Apoptotic osteocytes regulate osteoclast precursor recruitment and differentiation in vitro

Fatigue loading causes a spatial distribution of osteocyte apoptosis co-localized with bone resorption spaces peaking around microdamage sites. Since osteocytes have been shown to regulate osteoclast formation and activity, we hypothesize that osteocyte apoptosis regulates osteoclastogenesis. In this study, we used serum-starvation to mimic reduced nutrient transport in microdamaged bone and induce apoptosis in MLO-Y4 osteocyte-like cells; conditioned medium was used to apply soluble factors released by apoptotic osteocytes (aOCY) to healthy non-apoptotic MLO-Y4 cells. Osteoclast precursor (RAW264.7 monocyte) migration and differentiation were assessed in the presence of conditioned media (CM) from: (A) aOCY, (B) osteocytes treated with apoptosis conditioned medium (i.e., healthy osteocytes in the presence of apoptosis cues; apoptosis CM-treated osteocytes (atOCY)), and (C) osteocytes treated with non-apoptosis conditioned medium (i.e., healthy osteocytes in the absence of apoptosis cues; non-apoptosis CM-treated osteocytes (natOCY)). Receptor activator for nuclear factor-κB ligand (RANKL), macrophage colony stimulating factor (M-CSF), vascular endothelial growth factor (VEGF) and osteoprotegerin (OPG) mRNA, and protein expression were measured. Our findings indicate that soluble factors released by aOCY and atOCY promoted osteoclast precursor migration (up to 64% and 24% increase, respectively) and osteoclast formation (up to 450% and 265% increase, respectively). Osteoclast size increased up to 233% in the presence of aOCY and atOCY CM. Recruitment, formation and size were unaltered by natOCY. RANKL mRNA and protein expression were upregulated only in aOCY, while M-CSF and VEGF increased in atOCY. Addition of RANKL-blocking antibody abolished aOCY-induced osteoclast precursor migration and osteoclast formation. VEGF and M-CSF blocking antibodies abolished atOCY-induced osteoclastogenesis. These findings suggest that aOCY directly and indirectly (through atOCY) initiate targeted bone resorption by regulating osteoclast precursor recruitment and differentiation.     

Conditioned medium from osteocytes stimulates the proliferation of bone marrow mesenchymal stem cells and their differentiation into osteoblasts

Osteocytes are the most abundant cells in bone and there is increasing evidence that they control bone remodeling via direct cell-to-cell contacts and by soluble factors. In the present study, we have used the MLO-Y4 cell line to study the effect of osteocytes on the proliferation, differentiation and bone-forming capacity of bone marrow mesenchymal stem cells (MSC). Conditioned media (CM) from osteocytic MLO-Y4 and osteoblastic MC3T3-E1 cell lines were collected and added on mouse bone marrow cultures, in which MSC were induced to osteoblasts. There was a significant increase in alkaline phosphatase activity and osteocalcin expression in the presence of MLO-Y4 CM. No such stimulus could be observed with MC3T3-E1 CM. There was almost 4-fold increase in bone formation and up to 2-fold increase in the proliferation of MSC with MLO-Y4 CM. The highly proliferating bone marrow cells were negative for ALP and OCN, suggesting that they could represent early osteoblast precursors. MLO-Y4 CM did not enhance the viability of mature osteoblasts nor protected them of apoptosis. This is the first study to describe soluble signals between osteocytes and osteoblasts and there most likely are several still unidentified or unknown factors in osteocyte CM. We conclude that osteocytes have an active stimulatory role in controlling bone formation.     

Death of osteocytes turns off the inhibition of osteoclasts and triggers local bone resorption

Osteocytes have been suggested to play a role in the regulation of bone resorption, although their effect on bone turnover has remained controversial. In order to study this open question, we developed an organ culture system based on isolated rat calvaria, where the osteocyte viability and its effect on osteoclastic bone resorption can be monitored. Our results suggest that osteocytes are constitutively negative regulators of osteoclastic activity. Osteoclasts, which were cultured on calvarial slices with living osteocytes inside, failed to form actin rings which are the hallmarks of resorbing cells. A similar inhibitory effect was also achieved by the conditioned medium obtained from calvarial organ culture, suggesting that living osteocytes produce yet unrecognized osteoclast inhibitors. On the contrary, when osteocyte apoptosis was induced, this inhibitory effect disappeared and strong osteoclastic bone resorption activity was observed. Thus, local apoptosis of osteocytes may play a major role in triggering local bone remodeling.     

