Osteoclast bone resorption is enhanced in the presence of osteoblasts

Bone resorption activity by osteoclasts has been evaluated in a co-culture system in which osteoclasts have been plated in the presence of osteoblasts. The system prevents cell-cell contact but permits diffusion of molecules through the pores of a millipore membrane that separates the two compartments in which the two cell types have been plated. Results demonstrated that osteoblasts exert a stimulatory effect over osteoclast bone resorption due to soluble molecules capable of passing through the membrane pores. The effect is specific since periosteal cells, which do not express osteoblastic characteristics, fail to induce changes in the osteoclast activity. PTH does not affect osteoblast-mediated enhancement of bone resorption, indicating that the stimulatory effect that the hormone exert in vivo occurs via a different cellular system.     

Parathyroid hormone stimulates osteoblastic expression of MCP-1 to recruit and increase the fusion of pre/osteoclasts

The clinical findings that alendronate blunted the anabolic effect of human parathyroid hormone (PTH) on bone formation suggest that active resorption is involved and enhances the anabolic effect. PTH signals via its receptor on the osteoblast membrane, and osteoclasts are impacted indirectly via the products of osteoblasts. Microarray with RNA from rats injected with human PTH or vehicle showed a strong association between the stimulation of monocyte chemoattractant protein-1 (MCP-1) and the anabolic effects of PTH. PTH rapidly and dramatically stimulated MCP-1 mRNA in the femora of rats receiving daily injections of PTH or in primary osteoblastic and UMR 106-01 cells. The stimulation of MCP-1 mRNA was dose-dependent and a primary response to PTH signaling via the cAMP-dependent protein kinase pathway in vitro. Studies with the mouse monocyte cell line RAW 264.7 and mouse bone marrow proved that osteoblastic MCP-1 can potently recruit osteoclast monocyte precursors and facilitate receptor activator of NF-kappaB ligand-induced osteoclastogenesis and, in particular, enhanced fusion. Our model suggests that PTH-induced osteoblastic expression of MCP-1 is involved in recruitment and differentiation at the stage of multinucleation of osteoclast precursors. This information provides a rationale for increased osteoclast activity in the anabolic effects of PTH in addition to receptor activator of NF-kappaB ligand stimulation to initiate greater bone remodeling.     

Tumor necrosis factors alpha and beta induce osteoblastic cells to stimulate osteoclastic bone resorption

Antigen- or mitogen-stimulated leukocytes release bone-resorbing activity into culture supernatants in vitro. Among the agents likely to be present in such supernatants are monocyte-derived tumor necrosis factor (TNF-alpha) and lymphocyte-derived tumor necrosis factor (TNF-beta) (lymphotoxin), both of which have recently been shown to stimulate bone resorption in organ culture. To identify the mechanism of action of these agents, we compared bone resorption by isolated osteoclasts with bone resorption by osteoclasts cocultured with osteoblastic cells, and with bone resorption by osteoclasts incubated with supernatants from osteoblastic cells, in the presence and absence of recombinant TNF-alpha and TNF-beta. We found that neither TNF-alpha nor TNF-beta had any significant effect on bone resorption by isolated osteoclasts, but in the presence of osteoblasts the agents caused a twofold to threefold stimulation of bone resorption. A similar degree of stimulation was achieved by supernatants from osteoblasts incubated with TNF before addition to osteoclasts, compared with supernatants to which TNF were added after osteoblast incubation. These experiments suggest that TNF-alpha and TNF-beta stimulate bone resorption through a primary effect on osteoblastic cells, which are induced by TNF to produce a factor that stimulates osteoclastic resorption. Half-maximal stimulation of resorption occurred at 1.5 X 10(-10) M and 2.5 X 10(-10) M for TNF-alpha and TNF-beta, respectively. This degree of potency is comparable to that of parathyroid hormone, the major physiologic systemic regulator of bone resorption, and suggests that the TNF may exert a significant influence on osteoclastic bone resorption in vivo.     

