An atlas of genetic determinants of forearm fracture.

Osteoporotic fracture is among the most common and costly of diseases. While reasonably heritable, its genetic determinants have remained elusive. Forearm fractures are the most common clinically recognized osteoporotic fractures with a relatively high heritability. To establish an atlas of the genetic determinants of forearm fractures, we performed genome-wide association analyses including 100,026 forearm fracture cases. We identified 43 loci, including 26 new fracture loci. Although most fracture loci associated with bone mineral density, we also identified loci that primarily regulate bone quality parameters. Functional studies of one such locus, at TAC4, revealed that Tac4-/- mice have reduced mechanical bone strength. The strongest forearm fracture signal, at WNT16, displayed remarkable bone-site-specificity with no association with hip fractures. Tall stature and low body mass index were identified as new causal risk factors for fractures. The insights from this atlas may improve fracture prediction and enable therapeutic development to prevent fractures.

Transplantation of gut microbiota from old mice into young healthy mice reduces lean mass but not bone mass.

Aging is associated with low bone and lean mass as well as alterations in the gut microbiota (GM). In this study, we determined whether the reduced bone mass and relative lean mass observed in old mice could be transferred to healthy young mice by GM transplantation (GMT). GM from old (21-month-old) and young adult (5-month-old) donors was used to colonize germ-free (GF) mice in three separate studies involving still growing 5- or 11-week-old recipients and 17-week-old recipients with minimal bone growth. The GM of the recipient mice was similar to that of the donors, demonstrating successful GMT. GM from old mice did not have statistically significant effects on bone mass or bone strength, but significantly reduced the lean mass percentage of still growing recipient mice when compared with recipients of GM from young adult mice. The levels of propionate in the cecum of mice receiving old donor GM were significantly lower than those in mice receiving young adult donor GM. Bacteroides ovatus was enriched in the microbiota of recipient mice harboring GM from young adult donors. The presence of B. ovatus was not only significantly associated with high lean mass percentage in mice, but also with lean mass adjusted for fat mass in the large human HUNT cohort. In conclusion, GM from old mice reduces lean mass percentage but not bone mass in young, healthy, still growing recipient mice. Future studies are warranted to determine whether GM from young mice improves the musculoskeletal phenotype of frail elderly recipient mice.

Bone-Derived IGF-I Regulates Radial Bone Growth in Adult Male Mice.

Insulin-like growth factor-I (IGF-I) levels, which are reduced by age, and cortical bone dimensions are major determinants of fracture risk in elderly subjects. Inactivation of liver-derived circulating IGF-I results in reduced periosteal bone expansion in young and older mice. In mice with lifelong depletion of IGF-I in osteoblast lineage cells, the long bones display reduced cortical bone width. However, it has not previously been investigated whether inducible inactivation of IGF-I locally in bone in adult/old mice affects the bone phenotype. Adult tamoxifen-inducible inactivation of IGF-I using a CAGG-CreER mouse model (inducible IGF-IKO mice) substantially reduced IGF-I expression in bone (-55%) but not in liver. Serum IGF-I and body weight were unchanged. We used this inducible mouse model to assess the effect of local IGF-I on the skeleton in adult male mice, avoiding confounding developmental effects. After tamoxifen-induced inactivation of the IGF-I gene at 9 months of age, the skeletal phenotype was determined at 14 months of age. Computed tomography analyses of tibia revealed that the mid-diaphyseal cortical periosteal and endosteal circumferences and calculated bone strength parameters were decreased in inducible IGF-IKO mice compared with controls. Furthermore, 3-point bending showed reduced tibia cortical bone stiffness in inducible IGF-IKO mice. In contrast, the tibia and vertebral trabecular bone volume fraction was unchanged. In conclusion, inactivation of IGF-I in cortical bone with unchanged liver-derived IGF-I in older male mice resulted in reduced radial growth of cortical bone. This suggests that not only circulating IGF-I but also locally derived IGF-I regulates the cortical bone phenotype in older mice.

B4GALNT3 regulates glycosylation of sclerostin and bone mass.