Osteocytes Produce Interferon-β as a Negative Regulator of Osteoclastogenesis

Osteoclastogenesis is controlled by osteocytes; osteocytic osteoclastogenesis regulatory molecules are largely unknown. We searched for such factors using newly developed culture methods. Our culture system mimics the three-dimensional cellular structure of bone, consisting of collagen gel-embedded osteocytic MLO-Y4 cells, stromal ST2 cells on the gel as bone lining cells, and bone marrow cells. The gel-embedded MLO-Y4 cells inhibited the osteoclastogenesis induced by 1,25(OH)₂D₃ without modulating receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) production by ST2 cells, despite MLO-Y4 cells supported osteoclastogenesis in the absence of ST2 cells. In the bone marrow cell culture, the conditioned medium from MLO-Y4 cells decreased the capability of osteoclastic differentiation from the cells induced by macrophage colony-stimulating factor. This decreased capability was concomitant with an increase in protein kinase R mRNA expression and an inhibition of c-Fos translation. These changes were partially normalized by the simultaneous addition of an anti-interferon (IFN)-β neutralizing antibody to MLO-Y4 cell conditioned medium. To study primary osteocytes, we prepared non-osteocytic cell-free osteocyte-enriched bone fragments (OEBFs). When osteoclast precursors were induced by macrophage colony-stimulating factor in the presence of OEBFs, the generated cells exhibited a diminished capacity for osteoclastogenesis. OEBFs prepared from OPG-knock-out mice exhibited a similar effect, indicating OPG-independent inhibition. The addition of anti-IFN-β neutralizing antibody during the co-culture with OEBFs partially recovered the osteoclastogenic potential of the generated cells. The MLO-Y4 cells and OEBFs expressed IFN-β mRNA. Although osteocytic RANKL is known to be important for osteoclastogenesis, our data suggest that osteocytes also produce IFN-β as an inhibitor of osteoclastogenesis.     

Sclerostin stimulates osteocyte support of osteoclast activity by a RANKL-dependent pathway

Sclerostin is a product of mature osteocytes embedded in mineralised bone and is a negative regulator of bone mass and osteoblast differentiation. While evidence suggests that sclerostin has an anti-anabolic role, the possibility also exists that sclerostin has catabolic activity. To test this we treated human primary pre-osteocyte cultures, cells we have found are exquisitely sensitive to sclerostin, or mouse osteocyte-like MLO-Y4 cells, with recombinant human sclerostin (rhSCL) and measured effects on pro-catabolic gene expression. Sclerostin dose-dependently up-regulated the expression of receptor activator of nuclear factor kappa B (RANKL) mRNA and down-regulated that of osteoprotegerin (OPG) mRNA, causing an increase in the RANK:OPG mRNA ratio. To examine the effects of rhSCL on resulting osteoclastic activity, MLO-Y4 cells plated onto a bone-like substrate were primed with rhSCL for 3 days and then either mouse splenocytes or human peripheral blood mononuclear cells (PBMC) were added. This resulted in cultures with elevated osteoclastic resorption (approximately 7-fold) compared to untreated co-cultures. The increased resorption was abolished by co-addition of recombinant OPG. In co-cultures of MLO-Y4 cells with PBMC, SCL also increased the number and size of the TRAP-positive multinucleated cells formed. Importantly, rhSCL had no effect on TRAP-positive cell formation from monocultures of either splenocytes or PBMC. Further, rhSCL did not induce apoptosis of MLO-Y4 cells, as determined by caspase activity assays, demonstrating that the osteoclastic response was not driven by dying osteocytes. Together, these results suggest that sclerostin may have a catabolic action through promotion of osteoclast formation and activity by osteocytes, in a RANKL-dependent manner.     

