Targeted Disruption of Ephrin B1 in Cells of Myeloid Lineage Increases Osteoclast Differentiation and Bone Resorption in Mice

Disruption of ephrin B1 in collagen I producing cells in mice results in severe skull defects and reduced bone formation. Because ephrin B1 is also expressed during osteoclast differentiation and because little is known on the role of ephrin B1 reverse signaling in bone resorption, we examined the bone phenotypes in ephrin B1 conditional knockout mice, and studied the function of ephrin B1 reverse signaling on osteoclast differentiation and resorptive activity. Targeted deletion of ephrin B1 gene in myeloid lineage cells resulted in reduced trabecular bone volume, trabecular number and trabecular thickness caused by increased TRAP positive osteoclasts and bone resorption. Histomorphometric analyses found bone formation parameters were not changed in ephrin B1 knockout mice. Treatment of wild-type precursors with clustered soluble EphB2-Fc inhibited RANKL induced formation of multinucleated osteoclasts, and bone resorption pits. The same treatment of ephrin B1 deficient precursors had little effect on osteoclast differentiation and pit formation. Similarly, activation of ephrin B1 reverse signaling by EphB2-Fc treatment led to inhibition of TRAP, cathepsin K and NFATc1 mRNA expression in osteoclasts derived from wild-type mice but not conditional knockout mice. Immunoprecipitation with NHERF1 antibody revealed ephrin B1 interacted with NHERF1 in differentiated osteoclasts. Treatment of osteoclasts with exogenous EphB2-Fc resulted in reduced phosphorylation of ezrin/radixin/moesin. We conclude that myeloid lineage produced ephrin B1 is a negative regulator of bone resorption in vivo, and that activation of ephrin B1 reverse signaling inhibits osteoclast differentiation in vitro in part via a mechanism that involves inhibition of NFATc1 expression and modulation of phosphorylation status of ezrin/radixin/moesin

Targeted Overexpression of Osteoactivin in Cells of Osteoclastic Lineage Promotes Osteoclastic Resorption and Bone Loss in Mice

This study sought to test whether targeted overexpression of osteoactivin (OA) in cells of osteoclastic lineage, using the tartrate-resistant acid phosphase (TRAP) exon 1B/C promoter to drive OA expression, would increase bone resorption and bone loss in vivo. OA transgenic osteoclasts showed ∼2-fold increases in OA mRNA and proteins compared wild-type (WT) osteoclasts. However, the OA expression in transgenic osteoblasts was not different. At 4, 8, and 15.3 week-old, transgenic mice showed significant bone loss determined by pQCT and confirmed by μ-CT. In vitro, transgenic osteoclasts were twice as large, had twice as much TRAP activity, resorbed twice as much bone matrix, and expressed twice as much osteoclastic genes (MMP9, calciton receptor, and ADAM12), as WT osteoclasts. The siRNA-mediated suppression of OA expression in RAW264.7-derived osteoclasts reduced cell size and osteoclastic gene expression. Bone histomorphometry revealed that transgenic mice had more osteoclasts and osteoclast surface. Plasma c-telopeptide (a resorption biomarker) measurements confirmed an increase in bone resorption in transgenic mice in vivo. In contrast, histomorphometric bone formation parameters and plasma levels of bone formation biomarkers (osteocalcin and pro-collagen type I N-terminal peptide) were not different between transgenic mice and WT littermates, indicating the lack of bone formation effects. In conclusion, this study provides compelling in vivo evidence that osteoclast-derived OA is a novel stimulator of osteoclast activity and bone resorption

Lasting consequences of traumatic events on behavioral and skeletal parameters in a mouse model for post-traumatic stress disorder (PTSD).

