Cell autonomous requirement of connexin 43 for osteocyte survival: consequences for endocortical resorption and periosteal bone formation

Connexin 43 (Cx43) mediates osteocyte communication with other cells and with the extracellular milieu and regulates osteoblastic cell signaling and gene expression. We now report that mice lacking Cx43 in osteoblasts/osteocytes or only in osteocytes (Cx43(ΔOt) mice) exhibit increased osteocyte apoptosis, endocortical resorption, and periosteal bone formation, resulting in higher marrow cavity and total tissue areas measured at the femoral mid-diaphysis. Blockade of resorption reversed the increased marrow cavity but not total tissue area, demonstrating that endocortical resorption and periosteal apposition are independently regulated. Anatomical mapping of apoptotic osteocytes, osteocytic protein expression, and resorption and formation suggests that Cx43 controls osteoclast and osteoblast activity by regulating osteoprotegerin and sclerostin levels, respectively, in osteocytes located in specific areas of the cortex. Whereas empty lacunae and living osteocytes lacking osteoprotegerin were distributed throughout cortical bone in Cx43(ΔOt) mice, apoptotic osteocytes were preferentially located in areas containing osteoclasts, suggesting that osteoclast recruitment requires active signaling from dying osteocytes. Furthermore, Cx43 deletion in cultured osteocytic cells resulted in increased apoptosis and decreased osteoprotegerin expression. Thus, Cx43 is essential in a cell-autonomous fashion in vivo and in vitro for osteocyte survival and for controlling the expression of osteocytic genes that affect osteoclast and osteoblast function.     

Changes in bone sclerostin levels in mice after ovariectomy vary independently of changes in serum sclerostin levels

We examined the effects that ovariectomy had on sclerostin mRNA and protein levels in the bones of 8‐week‐old mice that were either sham‐operated (SHAM) or ovariectomized (OVX) and then euthanized 3 or 6 weeks later. In this model, bone loss occurred between 3 and 5 weeks postsurgery. In calvaria, ovariectomy significantly decreased sclerostin mRNA levels at 6 weeks postsurgery (by 52%) but had no significant effect at 3 weeks. In contrast, sclerostin mRNA levels were significantly lower in OVX femurs at 3 weeks postsurgery (by 53%) but equal to that of SHAM at 6 weeks. The effects of ovariectomy on sclerostin were not a global response of osteocytes because they were not mimicked by changes in the mRNA levels for two other relatively osteocyte‐specific genes: DMP‐1 and FGF‐23. Sclerostin protein decreased by 83% and 60%, at 3 and 6 weeks postsurgery in calvaria, respectively, and by 38% in lumbar vertebrae at 6 weeks. We also detected decreases in sclerostin by immunohistochemistry in cortical osteocytes of the humerus at 3 weeks postsurgery. However, there were no significant effects of ovariectomy on sclerostin protein in femurs or on serum sclerostin at 3 and 6 weeks postsurgery. These results demonstrate that ovariectomy has variable effects on sclerostin mRNA and protein in mice, which are dependent on the bones examined and the time after surgery. Given the discrepancy between the effects of ovariectomy on serum sclerostin levels and sclerostin mRNA and protein levels in various bones, these results argue that, at least in mice, serum sclerostin levels may not accurately reflect changes in the local production of sclerostin in bones. Additional studies are needed to evaluate whether this is also the case in humans. © 2013 American Society for Bone and Mineral Research.     

Patients with sclerosteosis and disease carriers: Human models of the effect of sclerostin on bone turnover

