High Bone Mass–Causing Mutant LRP5 Receptors Are Resistant to Endogenous Inhibitors In Vivo

Certain missense mutations affecting LRP5 cause high bone mass (HBM) in humans. Based on in vitro evidence, HBM LRP5 receptors are thought to exert their effects by providing resistance to binding/inhibition of secreted LRP5 inhibitors such as sclerostin (SOST) and Dickkopf homolog‐1 (DKK1). We previously reported the creation of two Lrp5 HBM knock‐in mouse models, in which the human p.A214V or p.G171V missense mutations were knocked into the endogenous Lrp5 locus. To determine whether HBM knock‐in mice are resistant to SOST‐ or DKK1‐induced osteopenia, we bred Lrp5 HBM mice with transgenic mice that overexpress human SOST in osteocytes (^8kbDmp1‐SOST) or mouse DKK1 in osteoblasts and osteocytes (^2.3kbCol1a1‐Dkk1). We observed that the ^8kbDmp1‐SOST transgene significantly lowered whole‐body bone mineral density (BMD), bone mineral content (BMC), femoral and vertebral trabecular bone volume fraction (BV/TV), and periosteal bone‐formation rate (BFR) in wild‐type mice but not in mice with Lrp5 p.G171V and p.A214V alleles. The ^2.3kbCol1a1‐Dkk1 transgene significantly lowered whole‐body BMD, BMC, and vertebral BV/TV in wild‐type mice and affected p.A214V mice more than p.G171V mice. These in vivo data support in vitro studies regarding the mechanism of HBM‐causing mutations, and imply that HBM LRP5 receptors differ in their relative sensitivity to inhibition by SOST and DKK1. © 2015 American Society for Bone and Mineral Research.     

Enhanced Wnt signaling improves bone mass and strength,but not brittleness,in the Col1a1+/mov13 mouse model of type I Osteogenesis Imperfecta

Osteogenesis Imperfecta (OI) comprises a group of genetic skeletal fragility disorders. The mildest form of OI, Osteogenesis Imperfecta type I, is frequently caused by haploinsufficiency mutations in COL1A1, the gene encoding the α₁(I) chain of type 1 collagen. Children with OI type I have a 95-fold higher fracture rate compared to unaffected children. Therapies for OI type I in the pediatric population are limited to anti-catabolic agents. In adults with osteoporosis, anabolic therapies that enhance Wnt signaling in bone improve bone mass, and ongoing clinical trials are determining if these therapies also reduce fracture risk. We performed a proof-of-principle experiment in mice to determine whether enhancing Wnt signaling in bone could benefit children with OI type I. We crossed a mouse model of OI type I (Col1a1^+/Mov13) with a high bone mass (HBM) mouse (Lrp5^+/p.A214V) that has increased bone strength from enhanced Wnt signaling. Offspring that inherited the OI and HBM alleles had higher bone mass and strength than mice that inherited the OI allele alone. However, OI + HBM and OI mice still had bones with lower ductility compared to wild-type mice. We conclude that enhancing Wnt signaling does not make OI bone normal, but does improve bone properties that could reduce fracture risk. Therefore, agents that enhance Wnt signaling are likely to benefit children and adults with OI type 1.     

Correlations Between Bone Mechanical Properties and Bone Composition Parameters in Mouse Models of Dominant and Recessive Osteogenesis Imperfecta and the Response to Anti‐TGF‐β Treatment

Osteogenesis imperfecta (OI) is a group of genetic disorders characterized by brittle bones that are prone to fracture. Although previous studies in animal models investigated the mechanical properties and material composition of OI bone, little work has been conducted to statistically correlate these parameters to identify key compositional contributors to the impaired bone mechanical behaviors in OI. Further, although increased TGF‐β signaling has been demonstrated as a contributing mechanism to the bone pathology in OI models, the relationship between mechanical properties and bone composition after anti‐TGF‐β treatment in OI has not been studied. Here, we performed follow‐up analyses of femurs collected in an earlier study from OI mice with and without anti‐TGF‐β treatment from both recessive (Crtap^‐/‐) and dominant (Col1a2^+/P.G610C) OI mouse models and WT mice. Mechanical properties were determined using three‐point bending tests and evaluated for statistical correlation with molecular composition in bone tissue assessed by Raman spectroscopy. Statistical regression analysis was conducted to determine significant compositional determinants of mechanical integrity. Interestingly, we found differences in the relationships between bone composition and mechanical properties and in the response to anti‐TGF‐β treatment. Femurs of both OI models exhibited increased brittleness, which was associated with reduced collagen content and carbonate substitution. In the Col1a2^+/P.G610C femurs, reduced hydroxyapatite crystallinity was also found to be associated with increased brittleness, and increased mineral‐to‐collagen ratio was correlated with increased ultimate strength, elastic modulus, and bone brittleness. In both models of OI, regression analysis demonstrated that collagen content was an important predictor of the increased brittleness. In summary, this work provides new insights into the relationships between bone composition and material properties in models of OI, identifies key bone compositional parameters that correlate with the impaired mechanical integrity of OI bone, and explores the effects of anti‐TGF‐β treatment on bone‐quality parameters in these models. © 2016 American Society for Bone and Mineral Research.     

