Survival Of Mature Osteoclasts Research Articles

Oleoylethanolamide Exhibits GPR119-Dependent Inhibition of Osteoclast Function and GPR119-Independent Promotion of Osteoclast Apoptosis.

Oleoylethanolamide (OEA), a bioactive lipid in bone, is known as an endogenous ligand for G protein-coupled receptor 119 (GPR119). Here, we explored the effects of OEA on osteoclast differentiation, function, and survival. While OEA inhibits osteoclast resorptive function by disrupting actin cytoskeleton, it does not affect receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. OEA attenuates osteoclast spreading, blocks actin ring formation, and eventually impairs bone resorption. Mechanistically, OEA inhibits Rac activation in response to macrophage colony-stimulating factor (M-CSF), but not RANKL. Furthermore, the OEA-mediated cytoskeletal disorganization is abrogated by GPR119 knockdown using small hairpin RNA (shRNA), indicating that GPR119 is pivotal for osteoclast cytoskeletal organization. In addition, OEA induces apoptosis in both control and GPR119 shRNA- transduced osteoclasts, suggesting that GPR119 is not required for osteoclast apoptosis. Collectively, our findings reveal that OEA has inhibitory effects on osteoclast function and survival of mature osteoclasts via GPR119-dependent and GPR119-independent pathways, respectively.

Pharmacological Inhibition of NF\u03baB Reduces Prostate Cancer Related Osteoclastogenesis In Vitro and Osteolysis Ex Vivo

NFκB is implicated in cancer and bone remodelling, and we have recently reported that the verified NFκB inhibitor Parthenolide (PTN) reduced osteolysis and skeletal tumour growth in models of metastatic breast cancer. Here, we took advantage of in vitro and ex vivo bone cell and organ cultures to study the effects of PTN on the ability of prostate cancer cells and their derived factors to regulate bone cell activity and osteolysis. PTN inhibited the in vitro growth of a panel of human, mouse and rat prostate cancer cells in a concentration-dependent manner with a varying degree of potency. In prostate cancer cell—osteoclast co-cultures, the rat Mat-Ly-Lu, but not human PC3 or mouse RM1-BT, enhanced RANKL stimulated osteoclast formation and PTN reduced these effects without affecting prostate cancer cell viability. In the absence of cancer cells, PTN reduced the support of Mat-Ly-Lu conditioned medium for the adhesion and spreading of osteoclast precursors, and survival of mature osteoclasts. Pre-exposure of osteoblasts to PTN prior to the addition of conditioned medium from Mat-Ly-Lu cells suppressed their ability to support the formation of osteoclasts by inhibition of RANKL/OPG ratio. PTN enhanced the ability of Mat-Ly-Lu derived factors to increase calvarial osteoblast differentiation and growth. Ex vivo, PTN enhanced bone volume in calvaria organ—Mat-Ly-Lu cell co-culture, without affecting Mat-Ly-Lu viability or apoptosis. Mechanistic studies in osteoclasts and osteoblasts confirmed that PTN inhibit NFκB activation related to derived factors from Mat-Ly-Lu cells. Collectively, these findings suggest that pharmacological inhibition of the skeletal NFκB signalling pathway reduces prostate cancer related osteolysis, but further studies in the therapeutic implications of NFκB inhibition in cells of the osteoblastic lineage are needed.

Lutein, a carotenoid, suppresses osteoclastic bone resorption and stimulates bone formation in cultures.

Lutein, a member of the xanthophyll family of carotenoids, suppressed IL-1-induced osteoclast differentiation and bone resorption. The survival of mature osteoclasts was also suppressed by lutein in cultures. When lutein was added to the cultures of osteoblasts, lutein enhanced the formation of mineralized bone nodules by elevating BMP2 expression and inhibiting sclerostin expression. Lutein may be beneficial for bone health.