Activation of the Cpx phosphorelay signal transduction system in acidic phospholipid-deficient pgsA mutant cells of Escherichia coli

Low-intensity pulsed ultrasound (LIPUS) has been used as a safe and effective modality to enhance fracture healing. As the most abundant cells in bone, osteocytes orchestrate biological activities of effector cells via direct cell-to-cell contacts and by soluble factors. In this study, we have used the osteocytic MLO-Y4 cells to study the effects of conditioned medium from LIPUS-stimulated MLO-Y4 cells on proliferation and differentiation of osteoblastic MC3T3-E1 cells. Conditioned media from LIPUS-stimulated MLO-Y4 cells (LIPUS-Osteocyte-CM) were collected and added on MC3T3-E1 cell cultures. MC3T3-E1 cells cultured in LIPUS-Osteocyte-CM demonstrated a significant inhibition of proliferation and an increased alkaline phosphatase activity. The results of PGE(2) and NO assay showed that LIPUS could enhance PGE(2) and NO secretion from MLO-Y4 cells at all time points within 24h after LIPUS stimulation. We conclude that LIPUS regulates proliferation and differentiation of osteoblasts through osteocytes in vitro. Increased secretion of PGE(2) from osteocytes may play a role in this effect.     

High mobility group box 1 protein regulates osteoclastogenesis through direct actions on osteocytes and osteoclasts in vitro

Old age and Cx43 deletion in osteocytes are associated with increased osteocyte apoptosis and osteoclastogenesis. We previously demonstrated that apoptotic osteocytes release elevated concentrations of the proinflammatory cytokine, high mobility group box 1 protein (HMGB1) and apoptotic osteocyte conditioned media (CM) promotes osteoclast differentiation. Further, prevention of osteocyte apoptosis blocks osteoclast differentiation and attenuates the extracellular release of HMGB1 and RANKL. Moreover, sequestration of HMGB1, in turn, reduces RANKL production/release by MLO-Y4 osteocytic cells silenced for Cx43 (Cx43^def), highlighting the possibility that HMGB1 promotes apoptotic osteocyte-induced osteoclastogenesis. However, the role of HMGB1 signaling in osteocytes has not been well studied. Further, the mechanisms underlying its release and the receptor(s) responsible for its actions is not clear. We now report that a neutralizing HMGB1 antibody reduces osteoclast formation in RANKL/M-CSF treated bone marrow cells. In bone marrow macrophages (BMMs), toll-like receptor 4 (TLR4) inhibition with LPS-RS, but not receptor for advanced glycation end products (RAGE) inhibition with Azeliragon attenuated osteoclast differentiation. Further, inhibition of RAGE but not of TLR4 in osteoclast precursors reduced osteoclast number, suggesting that HGMB1 produced by osteoclasts directly affects differentiation by activating TLR4 in BMMs and RAGE in preosteoclasts. Our findings also suggest that increased osteoclastogenesis induced by apoptotic osteocytes CM is not mediated through HMGB1/RAGE activation and that direct HMGB1 actions in osteocytes stimulate pro-osteoclastogenic signal release from Cx43^def osteocytes. Based on these findings, we propose that HMGB1 exerts dual effects on osteoclasts, directly by inducing differentiation through TLR4 and RAGE activation and indirectly by increasing pro-osteoclastogenic cytokine secretion from osteocytes.     

Secretion of a TGF-beta-like growth inhibitor by normal rat mammary epithelial cells in vitro

We have examined conditioned medium (CM) from cultures of normal rat mammary epithelial (RME) cells for growth factor activity on fresh RME cell cultures. RME cell-derived CM contained potent growth inhibitory activity toward fresh RME cell cultures when the medium was acidified by dialysis against 1% acetic acid prior to concentration. Dialysis of the CM at neutral pH resulted in CM that had growth stimulatory activity and no inhibitory activity. The acid-activated growth inhibitor was heat and acid stable, protease sensitive, and eluted from a Bio-Gel p60 column with a peak of activity in the 28 kDa range. Incubation of the acidified-concentrated CM with neutralizing antiserum (affinity purified IgG) against transforming growth factor (TGF)-beta completely abolished the inhibitory activity of the CM. Furthermore, RME cell growth in the presence of the growth inhibitor plus TGF-beta antiserum was greater than that observed in growth medium alone. Subsequent experiments demonstrated that addition of TGF-beta antiserum alone to serum-free medium enhanced RME cell growth, whereas addition of nonimmune IgG was without effect even at 25-fold higher concentrations. Zymographic analysis of RME-CM revealed the presence of plasminogen activator proteases that may mediate the partial activation of the latent growth factor. These results indicate that normal RME cells secrete a latent TGF-beta-like growth factor into conditioned medium. Furthermore, the results indicate that some of the latent growth factor is activated in situ and contributes to the growth potential of the cells in primary culture in an autocrine manner.     

