Talking among Ourselves: Paracrine Control of Bone Formation within the Osteoblast Lineage

While much research focuses on the range of signals detected by the osteoblast lineage that originate from endocrine influences, or from other cells within the body, there are also multiple interactions that occur within this family of cells. Osteoblasts exist as teams and form extensive communication networks both on, and within, the bone matrix. We provide four snapshots of communication pathways that exist within the osteoblast lineage between different stages of their differentiation, as follows: (1) PTHrP, a factor produced by early osteoblasts that stimulates the activity of more mature bone-forming cells and the most mature osteoblast embedded within the bone matrix, the osteocyte; (2) sclerostin, a secreted factor, released by osteocytes into their extensive communication network to restrict the activity of younger osteoblasts on the bone surface; (3) oncostatin M, a member of the IL-6/gp130 family of cytokines, expressed throughout osteoblast differentiation and acting to stimulate osteoblast activity that works on a different receptor in the mature osteocyte compared to the preosteoblast; and (4) Eph/ephrins, cell-contact-dependent kinases, and the osteoblast-lineage-specific interaction of EphB4 and ephrinB2, which provides a checkpoint for entry to the late stages of osteoblast differentiation and restricts RANKL expression.     

EphrinB2 Regulation by PTH and PTHrP Revealed by Molecular Profiling in Differentiating Osteoblasts

With the aim of identifying new pathways and genes regulated by PTH(1–34) and PTH‐related protein 1–141 [PTHrP(1–141)] in osteoblasts, this study was carried out using a mouse marrow stromal cell line, Kusa 4b10, that acquires features of the osteoblastic phenotype in long‐term culture conditions. After the appearance of functional PTH receptor 1 (PTHR1) in Kusa 4b10 cells, they were treated with either PTH(1–34) or PTHrP(1–141), and RNA was subjected to Affymetrix whole mouse genome array. The microarray data were validated using quantitative real‐time RT‐PCR on independently prepared RNA samples from differentiated Kusa 4b10, UMR106 osteosarcoma cells, and primary mouse calvarial osteoblasts, as well as in vivo using RNA from metaphyseal bone after a single PTH injection to 3‐wk‐old and 6‐mo‐old ovariectomized rats. Of the 45,101 probes used on the microarray, 4675 were differentially expressed by ≥1.5 fold, with a false discovery rate <0.1. Among the regulated genes, ephrinB2 mRNA was upregulated in response to both PTH and PTHrP. This was confirmed by quantitative real‐time PCR in vitro and in vivo. Increased ephrinB2 protein was also shown in vitro by Western blotting, and immunostaining of femur sections showed ephrinB2 in both osteoclasts and osteoblasts. Production of ephrinB2, as well as other ephrins or Eph family members, did not change during differentiation of Kusa 4b10 cells. Blockade of ephrinB2/EphB4 interaction resulted in inhibition of mineralization of Kusa 4b10 cells. Together with the shown effect of ephrinB2 promoting osteoblast differentiation and bone formation through action on EphB4, the data raise the possibility that PTH or PTHrP might regulate ephrinB2 to act in a paracrine or autocrine manner on EphB4 or EphB2 in the osteoblast, contributing as a local event to the anabolic action of PTH or PTHrP.     

Adiponectin stimulates RANKL and inhibits OPG expression in human osteoblasts through the MAPK signaling pathway.

