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.     

Bone density ligand, Sclerostin, directly interacts with LRP5 but not LRP5G171V to modulate Wnt activity.

We compared and contrasted the mechanism of action for the cysteine knot protein subfamily, Wise and Sost (Sclerostin). Our data suggest that functional interactions between Sost or Wise and LRP5/LRP6 have the potential to regulate bone deposition by modulating the Wnt pathway. INTRODUCTION: The human disease sclerosteosis exhibits an increase in bone mass thought to be caused by hyperactive osteoblasts. Sclerostin, SOST, the gene affected in this disease, has been postulated to exert its activity by functioning as a BMP antagonist. However, recent evidence indicates that SOST is highly related to Wise, which can also modulate the Wnt pathway by binding to LRP5 and LRP6. MATERIALS AND METHODS: For this study, we used cell culture to test the BMP and Wnt activity function of both Wise and Sost. In addition, we used Xenopus in vivo Wnt assays along with Xenopus in vitro Wnt assays to support our cell culture results. Epitope tagged cell supernatants containing either Sost or soluble mutant or wildtype LRP5/LRP6 were used for immunoprecipitation. Sost immunoprecipitation results were confirmed in vivo using cell culture. Finally, to support our in vitro data, we co-localized Sost, Wise, LRP5, and LRP6 in mouse long bone sections. Results: In this study, we report in vitro and in vivo evidence to show that Sost physically interacts with Lrp5 and Lrp6 and inhibits the canonical Wnt signaling pathway. Furthermore, using in vitro and in vivo assays, we showed that a variant of LRP5 (LRP5(G171V)) known to cause the human high bone mass (HBM) trait and a homologous change in LRP6 (LRP6(G158V)) abolished protein interactions with Sost. We used variants of Sost amino acids to further identify the contact points between Sost and LRP6. In Xenopus and mammalian cell culture assays, we showed that SOST is able to attenuate Wnt signaling and that this attenuation can be rescued by the addition of alpha-Sost antibodies or by the introduction of single amino acid substitution that alter its binding to LRP6. Sost differs from Wise in that it is unable to stimulate Wnt signaling. Using immunohistochemistry, we found that Sost and Wise are co-localized to osteoblasts, along with LRP5 and LRP6. CONCLUSIONS: Our data suggest that functional interactions between Sost or Wise and LRPs have the potential to regulate bone deposition by modulating Wnt signaling.     

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.     

High bone mass in mice expressing a mutant LRP5 gene.

A unique mutation in LRP5 is associated with high bone mass in man. Transgenic mice expressing this LRP5 mutation have a similar phenotype with high bone mass and enhanced strength. These results underscore the importance of LRP5 in skeletal regulation and suggest targets for therapies for bone disease. A mutation (G171V) in the low-density lipoprotein receptor related protein 5 (LRP5) has been associated with high bone mass (HBM) in two independent human kindreds. To validate the role of the mutation, several lines of transgenic mice were created expressing either the human LRP5 G171V substitution or the wildtype LRP5 gene in bone. Volumetric bone mineral density (vBMD) analysis by pQCT showed dramatic increases in both total vBMD (30-55%) and trabecular vBMD (103-250%) of the distal femoral metaphysis and increased cortical size of the femoral diaphysis in mutant G171V transgenics at 5, 9, 17, 26, and 52 weeks of age (p < 0.01 for all). In addition, high-resolution microcomputed tomography (microCT) analysis of the distal femorae and lumbar vertebrae revealed an increase (110-232%) in trabecular bone volume fraction caused by both increased trabecular number (41-74%) and increased trabecular thickness (34-46%; p < 0.01 for all) in the mutant G171V mice. The increased bone mass was associated with significant increases in vertebral compressive strength (80-140%) and the increased cortical size with significant increases in femoral bending strength (50-130%). There were no differences in osteoclast number at 17 weeks of age. However, compared with littermate controls, the mutant G171V transgenic mice showed an increase in actively mineralizing bone surface, enhanced alkaline phosphatase staining in osteoblasts, and a significant reduction in the number of TUNEL-positive osteoblasts and osteocytes. These results suggest that the increased bone mineral density in mutant G171V mice was caused by increased numbers of active osteoblasts, which could in part be because of their increased functional lifespan. While slight bone anabolic activity was observed from overexpression of the wildtype LRP5 gene, it is clear that the G171V mutation, rather than overexpression of the receptor itself, is primarily responsible for the dramatic HBM bone effects. Together, these findings establish the importance of this novel and unexpected role of a lipoprotein receptor in regulating bone mass and afford a new model to explore LRP5 and its recent association with Wnt signaling in bone biology.     

