Sustained RANKL response to parathyroid hormone in oncostatin M receptor-deficient osteoblasts converts anabolic treatment to a catabolic effect in vivo

Parathyroid hormone (PTH) is the only approved anabolic agent for osteoporosis treatment. It acts via osteoblasts to stimulate both osteoclast formation and bone formation, with the balance between these two activities determined by the mode of administration. Oncostatin M (OSM), a gp130‐dependent cytokine expressed by osteoblast lineage cells, has similar effects and similar gene targets in the osteoblast lineage. In this study, we investigated whether OSM might participate in anabolic effects of PTH. Microarray analysis and quantitative real‐time polymerase chain reaction (qPCR) of PTH‐treated murine stromal cells and primary calvarial osteoblasts identified significant regulation of gp130 and gp130‐dependent coreceptors and ligands, including a significant increase in OSM receptor (OSMR) expression. To determine whether OSMR signaling is required for PTH anabolic action, 6‐week‐old male Osmr^?/? mice and wild‐type (WT) littermates were treated with hPTH(1–34) for 3 weeks. In WT mice, PTH increased trabecular bone volume and trabecular thickness. In contrast, the same treatment had a catabolic effect in Osmr^?/? mice, reducing both trabecular bone volume and trabecular number. This was not explained by any alteration in the increased osteoblast formation and mineral apposition rate in response to PTH in Osmr^?/? compared with WT mice. Rather, PTH treatment doubled osteoclast surface in Osmr^?/? mice, an effect not observed in WT mice. Consistent with this finding, when osteoclast precursors were cultured in the presence of osteoblasts, more osteoclasts were formed in response to PTH when Osmr^?/? osteoblasts were used. Neither PTH1R mRNA levels nor cAMP response to PTH were modified in Osmr^?/? osteoblasts. However, RANKL induction in PTH‐treated Osmr^?/? osteoblasts was sustained at least until 24 hours after PTH exposure, an effect not observed in WT osteoblasts. These data indicate that the transient RANKL induction by intermittent PTH administration, which is associated with its anabolic action, is changed to a prolonged induction in OSMR‐deficient osteoblasts, resulting in bone destruction. © 2012 American Society for Bone and Mineral Research.     

Intermittent PTH Administration Stimulates Pre‐Osteoblastic Proliferation Without Leading to Enhanced Bone Formation in Osteoclast‐Less c‐fos−/− Mice

This study aimed to investigate the behavior and ultrastructure of osteoblastic cells after intermittent PTH treatment and attempted to elucidate the role of osteoclasts on the mediation of PTH‐driven bone anabolism. After administering PTH intermittently to wildtype and c‐fos^?/? mice, immunohistochemical, histomorphometrical, ultrastructural, and statistical examinations were performed. Structural and kinetic parameters related to bone formation were increased in PTH‐treated wildtype mice, whereas in the osteoclast‐deficient c‐fos^?/? mice, there were no significant differences between groups. In wildtype and knockout mice, PTH administration led to significant increases in the number of cells double‐positive for alkaline phosphatase and BrdU, suggesting active pre‐osteoblastic proliferation. Ultrastructural examinations showed two major pre‐osteoblastic subtypes: one rich in endoplasmic reticulum (ER), the hypER cell, and other with fewer and dispersed ER, the misER cell. The latter constituted the most abundant preosteoblastic phenotype after PTH administration in the wildtype mice. In c‐fos^?/? mice, misER cells were present on the bone surfaces but did not seem to be actively producing bone matrix. Several misER cells were shown to be positive for EphB4 and were eventually seen rather close to osteoclasts in the PTH‐administered wildtype mice. We concluded that the absence of osteoclasts in c‐fos^?/? mice might hinder PTH‐driven bone anabolism and that osteoclastic presence may be necessary for full osteoblastic differentiation and enhanced bone formation seen after intermittent PTH administration.     