BACKGROUND: Global sclerostin inhibition reduces fracture risk efficiently but has been associated with cardiovascular side effects. The strongest genetic signal for circulating sclerostin is in the B4GALNT3 gene region, but the causal gene is unknown. B4GALNT3 expresses the enzyme beta-1,4-N-acetylgalactosaminyltransferase 3 that transfers N-acetylgalactosamine onto N-acetylglucosaminebeta-benzyl on protein epitopes (LDN-glycosylation). METHODS: To determine if B4GALNT3 is the causal gene, B4galnt3-/- mice were developed and serum levels of total sclerostin and LDN-glycosylated sclerostin were analysed and mechanistic studies were performed in osteoblast-like cells. Mendelian randomization was used to determine causal associations. FINDINGS: B4galnt3-/- mice had higher circulating sclerostin levels, establishing B4GALNT3 as a causal gene for circulating sclerostin levels, and lower bone mass. However, serum levels of LDN-glycosylated sclerostin were lower in B4galnt3-/- mice. B4galnt3 and Sost were co-expressed in osteoblast-lineage cells. Overexpression of B4GALNT3 increased while silencing of B4GALNT3 decreased the levels of LDN-glycosylated sclerostin in osteoblast-like cells. Mendelian randomization demonstrated that higher circulating sclerostin levels, genetically predicted by variants in the B4GALNT3 gene, were causally associated with lower BMD and higher risk of fractures but not with higher risk of myocardial infarction or stroke. Glucocorticoid treatment reduced B4galnt3 expression in bone and increased circulating sclerostin levels and this may contribute to the observed glucocorticoid-induced bone loss. INTERPRETATION: B4GALNT3 is a key factor for bone physiology via regulation of LDN-glycosylation of sclerostin. We propose that B4GALNT3-mediated LDN-glycosylation of sclerostin may be a bone-specific osteoporosis target, separating the anti-fracture effect of global sclerostin inhibition, from indicated cardiovascular side effects. FUNDING: Found in acknowledgements.

Bone disturbances and progression of atherosclerosis in ApoE knockout mice.

OBJECTIVES: Epidemiological evidence supports a link between atherosclerosis and osteoporosis. These conditions might share common pathophysiological mechanisms, with inflammation being one of the hypotheses.Apolipoprotein E deficient mice (ApoE-/-) develop atherosclerotic lesions spontaneously, further aggravated by a high-fat diet. Their bone remodelling is also disturbed. We hypothesised that a proinflammatory state could be a common contributive factor for vessel and bone disturbances observed in this animal model. METHODS: We evaluated vessels and bones of ApoE-/- and control C57BL/6 (B6) female mice fed a high-fat diet in five time-points (8, 16, 20, 24 and 28 weeks of age) and quantified the development of atherosclerotic lesions, analysed gene expression of inflammatory and bone remodelling proteins (IL-1ß, IL-6, IL-17A, TNF, RANKL, and OPG), measured serum bone turnover markers (P1NP and CTX-I), performed bone (L3-L4 vertebras) histomorphometric analysis and evaluated biomechanical properties of bones. RESULTS: We compared the outcomes of B6 and ApoE-/- groups at each time-point and, within each group, over time. Atherosclerotic lesions developed as previously described for ApoE-/- mice, but no significant differences were found in bone histomorphometry or biomechanical properties between ApoE-/- and B6 mice. Also, gene expression (either in bones or aortas) and serum biomarkers were similar in both groups. When considering over time evaluations we found that bone histomorphometry changes were similar between ApoE-/- and B6 mice, but CTX-I/P1NP ratio was significantly increased (meaning higher resorption than bone formation) in ApoE-/- as compared to B6 mice. CONCLUSIONS: Our study suggests that inflammation is not the principal driver for atherosclerosis progression and bone disturbances in this animal model.

Estradiol and RSPO3 regulate vertebral trabecular bone mass independent of each other.

