SOST-KO Mice Research Articles

Sclerostin deficiency effectively promotes bone morphogenetic protein-2-induced ectopic bone formation.

Severe periodontitis causes alveolar bone resorption, resulting in tooth loss. Developments of tissue regeneration therapy that can restore alveolar bone mass are desired for periodontal disease. The application of bone morphogenetic protein-2 (BMP-2) has been attempted for bone fractures and severe alveolar bone loss. BMP-2 reportedly induces sclerostin expression, an inhibitor of Wnt signals, that attenuates bone acquisition. However, the effect of sclerostin-deficiency on BMP-2-induced bone regeneration has not been fully elucidated. We investigated BMP-2-induced ectopic bones in Sost-knockout (KO) mice. rhBMP-2 were implanted into the thighs of C57BL/6 (WT) and Sost-KO male mice at 8 weeks of age. The BMP-2-induced ectopic bones in these mice were examined on days 14 and 28 after implantation. Immunohistochemical and quantitative RT-PCR analyses showed that BMP-2-induced ectopic bones expressed sclerostin in osteocytes on days 14 and 28 after implantation in Sost-Green reporter mice. Micro-computed tomography analysis revealed that BMP-2-induced ectopic bones in Sost-KO mice showed a significant increased relative bone volume and bone mineral density (WT = 468 mg/cm3 , Sost-KO = 602 mg/cm3 ) compared with those in WT mice on day 14 after implantation. BMP-2-induced ectopic bones in Sost-KO mice showed an increased horizontal cross-sectional bone area on day 28 after implantation. Immunohistochemical staining showed that BMP-2-induced ectopic bones in Sost-KO mice had an increased number of osteoblasts with osterix-positive nuclei compared with those in WT mice on days 14 and 28 after implantation. Sclerostin deficiency increased bone mineral density in BMP-2-induced ectopic bones.

Evidence for the major contribution of remodeling-based bone formation in sclerostin-deficient mice.

Bone formation by osteoblasts is achieved through remodeling-based bone formation (RBBF) and modeling-based bone formation (MBBF). The former is when bone formation occurs after osteoclastic bone resorption to maintain bone mass and calcium homeostasis. The latter is when new bone matrices are added on the quiescent bone surfaces. Administration of anti-sclerostin neutralizing antibody promotes MBBF in ovariectomized rats and postmenopausal women. However, it remains to be elucidated which mode of bone formation mainly occurs in Sost-deficient mice under physiological conditions. Here, we show that two-thirds of bone formation involves RBBF in 12-week-old Sost-deficient mice (C57BL/6 background). Micro-computed tomography and histomorphometric analyses showed that the trabecular bone mass in Sost-KO mice was higher than that in Sost+/- mice. In contrast, the osteoclast number remained unchanged in Sost-KO mice, but the bone resorption marker TRAP5b in serum was slightly higher in those mice. Treatment with anti-RANKL antibody increased the trabecular bone mass of Sost+/- or Sost-KO mice. Bone formation markers such as osteoid surfaces, the mineral apposition rate, and bone formation rate were almost completely suppressed in Sost+/- mice treated with anti-RANKL antibody compared with vehicle-treated Sost+/- mice. In Sost-KO mice, treatment with anti-RANKL antibody suppressed those parameters by more than half. These findings indicate that RBBF accounts for most of the bone formation in Sost+/- mice, whereas approximately two-thirds of bone formation is estimated to be remodeling-based in 12-week-old Sost-deficient mice. Furthermore, anti-RANKL antibody may be useful for detecting MBBF on trabecular bone.

