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.     

Over-expression of TIMP-1 in osteoblasts increases the anabolic response to PTH

Intermittent PTH treatment induces structural changes that affect cancellous bone mass and have led to its indication for the treatment of osteoporosis. PTH is also known to upregulate the expression of matrix metalloproteinases (MMP) in osteoblasts. We wanted to find out whether inhibiting osteoblastic MMPs can affect the anabolic action of PTH in vivo. We had shown previously that mice over-expressing TIMP-1 (tissue inhibitor of MMPs) specifically in osteoblasts display an increase in bone mineral density and bone mass combined with an overall decrease in bone turnover. In the present study, 10-week-old wild-type (WT) and transgenic (TG) mice were treated with PTH at 40 microg/kg/day for 1.5 months. DEXA analysis was performed before and after treatment, and histomorphometric and molecular analysis were carried out at the end of the experiment. Our findings indicate that the transgene boosted the anabolic action of PTH. The femurs of PTH-treated TG mice displayed a greater increase in bone mineral density and trabecular bone volume than treated WT mice. Interestingly, the positive effect of the transgene on the action of PTH resulted from both reduced bone resorption activity and an increase in the bone formation rate. Osteoclastic surfaces that were increased in PTH-treated WT mice remained unchanged in TG mice, suggesting a decrease in osteoclastic differentiation. Histomorphometric data also indicate that PTH administration increased osteoblast activity in TG mice and affected the number of osteoblasts in WT mice. In conclusion, we demonstrate that inhibiting osteoblastic MMPs can potentiate the anabolic effect of PTH by decreasing osteoclast activity and increasing osteoblast activity. Our data also suggest that osteoblastic MMPs have some role in mediating the anabolic effects of PTH in vivo and indicate that inhibitors of MMPs could constitute a new therapy for degenerative diseases.     

Role of Bcl2 in Osteoclastogenesis and PTH Anabolic Actions in Bone

Introduction : B‐cell leukemia/lymphoma 2 (Bcl2) is a proto‐oncogene best known for its ability to suppress cell death. However, the role of Bcl2 in the skeletal system is unknown. Bcl2 has been hypothesized to play an important anti‐apoptotic role in osteoblasts during anabolic actions of PTH. Although rational, this has not been validated in vivo; hence, the impact of Bcl2 in bone remains unknown. Materials and Methods : The bone phenotype of Bcl2 homozygous mutant (Bcl2^?/?) mice was analyzed with histomorphometry and μCT. Calvarial osteoblasts were isolated and evaluated for their cellular activity. Osteoclastogenesis was induced from bone marrow cells using RANKL and macrophage‐colony stimulating factor (M‐CSF), and their differentiation was analyzed. PTH(1–3;34) (50 μg/kg) or vehicle was administered daily to Bcl2^+/+ and Bcl2^?/? mice (4 days old) for 9 days to clarify the influence of Bcl2 ablation on PTH anabolic actions. Western blotting and real‐time PCR were performed to detect Bcl2 expression in calvarial osteoblasts in response to PTH ex vivo. Results : There were reduced numbers of osteoclasts in Bcl2^?/? mice, with a resultant increase in bone mass. Bcl2^?/? bone marrow–derived osteoclasts ex vivo were significantly larger in size and short‐lived compared with wildtype, suggesting a pro‐apoptotic nature of Bcl2^?/? osteoclasts. In contrast, osteoblasts were entirely normal in their proliferation, differentiation, and mineralization. Intermittent administration of PTH increased bone mass similarly in Bcl2^+/+ and Bcl2^?/? mice. Finally, Western blotting and real‐time PCR showed that Bcl2 levels were not induced in response to PTH in calvarial osteoblasts. Conclusions : Bcl2 is critical in osteoclasts but not osteoblasts. Osteoclast suppression is at least in part responsible for increased bone mass of Bcl2^?/? mice, and Bcl2 is dispensable in PTH anabolic actions during bone growth.     

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     

The calcium‐sensing receptor and 25‐hydroxyvitamin D–1α‐hydroxylase interact to modulate skeletal growth and bone turnover

