Suppression of osteoprotegerin expression by prostaglandin E2 is crucially involved in lipopolysaccharide-induced osteoclast formation

LPS is a potent stimulator of bone resorption in inflammatory diseases. The mechanism by which LPS induces osteoclastogenesis was studied in cocultures of mouse osteoblasts and bone marrow cells. LPS stimulated osteoclast formation and PGE(2) production in cocultures of mouse osteoblasts and bone marrow cells, and the stimulation was completely inhibited by NS398, a cyclooxygenase-2 inhibitor. Osteoblasts, but not bone marrow cells, produced PGE(2) in response to LPS. LPS-induced osteoclast formation was also inhibited by osteoprotegerin (OPG), a decoy receptor of receptor activator of NF-kappaB ligand (RANKL), but not by anti-mouse TNFR1 Ab or IL-1 receptor antagonist. LPS induced both stimulation of RANKL mRNA expression and inhibition of OPG mRNA expression in osteoblasts. NS398 blocked LPS-induced down-regulation of OPG mRNA expression, but not LPS-induced up-regulation of RANKL mRNA expression, suggesting that down-regulation of OPG expression by PGE(2) is involved in LPS-induced osteoclast formation in the cocultures. NS398 failed to inhibit LPS-induced osteoclastogenesis in cocultures containing OPG knockout mouse-derived osteoblasts. IL-1 also stimulated PGE(2) production in osteoblasts and osteoclast formation in the cocultures, and the stimulation was inhibited by NS398. As seen with LPS, NS398 failed to inhibit IL-1-induced osteoclast formation in cocultures with OPG-deficient osteoblasts. These results suggest that IL-1 as well as LPS stimulates osteoclastogenesis through two parallel events: direct enhancement of RANKL expression and suppression of OPG expression, which is mediated by PGE(2) production.     

Effects of arachidonic acid, docosahexaenoic acid, prostaglandin E(2) and parathyroid hormone on osteoprotegerin and RANKL secretion by MC3T3-E1 osteoblast-like cells

Bone is continuously remodeled through resorption by osteoclasts and the subsequent synthesis of the bone matrix by osteoblasts. Cell-to-cell contact between osteoblasts and osteoclast precursors is required for osteoclast formation. RANKL (receptor activator of nuclear factor-kappaB ligand) expressed on osteoblastic cell membranes stimulates osteoclastogenesis, while osteoprotegerin (OPG) secreted by osteoblasts inhibits osteoclastogenesis. Although polyunsaturated fatty acids (PUFAs) have been implicated in bone homeostasis, the effects thereof on OPG and RANKL secretion have not been investigated. MC3T3-E1 osteoblasts were exposed to the n-6 PUFA arachidonic acid (AA) and the n-3 PUFA docosahexaenoic acid (DHA); furthermore, the bone-active hormone parathyroid hormone (PTH) and the effects thereof were tested on OPG and RANKL secretion. Prostaglandin E(2) (PGE(2)), a product of AA metabolism that was previously implicated in bone homeostasis, was included in the study. AA (5.0-20 microg/ml) inhibited OPG secretion by 25-30%, which was attenuated by pretreatment with the cyclooxygenase blocker indomethacin, suggesting that the inhibitory effect of AA on OPG could possibly be PGE(2)-mediated. MC3T3-E1 cells secreted very low basal levels of RANKL, but AA stimulated RANKL secretion, thereby decreasing the OPG/RANKL ratio. DHA suppressed OPG secretion to a smaller extent than AA. This could, however, be due to endogenous PGE(2) production. No RANKL could be detected after exposing the MC3T3-E1 cells to DHA. PTH did not affect OPG secretion, but stimulated RANKL secretion. This study demonstrates that AA and PTH reduce the OPG/RANKL ratio and may increase osteoclastogenesis. DHA, however, had no significant effect on OPG or RANKL in this model.     

Roles of stromal cell RANKL, OPG, and M-CSF expression in biphasic TGF-beta regulation of osteoclast differentiation