Role of Sclerostin in Multiple Myeloma Progression

Multiple Myeloma (MM) is a tumor of plasma cells that originates and expands within bone marrow (BM). The BM microenvironment, including its constituent stromal cells and cytokines contribute to disease progression. The BM stromal cells include osteolineage cells, osteoclasts, adipocytes, endothelial cells etc. Among these, cells of the osteoblast lineage have been identified as the most versatile regulators of hematopoietic cells. The osteolineage cells include preosteoblasts, committed osteoblasts, mature osteoblasts, and the terminally differentiated osteocytes, each with its own characteristic biological activity. The osteolineage cells have been shown to regulate commitment and differentiation of hematopoietic lineage cells including hematopoietic stem cells, B-cells, and myeloid lineage cells.Previously published data have shown that loss of parathyroid hormone (PTH) signaling in osteolineage cells adversely affects B-cell development (Panaroni et al., 2016). PTH is also a strong inhibitor of sclerostin, a Wnt-signaling inhibitor secreted by osteocytes. Moreover, mice lacking osteocytes show decreased lymphopoiesis, which may in part be regulated by sclerostin. A sclerostin-neutralizing antibody has shown promise for treatment for age-induced osteoporosis. We have recently demonstrated that MM patients have increased levels of sclerostin and that treatment with a SOST neutralizing antibody resulted in increased osteoblastic differentiation (Eda et al., 2015) and decreased tumor burden. Based upon these findings, we hypothesized that the osteolineage cells contribute to MM progression, and that this may in part occur via sclerostin. To test this hypothesis, we have used genetic mouse models of osteolineage or sclerostin deficiency. Mouse MM cell line 5TGM1 was used to examine MM progression in these mouse models.Global sclerostin deficient (SOST KO) mice were generated as described previously. Given that sclerostin acts as a negative regulator of bone formation, SOST KO mice displayed robust increase in trabecular and cortical bone density as analyzed by micro-computed tomography. Co-culture with 5TGM1 MM cells showed that the BM stromal cells from SOST KO mice significantly decreased proliferation of MM cells raising the possibility that sclerostin deficiency may decrease MM progression in vivo. The SOST KO mice showed significantly reduced BM cellularity compared to the controls. However, relevant to MM progression, overall B-lymphopoiesis, proliferation or apoptosis was unchanged during any stages of B-cell development in BM of 3-month old SOST KO mice.To determine if sclerostin deficiency alters MM engraftment in vivo, 5TGM1 cells were injected into the tibia of 3-month old control or SOST KO mice and engraftment was followed for 2-months. Complete blood counts (CBC) showed no significant changes between the control and SOST KO mice injected with the MM cells. The 5TGM1 cells were fluorescently tagged before in vivo injections allowing us to track the cells by whole body bioluminescence imaging and by contralateral tibia BM flow cytometry analysis. Both the analyses showed no changes in the distant MM engraftment between the control and the SOST KO mice. Ongoing work is examining local MM engraftment in the tibia with injected MM cells.These data suggest that the sclerostin deficiency does not alter systemic MM engraftment after 2 months of follow-up observations. It is possible, however, that the local progression of MM cells may have altered. We are investigating this possibility. We are also examining MM engraftment in mice lacking mature osteoblasts and osteocytes to identify sclerostin-dependent and independent factors controlling MM progression. DisclosuresRaje:Millenium: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Onyx: Consultancy.

Suppression of Sost/Sclerostin and Dickkopf-1 Augment Intervertebral Disc Structure in Mice.