Hindlimb Skeletal Muscle Function and Skeletal Quality and Strength in +/G610C Mice With and Without Weight‐Bearing Exercise

Osteogenesis imperfecta (OI) is a heterogeneous heritable connective tissue disorder associated with reduced bone mineral density and skeletal fragility. Bone is inherently mechanosensitive, with bone strength being proportional to muscle mass and strength. Physically active healthy children accrue more bone than inactive children. Children with type I OI exhibit decreased exercise capacity and muscle strength compared with healthy peers. It is unknown whether this muscle weakness reflects decreased physical activity or a muscle pathology. In this study, we used heterozygous G610C OI model mice (+/G610C), which model both the genotype and phenotype of a large Amish OI kindred, to evaluate hindlimb muscle function and physical activity levels before evaluating the ability of +/G610C mice to undergo a treadmill exercise regimen. We found +/G610C mice hindlimb muscles do not exhibit compromised muscle function, and their activity levels were not reduced relative to wild‐type mice. The +/G610C mice were also able to complete an 8‐week treadmill regimen. Biomechanical integrity of control and exercised wild‐type and +/G610C femora were analyzed by torsional loading to failure. The greatest skeletal gains in response to exercise were observed in stiffness and the shear modulus of elasticity with alterations in collagen content. Analysis of tibial cortical bone by Raman spectroscopy demonstrated similar crystallinity and mineral/matrix ratios regardless of sex, exercise, and genotype. Together, these findings demonstrate +/G610C OI mice have equivalent muscle function, activity levels, and ability to complete a weight‐bearing exercise regimen as wild‐type mice. The +/G610C mice exhibited increased femoral stiffness and decreased hydroxyproline with exercise, whereas other biomechanical parameters remain unaffected, suggesting a more rigorous exercise regimen or another exercise modality may be required to improve bone quality of OI mice. © 2015 American Society for Bone and Mineral Research.     

High-bone-mass-producing mutations in the Wnt signaling pathway result in distinct skeletal phenotypes

Mutations among genes that participate in the canonical Wnt signaling pathway can lead to drastically different skeletal phenotypes, ranging from severe osteoporosis to severe osteosclerosis. Many high-bone-mass (HBM) causing mutations that occur in the LRP5 gene appear to impart the HBM phenotype, in part, by increasing resistance to soluble Wnt signaling inhibitors, including sclerostin. Sost loss-of-function mutant mice (Sost knock-out) and Lrp5 gain-of-function mutant mice (Lrp5 HBM knock-in) have high bone mass. These mutants potentially would be predicted to be phenocopies of one another, because in both cases, the sclerostin-Lrp5 interaction is disrupted. We measured bone mass, size, geometry, architecture, and strength in bones from three different genetic mouse models (Sost knock-out, Lrp5 A214V knock-in, and Lrp5 G171V knock-in) of HBM. We found that all three mouse lines had significantly elevated bone mass in the appendicular skeleton and in the cranium. Sost mutants and Lrp5 A214V mutants were statistically indistinguishable from one another in most endpoints, whereas both were largely different from the Lrp5 G171V mutants. Lrp5 G171V mutants preferentially added bone endocortically, whereas Lrp5 A214V and Sost mutants preferentially added bone periosteally. Cranial thickness and cranial nerve openings were similarly altered in all three HBM models. We also assessed serum serotonin levels as a possible mechanism accounting for the observed changes in bone mass, but no differences in serum serotonin were found in any of the three HBM mouse lines. The skeletal dissimilarities of the Lrp5 G171V mutant to the other mutants suggest that other, non-sclerostin-associated mechanisms might account for the changes in bone mass resulting from this mutation.     