Our study indicates that recombinant adiponectin induced RANKL and inhibited OPG expression in human osteoblasts through the AdipoR1/p38 MAPK pathway, and these responses contributed to the adiponectin-induced osteoclasts formation in the co-culture of osteoblast and peripheral blood monocytes systems. These findings showed that adiponectin increased osteoclast formation indirectly through stimulating RANKL and inhibiting OPG production in osteoblasts. It also suggests the pharmacological nature of recombinant adiponectin that indirectly induces osteoclasts formation. INTRODUCTION: Recently, adiponectin has emerged as an element in the regulation of bone metabolism, but the mechanism remains. This study was undertaken to investigate the action of adiponectin on osteoclastogenesis through revealing RANKL and osteoprotegerin (OPG) expression in osteoblasts and osteoclast formation. MATERIALS AND METHODS: Real-time quantitative PCR and ELISA were used to detect RANKL and OPG mRNA and protein expression in cultured human osteoblasts. The involved signal pathway was studied using mitogen-activated protein kinase (MAPK) inhibitor and adiponectin receptor 1 (AdipoR1) siRNA. The effects of recombinant adiponectin on osteoclasts formation also were examined in the co-culture systems of osteoblast and peripheral blood monocytes (PBMCs) systems or purified CD14 + PBMCs cultures. RESULTS: Our study showed that recombinant adiponectin induced RANKL and inhibited OPG mRNA expression in human osteoblasts in a dose- and time-dependent manner. Adiponectin also increased soluble RANKL and decreased OPG secretion in osteoblasts conditioned media. Suppression of AdipoR1 with siRNA abolished the adiponectin-regulated RANKL and OPG mRNA expression in osteoblasts. Furthermore, pretreatment of osteoblasts with the MAPK inhibitor SB203580 abolished adiponectin-regulated RANKL and OPG mRNA expression. Adiponectin induced osteoclast formation in the co-culture systems of osteoblast and PBMCs systems, and OPG entirely blocked this response. However, adiponectin had no direct effect on the differentiation of osteoclast precursor purified CD14 + PBMCs. CONCLUSIONS: These data indicate that recombinant adiponectin induced RANKL and inhibited OPG expression in human osteoblasts through the AdipoR1/p38 MAPK pathway, and these responses contributed to the adiponectin-induced osteoclast formation in the co-culture of osteoblast and PBMCs systems. These findings showed that adiponectin increased osteoclast formation indirectly through stimulating RANKL and inhibiting OPG production in osteoblasts. It suggests the pharmacological nature of recombinant adiponectin that indirectly induces osteoclasts formation.     

Importance of membrane- or matrix-associated forms of M-CSF and RANKL/ODF in osteoclastogenesis supported by SaOS-4/3 cells expressing recombinant PTH/PTHrP receptors.

SaOS-4/3, a subclone of the human osteosarcoma cell line SaOS-2, established by transfecting the human parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) receptor complementary DNA (cDNA), supported osteoclast formation in response to PTH in coculture with mouse bone marrow cells. Osteoclast formation supported by SaOS-4/3 cells was completely inhibited by adding either osteoprotegerin (OPG) or antibodies against human macrophage colony-stimulating factor (M-CSF). Expression of messenger RNAs (mRNAs) for receptor activator of NF-kappaB ligand/osteoclast differentiation factor (RANKL/ODF) and both membrane-associated and secreted forms of M-CSF by SaOS-4/3 cells was up-regulated in response to PTH. SaOS-4/3 cells constitutively expressed OPG mRNA, expression of which was down-regulated by PTH. To elucidate the mechanism of PTH-induced osteoclastogenesis, SaOS-4/3 cells were spot-cultured for 2 h in the center of a culture well and then mouse bone marrow cells were uniformly plated over the well. When the spot coculture was treated for 6 days with both PTH and M-CSF, osteoclasts were induced exclusively inside the colony of SaOS-4/3 cells. Osteoclasts were formed both inside and outside the colony of SaOS-4/3 cells in coculture treated with a soluble form of RANKL/ODF (sRANKL/sODF) in the presence of M-CSF. When the spot coculture was treated with sRANKL/sODF, osteoclasts were formed only inside the colony of SaOS-4/3 cells. Adding M-CSF alone failed to support osteoclast formation in the spot coculture. PTH-induced osteoclast formation occurring inside the colony of SaOS-4/3 cells was not affected by the concentration of M-CSF in the culture medium. Mouse primary osteoblasts supported osteoclast formation in a similar fashion to SaOS-4/3 cells. These findings suggest that the up-regulation of RANKL/ODF expression is an essential step for PTH-induced osteoclastogenesis, and membrane- or matrix-associated forms of both M-CSF and RANKL/ ODF are essentially involved in osteoclast formation supported by osteoblasts/stromal cells.     

RANKL expression is related to the differentiation state of human osteoblasts.