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.     

Bone biomechanical properties in LRP5 mutant mice

The mutation responsible for the high bone mass (HBM) phenotype has been postulated to act through the adaptive response of bone to mechanical load resulting in denser and stronger skeletons in humans and animals. The bone phenotype of members of a HBM family is characterized by normally shaped bones that are exceptionally dense, particularly at load bearing sites [Cancer Res. 59 (1999) 1572]. The high bone mass (HBM) mutation was identified as a glycine to valine substitution at amino acid residue 171 in the gene coding for low-density lipoprotein receptor-related protein 5 (LRP5) [Bone Miner. Res. 16(4) (2001) 758]. Thus, efforts have focused on the examination of the role of LRP5 and the G171V mutation in bone mechanotransduction responses [J. Bone Miner. Res 18 (2002) 960]. Transgenic mice expressing the human G171V mutation have been shown to have skeletal phenotypes remarkably similar to those seen in affected individuals. In this study, we have identified differences in biomechanical (structural and apparent material) properties, bone mass/ash, and bone stiffness of cortical and cancellous bone driven by the G171V mutation in LRP5. As in humans, the LRP5 G171V plays an important role in regulating bone structural phenotypes in mice. These bone phenotypes include greater structural and apparent material properties in HBM HET as compared to non-transgenic littermates (NTG) mice. Body size and weight in HBM HET were similar to that in NTG control mice. However, the LRP5 G171V mutation in HET mice results in a skeleton that has greater structural (femoral shaft, femoral neck, tibiae, vertebral body) and apparent material (vertebral body) strength, percent bone ash weight (ulnae), and tibial stiffness. Despite similar body weight to NTG mice, the denser and stiffer bones in G171V mice may represent greater bone formation sensitivity to normal mechanical stimuli resulting in an overadaptation of skeleton to weight-related forces.     

Targeting the LRP5 Pathway Improves Bone Properties in a Mouse Model of Osteogenesis Imperfecta

The cell surface receptor low‐density lipoprotein receptor‐related protein 5 (LRP5) is a key regulator of bone mass and bone strength. Heterozygous missense mutations in LRP5 cause autosomal dominant high bone mass (HBM) in humans by reducing binding to LRP5 by endogenous inhibitors, such as sclerostin (SOST). Mice heterozygous for a knockin allele (Lrp5^p.A214V) that is orthologous to a human HBM‐causing mutation have increased bone mass and strength. Osteogenesis imperfecta (OI) is a skeletal fragility disorder predominantly caused by mutations that affect type I collagen. We tested whether the LRP5 pathway can be used to improve bone properties in animal models of OI. First, we mated Lrp5^+/p.A214V mice to Col1a2^+/p.G610C mice, which model human type IV OI. We found that Col1a2^+/p.G610C;Lrp5^+/p.A214V offspring had significantly increased bone mass and strength compared to Col1a2^+/p.G610C;Lrp5^+/+ littermates. The improved bone properties were not a result of altered mRNA expression of type I collagen or its chaperones, nor were they due to changes in mutant type I collagen secretion. Second, we treated Col1a2^+/p.G610C mice with a monoclonal antibody that inhibits sclerostin activity (Scl‐Ab). We found that antibody‐treated mice had significantly increased bone mass and strength compared to vehicle‐treated littermates. These findings indicate increasing bone formation, even without altering bone collagen composition, may benefit patients with OI. © 2014 American Society for Bone and Mineral Research.     

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).     

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.     