We examined parathyroid and skeletal function in 3‐month‐old mice expressing the null mutation for 25‐hydroxyvitamin D–1α‐hydroxylase [1α(OH)ase^?/?] and in mice expressing the null mutation for both the 1α(OH)ase and the calcium‐sensing receptor [Casr^?/?1α(OH)ase^?/?] genes. On a normal diet, all mice were hypocalcemic, with markedly increased parathyroid hormone (PTH), increased trabecular bone volume, increased osteoblast activity, poorly mineralized bone, enlarged and distorted cartilaginous growth plates, and marked growth retardation, especially in the compound mutants. Osteoclast numbers were reduced in the Casr^?/?1α(OH)ase^?/? mice. On a high‐lactose, high‐calcium, high‐phosphorus “rescue” diet, serum calcium and PTH were normal in the 1α(OH)ase^?/? mice but increased in the Casr^?/?1α(OH)ase^?/? mice with reduced serum phosphorus. Growth plate architecture and mineralization were improved in both mutants, but linear growth of the double mutants remained abnormal. Mineralization of bone improved in all mice, but osteoblast activity and trabecular bone volume remained elevated in the Casr^?/?1α(OH)ase^?/? mice. These studies support a role for calcium‐stimulated maturation of the cartilaginous growth plate and mineralization of the growth plate and bone and calcium‐stimulated CaSR‐mediated effects on bone resorption. PTH‐mediated bone resorption may require calcium‐stimulated CaSR‐mediated enhancement of osteoclastic activity. © 2010 American Society for Bone and Mineral Research. © 2010 American Society for Bone and Mineral Research     

Glycoprotein130 (Gp130)/interleukin-6 (IL-6) signalling in osteoclasts promotes bone formation in periosteal and trabecular bone

Interleukin-6 (IL-6) and interleukin-11 (IL-11) receptors (IL-6R and IL-11R, respectively) are both expressed in osteoclasts and transduce signal via the glycoprotein130 (gp130) co-receptor, but the physiological role of this pathway is unclear. To determine the critical roles of gp130 signalling in the osteoclast, we generated mice using cathepsin K Cre (CtskCre) to disrupt gp130 signalling in osteoclasts. Bone marrow macrophages from CtskCre.gp130^f/f mice generated more osteoclasts in vitro than cells from CtskCre.gp130^w/w mice; these osteoclasts were also larger and had more nuclei than controls. While no increase in osteoclast numbers was observed in vivo, osteoclasts on trabecular bone surfaces of CtskCre.gp130^f/f mice were more spread out than in control mice, but had no functional defect detectable by serum CTX1 levels or trabecular bone cartilage remnants. However, trabecular osteoblast number and mineralising surfaces were significantly lower in male CtskCre.gp130^f/f mice compared to controls, and this was associated with a significantly lower trabecular bone volume at 12 weeks of age. Furthermore, CtskCre.gp130^f/f mice exhibited greatly suppressed periosteal bone formation at this age, indicated by significant reductions in both double-labelled surface and mineral apposition rate. By 26 weeks of age, CtskCre.gp130^f/f mice exhibited narrower femora, with lower periosteal and endocortical perimeters than CtskCre.gp130^w/w controls. Since IL-6 and IL-11R global knockout mice exhibited a similar reduction in femoral width, we also assessed periosteal bone formation in those strains, and found bone forming surfaces were also reduced in male IL-6 null mice. These data suggest that IL-6/gp130 signalling in the osteoclast is not essential for normal bone resorption in vivo, but maintains both trabecular and periosteal bone formation in male mice by promoting osteoblast activity through the stimulation of osteoclast-derived "coupling factors" and "osteotransmitters", respectively.     

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.     

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     

Monocyte chemoattractant protein‐1 is a mediator of the anabolic action of parathyroid hormone on bone

Parathyroid hormone (PTH) has a significant role as an anabolic hormone in bone when administered by intermittent injection. Previous microarray studies in our laboratory have shown that the most highly regulated gene, monocyte chemoattractant protein‐1 (MCP‐1), is rapidly and transiently induced when hPTH(1‐34) is injected intermittently in rats. Through further in vivo studies, we found that rats treated with hPTH(1‐34) showed a significant increase in serum MCP‐1 levels 2 hours after PTH injection compared with basal levels. Using immunohistochemistry, increased MCP‐1 expression in osteoblasts and osteocytes is evident after PTH treatment. PTH also increased the number of marrow macrophages. MCP‐1 knockout mice injected daily with hPTH(1‐34) showed less trabecular bone mineral density and bone volume compared with wild‐type mice as measured by peripheral quantitative computed tomography (pQCT) and micro‐computed tomography (µCT). Histomorphometric analysis revealed that the increase in osteoclast surface and osteoclast number observed with intermittent PTH treatment in the wild‐type mice was completely eliminated in the MCP‐1 null mice, as well as much lower numbers of macrophages. Consequently, the lack of osteoclast and macrophage activity in the MCP‐1 null mice was paralleled by a reduction in bone formation. We conclude that osteoblast and osteocyte MCP‐1 expression is an important mediator for the anabolic effects of PTH on bone.     

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.     