Human osteoblast phenotypes that support osteoclast differentiation and bone formation are not well characterized. Osteoblast differentiation markers were examined in relation to RANKL expression. RANKL expression was induced preferentially in immature cells. These results support an important link between diverse osteoblast functions. Cells of the osteoblast lineage support two apparently distinct functions: bone formation and promotion of osteoclast formation. The aim of this study was to examine the relationship between these phenotypes in human osteoblasts (NHBC), in terms of the pre-osteoblast marker, STRO-1, and the mature osteoblast marker, alkaline phosphatase (AP), and the expression of genes involved in osteoclast formation, RANKL and OPG. The osteotropic stimuli, 1alpha,25(OH)2vitamin D3 (vitD3) and dexamethasone, were found to have profound proliferative and phenotypic effects on NHBCs. VitD3 inhibited NHBC proliferation and increased the percentage of cells expressing STRO-1 over an extended culture period, implying that vitD3 promotes and maintains an immature osteogenic phenotype. Concomitantly, RANKL mRNA expression was upregulated and maintained in NHBC in response to vitD3. Dexamethasone progressively promoted the proliferation of AP-expressing cells, resulting in the overall maturation of the cultures. Dexamethasone had little effect on RANKL mRNA expression and downregulated OPG mRNA expression in a donor-dependent manner. Regression analysis showed that RANKL mRNA expression was associated negatively with the percentage of cells expressing AP (p < 0.01) in vitD3- and dexamethasone-treated NHBCs. In contrast, RANKL mRNA expression was associated positively with the percentage of STRO-1+ cells (p < 0.01). In NHBCs sorted by FACS based on STRO-1 expression (STRO-1bright and STRO-1dim populations), it was found that vitD3 upregulated the expression of RANKL mRNA preferentially in STRO-1bright cells. The results suggest that immature osteoblasts respond to osteotropic factors in a potentially pro-osteoclastogenic manner. Additionally, the dual roles of osteoblasts, in supporting osteoclastogenesis or forming bone, may be performed by the same lineage of cells at different stages of their maturation.     

Regulatory Mechanisms of RANKL Presentation to Osteoclast Precursors

It is important to understand the molecular mechanisms regulating osteoclast formation, as excess activation of osteoclasts is associated with various osteopenic disorders. Receptor activator of nuclear factor kappa B (RANKL) is a central player in osteoclastogenesis. Recent findings suggest that osteocytes are the major supplier of RANKL to osteoclast precursors. It has also been suggested that osteocyte cell death upregulates the RANKL/osteoprotegerin (OPG) ratio in viable osteocytes adjacent to apoptotic osteocytes in areas of bone microdamage, thus, contributing to localized osteoclast formation. Indeed, viable osteocytes can provide RANKL through direct interactions with osteoclast precursors at osteocyte dendritic processes. In addition, OPG tightly regulates RANKL cell surface presentation in osteocytes, which contributes to the inhibition of RANKL signaling, as well as the decoy receptor function of OPG. By contrast, the physiological role of RANKL in osteoblasts is yet to be clarified, although similar mechanisms of regulation are observed in both osteocytes and osteoblasts.     

Focal adhesion protein Kindlin-2 regulates bone homeostasis in mice

Our recent studies demonstrate that the focal adhesion protein Kindlin-2 is critical for chondrogenesis and early skeletal development. Here, we show that deleting Kindlin-2 from osteoblasts using the 2.3-kb mouse Col1 a1-Cre transgene minimally impacts bone mass in mice, but deleting Kindlin-2 using the 10-kb mouse Dmp1-Cre transgene, which targets osteocytes and mature osteoblasts, results in striking osteopenia in mice. Kindlin-2 loss reduces the osteoblastic population but increases the osteoclastic and adipocytic populations in the bone microenvironment. Kindlin-2 loss upregulates sclerostin in osteocytes,downregulates β-catenin in osteoblasts, and inhibits osteoblast formation and differentiation in vitro and in vivo. Upregulation ofβ-catenin in the mutant cells reverses the osteopenia induced by Kindlin-2 deficiency. Kindlin-2 loss additionally increases the expression of RANKL in osteocytes and increases osteoclast formation and bone resorption. Kindlin-2 deletion in osteocytes promotes osteoclast formation in osteocyte/bone marrow monocyte cocultures, which is significantly blocked by an anti-RANKLneutralizing antibody. Finally, Kindlin-2 loss increases osteocyte apoptosis and impairs osteocyte spreading and dendrite formation.Thus, we demonstrate an important role of Kindlin-2 in the regulation of bone homeostasis and provide a potential target for the treatment of metabolic bone diseases.     

Role of IGF-I signaling in regulating osteoclastogenesis.