Sclerosteosis is a rare bone sclerosing dysplasia, caused by loss‐of‐function mutations in the SOST gene, encoding sclerostin, a negative regulator of bone formation. The purpose of this study was to determine how the lack of sclerostin affects bone turnover in patients with sclerosteosis and to assess whether sclerostin synthesis is decreased in carriers of the SOST mutation and, if so, to what extent this would affect their phenotype and bone formation. We measured sclerostin, procollagen type 1 amino‐terminal propeptide (P1NP), and cross‐linked C‐telopeptide (CTX) in serum of 19 patients with sclerosteosis, 26 heterozygous carriers of the C69T SOST mutation, and 77 healthy controls. Chips of compact bone discarded during routine surgery were also examined from 6 patients and 4 controls. Sclerostin was undetectable in serum of patients but was measurable in all carriers (mean 15.5 pg/mL; 95% confidence interval [CI] 13.7 to 17.2 pg/mL), in whom it was significantly lower than in healthy controls (mean 40.0 pg/mL; 95% CI 36.9 to 42.7 pg/mL; p < 0.001). P1NP levels were highest in patients (mean 153.7 ng/mL; 95% CI 100.5 to 206.9 ng/mL; p = 0.01 versus carriers, p = 0.002 versus controls), but carriers also had significantly higher P1NP levels (mean 58.3 ng/mL; 95% CI 47.0 to 69.6 ng/mL) than controls (mean 37.8 ng/mL; 95% CI 34.9 to 42.0 ng/mL; p = 0.006). In patients and carriers, P1NP levels declined with age, reaching a plateau after the age of 20 years. Serum sclerostin and P1NP were negatively correlated in carriers and age‐ and gender‐matched controls (r = 0.40, p = 0.008). Mean CTX levels were well within the normal range and did not differ between patients and disease carriers after adjusting for age (p = 0.22). Our results provide in vivo evidence of increased bone formation caused by the absence or decreased synthesis of sclerostin in humans. They also suggest that inhibition of sclerostin can be titrated because the decreased sclerostin levels in disease carriers did not lead to any of the symptoms or complications of the disease but had a positive effect on bone mass. Further studies are needed to clarify the role of sclerostin on bone resorption. © 2011 American Society for Bone and Mineral Research     

A Novel Osteogenic Cell Line That Differentiates Into GFP-Tagged Osteocytes and Forms Mineral With a Bone-Like Lacunocanalicular Structure

Osteocytes, the most abundant cells in bone, were once thought to be inactive, but are now known to have multifunctional roles in bone, including in mechanotransduction, regulation of osteoblast and osteoclast function and phosphate homeostasis. Because osteocytes are embedded in a mineralized matrix and are challenging to study, there is a need for new tools and cell models to understand their biology. We have generated two clonal osteogenic cell lines, OmGFP66 and OmGFP10, by immortalization of primary bone cells from mice expressing a membrane-targeted GFP driven by the Dmp1-promoter. One of these clones, OmGFP66, has unique properties compared with previous osteogenic and osteocyte cell models and forms 3-dimensional mineralized bone-like structures, containing highly dendritic GFP-positive osteocytes, embedded in clearly defined lacunae. Confocal and electron microscopy showed that structurally and morphologically, these bone-like structures resemble bone in vivo, even mimicking the lacunocanalicular ultrastructure and 3D spacing of in vivo osteocytes. In osteogenic conditions, OmGFP66 cells express alkaline phosphatase (ALP), produce a mineralized type I collagen matrix, and constitutively express the early osteocyte marker, E11/gp38. With differentiation they express osteocyte markers, Dmp1, Phex, Mepe, Fgf23, and the mature osteocyte marker, Sost. They also express RankL, Opg, and Hif1α, and show expected osteocyte responses to PTH, including downregulation of Sost, Dmp1, and Opg and upregulation of RankL and E11/gp38. Live cell imaging revealed the dynamic process by which OmGFP66 bone-like structures form, the motile properties of embedding osteocytes and the integration of osteocyte differentiation with mineralization. The OmGFP10 clone showed an osteocyte gene expression profile similar to OmGFP66, but formed less organized bone nodule-like mineral, similar to other osteogenic cell models. Not only do these cell lines provide useful new tools for mechanistic and dynamic studies of osteocyte differentiation, function, and biomineralization, but OmGFP66 cells have the unique property of modeling osteocytes in their natural bone microenvironment. © 2019 American Society for Bone and Mineral Research     

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     

Sclerostin antibody treatment improves bone mass,bone strength,and bone defect regeneration in rats with type 2 diabetes mellitus