Selective β2-adrenoreceptor signaling regulates osteoclastogenesis via modulating RANKL production and neuropeptides expression in osteocytic MLO-Y4 cells

The β2-adrenergic receptor (β2-AR) signaling on bone cells is the major contributor in the effect of the sympathetic nervous system on bone turnover. However, it remains unclear whether receptor activator of nuclear factor κ-Β ligand (RANKL) modulation and neuropeptides expression in osteocytes are responsible for the mechanism. This study used β2-AR stimulation to investigate cell cycle and proliferation, the gene and protein expression of RANKL, and osteoprotegerin (OPG), as well as neuropeptides regulation in osteocytic MLO-Y4 cells. Clenbuterol (CLE; a β2-AR agonist) slightly promoted the growth of MLO-Y4 cells in a concentration-dependent effect but had no effect on the proliferation index. And the concentration of 10⁻⁸ M showed a significant increase in the S-phase fraction on day 3 in comparison with the control. Additionally, CLE-promoted osteoclast formation and bone resorption in osteocytic MLO-Y4 cell-RAW264.7 cell cocultures. RANKL expression level and the ratio of RANKL to OPG in MLO-Y4 cells were enhanced in CLE treatment but were rescued by blocking β2-AR signaling. However, neuropeptide Y and α-calcitonin gene-related peptide, two neurogenic markers, were inhibited in CLE treatment of MLO-Y4 cells, which was reversed by a β2-AR blocker. The results indicate that osteocytic β2-AR plays an important role in the regulation of RANKL/OPG and neuropeptides expression, and β2-AR signaling in osteocytes can be used as a new valuable target for osteoclast-related pathologic disease.     

Irradiation-induced osteocyte damage promotes HMGB1-mediated osteoclastogenesis in vitro

Irradiation-induced bone loss is widely reported, especially in radiotherapy-induced osteoporosis. In addition to the mechanism of osteogenesis inhibition and osteoclastogenesis promotion, the regulation effect of osteocytes, which also send signals to modulate osteoclastogenesis, should be elucidated. In this study, the effect of irradiation on osteocyte and its accommodation to osteoclastogenesis via the release of high mobility group box 1 (HMGB1) was explored. Furthermore, the control response of HMGB1 inhibitor on receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) expression in osteocyte and osteocyte-induced osteoclastogenesis was assessed. It was observed that irradiated osteocyte-like MLO-Y4 cells exhibited polygonal-shaped morphological changes and shortened dendrites, inhibited cell viability and induced cellular apoptosis, along with the reduction in dendritic E11 protein/messenger RNA expression at a doses of 4 Gy. Additionally, the secretion of HMGB1 in supernatants was promoted, accompanied by the decreased OPG and elevated RANKL expression. When the RAW264.7 cells were cocultured with irradiated MLO-Y4 cells or its conditioned medium, enhanced migration and differentiation of osteoclast precursor was observed, and this difference was alleviated with anti-HMGB1 neutralizing antibody. In conclusion, this study demonstrated that irradiation deteriorated osteocytes’ potential to promote recruitment and differentiation of osteoclast precursor via stimulating HMGB1 release and subsequent elevation of RANKL/OPG level. This study will assist in designing the intervention programs for irradiation-induced bone loss.     