Bone sialoprotein deficiency impairs osteoclastogenesis and mineral resorption in vitro

Bone sialoprotein (BSP) and osteopontin (OPN) belong to the small integrin‐binding ligand N‐linked glycoprotein (SIBLING) family, whose members interact with bone cells and bone mineral. Previously, we showed that BSP knockout (BSP^?/?) mice have a higher bone mass than wild type (BSP^+/+) littermates, with very low bone‐formation activity and reduced osteoclast surfaces and numbers. Here we report that approximately twofold fewer tartrate‐resistant acid phosphatase (TRACP)–positive cells and approximately fourfold fewer osteoclasts form in BSP^?/? compared with BSP^+/+ spleen cell cultures. BSP^?/? preosteoclast cultures display impaired proliferation and enhanced apoptosis. Addition of RGD‐containing proteins restores osteoclast number in BSP^?/? cultures to BSP^+/+ levels. The expression of osteoclast‐associated genes is markedly altered in BSP^?/? osteoclasts, with reduced expression of cell adhesion and migration genes (αV integrin chain and OPN) and increased expression of resorptive enzymes (TRACP and cathepsin K). The migration of preosteoclasts and mature osteoclasts is impaired in the absence of BSP, but resorption pit assays on dentine slices show no significant difference in pit numbers between BSP^+/+ and BSP^?/? osteoclasts. However, resorption of mineral‐coated slides by BSP^?/? osteoclasts is markedly impaired but is fully restored by coating the mineral substrate with hrBSP and partly restored by hrOPN coating. In conclusion, lack of BSP affects both osteoclast formation and activity, which is in accordance with in vivo findings. Our results also suggest at least some functional redundancy between BSP and OPN that remains to be clarified. © 2010 American Society for Bone and Mineral Research.     

β‐Arrestin2 Regulates RANKL and Ephrins Gene Expression in Response to Bone Remodeling in Mice

PTH‐stimulated intracellular signaling is regulated by the cytoplasmic adaptor molecule β‐arrestin. We reported that the response of cancellous bone to intermittent PTH is reduced in β‐arrestin2^?/? mice and suggested that β‐arrestins could influence the bone mineral balance by controlling RANKL and osteoprotegerin (OPG) gene expression. Here, we study the role of β‐arrestin2 on the in vitro development and activity of bone marrow (BM) osteoclasts (OCs) and Ephrins ligand (Efn), and receptor (Eph) mRNA levels in bone in response to PTH and the changes of bone microarchitecture in wildtype (WT) and β‐arrestin2^?/? mice in models of bone remodeling: a low calcium diet (LoCa) and ovariectomy (OVX). The number of PTH‐stimulated OCs was higher in BM cultures from β‐arrestin2^?/? compared with WT, because of a higher RANKL/OPG mRNA and protein ratio, without directly influencing osteoclast activity. In vivo, high PTH levels induced by LoCa led to greater changes in TRACP5b levels in β‐arrestin2^?/? compared with WT. LoCa caused a loss of BMD and bone microarchitecture, which was most prominent in β‐arrestin2^?/?. PTH downregulated Efn and Eph genes in β‐arrestin2^?/?, but not WT. After OVX, vertebral trabecular bone volume fraction and trabecular number were lower in β‐arrestin2^?/? compared with WT. Histomorphometry showed that OC number was higher in OVX‐β‐arrestin2^?/? compared with WT. These results indicate that β‐arrestin2 inhibits osteoclastogenesis in vitro, which resulted in decreased bone resorption in vivo by regulating RANKL/OPG production and ephrins mRNAs. As such, β‐arrestins should be considered an important mechanism for the control of bone remodeling in response to PTH and estrogen deprivation.     

Endogenous parathyroid hormone–related protein compensates for the absence of parathyroid hormone in promoting bone accrual in vivo in a model of bone marrow ablation