We showed that IGF-I deficiency impaired osteoclastogenesis directly and/or indirectly by altering the interaction between stromal/osteoblastic cells and osteoclast precursors, reducing RANKL and M-CSF production. These changes lead to impaired bone resorption, resulting in high BV/TV in IGF-I null mice. INTRODUCTION: Although IGF-I has been clearly identified as an important growth factor in regulating osteoblast function, information regarding its role in osteoclastogenesis is limited. Our study was designed to analyze the role of IGF-I in modulating osteoclastogenesis using IGF-I knockout mice (IGF-I(-/-)). MATERIALS AND METHODS: Trabecular bone volume (BV/TV), osteoclast number, and morphology of IGF-I(-/-) or wildtype mice (IGF-I(+/+)) were evaluated in vivo by histological analysis. Osteoclast precursors from these mice were cultured in the presence of RANKL and macrophage-colony stimulating factor (M-CSF) or co-cultured with stromal/osteoblastic cells from either genotype. Osteoclast formation was assessed by measuring the number of multinucleated TRACP+ cells and pit formation. The mRNA levels of osteoclast regulation markers were determined by quantitative RT-PCR. RESULTS: In vivo, IGF-I(-/-) mice have higher BV/TV and fewer (76% of IGF-I(+/+)) and smaller osteoclasts with fewer nuclei. In vitro, in the presence of RANKL and M-CSF, osteoclast number (55% of IGF-I(+/+)) and resorptive area (30% of IGF-I(+/+)) in osteoclast precursor cultures from IGF-I(-/-) mice were significantly fewer and smaller than that from the IGF-I(+/+) mice. IGF-I (10 ng/ml) increased the size, number (2.6-fold), and function (resorptive area, 2.7-fold) of osteoclasts in cultures from IGF-I(+/+) mice, with weaker stimulation in cultures from IGF-I(-/-) mice. In co-cultures of IGF-I(-/-) osteoblasts with IGF-I(+/+) osteoclast precursors, or IGF-I(+/+) osteoblasts with IGF-I(-/-) osteoclast precursors, the number of osteoclasts formed was only 11% and 48%, respectively, of that from co-cultures of IGF-I(+/+) osteoblasts and IGF-I(+/+) osteoclast precursors. In the long bones from IGF-I(-/-) mice, mRNA levels of RANKL, RANK, M-CSF, and c-fms were 55%, 33%, 60%, and 35% of that from IGF-I(+/+) mice, respectively. CONCLUSIONS: Our results indicate that IGF-I regulates osteoclastogenesis by promoting their differentiation. IGF-I is required for maintaining the normal interaction between the osteoblast and osteoclast to support osteoclastogenesis through its regulation of RANKL and RANK expression.     

The Role of Osteoblast Density and Endogenous Interleukin-6 Production in Osteoclast Formation From the Hemopoietic Stem Cell Line FDCP-MIX C2GM in Coculture With Primary Osteoblasts

Osteoclast formation from the hemopoietic stem cell line FDCP-mix C₂GM was shown to be strongly dependent on osteoblast density. In cocultures of C₂GM cells with fetal mouse osteoblasts seeded at high density (i.e., 2.5 × 10⁴ cells/cm²), we found a significantly lower osteoclast formation compared with cocultures with osteoblasts seeded at low density (i.e., 1 × 10⁴ cells/cm²). The differentiation state of osteoblasts in high-density cultures resembled more than that of osteoblasts in low-density cultures, the differentiation state of mature osteoblasts, since the cells in the former cultures showed higher alkaline phosphatase (APase) activity than the cells in the latter cultures, and nodules were formed in high-density cultures but not in low-density cultures. Endogenous interleukin-6 (IL-6) production was found to be significantly lower in high-density cultures, which may partly explain the impaired osteoclast formation in high-density cocultures. Addition of IL-6 to the high-density cocultures indeed restored osteoclast formation. There appeared to be no overt difference in IL-6 receptor mRNA expression between high-density and low-density cultures. In conclusion, this paper suggests that mature, highly differentiated osteoblasts are not directly involved in osteoclastogenesis. In contrast, osteoblast-like cells lacking mature osteoblast markers induce osteoclast formation. Whether these low-density osteoblast-like cells represent an immature differentiation state or the lining cell phenotype is unclear. Received: 26 June 1997 / Accepted: 14 November 1997     