Type 2 diabetes mellitus results in increased risk of fracture and delayed fracture healing. ZDF fa/fa rats are an established model of type 2 diabetes mellitus with low bone mass and delayed bone healing. We tested whether a sclerostin‐neutralizing antibody (Scl‐AbVI) would reverse the skeletal deficits of diabetic ZDF rats. Femoral defects of 3 mm were created in 11‐week‐old diabetic ZDF fa/fa and nondiabetic ZDF +/+ rats and stabilized by an internal plate. Saline or 25 mg/kg Scl‐AbVI was administered subcutaneously (s.c.) twice weekly for 12 weeks (n = 9–10/group). Bone mass and strength were assessed using pQCT, micro–computed tomography (µCT), and biomechanical testing. Bone histomorphometry was used to assess bone formation, and the filling of the bone defect was analyzed by µCT. Diabetic rats displayed lower spinal and femoral bone mass compared to nondiabetic rats, and Scl‐AbVI treatment significantly enhanced bone mass of the femur and the spine of diabetic rats (p < 0.0001). Scl‐AbVI also reversed the deficit in bone strength in the diabetic rats, with 65% and 89% increases in maximum load at the femoral shaft and neck, respectively (p < 0.0001). The lower bone mass in diabetic rats was associated with a 65% decrease in vertebral bone formation rate, which Scl‐AbVI increased by sixfold, consistent with a pronounced anabolic effect. Nondiabetic rats filled 57% of the femoral defect, whereas diabetic rats filled only 21% (p < 0.05). Scl‐AbVI treatment increased defect regeneration by 47% and 74%, respectively (p < 0.05). Sclerostin antibody treatment reverses the adverse effects of type 2 diabetes mellitus on bone mass and strength, and improves bone defect regeneration in rats. © 2013 American Society for Bone and Mineral Research.     

Microtubule assembly affects bone mass by regulating both osteoblast and osteoclast functions: Stathmin deficiency produces an osteopenic phenotype in mice

Cytoskeleton microtubules regulate various cell signaling pathways that are involved in bone cell function. We recently reported that inhibition of microtubule assembly by microtubule‐targeting drugs stimulates osteoblast differentiation and bone formation. To further elucidate the role of microtubules in bone homeostasis, we characterized the skeletal phenotype of mice null for stathmin, an endogenous protein that inhibits microtubule assembly. In vivo micro–computed tomography (µCT) and histology revealed that stathmin deficiency results in a significant reduction of bone mass in adult mice concurrent with decreased osteoblast and increased osteoclast numbers in bone tissues. Phenotypic analyses of primary calvarial cells and bone marrow cells showed that stathmin deficiency inhibited osteoblast differentiation and induced osteoclast formation. In vitro overexpression studies showed that increased stathmin levels enhanced osteogenic differentiation of preosteoblast MC3T3‐E1 cells and mouse bone marrow–derived cells and attenuated osteoclast formation from osteoclast precursor Raw264.7 cells and bone marrow cells. Results of immunofluorescent studies indicated that overexpression of stathmin disrupted radial microtubule filaments, whereas deficiency of stathmin stabilized the microtubule network structure in these bone cells. In addition, microtubule‐targeting drugs that inhibit microtubule assembly and induce osteoblast differentiation lost these effects in the absence of stathmin. Collectively, these results suggest that stathmin, which alters microtubule dynamics, plays an essential role in maintenance of postnatal bone mass by regulating both osteoblast and osteoclast functions in bone. © 2011 American Society for Bone and Mineral Research     

Correlates of bone microarchitectural parameters and serum sclerostin levels in men: The STRAMBO study

Sclerostin is predominantly expressed by osteocytes. Serum sclerostin levels are positively correlated with areal bone mineral density (aBMD) measured by dual‐energy X‐ray absorptiometry (DXA) and bone microarchitecture assessed by high‐resolution peripheral quantitative computed tomography (HR‐pQCT) in small studies. We assessed the relation of serum sclerostin levels with aBMD and microarchitectural parameters based on HR‐pQCT in 1134 men aged 20 to 87 years using multivariable models adjusted for confounders (age, body size, lifestyle, comorbidities, hormones regulating bone metabolism, muscle mass and strength). The apparent age‐related increase in serum sclerostin levels was faster before the age of 63 years than afterward (0.43 SD versus 0.20 SD per decade). In 446 men aged ≤63 years, aBMD (spine, hip, whole body), trabecular volumetric BMD (Tb.vBMD), and trabecular number (Tb.N) at the distal radius and tibia were higher in the highest sclerostin quartile versus the three lower quartiles combined. After adjustment for aBMD, men in the highest sclerostin quartile had higher Tb.vBMD (mainly in the central compartment) and Tb.N at both skeletal sites (p < 0.05 to 0.001). In 688 men aged >63 years, aBMD was positively associated with serum sclerostin levels at all skeletal sites. Cortical vBMD (Ct.vBMD) and cortical thickness (Ct.Th) were lower in the first sclerostin quartile versus the three higher quartiles combined. Tb.vBMD increased across the sclerostin quartiles, and was associated with lower Tb.N and more heterogeneous trabecular distribution (higher Tb.Sp.SD) in men in the lowest sclerostin quartile. After adjustment for aBMD, men in the lowest sclerostin quartile had lower Tb.vBMD and Tb.N, but higher Tb.Sp.SD (p < 0.05 to 0.001) at both the skeletal sites. In conclusion, serum sclerostin levels in men are strongly positively associated with better bone microarchitectural parameters, mainly trabecular architecture, regardless of the potential confounders.     

