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.     

Disruption of BMP Signaling in Osteoblasts Through Type IA Receptor (BMPRIA) Increases Bone Mass

Bone morphogenetic proteins (BMPs) are known as ectopic bone inducers. The FDA approved BMPs (BMP2 and BMP7) for clinical use. However, direct effects of BMPs on endogenous bone metabolism are not yet well known. We conditionally disrupted BMP receptor type IA (BMPRIA) in osteoblasts during weanling and adult stages to show the impact of BMP signaling on endogenous bone modeling and remodeling. Cre recombination was detected in immature osteoblasts in the periosteum, osteoblasts, and osteocytes but not in chondrocytes and osteoclasts after tamoxifen administration. Bmpr1a conditional knockout mice (cKO) showed increased bone mass primarily in trabecular bone at P21 and 22 wk as determined by H&E staining. Vertebrae, tails, and ribs showed increased radiodensity at 22 wk, consistent with a significant increase in BMD. Both μCT and histomorphometry showed an increase in trabecular BV/TV and thickness of cKO adult bones, whereas osteoclast number, bone formation rate, and mineral apposition rate were decreased. Expression levels of bone formation markers (Runx2 and Bsp), resorption markers (Mmp9, Ctsk, and Tracp), and Rankl were decreased, and Opg was increased in adult bones, resulting in a reduction in the ratio of Rankl to osteoprotegerin (Opg). The reduction in osteoclastogenesis through the RANKL–OPG pathway was also observed in weanling stages and reproduced in newborn calvaria culture. These results suggest that Bmpr1a cKO increased endogenous bone mass primarily in trabecular bone with decreased osteoclastogenesis through the RANKL–OPG pathway. We conclude that BMPRIA signaling in osteoblasts affects both bone formation and resorption to reduce endogenous bone mass in vivo.     

Macrophage migration inhibitory factor inhibits osteoclastogenesis

MIF is an important regulator of innate and adaptive immunity, which is produced by a variety of cell types including activated T cells and macrophages. We examined the effects of MIF on osteoclastogenesis in bone marrow (BM) cultures from WT and MIF-deficient (KO) mice as well as the bone mass of MIF KO mice.Exogenous MIF inhibited osteoclast formation in BM cultures by decreasing fusion in cells that were treated with M-CSF and RANKL. However, inhibition of OCL formation by MIF treatment was not mediated by fusion-related molecules in heterogeneous bone marrow cultures. BM cultures from MIF KO mice that were treated with M-CSF and RANKL, PTH or vitamin D had significantly increased OCL number compared to cells from WT mice. MIF also significantly inhibited OCL formation in cultures of RAW 264.7 cells that were treated with RANKL. In addition, the number of CFU-GM and Mac-1⁺ cells in the BM of MIF KO mice was greater than in WT controls. Trabecular bone volume (TBV) in the femurs and vertebrae of MIF KO mice was decreased compared to WT mice. In addition, serum bone resorption and formation markers were decreased in MIF KO mice compared to WT mice.These results demonstrate that MIF has inhibitory effects on OCL formation in vitro. We also found that BM cell cultures from MIF KO mice had an increased capacity to form osteoclasts. Furthermore, MIF KO animals had significantly decreased TBV with low turnover. We conclude that MIF is an inhibitor of osteoclastogenesis in vitro, which may regulate bone turnover via indirect mechanism in vivo.     