Post-traumatic stress disorder (PTSD) is an anxiety disorder that not only affects mental health, but may also affect bone health. However, there have been no studies to examine the direct relationship between PTSD and bone.We employed electric shocks in mice to simulate traumatic events that cause PTSD. We also injected the anxiogenic drug FG-7142 prior to electric shocks. Electric shocks created lasting conditioned fear memory in all mice. In young mice, electric shocks elicited not only behavioral response but also skeletal response, and injection of FG-7142 appeared to increase both types of response. For example in behavioral response within the first week, mice shocked alone froze an average of 6.2 sec in 10 sec tests, and mice injected with FG-7142 froze 7.6 sec, both significantly different (P<0.05) from control mice, which only froze 1.3 sec. In skeletal response at week 2, shocks alone reduced 6% bone mineral content (BMC) in total body (P = 0.06), while shocks with FG-7142 injection reduced not only 11% BMC (P<0.05) but also 6% bone mineral density (BMD) (P<0.05). In addition, FG-7142 injection also caused significant reductions of BMC in specific bones such as femur, lumbar vertebra, and tibia at week 3. Strong negative correlations (R(2) = -0.56, P<0.05) and regression (y = 0.2527-0.0037 * x, P<0.01) between freezing behavior and total body BMC in young mice indicated that increased contextual PTSD-like behavior was associated with reduced bone mass acquisition.This is the first study to document evidence that traumatic events induce lasting consequences on both behavior and skeletal growth, and electric shocks coupled with injection of anxiogenic FG-7142 in young mice can be used as a model to study the effect of PTSD-like symptoms on bone development

Prepubertal OVX increases IGF-I expression and bone accretion in C57BL/6J mice

It is generally well accepted that the pubertal surge in estrogen is responsible for the rapid bone accretion that occurs during puberty and that this effect is mediated by an estrogen-induced increase in growth hormone (GH)/insulin-like growth factor (IGF) action. To test the cause and effect relationship between estrogen and GH/IGF, we evaluated the consequence of ovariectomy (OVX) in prepubertal mice (C57BL/6J mice at 3 wk of age) on skeletal changes and the GH/IGF axis during puberty. Contrary to our expectations, OVX increased body weight (12–18%), bone mineral content (11%), bone length (4%), bone size (3%), and serum, liver, and bone IGF-I (30–50%) and decreased total body fat (18%) at 3 wk postsurgery. To determine whether estrogen is the key ovarian factor responsible for these changes, we performed a second experiment in which OVX mice were treated with placebo or estrogen implants. In addition to observing similar results compared with our first experiment, estrogen treatment partially rescued the increased body weight and bone size and completely rescued body fat and IGF-I levels. The increased bone accretion in OVX mice was due to increased bone formation rate (as determined by bone histomorphometry) and increased serum procollagen peptide. In conclusion, contrary to the known estrogen effect as an initiator of GH/IGF surge and thereby pubertal growth spurt, our findings demonstrate that loss of estrogen and/or other hormones during the prepubertal growth period effect leads to an increase in IGF-I production and bone accretion in mice

Transgenic overexpression of ephrin b1 in bone cells promotes bone formation and an anabolic response to mechanical loading in mice.

To test if ephrin B1 overexpression enhances bone mass, we generated transgenic mice overexpressing ephrin B1 under the control of a 3.6 kb rat collagen 1A1 promoter (Col3.6-Tg (efnb1) ). Col3.6-Tg (efnb1) mice express 6-, 12- and 14-fold greater levels of full-length ephrin B1 protein in bone marrow stromal cells, calvarial osteoblasts, and osteoclasts, respectively. The long bones of both genders of Col3.6-Tg (efnb1) mice have increased trabecular bone volume, trabecular number, and trabecular thickness and decreased trabecular separation. Enhanced bone formation and decreased bone resorption contributed to this increase in trabecular bone mass in Col3.6-Tg (efnb1) mice. Consistent with these findings, our in vitro studies showed that overexpression of ephrin B1 increased osteoblast differentiation and mineralization, osterix and collagen 1A1 expression in bone marrow stromal cells. Interaction of ephrin B1 with soluble clustered EphB2-Fc decreased osteoclast precursor differentiation into multinucleated cells. Furthermore, we demonstrated that the mechanical loading-induced increase in EphB2 expression and newly formed bone were significantly greater in the Col3.6-Tg (efnb1) mice than in WT littermate controls. Our findings that overexpression of ephrin B1 in bone cells enhances bone mass and promotes a skeletal anabolic response to mechanical loading suggest that manipulation of ephrin B1 actions in bone may provide a means to sensitize the skeleton to mechanical strain to stimulate new bone formation