Osteoporosis is an age-dependent serious skeletal disease that leads to great suffering for the patient and high social costs, especially as the global population reaches higher age. Decreasing estrogen levels after menopause result in a substantial bone loss and increased fracture risk, whereas estrogen treatment improves bone mass in women. RSPO3, a secreted protein that modulates WNT signaling, increases trabecular bone mass and strength in the vertebrae of mice, and is associated with trabecular density and risk of distal forearm fractures in humans. The aim of the present study was to determine if RSPO3 is involved in the bone-sparing effect of estrogens. We first observed that estradiol (E2) treatment increases RSPO3 expression in bone of ovariectomized (OVX) mice, supporting a possible role of RSPO3 in the bone-sparing effect of estrogens. As RSPO3 is mainly expressed by osteoblasts in the bone, we used a mouse model devoid of osteoblast-derived RSPO3 (Runx2-creRspo3flox/flox mice) to determine if RSPO3 is required for the bone-sparing effect of E2 in OVX mice. We confirmed that osteoblast-specific RSPO3 inactivation results in a substantial reduction in trabecular bone mass and strength in the vertebrae. However, E2 increased vertebral trabecular bone mass and strength similarly in mice devoid of osteoblast-derived RSPO3 and control mice. Unexpectedly, osteoblast-derived RSPO3 was needed for the full estrogenic response on cortical bone thickness. In conclusion, although osteoblast-derived RSPO3 is a crucial regulator of vertebral trabecular bone, it is required for a full estrogenic effect on cortical, but not trabecular, bone in OVX mice. Thus, estradiol and RSPO3 regulate vertebral trabecular bone mass independent of each other.NEW & NOTEWORTHY Osteoblast-derived RSPO3 is known to be a crucial regulator of vertebral trabecular bone. Our new findings show that RSPO3 and estrogen regulate trabecular bone independent of each other, but that RSPO3 is necessary for a complete estrogenic effect on cortical bone.

Perfluoroalkyl Substances and Abdominal Aortic Calcification.

OBJECTIVE: To evaluate if serum perfluoroalkylated substances (PFAS) were associated with abdominal aortic calcification (AAC). METHODS: We used weighted logistic regression to investigate the gender-specific association between PFAS serum levels and AAC more than or equal to 6 from dual-energy X-ray absorptiometry (DXA) scans of the thoraco-lumbar spine from National Health and Nutrition Examination Survey 2013-2014 survey participants aged more than or equal to 40 years. RESULTS: After adjusting for confounding, none of log-transformed perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), perfluorohexane sulfonic acid (PFHxS), or perfluorononanoic acid (PFNA) were significantly associated with AAC for either men or women (adjusted odds ratios [ORs] ranged from 0.80 to 1.33, P  > 0.05 each). For PFOA and PFOS, the association was positive only in women (although the difference was not statistically significant in either case). CONCLUSION: These findings do not provide general support for a relationship of PFAS exposure to AAC, although the results show a need for gender-specific consideration in a larger dataset.

RSPO3 is important for trabecular bone and fracture risk in mice and humans.

With increasing age of the population, countries across the globe are facing a substantial increase in osteoporotic fractures. Genetic association signals for fractures have been reported at the RSPO3 locus, but the causal gene and the underlying mechanism are unknown. Here we show that the fracture reducing allele at the RSPO3 locus associate with increased RSPO3 expression both at the mRNA and protein levels, increased trabecular bone mineral density and reduced risk mainly of distal forearm fractures in humans. We also demonstrate that RSPO3 is expressed in osteoprogenitor cells and osteoblasts and that osteoblast-derived RSPO3 is the principal source of RSPO3 in bone and an important regulator of vertebral trabecular bone mass and bone strength in adult mice. Mechanistic studies revealed that RSPO3 in a cell-autonomous manner increases osteoblast proliferation and differentiation. In conclusion, RSPO3 regulates vertebral trabecular bone mass and bone strength in mice and fracture risk in humans.

Acute fat loss does not affect bone mass.