三维回转骨细胞条件培养基对成骨细胞功能的调节作用

骨细胞是骨组织中主要的力学感受器.研究失重条件下骨细胞对效应细胞的调控作用对于揭示失重引起的骨丢失机制具有重要意义.本研究拟采用三维回转器模拟失重,探讨模拟失重骨细胞条件培养基(RCM)对成骨功能的调节作用.小鼠骨细胞系MLO-Y4三维回转培养72h后,收集回转条件培养基(RCM)和未回转对照组的条件培养基(CCM),用四甲基偶氮唑盐比色(MTT)法、对硝基苯磷酸(pNPP)法和流式细胞术(FCM)分别检测RCM对小鼠成骨样细胞系MC3T3-E1增殖、周期及细胞分泌碱性磷酸酶(ALP)活性的影响.采用RT-PCR方法检测RCM对MC3T3-E1成骨相关基因表达的影响.结果显示,三维随机回转72h后的MLO-Y4RCM可促进MC3T3-E1增殖:条件培养基培养MC3T3-E124h和48h后,50%RCM组比CCM组分别增加了1.62和1.60倍,差异显著(*P0.05),培养72h后,100%RCM组比CCM组增加了1.69倍,差异显著(*P0.05);细胞周期检测结果表明,条件培养基培养24、48和72h后,RCM组部分恢复CCM引起的MC3T3-E1细胞周期阻滞;MC3T3-E1的ALP活性在RCM组和CCM组之间无差异;RT-PCR检测结果表明,100%MLO-Y4条件培养基培养MC3T3-E148h后,降低了成骨相关基因ALP、Runx2、OPN、OC的表达.差异显著(*P0.05,**P0.01,***P0.001).实验结果表明,三维随机回转模拟失重培养骨细胞72h后的条件培养基促进了成骨细胞增殖,抑制了成骨相关基因表达.     

Breast cancer cell line MDA-231 stimulates osteoclastogenesis and bone resorption in human osteoclasts

Breast cancers commonly cause osteolytic metastases in bone, a process that is dependent upon osteoclast-mediated bone resorption, but the mechanism responsible for tumor-mediated osteoclast activation has not yet been clarified. In the present study we utilized a well-known human breast cancer cell line (MDA-231) in order to assess its capability to influence osteoclastogenesis in human bone marrow cultures and bone resorption in fully differentiated osteoclasts. We demonstrated that conditioned medium (CM) harvested from MDA-231 increased the formation of multinucleated TRAP-positive cells in bone marrow cultures. Bone resorption activity of fully differentiated human osteoclasts and of osteoclast-like cell lines, from giant cell tumors of bone (GCT), was highly increased by the presence of MDA-231 CM. Moreover, while MDA-231 by themselves did not produce IL-6 tumor cell, CM increased the secretion of IL-6 by primary human osteoclasts and GCT cell lines compared to untreated controls. These data suggest that MDA-231 produce osteoclastic activating factor(s) that increase both osteoclast formation in bone marrow culture and bone resorption activity by mature cells. Moreover, breast cancer cells stimulate IL-6 secretion by osteoclasts that is one of the factors known to supports osteoclastogenesis.     

Adipose mesenchymal stem cell-derived exosomes ameliorate hypoxia/serum deprivation-induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro

Age-related skeletal changes is closely associated with imbalanced bone remodeling characterized by elevated osteocyte apoptosis and osteoclast activation. Since osteocytes are the commander of bone remodeling, attenuating increased osteocyte apoptosis may improve age-related bone loss. Exosomes, derived from mesenchymal stem cells, hold promising potential for cell-free therapy due to multiple abilities, such as promoting proliferation and suppressing apoptosis. We aimed to explore the effect of exosomes derived from adipose mesenchymal stem cell (ADSCs-exo) on osteocyte apoptosis and osteocyte-mediated osteoclastogenesis in vitro. The osteocyte-like cell line MLO-Y4 was used as a model, and apoptosis was induced by hypoxia and serum deprivation (H/SD). Our results showed that ADSCs-exo noticeably reduced H/SD-induced apoptosis in MLO-Y4 cells via upregulating the radio of Bcl-2/Bax, diminishing the production of reactive oxygen species and cytochrome c, and subsequent activation of caspase-9 and caspase-3. Additionally, ADSCs-exo lowered the expression of RANKL both at the mRNA and protein levels, as well as the ratio of RANKL/OPG at the gene level. As determined by tartrate-resistant acid phosphatase staining, reduced osteoclastogenesis was further validated in bone marrow monocytes cultured under conditioned medium from exosome-treated MLO-Y4. Together, ADSCs-exo could antagonize H/SD induced osteocyte apoptosis and osteocyte-mediated osteoclastogenesis, indicating the therapeutic potential of ADSCs-exo in age-related bone disease.     

