Amanda N. Farrugia

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.

Elevated Serum Levels of Stromal-Derived Factor-1α Are Associated with Increased Osteoclast Activity and Osteolytic Bone Disease in Multiple Myeloma Patients

Multiple myeloma (MM) is an incurable plasma cell (PC) malignancy able to mediate massive destruction of the axial and craniofacial skeleton. The aim of this study was to investigate the role of the potent chemokine, stromal-derived factor-1α (SDF-1α) in the recruitment of osteoclast precursors to the bone marrow. Our studies show that MM PC produce significant levels of SDF-1α protein and exhibit elevated plasma levels of SDF-1α when compared with normal, age-matched subjects. The level of SDF-1α positively correlated with the presence of multiple radiological bone lesions in individuals with MM, suggesting a potential role for SDF-1α in osteoclast precursor recruitment and activation. To examine this further, peripheral blood–derived CD14+ osteoclast precursors were cultured in an in vitro osteoclast-potentiating culture system in the presence of recombinant human SDF-1α. Although failing to stimulate an increase in TRAP+, multinucleated osteoclast formation, our studies show that SDF-1α mediated a dramatic increase in both the number and the size of the resorption lacunae formed. The increased osteoclast motility and activation in response to SDF-1α was associated with an increase in the expression of a number of osteoclast activation–related genes, including RANKL, RANK, TRAP, MMP-9, CA-II, and Cathepsin K. Importantly, the small-molecule CXCR4-specific inhibitor, 4F-Benzoyl-TE14011 (T140), effectively blocked osteoclast formation stimulated by the myeloma cell line, RPMI-8226. Based on these findings, we believe that the synthesis of high levels of SDF-1α by MM PC may serve to recruit osteoclast precursors to local sites within the bone marrow and enhance their motility and bone-resorbing activity. Therefore, we propose that inhibition of the CXCR4-SDF-1α axis may provide an effective means of treatment for MM-induced osteolysis.

Osteoprotegerin (OPG) is localized to the Weibel-Palade bodies of human vascular endothelial cells and is physically associated with von Willebrand factor†

Recent studies demonstrate roles for osteoprotegerin (OPG) in both skeletal and extra‐skeletal tissues. Although its role in preventing osteoclast (OC) formation and activity is well documented, emerging evidence suggests a role of OPG in endothelial cell survival and the prevention of arterial calcification. In this communication, we show that vascular endothelial cells in situ, and human umbilical vein endothelial cells (HUVEC) in vitro, express abundant OPG. In HUVEC, OPG co‐localizes with P‐selectin and von Willebrand factor (vWF), within the Weibel‐Palade bodies (WPB). Treatment of HUVEC with the pro‐inflammatory cytokines, tumor necrosis factor (TNF)‐α and IL‐1β, resulted in mobilization from the WPBs and subsequent secretion of OPG protein into the culture supernatant. Furthermore, TNF‐α treatment of HUVEC resulted in a sustained increase in OPG mRNA levels and protein secretion over the 24‐h treatment period. Reciprocal immunoprecipitation experiments revealed that while not associated with P‐Selectin, OPG is physically complexed with vWF both within the WPB and following secretion from endothelial cells. Interestingly, this association was also identified in human peripheral blood plasma. In addition to its interaction with vWF, we show that OPG also binds with high avidity to the vWF reductase, thrombospondin (TSP‐1), raising the intriguing possibility that OPG may provide a link between TSP‐1 and vWF. In summary, the intracellular localization of OPG in HUVEC, in association with vWF, together with its rapid and sustained secretory response to inflammatory stimuli, strongly support a modulatory role in vascular injury, inflammation and hemostasis.

The Nitrogen-Containing Bisphosphonate, Zoledronic Acid, Influences RANKL Expression in Human Osteoblast-Like Cells by Activating TNF-α Converting Enzyme (TACE)†

Bisphosphonates are used to prevent osteoclast‐mediated bone loss. Zoledronic acid inhibits osteoclast maturation indirectly by increasing OPG protein secretion and decreasing transmembrane RANKL expression in human osteoblasts. The decreased transmembrane RANKL expression seems to be related to the upregulation of the RANKL sheddase, TACE.

RANK Expression as a cell surface marker of human osteoclast precursors in peripheral blood, bone marrow, and giant cell tumors of bone.

RANK expression in vivo on hematopoietic subsets including pre‐osteoclasts, identified by monoclonal antibodies, has not been described. We describe the lineages that express RANK in bone marrow, peripheral blood, and GCTs. We show that CD14+RANKhigh cells constitute a circulating pre‐osteoclast pool.