Differential involvement of Wnt signaling in Bmp regulation of cancellous versus periosteal bone growth

Bone morphogenetic proteins(Bmp) are well-known to induce bone formation following chondrogenesis,but the direct role of Bmp signaling in the osteoblast lineage is not completely understood. We have recently shown that deletion of the receptor Bmpr1 a in the osteoblast lineage with Dmp1-Cre reduces osteoblast activity in general but stimulates proliferation of preosteoblasts specifically in the cancellous bone region,resulting in diminished periosteal bone growth juxtaposed with excessive cancellous bone formation.Because expression of sclerostin(SOST), a secreted Wnt antagonist, is notably reduced in the Bmpr1 adeficient osteocytes, we have genetically tested the hypothesis that increased Wnt signaling might mediate the increase in cancellous bone formation in response to Bmpr1 a deletion. Forced expression of human SOST from a Dmp1 promoter fragment partially rescues preosteoblast hyperproliferation and cancellous bone overgrowth in the Bmpr1 a mutant mice, demonstrating functional interaction between Bmp and Wnt signaling in the cancellous bone compartment. To test whether increased Wnt signaling can compensate for the defect in periosteal growth caused by Bmpr1 a deletion, we have generated compound mutants harboring a hyperactive mutation(A214 V) in the Wnt receptor Lrp5. However, the mutant Lrp5 does not restore periosteal bone growth in the Bmpr1 a-deficient mice. Thus, Bmp signaling restricts cancellous bone accrual partly through induction of SOST that limits preosteoblast proliferation, but promotes periosteal bone growth apparently independently of Wnt activation.     

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.     

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.     

Intrinsic material properties of cortical bone

The G171V mutation (high bone mass, HBM) is autosomal dominant and is responsible for high bone mass in humans. Transgenic HBM mice in which the human LRP5 G171V gene is inserted also show a similar phenotype with greater bone mass and biomechanical performance than wild-type mice, as determined by whole bone testing. Whole bone mechanics, however, depend jointly on bone mass, architecture, and intrinsic bone tissue mechanical properties. To determine whether the HBM mutation affects tissue-level biomechanical performance, we performed nano-indentation testing of unembedded cortical bone from HBM mice and their nontransgenic (NTG) littermates. Femora from 17-week-old mice (female, 8 mice/genotype) were subjected to nano-indentation using a Triboscope (Hysitron, Minneapolis, MN, USA). For each femoral specimen, approximately 10 indentations were made on the midshaft anterior surface with a target force of either 3 or 9 mN at a constant loading rate of 400 mN/s. The load–displacement data from each test were used to calculate indentation modulus and hardness for bone tissue. The intrinsic material property that reflected the bone modulus was greater (48%) in the HBM as compared to the NTG mice. Our results of intrinsic properties are consistent with the published structural and material properties of the midshaft femur in HBM and NTG mice. The greater intrinsic modulus in HBM reflects greater bone mineral content as compared to NTG (wild-type, WT) mice. This study suggests that the greater intrinsic property of cortical bone is derived from the greater bone mineral content and BMD, resulting in greater bone strength in HBM as compared to NTG (WT) mice.     

Decreased BMD and limb deformities in mice carrying mutations in both Lrp5 and Lrp6.

Humans and mice lacking Lrp5 have low BMD. To evaluate whether Lrp5 and Lrp6 interact genetically to control bone or skeletal development, we created mice carrying mutations in both Lrp5 and the related gene Lrp6. We found that compound mutants had dose-dependent deficits in BMD and limb formation, suggesting functional redundancy between these two genes in bone and limb development. INTRODUCTION: Lrp5 and Lrp6 are closely related members of the low density lipoprotein receptor family and are co-receptors for Wnt ligands. While Lrp5 mutations are associated with low BMD in humans and mice, the role of Lrp6 in bone formation has not been analyzed. MATERIALS AND METHODS: To address whether Lrp5 and Lrp6 play complimentary roles in bone and skeletal development, we created mice with mutations in both genes. We inspected limbs of mice from the different genotypic classes of compound mutants to identify abnormalities. DXA and muCT were used to evaluate the effect of mutations in Lrp5 and Lrp6 on BMD and microarchitecture. RESULTS: Mice heterozygous for mutations in Lrp6 and either heterozygous or homozygous for a mutation in Lrp5 (Lrp6(+/-);Lrp5(+/-) or Lrp6(+/-);Lrp5(-/-)) display limb defects with incomplete penetrance and variable expression. DXA analysis showed that BMD decreased as mice progressively were more deficient in Lrp5 and Lrp6. Lrp6(+/-);Lrp5(-/-) mice were more severely affected than Lrp6(+/+);Lrp5(-/-) mice, whereas Lrp6(+/-);Lrp5(+/-) mice had statistically higher BMD than Lrp6(+/+);Lrp5(-/-) mice and lower BMD compared with wildtype mice and mice heterozygous for either mutation alone. CONCLUSIONS: Lrp6 and Lrp5 genetically interact in limb development in mice. Furthermore, heterozygosity for an inactivating mutation in Lrp6 further reduces BMD in both male and female mice lacking Lrp5.     