Osteocyte-induced angiogenesis via VEGF–MAPK-dependent pathways in endothelial cells

Recently, it has been suggested osteocytes control the activities of bone formation (osteoblasts) and resorption (osteoclast), indicating their important regulatory role in bone remodelling. However, to date, the role of osteocytes in controlling bone vascularisation remains unknown. Our aim was to investigate the interaction between endothelial cells and osteocytes and to explore the possible molecular mechanisms during angiogenesis. To model osteocyte/endothelial cell interactions, we co-cultured osteocyte cell line (MLOY4) with endothelial cell line (HUVECs). Co-cultures were performed in 1:1 mixture of osteocytes and endothelial cells or by using the conditioned media (CM) transfer method. Real-time cell migration of HUVECs was measured with the transwell migration assay and xCELLigence system. Expression levels of angiogenesis-related genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The effect of vascular endothelial growth factor (VEGF) and mitogen-activated phosphorylated kinase (MAPK) signaling were monitored by western blotting using relevant antibodies and inhibitors. During the bone formation, it was noted that osteocyte dendritic processes were closely connected to the blood vessels. The CM generated from MLOY4 cells-activated proliferation, migration, tube-like structure formation, and upregulation of angiogenic genes in endothelial cells suggesting that secretory factor(s) from osteocytes could be responsible for angiogenesis. Furthermore, we identified that VEGF secreted from MLOY4-activated VEGFR2–MAPK–ERK-signaling pathways in HUVECs. Inhibiting VEGF and/or MAPK–ERK pathways abrogated osteocyte-mediated angiogenesis in HUVEC cells. Our data suggest an important role of osteocytes in regulating angiogenesis.     

Ability of breast cancer cell lines to stimulate bone resorbing activity of mature osteoclasts correlates with an anti-apoptotic effect mediated by macrophage colony stimulating factor

We compared the effect of conditioned medium (CM) from several human breast carcinoma cell lines on osteoclast bone resorbing activity and osteoclast apoptosis. Our findings indicate that ability of cancer cell line to increase the in vitro bone resorbing activity is linked to their potential to inhibit osteoclast apoptosis. Cancer cells producing the higher level of M-CSF have the higher osteolytic activity, suggesting that M-CSF originating from cancer cells may contribute, at least in part, to the osteoclast activity at the metastatic site by enhancing their survival. Given that M-CSF plays an important role in the anti-apoptotic effect, we speculated that blocking M-CSF pathway would prevent the CM effects. Small interfering RNA (siRNA) targeting M-CSF and imatinib, a protein tyrosine kinase inhibitor targeting M-CSF receptor, almost completely reversed the CM effect on both osteoclast apoptosis and bone resorption. Blockade of M-CSF pathway could be thus of clinical value in the treatment of breast cancer related bone destruction.     

Mechanical stimulation of gap junctions in bone osteocytes is mediated by prostaglandin E2

Gap junction-mediated intercellular communications are thought to transduce the effects of mechanical strain from osteocytes to cells on the bone surface to initiate remodeling. To determine whether gap junctions may co-ordinate the effects of mechanical loading, osteocyte-like MLO-Y4 cells were exposed to fluid flow-imposed shear stress. After exposure of MLO-Y4 to fluid flow, intercellular coupling increased in direct proportion to shear stress level. Interestingly, this stimulation is further enhanced during the post-stress period, indicating that released factor(s) is likely to be involved. The conditioned medium obtained from the fluid flow treated MLO-Y4 cells induced an increase in the number of functional gap junctions and Cx43 protein when added to non-sheer-stressed cells. Fluid flow was found to induce prostaglandin F2 (PGE2) release and increase cyclooxygenase 2 (COX-2) expression. When PGE2 was depleted from the fluid flow conditioned medium, the stimulatory effect on gap junctions was significantly decreased. Addition of the COX inhibitor indomethacin partially blocked the stimulatory effects of mechanical strain on gap junctions. Together, these studies suggest that the stimulatory effect of fluid flow on gap junctions is mediated in part by de novo synthesis and release of PGE2. Gap junctions may serve as channels for the signals generated by osteocytes in response to mechanical loading.     

Effects of transforming growth factor-beta on long-term human cord blood monocyte cultures.