ATP P2 receptors and regulation of bone effector cells

ATP, signaling through P2 receptors, is one of the most important extracellular regulatory molecules in the skeleton. P2 receptors are divided into two subclasses, P2Y which are G-protein coupled and P2X which are ligand-gated ion channels. There is molecular and functional evidence for widespread expression of both subclasses of receptors by bone cells. Co-activation of P2Y and PTH1 receptors on osteoblasts, leads to synergistic expression of osteoblastic genes, providing a mechanism for integrating local and systemic regulatory signals in bone. Activation of P2Y1 receptors on osteoblasts enhances expression of RANKL leading indirectly to an increase in osteoclast formation and resorption. Expression of P2X7 inducible pores on osteoclast precursor cell membranes allows fusion to form multinucleated osteoclasts and blockade of this receptor inhibits resorption. Bone cells release nucleotides into the extracellular environment to provide highly localized and transient signals that regulate bone formation and bone resorption.     

Bone cell interactions through Eph/ephrin

Bones cannot properly form or be maintained without cell-cell interactions through ephrin ligands and Eph receptors. Cell culture analysis and evaluation of genetic mouse models and human diseases reveal various ephrins and Eph functions in the skeletal system. Migration, attachment and spreading of mesenchymal stem cells are regulated by ephrinB ligands and EphB receptors. ephrinB1 loss-of-function is associated with craniofrontonasal syndrome (CFNS) in humans and mice. In bone remodeling, ephrinB2 is postulated to act as a “coupling stimulator.” In that case, bidirectional signaling between osteoclastic ephrinB2 and osteoblastic EphB4 suppresses osteoclastic bone resorption and enhances osteoblastic bone formation, facilitating the transition between these two states. Parathyroid hormone (PTH) induces ephrinB2 in osteoblasts and enhances osteoblastic bone formation. In contrast to ephrinB2, ephrinA2 acts as a “coupling inhibitor,” since ephrinA2 reverse signaling into osteoclasts enhances osteoclastogenesis and EphA2 forward signaling into osteoblasts suppresses osteoblastic bone formation and mineralization. Furthermore, ephrins and Ephs likely modulate pathological conditions such as osteoarthritis, rheumatoid arthritis, multiple myeloma and osteosarcoma. This review focuses on ephrin/Eph-mediated cell-cell interactions in bone biology.     

骨吸收与骨形成耦联中Eph/ephrin信号转导的研究进展

破骨细胞的骨吸收活动与成骨细胞的骨形成活动相互作用调节,形成一种特殊的耦联机制,影响骨骼生长、发育及正常骨组织结构的维持．最近几年提出的Eph/ephrin双向信号转导在骨吸收与骨形成耦联中的研究越来越受到关注．从Eph/ephrin分子结构、信号转导机制及生物学意义等几方面对该理论作一阐述．     

Parathyroid hormone stimulation and PKA signaling of latent transforming growth factor-beta binding protein-1 (LTBP-1) mRNA expression in osteoblastic cells

Parathyroid hormone (PTH) regulates bone remodeling and calcium homeostasis by acting on osteoblasts. Recently, the gene expression profile changes in the rat PTH (1-34, 10(-8)M)-treated rat osteoblastic osteosarcoma cell line, UMR 106-01, using DNA microarray analysis showed that mRNA for LTBP-1, a latent transforming growth factor (TGF-beta)-binding protein is stimulated by PTH. Latent TGF-beta binding proteins (LTBPs) are required for the proper folding and secretion of TGF-beta, thus modifying the activity of TGF-beta, which is a local factor necessary for bone remodeling. We show here by real time RT-PCR that PTH-stimulated LTBP-1 mRNA expression in rat and mouse preosteoblastic cells. PTH also stimulated LTBP-1 mRNA expression in all stages of rat primary osteoblastic cells but extended expression was found in differentiating osteoblasts. PTH also stimulated TGF-beta1 mRNA expression in rat primary osteoblastic cells, indicating a link between systemic and local factors for intracellular signaling in osteoblasts. An additive effect on LTBP-1 mRNA expression was found when UMR 106-01 cells were treated with PTH and TGF-beta1 together. We further examined the signaling pathways responsible for PTH-stimulated LTBP-1 and TGF-beta1 mRNA expression in UMR 106-01 cells. The PTH stimulation of LTBP-1 and TGF-beta1 mRNA expression was dependent on the PKA and the MAPK (MEK and p38 MAPK) pathways, respectively in these cells, suggesting that PTH mediates its effects on osteoblasts by several intracellular signaling pathways. Overall, we demonstrate here that PTH stimulates LTBP-1 mRNA expression in osteoblastic cells and this is PKA-dependent. This event may be important for PTH action via TGF-beta in bone remodeling.     