Combinatorial Inhibition of Myostatin and Activin A Improves Femoral Bone Properties in the G610C Mouse Model of Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a collagen-related bone disorder characterized by fragile osteopenic bone and muscle weakness. We have previously shown that the soluble activin receptor type IIB decoy (sActRIIB) molecule increases muscle mass and improves bone strength in the mild to moderate G610C mouse model of OI. The sActRIIB molecule binds multiple transforming growth factor-β (TGF-β) ligands, including myostatin and activin A. Here, we investigate the musculoskeletal effects of inhibiting activin A alone, myostatin alone, or both myostatin and activin A in wild-type (Wt) and heterozygous G610C (+/G610C) mice using specific monoclonal antibodies. Male and female Wt and +/G610C mice were treated twice weekly with intraperitoneal injections of monoclonal control antibody (Ctrl-Ab, Regn1945), anti-activin A antibody (ActA-Ab, Regn2476), anti-myostatin antibody (Mstn-Ab, Regn647), or both ActA-Ab and Mstn-Ab (Combo, Regn2476, and Regn647) from 5 to 16 weeks of age. Prior to euthanasia, whole body composition, metabolism and muscle force generation assessments were performed. Post euthanasia, hindlimb muscles were evaluated for mass, and femurs were evaluated for changes in microarchitecture and biomechanical strength using micro–computed tomography (μCT) and three-point bend analyses. ActA-Ab treatment minimally impacted the +/G610C musculoskeleton, and was detrimental to bone strength in male +/G610C mice. Mstn-Ab treatment, as previously reported, resulted in substantial increases in hindlimb muscle weights and overall body weights in Wt and male +/G610C mice, but had minimal skeletal impact in +/G610C mice. Conversely, the Combo treatment outperformed ActA-Ab alone or Mstn-Ab alone, consistently increasing hindlimb muscle and body weights regardless of sex or genotype and improving bone microarchitecture and strength in both male and female +/G610C and Wt mice. Combinatorial inhibition of activin A and myostatin more potently increased muscle mass and bone microarchitecture and strength than either antibody alone, recapturing most of the observed benefits of sActRIIB treatment in +/G610C mice. © 2022 American Society for Bone and Mineral Research (ASBMR).     

Gain-of-Function Lrp5 Mutation Improves Bone Mass and Strength and Delays Hyperglycemia in a Mouse Model of Insulin-Deficient Diabetes

High fracture rate and high circulating levels of the Wnt inhibitor, sclerostin, have been reported in diabetic patients. We studied the effects of Wnt signaling activation on bone health in a mouse model of insulin-deficient diabetes. We introduced the sclerostin-resistant Lrp5^A214V mutation, associated with high bone mass, in mice carrying the Ins2^Akita mutation (Akita), which results in loss of beta cells, insulin deficiency, and diabetes in males. Akita mice accrue less trabecular bone mass with age relative to wild type (WT). Double heterozygous Lrp5^A214V/Akita mutants have high trabecular bone mass and cortical thickness relative to WT animals, as do Lrp5^A214V single mutants. Likewise, the Lrp5^A214V mutation prevents deterioration of biomechanical properties occurring in Akita mice. Notably, Lrp5^A214V/Akita mice develop fasting hyperglycemia and glucose intolerance with a delay relative to Akita mice (7 to 8 vs. 5 to 6 weeks, respectively), despite lack of insulin production in both groups by 6 weeks of age. Although insulin sensitivity is partially preserved in double heterozygous Lrp5^A214V/Akita relative to Akita mutants up to 30 weeks of age, insulin-dependent phosphorylated protein kinase B (pAKT) activation in vitro is not altered by the Lrp5^A214V mutation. Although white adipose tissue depots are equally reduced in both compound and Akita mice, the Lrp5^A214V mutation prevents brown adipose tissue whitening that occurs in Akita mice. Thus, hyperactivation of Lrp5-dependent signaling fully protects bone mass and strength in prolonged hyperglycemia and improves peripheral glucose metabolism in an insulin independent manner. Wnt signaling activation represents an ideal therapeutic approach for diabetic patients at high risk of fracture. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

