Julian M. W. Quinn

Localization of RANKL (receptor activator of NF kappa B ligand) mRNA and protein in skeletal and extraskeletal tissues.

RANKL (receptor activator of NFkappaB ligand) is a membrane-associated osteoblastic molecule, and along with macrophage-colony-stimulating factor, is crucial for osteoclast formation. RANKL is known to be strongly expressed in osteoblasts and lymphoid tissues. We have sought to determine the skeletal and extraskeletal sites of production of RANKL mRNA and protein using the techniques of in situ hybridization and immunohistochemistry. Expression of RANKL mRNA and protein were determined in the developmental progression of endochondral bone formation in mouse, intramembranous bone formation in a rabbit model (mRNA only), in human giant cell tumors of bone, and at extraskeletal sites in the mouse. RANKL mRNA was expressed in prehypertrophic and hypertrophic chondrocytes at day E15 embryonic mouse long bone, and its expression was maintained at these sites throughout development. In newborn and adult mice, high levels of RANKL mRNA were expressed in mesenchymal cells of the periosteum and in mature osteoblasts, while megakaryocytes within the marrow microenvironment expressed RANKL mRNA from 1 week of age. Immunohistochemical analysis revealed a similar localization pattern of RANKL protein at the sites described. In the intramembranous bone formation model, RANKL mRNA was expressed in mesenchymal cells and in actively synthesizing osteoblasts, but not in flattened lining osteoblasts or late osteocytes. Expression of RANKL mRNA and protein in osteoclasts was variable with those within resorption lacunae showing the strongest signal/staining. Likewise, expression varied in osteoclasts from giant cell tumor of bone with a minority of tartrate-resistant acid phosphatase-positive multinucleated cells having no detectable RANKL mRNA or protein. In extraskeletal tissues, RANKL mRNA and protein were detected in the brain, heart, kidney, skeletal muscle, and skin throughout mouse development, suggesting the possibility of several other functions of the molecule. RANKL was also developmentally regulated, as evidenced by its expression in the intestine, liver, and lung at E15 and newborn mouse but not in the adult.

Transforming Growth Factor β Affects Osteoclast Differentiation via Direct and Indirect Actions

Transforming growth factor β (TGF‐β) is abundant in bone and has complex effects on osteolysis, with both positive and negative effects on osteoclast differentiation, suggesting that it acts via more than one mechanism. Osteoclastogenesis is determined primarily by osteoblast (OB) expression of the tumor necrosis factor (TNF)‐related molecule receptor activator of NF‐κB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG), which are increased and decreased, respectively, by osteolytic factors. A RANKL‐independent osteoclastogenic mechanism mediated by TNF‐α has also been shown. Therefore, we investigated TGF‐β effects on osteoclast formation in culture systems in which osteoclastogenic stimulus is dependent on OBs and culture systems where it was provided by exogenously added RANKL or TNF‐α. Both OPG and TGF‐β inhibited osteoclast formation in hemopoietic cell/OB cocultures, but the kinetics of their action differed. TGF‐β also inhibited osteoclastogenesis in cocultures of cells derived from OPG null (opg−/−) mice. TGF‐β strongly decreased RANKL messenger RNA (mRNA) expression in cultured osteoblasts, and addition of exogenous RANKL to TGFβ‐inhibited cocultures of opg−/− cells partially restored osteoclastogenesis. Combined, these data indicate that the inhibitory actions of TGF‐β were mediated mainly by decreased OB production of RANKL. In contrast, in the absence of OBs, TGF‐β greatly increased osteoclast formation in recombinant RANKL‐ or TNF‐α‐stimulated cultures of hemopoietic cells or RAW 264.7 macrophage‐like cells to levels several‐fold greater than attainable by maximal stimulation by RANKL or TNF‐α. These data suggest that TGF‐β may increase osteoclast formation via action on osteoclast precursors. Therefore, although RANKL (or TNF‐α) is essential for osteoclast formation, factors such as TGF‐β may powerfully modify these osteoclastogenic stimuli. Such actions may be critical to the control of physiological and pathophysiological osteolysis.

Bone Morphogenetic Protein 2 Stimulates Osteoclast Differentiation and Survival Supported by Receptor Activator of Nuclear Factor-κB Ligand