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     

Deletion of CD74, a putative MIF receptor,in mice enhances osteoclastogenesis and decreases bone mass

CD74 is a type II transmembrane protein that can act as a receptor for macrophage migration inhibitory factor (MIF) and plays a role in MIF‐regulated responses. We reported that MIF inhibited osteoclast formation and MIF knockout (KO) mice had decreased bone mass. We therefore examined if CD74 was involved in the ability of MIF to alter osteoclastogenesis in cultured bone marrow (BM) from wild‐type (WT) and CD74‐deficient (KO) male mice. We also measured the bone phenotype of CD74 KO male mice. Bone mass in the femur of 8‐week‐old mice was measured by micro–computed tomography and histomorphometry. Bone marrow cells from CD74 KO mice formed 15% more osteoclast‐like cells (OCLs) with macrophage colony‐stimulating factor (M‐CSF) and receptor activator of NF‐κB ligand (RANKL) (both at 30 ng/mL) compared to WT. Addition of MIF to WT cultures inhibited OCL formation by 16% but had no effect on CD74KO cultures. The number of colony forming unit granulocyte‐macrophage (CFU‐GM) in the bone marrow of CD74 KO mice was 26% greater than in WT controls. Trabecular bone volume (TBV) in the femurs of CD74 KO male mice was decreased by 26% compared to WT. In addition, cortical area and thickness were decreased by 14% and 11%, respectively. Histomorphometric analysis demonstrated that tartrate‐resistant acid phosphatase (TRAP)(+) osteoclast number and area were significantly increased in CD74 KO by 35% and 43%, respectively compared to WT. Finally, we examined the effect of MIF on RANKL‐induced‐signaling pathways in bone marrow macrophage (BMM) cultures. MIF treatment decreased RANKL‐induced nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) and c‐Fos protein in BMM cultures by 70% and 41%, respectively. Our data demonstrate that CD74 is required for MIF to affect in vitro osteoclastogenesis. Further, the bone phenotype of CD74 KO mice is similar to that of MIF KO mice. MIF treatment of WT cultures suppressed RANKL‐induced activator protein 1 (AP‐1) expression, which resulted in decreased osteoclast differentiation in vitro. We propose that CD74 plays a critical role in the MIF inhibition of osteoclastogenesis. © 2013 American Society for Bone and Mineral Research.     

Basal bone phenotype and increased anabolic responses to intermittent parathyroid hormone in healthy male COX-2 knockout mice

Cyclooxygenase-2 (COX-2) knockout (KO) mice in inbred strains can have renal dysfunction with secondary hyperparathyroidism (HPTH), making direct effects of COX-2 KO on bone difficult to assess. COX-2 KO mice in an outbred CD-1 background did not have renal dysfunction but still had two-fold elevated PTH compared to wild type (WT) mice. Compared to WT mice, KO mice had increased serum markers of bone turnover, decreased femoral bone mineral density (BMD) and cortical bone thickness, but no differences in trabecular bone volume by μCT or dynamic histomorphometry. Because PTH is a potent inducer of COX-2 and prostaglandin (PG) production, we examined the effects of COX-2 KO on bone responses after 3 weeks of intermittent PTH. Intermittent PTH increased femoral BMD and cortical bone area more in KO mice than in WT mice and increased trabecular bone volume in the distal femur in both WT and KO mice. Although not statistically significant, PTH-stimulated increases in trabecular bone tended to be greater in KO mice than in WT mice. PTH increased serum markers of bone formation and resorption more in KO than in WT mice but increased the ratio of osteoblastic surface-to-osteoclastic surface only in KO mice. PTH also increased femoral mineral apposition rates and bone formation rates in KO mice more than in WT mice. Acute mRNA responses to PTH of genes that might mediate some anabolic and catabolic effects of PTH tended to be greater in KO than WT mice. We conclude that (1) the basal bone phenotype in male COX-2 KO mice might reflect HPTH, COX-2 deficiency or both, and (2) increased responses to intermittent PTH in COX-2 KO mice, despite the presence of chronic HPTH, suggest that absence of COX-2 increased sensitivity to PTH. It is possible that manipulation of endogenous PGs could have important clinical implications for anabolic therapy with PTH.     