The Anti‐Osteoanabolic Function of Sclerostin Is Blunted in Mice Carrying a High Bone Mass Mutation of Lrp5

Activating mutations of the putative Wnt co‐receptor Lrp5 or inactivating mutations of the secreted molecule Sclerostin cause excessive bone formation in mice and humans. Previous studies have suggested that Sclerostin functions as an Lrp5 antagonist, yet clear in vivo evidence was still missing, and alternative mechanisms have been discussed. Moreover, because osteoblast‐specific inactivation of β‐catenin, the major intracellular mediator of canonical Wnt signaling, primarily affected bone resorption, it remained questionable, whether Sclerostin truly acts as a Wnt signaling antagonist by interacting with Lrp5. In an attempt to address this relevant question, we generated a mouse model (Col1a1‐Sost) with transgenic overexpression of Sclerostin under the control of a 2.3‐kb Col1a1 promoter fragment. These mice displayed the expected low bone mass phenotype as a consequence of reduced bone formation. The Col1a1‐Sost mice were then crossed with two mouse lines carrying different high bone mass mutations of Lrp5 (Lrp5^A170V and Lrp5^G213V), both of them potentially interfering with Sclerostin binding. Using µCT‐scanning and histomorphometry we found that the anti‐osteoanabolic influence of Sclerostin overexpression was not observed in Lrp5^A213V/A213V mice and strongly reduced in Lrp5^A170V/A170V mice. As a control we applied the same strategy with mice overexpressing the transmembrane Wnt signaling antagonist Krm2 and found that the anti‐osteoanabolic influence of the Col1a1‐Krm2 transgene was not affected by either of the Lrp5 mutations. Taken together, our data support the concept that Sclerostin inhibits bone formation through Lrp5 interaction, yet their physiological relevance remains to be established. © 2015 American Society for Bone and Mineral Research.     

Reversing LRP5‐Dependent Osteoporosis and SOST Deficiency–Induced Sclerosing Bone Disorders by Altering WNT Signaling Activity

The bone formation inhibitor sclerostin encoded by SOST binds in vitro to low‐density lipoprotein receptor‐related protein (LRP) 5/6 Wnt co‐receptors, thereby inhibiting Wnt/β‐catenin signaling, a central pathway of skeletal homeostasis. Lrp5/LRP5 deficiency results in osteoporosis‐pseudoglioma (OPPG), whereas Sost/SOST deficiency induces lifelong bone gain in mice and humans. Here, we analyzed the bone phenotype of mice lacking Sost (Sost^?/?), Lrp5 (Lrp5^?/?), or both (Sost^?/?;Lrp5^?/?) to elucidate the mechanism of action of Sost in vivo. Sost deficiency–induced bone gain was significantly blunted in Sost^?/?;Lrp5^?/? mice. Yet the Lrp5 OPPG phenotype was fully rescued in Sost^?/?;Lrp5^?/? mice and most bone parameters were elevated relative to wild‐type. To test whether the remaining bone increases in Sost^?/?;Lrp5^?/? animals depend on Lrp6, we treated wild‐type, Sost^?/?, and Sost^?/?;Lrp5^?/? mice with distinct Lrp6 function blocking antibodies. Selective blockage of Wnt1 class–mediated Lrp6 signaling reduced cancellous bone mass and density in wild‐type mice. Surprisingly, it reversed the abnormal bone gain in Sost^?/? and Sost^?/?;Lrp5^?/? mice to wild‐type levels irrespective of enhancement or blockage of Wnt3a class‐mediated Lrp6 activity. Thus, whereas Sost deficiency–induced bone anabolism partially requires Lrp5, it fully depends on Wnt1 class–induced Lrp6 activity. These findings indicate: first, that OPPG syndrome patients suffering from LRP5 loss‐of‐function should benefit from principles antagonizing SOST/sclerostin action; and second, that therapeutic WNT signaling inhibitors may stop the debilitating bone overgrowth in sclerosing disorders related to SOST deficiency, such as sclerosteosis, van Buchem disease, and autosomal dominant craniodiaphyseal dysplasia, which are rare disorders without viable treatment options. © 2014 American Society for Bone and Mineral Research.     

