Follicle-Stimulating Hormone Increases the Risk of Postmenopausal Osteoporosis by Stimulating Osteoclast Differentiation

Objective

The objectives of this study were to observe the changes in follicle-stimulating hormone (FSH) and bone mineral density (BMD) in postmenopausal women, to research the relationship between FSH and postmenopausal osteoporosis, and to observe the effects of FSH on osteoclast differentiation in RAW264.7 cells.

Methods

We analyzed 248 postmenopausal women with normal bone metabolism. A radioimmunoassay (RIA) was used to detect serum FSH, luteinizing hormone (LH), and estradiol (E₂). Dual-energy X-ray absorptiometry was used to measure forearm BMD. Then, we analyzed the age-related changes in serum FSH, LH and E₂. Additionally, FSH serum concentrations were compared between a group of postmenopausal women with osteoporosis and a control group. Osteoclasts were induced from RAW264.7 cells in vitro by receptor activator of nuclear factor kappa B ligand (RANKL), and these cells were treated with 0, 5, 10, and 20 ng/ml FSH. After the osteoclasts matured, tartrate-resistant acid phosphatase (TRAP) staining was used to identify osteoclasts, and the mRNA expression levels of genes involved in osteoclastic phenotypes and function, such as receptor activator of NF-κB (Rank), Trap, matrix metalloproteinase-9 (Mmp-9) and Cathepsin K, were detected in different groups using real-time PCR (polymerase chain reaction).

Results

1. FSH serum concentrations in postmenopausal women with osteoporosis increased notably compared with the control group. 2. RANKL induced RAW264.7 cell differentiation into mature osteoclasts in vitro. 3. FSH increased mRNA expression of genes involved in osteoclastic phenotypes and function, such as Rank, Trap, Mmp-9 and Cathepsin K, in a dose-dependent manner.

Conclusions

The circulating concentration of FSH may play an important role in the acceleration of bone loss in postmenopausal women. FSH increases osteoclastogenesis in vitro.     

Lysophosphatidic Acid Receptor Type 1 (LPA1) Plays a Functional Role in Osteoclast Differentiation and Bone Resorption Activity

Lysophosphatidic acid (LPA) is a natural bioactive lipid that acts through six different G protein-coupled receptors (LPA_1–6) with pleiotropic activities on multiple cell types. We have previously demonstrated that LPA is necessary for successful in vitro osteoclastogenesis of bone marrow cells. Bone cells controlling bone remodeling (i.e. osteoblasts, osteoclasts, and osteocytes) express LPA₁, but delineating the role of this receptor in bone remodeling is still pending. Despite Lpar1^−/− mice displaying a low bone mass phenotype, we demonstrated that bone marrow cell-induced osteoclastogenesis was reduced in Lpar1^−/− mice but not in Lpar2^−/− and Lpar3^−/− animals. Expression of LPA₁ was up-regulated during osteoclastogenesis, and LPA₁ antagonists (Ki16425, Debio0719, and VPC12249) inhibited osteoclast differentiation. Blocking LPA₁ activity with Ki16425 inhibited expression of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) and dendritic cell-specific transmembrane protein and interfered with the fusion but not the proliferation of osteoclast precursors. Similar to wild type osteoclasts treated with Ki16425, mature Lpar1^−/− osteoclasts had reduced podosome belt and sealing zone resulting in reduced mineralized matrix resorption. Additionally, LPA₁ expression markedly increased in the bone of ovariectomized mice, which was blocked by bisphosphonate treatment. Conversely, systemic treatment with Debio0719 prevented ovariectomy-induced cancellous bone loss. Moreover, intravital multiphoton microscopy revealed that Debio0719 reduced the retention of CX₃CR1-EGFP⁺ osteoclast precursors in bone by increasing their mobility in the bone marrow cavity. Overall, our results demonstrate that LPA₁ is essential for in vitro and in vivo osteoclast activities. Therefore, LPA₁ emerges as a new target for the treatment of diseases associated with excess bone loss.     

