Functionally improved bone in calbindin-D28k knockout mice

In vitro studies indicate that Calbindin-D28k, a calcium binding protein, is important in regulating the life span of osteoblasts as well as the mineralization of bone extracellular matrix. The recent creation of a Calbindin-D28k knockout mouse has provided the opportunity to study the physiological effects of the Calbindin-D28k protein on bone remodeling in vivo. In this experiment, histomorphometry, microCT, and bend testing were used to characterize bones in Calbindin-D28k KO (knockout) mice. The femora of Calbindin-D28k KO mice had significantly increased cortical bone volume (60.4% +/- 3.1) compared to wild-type (WT) mice (45.4% +/- 4.6). The increased bone volume was due to a decrease in marrow cavity area, and significantly decreased endosteal perimeters (3.397 mm +/- 0.278 in Calbindin-D28k KO mice, and 4.046 mm +/- 0.450 in WT mice). Similar changes were noted in the analysis of the tibias in both mice. The bone formation rates were similar in the femoral and tibial cortical bones of both mice. microCT analysis of the trabecular bone in the tibial plateau indicated that Calbindin-D28k KO mice had an increased bone volume (35.2% +/- 3.1) compared to WT mice (24.7% +/- 4.9) which was primarily due to increased trabecular number (8.99 mm(-1) +/- 0.94 in Calbindin-D28k KO mice compared to 6.75 mm(-1) +/- 0.85 in WT mice). Bone mineral content analysis of the tibias indicated that there is no difference in the calcium or phosphorus content between the Calbindin-D28k KO and WT mice. Cantilever bend testing of the femora demonstrated significantly lower strains in the bones of Calbindin-D28k KO mice (4135 micro strain/kg +/- 1266) compared to WT mice (6973 micro strain/kg +/- 998) indicating that the KO mice had stiffer bones. Three-point bending demonstrated increased failure loads in bones of Calbindin-D28k KO mice (31.6 N +/- 2.1) compared to WT mice (15.0 N +/- 1.7). In conclusion, Calbindin-D28k KO mice had increased bone volume and stiffness indicating that Calbindin-D28k plays an important role in bone remodeling.     

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.     

SRC-1 is necessary for skeletal responses to sex hormones in both males and females.

We created SRC-1(-/-) mice by mating floxed SRC-1 mice with CMV-Cre transgenic mice. The SRC-1(-/-) mice showed high turnover osteopenia under physiological conditions and hardly responded to osteoanabolic actions of exogenous androgen and estrogen in males and females, respectively, after gonadectomies, indicating that SRC-1 is essential for the maintenance of bone mass by sex hormones. INTRODUCTION: Steroid receptor coactivator-1 (SRC-1) is the first identified coactivator of nuclear receptors. This study investigated the role of SRC-1 in skeletal tissues of males and females using the deficient (SRC-1(-/-)) mice. MATERIALS AND METHODS: SRC-1(-/-) mice were generated by mating our original floxed SRC-1 mice with CMV-Cre transgenic mice. Bone metabolism between 24-week-old SRC-1(-/-) and wildtype (WT) littermates under physiological conditions was compared in males and females by radiological, histological, and biochemical analyses. Difference of skeletal responses to steroid hormones was examined by gonadectomies and exogenous administration experiments with the hormones. Statistical analysis was performed by ANOVA determined by posthoc testing using Bonferroni's method. RESULTS AND CONCLUSIONS: Although SRC-1(-/-) mice showed no abnormality in growth or major organs, both males and females showed osteopenia with high bone turnover in the trabecular bones, but not in the cortical bones, compared with WT littermates. Their serum levels of sex hormones were upregulated, suggesting a compensatory reaction for the insensitivity to these hormones. Gonadectomies caused decreases in BMDs of SRC-1(-/-) and WT mice to the same levels; however, replacement with 5 alpha-dihydrotestosterone and 17 beta-estradiol in males and females, respectively, failed to restore the bone loss in SRC-1(-/-), whereas the WT bone volume was increased to the sham-operated levels. In contrast, bone loss by administered prednisolone was similarly seen in SRC-1(-/-) and WT mice. We conclude that SRC-1 is essential for the maintenance of bone mass by sex hormones, but not for the catabolic action of glucocorticoid, under both physiological and pathological conditions.     