To assess the effect of hypoparathyroidism on osteogenesis and bone turnover in vivo, bone marrow ablation (BMXs) were performed in tibias of 8‐week‐old wild‐type and parathyroid hormone–null (PTH^?/?) mice and newly formed bone tissue was analyzed from 5 days to 3 weeks after BMX. At 1 week after BMX, trabecular bone volume, osteoblast numbers, alkaline phosphatase‐positive areas, type I collagen‐positive areas, PTH receptor–positive areas, calcium sensing receptor–positive areas, and expression of bone formation–related genes were all decreased significantly in the diaphyseal regions of bones of PTH^?/? mice compared to wild‐type mice. In contrast, by 2 weeks after BMX, all parameters related to osteoblastic bone accrual were increased significantly in PTH^?/? mice. At 5 days after BMX, active tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts had appeared in wild‐type mice but were undetectable in PTH^?/? mice, Both the ratio of mRNA levels of receptor activator of NF‐κB ligand (RANKL)/osteoprotegerin (OPG) and TRAP‐positive osteoclast surface were still reduced in PTH^?/? mice at 1 week but were increased by 2 weeks after BMX. The expression levels of parathyroid hormone–related protein (PTHrP) at both mRNA and protein levels were upregulated significantly at 1 week and more dramatically at 2 weeks after BMX in PTH^?/? mice. To determine whether the increased newly formed bones in PTH^?/? mice at 2 weeks after BMX resulted from the compensatory action of PTHrP, PTH^?/?PTHrP^+/? mice were generated and newly formed bone tissue was compared in these mice with PTH^?/? and wild‐type mice at 2 weeks after BMX. All parameters related to osteoblastic bone formation and osteoclastic bone resorption were reduced significantly in PTH^?/?PTHrP^+/? mice compared to PTH^?/? mice. These results demonstrate that PTH deficiency itself impairs osteogenesis, osteoclastogenesis, and osteoclastic bone resorption, whereas subsequent upregulation of PTHrP in osteogenic cells compensates by increasing bone accrual. © 2013 American Society for Bone and Mineral Research     

Hyperlipidemia induces resistance to PTH bone anabolism in mice via oxidized lipids

In hyperlipidemia, oxidized lipids accumulate in vascular tissues and trigger atherosclerosis. Such lipids also deposit in bone tissues, where they may promote osteoporosis. We found previously that oxidized lipids attenuate osteogenesis and that parathyroid hormone (PTH) bone anabolism is blunted in hyperlipidemic mice, suggesting that osteoporotic patients with hyperlipidemia may develop resistance to PTH therapy. To determine if oxidized lipids account for this PTH resistance, we blocked lipid oxidation products in hyperlipidemic mice with an ApoA‐I mimetic peptide, D‐4F, and the bone anabolic response to PTH treatment was assessed. Skeletally immature Ldlr^?/? mice were placed on a high‐fat diet and treated with D‐4F peptide and/or with intermittent PTH(1–34) injections. As expected, D‐4F attenuated serum lipid oxidation products and tissue lipid deposition induced by the diet. Importantly, D‐4F treatment attenuated the adverse effects of dietary hyperlipidemia on PTH anabolism by restoring micro–computed tomographic parameters of bone quality—cortical mineral content, area, and thickness. D‐4F significantly reduced serum markers of bone resorption but not bone formation. PTH and D‐4F, together but not separately, also promoted bone anabolism in an alternative model of hyperlipidemia, Apoe^?/? mice. In normolipemic mice, D‐4F cotreatment did not further enhance the anabolic effects of PTH, indicating that the mechanism is through its effects on lipids. These findings suggest that oxidized lipids mediate hyperlipidemia‐induced PTH resistance in bone through modulation of bone resorption. © 2011 American Society for Bone and Mineral Research.     

Regulation of bone mass and osteoclast function depend on the F-actin modulator SWAP-70

Bone remodeling involves tightly regulated bone‐resorbing osteoclasts and bone‐forming osteoblasts. Determining osteoclast function is central to understanding bone diseases such as osteoporosis and osteopetrosis. Here, we report a novel function of the F‐actin binding and regulatory protein SWAP‐70 in osteoclast biology. F‐actin ring formation, cell morphology, and bone resorption are impaired in Swap‐70^?/? osteoclasts, whereas the expression of osteoclast differentiation markers induced in vitro by macrophage colony‐stimulating factor (M‐CSF) and receptor activator of NF‐κB ligand (RANKL) remains unaffected. Swap‐70^?/? mice develop osteopetrosis with increased bone mass, abnormally dense bone, and impaired osteoclast function. Ectopic expression of SWAP‐70 in Swap‐70^?/? osteoclasts in vitro rescues their deficiencies in bone resorption and F‐actin ring formation. Rescue requires a functional pleckstrin homology (PH) domain, known to support membrane localization of SWAP‐70, and the F‐actin binding domain. Transplantation of SWAP‐70–proficient bone marrow into Swap‐70^?/? mice restores osteoclast resorption capacity in vivo. The identification of the role of SWAP‐70 in promoting osteoclast function through modulating membrane‐proximal F‐actin rearrangements reveals a new pathway to control osteoclasts and bone homeostasis. © 2012 American Society for Bone and Mineral Research.     