In vivo anabolic effect of strontium on trabecular bone was associated with increased osteoblastogenesis of bone marrow stromal cells

In vitro studies have demonstrated that strontium (Sr) could increase osteogenic differentiation of bone marrow stromal cells (BMSCs). We investigated the in vivo effect of Sr on BMSCs. Thirty‐six female rats were randomly divided into the following groups: sham operated and treated with either vehicle (Sham + Veh) or Sr compound (Sham + Sr) and ovariectomized and treated with either vehicle (OVX + Veh) or Sr compound (OVX + Sr). Vehicle and Sr were orally administrated daily starting immediately after the surgery and continuing for 12 weeks. The anabolic effect of Sr on trabecular bone was determined at the structural and tissue level by microCT and histomorphometry, respectively. Colony formation assays demonstrated that BMSCs exhibited higher osteogenic colony but lower adipogenic colony in Sr‐treated versus Veh‐treated OVX rats. The mRNA level of osteogenic genes was higher, while the mRNA level of adipogenic genes was lower in BMSCs from Sr‐treated versus Veh‐treated Sham and OVX rats. The effect of Sr on rat BMSCs was reproducible in human BMSCs. Taken together, this study suggests that the anabolic effect of Sr on normal or osteoporotic bones is associated with increased osteoblastic differentiation of BMSCs. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1208–1214, 2010     

Regulation of postnatal trabecular bone formation by the osteoblast endothelin A receptor

Endothelin‐1 (ET‐1) is a potent vasoconstrictor that also stimulates cells in the osteoblast lineage by binding to the endothelin A receptor (ETAR). ET‐1 ligand is widely secreted, particularly by the vasculature. However, the contributions of ETAR signaling to adult bone homeostasis have not been defined. ETAR was inactivated in osteoblasts by crossing ETAR‐floxed and osteocalcin‐Cre mice. Histomorphometric analyses were performed on 4‐, 8‐, and 12‐week‐old osteoblast‐targeted ETAR knockout (KO) and wild‐type (WT) male and female mice. Tibial trabecular bone volume was significantly lower from 12 weeks in KO versus WT mice in both males and females. Bone‐formation rate, osteoblast density, and in vitro osteoblast differentiation were reduced by targeted inactivation of ETAR. A separate longitudinal analysis was performed between 8 and 64 weeks to examine the effect of aging and castration on bone metabolism in ETAR KO mice. Hypogonadism did not change the rate of bone accrual in WT or KO females. However, eugonadal KO males had a significantly larger increase in tibial and femoral bone acquisition than WT mice. Male mice castrated at 8 weeks of age showed the reverse: KO mice had reduced rates of tibial and femoral BMD acquisition compared with WT mice. In vitro, ET‐1 increased osteoblast proliferation, survival, and differentiation. Dihydrotestosterone also increased osteoblast differentiation using a mechanism distinct from the actions of ET‐1. These results demonstrate that endothelin signaling in osteoblasts is an important regulator of postnatal trabecular bone remodeling and a modulator of androgen effects on bone. © 2011 American Society for Bone and Mineral Research     

Interleukin-7 influences osteoclast function in vivo but is not a critical factor in ovariectomy-induced bone loss.