Obesity has previously been thought to protect bone since high body weight and body mass index are associated with high bone mass. However, some more recent studies suggest that increased adiposity negatively impacts bone mass. Here, we aimed to test whether acute loss of adipose tissue, via adipocyte apoptosis, alters bone mass in age-related obese mice. Adipocyte apoptosis was induced in obese male FAT-ATTAC mice through AP20187 dimerizer-mediated activation of caspase 8 selectively in adipocytes. In a short-term experiment, dimerizer was administered to 5.5 month-old mice that were terminated 2 weeks later. At termination, the total fat mass weighed 58% less in dimerizer-treated mice compared with vehicle-treated controls, but bone mass did not differ. To allow for the detection of long-term effects, we used 9-month-old mice that were terminated six weeks after dimerizer administration. In this experiment, the total fat mass weighed less (- 68%) in the dimerizer-treated mice than in the controls, yet neither bone mass nor biomechanical properties differed between groups. Our findings show that adipose tissue loss, despite the reduced mechanical loading, does not affect bone in age-related obese mice. Future studies are needed to test whether adipose tissue loss is beneficial during more severe obesity.

Endocrine, metabolic and apical effects of in utero and lactational exposure to non-dioxin-like 2,2',3,4,4',5,5'-heptachlorobiphenyl (PCB 180): A postnatal follow-up study in rats.

PCB 180 is a persistent and abundant non-dioxin-like PCB (NDL-PCB). We determined the developmental toxicity profile of ultrapure PCB 180 in developing offspring following in utero and lactational exposure with the focus on endocrine, metabolic and retinoid system alterations. Pregnant rats were given total doses of 0, 10, 30, 100, 300 or 1000 mg PCB 180/kg bw on gestational days 7-10 by oral gavage, and the offspring were sampled on postnatal days (PND) 7, 35 and 84. Decreased serum testosterone and triiodothyronine concentrations on PND 84, altered liver retinoid levels, increased liver weights and induced 7-pentoxyresorufin O-dealkylase (PROD) activity were the sensitive effects used for margin of exposure (MoE) calculations. Liver weights were increased together with induction of the metabolizing enzymes cytochrome P450 (CYP) 2B1, CYP3A1, and CYP1A1. Less sensitive effects included decreased serum estradiol and increased luteinizing hormone levels in females, decreased prostate and seminal vesicle weight and increased pituitary weight in males, increased cortical bone area and thickness of tibial diaphysis in females and decreased cortical bone mineral density in males. Developmental toxicity profiles were partly different in male and female offspring, males being more sensitive to increased liver weight, PROD induction and decreased thyroxine concentrations. MoE assessment indicated that the 95th percentile of current maternal PCB 180 concentrations do not exceed the estimated tolerable human lipid-based PCB 180 concentration. Although PCB 180 is much less potent than dioxin-like compounds, it shares several toxicological targets suggesting a potential for interactions.

Osteocyte- and late osteoblast-derived NOTUM reduces cortical bone mass in mice.

Osteoporosis is a common skeletal disease, with increased risk of fractures. Currently available osteoporosis treatments reduce the risk of vertebral fractures, mainly dependent on trabecular bone, whereas the effect on nonvertebral fractures, mainly dependent on cortical bone, is less pronounced. WNT signaling is a crucial regulator of bone homeostasis, and the activity of WNTs is inhibited by NOTUM, a secreted WNT lipase. We previously demonstrated that conditional inactivation of NOTUM in all osteoblast lineage cells increases the cortical but not the trabecular bone mass. The aim of the present study was to determine if NOTUM increasing cortical bone is derived from osteoblast precursors/early osteoblasts or from osteocytes/late osteoblasts. First, we demonstrated Notum mRNA expression in Dmp1-expressing osteocytes and late osteoblasts in cortical bone using in situ hybridization. We then developed a mouse model with inactivation of NOTUM in Dmp1-expressing osteocytes and late osteoblasts (Dmp1-creNotumflox/flox mice). We observed that the Dmp1-creNotumflox/flox mice displayed a substantial reduction of Notum mRNA in cortical bone, resulting in increased cortical bone mass and decreased cortical porosity in femur but no change in trabecular bone volume fraction in femur or in the lumbar vertebrae L5 in Dmp1-creNotumflox/flox mice as compared with control mice. In conclusion, osteocytes and late osteoblasts are the principal source of NOTUM in cortical bone, and NOTUM derived from osteocytes/late osteoblasts reduces cortical bone mass. These findings demonstrate that inhibition of osteocyte/late osteoblast-derived NOTUM might be an interesting pharmacological target to increase cortical bone mass and reduce nonvertebral fracture risk.NEW & NOTEWORTHY NOTUM produced by osteoblasts is known to regulate cortical bone mass. Our new findings show that NOTUM specifically derived by DMP1-expressing osteocytes and late osteoblasts regulates cortical bone mass and not trabecular bone mass.

