EphrinB2/EphB4 inhibition in the osteoblast lineage modifies the anabolic response to parathyroid hormone

Previous reports indicate that ephrinB2 expression by osteoblasts is stimulated by parathyroid hormone (PTH) and its related protein (PTHrP) and that ephrinB2/EphB4 signaling between osteoblasts and osteoclasts stimulates osteoblast differentiation while inhibiting osteoclast differentiation. To determine the role of the ephrinB2/EphB4 interaction in the skeleton, we used a specific inhibitor, soluble EphB4 (sEphB4), in vitro and in vivo. sEphB4 treatment of cultured osteoblasts specifically inhibited EphB4 and ephrinB2 phosphorylation and reduced mRNA levels of late markers of osteoblast/osteocyte differentiation (osteocalcin, dentin matrix protein‐1 [DMP‐1], sclerostin, matrix‐extracellular phosphoglycoprotein [MEPE]), while substantially increasing RANKL. sEphB4 treatment in vivo in the presence and absence of PTH increased osteoblast formation and mRNA levels of early osteoblast markers (Runx2, alkaline phosphatase, Collagen 1α1, and PTH receptor [PTHR1]), but despite a substantial increase in osteoblast numbers, there was no significant change in bone formation rate or in late markers of osteoblast/osteocyte differentiation. Rather, in the presence of PTH, sEphB4 treatment significantly increased osteoclast formation, an effect that prevented the anabolic effect of PTH, causing instead a decrease in trabecular number. This enhancement of osteoclastogenesis by sEphB4 was reproduced in vitro but only in the presence of osteoblasts. These data indicate that ephrinB2/EphB4 signaling within the osteoblast lineage is required for late stages of osteoblast differentiation and, further, restricts the ability of osteoblasts to support osteoclast formation, at least in part by limiting RANKL production. This indicates a key role for the ephrinB2/EphB4 interaction within the osteoblast lineage in osteoblast differentiation and support of osteoclastogenesis. © 2013 American Society for Bone and Mineral Research.     

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.     

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.     

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.     

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。     

Inhibition of Lamin A/C Attenuates Osteoblast Differentiation and Enhances RANKL‐Dependent Osteoclastogenesis

Age‐related osteoporosis is characterized by low bone mass, poor bone quality, and impaired osteoblastogenesis. Recently, the Hutchinson‐Gilford progeria syndrome (HGPS), a disease of accelerated aging and premature osteoporosis, has been linked to mutations in the gene encoding for the nuclear lamina protein lamin A/C. Here, we tested the hypothesis that inhibition of lamin A/C in osteoblastic lineage cells impairs osteoblastogenesis and accelerates osteoclastogenesis. Lamin A/C was knocked‐down with small interfering (si)RNA molecules in human bone marrow stromal cells (BMSCs) differentiating toward osteoblasts. Lamin A/C knockdown led to an inhibition of osteoblast proliferation by 26% and impaired osteoblast differentiation by 48% based on the formation of mineralized matrix. In mature osteoblasts, expression levels of runx2 and osteocalcin mRNA were decreased by lamin A/C knockdown by 44% and 78%, respectively. Furthermore, protein analysis showed that osteoblasts with diminished levels of lamin A/C also secreted less osteocalcin and expressed a lower alkaline phosphatase activity (?50%). Lamin A/C inhibition increased RANKL mRNA and protein levels, whereas osteoprotegerin (OPG) expression was decreased, resulting in an increased RANKL/OPG ratio and an enhanced ability to support osteoclastogenesis, as reflected by a 34% increase of TRACP⁺ multinucleated cells. Our data indicate that lamin A/C is essential for proper osteoblastogenesis. Moreover, lack of lamin A/C favors an osteoclastogenic milieu and contributes to enhanced osteoclastogenesis.     

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.     

Vitamin D action and regulation of bone remodeling: suppression of osteoclastogenesis by the mature osteoblast.