Inhibitory Effects of KP-A159, a Thiazolopyridine Derivative,on Osteoclast Differentiation,Function, and Inflammatory Bone Loss via Suppression of RANKL-Induced MAP Kinase Signaling Pathway

Abnormally elevated formation and activation of osteoclasts are primary causes for a majority of skeletal diseases. In this study, we found that KP-A159, a newly synthesized thiazolopyridine derivative, inhibited osteoclast differentiation and function in vitro, and inflammatory bone loss in vivo. KP-A159 did not cause a cytotoxic response in bone marrow macrophages (BMMs), but significantly inhibited the formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). KP-A159 also dramatically inhibited the expression of marker genes related to osteoclast differentiation, including TRAP (Acp5), cathepsin K (Ctsk), dendritic cell-specific transmembrane protein (Dcstamp), matrix metallopeptidase 9 (Mmp9), and nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1). Moreover, actin ring and resorption pit formation were inhibited by KP-A159. Analysis of the signaling pathway involved showed that KP-A159 inhibited RANKL-induced activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and mitogen-activated protein kinase kinase1/2 (MEK1/2). In a mouse inflammatory bone loss model, KP-A159 significantly rescued lipopolysaccharide (LPS)-induced bone loss by suppressing osteoclast numbers. Therefore, KP-A159 targets osteoclasts, and may be a potential candidate compound for prevention and/or treatment of inflammatory bone loss.     

Uncaria tomentosa reduces osteoclastic bone loss in vivo

BackgroundThe genus Uncaria (Rubiaceae) has several biological properties significant to human health. However, the mechanisms underlying the protective effect of this plant on bone diseases are uncertain.PurposeThe present study investigated the role of Uncaria tomentosa extract (UTE) on alveolar bone loss in rats and on osteoclastogenesis in vitro.MaterialsUTE was characterized by an Acquity UPLC (Waters) system, coupled to an Electrospray Ionization (ESI) interface and Quadrupole/Flight Time (QTOF, Waters) Mass Spectrometry system (MS). The effect of UTE treatment for 11 days on the ligature-induced bone loss was assessed focusing on several aspects: macroscopic and histological analysis of bone loss, neutrophil and osteoclast infiltration, and anabolic effect. The effect of UTE on bone marrow cell differentiation to osteoclasts was assessed in vitro.ResultsThe analysis of UTE by UPLC-ESI-QTOF-MS/MS identified 24 compounds, among pentacyclic or tetracyclic oxindole alkaloids and phenols. The administration of UTE for 11 days on ligature-induced rat attenuated the periodontal attachment loss and alveolar bone resorption. It also diminished neutrophil migration to the gingiva tissue, demonstrated by a lower level of MPO. UTE treatment also decreased the level of RANKL/OPG ratio, the main osteoclast differentiation-related genes, followed by reduced TRAP-positive cell number lining the alveolar bone. Additionally, the level of bone-specific alkaline phosphatase, an anabolic bone marker, was elevated in the plasma of UTE treated rats. Next, we determined a possible direct effect of UTE on osteoclast differentiation in vitro. The incubation of primary osteoclast with UTE decreased RANKL-induced osteoclast differentiation without affecting cell viability. This effect was supported by downregulation of the nuclear factor activated T-cells, cytoplasmic 1 expression, a master regulator of osteoclast differentiation, and other osteoclast-specific activity markers, such as cathepsin K and TRAP.ConclusionUTE exhibited an effective anti-resorptive and anabolic effects, which highlight it as a potential natural product for the treatment of certain osteolytic diseases, such as periodontitis.     

FSH directly regulates bone mass

Postmenopausal osteoporosis, a global public health problem, has for decades been attributed solely to declining estrogen levels. Although FSH levels rise sharply in parallel, a direct effect of FSH on the skeleton has never been explored. We show that FSH is required for hypogonadal bone loss. Neither FSHbeta nor FSH receptor (FSHR) null mice have bone loss despite severe hypogonadism. Bone mass is increased and osteoclastic resorption is decreased in haploinsufficient FSHbeta+/- mice with normal ovarian function, suggesting that the skeletal action of FSH is estrogen independent. Osteoclasts and their precursors possess G(i2alpha)-coupled FSHRs that activate MEK/Erk, NF-kappaB, and Akt to result in enhanced osteoclast formation and function. We suggest that high circulating FSH causes hypogonadal bone loss.     