Impact of Genetic and Pharmacologic Inhibition of Myostatin in a Murine Model of Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a genetic connective tissue disorder characterized by compromised skeletal integrity, altered microarchitecture, and bone fragility. Current OI treatment strategies focus on bone antiresorptives and surgical intervention with limited effectiveness, and thus identifying alternative therapeutic options remains critical. Muscle is an important stimulus for bone formation. Myostatin, a TGF-β superfamily myokine, acts through ActRIIB to negatively regulate muscle growth. Recent studies demonstrated the potential benefit of myostatin inhibition with the soluble ActRIIB fusion protein on skeletal properties, although various OI mouse models exhibited variable skeletal responses. The genetic and clinical heterogeneity associated with OI, the lack of specificity of the ActRIIB decoy molecule for myostatin alone, and adverse events in human clinical trials further the need to clarify myostatin's therapeutic potential and role in skeletal integrity. In this study, we determined musculoskeletal outcomes of genetic myostatin deficiency and postnatal pharmacological myostatin inhibition by a monoclonal anti-myostatin antibody (Regn647) in the G610C mouse, a model of mild–moderate type I/IV human OI. In the postnatal study, 5-week-old wild-type and +/G610C male and female littermates were treated with Regn647 or a control antibody for 11 weeks or for 7 weeks followed by a 4-week treatment holiday. Inhibition of myostatin, whether genetically or pharmacologically, increased muscle mass regardless of OI genotype, although to varying degrees. Genetic myostatin deficiency increased hindlimb muscle weights by 6.9% to 34.4%, whereas pharmacological inhibition increased them by 13.5% to 29.6%. Female +/mstn +/G610C (Dbl.Het) mice tended to have similar trabecular and cortical bone parameters as Wt showing reversal of +/G610C characteristics but with minimal effect of +/mstn occurring in male mice. Pharmacologic myostatin inhibition failed to improve skeletal bone properties of male or female +/G610C mice, although skeletal microarchitectural and biomechanical improvements were observed in male wild-type mice. Four-week treatment holiday did not alter skeletal outcomes. © 2020 American Society for Bone and Mineral Research (ASBMR).     

The Lrp4R1170Q Homozygous Knock‐In Mouse Recapitulates the Bone Phenotype of Sclerosteosis in Humans

Sclerosteosis is a rare autosomal recessive bone disorder marked by hyperostosis of the skull and tubular bones. Initially, we and others reported that sclerosteosis was caused by loss‐of‐function mutations in SOST, encoding sclerostin. More recently, we identified disease‐causing mutations in LRP4, a binding partner of sclerostin, in three sclerosteosis patients. Upon binding to sclerostin, LRP4 can inhibit the canonical WNT signaling that is known to be an important pathway in the regulation of bone formation. To further investigate the role of LRP4 in the bone formation process, we generated an Lrp4 mutated sclerosteosis mouse model by introducing the p.Arg1170Gln mutation in the mouse genome. Extensive analysis of the bone phenotype of the Lrp4R1170Q/R1170Q knock‐in (KI) mouse showed the presence of increased trabecular and cortical bone mass as a consequence of increased bone formation by the osteoblasts. In addition, three‐point bending analysis also showed that the increased bone mass results in increased bone strength. In contrast to the human sclerosteosis phenotype, we could not observe syndactyly in the forelimbs or hindlimbs of the Lrp4 KI animals. Finally, we could not detect any significant changes in the bone formation and resorption markers in the serum of the mutant mice. However, the serum sclerostin levels were strongly increased and the level of sclerostin in the tibia was decreased in Lrp4R1170Q/R1170Q mice, confirming the role of LRP4 as an anchor for sclerostin in bone. In conclusion, the Lrp4R1170Q/R1170Q mouse is a good model for the human sclerosteosis phenotype caused by mutations in LRP4 and can be used in the future for further investigation of the mechanism whereby LRP4 regulates bone formation. © 2017 American Society for Bone and Mineral Research.     