IL-7 is produced by stromal cells in bone marrow and is a major regulator of B and T lymphopoiesis. It is also a direct inhibitor of osteoclastogenesis in vitro. In this study we show that IL-7-deficient mice have increased OC and decreased trabecular bone volume compared with WT mice but mimic WT mice in the amount of trabecular but not cortical bone lost after ovariectomy. INTRODUCTION: Interleukin (IL)-7 is a potent regulator of lymphocyte development, which has significant effects on bone. Bone marrow cell cultures from IL-7 deficient (IL-7KO) mice produced significantly more TRACP(+) osteoclasts (OCs) than did cells from wildtype (WT) mice. A previous study found that treatment of mice with a neutralizing antibody to IL-7 blocked ovariectomy (OVX)-induced bone loss. We examined if differences exist between the bones of WT and IL-7KO mice and if OVX altered bone mass in IL-7KO mice. MATERIALS AND METHODS: Studies were in 2-month-old sham-operated (SHAM) and OVX female mice that were killed 4 weeks after surgery. IL-7KO mice and WT controls were in a C57BL/6 background. Both vertebrae (L(1)) and femora were evaluated by DXA, muCT, and histomorphometry. IL-7KO mice were confirmed as IL-7 deficient by their almost total lack of mature B cells in their bone marrow. RESULTS: There was significantly less trabecular bone volume in the vertebrae of IL-7KO mice than in WT mice. In addition, IL-7KO mice had significantly decreased (p < 0.05) trabecular number (13%) and increased trabecular spacing (15%). OVX decreased vertebral trabecular bone volume (TBV) by 21% (p < 0.05) in WT mice and by 22% (p < 0.05) in IL-7KO mice compared with SHAM. IL-7KO SHAM mice also had significantly less (30%) TBV (TA/TTA) in their femurs, as measured histomorphometrically, than did WT SHAM mice. Femurs from IL-7KO SHAM mice had significantly increased percent OC surface (23%) compared with WT SHAM. As in the vertebrae, OVX significantly decreased femoral TBV in both WT and IL-7KO mice by similar amounts (47% and 48%, respectively, p < 0.05 for both) compared with SHAM. However, OVX decreased cortical bone mass in WT but not in IL-7KO bones. We also examined bone marrow cells from WT and IL-7KO mice. Bone marrow cells from IL-7KO animals showed a significant increase in the number of TRACP(+) osteoclast-like cells (OCLs), which formed in cultures that were stimulated with macrophage-colony stimulating factor (M-CSF) and RANKL (both at 30 ng/ml). However, there was no significant difference in the number of OCLs that formed in B lymphocyte-depleted (B220(-)) bone marrow cell cultures from WT and IL-7KO mice. CONCLUSIONS: IL-7 deficiency in mice caused increased OC number in bone and decreased bone mass. OVX-induced bone loss in IL-7-deficient mice was selective and occurred in trabecular but not cortical bone.     

Thrombin receptor deficiency leads to a high bone mass phenotype by decreasing the RANKL/OPG ratio

Thrombin and its receptor (TR) are, respectively, expressed in osteoclasts and osteoblasts. However, their physiological roles on bone metabolism have not been fully elucidated. Here we investigated the bone microarchitecture by micro-computed tomography (μCT) and demonstrated increased trabecular and cortical bone mass in femurs of TR KO mice compared to WT littermates. Trabecular thickness and connectivity were significantly enhanced. The physiological role of TR on both inorganic and organic phases of bone is illustrated by a significant increase in BMD and a decrease in urinary deoxypyridinoline (DPD) crosslink concentration in TR KO mice. Moreover, TR KO cortical bone expanded and had a higher polar moment of inertia (J), implying stronger bone. Bone histomorphometry illustrated unaltered osteoblast and osteoclast number and surface in femoral metaphyses, indicating that thrombin/TR regulates osteoblasts and osteoclasts at functional levels. Serum analysis showed a decrease in RANKL and an increase in osteoprotegerin (OPG) levels and reflected a reduced RANKL/OPG ratio in the TR KO group. In vitro experiments using MC3T3 pre-osteoblasts demonstrated a TR-dependent stimulatory effect of thrombin on the RANKL/OPG ratio. This effect was blocked by TR antagonist and p42/p44-ERK inhibitor. In addition, thrombin also intensified p42/p44-ERK expression and phosphorylation. In conclusion, the thrombin/TR system maintains normal bone remodeling by activating RANKL and limiting OPG synthesis by osteoblasts through the p42/44-ERK signaling pathway. Consequently, TR deficiency inhibits osteoclastogenesis, resulting in a high bone mass phenotype.     

T lymphocyte-deficient mice lose trabecular bone mass with ovariectomy.