PTHrP‐Derived Peptides Restore Bone Mass and Strength in Diabetic Mice: Additive Effect of Mechanical Loading

There is an unmet need to understand the mechanisms underlying skeletal deterioration in diabetes mellitus (DM) and to develop therapeutic approaches to treat bone fragility in diabetic patients. We demonstrate herein that mice with type 1 DM induced by streptozotocin exhibited low bone mass, inferior mechanical and material properties, increased bone resorption, decreased bone formation, increased apoptosis of osteocytes, and increased expression of the osteocyte‐derived bone formation inhibitor Sost/sclerostin. Further, short treatment of diabetic mice with parathyroid hormone related protein (PTHrP)‐derived peptides corrected these changes to levels undistinguishable from non‐diabetic mice. In addition, diabetic mice exhibited reduced bone formation in response to mechanical stimulation, which was corrected by treatment with the PTHrP peptides, and higher prevalence of apoptotic osteocytes, which was reduced by loading or by the PTHrP peptides alone and reversed by a combination of loading and PTHrP peptide treatment. In vitro experiments demonstrated that the PTHrP peptides or mechanical stimulation by fluid flow activated the survival kinases ERKs and induced nuclear translocation of the canonical Wnt signaling mediator β‐catenin, and prevented the increase in osteocytic cell apoptosis induced by high glucose. Thus, PTHrP‐derived peptides cross‐talk with mechanical signaling pathways to reverse skeletal deterioration induced by DM in mice. These findings suggest a crucial role of osteocytes in the harmful effects of diabetes on bone and raise the possibility of targeting these cells as a novel approach to treat skeletal deterioration in diabetes. Moreover, our study suggests the potential therapeutic efficacy of combined pharmacological and mechanical stimuli to promote bone accrual and maintenance in diabetic subjects. © 2016 American Society for Bone and Mineral Research.     

Sclerostin Regulates Release of Bone Mineral by Osteocytes by Induction of Carbonic Anhydrase 2

The osteocyte product sclerostin is emerging as an important paracrine regulator of bone mass. It has recently been shown that osteocyte production of receptor activator of NF‐κB ligand (RANKL) is important in osteoclastic bone resorption, and we reported that exogenous treatment of osteocytes with sclerostin can increase RANKL‐mediated osteoclast activity. There is good evidence that osteocytes can themselves liberate mineral from bone in a process known as osteocytic osteolysis. In the current study, we investigated sclerostin‐stimulated mineral dissolution by human primary osteocyte‐like cells (hOCy) and mouse MLO‐Y4 cells. We found that sclerostin upregulated osteocyte expression of carbonic anhydrase 2 (CA2/Car2), cathepsin K (CTSK/Ctsk), and tartrate‐resistant acid phosphatase (ACP5/Acp5). Because acidification of the extracellular matrix is a critical step in the release of mineral from bone, we further examined the regulation by sclerostin of CA2. Sclerostin stimulated CA2 mRNA and protein expression in hOCy and in MLO‐Y4 cells. Sclerostin induced a decrease in intracellular pH (pHi) in both cell types as well as a decrease in extracellular pH (pHo) and the release of calcium ions from mineralized substrate. These effects were reversed in the co‐presence of the carbonic anhydrase inhibitor, acetozolamide. Car2‐siRNA knockdown in MLO‐Y4 cells significantly inhibited the ability of sclerostin to both reduce the pHo and release calcium from a mineralized substrate. Knockdown in MLO‐Y4 cells of each of the putative sclerostin receptors, Lrp4, Lrp5 and Lrp6, using siRNA, inhibited the sclerostin induction of Car2, Catk and Acp5 mRNA, as well as pHo and calcium release. Consistent with this activity of sclerostin resulting in osteocytic osteolysis, human trabecular bone samples treated ex vivo with recombinant human sclerostin for 7 days exhibited an increased osteocyte lacunar area, an effect that was reversed by the co‐addition of acetozolamide. These findings suggest a new role for sclerostin in the regulation of perilacunar mineral by osteocytes. © 2013 American Society for Bone and Mineral Research.     