Bone invasion in oral squamous cell carcinoma (OSCC)

Background: Bone invasion in OSCC has poor outcomes. We hypothesise stimulation of osteoclasts directly by cancer cells; indirectly via osteoblasts. Methods and Results: Cross-over co-culture of OSCC and bone cell-lines shows: matrix-metalloproteinases (MMPs) from OSCC affect osteoblast differentiation and osteoclastogenesis; conditioned medium (CM) from osteoblasts increases MMPs in OSCC cells. Western blotting showed increased Twistl protein (not Runx2). RANKL/OPG ratio, zymogen and protein expression of MMP-9 were increased in bone cells cultured with CM from OSCC cell lines; rtPCR shows mRNA consistent with protein. All these molecules were expressed in invading malignant keratinocytes; all but OPG in osteoclasts. OSCC cell-lines were induced to epithelial-mesenchymal-transition (EMT) by TGF-β. Whilst this did not affect proliferation, Vimentin was induced and EMT markers Twist1 and N-cadherin up-regulated; Snaill and E-cadherin down-regulated. Of factors associated with invasion, MMP-2,9 were increased/unchanged in OSCC cell-lines. For osteo-clast-related molecules, membrane-type-1-MMP and RANKL were up-regulated; OPG down-regulated in all cells. CM from OSCC cells pre-treated with TGF-β prolonged survival of mature osteoclasts. For future biotherapies, we asked whether inhibition of monocyte-chemotactic-protein (MCP)-l, a chemokine which recruits/activates osteoclasts, might inhibit bone invasion. MCP-1 protein was observed in tumour cells and osteoclasts from 10 patients. Real-time-PCR demonstrated MCP-1 mRNA highly expressed by OSCC cell-lines. A plasmid with inhibitor of MCP-1 (7ND vector) was transfected into SCC25 cells: CM of SCC25-7ND efficiently inhibited formation of human osteoclasts from a CD14+ monocyte subpopulation. Conclusion: Extensive crosstalk occurs between cancer and bone cells, suggesting pathways for inhibition of bone invasion.

IL-11 produced by breast cancer cells augments osteoclastogenesis by sustaining the pool of osteoclast progenitor cells

BackgroundInterleukin (IL)-11, a cytokine produced by breast cancer, has been implicated in breast cancer-induced osteolysis (bone destruction) but the mechanism(s) of action remain controversial. Some studies show that IL-11 is able to promote osteoclast formation independent of the receptor activator of NF-κB ligand (RANKL), while others demonstrate IL-11 can induce osteoclast formation by inducing osteoblasts to secrete RANKL. This work aims to further investigate the role of IL-11 in metastasis-induced osteolysis by addressing a new hypothesis that IL-11 exerts effects on osteoclast progenitor cells.MethodsTo address the precise role of breast cancer-derived IL-11 in osteoclastogenesis, we determined the effect of breast cancer conditioned media on osteoclast progenitor cells with or without an IL-11 neutralizing antibody. We next investigated whether recombinant IL-11 exerts effects on osteoclast progenitor cells and survival of mature osteoclasts. Finally, we examined the ability of IL-11 to mediate osteoclast formation in tissue culture dishes and on bone slices in the absence of RANKL, with suboptimal levels of RANKL, or from RANKL-pretreated murine bone marrow macrophages (BMMs).ResultsWe found that freshly isolated murine bone marrow cells cultured in the presence of breast cancer conditioned media for 6 days gave rise to a population of cells which were able to form osteoclasts upon treatment with RANKL and M-CSF. Moreover, a neutralizing anti-IL-11 antibody significantly inhibited the ability of breast cancer conditioned media to promote the development and/or survival of osteoclast progenitor cells. Similarly, recombinant IL-11 was able to sustain a population of osteoclast progenitor cells. However, IL-11 was unable to exert any effect on osteoclast survival, induce osteoclastogenesis independent of RANKL, or promote osteoclastogenesis in suboptimal RANKL conditions.ConclusionsOur data indicate that a) IL-11 plays an important role in osteoclastogenesis by stimulating the development and/or survival of osteoclast progenitor cells and b) breast cancer may promote osteolysis in part by increasing the pool of osteoclast progenitor cells via tumor cell-derived IL-11. However, given the heterogeneous nature of the bone marrow cells, the precise mechanism by which IL-11 treatment gives rise to a population of osteoclast progenitor cells warrants further investigation.

Coptisine inhibits RANKL-induced NF-κB phosphorylation in osteoclast precursors and suppresses function through the regulation of RANKL and OPG gene expression in osteoblastic cells