Choline Kinase β Mutant Mice Exhibit Reduced Phosphocholine,Elevated Osteoclast Activity,and Low Bone Mass

The maintenance of bone homeostasis requires tight coupling between bone-forming osteoblasts and bone-resorbing osteoclasts. However, the precise molecular mechanism(s) underlying the differentiation and activities of these specialized cells are still largely unknown. Here, we identify choline kinase β (CHKB), a kinase involved in the biosynthesis of phosphatidylcholine, as a novel regulator of bone homeostasis. Choline kinase β mutant mice (flp/flp) exhibit a systemic low bone mass phenotype. Consistently, osteoclast numbers and activity are elevated in flp/flp mice. Interestingly, osteoclasts derived from flp/flp mice exhibit reduced sensitivity to excessive levels of extracellular calcium, which could account for the increased bone resorption. Conversely, supplementation of cytidine 5′-diphosphocholine in vivo and in vitro, a regimen that bypasses CHKB deficiency, restores osteoclast numbers to physiological levels. Finally, we demonstrate that, in addition to modulating osteoclast formation and function, loss of CHKB corresponds with a reduction in bone formation by osteoblasts. Taken together, these data posit CHKB as a new modulator of bone homeostasis.     

Mechanism and Function of Monoclonal Antibodies Targeting Siglec-15 for Therapeutic Inhibition of Osteoclastic Bone Resorption

The use of monoclonal antibodies to target functionally important cell-surface proteins on bone-resorbing osteoclasts represents a promising approach for treatment of cancer-associated bone loss and other skeletal pathologies. Previously, we identified Siglec-15, a little studied sialic acid-binding receptor, as a candidate target that is highly up-regulated during osteoclast differentiation induced by the cytokine receptor activator of NF-κB ligand (RANKL). In this report, we confirm that Siglec-15 is localized to the plasma membrane where it can be targeted by monoclonal antibodies to inhibit differentiation of functional osteoclasts in vitro. Furthermore, we found that treatment of mice with these antibodies led to a marked increase in bone mineral density, consistent with inhibition of osteoclast activity. Interestingly, osteoblast numbers were maintained despite the anti-resorptive activity. At the molecular level, Siglec-15 interacts with the adapter protein DAP12 and can induce Akt activation when clustered on the osteoclast cell surface, which likely represents its normal signaling function. Importantly, we discovered that monoclonal antibodies induce rapid internalization, lysosomal targeting, and degradation of Siglec-15 by inducing receptor dimerization. This study defines a key regulatory node that controls osteoclast differentiation and activity downstream of RANKL and supports further development of Siglec-15 antibodies as a novel class of bone loss therapeutics.     

Bone loss in ovariectomized rats: dominant role for estrogen but apparently not for FSH

Estrogen deficiency as the sole factor underlying post‐menopausal osteoporosis was challenged, in light of reports that both follicular stimulation hormone (FSH) receptor and FSHβ knockout mice were resistant to bone loss, suggesting a detrimental role for FSH. We assessed whether lowering FSH levels by gonadotropin realizing (GnRH) analog decapeptyl in ovariectomized female rats (OVX) affects bone. Wistar‐derived 25 days old OVX female rats were injected for 10 weeks with estradiol‐17β (E₂), with GnRH analog (decapeptyl) or with both. FSH and luteinizing hormone (LH) serum levels were markedly increased in OVX rats, with smaller growth plates with disrupted architecture; heavy infiltration of bone marrow with numerous adipocytes and reduced thickness of cortical bone. In OVX rats treated with E₂, FSH, and LH levels were intermediate, the tibia was similar to that of intact rats, but there was reduced thickness of cortical bone. In decapeptyl treated OVX rats, FSH and LH levels were suppressed, the organization of growth plate and the trabecular bone were disrupted, and there were fewer proliferative and chondroblastic cells and a large adipocytes population in bone marrow, but an increased trabecular bone volume (TBV). In the E₂ + decapeptyl treatment, FSH and LH levels were suppressed, with partially restored growth plate architecture and improved TBV. In conclusion, E₂ deficiency is the dominant factor impairing bone loss in OVX and concomitant changes in FSH/LH levels achieved by decapeptyl have some modulating, though complex role in this setting. The role of high FSH levels in post‐menopausal bone loss requires further investigation using combined sub‐optimal doses of the different hormones. J. Cell. Biochem. 112: 128–137, 2011. © 2010 Wiley‐Liss, Inc.     