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.     

Physiological Functions of Osteoblast Lineage and T Cell–Derived RANKL in Bone Homeostasis

The cytokine RANKL is essential for osteoclast development in bone. The cellular sources of RANKL for support of osteoclast generation under various pathophysiological conditions have remained unclear, however. Here we show that inactivation of Rankl specifically in osteoblast lineage cells of mice with the use of an Osterix‐Cre transgene results in typical osteopetrosis in the trabecular compartment of the tibia, with the phenotype being progressively less marked in the femur and vertebrae. In contrast to its effects on trabecular bone, RANKL deficiency in osteoblast lineage resulted in thinning of the femoral cortex in association with suppression of bone formation during the modeling process. Ablation of RANKL specifically in T cells resulted in a moderate but significant increase in tibial trabecular bone. Mice with RANKL deficiency in osteoblast lineage were protected from bone loss induced by ovariectomy as well as from joint destruction associated with arthritis, whereas loss of RANKL in T cells did not confer such protection. Finally, inducible deletion of Rankl selectively in the osteoblasts from 6 to 12 weeks of age resulted in an increase in bone mass in association with reduced bone resorption and formation. Our results thus suggest that RANKL produced by osteoblasts contributes to osteoclast development in vivo. © 2014 American Society for Bone and Mineral Research.     

Regulation of circulating sclerostin levels by sex steroids in women and in men

Sex steroids are important regulators of bone turnover, but the mechanisms of their effects on bone remain unclear. Sclerostin is an inhibitor of Wnt signaling, and circulating estrogen (E) levels are inversely associated with sclerostin levels in postmenopausal women. To directly test for sex steroid regulation of sclerostin levels, we examined effects of E treatment of postmenopausal women or selective withdrawal of E versus testosterone (T) in elderly men on circulating sclerostin levels. E treatment of postmenopausal women (n = 17) for 4 weeks led to a 27% decrease in serum sclerostin levels [versus +1% in controls (n = 18), p < .001]. Similarly, in 59 elderly men, we eliminated endogenous E and T production and studied them under conditions of physiologic T and E replacement, and then following withdrawal of T or E, we found that E, but not T, prevented increases in sclerostin levels following induction of sex steroid deficiency. In both sexes, changes in sclerostin levels correlated with changes in bone‐resorption, but not bone‐formation, markers (r = 0.62, p < .001, and r = 0.33, p = .009, for correlations with changes in serum C‐terminal telopeptide of type 1 collagen in the women and men, respectively). Our studies thus establish that in humans, circulating sclerostin levels are reduced by E but not by T. Moreover, consistent with recent data indicating important effects of Wnts on osteoclastic cells, our findings suggest that in humans, changes in sclerostin production may contribute to effects of E on bone resorption. © 2011 American Society for Bone and Mineral Research.     

Osteocyte RANKL: new insights into the control of bone remodeling

The idea that osteoblasts, or their progenitors, support osteoclast formation by expressing the cytokine receptor activator of NFkB ligand (RANKL) is a widely held tenet of skeletal biology. Two recent studies provide evidence that osteocytes, and not osteoblasts or their progenitors, are the major source of RANKL driving osteoclast formation in cancellous bone. The goal of this review is to highlight the results of these new studies and discuss their implications for our understanding of bone remodeling.     