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.     

Protein Kinase G2 Is Essential for Skeletal Homeostasis and Adaptation to Mechanical Loading in Male but Not Female Mice

We previously showed that the NO/cGMP/protein kinase G (PKG) signaling pathway positively regulates osteoblast proliferation, differentiation, and survival in vitro, and that cGMP-elevating agents have bone-anabolic effects in mice. Here, we generated mice with an osteoblast-specific (OB) knockout (KO) of type 2 PKG (gene name Prkg2) using a Col1a1(2.3 kb)-Cre driver. Compared to wild type (WT) littermates, 8-week-old male OB Prkg2-KO mice had fewer osteoblasts, reduced bone formation rates, and lower trabecular and cortical bone volumes. Female OB Prkg2-KO littermates showed no bone abnormalities, despite the same degree of PKG2 deficiency in bone. Expression of osteoblast differentiation- and Wnt/β-catenin-related genes was lower in primary osteoblasts and bones of male KO but not female KO mice compared to WT littermates. Osteoclast parameters were unaffected in both sexes. Since PKG2 is part of a mechano-sensitive complex in osteoblast membranes, we examined its role during mechanical loading. Cyclical compression of the tibia increased cortical thickness and induced mechanosensitive and Wnt/β-catenin-related genes to a similar extent in male and female WT mice and female OB Prkg2-KO mice, but loading had a minimal effect in male KO mice. We conclude that PKG2 drives bone acquisition and adaptation to mechanical loading via the Wnt/β-catenin pathway in male mice. The striking sexual dimorphism of OB Prkg2-KO mice suggests that current U.S. Food and Drug Administration-approved cGMP-elevating agents may represent novel effective treatment options for male osteoporosis. © 2022 American Society for Bone and Mineral Research (ASBMR).     

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.     

Osteopenia and impaired fracture healing in aged EP4 receptor knockout mice

The EP4 receptor, one of the subtypes of the prostaglandin E2 (PGE2) receptor, plays a critical role in the anabolic effects of PGE2 on bone. However, its role in the maintenance of bone mass in aged animals and its role in fracture healing is not well known. Our studies addressed these issues by characterizing the skeletal phenotype of aged, EP4 receptor knockout (KO) mice, and by comparing fracture healing in aged KO mice versus wild type (WT) mice. There was no significant difference in body weight and femoral length between KO and WT mice at 15 to 16 months of age. Lower bone mass was seen radiographically in both axial and long bones of KO mice relative to WT mice. Micro-CT images of the distal femurs showed thinner cortices, fewer trabeculae, and a deteriorated trabecular network in KO mice. Total bone content, trabecular content, and cortical content, as assessed by pQCT in the distal femur, were lower in KO mice than WT controls. Histomorphometric measurements showed that trabecular bone volume and bone formation rate were significantly decreased whereas osteoclast number on trabecular surface and eroded surface on endocortical surface were significantly increased in KO mice. These data indicated that deleting the EP4 receptor resulted in an imbalance in bone resorption over formation, leading to a negative bone balance. The lower bone formation rate in EP4 KO mice was primarily due to decreased mineralizing surface, suggesting that the defect in overall bone formation was mainly due to the defect in osteoblastogenesis. Fracture healing was examined in KO and WT mice subjected to a transverse femoral fracture. Callus formation was significantly delayed as evidenced both radiographically and histologically in the fractured femurs of KO mice compared with those of WT mice. KO mice had significant decreases in total callus area, cartilaginous callus area, and bony callus area 2 weeks after fracture. By 4 weeks, complete bony bridging was seen in WT mice but not in KO mice. These data demonstrate that the absence of the EP4 receptor decreases bone mass and impairs fracture healing in aged male mice. Our findings indicate that the EP4 receptor is a positive regulator in the maintenance of bone mass and fracture healing.     

