramide (LacCer). Following treatment with RANKL, ganglioside GM3 and GM1 were increased in the treated bone marrow cells, whereas other types were not detected using thin layer chromatography analysis. In cells cultured with those glycolipids, exogenously added LacCer rescued osteoclastogenesis blocking by D-PDMP. Furthermore, receptor activator of nuclear factor kappaB (RANK) induced the recruitment of tumor necrosis factor (TNF)-associated factors 2 and 6 (TRAF2 and 6, respectively) to the cytoplasmic tail of RANKL with activated IkappaB kinase and IkappaB phosphorylation, while D-PDMP treatment inhibited RANKL and induced IkappaB phosphorylation, and that inhibition was recovered by LacCer. In addition, RANK, TRAF2, TRAF6, and LacCer were found localized in lipid rafts on the cell surfaces. These results suggest that glycosphingolipids, especially LacCer, are important for the initial step of RANKL-induced osteoclastogenesis via lipid rafts.     

Inhibition of osteoclast differentiation and bone resorption by a novel lysophosphatidylcholine derivative, SCOH

Osteoclasts are multinucleated cells formed by multiple steps of cell differentiation from progenitor cells of hematopoietic origin. Intervention in osteoclast differentiation is considered as an effective therapeutic approach to the treatment for bone diseases involving osteoclasts. In this study, we found that the organic compound (S)-1-lyso-2-stearoylamino-2-deoxy-sn-glycero-3-phosphatidylcholine (SCOH) inhibited osteoclast differentiation. The inhibitory effect of SCOH was observed in mouse bone marrow cell cultures supported either by coculturing with osteoblasts or by adding macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL). M-CSF and RANKL activate the ERK, Akt, and NF-kappaB signal transduction pathways, and SCOH suppressed this activation. SCOH also inhibited the bone resorptive activity of differentiated osteoclasts. It attenuated bone resorption, actin ring formation, and survival of mature osteoclasts. Reduced activation of Akt and NF-kappaB and decreased induction of XIAP were observed in mature osteoclasts treated with SCOH. Thus, this novel phosphatidylcholine derivative may be useful for treating bone-resorption diseases.     

Comparative effect of cadmium on osteoblastic cells and osteoclastic cells

Cadmium(Cd) has been thought to disturb the bone metabolism directly. The mechanism for the bone lesion is unknown, however. To examine the effects of cadmium on bone metabolism, we compared its effects on osteoblasts and osteoclasts in vitro. We used an established cell line, MC3T3-E₁, as osteoblasts and tartrate resistant acid phosphatase (TRACP)-positive multi-nucleated cells (MNC) formed by a bone marrow culture system as osteoclasts. Alkaline phosphatase (ALP) activity was decreased by 10^–7 M Cd and DNA content and hydroxyproline content of osteoblastic cells were decreased by 10^–5 M Cd. Cadmium at 10^–7 M inhibited the osteoclastic cell formation from mouse bone marrow in the presence of 10^–8 M 1,25(OH)₂ vitamin D₃. A 100-fold higher concentration of zinc(Zn) simultaneously added to the cadmium-containing medium prevented the toxicity of cadmium to osteoclastic cells as observed in the culture of osteoblastic cells. These results indicate that both bone formation and bone resorption are inhibited by cadmium. The responses of osteoclasts and osteoblasts to cadmium in this culture system were the same and the responses of cadmium-damaged osteoblasts and osteoclasts to zinc were also similar. These results suggest that another mechanism by which cadmium could cause bone damage should be considered in addition to the specific induction of osteoclastic cells by Cd.     

Rutaecarpine attenuates osteoclastogenesis by impairing macrophage colony stimulating factor and receptor activator of nuclear factor κ‐B ligand‐stimulated signalling pathways

Rutaecarpine is a major alkaloid isolated from Evodia rutaecarpa. Here, we investigated the effects of rutaecarpine on osteoclast differentiation induced by macrophage colony stimulating factor (M‐CSF) and receptor activator of nuclear factor κ‐B ligand (RANKL) in bone marrow‐derived macrophages (BMMs). Treatment with rutaecarpine significantly inhibited osteoclastogenesis and prevented bone resorption of BMM‐derived osteoclasts. Mechanistically, rutaecarpine decreased the protein level of nuclear factor of activated T cells cytoplasmic‐1 (NFATc1) and the phosphorylation of other signalling pathways during the osteoclast differentiation. Thus, rutaecarpine may be useful as a therapeutic agent for the treatment of bone diseases.     

淫羊藿苷和仙茅苷协同抑制破骨细胞的形成、分化和骨吸收功能

目的 研究淫羊藿苷和仙茅苷配伍抑制破骨细胞形成、分化和骨吸收的相互作用关系.方法 用1,25-(0H)2Vitamin D3和地塞米松;或人巨噬细胞集落刺激因子(MCSF)和核因子κB受体活化因子配体(RANKL)诱导骨髓单核细胞使其分化为破骨细胞抗酒石酸酸性磷酸酶(TRAP)染色进行阳性破骨细胞鉴定;磷酸苯二钠法测定抗酒石酸酸性磷酸酶活性;将破骨细胞和骨片共同培养,以计算机图像分析测定骨吸收陷窝的面积;以Hoechst 33258和罗丹明-鬼笔环肽染色,激光共聚焦显微镜观察破骨细胞肌动蛋白环(F-actin Ring)的形态;用Western-blot分析细胞骨架相关蛋白的表达.结果 淫羊藿苷和仙茅苷在1×10-6mol/L浓度下可协同抑制破骨细胞的形成、降低抗酒石酸酸性磷酸酶的活性,减少破骨细胞在骨片上形成的骨吸收陷窝面积,抑制破骨细胞骨架F-actin环的构建及其调控因子Rho GTPases和灶性黏附激酶(focal adhesion kinase,FAK)的表达.结论 淫羊藿苷和仙茅苷协同抑制破骨细胞性的骨吸收,为药对淫羊藿仙茅的配伍提供了实验依据.