Osteoblast-derived NOTUM reduces cortical bone mass in mice and the NOTUM locus is associated with bone mineral density in humans.

Osteoporosis is a common skeletal disease, affecting millions of individuals worldwide. Currently used osteoporosis treatments substantially reduce vertebral fracture risk, whereas nonvertebral fracture risk, mainly caused by reduced cortical bone mass, has only moderately been improved by the osteoporosis drugs used, defining an unmet medical need. Because several wingless-type MMTV integration site family members (WNTs) and modulators of WNT activity are major regulators of bone mass, we hypothesized that NOTUM, a secreted WNT lipase, might modulate bone mass via an inhibition of WNT activity. To characterize the possible role of endogenous NOTUM as a physiologic modulator of bone mass, we developed global, cell-specific, and inducible Notum-inactivated mouse models. Notum expression was high in the cortical bone in mice, and conditional Notum inactivation revealed that osteoblast lineage cells are the principal source of NOTUM in the cortical bone. Osteoblast lineage-specific Notum inactivation increased cortical bone thickness via an increased periosteal circumference. Inducible Notum inactivation in adult mice increased cortical bone thickness as a result of increased periosteal bone formation, and silencing of Notum expression in cultured osteoblasts enhanced osteoblast differentiation. Large-scale human genetic analyses identified genetic variants mapping to the NOTUM locus that are strongly associated with bone mineral density (BMD) as estimated with quantitative ultrasound in the heel. Thus, osteoblast-derived NOTUM is an essential local physiologic regulator of cortical bone mass via effects on periosteal bone formation in adult mice, and genetic variants in the NOTUM locus are associated with BMD variation in adult humans. Therapies targeting osteoblast-derived NOTUM may prevent nonvertebral fractures.-Movérare-Skrtic, S., Nilsson, K. H., Henning, P., Funck-Brentano, T., Nethander, M., Rivadeneira, F., Coletto Nunes, G., Koskela, A., Tuukkanen, J., Tuckermann, J., Perret, C., Souza, P. P. C., Lerner, U. H., Ohlsson, C. Osteoblast-derived NOTUM reduces cortical bone mass in mice and the NOTUM locus is associated with bone mineral density in humans.

The androgen receptor is required for maintenance of bone mass in adult male mice.

Previous studies evaluating the role of the androgen receptor (AR) for bone mass have used mouse models with global or tissue-specific lifelong inactivation of the AR. However, these mouse models have the AR inactivated already early in life and the relative roles of the AR during development, sexual maturation and in adult mice cannot be evaluated separately. The aim of the present study was to determine the specific roles of the AR in bone during sexual maturation and in adult mice. The AR was conditionally ablated at four (pre-pubertal) or ten (post-pubertal) weeks of age in male mice using tamoxifen-inducible Cre-mediated recombination. Both the pre-pubertal and the post-pubertal AR inactivation were efficient demonstrated by substantially lower AR mRNA levels in seminal vesicle, bone and white adipose tissue as well as markedly reduced weights of reproductive tissues when comparing inducible ARKO mice and control mice at 14 weeks of age. Total body BMD, as analyzed by DXA, as well as tibia diaphyseal cortical bone thickness and proximal metaphyseal trabecular bone volume fraction, as analyzed by µCT, were significantly reduced by both pre-pubertal and post-pubertal AR inactivation. These bone effects were associated with an increased bone turnover, indicating a high bone turnover osteoporosis. Pre-pubertal but not post-pubertal AR inactivation resulted in substantially increased fat mass. In conclusion, the AR is required for maintenance of both trabecular and cortical bone in adult male mice while AR expression during puberty is crucial for normal fat mass homeostasis in adult male mice.