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     

Targeted Deletion of the Sclerostin Gene in Mice Results in Increased Bone Formation and Bone Strength

Introduction: Sclerosteosis is a rare high bone mass genetic disorder in humans caused by inactivating mutations in SOST, the gene encoding sclerostin. Based on these data, sclerostin has emerged as a key negative regulator of bone mass. We generated SOST knockout (KO) mice to gain a more detailed understanding of the effects of sclerostin deficiency on bone. Materials and Methods: Gene targeting was used to inactivate SOST and generate a line of SOST KO mice. Radiography, densitometry, μCT, histomorphometry, and mechanical testing were used to characterize the impact of sclerostin deficiency on bone in male and female mice. Comparisons were made between same sex KO and wildtype (WT) mice. Results: The results for male and female SOST KO mice were similar, with differences only in the magnitude of some effects. SOST KO mice had increased radiodensity throughout the skeleton, with general skeletal morphology being normal in appearance. DXA analysis of lumbar vertebrae and whole leg showed that there was a significant increase in BMD (>50%) at both sites. μCT analysis of femur showed that bone volume was significantly increased in both the trabecular and cortical compartments. Histomorphometry of trabecular bone revealed a significant increase in osteoblast surface and no significant change in osteoclast surface in SOST KO mice. The bone formation rate in SOST KO mice was significantly increased for trabecular bone (>9‐fold) at the distal femur, as well as for the endocortical and periosteal surfaces of the femur midshaft. Mechanical testing of lumbar vertebrae and femur showed that bone strength was significantly increased at both sites in SOST KO mice. Conclusions: SOST KO mice have a high bone mass phenotype characterized by marked increases in BMD, bone volume, bone formation, and bone strength. These results show that sclerostin is a key negative regulator of a powerful, evolutionarily conserved bone formation pathway that acts on both trabecular and cortical bone.     

Androgen receptor disruption increases the osteogenic response to mechanical loading in male mice

In female mice, estrogen receptor‐alpha (ERα) mediates the anabolic response of bone to mechanical loading. Whether ERα plays a similar role in the male skeleton and to what extent androgens and androgen receptor (AR) affect this response in males remain unaddressed. Therefore, we studied the adaptive response of in vivo ulna loading in AR‐ERα knockout (KO) mice and corresponding male and female single KO and wild‐type (WT) littermates using dynamic histomorphometry and immunohistochemistry. Additionally, cultured bone cells from WT and AR KO mice were subjected to mechanical loading by pulsating fluid flow in the presence or absence of testosterone. In contrast with female mice, ERα inactivation in male mice had no effect on the response to loading. Interestingly, loading induced significantly more periosteal bone formation in AR KO (+320%) and AR‐ERα KO mice (+256%) compared with male WT mice (+114%) and had a stronger inhibitory effect on SOST/sclerostin expression in AR KO versus WT mice. In accordance, the fluid flow‐induced nitric oxide production was higher in the absence of testosterone in bone cells from WT but not AR KO mice. In conclusion, AR but not ERα activation limits the osteogenic response to loading in male mice possibly via an effect on WNT signaling. © 2010 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.     

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.     

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.     

Relative impact of androgen and estrogen receptor activation in the effects of androgens on trabecular and cortical bone in growing male mice: a study in the androgen receptor knockout mouse model.

