IL-12 inhibits TNF-α induced osteoclastogenesis via a T cell-independent mechanism in vivo

It has been reported that TNF-α plays an important role in bone resorption in pathological conditions. IL-12, which is a T cell mediator, is also an important inflammatory cytokine. We previously reported that IL-12 induces apoptosis in bone marrow cells treated with TNF-α in vitro via an interaction between TNF-α-induced Fas and IL-12-induced Fas ligand (FasL), and that, as a result, osteoclastogenesis is inhibited. The purpose of this study was to investigate the effects of IL-12 on TNF-α-mediated osteoclastogenesis in vivo. We administered TNF-α with and without IL-12 into the supracalvaria in mice. The numbers of osteoclasts in the sutures in the calvaria were higher in mice administered TNF-α than in control mice not administered TNF-α. The numbers of osteoclasts in mice administered both TNF-α and IL-12 were lower than those in mice administered only TNF-α. Next, we determined the levels of mRNAs for cathepsin K and tartrate-resistant acid phosphatase (TRAP). mRNA levels were increased in mice administered TNF-α compared with control mice, but not in mice administered both TNF-α and IL-12. We also evaluated the amounts of tartrate-resistant acid phosphatase 5b (TRACP 5b) in mouse sera. The levels of TRACP 5b in mice administered TNF-α were higher than those in control mice. On the other hand, in mice administered both TNF-α and IL-12, the levels were lower than those in mice administered TNF-α alone. Fas and FasL expression levels were analyzed by real-time RT-PCR. The levels of Fas mRNA were increased in the calvaria of mice administered TNF-α compared with control mice, while those of FasL mRNAs were increased in the calvaria of mice administered IL-12. In TdT-mediated dUTP-biotin nick end-labeling (TUNEL) assays, many apoptotic cells were found in the sutures in the calvaria of mice administered both TNF-α and IL-12. IL-12 also inhibited TNF-α-induced osteoclastogenesis in mice whose T cells were blocked by anti-CD4 and anti-CD8 antibodies. These results suggest that IL-12 inhibits TNF-α-mediated osteoclastogenesis and induces apoptotic changes through an interaction between TNF-α-induced Fas and IL-12-induced FasL, in vivo, via a T cell-independent mechanism.     

Docetaxel inhibits bone resorption through suppression of osteoclast formation and function in different manners

Osteoclasts are formed from the monocyte-macrophage lineage in response to receptor activator of nuclear factor κB ligand (RANKL) expressed by osteoblasts. Bone is the most common site of breast cancer metastasis, and osteoclasts play roles in the metastasis. The taxane-derived compounds paclitaxel and docetaxel are used for the treatment of malignant diseases, including breast cancer. Here we explored the effects of docetaxel on osteoclastic bone resorption in mouse culture systems. Osteoclasts were formed within 6 days in cocultures of osteoblasts and bone marrow cells treated with 1,25-dihydroxyvitamin D₃ plus prostaglandin E₂. Docetaxel at 10⁻⁸ M inhibited osteoclast formation in the coculture when added for the entire culture period or for the first 3 days. Docetaxel, even at 10⁻⁶ M added for the final 3 days, failed to inhibit osteoclast formation. Osteoprotegerin, a decoy receptor of RANKL, completely inhibited osteoclast formation when added for the final 3 days. Docetaxel at 10⁻⁸ M inhibited the proliferation of osteoblasts and bone marrow cells. RANKL mRNA expression induced by 1,25-dihydroxyvitamin D₃ plus prostaglandin E₂ in osteoblasts was not affected by docetaxel even at 10⁻⁶ M. Docetaxel at 10⁻⁶ M, but not at 10⁻⁸ M, inhibited pit-forming activity of osteoclasts cultured on dentine. Actin ring formation and l-glutamate secretion by osteoclasts were also inhibited by docetaxel at 10⁻⁶ M. Thus, docetaxel inhibits bone resorption in two different manners: inhibition of osteoclast formation at 10⁻⁸ M and of osteoclast function at 10⁻⁶ M. These results suggest that taxanes have beneficial effects in the treatment of bone metastatic cancers. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The generation of osteoclasts from RAW 264.7 precursors in defined,serum-free conditions

Osteoclasts are the unique cell type capable of resorbing bone. The discovery of the TNF-ligand family member, RANKL, has allowed more reliable study of these important cells. The mouse monocytic cell line, RAW 264.7, has been shown to readily differentiate into osteoclasts upon exposure to recombinant RANKL. Unlike primary osteoclast precursors, there is no requirement for the addition of macrophage colony stimulating factor (M-CSF). However, to date, their differentiation has always been studied in the context of added foetal calf serum (FCS). FCS is a complex and largely undefined mixture of growth factors and matrix proteins, and varies between batches. For this reason, osteoclastogenesis would ideally be studied in the context of a defined, serum-free medium. RAW 264.7 cells were cultured in serum-replete α-MEM or serum-deprived medium (SDM) shown previously to support the growth of human osteoclasts in a co-culture with normal osteoblasts. In SDM, in the presence of recombinant RANKL, RAW 264.7 cells readily differentiated into tartrate resistant acid phosphatase (TRAP) positive multinucleated osteoclast-like cells, a process that was enhanced with the addition of 1α,25-dihydroxyvitamin D₃ (1,25D). While the osteoclasts grown in SDM were smaller in size compared with those derived in serum-replete media, their resorptive capacity was significantly increased as indicated by a twofold increase in average resorption pit size. In conclusion, we describe a defined model for studying osteoclast differentiation and activity in the absence of serum, which will be ideal for studying the role of agonistic and antagonistic molecules in this process.     