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.     

A Comprehensive Overview of Skeletal Phenotypes Associated with Alterations in Wnt/β-catenin Signaling in Humans and Mice

The Wnt signaling pathway plays key roles in differentiation and development and alterations in this signaling pathway are causally associated with numerous human diseases. While several laboratories were examining roles for Wnt signaling in skeletal development during the 1990s, interest in the pathway rose exponentially when three key papers were published in 2001–2002. One report found that loss of the Wnt co-receptor, Low-density lipoprotein related protein-5 (LRP5), was the underlying genetic cause of the syndrome Osteoporosis pseudoglioma (OPPG). OPPG is characterized by early-onset osteoporosis causing increased susceptibility to debilitating fractures. Shortly thereafter, two groups reported that individuals carrying a specific point mutation in LRP5 (G171V) develop high-bone mass. Subsequent to this, the causative mechanisms for these observations heightened the need to understand the mechanisms by which Wnt signaling controlled bone development and homeostasis and encouraged significant investment from biotechnology and pharmaceutical companies to develop methods to activate Wnt signaling to increase bone mass to treat osteoporosis and other bone disease. In this review, we will briefly summarize the cellular mechanisms underlying Wnt signaling and discuss the observations related to OPPG and the high-bone mass disorders that heightened the appreciation of the role of Wnt signaling in normal bone development and homeostasis. We will then present a comprehensive overview of the core components of the pathway with an emphasis on the phenotypes associated with mice carrying genetically engineered mutations in these genes and clinical observations that further link alterations in the pathway to changes in human bone.     

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     

Where Wnts Went: The Exploding Field of Lrp5 and Lrp6 Signaling in Bone

Wnt signaling has emerged as a central regulator of skeletal modeling and remodeling. Loss‐ or gain‐of‐function mutations in two Wnt co‐receptors, Lrp5 and (more recently) Lrp6, have drawn attention to the importance of the Wnt pathway in bone biology. This review summarizes our current understanding of how the Wnt pathway operates on bone and the implications this has for skeletal physiology and drug discovery. Over the past 9 yr, rapid advances have been made in our understanding of the cellular targets for Wnt signaling and of the important regulatory molecules in this metabolic pathway. Both canonical and noncanonical signaling pathways seem to be important for mediating the effects of Wnt in bone. A rapidly expanding catalog of genetically engineered mice has been used to establish the importance of downstream effector molecules (such as β‐catenin) in the Wnt pathway, as well as the critical role of endogenous inhibitors of Wnt signaling (such as Dkk1 and sclerostin) in bone metabolism. Indeed, regulation of sclerostin in osteocytes is emerging as an important final pathway for regulating bone anabolism in response to diverse trophic stimuli, from mechnotransduction to the anabolic actions of PTH. From the outset, it had been assumed that the effects of Wnt signaling in bone were caused by direct actions in osteoblast precursors, osteoblasts, and osteocytes. However, startling recent findings have challenged this view and suggest that a key target, at least in mice, is the duodenal enterochromaffin cell. There, Wnt signaling transduced by Lrp5 regulates serotonin synthesis, which acts in an endocrine fashion to regulate bone cell metabolism. It will take time to reconcile this new information with the considerable body of information we already have regarding the actions of Wnt in bone. The Wnt pathway has rapidly emerged as a therapeutic target for drug discovery. Neutralizing antibodies and small‐molecule inhibitors of endogenous Wnt inhibitors have shown early promise as bone anabolic agents. However, given the central role of the Wnt pathway in regulating growth and development in extraskeletal tissues, as well as our still rudimentary understanding of how this signaling cascade actually affects bone metabolism, considerable work will be needed to ensure the safety of these new therapies.