We examined OVX-induced bone loss in three TLD mouse models. In TLD mice, OVX caused trabecular bone loss equivalent to that of WT. In contrast, cortical bone loss with OVX was variable. We conclude that T lymphocytes do not influence OVX-induced trabecular bone loss. INTRODUCTION: We examined ovariectomy (OVX)-induced bone loss in three T lymphocyte-deficient (TLD) mouse models: nude mice, recombination activating gene 2-deficient (RAG2 KO) mice, and T cell receptor alpha chain-deficient (TCRalpha KO) mice. MATERIALS AND METHODS: Bone mass was examined by DXA, microCT, and histomorphometry. We also examined the effect of OVX on T lymphocytes in the bone marrow and spleens of wildtype (WT) mice and on in vitro osteoclastogenesis and colony forming unit-granulocyte macrophage (CFU-GM) activity in the bone marrow of WT and nude mice. RESULTS: In WT mice, OVX did not alter T lymphocyte number in the bone marrow but did increase T lymphocytes in the spleen. Comparison of bone mass in nude, RAG2 KO, and TCRalpha KO mice with WT as measured by DXA showed decreased femoral bone mass in nude mice and increased vertebral bone mass in RAG2 KO mice. In TCRalpha KO mice, femoral, tibial, and vertebral bone mass were decreased. In vertebrae and long bones, bone loss with OVX was consistently present in WT mice but variably present in TLD mice as measured by DXA. In contrast, microCT and histomorphometry showed similar trabecular bone loss after OVX in all mice. However, femoral cortical bone loss occurred only in WT and RAG2 KO mice. OVX produced similar trabecular bone loss in WT and TCRalpha KO mice and also induced cortical bone loss in both. Histomorphometry showed that TRACP(+) area in bones was increased by OVX in femurs from both WT and nude mice as was in vitro osteoclast-like cell formation and CFU-GM activity. CONCLUSIONS: These results show that OVX caused similar trabecular bone loss in both WT and TLD mice. The ability of DXA and measurement of cortical bone loss to show OVX-induced effects on bone mass was variable. It seems that T lymphocytes are not critical for OVX-induced trabecular bone loss in these mouse models.     

Genetic background influences fluoride's effects on osteoclastogenesis

Excessive fluoride (F) can lead to abnormal bone biology. Numerous studies have focused on the anabolic action of F yet little is known regarding any action on osteoclastogenesis. Little is known regarding the influence of an individual's genetic background on the responses of bone cells to F. Four-week old C57BL/6J (B6) and C3H/HeJ (C3H) female mice were treated with NaF in the drinking water (0 ppm, 50 ppm and 100 ppm F ion) for 3 weeks. Bone marrow cells were harvested for osteoclastogenesis and hematopoietic colony-forming cell assays. Sera were analyzed for biochemical and bone markers. Femurs, tibiae, and lumbar vertebrae were subjected to microCT analysis. Tibiae and femurs were subjected to histology and biomechanical testing, respectively. The results demonstrated new actions of F on osteoclastogenesis and hematopoietic cell differentiation. Strain-specific responses were observed. The anabolic action of F was favored in B6 mice exhibiting dose-dependent increases in serum ALP activity (p < 0.001); in proximal tibia trabecular and vertebral BMD (tibia at 50&100 ppm, p = 0.001; vertebrae at 50 and 100 ppm, p = 0.023&0.019, respectively); and decrease in intact PTH and sRANKL (p = 0.045 and p < 0.001, respectively). F treatment in B6 mice also resulted in increased numbers of CFU-GEMM colonies (p = 0.025). Strain-specific accumulations in bone [F] were observed. For C3H mice, dose-dependent increases were observed in osteoclast potential (p < 0.001), in situ trabecular osteoclast number (p = 0.007), hematopoietic colony forming units (CFU-GEMM: p < 0.001, CFU-GM: p = 0.006, CFU-M: p < 0.001), and serum markers for osteoclastogenesis (intact PTH: p = 0.004, RANKL: p = 0.022, TRAP5b: p < 0.001). A concordant decrease in serum OPG (p = 0.005) was also observed. Fluoride treatment had no significant effects on bone morphology, BMD, and serum PYD cross-links in C3H suggesting a lack of significant bone resorption. Mechanical properties were also unaltered in C3H. In conclusion, short term F treatment at physiological levels has strain-specific effects in mice. The expected anabolic effects were observed in B6 and novel actions hallmarked by enhanced osteoclastogenesis shifts in hematopoietic cell differentiation in the C3H strain.     