Bone structure and metabolism in a rodent model of male senile osteoporosis

Osteoporosis is a common and severe condition in elderly men, which is poorly characterized. In order to identify the hallmarks of age-related bone loss in the male mammalian skeleton, we studied several aspects of bone structure and metabolism in 23-month-old male Sprague-Dawley rats and compared them to 5-month-old animals. Cancellous bone mineral density, bone volume and trabecular number were markedly reduced in the proximal tibia of aged rats when compared to the young rats. An increase in bone matrix material density indicating a reduced deposition of new bone matrix was seen. Also, serum levels of osteocalcin, a marker of bone formation, were reduced in old males. The decreased bone formation could in part be linked to the decreased serum insulin-like growth factor 1 (IGF-1) levels which were observed in these animals. Serum levels of RatLaps (c-terminal telopeptide of type I collagen) were increased. Interestingly, an ex vivo osteoclast generation assay revealed that bone marrow from aged rats formed fewer osteoclasts than that from young rats. Consistent with this observation, serum levels of soluble RANKL, a critical osteoblast derived factor for osteoclastogenesis, were decreased in aged rats and RANKL mRNA expression was slightly reduced in bone marrow cells. Elevated leptin and adiponectin levels present in these animals could have contributed further to impaired osteoclastogenesis. We conclude that aged male rodents are characterized by a severely diminished cancellous bone network and a bone turnover situation in which bone formation is decreased to such an extent that it is outweighed by bone resorption, despite a blunted osteoclast generation potential of the bone marrow.     

Activation of MCP-1/CCR2 axis promotes prostate cancer growth in bone

Prostate cancer (PCa) frequently metastasizes to bone resulting in a mixture of osteolytic and osteoblastic lesions. We have previously reported that monocyte chemotactic protein-1 (MCP-1) is chemotactic for PCa cells, and its receptor, CCR2 expression, correlates with pathological stages. However, the role of MCP-1/CCR2 axis on PCa progression in bone remains unclear. We first evaluated the serum levels of MCP-1 in patients with bone metastases or localized PCa by enzyme-linked immunosorbent assay. We found that MCP-1 levels were elevated in patients with bone metastases compared to localized PCa. We further determined the effects of knockdown CCR2 or MCP-1 on PCa cell invasion and the tumor cell-induced osteoclast activity in vitro, respectively. PCa C4-2B and PC3 cells were transfected stably with either CCR2 short hairpin RNA (shRNA) or a scrambled RNA. CCR2 knockdown significantly diminished the MCP-1-induced PCa cell invasion. In addition, the MCP-1 production was knocked down by MCP-1 shRNA in C4-2B and PC3 cells. Conditioned media (CM) was collected and determined for the CM-induced osteoclast formation in vitro. MCP-1 knockdown significantly decreased the PCa CM-induced osteoclast formation. Finally, MCP-1 knockdown PC3 cells were implanted into the tibia of SCID mice for 4 weeks. Tumor volume was determined by histopathology and bone histomorphometry. MCP-1 knockdown diminished PC3 tumor growth in bone. We concluded that activation of MCP-1/CCR2 axis promotes PCa growth in bone. This study suggests that MCP-1 may be a target for PCa progression.     

Estrogen deficiency increases osteoclastogenesis up-regulating T cells activity: a key mechanism in osteoporosis

Compelling evidences suggest that increased production of osteoclastogenic cytokines by activated T cells plays a relevant role in the bone loss induced by estrogen deficiency in the mouse. However, little information is available on the role of T cells in post-menopausal bone loss in humans. To investigate this issue we have assessed the production of cytokines involved in osteoclastogenesis (RANKL, TNFalpha and OPG), in vitro osteoclast (OC) formation in pre and post-menopausal women, the latter with or without osteoporosis. We evaluated also OC precursors in peripheral blood and the ability of peripheral blood mononuclear cells to produce TNFalpha in both basal and stimulated condition by flow cytometry in these subjects. Our data demonstrate that estrogen deficiency enhances the production of the pro-osteoclastogenetic cytokines TNFalpha and RANKL and increases the number of circulating OC precursors. Furthermore, we show that T cells and monocytes from women with osteoporosis exhibit a higher production of TNFalpha than those from the other two groups. Our findings suggest that estrogen deficiency stimulates OC formation both by increasing the production of TNFalpha and RANKL and increasing the number of OC precursors. Women with post-menopausal osteoporosis have a higher T cell activity than healthy post-menopausal subjects; T cells thus contribute to the bone loss induced by estrogen deficiency in humans as they do in the mouse.     