The relative importance of AR and ER activation has been studied in pubertal male AR knockout and WT mice after orchidectomy and androgen replacement therapy, either with or without an aromatase inhibitor. AR activation dominates normal trabecular bone development and cortical bone modeling in male mice. Moreover, optimal periosteal bone expansion is only observed in the presence of both AR and ER activation. INTRODUCTION: Androgen receptor (AR)-mediated androgen action has traditionally been considered a key determinant of male skeletal growth. Increasing evidence, however, suggests that estrogens are also essential for normal male bone growth. Therefore, the relative importance of AR-mediated and estrogen receptor (ER)-mediated androgen action after aromatization remains to be clarified. MATERIALS AND METHODS: Trabecular and cortical bone was studied in intact or orchidectomized pubertal AR knockout (ARKO) and male wildtype (WT) mice, with or without replacement therapy (3-8 weeks of age). Nonaromatizable (dihydrotestosterone [DHT]) and aromatizable (testosterone [T]) androgens and T plus an aromatase inhibitor (anastrazole) were administered to orchidectomized ARKO and WT mice. Trabecular and cortical bone modeling were evaluated by static and dynamic histomorphometry, respectively. RESULTS: AR inactivation or orchidectomy induced a similar degree of trabecular bone loss (-68% and -71%, respectively). Both DHT and T prevented orchidectomy-induced bone loss in WT mice but not in ARKO mice. Administration of an aromatase inhibitor did not affect T action on trabecular bone. AR inactivation and orchidectomy had similar negative effects on cortical thickness (-13% and -8%, respectively) and periosteal bone formation (-50% and -26%, respectively). In orchidectomized WT mice, both DHT and T were found to stimulate periosteal bone formation and, as a result, to increase cortical thickness. In contrast, the periosteum of ARKO mice remained unresponsive to either DHT or T. Interestingly, administration of an aromatase inhibitor partly reduced T action on periosteal bone formation in orchidectomized WT mice (-34% versus orchidectomized WT mice on T), but not in ARKO mice. This effect was associated with a significant decrease in serum IGF-I (-21% versus orchidectomized WT mice on T). CONCLUSIONS: These findings suggest a major role for AR activation in normal development of trabecular bone and periosteal bone growth in male mice. Moreover, optimal stimulation of periosteal growth is only obtained in the presence of both AR and ER activation.     

Effects of Amylin Deficiency on Trabecular Bone in Young Mice Are Sex-Dependent

Amylin deficiency in mice results in late-onset osteopenia. Sex differences have been identified in insulin secretion in Amylin-overexpressing transgenic mice, suggesting a possible interaction of sex steroids, growth factors, or cytokines and amylin. The aim of the current study was to compare the effects of amylin deficiency on bone in young and adult male and female mice. The metaphyses of the distal femora from male and female Amylin-deficient mice at 4, 6, and 26 weeks of age were assessed by bone histomorphometry. Femoral length was increased in Amylin-deficient male mice compared to wild-type (WT) mice at 26 weeks of age (P < 0.005) but not in females. This was associated with an increase in growth plate height in Amylin-deficient males at 4 (P < 0.01) and 6 (P < 0.05) weeks of age. Furthermore, young Amylin-deficient males had decreased trabecular number at 4 weeks of age (P < 0.05) and increased trabecular thickness at 4 and 6 weeks of age (P < 0.05) compared to WT mice, with no net change in trabecular bone volume. These effects of amylin deficiency were not observed in female mice. In conclusion, this study demonstrates that amylin deficiency exerts effects on bone during growth that are sex-dependent and suggest a possible interaction between amylin and testosterone, growth factors, or cytokines to regulate bone cell metabolism.     

Disruption of the insulin-like growth factor-1 gene in osteocytes impairs developmental bone growth in mice

This study evaluated the role of osteocyte-derived insulin-like growth factor 1 (IGF-1) in developmental bone growth by assessing the bone phenotype of osteocyte Igf1 conditional knockout (KO) mice, generated by crossing the Dmp1-driven Cre-expressing transgenic mice with Igf1 floxed mice containing loxP sites that flank exon 4 of the Igf1 gene. The periosteal diameter of femurs of homozygous conditional KO mutants was 8–12% smaller than wild-type (WT) littermates. The conditional mutants had 14–20%, 10–21%, and 15–31% reduction in total, trabecular, and cortical bone mineral contents, respectively. However, there were no differences in the total, trabecular, or cortical bone mineral densities, or in trabecular bone volume, thickness, number, and separation at secondary spongiosa between the mutants and WT littermates. The conditional KO mutants showed reduction in dynamic bone formation parameters at both periosteal and endosteal surfaces at the mid-diaphysis and in trabecular bone formation rate and resorption parameters at secondary spongiosa. The lower plasma levels of PINP and CTx in conditional KO mice support a regulatory role of osteocyte-derived IGF-1 in the bone turnover. The femur length of conditional KO mutants was 4–7% shorter due to significant reduction in the length of growth plate and hypertropic zone. The effect on periosteal expansion appeared to be bigger than that on longitudinal bone growth. The conditional KO mice had 14% thinner calvaria than WT littermates, suggesting that deficient osteocyte IGF-1 production also impairs developmental growth of intramembraneous bone. Conditional disruption of Igf1 in osteocytes did not alter plasma levels of IGF-1, calcium, or phosphorus. In summary, this study shows for the first time that osteocyte-derived IGF-1 plays an essential role in regulating bone turnover during developmental bone growth.     