Endosomal TLR3 signaling in stromal osteoblasts induces prostaglandin E2–mediated inflammatory periodontal bone resorption

Toll-like receptors (TLRs) are pattern recognition receptors that play a critical role in innate immune diseases. TLR3, which is localized in the endosomal compartments of hematopoietic immune cells, is able to recognize double-stranded RNA (dsRNA) derived from viruses and bacteria and thereby induce innate immune responses. Inflammatory periodontal bone resorption is caused by bacterial infections, which initially is regulated by innate immunity; however, the roles of TLR3 signaling in bone resorption are still not known. We examined the roles of TLR3 signaling in bone resorption using poly(I:C), a synthetic dsRNA analog. In cocultures of mouse bone marrow cells and stromal osteoblasts, poly(I:C) clearly induced osteoclast differentiation. In osteoblasts, poly(I:C) increased PGE₂ production and upregulated the mRNA expression of PGE₂-related genes, Ptgs2 and Ptges, as well as that of a gene related to osteoclast differentiation, Tnfsf11. In addition, we found that indomethacin (a COX-2 inhibitor) or an antagonist of the PGE₂ receptor EP4 attenuated the poly(I:C)-induced PGE₂ production and subsequent Tnfsf11 expression. Poly(I:C) also prolonged the survival of the mature osteoclasts associated with the increased mRNA expression of osteoclast marker genes, Nfatc1 and Ctsk. In ex vivo organ cultures of periodontal alveolar bone, poly(I:C) induced bone-resorbing activity in a dose-dependent manner, which was attenuated by the simultaneous administration of either indomethacin or an EP4 antagonist. These data suggest that TLR3 signaling in osteoblasts controls PGE₂ production and induces the subsequent differentiation and survival of mature osteoclasts. Endogenous TLR3 in stromal osteoblasts and osteoclasts synergistically induces inflammatory alveolar bone resorption in periodontitis.     

Blocking FSH action attenuates osteoclastogenesis

A direct effect of FSH on bone turnover via stimulation of osteoclast formation has been reported. Here we show that monoclonal or polyclonal antibodies to FSH inhibit osteoclast formation induced by FSH to an extent similar to that noted in FSH receptor (FSHR) knockout cells. Furthermore, we document the amplification of FSHR cDNA from well-characterized human CD14+ osteoclast precursors and osteoclasts, and the direct sequencing of the PCR products to definitively establish the expression of FSHRs. At these sites, the FSHR was expressed predominantly as an isoform that omits exon 9, a linker between the FSH-binding region and a long, invariant signaling domain of the receptor. These data provide compelling evidence for expression of a FSH receptor isoform in osteoclasts and their precursors.     

The effects of luteolin on osteoclast differentiation, function in vitro and ovariectomy-induced bone loss

Flavonoids, a group of polyphenolic compounds abundant in plants, are known to prevent bone loss in ovariectomized (OVX) animal models. Inhibition of osteoclast differentiation and bone resorption is considered as an effective therapeutic approach in the treatment of postmenopausal bone loss. Luteolin, a plant flavonoid, has potent anti-inflammatory properties both in vivo and vitro. In this study, we found that luteolin markedly decreased the differentiation of both bone marrow mononuclear cells and Raw264.7 cells into osteoclasts. Luteolin also inhibited the bone resorptive activity of differentiated osteoclasts. We further investigated the effects of luteolin on ovariectomy-induced bone loss using micro-computed tomography, biomechanical tests and serum markers assay for bone remodeling. Oral administration of luteolin (5 and 20 mg/kg per day) to OVX mice caused significant increase in bone mineral density and bone mineral content of trabecular and cortical bones in the femur as compared to those of OVX controls, and prevented decreases of bone strength indexes induced by OVX surgery. Serum biochemical markers assays revealed that luteolin prevents OVX-induced increases in bone turnover. These data strongly suggest that luteolin has the potential for prevention of bone loss in postmenopausal osteoporosis by reducing both osteoclast differentiation and function.     