Mutations in Known Monogenic High Bone Mass Loci Only Explain a Small Proportion of High Bone Mass Cases

High bone mass (HBM) can be an incidental clinical finding; however, monogenic HBM disorders (eg, LRP5 or SOST mutations) are rare. We aimed to determine to what extent HBM is explained by mutations in known HBM genes. A total of 258 unrelated HBM cases were identified from a review of 335,115 DXA scans from 13 UK centers. Cases were assessed clinically and underwent sequencing of known anabolic HBM loci: LRP5 (exons 2, 3, 4), LRP4 (exons 25, 26), SOST (exons 1, 2, and the van Buchem's disease [VBD] 52‐kb intronic deletion 3′). Family members were assessed for HBM segregation with identified variants. Three‐dimensional protein models were constructed for identified variants. Two novel missense LRP5 HBM mutations ([c.518C>T; p.Thr173Met], [c.796C>T; p.Arg266Cys]) were identified, plus three previously reported missense LRP5 mutations ([c.593A>G; p.Asn198Ser], [c.724G>A; p.Ala242Thr], [c.266A>G; p.Gln89Arg]), associated with HBM in 11 adults from seven families. Individuals with LRP5 HBM (～prevalence 5/100,000) displayed a variable phenotype of skeletal dysplasia with increased trabecular BMD and cortical thickness on HRpQCT, and gynoid fat mass accumulation on DXA, compared with both non‐LRP5 HBM and controls. One mostly asymptomatic woman carried a novel heterozygous nonsense SOST mutation (c.530C>A; p.Ser177X) predicted to prematurely truncate sclerostin. Protein modeling suggests the severity of the LRP5‐HBM phenotype corresponds to the degree of protein disruption and the consequent effect on SOST‐LRP5 binding. We predict p.Asn198Ser and p.Ala242Thr directly disrupt SOST binding; both correspond to severe HBM phenotypes (BMD Z‐scores +3.1 to +12.2, inability to float). Less disruptive structural alterations predicted from p.Arg266Cys, p.Thr173Met, and p.Gln89Arg were associated with less severe phenotypes (Z‐scores +2.4 to +6.2, ability to float). In conclusion, although mutations in known HBM loci may be asymptomatic, they only account for a very small proportion (～3%) of HBM individuals, suggesting the great majority are explained by either unknown monogenic causes or polygenic inheritance. © 2015 The Authors Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).     

Mechanotransduction in bone tissue: The A214V and G171V mutations in Lrp5 enhance load-induced osteogenesis in a surface-selective manner

Mechanotransduction in bone requires components of the Wnt signaling pathway to produce structurally adapted bone elements. In particular, the Wnt co-receptor LDL-receptor-related protein 5 (LRP5) appears to be a crucial protein in the mechanotransduction cascades that translate physical tissue deformation into new bone formation. Recently discovered missense mutations in LRP5 are associated with high bone mass (HBM), and the altered function of these proteins provide insight into LRP5 function in many skeletal processes, including mechanotransduction. We further investigated the role of LRP5 in bone cell mechanotransduction by applying mechanical stimulation in vivo to two different mutant mouse lines, which harbor HBM-causing missense mutations in Lrp5. Axial tibia loading was applied to mature male Lrp5 G171V and Lrp5 A214V knock-in mice, and to their wild type controls. Fluorochrome labeling revealed that 3days of loading resulted in a significantly enhanced periosteal response in the A214V knock in mice, whereas the G171V mice exhibited a lowered osteogenic threshold on the endocortical surface. In summary, our data further highlight the importance of Lrp5 in bone cell mechanotransduction, and indicate that the HBM-causing mutations in Lrp5 can alter the anabolic response to mechanical stimulation in favor of increased bone gain.     

Levels of serotonin,sclerostin, bone turnover markers as well as bone density and microarchitecture in patients with high‐bone‐mass phenotype due to a mutation in Lrp5