Bone is a major storage site for TGFbeta superfamily members, including TGFbeta and bone morphogenetic proteins. It is believed that these cytokines are released from bone during bone resorption. Recent studies have shown that both RANKL and macrophage colony-stimulating factor are two essential factors produced by osteoblasts for inducing osteoclast differentiation. In the present study we examined the effects of bone morphogenetic protein-2 on osteoclast differentiation and survival supported by RANKL and/or macrophage colony-stimulating factor. Mouse bone marrow-derived macrophages differentiated into osteoclasts in the presence of RANKL and macrophage colony-stimulating factor. TGFbeta superfamily members such as bone morphogenetic protein-2, TGFbeta, and activin A markedly enhanced osteoclast differentiation induced by RANKL and macrophage colony-stimulating factor, although each cytokine alone failed to induce osteoclast differentiation in the absence of RANKL. Addition of a soluble form of bone morphogenetic protein receptor type IA to the culture markedly inhibited not only osteoclast formation induced by RANKL and bone morphogenetic protein-2, but also the basal osteoclast formation supported by RANKL alone. Either RANKL or macrophage colony-stimulating factor stimulated the survival of purified osteoclasts. Bone morphogenetic protein-2 enhanced the survival of purified osteoclasts supported by RANKL, but not by macrophage colony-stimulating factor. Both bone marrow macrophages and mature osteoclasts expressed bone morphogenetic protein-2 and bone morphogenetic protein receptor type IA mRNAs. An EMSA revealed that RANKL activated nuclear factor-kappaB in purified osteoclasts. Bone morphogenetic protein-2 alone did not activate nuclear factor-kappaB, but rather inhibited the activation of nuclear factor-kappaB induced by RANKL in purified osteoclasts. These findings suggest that bone morphogenetic protein-mediated signals cross-communicate with RANKL-mediated ones in inducing osteoclast differentiation and survival. The enhancement of RANKL-induced survival of osteoclasts by bone morphogenetic protein-2 appears unrelated to nuclear factor-kappaB activation.

Expression of osteoclast differentiation factor at sites of bone erosion in collagen‐induced arthritis

OBJECTIVE To investigate the cellular mechanism of bone destruction in collagen-induced arthritis (CIA). METHODS After induction of CIA in DA rats, a histologic study of the advanced arthritic lesion was carried out on whole, decalcified joints from the hindpaws of affected animals. To conclusively identify osteoclasts, joint tissue sections were stained for tartrate-resistant acid phosphatase (TRAP) enzyme activity, and calcitonin receptors (CTR) were identified using a specific rabbit polyclonal antibody. The expression of messenger RNA (mRNA) for the osteoclast differentiation factor (also known as receptor activator of nuclear factor kappaB ligand [RANKL]) was investigated using in situ hybridization with a specific riboprobe. RESULTS TRAP-positive and CTR-positive multinucleated cells were invariably detected in arthritic lesions that were characterized by bone destruction. Osteoclasts were identified at the pannus-bone and pannus-subchondral bone junctions of arthritic joints, where they formed erosive pits in the bone. TRAP-positive multinucleated cells were detected within synovium and at the bone erosive front; however, CTR-positive multinucleated cells were present only at sites adjacent to bone. RANKL mRNA was highly expressed in the synovial cell infiltrate in arthritic joints, as well as by osteoclasts at sites of bone erosion. CONCLUSION Focal bone erosion in CIA is attributed to cells expressing definitive features of osteoclasts, including CTR. The expression of RANKL by cells within inflamed synovium suggests a mechanism for osteoclast differentiation and activation at sites of bone erosion. Inhibitors of RANKL may represent a novel approach to treatment of bone loss in rheumatoid arthritis.

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.

Fibroblastic stromal cells express receptor activator of NF-kappa B ligand and support osteoclast differentiation.

Osteoclast formation in bone is supported by osteoblasts expressing receptor activator of NF‐κB ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF) expression. Numerous osteotropic factors regulate expression levels of RANKL and the RANKL decoy receptor osteoprotegerin (OPG) in osteoblasts, thereby affecting osteoclast differentiation. However, not only is RANKL widely expressed in soft tissues, but osteoclasts have been noted in extraskeletal lesions. We found that cultured skin fibroblastic cells express RANKL, M‐CSF, and OPG messenger (mRNA). Stimulation by 1α,25 dihydroxyvitamin D3 [1,25(OH)2D3] plus dexamethasone (Dex) augmented RANKL and diminished OPG mRNA expression in fibroblastic cells and caused the formation of numerous osteoclasts in cocultures of skin fibroblastic cells with hemopoietic cells or monocytes. The osteoclasts thus formed expressed tartrate‐resistant acid phosphatase (TRAP) and calcitonin (CT) receptors and formed resorption pits in cortical bone. Osteoclast formation also was stimulated (in the presence of Dex) by prostaglandin E2 (PGE2), interleukin‐11 (IL‐11), IL‐1, tumor necrosis factor‐α (TNF‐α), and parathyroid hormone‐related protein (PTHrP), factors which also stimulate osteoclast formation supported by osteoblasts. In addition, granulocyte‐macrophage‐CSF (GM‐CSF), transforming growth factor‐β (TGF‐β), and OPG inhibited osteoclast formation in skin fibroblastic cell‐hemopoietic cell cocultures; CT reduced only osteoclast nuclearity. Fibroblastic stromal cells from other tissues (lung, respiratory diaphragm, spleen, and tumor) also supported osteoclast formation. Thus, RANKL‐positive fibroblastic cells in extraskeletal tissues can support osteoclastogenesis if osteolytic factors and osteoclast precursors are present. Such mesenchymally derived cells may play a role in pathological osteolysis and may be involved in osteoclast formation in extraskeletal tissues.