Targeted Disruption of the CXCL12/CXCR4 Axis Inhibits Osteolysis in a Murine Model of Myeloma-Associated Bone Loss†‡

The plasma cell (PC) malignancy, multiple myeloma (MM), is unique among hematological malignancies in its capacity to cause osteoclast (OC)‐mediated skeletal destruction. We have previously shown that elevated plasma levels of PC‐derived CXCL12 are associated with presence of X‐ray detectable osteolytic lesions in MM patients. To further investigate this relationship, plasma levels of CXCL12 and βCrossLaps, a marker of bone loss, were measured. A strong correlation between levels of CXCL12 and OC‐mediated bone resorption was identified. To confirm the OC‐activating potential of MM PC‐derived CXCL12 in vivo, we established a model of MM‐mediated focal osteolysis, wherein MM PC lines, such as RPMI‐8226, were injected into the tibias of nude mice. Implanting RPMI‐8226 gave rise to osteolytic lesions proximal to the tumor, resulting in a 5% decrease in bone volume (BV) compared with vehicle control. Importantly, bone loss was significantly inhibited with systemic administration of the CXCL12/CXCR4 antagonist T140. Furthermore, implanting CXCL12‐overexpressing RPMI‐8226 cells resulted in a 13% decrease in BV and was associated with increased OC recruitment proximal to the tumor, increased serum matrix metalloproteinase activity, and increased levels of collagen I degradation products. These findings confirm our hypothesis that MM PC‐derived CXCL12 stimulates the recruitment and activity of OC, thereby contributing to the formation of MM osteolytic lesions.

Therapeutic concentrations of dasatinib inhibit in vitro osteoclastogenesis

Dasatinib (BMS-354825, SPRYCEL; Bristol-Myers Squibb, New York, USA) is a second-generation ATP-competitive inhibitor of a subset of protein tyrosine kinases, including abl, Src-family kinases (c-Src, Lck, Hck, Yes, Fgr, Lyn and Fyn), and the plateletderived growth factor family members c-kit and platelet-derived growth factor-a and -b. Dasatinib displays clinical activity in chronic myeloid leukaemia patients who develop resistance to or are intolerant to the frontline chronic myeloid leukaemia chemotherapeutic imatinib mesylate (STI571, Gleevec; Novartis, Basel, Switzerland). Although designed to inhibit abl and/or Src protein kinases, tyrosine kinase inhibitors, like dasatinib and imatinib, show off-target effects. We have shown earlier that therapeutic concentrations of imatinib decrease osteoclast numbers and activity, at least in part through inhibition of the macrophage colony-stimulating factor (M-CSF) receptor, c-fms. Signalling through c-fms plays an essential role in the survival and activity of bone-resorbing osteoclasts, which act in conjunction with bone-forming osteoblasts to maintain skeletal health. In in vitro osteoclast cultures and animal models, such as op/op mice, an absence of signalling through c-fms results in an osteopetrotic phenotype because of deficient osteoclasts and osteoclast precursors. Recent evidence suggests that dasatinib, like imatinib, may potentially inhibit signal transduction through c-fms. In cultures of M-CSF-dependent Ba/F3 cells that ectopically expressed human c-fms, cell numbers were decreased by dasatinib treatment. In light of this observation, this study examined whether dasatinib could modulate osteoclast formation and/or activity, and whether any such effects were attributable to an inhibition of c-fms signal transduction. We examined the effect of dasatinib on osteoclast formation and activity in human and murine systems in vitro. Human CD14þ mononuclear cells (huCD14þ ) were isolated from the peripheral blood of healthy volunteers using Percoll gradient separation and a MACS negative selection monocyte kit (Miltenyi Biotech, Bergisch Gladbach, Germany). The cells were cultured with recombinant human (rh)M-CSF and rhRANKL, to induce osteoclastogenesis, supplemented with dasatinib or vehicle. After 14 days of culture, a significant decrease in the number of tartrate-resistant acid phosphatasepositive multinucleated cells was observed at 20 nM dasatinib (Po0.001; IC501⁄4 10.5 nM; Figure 1a). Murine osteoclastogenesis assays were established by explanting mouse bone marrow from the tibiae and femora of C57 BL/6 mice. Cells were incubated overnight at 37 1C in 5% CO2, to allow stromal cells to adhere, and non-adherent mouse bone marrow cells (mBM) were collected and used as monocyte/macrophage osteoclast precursor cells. In mBM cultures treated with rhM-CSF and rhRANKL for 6 days, osteoclast numbers were decreased at 10 nM dasatinib (Po0.01; Figure 1b) and osteoclast formation was completely abrogated at 20 nM dasatinib (IC501⁄4 8.0 nM; Figure 1b). Osteoclast size was also substantially reduced in cultures treated with 10 nM dasatinib compared with controls, in both human and murine systems (Figure 1c). The effect of dasatinib on osteoclast activity was also assessed in huCD14þ and mBM cultures established on calcium phosphate-coated slides. A significant inhibition of resorption was observed at X1.25 nM dasatinib in huCD14þ cultures (Po0.001; IC501⁄4 2.4 nM; Figures 1d and f), and at X2.5 nM dasatinib in mBM cultures (Po0.05; IC501⁄4 3.5 nM; Figures 1e and f), relative to vehicle controls. Thus, therapeutically relevant concentrations of dasatinib (Cmax1⁄4 110.0 nM) significantly reduced the formation and activity of osteoclasts from huCD14þ and mBM at IC50 concentrations of p10 nM dasatinib. As dasatinib may affect osteoclastogenesis through inhibition of c-fms, we investigated whether dasatinib could specifically inhibit c-fms kinase activity. Initially, the effect of dasatinib on c-fms-dependent cell proliferation was examined using FDC-P1 cells expressing c-fms protein (FDC-cfms). FDC-P1 cells are dependent on interleukin-3 for proliferation and are also rendered M-CSF-responsive by transfection with a c-fms expression construct. The resulting cells are dependent on either interleukin-3 or M-CSF for survival and proliferation. Treatment of rhM-CSF-stimulated FDC-cfms cells with dasatinib for 24 h significantly decreased cell numbers at concentrations of X50 nM (Po0.001; IC501⁄4 57.8 nM; Figure 2a). In contrast, the proliferation of FDC-P1 cells or FDC-cfms cells cultured in the presence of interleukin-3 (0.5 ng/ ml) was not significantly affected by dasatinib treatment at concentrations up to 100 nM (P1⁄4 0.9012 and P1⁄4 0.2008, respectively; Figure 2a). To determine whether dasatinib treatment could also inhibit the M-CSF-dependent proliferation/survival of primary bone marrow cells, the effect of dasatinib on mBM cells was assessed using a WST-1 assay (Tanaka, Madison, WI, USA). The relative number of viable, metabolically active cells per well, as detected by the quantification of mitochondrial dehydrogenase