Enhancement of Crude Bone Morphogenetic Protein-Induced New Bone Formation and Normalization of Endochondral Ossification by Bisphosphonate Treatment in Osteoprotegerin-Deficient Mice

Osteoprotegerin (OPG) is a novel secreted member of the tumor necrosis factor receptor family which plays a crucial role in negative regulation of osteoclastic bone resorption. We investigated both the quantity and quality of heterotopic new bone induced by crude bone morphogenetic protein (BMP) as a means of examining bone metabolism by bisphosphonate administration in OPG^−/− mice. Four weeks after implantation of crude BMP, the volume of heterotopic new bone in OPG^−/− mice without alendronate was significantly less than in wild-type (WT) mice. Alendronate treatment of OPG^−/− mice resulted in enhancement of the volume of heterotopic new bone. Histological findings revealed that WT mice showed normal bone formation with persistent cartilage that was interspersed with islands of bone. In contrast, the cartilage was replaced by trabecular bone and bone marrow adipocytes in OPG^−/− mice without alendronate. However, some cartilage was still present in OPG^−/− mice with alendronate compared to those without alendronate. All bone formation-related parameters and bone resorption-related parameters were significantly lower in OPG^−/− mice with alendronate than in those without alendronate. These findings suggest that in stimulated osteoclastogenesis without OPG, osteoinductive activity induced by crude BMP is inhibited and endochondral ossification induced by crude BMP is accelerated. On the other hand, alendronate treatment of OPG^−/− mice caused osteoinductive activity induced by crude BMP to increase and endochondral ossification induced by crude BMP to be decelerated. In conclusion, inhibition of stimulated osteoclastogenesis results in the enhancement of new bone formation and normalization of endochondral ossification.     

Loss of milk fat globule-epidermal growth factor 8 (MFG-E8) in mice leads to low bone mass and accelerates ovariectomy-associated bone loss by increasing osteoclastogenesis

Milk fat globule-epidermal growth factor 8 (MFG-E8) is a glycoprotein that controls the engulfment of apoptotic cells and exerts inflammation-modulatory effects. Recently, it has been implicated in osteoclastogenesis and the pathogenesis of inflammatory periodontal bone loss, but its role in physiological bone homeostasis is still not well defined. Here, we evaluated the influence of MFG-E8 on osteoblasts and osteoclasts and its impact on bone remodeling in healthy and ovariectomized mice as a model for post-menopausal osteoporosis.Total and trabecular bone mineral densities at the lumbar spine in 6-week-old MFG-E8 KO mice were reduced by 11% (p < 0.05) and 17% (p < 0.01), respectively, as compared to wild-type (WT) mice. Accordingly, serum levels of the bone formation marker P1NP were decreased by 37% (p < 0.01) in MFG-E8 KO mice as were the ex vivo mineralization capacity and expression of osteoblast genes (Runx2, alkaline phosphatase, osteocalcin) in MFG-E8 KO osteoblasts. In contrast, serum bone resorption markers CTX1 and TRAP5b were increased by 30% and 60% (p < 0.05), respectively, in MFG-E8 KO mice. Furthermore, bone marrow macrophages from MFG-E8-KO mice differentiated more effectively into osteoclasts, as compared to WT cells. MFG-E8-deficient osteoclasts displayed increased bone resorption ex vivo, which could be reversed by the presence of recombinant MFG-E8. To determine the significance of the enhanced osteoclastogenesis in MFG-E8 KO mice, we performed an ovariectomy, which is associated with bone loss due to increased osteoclast activity. Indeed, MFG-E8 KO mice lost 12% more trabecular bone density than WT mice after ovariectomy.Together, these data indicate that MFG-E8 controls steady-state and pathological bone turnover and may therefore represent a new target gene in the treatment of bone diseases.     