To better understand the complex roles of transforming growth factor-beta (TGF-beta) in bone metabolism, we examined the impact of a range of TGF-beta concentrations on osteoclast differentiation. In co-cultures of support cells and spleen or marrow osteoclast precursors, low TGF-beta concentrations stimulated while high concentrations inhibited differentiation. We investigated the influences of TGF-beta on macrophage colony stimulating factor (M-CSF), receptor activator of NF-kappaB ligand (RANKL), and osteoprotegerin (OPG) expression and found a dose dependent inhibition of M-CSF expression. RANKL expression was elevated at low TGF-beta concentrations with a less dramatic increase in OPG. Addition of OPG blocked differentiation at the stimulatory TGF-beta dose. Thus, low TGF-beta concentrations elevated the RANKL/OPG ratio while high concentrations did not, supporting that, at low TGF-beta concentrations, there is sufficient M-CSF and a high RANKL/OPG ratio to stimulate differentiation. At high TGF-beta concentrations, the RANKL/OPG ratio and M-CSF expression were both repressed and there was no differentiation. We examined whether TGF-beta-mediated repression of osteoclasts differentiation is due to these changes by adding M-CSF and/or RANKL and did not observe any impact on differentiation repression. We studied direct TGF-beta impacts on osteoclast precursors by culturing spleen or marrow cells with M-CSF and RANKL. TGF-beta treatment dose-dependently stimulated osteoclast differentiation. These data indicate that low TGF-beta levels stimulate osteoclast differentiation by impacting the RANKL/OPG ratio while high TGF-beta levels repress osteoclast differentiation by multiple avenues including mechanisms independent of the RANKL/OPG ratio or M-CSF expression regulation.     

Metformin stimulates osteoprotegerin and reduces RANKL expression in osteoblasts and ovariectomized rats

Anti-diabetic drug metformin has been shown to enhance osteoblasts differentiation and inhibit osteoclast differentiation in vitro and prevent bone loss in ovariectomized (OVX) rats. But the mechanisms through which metformin regulates osteoclastogensis are not known. Osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) are cytokines predominantly secreted by osteoblasts and play critical roles in the differentiation and function of osteoclasts. In this study, we demonstrated that metformin dose-dependently stimulated OPG and reduced RANKL mRNA and protein expression in mouse calvarial osteoblasts and osteoblastic cell line MC3T3-E1. Inhibition of AMP-activated protein kinase (AMPK) and CaM kinase kinase (CaMKK), two targets of metformin, suppressed endogenous and metformin-induced OPG secretion in osteoblasts. Moreover, supernatant of osteoblasts treated with metformin reduced formation of tartrate resistant acid phosphatase (TRAP)-positive multi-nucleated cells in Raw264.7 cells. Most importantly, metformin significantly increased total body bone mineral density, prevented bone loss and decreased TRAP-positive cells in OVX rats proximal tibiae, accompanied with an increase of OPG and decrease of RANKL expression. These in vivo and in vitro studies suggest that metformin reduces RANKL and stimulates OPG expression in osteoblasts, further inhibits osteoclast differentiation and prevents bone loss in OVX rats.     

Impact of the microgravity environment in a 3-dimensional clinostat on osteoblast- and osteoclast-like cells

Mechanical unloading conditions result in decreases in bone mineral density and quantity, which may be partly attributed to an imbalance in bone formation and resorption. To investigate the effect of mechanical unloading on osteoblast and osteoclast differentiation, and the expression of RANKL and OPG genes in osteoblasts, we used a three-dimensional (3D) clinostat system simulating microgravity to culture MC3T3-E1 and RAW264.7 cells. Long-term exposure (7 days) of MC3T3-E1 cells to microgravity in the 3D clinostat inhibited the expression of Runx2, Osterix, type I collagen alphaI chain, RANKL and OPG genes. Similarly, 3D clinostat exposure inhibited the enhancement of beta3-integrin gene expression, which normally induced by sRANKL stimulation in RAW264.7 cells. These results, taken together, demonstrate that long-term 3D clinostat exposure inhibits the differentiation of MC3T3-E1 cells together with suppression of RANKL and OPG gene expression, as well as the RANKL-dependent cellular fusion of RAW264.7 cells, suggesting that long-term mechanical unloading suppresses bone formation and resorption.     

Runx-2 is not essential for the vitamin D-regulated expression of RANKL and osteoprotegerin in osteoblastic cells