Effect of a V-ATPase inhibitor, FR202126, in syngeneic mouse model of experimental bone metastasis

Purpose It has been demonstrated that vacuolar ATPase (V-ATPase) is involved in various aspects of bone metastasis. The aim of this study is to investigate the effect of the anti-bone resorptive activity of the V-ATPase inhibitor FR202126 on bone metastases in mice with metastatic breast cancer. Method As a spontaneous model of breast cancer metastasis to bone, mouse breast cancer cells, 4T1, were injected into the mammary fat pad in immunocompetent syngeneic mice. The mice were orally treated with FR202126 for 29 days. Tumor volume was measured once a week. Thirty days after the injection of the cells, the bone mineral density (BMD) of the proximal tibia was measured using peripheral quantitative computed tomography. Histomorphometric analysis of the distal femurs and the proximal tibiae was performed. To elucidate the mechanism behind the anti-osteolytic effect of FR202126, 4T1 cells were treated directly in vitro with FR202126. Cell viability was measured, and cell invasion was assessed using matrigel. Results Oral administration of FR202126 significantly increased BMD by reducing the eroded bone surface ratio. While FR202126 is known to potently inhibit osteoclast mediated bone resorption, it did not prevent invasion by cancer cells or their proliferation. Conclusion The V-ATPase inhibitor FR202126 was found to be effective at ameliorating osteolysis induced by metastatic breast cancer, even when the cancer cells themselves are not significantly affected by it. These results suggest that the anti-bone resorptive effect of the V-ATPase inhibitor might be useful for treating bone metastases associated with breast cancer.     

Histochemical assessment on osteoclasts in long bones of toll-like receptor 2 (TLR2) deficient mice

ObjectiveToll-like receptor 2 (TLR2), recognizes a wide variety of pathogen-associated molecular patterns such as lipopolysaccharides, peptidoglycans, and lipopeptides, and is generally believed to be present in monocytes, macrophages, dendritic cells, and vascular endothelial cells. However, no histological examination of osteoclasts, which differentiate from precursors common to macrophages/monocytes, has been performed in a non-infected state of TLR2 deficiency. The objective of this study was to examine the histological properties and function of osteoclasts in the long bones of 8-week-old male TLR2 deficient (TLR2^−/−) mice to gain insight into TLR2 function in biological circumstances without microbial infection.MethodsEight-week-old male wild-type and TLR2^−/− mice were fixed with paraformaldehyde solution, and their tibiae and femora were used for micro-CT analysis, immunohistochemistry, transmission electron microscopy, and real-time PCR analysis.ResultsTLR2^−/− tibiae and femora exhibited increased bone volume of metaphyseal trabeculae and elevated numbers of TRAP-positive osteoclasts. However, the number of multinucleated TRAP-positive osteoclasts was reduced, whereas mononuclear TRAP-positive cells increased, despite the high expression levels of Dc-Stamp and Oc-Stamp. Although TRAP-positive multinucleated and mononuclear osteoclasts showed the immunoreactivity and elevated expression of RANK and siglec-15, they revealed weak cathepsin K-positivity and less incorporation of the mineralized bone matrix, and often missing ruffled borders. It seemed likely that, despite the increased numbers, TLR2^−/− osteoclasts reduced cell fusion and bone resorption activity.ConclusionIt seems likely that even without bacterial infection, TLR2 might participate in cell fusion and subsequent bone resorption of osteoclasts.     

RANKL-dependent and RANKL-independent mechanisms of macrophage-osteoclast differentiation in breast cancer

The cellular and humoral mechanisms accounting for tumour osteolysis in metastatic breast cancer are uncertain. Osteoclasts, the specialised multinucleated cells responsible for tumour osteolysis, are derived from monocyte/macrophage precursors. Breast cancer-derived tumour-associated macrophages (TAMs) are capable of osteoclast differentiation but the cellular and humoral mechanisms controlling this activity are uncertain. In this study, TAMs were isolated from primary breast cancers and cultured in the presence and absence of cytokines/growth factors influencing osteoclastogenesis. Extensive TAM-osteoclast differentiation occurred only in the presence of RANKL and M-CSF; this process was inhibited by OPG and RANK:Fc, decoy receptors for RANKL. Breast cancer-derived fibroblasts and human bone stromal cells expressed mRNA for RANKL, OPG and TRAIL, and co-culture of these fibroblasts with human monocytes stimulated osteoclast formation by a RANKL-dependent mechanism. Osteoclast formation and lacunar resorption also occurred by a RANKL-independent mechanism when the conditioned medium from breast cancer cells, MDA-MB-231 and MCF-7, was added (with M-CSF) to monocyte cultures. Our findings indicate that TAMs in breast cancer are capable of osteoclast differentiation and that breast cancer-derived fibroblasts and breast cancer cells contribute to this process by producing soluble factors that influence osteoclast formation by RANKL-dependent and RANKL-independent mechanisms respectively.