Osteoclasts secrete non-bone derived signals that induce bone formation

Bone turnover is a highly regulated process, where bone resorption in the normal healthy individual always is followed by bone formation in a manner referred to as coupling. Patients with osteopetrosis caused by defective acidification of the resorption lacuna have severely decreased resorption, in face of normal or even increased bone formation. This suggests that osteoclasts, not their resorptive activity, are important for sustaining bone formation. To investigate whether osteoclasts mediate control of bone formation by production of bone anabolic signals, we collected conditioned media (CM) from human osteoclasts cultured on either bone or plastic, and tested their effects on bone nodule formation by osteoblasts. Both types of CM were shown to dose-dependently induce bone nodule formation, whereas non-conditioned osteoclast culture medium had no effects. These data show that osteoclasts secrete non-bone derived factors, which induce preosteoblasts to form bone-like nodules, potentially explaining the imbalanced coupling seen in osteopetrotic patients.     

Normal human osteoclasts formed from peripheral blood monocytes express PTH type 1 receptors and are stimulated by PTH in the absence of osteoblasts

The prevailing view for many years has been that osteoclasts do not express parathyroid hormone (PTH) receptors and that PTH's effects on osteoclasts are mediated indirectly via osteoblasts. However, several recent reports suggest that osteoclasts express PTH receptors. In this study, we tested the hypothesis that human osteoclasts formed in vitro express functional PTH type 1 receptors (PTH1R). Peripheral blood monocytes (PBMC) were cultured on bone slices or plastic culture dishes with human recombinant RANK ligand (RANKL) and recombinant human macrophage colony-stimulating factor (M-CSF) for 16-21 days. This resulted in a mixed population of mono- and multi-nucleated cells, all of which stained positively for the human calcitonin receptor. The cells actively resorbed bone, as assessed by release of C-terminal telopeptide of type I collagen and the formation of abundant resorption pits. We obtained evidence for the presence of PTH1R in these cells by four independent techniques. First, using immunocytochemistry, positive staining for PTH1R was observed in both mono- and multi-nucleated cells intimately associated with resorption cavities. Second, PTH1R protein expression was demonstrated by Western blot analysis. Third, the cells expressed PTH1R mRNA at 21 days and treatment with 10(-7) M hPTH (1-34) reduced PTH1R mRNA expression by 35%. Finally, bone resorption was reproducibly increased by two to threefold when PTH (1-34) was added to the cultures. These findings provide strong support for a direct stimulatory action of PTH on human osteoclasts mediated by PTH1R. This suggests a dual regulatory mechanism, whereby PTH acts both directly on osteoclasts and also, indirectly, via osteoblasts.     