Vitamin D acts through the immature osteoblast to stimulate osteoclastogenesis. Transgenic elevation of VDR in mature osteoblasts was found to inhibit osteoclastogenesis associated with an altered OPG response. This inhibition was confined to cancellous bone. This study indicates that vitamin D-mediated osteoclastogenesis is regulated locally by OPG production in the mature osteoblast. INTRODUCTION: Vitamin D stimulates osteoclastogenesis acting through its nuclear receptor (VDR) in immature osteoblast/stromal cells. This mobilization of calcium stores does not occur in a random manner, with bone preferentially removed from cancellous bone. The process whereby the systemic, humoral regulator is targeted to a particular region of the skeleton is unclear. MATERIALS AND METHODS: Bone resorption was assessed in mice with vitamin D receptor transgenically elevated in mature osteoblasts (OSVDR). Vitamin D-mediated osteoclastogenesis was examined in vitro using OSVDR osteoblasts and osteoblastic RANKL: osteoprotegerin (OPG) examined in vivo and in vitro after vitamin D treatment. RESULTS: Vitamin D-mediated osteoclastogenesis was reduced in OSVDR mice on chow and calcium-restricted diets, with effects confined to cancellous bone. OSVDR osteoblasts had a reduced capacity to support osteoclastogenesis in culture. The vitamin D-mediated reduction in OPG expression was reduced in OSVDR osteoblasts in vivo and in vitro, resulting in a reduced RANKL/OPG ratio in OSVDR compared with wildtype, after exposure to vitamin D. CONCLUSIONS: Mature osteoblasts play an inhibitory role in bone resorption, with active vitamin D metabolites acting through the VDR to increase OPG. This inhibition is less active in cancellous bone, effectively targeting this region for resorption after the systemic release of activated vitamin D metabolites.     

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.     

Effects of 17β-estradiol on adiponectin regulation of the expression of osteoprotegerin and receptor activator of nuclear factor-κB ligand

Adiponectin may exert a negative effect on bone metabolism by regulating osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) expression. However, the action of adiponectin on bone may be influenced by estrogen in women. The present study was undertaken to investigate the effects of 17β-estradiol (E2) on adiponectin-regulated OPG and RANKL expression in human osteoblast. Human osteoblasts were treated with α-MEM containing 10μg/ml adiponectin alone or together with 10(-10) to 10(-8)M E2 for 12-48h. Cells were also treated with α-MEM containing 10μg/ml adiponectin together with 10(-8)M E2 plus p38 agonist-anisomycin or estrogen receptor (ER) antagonist ICI182780 for 48h. The effects of E2 were also investigated by knockdown of ERs or overexpression of p38 MAPK in osteoblasts. Further, we examined the effects of E2 on adiponectin-dependent osteoclastogenesis by the co-culture systems of osteoblast and CD14+ peripheral blood monocytes (PBMCs). Real-time quantitative PCR (RT-PCR) and ELISA were used to detect OPG/RANKL mRNA and their corresponding protein expression, Western Blot was used to analyze the phosphorylated p38 (p-p38) levels. The results showed that E2 blocked adiponectin-induced p38 phosphorylation, decreased adiponectin-regulated OPG/RANKL mRNA and protein expression in a dose- and time-dependent manner. ICI182780 or knockdown of ERs abolished the effects of E2 on adiponectin-dependent p38 phosphorylation and OPG/RANKL expression. Furthermore, anisomycin or overexpression of p38 also reserved the effects of E2 on adiponectin-dependent p38 phosphorylation and OPG/RANKL expression. E2 inhibited adiponectin-dependent osteoclastogenesis in the co-culture systems of osteoblast and CD14+ PBMCs, whereas anisomycin, ICI182780, knockdown of ERs and overexpression of p38 significantly reversed this response. In conclusions, our findings demonstrated, through blocking the activation of adiponectin-induced p38 MAPK, E2 suppressed the adiponectin-regulated OPG/RANKL expression and then inhibited osteoclastogenesis, which suggested that estrogen would suppress the effect of adiponectin on bone metabolism.     