Deficiency of Adiponectin Protects against Ovariectomy-Induced Osteoporosis in Mice

Adipokine adiponectin (APN) has been recently reported to play a role in regulating bone mineral density (BMD). To explore the mechanism by which APN affects BMD, we investigated BMD and biomechanical strength properties of the femur and vertebra in sham-operated (Sham) and ovariectomized (OVX) APN knockout (KO) mice as compared to their operated wild-type (WT) littermates. The results show that APN deficiency has no effect on BMD but induces increased ALP activity and osteoclast cell number. While OVX indeed leads to significant bone loss in both femora and vertebras of WT mice with comparable osteogenic activity and a significant increase in osteoclast cell number when compared to that of sham control. However, no differences in BMD, ALP activity and osteoclast cell number were found between Sham and OVX mice deficient for APN. Further studies using bone marrow derived mesenchymal stem cells (MSCs) demonstrate an enhanced osteogenic differentiation and extracellular matrix calcification in APN KO mice. The possible mechanism for APN deletion induced acceleration of osteogenesis could involve increased proliferation of MSCs and higher expression of Runx2 and Osterix genes. These findings indicate that APN deficiency can protect against OVX-induced osteoporosis in mice, suggesting a potential role of APN in regulating the balance of bone formation and bone resorption, especially in the development of post-menopausal osteoporosis.     

5-Azacytidine-induced Protein 2 (AZI2) Regulates Bone Mass by Fine-tuning Osteoclast Survival

5-Azacytidine-induced protein 2 (AZI2) is a TNF receptor (TNFR)-associated factor family member-associated NF-κB activator-binding kinase 1-binding protein that regulates the production of IFNs. A previous in vitro study showed that AZI2 is involved in dendritic cell differentiation. However, the roles of AZI2 in immunity and its pleiotropic functions are unknown in vivo. Here we report that AZI2 knock-out mice exhibit normal dendritic cell differentiation in vivo. However, we found that adult AZI2 knock-out mice have severe osteoporosis due to increased osteoclast longevity. We revealed that the higher longevity of AZI2-deficient osteoclasts is due to an augmented activation of proto-oncogene tyrosine-protein kinase Src (c-Src), which is a critical player in osteoclast survival. We found that AZI2 inhibits c-Src activity by regulating the activation of heat shock protein 90 (Hsp90), a chaperone involved in c-Src dephosphorylation. Furthermore, we demonstrated that AZI2 indirectly inhibits c-Src by interacting with the Hsp90 co-chaperone Cdc37. Strikingly, administration of a c-Src inhibitor markedly prevented bone loss in AZI2 knock-out mice. Together, these findings indicate that AZI2 regulates bone mass by fine-tuning osteoclast survival.     

Pyrroloquinoline Quinine Inhibits RANKL-Mediated Expression of NFATc1 in Part via Suppression of c-Fos in Mouse Bone Marrow Cells and Inhibits Wear Particle-Induced Osteolysis in Mice

The effects of pyrroloquinoline quinine (PQQ) on RANKL-induced osteoclast differentiation and on wear particle-induced osteolysis were examined in this study. PQQ inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages (BMMs) in a dose-dependent manner without any evidence of cytotoxicity. The mRNA expression of c-Fos, NFATc1, and TRAP in RANKL-treated BMMs was inhibited by PQQ treatment. Moreover, RANKL-induced c-Fos and NFATc1 protein expression was suppressed by PQQ. PQQ additionally inhibited the bone resorptive activity of differentiated osteoclasts. Further a UHMWPE-induced murine calvaria erosion model study was performed to assess the effects of PQQ on wear particle-induced osteolysis in vivo. Mice treated with PQQ demonstrated marked attenuation of bone erosion based on Micro-CT and histologic analysis of calvaria. These results collectively suggested that PQQ demonstrated inhibitory effects on osteoclast differentiation in vitro and may suppress wear particle-induced osteolysis in vivo, indicating that PQQ may therefore serve as a useful drug in the prevention of bone loss.     