The Heat Shock Protein 90 Inhibitor, 17-Allylamino-17-demethoxygeldanamycin, Enhances Osteoclast Formation and Potentiates Bone Metastasis of a Human Breast Cancer Cell Line

Breast cancer metastasis to the bone occurs frequently, causing numerous complications including severe pain, fracture, hypercalcemia, and paralysis. Despite its prevalence and severity, few effective therapies exist. To address this, we examined whether the heat shock protein 90 (Hsp90) inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), would be efficacious in inhibiting breast cancer metastasis to bone. Utilizing the human breast cancer subline, MDA-MB-231SA, previously in vivo selected for its enhanced ability to generate osteolytic bone lesions, we determined that 17-AAG potently inhibited its in vitro proliferation and migration. Moreover, 17-AAG significantly reduced MDA-MB-231SA tumor growth in the mammary-fat pad of nude mice. Despite these findings, 17-AAG enhanced the incidence of bone metastasis and osteolytic lesions following intracardiac inoculation in the nude mouse. Consistent with these findings, 17-AAG enhanced osteoclast formation 2- to 4-fold in mouse bone marrow/osteoblast cocultures, receptor activator of nuclear factor kappaB ligand (RANKL)-stimulated bone marrow, and RAW264.7 cell models of in vitro osteoclastogenesis. Moreover, the drug enhanced osteoclastogenesis in human cord blood progenitor cells, demonstrating that its effects were not limited to mouse models. In addition to 17-AAG, other Hsp90 inhibitors, such as radicicol and herbimycin A, also enhanced osteoclastogenesis. A pro-osteolytic action of 17-AAG independent of tumor presence was also determined in vivo, in which 17-AAG-treated tumor-naive mice had reduced trabecular bone volume with an associated increase in osteoclast number. Thus, HSP90 inhibitors can stimulate osteoclast formation, which may underlie the increased incidence of osteolysis and skeletal tumor incidence caused by 17-AAG in vivo. These data suggest an important contraindication to the Hsp90 targeted cancer therapy currently undergoing clinical trial.

Cardiotrophin‐1 Is an Osteoclast‐Derived Stimulus of Bone Formation Required for Normal Bone Remodeling

Cardiotrophin (CT‐1) signals through gp130 and the LIF receptor (LIFR) and plays a major role in cardiac, neurological, and liver biology. We report here that CT‐1 is also expressed within bone in osteoclasts and that CT‐1 is capable of increasing osteoblast activity and mineralization both in vitro and in vivo. Furthermore, CT‐1 stimulated CAAT/enhancer‐binding protein‐δ (C/EBPδ) expression and runt‐related transcription factor 2 (runx2) activation. In neonate CT‐1−/− mice, we detected low bone mass associated with reduced osteoblasts and many large osteoclasts, but increased cartilage remnants within the bone, suggesting impaired resorption. Cultured bone marrow (BM) from CT‐1−/− mice generated many oversized osteoclasts and mineralized poorly compared with wildtype BM. As the CT‐1−/− mice aged, the reduced osteoblast surface (ObS/BS) was no longer detected, but impaired bone resorption continued resulting in an osteopetrotic phenotype in adult bone. CT‐1 may now be classed as an essential osteoclast‐derived stimulus of both bone formation and resorption.

Interleukin-11 receptor signaling is required for normal bone remodeling.

IL‐6 and ‐11 regulate bone turnover and have been implicated in estrogen deficiency‐related bone loss. In this study, deletion of IL‐11 signaling, but not that of IL‐6, suppressed osteoclast differentiation, resulting in high trabecular bone volume and reduced bone formation. Furthermore, IL‐11 signaling was not required for the effects of estradiol or estrogen deficiency on the mouse skeleton.

Insulin receptor expression in primary and cultured osteoclast-like cells

Skeletal growth is the net product of coordinated bone formation and resorption. Insulin is known to stimulate bone formation by actions on osteoblasts. It is not known whether insulin receptors are present on osteoclasts, or whether insulin regulates osteoclastic function. We present here immunocytochemical evidence of insulin receptor expression by mature mono- and multinucleated murine osteoclast-like cells generated in vitro, and in primary neonatal rat and mouse osteoclasts. Radiolabeled studies indicated that progressive enrichment of osteoclast-like cells in coculture was associated with increased insulin binding. When osteoclast-like cells generated in vitro were plated onto dentine slices, insulin dose-dependently inhibited pit formation by up to 80%, suggesting a role for insulin in osteoclast function. These data are consistent with an effect of insulin on bone resorption in addition to those previously recognized on bone formation, actions that together result in net bone growth.