Transforming growth factor-beta (TGF-beta) modulates growth and differentiation in many cell types and is abundant in bone matrix. We recently showed that human cord blood monocytes cultured in the presence of 1,25(OH)2D3 acquire some features of osteoclast precursors. Since TGF-beta has been shown to influence bone resorption in organ culture, we have studied the effect of TGF-beta (1-1,000 pg/ml) on cord blood monocyte cultures. These cells were cultured on plastic substrate during 3 weeks in the presence of 20% horse serum and 10(-9) M 1,25(OH)2D3. TGF-beta, from a concentration of 10 pg/ml in the culture medium, decreased in a dose dependent manner the formation of multinucleated cells. At a concentration of TGF-beta of 1 ng/ml, the multinucleated cells were reduced to 2.1% +/- 0.3%, compared to 19.3% +/- 1.5% in control cultures. TGF-beta inhibited in a dose-dependent manner the proliferation of cord blood monocytes as assessed by 3H-thymidine incorporation at 7 and 14 days of culture. The fusion index was also decreased by 3 weeks of treatment with TGF-beta. Indomethacin did not reverse the inhibitory effects of TGF-beta. The expression of the osteoclastic phenotype was assessed using two different antibodies: 23C6, a monoclonal antibody directed against the vitronectin receptor, which is highly expressed by osteoclasts but not by adult monocytes, and an antibody to HLA-DR, which is not present on osteoclast. TGF-beta decreased the expression of HLA-DR and increased in a dose-dependent manner the proportion of 23C6-labeled cells; these results suggest that TGF-beta could modulate a differentiation effect to the osteoclastic phenotype. However, when cord blood monocytes were cultured on devitalized rat calvariae prelabeled with 45Ca, TGF-beta did not induce any 45Ca release from bone cultured with monocytes, suggesting that full osteoclastic differentiation was not achieved. These results emphasize the complex role of TGF-beta in the local regulation of bone cell differentiation and in bone remodeling.     

Pulsed electromagnetic fields regulate osteocyte apoptosis,RANKL/OPG expression,and its control of osteoclastogenesis depending on the presence of primary cilia

Growing evidence has shown that pulsed electromagnetic fields (PEMF) can modulate bone metabolism in vivo and regulate the activities of osteoblasts and osteoclasts in vitro. Osteocytes, accounting for 95% of bone cells, act as the major mechanosensors in bone for transducing external mechanical signals and producing cytokines to regulate osteoblastic and osteoclastic activities. Targeting osteocytic signaling pathways is becoming an emerging therapeutic strategy for bone diseases. We herein systematically investigated the changes of osteocyte behaviors, functions, and its regulation on osteoclastogenesis in response to PEMF. The osteocyte-like MLO-Y4 cells were exposed to 15 Hz PEMF stimulation with different intensities (0, 5, and 30 Gauss [G]) for 2 hr. We found that the cell apoptosis and cytoskeleton organization of osteocytes were regulated by PEMF with an intensity-dependent manner. Moreover, PEMF exposure with 5 G significantly inhibited apoptosis-related gene expression and also suppressed the gene and protein expression of the receptor activator of nuclear factor κB ligand/osteoprotegerin (RANKL/OPG) ratio in MLO-Y4 cells. The formation, maturation, and osteoclastic bone-resorption capability of in vitro osteoclasts were significantly suppressed after treated with the conditioned medium from PEMF-exposed (5 G) osteocytes. Our results also revealed that the inhibition of osteoclastic formation, maturation, and bone-resorption capability induced by the conditioned medium from 5 G PEMF-exposed osteocytes was significantly attenuated after abrogating primary cilia in osteocytes using the polaris siRNA transfection. Together, our findings highlight that PEMF with 5 G can inhibit cellular apoptosis, modulate cytoskeletal distribution, and decrease RANKL/OPG expression in osteocytes, and also inhibit osteocyte-mediated osteoclastogenesis, which requires the existence of primary cilia in osteocytes. This study enriches our basic knowledge for further understanding the biological behaviors of osteocytes and is also helpful for providing a more comprehensive mechanistic understanding of the effect of electromagnetic stimulation on bone and relevant skeletal diseases (e.g., bone fracture and osteoporosis).