Porphyromonas gingivalis Stimulates Bone Resorption by Enhancing RANKL (Receptor Activator of NF-κB Ligand) through Activation of Toll-like Receptor 2 in Osteoblasts

Periodontitis has been associated with rheumatoid arthritis. In experimental arthritis, concomitant periodontitis caused by oral infection with Porphyromonas gingivalis enhances articular bone loss. The aim of this study was to investigate how lipopolysaccharide (LPS) from P. gingivalis stimulates bone resorption. The effects by LPS P. gingivalis and four other TLR2 ligands on bone resorption, osteoclast formation, and gene expression in wild type and Tlr2-deficient mice were assessed in ex vivo cultures of mouse parietal bones and in an in vivo model in which TLR2 agonists were injected subcutaneously over the skull bones. LPS P. gingivalis stimulated mineral release and matrix degradation in the parietal bone organ cultures by increasing differentiation and formation of mature osteoclasts, a response dependent on increased RANKL (receptor activator of NF-κB ligand). LPS P. gingivalis stimulated RANKL in parietal osteoblasts dependent on the presence of TLR2 and through a MyD88 and NF-κB-mediated mechanism. Similarly, the TLR2 agonists HKLM, FSL1, Pam2, and Pam3 stimulated RANKL in osteoblasts and parietal bone resorption. LPS P. gingivalis and Pam2 robustly enhanced osteoclast formation in periosteal/endosteal cell cultures by increasing RANKL. LPS P. gingivalis and Pam2 also up-regulated RANKL and osteoclastic genes in vivo, resulting in an increased number of periosteal osteoclasts and immense bone loss in wild type mice but not in Tlr2-deficient mice. These data demonstrate that LPS P. gingivalis stimulates periosteal osteoclast formation and bone resorption by stimulating RANKL in osteoblasts via TLR2. This effect might be important for periodontal bone loss and for the enhanced bone loss seen in rheumatoid arthritis patients with concomitant periodontal disease.     

Expression of estrogen receptor-alpha in cells of the osteoclastic lineage

 Estrogen deficiency at the menopause is associated with an increased rate of bone loss and subsequent risk of skeletal fracture. Whilst cells of the osteoblastic lineage are known to express estrogen receptors, the presence of estrogen receptors in osteoclasts remains controversial. We have examined expression of the classic estrogen receptor, estrogen receptor-alpha (ERα), during osteoclast differentiation. In situ mRNA hybridisation with a digoxygenin-labelled riboprobe to ERα mRNA, together with immunocytochemical analysis using a human ERα-specific monoclonal antibody demonstrated similar findings and confirmed the expression of ERα in chondroblasts and osteoblasts from human fetal bone and mineralising human bone marrow cultures. ERα expression was detected in human bone marrow cultures treated with 1,25(OH)₂D₃ and macrophage colony-stimulating factor and in macrophage cultures treated with 1,25(OH)₂D₃. However, in an in vitro model of human osteoclast formation, no ERα expression was observed in the osteoclasts that developed. The human preosteoclast TCG 51 cell line showed strong expression of ERα in contrast to the low levels observed in the more mature bone resorptive TCG 23 cell line. No expression was detectable in osteoclasts cultured from giant cell tumour of bone (GCTB) tissue or in osteoclasts in Pagetic, GCTB, or hyperparathyroid bone tissues. In conclusion, preosteoclasts express detectable levels of ERα, but osteoclast maturation and bone resorption is associated with loss of ERα expression. This indicates that ERα expression and regulation may play a role in osteoclast formation. Accepted: 4 November 1998     

Geranylgeranyl transferase type II inhibition prevents myeloma bone disease

Geranylgeranyl transferase II (GGTase II) is an enzyme that plays a key role in the isoprenylation of proteins. 3-PEHPC, a novel GGTase II inhibitor, blocks bone resorption and induces myeloma cell apoptosis in vitro. Its effect on bone resorption and tumor growth in vivo is unknown. We investigated the effect of 3-PEHPC on tumor burden and bone disease in the 5T2MM model of multiple myeloma in vivo. 3-PEHPC significantly reduced osteoclast numbers and osteoclast surface. 3-PEHPC prevented the bone loss and the development of osteolytic bone lesions induced by 5T2MM myeloma cells. Treatment with 3-PEHPC also significantly reduced myeloma burden in bone. The magnitude of response was similar to that seen with the bisphosphonate, risedronate. These data show that targeting GGTase II with 3-PEHPC can prevent osteolytic bone disease and reduce tumor burden in vivo, and represents a novel approach to treating tumors that grow in bone.