The differentiation and activity of osteoclasts are positively and negatively controlled by receptor activator of nuclear factor-kappaB ligand (RANKL), which is expressed on the surface of osteoblasts and stromal cells, and its decoy receptor osteoprotegerin (OPG), which is secreted by osteoblasts and stromal cells, respectively. The expression of the genes for RANKL and OPG is regulated by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)]. Runt-related gene-2 (Runx-2) is essential for osteoblast differentiation and there are several reports that Runx-2 is involved in osteoclast formation. Therefore, to clarify the role of Runx-2 in osteoclastogenesis, we designed a series of experiments using C2 cells and C6 cells, which are derived from calvariae of runx2-deficient mice. Treatment of C2 cells and C6 cells with 1alpha,25(OH)(2)D(3) for 2-4 days increased and decreased the levels of expression of the mRNAs for RANKL and OPG, respectively, and the effects were dose-dependent. However, by day 8, the level of RANKL mRNA had fallen and that of OPG mRNA had risen. Furthermore, C6 cells induced the differentiation of mouse spleen cells into tartrate-resistant acid phosphatase-positive (TRAP-positive) multinucleated cells (osteoclast-like cells) in the presence of 10(-7)M 1alpha,25(OH)(2)D(3). Such formation of osteoclast-like cells was inhibited by exogenous OPG in a dose-dependent manner. Thus, our findings indicate that Runx-2 is not essential for the expression of RANKL and OPG, and the formation of osteoclast-like cells.     

Modeled microgravity stimulates osteoclastogenesis and bone resorption by increasing osteoblast RANKL/OPG ratio

Mechanical unloading causes detrimental effects on the skeleton, but the underlying mechanisms are still unclear. We investigated the effect of microgravity on osteoblast ability to regulate osteoclastogenesis. Mouse osteoblast primary cultures were grown for 24 h at unit gravity or under simulated microgravity, using the NASA-developed Rotating Wall Vessel bioreactor. Conditioned media (CM) from osteoblasts subjected to microgravity increased osteoclastogenesis and bone resorption in mouse bone marrow cultures. In these osteoblasts, the RANKL/OPG ratio was higher relative to 1g. Consistently, treatment with high concentrations of OPG-inhibited osteoclastogenesis and bone resorption in the presence of CM arising from osteoblasts cultured under microgravity. Microgravity failed to affect osteoblast differentiation and function in the time frame of the experiment, as we found no effect on alkaline phosphatase mRNA and activity, nor on Runx2, osteocalcin, osteopontin, and collagen1A2 mRNA expression. In contrast, microgravity induced a time dependent increase of ERK-1/2 phosphorylation, while phospho-p38 and phospho-JNK remained unchanged. Apoptosis, revealed by bis-benzimide staining, was similar among the various gravity conditions, while it was increased under microgravity after treatment with the MEK-1/2 inhibitor, PD98059, suggesting a protection role by ERK-1/2 against cell death. In conclusion, microgravity is capable to indirectly stimulate osteoclast formation and activity by regulating osteoblast secretion of crucial regulatory factors such as RANKL and OPG. We hypothesize that this mechanism could contribute to bone loss in individuals subjected to weightlessness and other unloading conditions.     

Protein expression and functional difference of membrane-bound and soluble receptor activator of NF-kappaB ligand: modulation of the expression by osteotropic factors and cytokines

A variety of humoral factors modulate the osteoclastogenesis. Receptor activator of NF-kappaB ligand (RANKL) expressed on osteoblast/stromal lineage cells plays a pivotal role to transduce an essential differentiation signal to osteoclast lineage cells through binding to its receptor, RANK, expressed on the latter cell population; however, the difficulty to detect RANKL protein expression hampers us in investigating the regulation of RANKL expression by humoral factors. To determine protein expression of RANKL, we have established a new method, named as a ligand-receptor precipitation (LRP) Western blot analysis, which can specifically concentrate the target protein by the use of specific binding characteristic between RANKL and RANK/osteoprotegrin (OPG). RANKL protein expression in the postnuclear supernatant was not detected by common Western blotting, but LRP Western blot analysis clearly showed that RANKL is produced as a membrane-bound protein on murine osteoblasts/stromal cells, and cleaved into a soluble form by metalloprotease. Cytokines stimulating the osteoclastogenesis, such as IL-1beta, IL-6, IL-11, IL-17, and TNF-alpha, increased the expression of RANKL with decrease of OPG expression in osteoblasts/stromal cells. In contrast, cytokines inhibiting the osteoclastogenesis, such as IL-13, INF-gamma, and TGF-beta1 suppressed the expression of RANKL and/or augmented OPG expression. Functional difference between membrane-bound and soluble RANKL was demonstrated, which showed that membrane-bound RANKL works more efficiently than soluble RANKL in the osteoclastogenesis developed from murine bone marrow cell culture. The present study indicates the usefulness of LRP Western blot analysis, which shows that the modulation of osteoclastogenesis by humoral factors is achieved, in part, by regulation of the expression of RANKL and OPG in osteoblast/stromal lineage cells.     