An essential role for the association of CD47 to SHPS‐1 in skeletal remodeling

Integrin‐associated protein (IAP/CD47) has been implicated in macrophage‐macrophage fusion. To understand the actions of CD47 on skeletal remodeling, we compared Cd47^?/? mice with Cd47^+/+ controls. Cd47^?/? mice weighed less and had decreased areal bone mineral density compared with controls. Cd47^?/? femurs were shorter in length with thinner cortices and exhibited lower trabecular bone volume owing to decreased trabecular number and thickness. Histomorphometry revealed reduced bone‐formation and mineral apposition rates, accompanied by decreased osteoblast numbers. No differences in osteoclast number were observed despite a nonsignificant but 40% decrease in eroded surface/bone surface in Cd47^?/? mice. In vitro, the number of functional osteoclasts formed by differentiating Cd47^?/? bone marrow cells was significantly decreased compared with wild‐type cultures and was associated with a decrease in bone‐resorption capacity. Furthermore, by disrupting the CD47–SHPS‐1 association, we found that osteoclastogenesis was markedly impaired. Assays for markers of osteoclast maturation suggested that the defect was at the point of fusion and not differentiation and was associated with a lack of SHPS‐1 phosphorylation, SHP‐1 phosphatase recruitment, and subsequent dephosphorylation of non–muscle cell myosin IIA. We also demonstrated a significant decrease in osteoblastogenesis in bone marrow stromal cells derived from Cd47^?/? mice. Our finding of cell‐autonomous defects in Cd47^?/? osteoblast and osteoclast differentiation coupled with the pronounced skeletal phenotype of Cd47^?/? mice support the conclusion that CD47 plays an important role in regulating skeletal acquisition and maintenance through its actions on both bone formation and bone resorption. © 2011 American Society for Bone and Mineral Research     

Interferon‐γ plays a role in bone formation in vivo and rescues osteoporosis in ovariectomized mice

Interferon γ (IFN‐γ) is a cytokine produced locally in the bone microenvironment by cells of immune origin as well as mesenchymal stem cells. However, its role in normal bone remodeling is still poorly understood. In this study we first examined the consequences of IFN‐γ ablation in vivo in C57BL/6 mice expressing the IFN‐γ receptor knockout phenotype (IFNγR1^?/?). Compared with their wild‐type littermates (IFNγR1^+/+), IFNγR1^?/? mice exhibit a reduction in bone volume associated with significant changes in cortical and trabecular structural parameters characteristic of an osteoporotic phenotype. Bone histomorphometry of IFNγR1^?/? mice showed a low‐bone‐turnover pattern with a decrease in bone formation, a significant reduction in osteoblast and osteoclast numbers, and a reduction in circulating levels of bone‐formation and bone‐resorption markers. Furthermore, administration of IFN‐γ (2000 and 10,000 units) to wild‐type C57BL/6 sham‐operated (SHAM) and ovariectomized (OVX) female mice significantly improved bone mass and microarchitecture, mechanical properties of bone, and the ratio between bone formation and bone resorption in SHAM mice and rescued osteoporosis in OVX mice. These data therefore support an important physiologic role for IFN‐γ signaling as a potential new anabolic therapeutic target for osteoporosis. © 2011 American Society for Bone and Mineral Research.     