Sclerostin Expression in Bile Ducts of Patients With Chronic Cholestasis May Influence the Bone Disease in Primary Biliary Cirrhosis

Sclerostin is involved in the regulation of osteoblastogenesis and little is known about its role in the development of bone disease in primary biliary cirrhosis (PBC), characterized by low bone formation. Therefore, we have assessed the circulating levels and the liver expression of sclerostin in this cholestatic disease. Serum sclerostin levels were measured in 79 women with PBC (mean age 60.6 ± 1.2 years) and in 80 control women. Lumbar and femoral bone mineral density (BMD), as well as parameters of mineral metabolism and bone remodeling, were measured. Moreover, sclerostin gene (SOST) expression in the liver was assessed by real‐time PCR in samples of liver tissue taken by biopsy in 11 PBC patients and in 5 normal liver specimens. Presence and distribution of sclerostin was evaluated in liver slices from 11 patients by immunohistochemistry. The severity of histologic lesions was assessed semiquantitatively in the same liver samples. PBC patients had higher sclerostin levels than controls (75.6 ± 3.9 versus 31.7 ± 1.6 pmol/L, p < 0.001). Serum sclerostin correlated inversely with markers of bone formation and resorption. Sclerostin mRNA in the liver was overexpressed compared with control samples (2.7‐fold versus healthy liver). Sclerostin was detected by immunohistochemistry in 7 of the 11 liver samples, mainly located in the bile ducts. Liver sclerostin was associated with the severity of cholangitis (p = 0.02) and indirectly with the degree of lobular inflammation (p = 0.03). Sclerostin mRNA expression was higher in samples that tested positive by immunohistochemistry and particularly in those with lobular granuloma (p = 0.02). The increased expression of sclerostin in the liver and the association with histologic cholangitis may explain the high serum levels of this protein in patients with PBC, thus suggesting that sclerostin may influence the decreased bone formation in this cholestatic disease. © 2016 American Society for Bone and Mineral Research.     

Determinants of serum sclerostin in healthy pre‐ and postmenopausal women

Sclerostin is a secreted Wnt antagonist produced almost exclusively by osteocytes that regulates bone mass. However, there is currently limited information on the determinants of sclerostin in a large population‐based study. The main objectives of the present study were to: (1) establish reference normative interval values for serum sclerostin in randomly selected healthy premenopausal women; (2) study the changes in serum sclerostin in relation to age in premenopausal and postmenopausal women and the factors that may influence bone turnover; and (3) determine the effect of menopausal status on serum sclerostin. A total of 1803 women were studied (including [n = 1235] premenopausal, and [n = 568] postmenopausal women, respectively, aged 20 to 79 years). A total of 443 healthy premenopausal women (aged 35 to 45 years) were used to establish reference normative intervals for serum sclerostin. All women studied were medically examined and had their bone mineral density values obtained for the lumbar spine (L₁–L₄) and femoral neck according to a detailed inclusion criteria. In all women, values of serum sclerostin increased with increasing age up to the age of 45 years, and remained increased in postmenopausal women. Significant increases were evident in serum sclerostin in postmenopausal women with increasing years since menopause. Using stepwise multiple linear regression analysis, several variables were identified as determinants of serum sclerostin, including age, parathyroid hormone, estradiol (E₂), and follicle‐stimulating hormone (FSH) for premenopausal women; age, FSH, and E₂ for postmenopausal women; and age, serum osteocalcin, FSH, and E₂ in the entire sample studied. Further studies are needed to establish the potential role of this increase in mediating the known age‐related impairment in bone formation. © 2011 American Society for Bone and Mineral Research     

Single‐dose,placebo‐controlled,randomized study of AMG 785, a sclerostin monoclonal antibody