Expression and function of P2 receptors in bone

ATP (adenosine 5'-triphosphate) is one of the most important extracellular regulatory molecules in the skeleton. Extracellular ATP and other nucleotides signal through P2 receptors, a diverse group of receptors that are widely expressed by bone cells. P2 receptors are divided into two subclasses; P2Y G-protein coupled receptors, and P2X ligand-gated ion channels, and there is functional and molecular evidence for the expression of these receptors on both osteoblasts and osteoclasts. In order to activate P2 receptors, nucleotides must be released into the bone microenvironment. ATP is present in mmol concentrations in cells and can be released by cell lysis, cell trauma or physiological mechanisms, possibly through ABC transporters. Following co-activation of P2Y and PTH1 receptors on osteoblasts, there are multiple levels of interaction in downstream signalling that eventually lead to synergistic expression of osteoblastic genes, providing a mechanism for integrating local and systemic regulatory signals in bone particularly with regard to the activation of bone remodelling. Activation of P2Y1 receptors on osteoblasts enhances expression of RANKL leading indirectly to an increase in osteoclast formation and resorption. Expression of P2X7 inducible pores on osteoclast precursor cell membranes allows fusion to form multinucleated osteoclasts and blockade of this receptor inhibits resorption. The capacity of extracellular nucleotides to provide a highly localized and transient signal coupled with the profound effects of P2 receptor activation on osteoblastic and osteoclastic cells and the synergistic interactions with systemic hormones, indicate that nucleotides have a strong influence over bone tissue growth and regeneration.     

Osteoblastic regulation of osteoclast survival: effect of calcitriol

Throughout life, bone is remodelled in a dynamic process which results in a balance between bone formation by osteoblasts and bone resorption by osteoclasts. It is now clearly established that osteoblasts/stromal cells are crucial for differentiation of osteoclasts, through a mechanism involving cell-to-cell contact. However, the possible involvement of osteoblasts and stromal cells in the survival of osteoclasts has not yet been clearly demonstrated. In this study, we assessed the influence of cellular microenvironment, especially osteoblasts, on the osteoclast survival. Our results have shown significant differences in osteoclastic survival between unfractionated bone cells and pure osteoclasts. Furthermore, we have shown that addition of 1.25(OH)2D3 to unfractionated bone cells resulted in a dose-dependent increase in osteoclast survival. Finally, we have shown that a conditioned medium obtained from rat osteoblastic cells cultured with calcitriol was able to increase significantly survival of pure osteoclasts. Taken together, these results strongly suggest that osteoblastic cells present in the bone microenvironment might play a role in the osteoclastic survival by producing soluble factor which modulate osteoclast apoptosis.     

Imbalance of local bone metabolism in inflammatory arthritis and its reversal upon tumor necrosis factor blockade: direct analysis of bone turnover in murine arthritis

Chronic arthritis typically leads to loss of periarticular bone, which results from an imbalance between bone formation and bone resorption. Recent research has focused on the role of osteoclastogenesis and bone resorption in arthritis. Bone resorption cannot be observed isolated, however, since it is closely linked to bone formation and altered bone formation may also affect inflammatory bone loss. To simultaneously assess bone resorption and bone formation in inflammatory arthritis, we developed a histological technique that allows visualization of osteoblast function by in-situ hybridization for osteocalcin and osteoclast function by histochemistry for tartrate-resistant acid phosphatase. Paw sections from human tumor necrosis factor transgenic mice, which develop an erosive arthritis, were analyzed at three different skeletal sites: subchondral bone erosions, adjacent cortical bone channels, and endosteal regions distant from bone erosions. In subchondral bone erosions, osteoclasts were far more common than osteoblasts. In contrast, cortical bone channels underneath subchondral bone erosions showed an accumulation of osteoclasts but also of functional osteoblasts resembling a status of high bone turnover. In contrast, more distant skeletal sites showed only very low bone turnover with few scattered osteoclasts and osteoblasts. Within subchondral bone erosions, osteoclasts populated the subchondral as well as the inner wall, whereas osteoblasts were almost exclusively found along the cortical surface. Blockade of tumor necrosis factor reversed the negative balance of bone turnover, leading to a reduction of osteoclast numbers and enhanced osteoblast numbers, whereas the blockade of osteoclastogenesis by osteoprotegerin also abrogated the osteoblastic response. These data indicate that bone resorption dominates at skeletal sites close to synovial inflammatory tissue, whereas bone formation is induced at more distant sites attempting to counter-regulate bone resorption.     