Continuous elevation of PTH increases the number of osteoblasts via both osteoclast‐dependent and ‐independent mechanisms

Sustained parathyroid hormone (PTH) elevation stimulates bone remodeling (ie, both resorption and formation). The former results from increased RANKL synthesis, but the cause of the latter has not been established. Current hypotheses include release of osteoblastogenic factors from osteoclasts or from the bone matrix during resorption, modulation of the production and activity of osteoblastogenic factors from cells of the osteoblast lineage, and increased angiogenesis. To dissect the contribution of these mechanisms, 6‐month‐old Swiss‐Webster mice were infused for 5 days with 470 ng/h PTH(1‐84) or 525 ng/h soluble RANKL (sRANKL). Both agents increased osteoclasts and osteoblasts in vertebral cancellous bone, but the ratio of osteoblasts to osteoclasts and the increase in bone formation was greater in PTH‐treated mice. Cancellous bone mass was maintained in mice receiving PTH but lost in mice receiving sRANKL, indicating that maintenance of balanced remodeling requires osteoblastogenic effects beyond those mediated by osteoclasts. Consistent with this contention, PTH, but not sRANKL, decreased the level of the Wnt antagonist sclerostin and increased the expression of the Wnt target genes Nkd2, Wisp1, and Twist1. Furthermore, PTH, but not sRANKL, increased the number of blood vessels in the bone marrow. Weekly injections of the RANKL antagonist osteoprotegerin at 10 µg/g for 2 weeks prior to PTH infusion eliminated osteoclasts and osteoblasts and prevented the PTH‐induced increase in osteoclasts, osteoblasts, and blood vessels. These results indicate that PTH stimulates osteoclast‐dependent as well as osteoclast‐independent (Wnt signaling) pro‐osteoblastogenic pathways, both of which are required for balanced focal bone remodeling in cancellous bone. © 2010 American Society for Bone and Mineral Research.     

Hyperlipidemia Impairs Osteoanabolic Effects of PTH

Epidemiological and in vitro studies have suggested that hyperlipidemia/oxidized phospholipids adversely affect bone. We recently found that oxidized phospholipids attenuate PTH‐induced cAMP and immediate‐early gene (IEG) expression in MC3T3‐E1 cells, raising concerns that clinical hyperlipidemia may attenuate osteoanabolic effects of PTH in vivo. Thus, we studied whether intermittent PTH treatment has differential osteoanabolic effects in wildtype (C57BL/6) and hyperlipidemic (LDLR^?/?) mice. Consistent with our previous in vitro studies, induction of IEGs in calvarial tissue, 45 min after a single dose of recombinant hPTH(1‐34), was attenuated in LDLR^?/? mice compared with C57BL/6 mice. Daily hPTH(1‐34) injections for 5 wk significantly increased total and cortical BMD and BMC, assessed by pQCT, in C57BL/6 mice. However, this induction was completely abrogated in LDLR^?/? mice. Similarly, PTH(1‐34) failed to increase BMD in another hyperlipidemic mouse model, ApoE^?/? mice. Histomorphometric analysis showed that trabecular bone of both mice responded similarly to PTH(1‐34). Structural parameters improved significantly in response to PTH(1‐34) in both mouse strains, although to a lesser degree in LDLR^?/? mice. With PTH(1‐34) treatment, osteoblast surface trended toward an increase in C57BL/6 mice and increased significantly in LDLR^?/? mice. PTH(1‐34) did not alter resorption parameters significantly, except for the eroded surface (ES/BS), which was reduced in the C57BL/6 but not in the LDLR^?/? mice. These results show that PTH(1‐34) has adverse effects on cortical bones of the hyperlipidemic mice, suggesting that the therapeutic effects of PTH may be compromised in the presence of hyperlipidemia.     

Exogenous PTH and Endogenous 1,25‐Dihydroxyvitamin D Are Complementary in Inducing an Anabolic Effect on Bone