RANKL与骨重建

骨是一种动态更新的组织,它不断进行骨吸收（bone resorption）与骨形成（bone formation）的平衡,这个过程称之为骨重建（bone remodeling）．核因子κB受体活化因子配体（receptor activator of nuclear factor κB ligand,RANKL）是骨吸收和骨形成耦联的关键,具有诱导破骨细胞（osteoclast, OC）生成、活化,抑制破骨细胞凋亡的作用．RANKL最初发现于活化的T细胞,但骨重建过程中RANKL主要来源于骨细胞、成骨细胞和骨髓基质细胞．RANKL/核因子κB受体活化因子（receptor activator of nuclear factor κB,RANK）/骨保护素（osteoprotegerin, OPG）信号通路在成骨细胞调控破骨细胞生成的过程中起着重要的调节作用,是维持骨重建平衡的关键．本文就RANKL及其在骨中的分子作用机制作一综述.     

RANKL synthesized by articular chondrocytes contributes to juxta-articular bone loss in chronic arthritis

Introduction

The receptor activator nuclear factor-kappaB ligand (RANKL) diffuses from articular cartilage to subchondral bone. However, the role of chondrocyte-synthesized RANKL in rheumatoid arthritis-associated juxta-articular bone loss has not yet been explored. This study aimed to determine whether RANKL produced by chondrocytes induces osteoclastogenesis and juxta-articular bone loss associated with chronic arthritis.

Methods

Chronic antigen-induced arthritis (AIA) was induced in New Zealand (NZ) rabbits. Osteoarthritis (OA) and control groups were simultaneously studied. Dual X-ray absorptiometry of subchondral knee bone was performed before sacrifice. Histological analysis and protein expression of RANKL and osteoprotegerin (OPG) were evaluated in joint tissues. Co-cultures of human OA articular chondrocytes with peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with macrophage-colony stimulating factor (M-CSF) and prostaglandin E₂ (PGE₂), then further stained with tartrate-resistant acid phosphatase.

Results

Subchondral bone loss was confirmed in AIA rabbits when compared with controls. The expression of RANKL, OPG and RANKL/OPG ratio in cartilage were increased in AIA compared to control animals, although this pattern was not seen in synovium. Furthermore, RANKL expression and RANKL/OPG ratio were inversely related to subchondral bone mineral density. RANKL expression was observed throughout all cartilage zones of rabbits and was specially increased in the calcified cartilage of AIA animals. Co-cultures demonstrated that PGE₂-stimulated human chondrocytes, which produce RANKL, also induce osteoclasts differentiation from PBMCs.

Conclusions

Chondrocyte-synthesized RANKL may contribute to the development of juxta-articular osteoporosis associated with chronic arthritis, by enhancing osteoclastogenesis. These results point out a new mechanism of bone loss in patients with rheumatoid arthritis.     

Prolactin decreases expression of Runx2, osteoprotegerin, and RANKL in primary osteoblasts derived from tibiae of adult female rats

Hyperprolactinemia caused by physiological or pathological conditions, such as those occurring during lactation and prolactinoma, respectively, results in progressive osteopenia. The underlying mechanisms, however, are controversial. Prolactin (PRL) may directly attenuate the functions of osteoblasts, since these bone cells express PRL receptors. The present study therefore aimed to investigate the effects of PRL on the expression of genes related to the osteoblast functions by using quantitative real-time PCR technique. Herein, we used primary osteoblasts that were derived from the tibiae of adult rats and displayed characteristics of differentiated osteoblasts, including in vitro mineralization. Osteoblasts exposed for 48 h to 1000 ng/mL PRL, but not to 10 or 100 ng/mL PRL, showed decreases in the mRNA expression of Runx2, osteoprotegerin (OPG), and receptor activator of nuclear factor kappaBeta ligand (RANKL) by 60.49%, 72.74%, and 87.51%, respectively. Nevertheless, PRL did not change the RANKL/OPG ratio, since expression of OPG and RANKL were proportionally decreased. These concentrations of PRL had no effect on the mRNA expression of osteocalcin and osteopontin, nor on mineralization. High pathologic concentrations of PRL (1000 ng/mL) may downregulate expression of genes that are essential for osteoblast differentiation and functions. The present results explained the clinical findings of hyperprolactinemia-induced bone loss.     