Excessive receptor activator of NF-κB ligand (RANKL) signaling causes enhanced osteoclast formation and bone resorption. The downregulation of RANKL expression and its downstream signals may be an effective therapeutic approach to the treatment of bone loss diseases such as osteoporosis. Here, we found that coptisine, one of the isoquinoline alkaloids from Coptidis Rhizoma, exhibited inhibitory effects on osteoclastogenesis in vitro. Although coptisine has been studied for its antipyretic, antiphotooxidative, dampness dispelling, antidote, antinociceptive, and anti-inflammatory activities in vitro and in vivo, its effects on osteoclastogenesis have not been investigated. Therefore, we evaluated the effects of coptisine on osteoblastic cells as well as osteoclast precursors for osteoclastogenesis in vitro. The addition of coptisine to cocultures of mouse bone marrow cells and primary osteoblastic cells with 10(-8) M 1α,25(OH)(2)D(3) caused significant inhibition of osteoclast formation in a dose-dependent manner. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed that coptisine inhibited RANKL gene expression and stimulated the osteoprotegerin gene expression induced by 1α,25(OH)(2)D(3) in osteoblastic cells. Coptisine strongly inhibited RANKL-induced osteoclast formation when added during the early stage of bone marrow macrophage (BMM) cultures, suggesting that it acts on osteoclast precursors to inhibit RANKL/RANK signaling. Among the RANK signaling pathways, coptisine inhibited NF-κB p65 phosphorylations, which are regulated in response to RANKL in BMMs. Coptisine also inhibited the RANKL-induced expression of NFATc1, which is a key transcription factor. In addition, 10 μM coptisine significantly inhibited both the survival of mature osteoclasts and their pit-forming activity in cocultures. Thus, coptisine has potential for the treatment or prevention of several bone diseases characterized by excessive bone destruction.

Inhibitory effects of mevastatin and a geranylgeranyl transferase I inhibitor (GGTI-2166) on mononuclear osteoclast formation induced by receptor activator of NFκB ligand (RANKL) or tumor necrosis factor-α (TNF-α)

We have previously reported that the statin mevastatin (compactin) reversibly inhibits the fusion of TRAP-positive mononuclear preosteoclasts (pOCs) into multinucleated osteoclasts and disrupts the actin ring in mature osteoclasts through the inhibition of protein prenylation. Protein geranylgeranylation, specifically, is known to be required for pOC fusion and for the function and survival of mature osteoclasts. However, it has not been determined whether protein geranylgeranylation is involved in early differentiation of osteoclasts (pOC formation). The current study shows that statins and the geranylgeranyl transferase I inhibitor GGTI-2166 inhibit the pOC formation induced by RANKL or TNF-α in cultures of both mouse marrow-derived macrophage-colony-stimulating factor (M-CSF) dependent monocytes (MD cells) and the mouse monocyte cell line RAW 264.7 (RAW cells). Mevastatin, 0.1–0.6 μM, inhibited the formation of pOCs induced by receptor activator of nuclear factor-κB ligand (RANKL) or tumor necrosis factor (TNF-α) in both cell cultures. The inhibitory effects of mevastatin were overcome by the addition of mevalonate, farnesyl pyrophosphate or geranylgeranyl pyrophosphate. GGTI-2166 inhibited TRAP activity induced by RANKL or TNF-α in both cell cultures and prevented the incorporation of [ 3H]all- trans geranylgeraniol into prenylated proteins in RAW cells. However, the farnesyl transferase inhibitor FTI-2153 did not inhibit TRAP activity although FTI prevented the incorporation of [ 14C]mevalonate into farnesylated proteins in RAW cells. Clostridium difficile cytotoxin B (toxin B) inhibited pOC formation induced by RANKL or TNF-α in both cell cultures. The inhibitory effects of statins and GGTI-2166 on pOC formation may result from the inhibition of the geranylgeranylation of G-proteins, such as Rho or Rac, suggesting that the geranylgeranylation of these proteins is involved in the early differentiation of progenitor cells into pOCs.

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 κB ligand (RANKL). M-CSF and RANKL activate the ERK, Akt, and NF-κB 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-κB 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.

Decoy oligodeoxynucleotides targeting NF-kappaB transcription factors: induction of apoptosis in human primary osteoclasts