Pregnancy-associated plasma protein-A modulates the anabolic effects of parathyroid hormone in mouse bone

Intermittent parathyroid hormone (PTH) is a potent anabolic therapy for bone, and several studies have implicated local insulin-like growth factor (IGF) signaling in mediating this effect. The IGF system is complex and includes ligands and receptors, as well as IGF binding proteins (IGFBPs) and IGFBP proteases. Pregnancy-associated plasma protein-A (PAPP-A) is a metalloprotease expressed by osteoblasts in vitro that has been shown to enhance local IGF action through cleavage of inhibitory IGFBP-4. This study was set up to test two specific hypotheses: 1) Intermittent PTH treatment increases the expression of IGF-I, IGFBP-4 and PAPP-A in bone in vivo, thereby increasing local IGF activity. 2) In the absence of PAPP-A, local IGF activity and the anabolic effects of PTH on bone are reduced. Wild-type (WT) and PAPP-A knock-out (KO) mice were treated with 80 μg/kg human PTH 1-34 or vehicle by subcutaneous injection five days per week for six weeks. IGF-I, IGFBP-4 and PAPP-A mRNA expression in bone were significantly increased in response to PTH treatment. PTH treatment of WT mice, but not PAPP-A KO mice, significantly increased expression of an IGF-responsive gene. Bone mineral density (BMD), as measured by DEXA, was significantly decreased in femurs of PAPP-A KO compared to WT mice with PTH treatment. Volumetric BMD, as measured by pQCT, was significantly decreased in femoral midshaft (primarily cortical bone), but not metaphysis (primarily trabecular bone), of PAPP-A KO compared to WT mice with PTH treatment. These data suggest that stimulation of PAPP-A expression by intermittent PTH treatment contributes to PTH bone anabolism in mice.     

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.     

Elevated serum IGF‐1 levels synergize PTH action on the skeleton only when the tissue IGF‐1 axis is intact

There is growing evidence that insulin‐like growth factor 1 (IGF‐1) and parathyroid hormone (PTH) have synergistic actions on bone and that part of the anabolic effects of PTH is mediated by local production of IGF‐1. In this study we analyzed the skeletal response to PTH in mouse models with manipulated endocrine or autocrine/paracrine IGF‐1. We used mice carrying a hepatic IGF‐1 transgene (HIT), which results in a threefold increase in serum IGF‐1 levels and normal tissue IGF‐1 expression, and Igf1 null mice with blunted IGF‐1 expression in tissues but threefold increases in serum IGF‐1 levels (KO‐HIT). Evaluation of skeletal growth showed that elevations in serum IGF‐1 in mice with Igf1 gene ablation in all tissues except the liver (KO‐HIT) resulted in a restoration of skeletal morphology and mechanical properties by adulthood. Intermittent PTH treatment of adult HIT mice resulted in increases in serum osteocalcin levels, femoral total cross‐sectional area, cortical bone area and cortical bone thickness, as well as bone mechanical properties. We found that the skeletal response of HIT mice to PTH was significantly higher than that of control mice, suggesting synergy between IGF‐1 and PTH on bone. In sharp contrast, although PTH‐treated KO‐HIT mice demonstrated an anabolic response in cortical and trabecular bone compartments compared with vehicle‐treated KO‐HIT mice, their response was identical to that of PTH‐treated control mice. We conclude that (1) in the presence of elevated serum IGF‐1 levels, PTH can exert an anabolic response in bone even in the total absence of tissue IGF‐1, and (2) elevations in serum IGF‐1 levels synergize PTH action on bone only if the tissue IGF‐1 axis is intact. Thus enhancement of PTH anabolic actions depends on tissue IGF‐1. © 2010 American Society for Bone and Mineral Research.     