Imbalance of local bone metabolism in inflammatory arthritis and its reversal upon tumor necrosis factor blockade: direct analysis of bone turnover in murine arthritis

Chronic arthritis typically leads to loss of periarticular bone, which results from an imbalance between bone formation and bone resorption. Recent research has focused on the role of osteoclastogenesis and bone resorption in arthritis. Bone resorption cannot be observed isolated, however, since it is closely linked to bone formation and altered bone formation may also affect inflammatory bone loss. To simultaneously assess bone resorption and bone formation in inflammatory arthritis, we developed a histological technique that allows visualization of osteoblast function by in-situ hybridization for osteocalcin and osteoclast function by histochemistry for tartrate-resistant acid phosphatase. Paw sections from human tumor necrosis factor transgenic mice, which develop an erosive arthritis, were analyzed at three different skeletal sites: subchondral bone erosions, adjacent cortical bone channels, and endosteal regions distant from bone erosions. In subchondral bone erosions, osteoclasts were far more common than osteoblasts. In contrast, cortical bone channels underneath subchondral bone erosions showed an accumulation of osteoclasts but also of functional osteoblasts resembling a status of high bone turnover. In contrast, more distant skeletal sites showed only very low bone turnover with few scattered osteoclasts and osteoblasts. Within subchondral bone erosions, osteoclasts populated the subchondral as well as the inner wall, whereas osteoblasts were almost exclusively found along the cortical surface. Blockade of tumor necrosis factor reversed the negative balance of bone turnover, leading to a reduction of osteoclast numbers and enhanced osteoblast numbers, whereas the blockade of osteoclastogenesis by osteoprotegerin also abrogated the osteoblastic response. These data indicate that bone resorption dominates at skeletal sites close to synovial inflammatory tissue, whereas bone formation is induced at more distant sites attempting to counter-regulate bone resorption.     

A two-receptor model for the action of parathyroid hormone on osteoblasts: a role for intracellular free calcium and cAMP

It has been suggested that intracellular Ca2+, in addition to cAMP, plays an important role in PTH-stimulated bone resorption. There is now strong evidence indicating that the osteoblast is the main target cell for PTH action, regulating indirectly, via cell-cell communication, osteoclastic bone resorption. In order to investigate the possible role of free cytosolic calcium in stimulated bone resorption, we studied the effects of the intact hormone (bPTH 1-84) and some of its fragments (bPTH (1-34), bPTH(3-34,) (Nle-8, Nle-18,Tyr-34) bPTH (3-34) amide) on their capacity to modify the cytosolic Ca2+ concentration in rat osteoblast-like cells. The experiments were performed using Quin-2, a fluorescent indicator of free calcium. We found an excellent correlation between the ability of PTH and PTH fragments to transiently increase cytosolic Ca2+ concentration in rat osteoblast-like cells and their ability to stimulate bone resorption in embryonic rat calvaria in vitro. On the other hand, no direct correlation was found for the cAMP and bone-resorbing responses. On the ground of these data we propose a two-receptor model for PTH action in osteoblasts, in which one receptor is coupled to the production of cAMP, whereas the other is involved in the increase of cytosolic Ca2+. Activation of both receptors by PTH (1-84) or PTH (1-34) leads to the full physiological response in osteoblasts, most probably the release of one or more factors which stimulate the activity of existing osteoclasts and others which stimulate the recruitment of additional osteoclasts.     

Synaptotagmin VII regulates bone remodeling by modulating osteoclast and osteoblast secretion

Maintenance of bone mass and integrity requires a tight balance between resorption by osteoclasts and formation by osteoblasts. Exocytosis of functional proteins is a prerequisite for the activity of both cells. In the present study, we show that synaptotagmin VII, a calcium sensor protein that regulates exocytosis, is associated with lysosomes in osteoclasts and bone matrix protein-containing vesicles in osteoblasts. Absence of synaptotagmin VII inhibits cathepsin K secretion and formation of the ruffled border in osteoclasts and bone matrix protein deposition in osteoblasts, without affecting the differentiation of either cell. Reflecting these in vitro findings, synaptotagmin VII-deficient mice are osteopenic due to impaired bone resorption and formation. Therefore, synaptotagmin VII plays an important role in bone remodeling and homeostasis by modulating secretory pathways functionally important in osteoclasts and osteoblasts.     