Patients with an activation mutation of the Lrp5 gene exhibit high bone mass (HBM). Limited information is available regarding compartment‐specific changes in bone. The relationship between the phenotype and serum serotonin is not well documented. To evaluate bone, serotonin, and bone turnover markers (BTM) in Lrp5‐HBM patients, we studied 19 Lrp5‐HBM patients (T253I) and 19 age‐ and sex‐matched controls. DXA and HR‐pQCT were used to assess BMD and bone structure. Serum serotonin, sclerostin, dickkopf‐related protein 1 (DKK1), and BTM were evaluated. Z‐scores for the forearm, total hip, lumbar spine, forearm, and whole body were significantly increased (mean ± SD) between 4.94 ± 1.45 and 7.52 ± 1.99 in cases versus ?0.19 ± 1.19 to 0.58 ± 0.84 in controls. Tibial and radial cortical areas, thicknesses, and BMD were significantly higher in cases. In cases, BMD at the lumbar spine and forearm and cortical thickness were positively associated and trabecular area negatively associated with age (r = 0.49, 0.57, 0.74, and ?0.61, respectively, p < .05). Serotonin was lowest in cases (69.5 [29.9–110.4] ng/mL versus 119.4 [62.3–231.0] ng/mL, p < .001) and inversely associated with tibial cortical density (r = ?0.49, p < .05) and directly with osteocalcin (OC), bone‐specific alkaline phosphatase (B‐ALP), and procollagen type 1 amino‐terminal propeptide (PINP) (r = 0.52–0.65, p < .05) in controls only. OC and S‐CTX were lower and sclerostin higher in cases, whereas B‐ALP, PINP, tartrate‐resistant acid phosphatase (TRAP), and dickkopf‐related protein 1 (DKK1) were similar in cases and controls. In conclusion, increased bone mass in Lrp5‐HBM patients seems to be caused primarily by changes in trabecular and cortical bone mass and structure. The phenotype appeared to progress with age, but BTM did not suggest increased bone formation. © 2011 American Society for Bone and Mineral Research     

N‐cadherin Regulation of Bone Growth and Homeostasis Is Osteolineage Stage–Specific

N‐cadherin inhibits osteogenic cell differentiation and canonical Wnt/β‐catenin signaling in vitro. However, in vivo both conditional Cdh2 ablation and overexpression in osteoblasts lead to low bone mass. We tested the hypothesis that N‐cadherin has different effects on osteolineage cells depending upon their differentiation stage. Embryonic conditional osteolineage Cdh2 deletion in mice results in defective growth, low bone mass, and reduced osteoprogenitor number. These abnormalities are prevented by delaying Cdh2 ablation until 1 month of age, thus targeting only committed and mature osteoblasts, suggesting they are the consequence of N‐cadherin deficiency in osteoprogenitors. Indeed, diaphyseal trabecularization actually increases when Cdh2 is ablated postnatally. The sclerostin‐insensitive Lrp5^A214V mutant, associated with high bone mass, does not rescue the growth defect, but it overrides the low bone mass of embryonically Cdh2‐deleted mice, suggesting N‐cadherin interacts with Wnt signaling to control bone mass. Finally, bone accrual and β‐catenin accumulation after administration of an anti‐Dkk1 antibody are enhanced in N‐cadherin–deficient mice. Thus, although lack of N‐cadherin in embryonic and perinatal age is detrimental to bone growth and bone accrual, in adult mice loss of N‐cadherin in osteolineage cells favors bone formation. Hence, N‐cadherin inhibition may widen the therapeutic window of osteoanabolic agents. © 2017 American Society for Bone and Mineral Research.     

The Binding Between Sclerostin and LRP5 is Altered by DKK1 and by High-Bone Mass <Emphasis Type="Italic">LRP5</Emphasis> Mutations

Low-density lipoprotein receptor–related protein 5 (LRP5), a Wnt coreceptor, plays an important role in bone metabolism as loss-of-function and gain-of-function mutations in LRP5 result in the autosomal recessive osteoporosis-pseudoglioma syndrome and autosomal dominant high–bone mass (HBM) phenotypes, respectively. Prior studies suggested that the presence of HBM-associated LRP5 mutations results in decreased antagonism of LRP5-mediated Wnt signaling. In the present study, we investigated six different HBM-LRP5 mutations and confirm that neither Dickkopf1 (DKK1) nor sclerostin efficiently inhibits HBM-LRP5 signaling. In addition, when coexpressed, DKK1 and sclerostin do not inhibit HBM-LRP5 mutants better than either inhibitor by itself. Also, DKK1 and sclerostin do not simultaneously bind to wild-type LRP5, and DKK1 is able to displace sclerostin from previously formed sclerostin–LRP5 complexes. In conclusion, our results indicate that DKK1 and sclerostin are independent, and not synergistic, regulators of LRP5 signaling and that the function of each is impaired by HBM-LRP5 mutations.     