Overexpression of secreted frizzled‐related protein 1 inhibits bone formation and attenuates parathyroid hormone bone anabolic effects

Secreted frizzled‐related protein 1 (sFRP1) is an antagonist of Wnt signaling, an important pathway in maintaining bone homeostasis. In this study we evaluated the skeletal phenotype of mice overexpressing sFRP1 (sFRP1 Tg) and the interaction of parathyroid hormone (PTH) treatment and sFRP1 (over)expression. Bone mass and microarchitecture were measured by micro‐computed tomography (µCT). Osteoblastic and osteoclastic cell maturation and function were assessed in primary bone marrow cell cultures. Bone turnover was assessed by biochemical markers and dynamic bone histomorphometry. Real‐time PCR was used to monitor the expression of several genes that regulate osteoblast maturation and function in whole bone. We found that trabecular bone mass measurements in distal femurs and lumbar vertebral bodies were 22% and 51% lower in female and 9% and 33% lower in male sFRP1 Tg mice, respectively, compared with wild‐type (WT) controls at 3 months of age. Genes associated with osteoblast maturation and function, serum bone formation markers, and surface based bone formation were significantly decreased in sFRP1 Tg mice of both sexes. Bone resorption was similar between sFRP1 Tg and WT females and was higher in sFRP1 Tg male mice. Treatment with hPTH(1‐34) (40 µg/kg/d) for 2 weeks increased trabecular bone volume in WT mice (females: +30% to 50%; males: +35% to 150%) compared with sFRP1 Tg mice (females: +5%; males: +18% to 54%). Percentage increases in bone formation also were lower in PTH‐treated sFRP1 Tg mice compared with PTH‐treated WT mice. In conclusion, overexpression of sFRP1 inhibited bone formation as well as attenuated PTH anabolic action on bone. The gender differences in the bone phenotype of the sFRP1 Tg animal warrants further investigation. © 2010 American Society for Bone and Mineral Research     

Osteoblast‐Derived Extracellular Vesicles Are Biological Tools for the Delivery of Active Molecules to Bone

Extracellular vesicles (EVs) are newly appreciated regulators of tissue homeostasis and a means of intercellular communication. Reports have investigated the role of EVs and their cargoes in cellular regulation and have tried to fine‐tune their biotechnological use, but to date very little is known on their function in bone biology. To investigate the relevance of EV‐mediated communication between bone cells, we isolated EVs from primary mouse osteoblasts and assessed membrane integrity, size, and structure by transmission electron microscopy (TEM) and fluorescence‐activated cell sorting (FACS). EVs actively shuttled loaded fluorochromes to osteoblasts, monocytes, and endothelial cells. Moreover, osteoblast EVs contained mRNAs shared with donor cells. Osteoblasts are known to regulate osteoclastogenesis, osteoclast survival, and osteoclast function by the pro‐osteoclastic cytokine, receptor activator of nuclear factor κ‐B ligand (Rankl). Osteoblast EVs were enriched in Rankl, which increased after PTH treatment. These EVs were biologically active, supporting osteoclast survival. EVs isolated from rankl^–/– osteoblasts lost this pro‐osteoclastic function, indicating its Rankl‐dependence. They integrated ex vivo into murine calvariae, and EV‐shuttled fluorochromes were quickly taken up by the bone upon in vivo EV systemic administration. Rankl^–/– mice lack the osteoclast lineage and are negative for its specific marker tartrate‐resistant acid phosphatase (TRAcP). Treatment of rankl^–/– mice with wild‐type osteoblast EVs induced the appearance of TRAcP‐positive cells in an EV density‐dependent manner. Finally, osteoblast EVs internalized and shuttled anti‐osteoclast drugs (zoledronate and dasatinib), inhibiting osteoclast activity in vitro and in vivo. We conclude that osteoblast EVs are involved in intercellular communication between bone cells, contribute to the Rankl pro‐osteoclastic effect, and shuttle anti‐osteoclast drugs, representing a potential means of targeted therapeutic delivery. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.     

Ciliary Neurotrophic Factor Inhibits Bone Formation and Plays a Sex-Specific Role in Bone Growth and Remodeling