Bone morphogenic protein 2 directly enhances differentiation of murine osteoclast precursors

Previous studies found that bone morphogenic proteins (BMPs) support osteoclast formation, but it is not clear whether this is a direct effect on osteoclasts or mediated indirectly through osteoblasts. We have shown that a mouse deficient for the BMP antagonist Twisted gastrulation suggested a direct positive role for BMPs on osteoclastogenesis. In this report, we further determine the significance of BMP signaling on osteoclast formation in vitro. We find that BMP2 synergizes with suboptimal levels of receptor activator of NF‐κB ligand (RANKL) to enhance in vitro differentiation of osteoclast‐like cells. The enhancement by BMP2 is not a result of changes in the rate of proliferation or survival of the bone marrow‐derived cultures, but is accompanied by an increase in expression of genes involved in osteoclast differentiation and fusion. Treatment with BMP2 did not significantly alter expression of RANKL or OPG in our osteoclast cultures, suggesting that the enhancement of osteoclastogenesis is not mediated indirectly through osteoblasts or stromal cells. Consistent with this, we detected phosphorylated SMAD1,5,8 (p‐SMAD) in the nuclei of mononuclear and multinucleated cells in osteoclast cultures. Levels of p‐SMAD, BMP2, and BMP receptors increased during differentiation. RNAi suppression of Type II BMP receptor inhibited RANKL‐stimulated formation of multinuclear TRAP‐positive cells. The BMP antagonist noggin inhibited RANKL‐mediated osteoclast differentiation when added prior to day 3, while addition of noggin on day 3 or later failed to inhibit their differentiation. Taken together, these data indicate that osteoclasts express BMP2 and BMP receptors, and that autocrine BMP signaling directly promotes the differentiation of osteoclasts‐like cells. J. Cell. Biochem. 109: 672–682, 2010. © 2009 Wiley‐Liss, Inc.     

Human interleukin-1-induced murine osteoclastogenesis is dependent on RANKL, but independent of TNF-alpha

Although interleukin-1 (IL-1) has been implicated in the pathogenesis of inflammatory osteolysis, the means by which it recruits osteoclasts and promotes bone destruction are largely unknown. Recently, a cytokine-driven, stromal cell-free mouse osteoclastogenesis model was established. A combination of macrophage colony stimulating factor (M-CSF) and receptor activator of NFkappaB ligand (RANKL) was proven to be sufficient in inducing differentiation of bone marrow hematopoietic precursor cells to bone-resorbing osteoclasts in the absence of stromal cells or osteoblasts. This study utilizes this model to examine the impact of human IL-1beta on in vitro osteoclastogenesis of bone marrow progenitor cells. We found that osteoclast precursor cells failed to undergo osteoclastogenesis when treated with IL-1 alone. In contrast, IL-1 dramatically up-regulated osteoclastogenesis by 2.5- to 4-folds in the presence of RANKL and M-CSF. The effect can be significantly blocked by IL-1 receptor antagonist (p < 0.01). Tumor necrosis factor-alpha (TNF-alpha) was undetectable in the culture medium of differentiating osteoclasts induced by IL-1. Adding exogenous TNF-alpha neutralizing antibody had no influence on the IL-1-induced effect as well. These results show that in the absence of stromal cells, IL-1 exacerbates osteoclastogenesis by cooperating with RANKL and M-CSF, while TNF-alpha is not involved in this IL-1-stimulated osteoclast differentiation pathway.     

Role of Runx genes in chondrocyte differentiation

Runx2/Cbfa1 plays a central role in skeletal development as demonstrated by the absence of osteoblasts/bone in mice with inactivated Runx2/Cbfa1 alleles. To further investigate the role of Runx2 in cartilage differentiation and to assess the potential of Runx2 to induce bone formation, we cloned chicken Runx2 and overexpressed it in chick embryos using a retroviral system. Infected chick wings showed multiple phenotypes consisting of (1) joint fusions, (2) expansion of carpal elements, and (3) shortening of skeletal elements. In contrast, bone formation was not affected. To investigate the function of Runx2/Cbfa1 during cartilage development, we have generated transgenic mice that express a dominant negative form of Runx2 in cartilage. The selective inactivation of Runx2 in chondrocytes results in a severe shortening of the limbs due to a disturbance in chondrocyte differentiation, vascular invasion, osteoclast differentiation, and periosteal bone formation. Analysis of the growth plates in transgenic mice and in chick limbs shows that Runx2 is a positive regulator of chondrocyte differentiation and vascular invasion. The results further indicate that Runx2 promotes chondrogenesis either by maintaining or by initiating early chondrocyte differentiation. Furthermore, Runx2 is essential but not sufficient to induce osteoblast differentiation. To analyze the role of runx genes in skeletal development, we performed in situ hybridization with Runx2- and Runx3-specific probes. Both genes were coexpressed in cartilaginous condensations, indicating a cooperative role in the regulation of early chondrocyte differentiation and thus explaining the expansion/maintenance of cartilage in the carpus and joints of infected chick limbs.