Proteins belonging to the nuclear factor kappaB (NF-kappaB) superfamily are involved in osteoclast formation, playing a very important role for both differentiation of osteoclast precursors and survival of mature osteoclasts. Several drugs used to fight bone loss in a variety of human pathologies, including osteoporosis, act by increasing the frequency of osteoclast apoptosis, since it was demonstrated that small changes in osteoclast apoptosis can result in large changes in bone formation. In this respect, targeting of NF-kappaB transcription factor could be of great interest. Among nonviral gene therapy strategies recently proposed to inhibit or even block NF-kappaB activity, the transcription factor decoy (TFD) should be taken in great consideration. The main issue of the present study was to examine the effects of decoy DNA/DNA molecules targeting NF-kappaB on apoptosis of human osteoclasts (OCs), with the aim to interfere with the pathway regulating osteoclast differentiation and programmed cell death. To this aim, we used a mixture of receptor activator of NF-kappaB ligand (RANKL), macrophage colony-stimulating factor (M-CSF) and parathyroid hormone (PTH) to prepare human OCs from peripheral blood cells. Then, transfection with the decoy molecules targeting NF-kappaB was performed. The results obtained demonstrate that in primary cells expressing typical osteoclast markers such as TRAP and MMP9, NF-kappaB decoy significantly stimulated apoptosis. Inhibition of IL-6 expression and induction of Caspase 3 were found in OCs treated with NF-kappaB DNA/DNA decoys. We consider these data as the basis for setting up experimental conditions allowing nonviral gene therapy of several bone disorders.

Signal transduction by receptor activator of nuclear factor kappa B in osteoclasts

Receptor activator of nuclear factor kappa B (RANK) is a tumor necrosis factor receptor (TNFR) family protein that plays an important role in the regulation of bone and immune systems. Cellular responses to RANK ligand (RANKL) and the signal transduction pathways of RANK have been well characterized in osteoclasts and osteoclast precursor cells. RANKL induces the differentiation of osteoclast precursor cells and stimulates the resorption function and survival of mature osteoclasts. The RANK signaling mechanisms involved in these responses include the recruitment of TNF receptor-associated factor proteins, the activation of transcription factors (NF-κB, AP-1, and NFAT2), the cascades of mitogen-activated protein kinases (ERK, JNK, and p38), and the induction of Src- and phosphatidylinositol 3-kinase-dependent Akt activation. Despite the identification of several molecular targets, a comprehensive understanding of RANK signaling requires further studies on more complicated issues such as the temporal and spatial pattern of the engagement of signaling molecules and the precise relationship between the signaling pathway and the cellular response.

Mechanisms involved in bone resorption.

Osteoclasts, which are present only in bone, are multinucleated giant cells with the capacity to resorb mineralized tissues. These osteoclasts are derived from hemopoietic progenitors of the monocyte-macrophage lineage. Osteoblasts or bone marrow-derived stromal cells are involved in osteoclastogenesis through a mechanism involving cell-to-cell contact with osteoclast progenitors. Experiments on the osteopetrotic op/op mouse model have established that a product of osteoblasts, macrophage colony-stimulating factor (M-CSF), regulates differentiation of osteoclast progenitors into osteoclasts. Recent discovery of osteoclast differentiation factor (ODF)/receptor activator of NF-kappa B ligand (RANKL) allowed elucidation of the precise mechanism by which osteoblasts regulate osteoclastic bone resorption. Treatment of osteoblasts with bone-resorbing factors up-regulated expression of RANKL mRNA. In contrast, TNF alpha stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the RANKL system. IL-1 also directly acts on mature osteoclasts as a potentiator of osteoclast activation. In addition, TGF-beta super family members, such as bone morphogenetic proteins (BMPs) strikingly enhanced osteoclast differentiation from their progenitors and survival of mature osteoclasts induced by RANKL. These results suggest that BMP-mediated signals cross-communicate with RANKL-mediated ones in inducing osteoclast differentiation and function.

Hypocalcemic effect of osteoclastogenesis inhibitory factor/osteoprotegerin in the thyroparathyroidectomized rat.

Osteoclastogenesis inhibitory factor (OCIF), also termed as osteoprotegerin (OPG), is a soluble member of the tumor necrosis factor receptor family. Although OCIF/OPG is shown to inhibit osteoclast formation in vitro and prevent ovariectomy-induced bone loss in vivo, its effect on serum calcium level remains to be determined. In this study we examined the acute effect of OCIF on thyroparathyroidectomized rats whose serum calcium concentrations were raised either by exogenous PTH or 1,25-(OH)2D3. When OCIF was administered at the start of PTH infusion, it attenuated the initial rise in serum calcium. When OCIF was administered into rats with established hypercalcemia, it decreased serum calcium rapidly (within 2 hr) and dramatically. OCIF did not increase urinary calcium excretion. These findings, especially the rapid onset of its hypocalcemic effect, suggest that OCIF not only inhibits the formation of osteoclasts but also affects the function and/or survival of mature osteoclasts at doses used in this study.

Regulation of osteoclast function.

Regulation of osteoclast function.