Androgen receptor SUMOylation regulates bone mass in male mice.

The crucial effects of androgens on the male skeleton are at least partly mediated via the androgen receptor (AR). In addition to hormone binding, the AR activity is regulated by post-translational modifications, including SUMOylation. SUMOylation is a reversible modification in which Small Ubiquitin-related MOdifier proteins (SUMOs) are attached to the AR and thereby regulate the activity of the AR and change its interactions with other proteins. To elucidate the importance of SUMOylation of AR for male bone metabolism, we used a mouse model devoid of the two AR SUMOylation sites (ARSUM-; K381R and K500R are substituted). Six-month-old male ARSUM- mice displayed significantly reduced trabecular bone volume fraction in the distal metaphyseal region of femur compared with wild type (WT) mice (BV/TV, -19.1 ±â€¯4.9%, P < 0.05). The number of osteoblasts per bone perimeter was substantially reduced (-60.5 ±â€¯7.2%, P < 0.001) while no significant effect was observed on the number of osteoclasts in the trabecular bone of male ARSUM- mice. Dynamic histomorphometric analysis of trabecular bone revealed a reduced bone formation rate (-32.6 ±â€¯7.4%, P < 0.05) as a result of reduced mineralizing surface per bone surface in ARSUM- mice compared with WT mice (-24.3 ±â€¯3.6%, P < 0.001). Furthermore, cortical bone thickness in the diaphyseal region of femur was reduced in male ARSUM- mice compared with WT mice (-7.3 ±â€¯2.0%, P < 0.05). In conclusion, mice devoid of AR SUMOylation have reduced trabecular bone mass as a result of reduced bone formation. We propose that therapies enhancing AR SUMOylation might result in bone-specific anabolic effects with minimal adverse effects in other tissues.

Clinically relevant doses of vitamin A decrease cortical bone mass in mice

Excess vitamin A has been associated with decreased cortical bone thickness and increased fracture risk. While most studies in rodents have employed high dosages of vitamin A for short periods of time, we investigated the bone phenotype in mice after longer exposure to more clinically relevant doses. For 1, 4 and 10 weeks, mice were fed a control diet (4.5 µg retinyl acetate/g chow), a diet modeled from the human upper tolerable limit (UTL; 20 µg retinyl acetate/g chow) and a diet three times UTL (supplemented; 60 µg retinyl acetate/g chow). Time-dependent decreases in periosteal circumference and bone mineral content were noted with the supplemented dose. These reductions in cortical bone resulted in a significant time-dependent decrease of predicted strength and a non-significant trend toward reduced bone strength as analyzed by three-point bending. Trabecular bone in tibiae and vertebrae remained unaffected when vitamin A was increased in the diet. Dynamic histomorphometry demonstrated that bone formation was substantially decreased after 1 week of treatment at the periosteal site with the supplemental dose. Increasing amount of vitamin A decreased endocortical circumference, resulting in decreased marrow area, a response associated with enhanced endocortical bone formation. In the presence of bisphosphonate, vitamin A had no effect on cortical bone, suggesting that osteoclasts are important, even if effects on bone resorption were not detected by osteoclast counting, genes in cortical bone or analysis of serum TRAP5b and CTX. In conclusion, our results indicate that even clinically relevant doses of vitamin A have a negative impact on the amount of cortical bone.

Porcupine inhibitors impair trabecular and cortical bone mass and strength in mice.