PTH and 1,25(OH)₂D each exert dual anabolic and catabolic skeletal effects. We assessed the potential interaction of PTH and 1,25(OH)₂D in promoting skeletal anabolism by comparing the capacity of exogenous, intermittently injected PTH(1‐34) to produce bone accrual in mice homozygous for the 1α(OH)ase‐null allele [1α(OH)ase^?/? mice] and in wildtype mice. In initial studies, 3‐mo‐old wildtype mice were either injected once daily (40 μg/kg) or infused continuously (120 μg/kg/d) with PTH(1–34) for up to 1 mo. Infused PTH reduced BMD, increased the bone resorption marker TRACP‐5b, and raised serum calcium but did not increase serum 1,25(OH)₂D. Injected PTH increased serum 1,25(OH)₂D and BMD, raised the bone formation marker osteocalcin more than did infused PTH, and did not produce sustained hypercalcemia as did PTH infusion. In subsequent studies, 3‐mo‐old 1α(OH)ase^?/? mice, raised on a rescue diet, and wildtype littermates were injected with PTH(1–34) (40 μg/kg) either once daily or three times daily for 1 mo. In 1α(OH)ase^?/? mice, baseline bone volume (BV/TV) and bone formation (BFR/BS) were lower than in wildtype mice. PTH administered intermittently increased BV/TV and BFR/BS in a dose‐dependent manner, but the increases were always less than in wildtype mice. These studies show that exogenous PTH administered continuously resorbs bone without raising endogenous 1,25(OH)₂D. Intermittently administered PTH can increase bone accrual in the absence of 1,25(OH)₂D, but 1,25(OH)₂D complements this PTH action. An increase in endogenous 1,25(OH)₂D may therefore facilitate an optimal skeletal anabolic response to PTH and may be relevant to the development of improved therapeutics for enhancing skeletal anabolism.     

Differential effects between the loss of MMP‐2 and MMP‐9 on structural and tissue‐level properties of bone

Matrix metalloproteinases (MMPs) are capable of processing certain components of bone tissue, including type 1 collagen, a determinant of the biomechanical properties of bone tissue, and they are expressed by osteoclasts and osteoblasts. Therefore, we posit that MMP activity can affect the ability of bone to resist fracture. To explore this possibility, we determined the architectural, compositional, and biomechanical properties of bones from wild‐type (WT), Mmp2^?/?, and Mmp9^?/? female mice at 16 weeks of age. MMP‐2 and MMP‐9 have similar substrates but are expressed primarily by osteoblasts and osteoclasts, respectively. Analysis of the trabecular compartment of the tibia metaphysis by micro–computed tomography (µCT) revealed that these MMPs influence trabecular architecture, not volume. Interestingly, the loss of MMP‐9 improved the connectivity density of the trabeculae, whereas the loss of MMP‐2 reduced this parameter. Similar differential effects in architecture were observed in the L₅ vertebra, but bone volume fraction was lower for both Mmp2^?/? and Mmp9^?/? mice than for WT mice. The mineralization density and mineral‐to‐collagen ratio, as determined by µCT and Raman microspectroscopy, were lower in the Mmp2^?/? bones than in WT control bones. Whole‐bone strength, as determined by three‐point bending or compression testing, and tissue‐level modulus and hardness, as determined by nanoindentation, were less for Mmp2^?/? than for WT bones. In contrast, the Mmp9^?/? femurs were less tough with lower postyield deflection (more brittle) than the WT femurs. Taken together, this information reveals that MMPs play a complex role in maintaining bone integrity, with the cell type that expresses the MMP likely being a contributing factor to how the enzyme affects bone quality. © 2011 American Society for Bone and Mineral Research.     

Proteoglycan 4: A dynamic regulator of skeletogenesis and parathyroid hormone skeletal anabolism

Proteoglycan 4 (Prg4), known for its lubricating and protective actions in joints, is a strong candidate regulator of skeletal homeostasis and parathyroid hormone (PTH) anabolism. Prg4 is a PTH‐responsive gene in bone and liver. Prg4 null mutant mice were used to investigate the impact of proteoglycan 4 on skeletal development, remodeling, and PTH anabolic actions. Young Prg4 mutant and wild‐type mice were administered intermittent PTH(1–34) or vehicle daily from 4 to 21 days. Young Prg4 mutant mice had decreased growth plate hypertrophic zones, trabecular bone, and serum bone formation markers versus wild‐type mice, but responded with a similar anabolic response to PTH. Adult Prg4 mutant and wild‐type mice were administered intermittent PTH(1–34) or vehicle daily from 16 to 22 weeks. Adult Prg4 mutant mice had decreased trabecular and cortical bone, and blunted PTH‐mediated increases in bone mass. Joint range of motion and animal mobility were lower in adult Prg4 mutant versus wild‐type mice. Adult Prg4 mutant mice had decreased marrow and liver fibroblast growth factor 2 (FGF‐2) mRNA and reduced serum FGF‐2, which were normalized by PTH. A single dose of PTH decreased the PTH/PTHrP receptor (PPR), and increased Prg4 and FGF‐2 to a similar extent in liver and bone. Proteoglycan 4 supports endochondral bone formation and the attainment of peak trabecular bone mass, and appears to support skeletal homeostasis indirectly by protecting joint function. Bone‐ and liver‐derived FGF‐2 likely regulate proteoglycan 4 actions supporting trabeculae formation. Blunted PTH anabolic responses in adult Prg4 mutant mice are associated with altered biomechanical impact secondary to joint failure. © 2012 American Society for Bone and Mineral Research     