The Proteasome Inhibitor Carfilzomib Suppresses Parathyroid Hormone-induced Osteoclastogenesis through a RANKL-mediated Signaling Pathway

Parathyroid hormone (PTH) induces osteoclast formation and activity by increasing the ratio of RANKL/OPG in osteoblasts. The proteasome inhibitor carfilzomib (CFZ) has been used as an effective therapy for multiple myeloma via the inhibition of pathologic bone destruction. However, the effect of combination of PTH and CFZ on osteoclastogenesis is unknown. We now report that CFZ inhibits PTH-induced RANKL expression and secretion without affecting PTH inhibition of OPG expression, and it does so by blocking HDAC4 proteasomal degradation in osteoblasts. Furthermore, we used different types of culture systems, including co-culture, indirect co-culture, and transactivation, to assess the effect of CFZ on PTH action to induce osteoclastogenesis. Our results demonstrated that CFZ blocks PTH-induced osteoclast formation and bone resorption by its additional effect to inhibit RANKL-mediated IκB degradation and NF-κB activation in osteoclasts. This study showed for the first time that CFZ targets both osteoblasts and osteoclasts to suppress PTH-induced osteoclast differentiation and bone resorption. These findings warrant further investigation of this novel combination in animal models of osteoporosis and in patients.     

Bone cell interactions through Eph/ephrin: Bone modeling,remodeling and associated diseases

Bones cannot properly form or be maintained without cell-cell interactions through ephrin ligands and Eph receptors. Cell culture analysis and evaluation of genetic mouse models and human diseases reveal various ephrins and Eph functions in the skeletal system. Migration, attachment and spreading of mesenchymal stem cells are regulated by ephrinB ligands and EphB receptors. ephrinB1 loss-of-function is associated with craniofrontonasal syndrome (CFNS) in humans and mice. In bone remodeling, ephrinB2 is postulated to act as a “coupling stimulator.” In that case, bidirectional signaling between osteoclastic ephrinB2 and osteoblastic EphB4 suppresses osteoclastic bone resorption and enhances osteoblastic bone formation, facilitating the transition between these two states. Parathyroid hormone (PTH) induces ephrinB2 in osteoblasts and enhances osteoblastic bone formation. In contrast to ephrinB2, ephrinA2 acts as a “coupling inhibitor,” since ephrinA2 reverse signaling into osteoclasts enhances osteoclastogenesis and EphA2 forward signaling into osteoblasts suppresses osteoblastic bone formation and mineralization. Furthermore, ephrins and Ephs likely modulate pathological conditions such as osteoarthritis, rheumatoid arthritis, multiple myeloma and osteosarcoma. This review focuses on ephrin/Eph-mediated cell-cell interactions in bone biology.     

Nutritional factors and bone homeostasis: synergistic effect with zinc and genistein in osteogenesis

Bone homeostasis is regulated through osteoclasts and osteoblasts. Osteoporosis, which is induced with its accompanying decrease in bone mass with increasing age, is widely recognized as a major public health problem. Bone loss may be due to decreased osteoblastic bone formation and increased osteoclastic bone resorption. There is growing evidence that nutritional and food factors may play a part in the prevention of bone loss with aging and have been to be worthy of notice in the prevention of osteoporosis. Zinc, an essential trace element, or genistein, which are contained in soybeans, has been shown to have a stimulatory effect on osteoblastic bone formation and an inhibitory effect on osteoclastic bone resorption, thereby increasing bone mass. These factors have an effect on protein synthesis and gene expression, which are related to bone formation in osteoblastic cells and bone resorption in osteoclastic cells. The combination of zinc and genistein is found to reveal the synergistic effect on bone anabolic effect. The oral administration of those factors has been shown to prevent on bone loss in ovariectomized rats, an animal model for osteoporosis, indicating a role in the prevention of osteoporosis. Supplemental intake of ingredient with the combination of zinc and genistein has been shown to have a preventive effect on osteoporosis in human subjects, suggesting a role in the prevention of bone loss.