Osteoblasts of calvaria induce higher numbers of osteoclasts than osteoblasts from long bone

Several studies have demonstrated the existence of functional differences between osteoclasts harbored in different bones. The mechanisms involved in the occurrence of such a heterogeneity are not yet understood. Since cells of the osteoblast lineage play a critical role in osteoclastogenesis, osteoclast heterogeneity may be due to osteoblasts that differ at the different bone sites. In the present study we evaluated possible differences in the capacity of calvaria and long bone osteoblasts to induce osteoclastogenesis. Osteoblasts were isolated from calvaria and long bone of mice and co-cultured with osteoclast precursors obtained from bone marrow of both types of bone, spleen and peripheral blood. Irrespective of the source of the precursors, a significantly higher number of TRACP-positive multinucleated cells were formed with calvaria osteoblasts. The expression of osteoclastogenesis related genes was analyzed by qPCR. OPG was significantly higher expressed by long bone osteoblasts. The RANKL/OPG ratio and TNF-α gene expression were significantly higher in calvaria osteoblast cultures. OPG added to the culture system inhibited osteoclastogenesis in both groups. Blocking TNF-α had no effect on osteoclastogenesis. Calvaria and long bone osteoblasts were pre-stimulated with VitD3 for 5 days. Subsequently, osteoclast precursors were added to these cultures. After a co-culture of 6 days, it was shown that VitD3 pre-stimulation of long bone osteoblasts strongly improved their capacity to induce osteoclast formation. This coincided with an increased ratio of RANKL/OPG. Taken together, the data demonstrated differences in the capacity of calvaria and long bone osteoblasts to induce osteoclastogenesis. This appeared to be due to differences in the expression of RANKL and OPG. VitD3 pre-stimulation improved the ability of long bone osteoblasts to induce osteoclast formation. Our findings demonstrate bone-site specific differences in osteoblast-mediated formation of osteoclasts. The data may suggest that the heterogeneity of osteoclasts is partially due to the way the osteoblasts induce their formation.     

SC-19220, antagonist of prostaglandin E2 receptor EP1, inhibits osteoclastogenesis by RANKL.

We examined the direct effect of SC-19220, an EP1 prostaglandin (PG) E2 receptor antagonist, on osteoclastogenesis induced by RANK/RANKL signaling in mouse cell cultures. We found that SC-19220 inhibited RANKL-induced osteoclastogenesis by suppression of the RANK/RANKL signaling pathway in osteoclast precursors. INTRODUCTION: Bone growth is accomplished by a dynamic equilibrium between formation by osteoblasts and resorption by osteoclasts, which are regulated by many systemic and local osteotropic factors that induce osteoclast formation from hematopoietic precursors through RANK/RANKL signaling. There are four subtypes of prostaglandin E (PGE) receptors, EP1, EP2, EP3, and EP4, and PGE2 facilitates bone resorption by a mechanism mediated by EP2/EP4. It is well known that SC-19220 is an EP1-specific antagonist. We previously found that SC-19220 inhibited osteoclastogenesis induced by osteotropic factors, including PGE2; however, the inhibitory mechanism is not clear. In this study, we investigated the inhibitory effects of SC-19220 on osteoclastogenesis induced by RANK/RANKL signaling in mouse cell cultures and analyzed the mechanism involved. MATERIALS AND METHODS: A bone marrow culture system and bone marrow macrophages were used to examine the effects of SC-19220 on PGE2-, 11-deoxy-PGE1-, and RANKL-induced osteoclastogenesis. We analyzed RANKL expression in osteoblasts induced by PGE2 using RT-PCR. We also examined the effects of SC-19220 on the macrophage-colony-stimulating factor (M-CSF) receptor (c-Fms) and RANK expression in osteoclast precursors as well as RANK/RANKL signaling using RT-PCR and Western blotting analyses. RESULTS AND CONCLUSION: SC-19220 dose-dependently inhibited osteoclast formation induced by PGE2, 11-deoxy-PGE1, and RANKL in the mouse culture system; however, it had no influence on RANKL expression in osteoblasts induced by PGE2. Furthermore, the expression of RANK and c-Fms in osteoclast precursors was decreased by SC-19220 at the mRNA and protein levels. In RANK signaling networks, SC-19220 inhibited c-Src and NFAT2 expression. Our findings indicated that SC-19220 inhibits RANKL-induced osteoclastogenesis through the suppression of RANK, c-Fms, c-Src, and NFAT2, suggesting that this EP1-specific antagonist inhibits osteoclast formation induced by RANKL from the early stage of osteoclastogenesis.     