Fracture Healing in Collagen-Related Preclinical Models of Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a genetic bone dysplasia characterized by bone deformities and fractures caused by low bone mass and impaired bone quality. OI is a genetically heterogeneous disorder that most commonly arises from dominant mutations in genes encoding type I collagen (COL1A1 and COL1A2). In addition, OI is recessively inherited with the majority of cases resulting from mutations in prolyl-3-hydroxylation complex members, which includes cartilage-associated protein (CRTAP). OI patients are at an increased risk of fracture throughout their lifetimes. However, non-union or delayed healing has been reported in 24% of fractures and 52% of osteotomies. Additionally, refractures typically go unreported, making the frequency of refractures in OI patients unknown. Thus, there is an unmet need to better understand the mechanisms by which OI affects fracture healing. Using an open tibial fracture model, our study demonstrates delayed healing in both Col1a2 ^G610c/+ and Crtap ^−/− OI mouse models (dominant and recessive OI, respectively) that is associated with reduced callus size and predicted strength. Callus cartilage distribution and chondrocyte maturation were altered in OI, suggesting accelerated cartilage differentiation. Importantly, we determined that healed fractured tibia in female OI mice are biomechanically weaker when compared with the contralateral unfractured bone, suggesting that abnormal OI fracture healing OI may prime future refracture at the same location. We have previously shown upregulated TGF-β signaling in OI and we confirm this in the context of fracture healing. Interestingly, treatment of Crtap ^−/− mice with the anti-TGF-β antibody 1D11 resulted in further reduced callus size and predicted strength, highlighting the importance of investigating dose response in treatment strategies. These data provide valuable insight into the effect of the extracellular matrix (ECM) on fracture healing, a poorly understood mechanism, and support the need for prevention of primary fractures to decrease incidence of refracture and deformity in OI patients. © 2020 American Society for Bone and Mineral Research.     

Measurement of Plasma,Serum, and Platelet Serotonin in Individuals With High Bone Mass and Mutations in LRP5

It has recently been suggested that the low‐density lipoprotein receptor‐related protein 5 (LRP5) regulates bone mass by suppressing secretion of serotonin from duodenal enterochromaffin cells. In mice with targeted expression of a high bone mass–causing (HBM‐causing) LRP5 mutation and in humans with HBM LRP5 mutations, circulating serotonin levels have been reported to be lower than in controls whereas individuals with loss‐of‐function mutations in LRP5 have high blood serotonin. In contrast, others have reported that conditionally activating a knock‐in allele of an HBM‐causing LRP5 mutation in several tissues, or genetic deletion of LRP5 in mice has no effect on serum serotonin levels. To further explore the possible association between HBM‐causing LRP5 mutations and circulating serotonin, levels of the hormone were measured in the platelet poor plasma (PPP), serum, and platelet pellet (PP) of 16 affected individuals from 2 kindreds with HBM‐causing LRP5 mutations (G171V and N198S) and 16 age‐matched controls. When analyzed by HPLC, there were no differences in levels of serotonin in PPP and PP between affected individuals and age‐matched controls. Similarly, when analyzed by ELISA, there were no differences in PPP or PP between these two groups. By ELISA, serum levels of serotonin were higher in the affected individuals when compared to age‐matched controls. A subgroup analysis of only the G171V subjects (n = 14) demonstrated that there were no differences in PPP and PP serotonin between affected individuals and controls when analyzed by HPLC. PP serotonin was lower in the affected individuals when measured by ELISA but serum serotonin levels were not different. We conclude that there is no change in PPP serotonin in individuals with HBM‐causing mutations in LRP5. © 2014 American Society for Bone and Mineral Research.     

Patients with high‐bone‐mass phenotype owing to Lrp5‐T253I mutation have low plasma levels of serotonin

The Lrp5 gene is a major determinant of bone mass accrual. It has been demonstrated recently to achieve this function by hampering the synthesis of gut‐derived serotonin, which is a powerful inhibitor of bone formation. In this study we analyzed plasma serotonin levels in patients with a high‐bone‐mass (HBM) phenotype owing to gain‐of‐function mutation of Lrp5 (T253I). A total of 9 HBM patients were compared with 18 sex‐ and age‐matched controls. In HBM patients, the serotonin concentrations in platelet‐poor plasma were significantly lower than in the controls (mean ± SEM: 2.16 ± 0.28 ng/mL versus 3.51 ± 0.49 ng/mL, respectively, p < .05). Our data support the hypothesis that circulating serotonin levels mediate the increased bone mass resulting from gain‐of‐function mutations in Lrp5 in humans. © 2010 American Society for Bone and Mineral Research.