Sclerostin, an osteocyte‐secreted protein, negatively regulates osteoblasts and inhibits bone formation. In this first‐in‐human study, a sclerostin monoclonal antibody (AMG 785) was administered to healthy men and postmenopausal women. In this phase I, randomized, double‐blind, placebo‐controlled, ascending, single‐dose study, 72 healthy subjects received AMG 785 or placebo (3:1) subcutaneously (0.1, 0.3, 1, 3, 5, or 10 mg/kg) or intravenously (1 or 5 mg/kg). Depending on dose, subjects were followed for up to 85 days. The effects of AMG 785 on safety and tolerability (primary objectives) and pharmacokinetics, bone turnover markers, and bone mineral density (secondary objectives) were evaluated. AMG 785 generally was well tolerated. One treatment‐related serious adverse event of nonspecific hepatitis was reported and was resolved. No deaths or study discontinuations occurred. AMG 785 pharmacokinetics were nonlinear with dose. Dose‐related increases in the bone‐formation markers procollagen type 1 N‐propeptide (P1NP), bone‐specific alkaline phosphatase (BAP), and osteocalcin were observed, along with a dose‐related decrease in the bone‐resorption marker serum C‐telopeptide (sCTx), resulting in a large anabolic window. In addition, statistically significant increases in bone mineral density of up to 5.3% at the lumbar spine and 2.8% at the total hip compared with placebo were observed on day 85. Six subjects in the higher‐dose groups developed anti‐AMG 785 antibodies, 2 of which were neutralizing, with no discernible effect on the pharmacokinetics or pharmacodynamics. In summary, single doses of AMG 785 generally were well tolerated, and the data support further clinical investigation of sclerostin inhibition as a potential therapeutic strategy for conditions that could benefit from increased bone formation. © 2011 American Society for Bone and Mineral Research.     

Sclerostin antibody improves skeletal parameters in a Brtl/+ mouse model of osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a genetic bone dysplasia characterized by osteopenia and easy susceptibility to fracture. Symptoms are most prominent during childhood. Although antiresorptive bisphosphonates have been widely used to treat pediatric OI, controlled trials show improved vertebral parameters but equivocal effects on long‐bone fracture rates. New treatments for OI are needed to increase bone mass throughout the skeleton. Sclerostin antibody (Scl‐Ab) therapy is potently anabolic in the skeleton by stimulating osteoblasts via the canonical wnt signaling pathway, and may be beneficial for treating OI. In this study, Scl‐Ab therapy was investigated in mice heterozygous for a typical OI‐causing Gly→Cys substitution in col1a1. Two weeks of Scl‐Ab successfully stimulated osteoblast bone formation in a knock‐in model for moderately severe OI (Brtl/+) and in WT mice, leading to improved bone mass and reduced long‐bone fragility. Image‐guided nanoindentation revealed no alteration in local tissue mineralization dynamics with Scl‐Ab. These results contrast with previous findings of antiresorptive efficacy in OI both in mechanism and potency of effects on fragility. In conclusion, short‐term Scl‐Ab was successfully anabolic in osteoblasts harboring a typical OI‐causing collagen mutation and represents a potential new therapy to improve bone mass and reduce fractures in pediatric OI. © 2013 American Society for Bone and Mineral Research     

High serum sclerostin predicts the occurrence of osteoporotic fractures in postmenopausal women: The center of excellence for osteoporosis research study

Sclerostin regulates bone formation by inhibiting Wnt pathway signaling. Low circulating sclerostin levels cause high bone mass. We hypothesized that postmenopausal women with increased sclerostin levels have a greater risk for osteoporosis‐related fractures. We examined the association between circulating sclerostin together with bone turnover markers and osteoporosis‐related fracture risk in 707 postmenopausal women, in a population‐based study with a mean follow‐up period of 5.2 ± 1.3 years. Multivariate Cox proportional hazards regression models were used to analyze fracture risk, adjusted for age, body mass index, and other confounding risk factors. High sclerostin levels were strongly associated with increased fracture risk. After adjustment for age and other confounders, the relative fracture risk was more than sevenfold among postmenopausal women for each 1‐SD increment increase in sclerostin level. Women in the highest quartile of sclerostin levels had about a 15‐fold increase in fracture risk. Results were similar when we compared sclerostin at the 1‐year visit to an average of two to three annual measurements. Fracture risk attributable to sclerostin levels was 56.6% in the highest quartile. Only high levels of bone resorption markers (plasma cross‐linked C‐terminal telopeptide of type 1 collagen [p‐CTx], urinary CTx [u‐CTx], and urinary N‐telopeptide of type 1 collagen [u‐NTx]) were predictive of osteoporosis‐related fractures but at much lower hazard ratio (HR) values than that of serum sclerostin. Associations between sclerostin levels and fracture risk were independent of bone mineral density and other confounding risk factors. High sclerostin levels are a strong and independent risk factor for osteoporosis‐related fractures among postmenopausal women. © 2012 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.