Osteoprotegerin deficiency attenuates strontium‐mediated inhibition of osteoclastogenesis and bone resorption

Strontium (Sr) exerts an anabolic and antiresorptive effect on bone, but the mechanism remains unknown. Osteoprotegerin (OPG) expressed by osteoblasts plays an important role in regulating bone homeostasis by inhibiting osteoclastogenesis and bone resorption. This study aims at evaluating the role of OPG in Sr‐mediated inhibition of osteoclastogenesis and bone resorption. Six‐week‐old Opg knockout (KO) male mice and their wild‐type (WT) littermates were treated orally with vehicle (Veh) or Sr compound (4 mmol/kg) daily for 8 weeks. Bone mass and microstructure in the lumbar spine (L₄) and proximal tibia were analyzed with micro–computed tomography (µCT). Bone remodeling was evaluated with serum biochemical analysis and static and dynamic bone histomorphometry. Osteoclast differentiation potential and gene expression were analyzed in bone marrow cells. The findings demonstrate that Sr compound treatment results in greater bone volume and trabecular number than Veh treatment in WT mice. The anabolic response of trabecular bone to Sr treatment is attenuated in KO mice. Although Sr treatment significantly decreases in vitro osteoclastogenesis and bone resorption in WT mice, these effects are attenuated in KO mice. Furthermore, Sr treatment profoundly increases Opg gene expression in the tibias and OPG protein levels in the sera of WT mice. This study concludes that the inhibition of osteoclastogenesis and bone resorption is possibly associated with OPG upregulation by Sr treatment. © 2011 American Society for Bone and Mineral Research.     

PTH stimulates bone formation in mice deficient in Lrp5.

Lrp5 deficiency decreases bone formation and results in low bone mass. This study evaluated the bone anabolic response to intermittent PTH treatment in Lrp5-deficient mice. Our results indicate that Lrp5 is not essential for the stimulatory effect of PTH on cancellous and cortical bone formation. INTRODUCTION: Low-density lipoprotein receptor-related protein 5 (Lrp5), a co-receptor in canonical Wnt signaling, increases osteoblast proliferation, differentiation, and function. The purpose of this study was to use Lrp5-deficient mice to evaluate the potential role of this gene in mediating the bone anabolic effects of PTH. MATERIALS AND METHODS: Adult wildtype (WT, 23 male and 25 female) and Lrp5 knockout (KO, 27 male and 26 female) mice were treated subcutaneously with either vehicle or 80 microg/kg human PTH(1-34) on alternate days for 6 weeks. Femoral BMC and BMD were determined using DXA. Lumbar vertebrae were processed for quantitative bone histomorphometry. Bone architecture was evaluated by microCT. Data were analyzed using a multiway ANOVA. RESULTS: Cancellous and cortical bone mass were decreased with Lrp5 deficiency. Compared with WT mice, cancellous bone volume in the distal femur and the lumbar vertebra in Lrp5 KO mice was 54% and 38% lower, respectively (p<0.0001), whereas femoral cortical thickness was 11% lower in the KO mice (p<0.0001). The decrease in cancellous bone volume in the lumbar vertebrae was associated with a 45% decrease in osteoblast surface (p<0.0001) and a comparable decrease in bone formation rate (p<0.0001). Osteoclast surface, an index of bone resorption, was 24% lower in Lrp5 KO compared with WT mice (p<0.007). Treatment of mice with PTH for 6 weeks resulted in a 59% increase in osteoblast surface (p<0.0001) and a 19% increase in osteoclast surface (p=0.053) in both genotypes, but did not augment cancellous bone volume in either genotype. Femur cortical thickness was 11% higher in PTH-treated mice in comparison with vehicle-treated mice (p<0.0001), regardless of genotype. CONCLUSIONS: Whereas disruption of Lrp5 results in decreased bone mass because of decreased bone formation, Lrp5 does not seem to be essential for the stimulatory effects of PTH on cancellous and cortical bone formation.