Biglycan deficiency interferes with ovariectomy-induced bone loss.

Biglycan is a matrix proteoglycan with a possible role in bone turnover. In a 4-week study with sham-operated or OVX biglycan-deficient or wildtype mice, we show that biglycan-deficient mice are resistant to OVX-induced trabecular bone loss and that there is a gender difference in the response to biglycan deficiency. INTRODUCTION: Biglycan (bgn) is a small extracellular matrix proteoglycan enriched in skeletal tissues, and biglycan-deficient male mice have decreased trabecular bone mass and bone strength. The purpose of this study was to investigate the bone phenotype of the biglycan-deficient female mice and to investigate the effect of estrogen depletion by ovariectomy (OVX). MATERIALS AND METHODS: OVX or sham operations were performed on 21-week-old mice that were divided into four groups: wt sham (n = 7), wt OVX (n = 9), bgn-deficient sham (n = 10) and bgn-deficient OVX (n = 10). The mice were killed 4 weeks after surgery. Bone mass and bone turnover were analyzed by peripheral quantitative computed tomography (pQCT), biochemical markers, and histomorphometry. RESULTS AND CONCLUSIONS: In contrast to the male mice, there were only few effects of bgn deficiency on bone metabolism in female mice, showing a clear gender difference. However, when stressed by OVX, the female bgn knockout (KO) mice were resistant to the OVX-induced trabecular bone loss. The wt mice showed a decrease in trabecular bone mineral density by pQCT measurements, a decrease in trabecular bone volume (BV/TV), and an increase in mineral apposition rate. In contrast, no significant changes were detected in bgn KO mice after OVX. In addition, analysis of the bone resorption marker deoxypyridinoline showed no significant increase in the bgn KO OVX mice compared with bgn KO sham mice. Measurements of serum osteoprotegerin (OPG) and RANKL revealed increased levels of OPG and decreased levels of RANKL in the bgn KO mice compared with wt mice. In conclusion, the bgn deficiency protects against increased trabecular bone turnover and bone loss in response to estrogen depletion, supporting the concept that bgn has dual roles in bone, where it may modulate both formation and resorption ultimately influencing the bone turnover process.     

Bone Biomechanical Properties in EP4 Knockout Mice

Among the four prostaglandin E receptor subtypes, EP₄ has been implicated as an important regulator of both bone formation and bone resorption; however, the integrated activities of this receptor on bone biomechanical properties have not been examined previously. This study compared the bone biomechanical properties of EP₄ knockout (KO) transgenic mice to strain-matched wild-type (WT) controls. We examined two groups of adult female mice: WT (n = 12) and EP₄ KO (n = 12). Femurs were tested in three-point bending and the lumbar-4 (L4) vertebral body by compression. Distal femur and vertebral body trabecular bone architecture were quantified using micro-computed tomography. Biomechanical structural parameters (ultimate/yield load, stiffness) were measured and apparent material parameters (ultimate/yield stress, modulus) calculated. Body weights and bone sizes were not different between EP₄ KO and WT mice (P > 0.05, Student’s t-test). EP₄ KO mice exhibited reduced structural (ultimate/yield load) and apparent material (ultimate/yield stress) strength in the femoral shaft and vertebral body compared to WT (P < 0.05). Vertebral body stiffness and femoral neck ultimate load (structural strength) were marginally lower in EP₄ KO than that in WT mice (P < 0.1). In addition, EP₄ KO mice have smaller distal femur and vertebral bone volume to total volume (BV/TV) trabecular thickness than WT mice (P < 0.05). These results suggest that the prostaglandin receptor EP₄ has an important role in determining biomechanical competence in the mouse skeleton. Despite similar bone size, the absence of an EP₄ receptor may have removed a necessary link for bone adaptation pathways, which resulted in relatively weaker bone properties.