Modeling bone morphogenetic protein and bisphosphonate combination therapy in wild‐type and Nf1 haploinsufficient mice

Recombinant bone morphogenetic proteins (BMPs) show promise in treating the orthopedic complications associated with neurofibromatosis type 1 (NF1), such as congenital pseudarthrosis of the tibia. Minimal scientific information regarding the effects of BMP in the context of NF1 is available. As abnormalities in both bone formation and resorption have been documented in Nf1‐deficient mice, we hypothesized that inadequate BMP‐induced bone formation could be augmented by cotreatment with the bisphosphonate zoledronic acid (ZA). First, primary osteoblasts isolated from wild type (Nf1^+/+) and Nf1‐deficient (Nf1^+/?) mice were cultured in the presence and absence of BMP‐2. While Nf1^+/? cells exhibited less osteogenic potential than Nf1^+/+ cells, alkaline phosphatase expression and matrix mineralization for both genotypes were enhanced by BMP‐2 treatment. To model this response in vivo, 20 µg BMP‐2 was implanted intramuscularly into the quadriceps of mice to induce heterotopic bone. Radiographs revealed significantly less net bone formation in Nf1^+/? mice compared to Nf1^+/+ controls. To test the effect of an antiresorptive agent, mice were cotreated twice weekly from postoperative day 3 with 0.02 mg/kg ZA or with saline. ZA treatment led to a synergistic increase in the amount of heterotopic bone in both Nf1^+/+ and Nf1^+/? mice compared with saline controls, as measured by DEXA and histomorphometry. Thus, the anabolic deficiency noted in Nf1^+/? mice is amenable to stimulation by BMP‐2, but mineralized tissue formation remains below that of Nf1^+/+ controls. Bisphosphonate combination therapy is superior to BMP therapy alone in terms of net bone production in vivo in both wild‐type and Nf1‐deficient mice. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:65–74, 2008     

Targeted overexpression of Dkk1 in osteoblasts reduces bone mass but does not impair the anabolic response to intermittent PTH treatment in mice

Parathyroid hormone (PTH) is a potent anabolic agent, but the cellular mechanisms by which it increases bone mass are not fully understood. Dickkopf 1 (Dkk1) is an endogenous inhibitor of Wnt signaling and suppresses bone formation in vivo. We sought to determine if Dkk1 and anabolic PTH treatment interact in regulating bone mass. PTH treatment of primary murine osteoblasts for 24 h reduced Dkk1 expression by 90% as quantified by real-time PCR, whereas PTH treatment in vivo reduced Dkk1 expression by 30% when given as a single daily subcutaneous dose. To directly determine whether Dkk1 modulates the anabolic response of PTH in vivo, we engineered transgenic (TG) mice expressing murine Dkk1 under the control of the 2.3-kb rat collagen alpha-1 promoter. TG mice had significantly reduced bone mass, which was accompanied by reduced histomorphometric parameters of bone formation (reduced OV/TV, ObS/OS, and NOb/TAR). Treatment of TG mice and wild-type (WT) littermates with 95 ng/g body weight of human (1–34) PTH daily for 34 days resulted in comparable increases in bone mass at all skeletal sites. Histomorphometric analyses indicated that PTH treatment increased the numbers of both osteoblasts and osteoclasts in WT mice but only increased the numbers of osteoblasts in TG mice. We conclude that overexpression of Dkk1 does not attenuate the anabolic response to PTH in vivo.