Tumor Angiogenesis Is Associated with Plasma Levels of Stromal-Derived Factor-1α in Patients with Multiple Myeloma

Purpose: Multiple myeloma is an incurable hematologic malignancy characterized by increased bone marrow angiogenesis and extensive lytic bone disease. We have previously shown that elevated levels of stromal-derived factor-1α (SDF-1α) in peripheral blood plasma are associated with osteolysis in multiple myeloma patients. We have now examined whether SDF-1α levels also correlate with angiogenesis. Experimental Design: We examined the contribution of multiple myeloma plasma cell–derived SDF-1α in the stimulation of in vitro angiogenesis using a tube formation assay. We also collected trephine and peripheral blood plasma samples from patients with multiple myeloma to analyze microvessel density and SDF-1α levels, respectively. Results: We show that multiple myeloma plasma cell line–derived conditioned medium containing SDF-1α stimulates in vitro angiogenesis. In addition, in a large cohort of patients with multiple myeloma and its precursor condition monoclonal gammopathy of undetermined significance, we confirm previous findings that plasma cell burden correlates with both angiogenesis and plasma levels of SDF-1α. We now extend these observations and show the novel finding that peripheral blood plasma levels of SDF-1α positively correlate with the degree of bone marrow angiogenesis in multiple myeloma and monoclonal gammopathy of undetermined significance patients. Conclusions: High levels of SDF-1α produced by multiple myeloma plasma cells promote osteolysis and bone marrow angiogenesis. Therefore, we propose that inhibition of SDF-1α may be an effective mechanism by which angiogenesis and osteolysis can be reduced in multiple myeloma patients.

An in vitro osteoclast-forming assay to measure myeloma cell-derived osteoclast-activating factors.

Much of the morbidity and mortality associated with the plasma cell (PC) malignancy, multiple myeloma (MM), is owing to the severe osteolytic bone disease seen in patients with this disease. Although the molecular mechanisms responsible for osteolysis remain to be fully elucidated, it is clear from numerous studies that it is owing, in part, to an increase in osteoclastic bone resorption. Several known osteoclast (OC)-activating factors (OAFs) are produced by myeloma PCs (MPCs), or by stromal cells in response to MPCs and include interleukin-1beta (IL-1beta); tumor necrosis factor-alpha (TNF-alpha); IL-6; parathyroid hormone-related protein; macrophage inflammatory protein-1alpha; and, most recently, the TNF-ligand family member receptor activator of nuclear factor-kappaB ligand (RANKL). The identification and significance of any one of these myeloma-derived OAFs is dependent on robust and reliable assays that measure the de novo formation and activation of OCs. A number of in vitro assay systems have been described that examine the requirements for normal OC formation and are easily adaptable for examining which MM-derived OAF and to what extent it is responsible for the bone loss observed in individuals with myeloma. This chapter describes one such in vitro model system.