Production of IL-7 is increased in ovariectomized mice,but not RANKL mRNA expression by osteoblasts/stromal cells in bone,and IL-7 enhances generation of osteoclast precursors in vitro

Osteoclastogenic cytokines produced by T and B lineage cells and interleukin (IL)-7-induced expansion of the pool size of osteoclast precursors have been suggested to play an important role in acceleration of osteoclastogenesis induced by estrogen deficiency. However, the contribution of increased RANKL produced by osteoblasts/stromal cells to increase osteoclastogenesis in a mouse model of estrogen-deficient osteoporosis and in vitro effects of IL-7 on osteoclast precursor generation remain controversial. Thus, we investigated the effect of ovariectomy (OVX) of mice on production of RANKL, osteoprotegerin (OPG), and IL-7 in bone and the effect of IL-7 on osteoclast precursor generation in vitro. OVX did not significantly stimulate mRNA expressions of RANKL and OPG in whole femurs. Because the epiphysis, but not the femoral shaft (diaphysis) or bone marrow, is the main site of osteoclastogenesis, it is important to specifically analyze mRNA expression by osteoblasts/stromal cells at these parts of the femur. Therefore, we isolated RNA from bone marrow cell-free epiphysis, diaphysis, and flushed-out bone marrow and examined mRNA expression. The results showed no significant changes of RANKL and OPG mRNA expression in any part of the femur. In addition, OVX did not significantly affect RANKL and OPG mRNA expression by the adherent stromal cells isolated from flushed-out bone marrow cells but did stimulate RANKL mRNA expression by B220⁺ cells in the nonadherent cell fraction. On the other hand, OVX increased IL-7 mRNA expression in the femur as well as IL-7 concentrations in bone fluid. In cultures of unfractionated bone cells isolated by vigorous agitation of minced whole long bones to release the cells tightly attached to the bone surfaces, but not in cocultures of clonal osteoblasts/stromal cells and flushed-out bone marrow cells, IL-7 stimulated generations of osteoclasts as well as osteoclast precursors. These data suggest that increased RANKL production by osteoblasts/stromal cells is unlikely to play a central role in acceleration of osteoclastogenesis in estrogen deficiency of mice and that IL-7 stimulates osteoclast precursor generation, presumably through an action of IL-7 on the cells attached to bone rather than on cells contained in the bone marrow cell population.     

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.     

Regulation of receptor activator of nuclear factor-kappa B ligand and osteoprotegerin mRNA expression by parathyroid hormone is predominantly mediated by the protein kinase a pathway in murine bone marrow cultures

Parathyroid hormone (PTH) stimulates receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA and inhibits osteoprotegerin (OPG) mRNA expression in murine bone marrow cultures. To understand the mechanisms influencing these responses, we investigated the role of the protein kinase A (PKA) and protein kinase C (PKC) pathways in the regulation of RANKL and OPG mRNA expression in murine bone marrow cultures. Murine bone marrow cells were stimulated with bovine PTH(1-34) and (1-34) amide, which activate both pathways; PTH(3-34), which more selectively activates the PKC and calcium pathways; and human PTH (1-31), which stimulates adenylyl cyclase, but not protein kinase C. We also examined agents that more directly activate either the PKA pathway (forskolin [FSK] and 8-bromo cAMP [8-Br-cAMP]) or the PKC pathway (phorbol 12-myristate 13-acetate [PMA]) in murine bone marrow cultures. After 1 h, RANKL mRNA expression was stimulated to a similar degree by agents that activate either or both the PKA and PKC pathways. However, this effect was sustained for 24 h only with agents that stimulated PKA. OPG mRNA expression was inhibited by all agents that stimulated PKA at 6 h. In contrast, PKC-specific stimulators [PMA and bPTH(3-34)] had no effect on OPG regulation in this culture system. To determine the involvement of the PKC signaling pathway in responses of RANKL, bone marrow cells were pretreated with PMA for 24 h and then treated with PTH(1-34) or FSK for 2 h. PMA pretreatment did not alter the ability of PTH or FSK to stimulate RANKL or inhibit OPG mRNA expression. Treatment of cells with H-89, a PKA inhibitor, significantly reduced the ability of PTH and FSK to induce RANKL and inhibit OPG mRNA expression. Calphostin C, a PKC inhibitor, significantly reduced PMA-stimulated RANKL mRNA expression without altering PTH- or FSK-mediated effects on RANKL or OPG mRNA. Cycloheximide, an inhibitor for protein synthesis, inhibited PTH-stimulated RANKL mRNA expression by 60% without altering the effect of PTH on OPG mRNA expression. To examine the involvement of prostaglandin in PMA-mediated responses, cells were treated with indomethacin, a nonspecific prostaglandin G/H synthase (PGHS) inhibitor, or NS-398, a selective inhibitor of PGHS-2. Neither PGHS inhibitor altered PMA-induced effects on RANKL and OPG mRNA expression. These results demonstrate that the PKA pathway is predominantly involved in the effects of PTH on RANKL mRNA expression in murine bone marrow cultures, but there is also a PKC-mediated response, which is not sustained. Inhibition of OPG by PTH appears to be a selective PKA response.     