WNT signaling is involved in the tumorigenesis of various cancers and regulates bone homeostasis. Palmitoleoylation of WNTs by Porcupine is required for WNT activity. Porcupine inhibitors are under development for cancer therapy. As the possible side effects of Porcupine inhibitors on bone health are unknown, we determined their effects on bone mass and strength. Twelve-week-old C57BL/6N female mice were treated by the Porcupine inhibitors LGK974 (low dose = 3 mg/kg/day; high dose = 6 mg/kg/day) or Wnt-C59 (10 mg/kg/day) or vehicle for 3 weeks. Bone parameters were assessed by serum biomarkers, dual-energy X-ray absorptiometry, µCT and histomorphometry. Bone strength was measured by the 3-point bending test. The Porcupine inhibitors were well tolerated demonstrated by normal body weight. Both doses of LGK974 and Wnt-C59 reduced total body bone mineral density compared with vehicle treatment (P < 0.001). Cortical thickness of the femur shaft (P < 0.001) and trabecular bone volume fraction in the vertebral body (P < 0.001) were reduced by treatment with LGK974 or Wnt-C59. Porcupine inhibition reduced bone strength in the tibia (P < 0.05). The cortical bone loss was the result of impaired periosteal bone formation and increased endocortical bone resorption and the trabecular bone loss was caused by reduced trabecular bone formation and increased bone resorption. Porcupine inhibitors exert deleterious effects on bone mass and strength caused by a combination of reduced bone formation and increased bone resorption. We suggest that cancer targeted therapies using Porcupine inhibitors may increase the risk of fractures.

Inducible Wnt16 inactivation: WNT16 regulates cortical bone thickness in adult mice.

Substantial progress has been made in the therapeutic reduction of vertebral fracture risk in patients with osteoporosis, but non-vertebral fracture risk has been improved only marginally. Human genetic studies demonstrate that the WNT16 locus is a major determinant of cortical bone thickness and non-vertebral fracture risk and mouse models with life-long Wnt16 inactivation revealed that WNT16 is a key regulator of cortical thickness. These studies, however, could not exclude that the effect of Wnt16 inactivation on cortical thickness might be caused by early developmental and/or growth effects. To determine the effect of WNT16 specifically on adult cortical bone homeostasis, Wnt16 was conditionally ablated in young adult and old mice through tamoxifen-inducible Cre-mediated recombination using CAG-Cre-ER; Wnt16flox/flox (Cre-Wnt16flox/flox) mice. First, 10-week-old Cre-Wnt16flox/flox and Wnt16flox/flox littermate control mice were treated with tamoxifen. Four weeks later, Wnt16 mRNA levels in cortical bone were reduced and cortical thickness in femur was decreased in Cre-Wnt16flox/flox mice compared to Wnt16flox/flox mice. Then, inactivation of Wnt16 in 47-week-old mice (evaluated four weeks later) resulted in a reduction of Wnt16 mRNA levels, cortical thickness and cortical bone strength with no effect on trabecular bone volume fraction. Mechanistic studies demonstrated that the reduced cortical bone thickness was caused by a combination of increased bone resorption and reduced periosteal bone formation. In conclusion, WNT16 is a crucial regulator of cortical bone thickness in young adult and old mice. We propose that new treatment strategies targeting the adult regulation of WNT16 might be useful to reduce fracture risk at cortical bone sites.

Perfluoroalkyl substances, bone density, and cardio-metabolic risk factors in obese 8-12 year old children: A pilot study.

BACKGROUND AND OBJECTIVE: Perfluoroalkyl substances (PFASs), including perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), perfluorohexane sulfonic acid (PFHxS), and perfluorononanoic acid (PFNA), have been associated with adverse bone, and metabolic changes in adults. However association of PFASs with bone health in children is understudied. Considering their role as endocrine disruptors, we examined relationships of four PFASs with bone health in children. METHODS: In a cross sectional pilot study, 48 obese children aged 8-12 years were enrolled from Dayton's Children Hospital, Ohio. Anthropometric, clinical and biochemical assessments of serum were completed. Serum PFASs were measured by UPLC-ESI-MS/MS. In a subset of 23 children, bone health parameters were measured using calcaneal quantitative ultrasound (QUS). RESULTS: While PFASs exposure was associated with a consistent negative relationship with bone health parameters, among four PFASs tested, only PFNA showed a significant negative relationship with bone parameter (ß [95% CI], = - 72.7 [- 126.0, - 19.6], p = .010). PFNA was also associated with raised systolic blood pressure (p = .008), low density lipoprotein cholesterol (LDL-C; p < .001), and total cholesterol (TC; p = .014). In addition, both PFOA and PFOS predicted elevation in LDL-C, and PFOA predicted increased TC, as well. In this analysis, PFASs were not strongly related to thyroid hormones, 25-hydroxy vitamin D, liver enzymes, or glucose homeostasis. CONCLUSION: PFASs exposure in obese children may play a role in adverse skeletal and cardiovascular risk profiles.