Secreted frizzled-related protein-1 inhibits RANKL-dependent osteoclast formation.

We determined that sFRP-1 mRNA was differentially expressed by osteoblast/stromal cell lines and that sFRP-1 neutralizing antibodies and siRNA complementary to sFRP-1 coding sequence enhanced, while recombinant sFRP-1 inhibited, osteoclast formation. In studying the mechanism of action for sFRP-1, we found that sFRP-1 could bind recombinant RANKL. These results suggest potential cross-talk between Wnt and RANKL pathways. INTRODUCTION: Osteoclast formation in normal bone remodeling requires the presence of osteoblast lineage cells that express RANKL and macrophage-colony-stimulating factor (M-CSF), which interact with their cognate receptors on the osteoclast precursor. We identified secreted Frizzled-related protein-1 (sFRP-1), which is known to bind to Wnt and inhibit the Wnt signaling pathway, as an osteoblast-derived factor that impinges on osteoclast formation and activity. MATERIALS AND METHODS: Differential display of mRNA from osteoblast lineage cell lines established sFRP-1 to be highly expressed in an osteoclast supporting cell line. sFRP-1 expression in bone was determined by in situ hybridization, and the effects of sFRP-1 on osteoclast formation were determined using a neutralizing antibody, siRNA, for sFRP-1 and recombinant protein. RESULTS: In situ hybridization revealed sFRP-1 mRNA expression in osteoblasts and chondrocytes in murine bone. sFRP-1 mRNA expression could be elevated in calvarial primary osteoblasts in response to prostaglandin E2 (PGE2) or interleukin (IL)-11, whereas many other osteotropic agents (e.g., IL-1, IL-6, calcitrol, parathyroid hormone) were without any effect. In vitro assays of osteoclast formation established sFRP-1 to be an inhibitor of osteoclast formation. Neutralizing antibodies against sFRP-1 enhanced TRACP+ mononuclear and multinuclear osteoclast formation (3- and 2-fold, respectively) in co-cultures of murine osteoblasts with spleen cells, whereas siRNA complementary to sFRP-1 coding sequence significantly enhanced osteoclast formation in co-cultures of KUSA O (osteoblast/stromal cell line) and bone marrow cells, cultured in the presence of PGE2 and 1,25(OH)2 vitamin D3. Recombinant sFRP-1 dose-dependently inhibited osteoclast formation in osteoblast/spleen co-cultures, RANKL + M-CSF-treated splenic cultures, and RANKL-treated RAW264.7 cell cultures, indicating a direct action of sFRP-1 on hematopoietic cells. Consistent with this, sFRP-1 was found to bind to RANKL in ELISAs. CONCLUSION: sFRP-1 is expressed by osteoblasts and inhibits osteoclast formation. While sFRP-1 activity might involve the blocking of endogenous Wnt signaling, our results suggest that, alternatively, it could be because of direct binding to RANKL. This study describes a new mechanism whereby osteoblasts regulate osteoclastogenesis through the expression and release of sFRP-1.     

护骨素及配体在卵巢切除大鼠骨组织的蛋白表达和细胞定位

目的对卵巢切除和假切大鼠骨组织中护骨素（OPG）和配体（RANKL）的表达进行比较，观察不同分化阶段成骨细胞的OPG和RANKL表达变化，深入地探讨成骨细胞对破骨细胞发生的调控作用。方法9月龄雌性大鼠分为卵巢切除组和假切组，相同条件喂养3月后处死，取材制作骨病理切片，用免疫组织化学方法测定大鼠股骨OPG和RANKL的蛋白表达，用图像分析软件对蛋白表达情况半定量分析，对各组数据和组织形态进行分析比较。结果OPG和RANKL蛋白在骨组织表达相对稳定。RANKL主要表达在增殖活跃的成骨细胞和幼稚的骨细胞，OPG主要表达在成熟骨细胞和静息骨衬里细胞。与假切组相比，卵巢切除组骨组织内RANKL表达升高（P〈0．01），OPG表达降低（P〈0．05）。结论卵巢切除后骨组织中RANKL/OPG升高，破骨细胞活性增强，骨转换加快。不同发育阶段的成骨细胞对破骨细胞有不同的调节作用，幼稚阶段表现出对破骨细胞的诱导作用，而成熟阶段则表现为抑制作用。     