Expression profiles of receptor activator of nuclear factor kappaB ligand, receptor activator of nuclear factor kappaB, and osteoprotegerin messenger RNA in aged and ovariectomized rat bones.

The receptor activator of nuclear factor-kappaB ligand (RANKL; also known as tumor necrosis factor-related activation-induced cytokine [TRANCE], osteoprotegerin ligand [OPGL], and osteoclast differentiation factor [ODF]) is a transmembrane ligand expressed in osteoblasts and bone marrow stromal cells. It binds to RANK, which is expressed in osteoclast progenitor cells, and induces osteoclastogenesis. OPG, a decoy receptor for RANKL, also binds to RANKL, and competitive binding of RANKL with RANK or OPG is thought to regulate bone metabolism. To investigate roles of the RANKL/RANK/OPG system in pathophysiological conditions, the expression of RANKL, RANK, and OPG messenger RNA (mRNA) was analyzed in bones of aged and ovariectomized rats by means of in situ hybridization. In the control 8-week-old male and sham-operated female rat bones, the expression of RANKL mRNA was detected in hypertrophic chondrocytes of the growth plate and some periosteal and endosteal mesenchymal cells. The expression of RANK mRNA was detected in osteoclast-like cells and mononuclear cells in contact with the cortical and trabecular bones. The expression of OPG mRNA was detected in proliferating chondrocytes and osteocytes. In the 2.5-year-old rat bones, the expression of RANKL, RANK, and OPG mRNA tended to decrease except for the endosteal region. In the ovariectomized rat bones, the expression of RANKL, RANK, and OPG mRNA increased, and high expression of OPG mRNA was induced in resting chondrocytes and osteocytes. These results suggest that estrogen deficiency stimulates the RANKL/RANK/OPG system and induces OPG in cells that have been thought to be less important for bone metabolism.     

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.     

Expression of RANKL and OPG correlates with age-related bone loss in male C57BL/6 mice.

Osteoblasts regulate the recruitment and activity of osteoclasts through expression of RANKL and osteoprotegerin (OPG). To determine whether expression of RANKL and OPG change with age and how these changes relate to the bone loss of aging, we measured bone mass and cancellous volume, and expression of RANKL, OPG, alkaline phosphatase (AP), osteocalcin (OC), and alpha I collagen (COLL) in whole bone and osteoblast-like cells in culture using 6-week- (young), 6-month- (adult), and 24-month-old (old) mice. Cancellous volume decreased by 20% from young to adult and by 52% from adult to old. RANKL mRNA levels in whole bone were 2.1-fold and 4.4-fold higher in adult and old mice, respectively, compared with young mice, whereas OPG mRNA levels decreased with age slightly. RANKL expression was negatively (r = -0.99) and OPG was positively (r = 0.92) correlated with cancellous bone volume. Expression of RANKL was higher and OPG lower in cells from older animals early in culture (day 7). With cell maturation, RANKL mRNA levels in cells from young and adult mice increased, whereas levels in cells from old animals decreased. By 21 and 28 days of culture, no differences were found in RANKL mRNA in osteoblast-like cells among different age groups. We conclude that expression of RANKL and OPG change with age in whole bone and in cultured osteoblast-like cells. These changes favor increased osteoclast over osteoblast activity, and may explain, in part, the imbalance in bone formation and resorption associated with aging.     

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.