Ciliary neurotrophic factor (CNTF) receptor (CNTFR) expression has been described in osteoblast-like cells, suggesting a role for CNTF in bone metabolism. When bound to CNTF, neuropoietin (NP), or cardiotrophin-like-cytokine (CLC), CNTFR forms a signaling complex with gp130 and the leukemia inhibitory factor receptor, which both play critical roles in bone cell biology. This study aimed to determine the role of CNTFR-signaling cytokines in bone. Immunohistochemistry detected CNTF in osteoblasts, osteocytes, osteoclasts, and proliferating chondrocytes. CNTFR mRNA was detected in primary calvarial osteoblasts and was upregulated during osteoblast differentiation. Treatment of osteoblasts with CNTF or CLC, but not NP, significantly inhibited mineralization and osterix mRNA levels. Twelve-week-old male CNTF ^−/− mice demonstrated reduced femoral length, cortical thickness, and periosteal circumference; but femoral trabecular bone mineral density (Tb.BMD) and tibial trabecular bone volume (BV/TV) were not significantly different from wild-type, indicating a unique role for CNTF in bone growth in male mice. In contrast, female CNTF ^−/− femora were of normal width, but femoral Tb.BMD, tibial BV/TV, trabecular number, and trabecular thickness were all increased. Female CNTF ^−/− tibiae also demonstrated high osteoblast number and mineral apposition rate compared to wild-type littermates, and this was intrinsic to the osteoblast lineage. CNTF is expressed locally in bone and plays a unique role in female mice as an inhibitor of trabecular bone formation and in male mice as a stimulus of cortical growth.     

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.     

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.     

EphrinB2/EphB4 inhibition in the osteoblast lineage modifies the anabolic response to parathyroid hormone

Previous reports indicate that ephrinB2 expression by osteoblasts is stimulated by parathyroid hormone (PTH) and its related protein (PTHrP) and that ephrinB2/EphB4 signaling between osteoblasts and osteoclasts stimulates osteoblast differentiation while inhibiting osteoclast differentiation. To determine the role of the ephrinB2/EphB4 interaction in the skeleton, we used a specific inhibitor, soluble EphB4 (sEphB4), in vitro and in vivo. sEphB4 treatment of cultured osteoblasts specifically inhibited EphB4 and ephrinB2 phosphorylation and reduced mRNA levels of late markers of osteoblast/osteocyte differentiation (osteocalcin, dentin matrix protein‐1 [DMP‐1], sclerostin, matrix‐extracellular phosphoglycoprotein [MEPE]), while substantially increasing RANKL. sEphB4 treatment in vivo in the presence and absence of PTH increased osteoblast formation and mRNA levels of early osteoblast markers (Runx2, alkaline phosphatase, Collagen 1α1, and PTH receptor [PTHR1]), but despite a substantial increase in osteoblast numbers, there was no significant change in bone formation rate or in late markers of osteoblast/osteocyte differentiation. Rather, in the presence of PTH, sEphB4 treatment significantly increased osteoclast formation, an effect that prevented the anabolic effect of PTH, causing instead a decrease in trabecular number. This enhancement of osteoclastogenesis by sEphB4 was reproduced in vitro but only in the presence of osteoblasts. These data indicate that ephrinB2/EphB4 signaling within the osteoblast lineage is required for late stages of osteoblast differentiation and, further, restricts the ability of osteoblasts to support osteoclast formation, at least in part by limiting RANKL production. This indicates a key role for the ephrinB2/EphB4 interaction within the osteoblast lineage in osteoblast differentiation and support of osteoclastogenesis. © 2013 American Society for Bone and Mineral Research.     

The beneficial effect of Icariin on bone is diminished in osteoprotegerin-deficient mice

BackgroundOsteoprogeterin (OPG) plays an important role in regulating bone homeostasis by inhibiting osteoclastogenesis and bone resorption. Icariin is the major ingredient of Herba Epimedii, which exerts anabolic and anti-resorptive effects on bone, but the mechanism remains unknown. In this study, we evaluated the role of OPG in Icariin-mediated beneficial effects on bone.Materials and methodsTwelve-week-old Opg knockout (KO) male mice and their wild type (WT) littermates were orally administered with Icariin (0.3 mg/g) everyday for 8 weeks. Bone mass and microstructure in the right proximal tibiae were analyzed with micro-computed tomography (μCT). Bone remodeling was evaluated with serum biochemical analyses and bone histomorphometry. The colonies of fibroblast and osteoblast from bone marrow derived cells were quantified. The mRNA expressions of osteoblast and osteoclast related genes in trabecular bone from the femora were analyzed by real-time PCR.ResultsIcariin treatment led to greater trabecular bone volume and trabecular number compared with vehicle treatment in WT mice. Icariin treatment increased bone formation parameters while it decreased bone resorption parameters in WT mice; however, the anabolic response of trabecular bone to Icariin treatment was diminished in KO mice. At cellular and molecular levels, Icariin significantly increased the formation of osteoblast colonies from bone marrow derived cells and the Opg gene expression in trabecular bone of WT mice.ConclusionsThese data suggest that Icariin treatment exerted anabolic and anti-resorptive effects on trabecular bone of WT mice, in which the effects were diminished in KO mice. The effects of Icariin treatment on bone are dependent on up-regulation of Opg, therefore, OPG plays an essential role in Icariin-mediated beneficial effects on trabecular bone.