The Bone Sparing Effects of 2-Methoxyestradiol Are Mediated via Estrogen Receptor-α in Male Mice.

2-Methoxyestradiol (2ME2), a metabolite of 17ß-estradiol (E2), exerts bone sparing effects in animal models. We hypothesized that the underlying mechanism is back conversion of 2ME2 to E2, which subsequently acts via estrogen receptor (ER)α. We measured serum E2 levels in orchidectomized wild-type (WT) mice treated with 2ME2 66.6 µg/d or placebo. In placebo-treated animals, E2 was below the detection limit. In 2ME2-treated mice, the serum E2 level was 4.97 ± 0.68 pg/mL. This corresponds to the level found in diesterus in cycling female mice. Next, we investigated bone parameters in orchidectomized WT and ERα knockout mice treated with 2ME2 or placebo for 35 days. 2ME2 (6.66 µg/d) preserved trabecular and cortical bone in WT mice. Trabecular volumetric-bone mineral density was 64 ± 20%, and trabecular bone volume/total volume was 60 ± 20% higher in the metaphyseal region of the femur in the 2ME2 group, compared with placebo (P < .01). Both trabecular number and trabecular thickness were increased (P < .01). Cortical bone mineral content in the diaphyseal region of the femur was 31 ± 3% higher in the 2ME2 group, compared with placebo (P < .001). This was due to larger cortical area (P < .001). Three-point bending showed an increased bone strength in WT 2ME2-treated animals compared with placebo (maximum load [Fmax] +19±5% in the 2ME2 group, P < .05). Importantly, no bone parameter was affected by 2ME2 treatment in ERα knockout mice. In conclusion, 2ME2 treatment of orchidectomized mice results in increased serum E2. ERα mediates the bone sparing effects of 2ME2. The likely mediator of this effect is E2 resulting from back conversion of 2ME2.

Liver-derived IGF-I regulates cortical bone mass but is dispensable for the osteogenic response to mechanical loading in female mice.

Low circulating IGF-I is associated with increased fracture risk. Conditional depletion of IGF-I produced in osteoblasts or osteocytes inhibits the bone anabolic effect of mechanical loading. Here, we determined the role of endocrine IGF-I for the osteogenic response to mechanical loading in young adult and old female mice with adult, liver-specific IGF-I inactivation (LI-IGF-I(-/-) mice, serum IGF-I reduced by ≈70%) and control mice. The right tibia was subjected to short periods of axial cyclic compressive loading three times/wk for 2 wk, and measurements were performed using microcomputed tomography and mechanical testing by three-point bending. In the nonloaded left tibia, the LI-IGF-I(-/-) mice had lower cortical bone area and increased cortical porosity, resulting in reduced bone mechanical strength compared with the controls. Mechanical loading induced a similar response in LI-IGF-I(-/-) and control mice in terms of cortical bone area and trabecular bone volume fraction. In fact, mechanical loading produced a more marked increase in cortical bone mechanical strength, which was associated with a less marked increase in cortical porosity, in the LI-IGF-I(-/-) mice compared with the control mice. In conclusion, liver-derived IGF-I regulates cortical bone mass, cortical porosity, and mechanical strength under normal (nonloaded) conditions. However, despite an ∼70% reduction in circulating IGF-I, the osteogenic response to mechanical loading was not attenuated in the LI-IGF-I(-/-) mice.