Osteoprotegerin and its Ligand: A New Paradigm for Regulation of Osteoclastogenesis and Bone Resorption

In just 3 years, striking new advances have been made in understanding the molecular mechanisms that govern the crosstalk between osteoblasts/stromal cells and hematopoietic osteoclast precursor cells that leads to osteoclastogenesis. Led first by the discovery of osteoprotegerin (OPG), a naturally occurring protein with potent osteoclastogenesis inhibitory activity, rapid progress was made to the isolation of RANKL, a transmembrane ligand expressed on osteoblasts/stromal cells that binds to RANK, a transmembrane receptor on hematopoietic osteoclast precursor cells. The interaction of RANK and RANKL initiates a signaling and gene expression cascade that results in differentiation and maturation of osteoclast precursor cells to active osteoclasts capable of resorbing bone. OPG acts as a decoy receptor, binding to RANKL and blocking its interaction with RANK, inhibiting osteoclast development. Many of the calciotropic hormones and cytokines, including 1,25(OH)₂D₃, PTH, PGE₂ and IL-11, appear to act through a dual capacity to inhibit production of OPG and stimulate production of RANKL. Estrogen, on the other hand, appears to inhibit production of RANKL and RANKL-stimulated osteoclastogenesis. Recently, the results of the first clinical trial with OPG supported its potential as a therapeutic agent for diseases such as osteoporosis. The new understanding provided by the RANK/RANKL/OPG paradigm for both differentiation of osteoclasts and their activation has had tremendous impact on the field and opened new avenues for development of possible treatments of diseases characterized by excessive bone resorption.     

Strontium ranelate treatment of human primary osteoblasts promotes an osteocyte-like phenotype while eliciting an osteoprotegerin response

Summary  The effect of strontium ranelate (SR) on human osteoblast differentiation was tested. SR induced osteoblastic proliferation, in vitro mineralization, and increased the expression of osteocyte markers. SR also elicited an osteoprotegerin (OPG) secretory response. We conclude that SR promotes the osteoblast maturation and osteocyte differentiation while promoting an additional antiresorptive effect. Introduction  SR is a new treatment for osteoporosis that reduces the risk of hip and vertebral fractures in postmenopausal women. This study sought to investigate the extent, to which SR modulates human osteoblast differentiation. Methods  Adult human primary osteoblasts (NHBC) were exposed to SR under mineralizing conditions in long-term cultures. Osteoblast differentiation status was investigated by cell-surface phenotypic analysis. Expression of genes associated with osteoblast/osteocyte differentiation was examined using real-time RT-PCR. Secreted OPG was assayed by enzyme-linked immunosorbent assay. Results  SR significantly increased osteoblast replication. SR time- and dose-dependently induced an osteocyte-like phenotype, as determined by cell surface alkaline phosphatase and STRO-1 expression. SR at 5 mM or greater dramatically increased in vitro mineralization. In parallel, mRNA levels of dentin matrix protein (DMP)-1 and sclerostin were higher under SR treatment, strongly suggestive of the presence of osteocytes. SR also increased the OPG/RANKL ratio throughout the culture period, consistent with an effect to inhibit osteoblast-induced osteoclastogenesis. Conclusions  This study suggests that SR can promote osteoblast maturation and an osteocyte-like phenotype. Coupled with its effect on the OPG/RANKL system, these findings are consistent with in vivo effects in patients receiving SR for the treatment of osteoporosis.     

Cyclic adenosine monophosphate/protein kinase A mediates parathyroid hormone/parathyroid hormone-related protein receptor regulation of osteoclastogenesis and expression of RANKL and osteoprotegerin mRNAs by marrow stromal cells.