Identification of mouse Duffy Antigen Receptor for Chemokines (Darc) as a BMD QTL gene

It is now well known that bone mineral density (BMD) variance is determined by both genetic and environmental factors. Accordingly, studies in human and animal models have revealed evidence for the presence of several quantitative trait loci (QTL) that contribute to BMD variations. However, the identification of BMD QTL genes remains a big challenge. In the current study, we focused our efforts to identify the BMD candidate gene in chromosome 1 (Chr 1) QTL that was detected from a cross involving high BMD CAST/EiJ (CAST) and low BMD C57BL/6J (B6) mice. To this end, we have combined several approaches including: (1) fine mapping the BMD QTL in Chr 1 of the B6.CAST F(2) female mice using a large number of polymorphic markers; (2) the generation of congenic sublines of mice by repeated backcrossing of CAST with B6 mice and phenotype characterization; (3) expression profiling genes in the QTL region; and (4) SNP analyses to identify the mouse Duffy Antigen Receptor for Chemokines (Darc) as a candidate gene for Chr 1 BMD QTL2. We verified the involvement of the Darc protein in BMD variation by evaluating the skeletal phenotype of Darc-knockout mice and congenic sublines of mice carrying small chromosomal segments from CAST BMD QTL. Based on the findings that Darc-antibody blocked formation of multinucleated osteoclasts in vitro and that Darc from CAST binds chemokines, known to regulate osteoclast formation, with reduced affinity compared with Darc from B6 mice, we conclude that Darc regulates BMD negatively by increasing osteoclast formation, and that the genetic association between Darc gene polymorphism and BMD variations in humans merits investigation

Direct lentiviral-cyclooxygenase 2 application to the tendon-bone interface promotes osteointegration and enhances return of the pull-out tensile strength of the tendon graft in a rat model of biceps tenodesis.

This study sought to determine if direct application of the lentiviral (LV)-cyclooxygenase 2 (COX2) vector to the tendon-bone interface would promote osteointegration of the tendon graft in a rat model of biceps tenodesis. The LV-COX2 gene transfer strategy was chosen for investigation because a similar COX2 gene transfer strategy promoted bony bridging of the fracture gap during bone repair, which involves similar histologic transitions that occur in osteointegration. Briefly, a 1.14-mm diameter tunnel was drilled in the mid-groove of the humerus of adult Fischer 344 rats. The LV-COX2 or βgal control vector was applied directly into the bone tunnel and onto the end of the tendon graft, which was then pulled into the bone tunnel. A poly-L-lactide pin was press-fitted into the tunnel as interference fixation. Animals were sacrificed at 3, 5, or 8 weeks for histology analysis of osteointegration. The LV-COX2 gene transfer strategy enhanced neo-chondrogenesis at the tendon-bone interface but with only marginal effect on de novo bone formation. The tendon-bone interface of the LV-COX2-treated tenodesis showed the well-defined tendon-to-fibrocartilage-to-bone histologic transitions that are indicative of osteointegration of the tendon graft. The LV-COX2 in vivo gene transfer strategy also significantly enhanced angiogenesis at the tendon-bone interface. To determine if the increased osteointegration was translated into an improved pull-out mechanical strength property, the pull-out tensile strength of the LV-COX2-treated tendon grafts was determined with a pull-out mechanical testing assay. The LV-COX2 strategy yielded a significant improvement in the return of the pull-out strength of the tendon graft after 8 weeks. In conclusion, the COX2-based in vivo gene transfer strategy enhanced angiogenesis, osteointegration and improved return of the pull-out strength of the tendon graft. Thus, this strategy has great potential to be developed into an effective therapy to promote tendon-to-bone healing after tenodesis or related surgeries