Parathyroid hormone (PTH) is a major regulator of osteoclast formation and activation, effects that are associated with reciprocal up- and down-regulation of RANKL and osteoprotegerin (OPG), respectively. The roles of specific downstream signals generated by the activated PTH/PTH-related protein (PTHrP) receptor (PTH1R), such as cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) and phospholipase C/protein kinase C (PLC/PKC), in controlling RANKL and OPG expression and osteoclastogenesis remain uncertain. In MS1 conditionally transformed clonal murine marrow stromal cells, which support PTH-induced osteoclast formation from cocultured normal spleen cells, PTH(1-34) increased RANKL and macrophage colony-stimulating factor (M-CSF) mRNA expression and decreased that of OPG when present continuously for 7-20 days at 37 degrees C in the presence of dexamethasone (Dex). In cells precultured for 7 days and then treated with PTH(1-34), similar reciprocal regulation of RANKL and OPG occurred, maximally at 6-24 h, that was of greater amplitude than the changes induced by chronic (7-10 days) PTH exposure. These acute effects of PTH(1-34) were mimicked by PKA stimulators (8-bromoadenosine [8Br]-cAMP or forskolin [FSK]), blocked by the PKA inhibitor Rp-cAMPs but unaffected by the PKC inhibitor GF109203X. Amino-truncated PTH(1-34) analogs PTH(5-34) and PTH(7-34) neither increased cAMP production in MS1 cells nor regulated RANKL or OPG mRNA. Reciprocal RANKL/OPG mRNA regulation was induced in MS1 cells by PTH(3-34) but only at high concentrations that also increased cAMP. The highly PKA-selective PTH analog [Gly1,Arg19]human PTH(1-28) exerted effects similar to PTH(1-34) on RANKL and OPG mRNAs and on osteoclast formation, both in MS1/spleen cell cocultures and in normal murine bone marrow cultures. The direct PKC stimulator 12-O-tetradecanoylphorbol-13-acetate (PMA) did not induce RANKL mRNA in MS1 cells, but it did up-regulate OPG mRNA and also antagonized osteoclast formation induced by PTH(1-34) in both MS1/spleen cocultures and normal bone marrow cultures. Thus, cAMP/PKA signaling via the PTH1R is the primary mechanism for controlling RANKL-dependent osteoclastogenesis, although direct PKC activation may negatively regulate this effect of PTH by inducing expression of OPG.     

Ephrin B2/EphB4 Mediates the Actions of IGF‐I Signaling in Regulating Endochondral Bone Formation

Ephrin B2/EphB4 mediates interactions among osteoblasts (OBs), osteoclasts (OCLs), and chondrocytes to regulate their differentiation. We investigated the role of ephrin B2/EphB4 signaling in mediating the anabolic effects of insulin‐like growth factor‐I (IGF‐I) and parathyroid hormone (PTH) on those cells and overall endochondral bone formation. Immunohistochemistry demonstrated that the expression of ephrin B2 in OBs, OCLs, and osteocytes, and the expression of EphB4 in OBs and osteocytes was dramatically decreased in global IGF‐I knockout mice. Inactivation of EphB4 by EphB4 small, interfering RNA (siRNA) in cultured bone marrow stromal cells significantly decreased the mRNA levels of OB differentiation markers and abolished the stimulatory effects of IGF‐I on these markers. Blocking the interaction of EphB4 and ephrin B2 in the OB‐OCL cocultures with the EphB4 specific peptide TNYL‐RAW or deletion of ephrin B2 in OCL prior to coculture led to fewer and smaller tartrate‐resistant acid phosphatase (TRAP)‐positive cells, decreased expression of OB differentiation markers, and blunted response to IGF‐I for both OCL and OB differentiation. In the growth plate, both ephrin B2 and EphB4 are expressed in late stage proliferating and prehypertrophic chondrocytes, and their expression was decreased in mice lacking the IGF‐I receptor specifically in chondrocytes. In vitro, blocking the interaction of EphB4 and ephrin B2 in chondrogenic ATDC5 cells with TNYL‐RAW significantly decreased both basal and IGF1‐induced expression of type II and type X collagen. In the cocultures of ATDC5 cells and spleen cells (osteoclast precursors), TNYL‐RAW decreased the numbers of TRAP‐positive cells and the expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) and receptor activator of NF‐κB (RANK), and blocked their stimulation by IGF‐I. Our data indicate that IGF‐I/IGF‐IR signaling promotes OB, OCL, and chondrocyte differentiation via ephrin B2/EphB4 mediated cell‐cell communication. © 2014 American Society for Bone and Mineral Research.