ABSTRACTIntervertebral disc (IVD) degeneration is a leading cause of low back pain, characterized by accelerated extracellular matrix breakdown and IVD height loss, but there is no approved pharmacological therapeutic. Deletion of Wnt ligand competitor Lrp5 induces IVD degeneration, suggesting that Wnt signaling is essential for IVD homeostasis. Therefore, the IVD may respond to neutralization of Wnt ligand competitors sost(gene)/sclerostin(protein) and/or dickkopf‐1 (dkk1). Anti‐sclerostin antibody (scl‐Ab) is an FDA‐approved bone therapeutic that activates Wnt signaling. We aimed to (i) determine if pharmacological neutralization of sclerostin, dkk1, or their combination would stimulate Wnt signaling and augment IVD structure and (ii) determine the prolonged adaptation of the IVD to global, persistent deletion of sost. Nine‐week‐old C57Bl/6J female mice (n = 6–7/group) were subcutaneously injected 2×/week for 5.5 weeks with scl‐Ab (25 mg/kg), dkk1‐Ab (25 mg/kg), 3:1 scl‐Ab/dkk1‐Ab (18.75:6.25 mg/kg), or vehicle (veh). Separately, IVD of sost KO and wild‐type (WT) mice (n = 8/group) were harvested at 16 weeks of age. First, compared with vehicle, injection of scl‐Ab, dkk1‐Ab, and 3:1 scl‐Ab/dkk1‐Ab similarly increased lumbar IVD height and β‐catenin gene expression. Despite these similarities, only injection of scl‐Ab alone strengthened IVD mechanical properties and decreased heat shock protein gene expressions. Genetically and compared with WT, sost KO enlarged IVD height, increased proteoglycan staining, and imbibed IVD hydration. Notably, persistent deletion of sost was compensated by upregulation of dkk1, which consequently reduced the cell nuclear fraction for Wnt signaling co‐transcription factor β‐catenin in the IVD. Lastly, RNA‐sequencing pathway analysis confirmed the compensatory suppression of Wnt signaling and revealed a reduction of cellular stress‐related pathways. Together, suppression of sost/sclerostin or dkk1 each augmented IVD structure by stimulating Wnt signaling, but scl‐Ab outperformed dkk1‐Ab in strengthening the IVD. Ultimately, postmenopausal women prescribed scl‐Ab injections to prevent vertebral fracture may also benefit from a restoration of IVD height and health. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

P1244MILD DIFFERENCES IN CARDIAC CALCIFICATION BETWEEN WILD TYPE AND SCLEROSTIN KNOCKOUT MICE WITH CKD

Abstract Background and Aims Ectopic soft tissue calcifications, especially cardiovascular calcifications, are frequently observed in patients with chronic kidney disease (CKD) and are associated with increased morbidity and mortality. Sclerostin, a well-known inhibitor of bone formation, shows significantly increased serum levels in CKD patients. Since the process of ectopic calcification shares many similarities with physiological bone formation, we investigated a possible role for sclerostin in ectopic calcification development. Method CKD was induced in wild type (WT) (n=21) and sclerostin knockout (Sost ko) mice (n = 21), both on a C57Bl6/J background, by the alternate administration of a 0.30% and 0.15% adenine supplemented diet, containing 1% phosphate. A normal renal function control group receiving standard mouse chow (WT C57Bl6/J n = 8) was also included in the study. Serum creatinine and phosphate levels were measured at the start and end of the study to assess CKD development. At sacrifice, the calcium content of the kidneys, heart and aorta was determined by flame atomic absorption spectrometry. Results The adenine-supplemented diet led to a clear induction of CKD in both the Sost ko and wild type mice, as reflected by increased serum creatinine (0.38 ± 0.09 mg/dL in control mice vs 0.65 ± 0.18 mg/dL in adenine-exposed mice, p<0.0001) and phosphate levels (9.12 ± 1.16 mg/dL in control mice vs 19.03 ± 3.11 mg/dL in adenine-exposed mice, p<0.0001). The Sost ko and WT mice receiving the adenine diet, developed the same degree of renal failure, since there were no differences in serum levels of creatinine (p=0.0507) and phosphate (p=0.4890). Significantly higher amounts of calcium were measured in the kidney (0.05 ± 0.01 mg/g wet tissue vs 3.70 ± 2.00 mg/g wet tissue, p<0.0001) and the heart (0.03 ± 0.01 mg/g wet tissue vs 0.07 ± 0.04 mg/g wet tissue, p<0.0001) of adenine-exposed mice, compared to mice receiving the control diet. Furthermore, Sost ko mice had a significantly higher calcium content in the heart compared to the WT mice with renal failure (0.05 ± 0.03 mg/g wet tissue in WT mice vs 0.08 ± 0.05 mg/g wet tissue in Sost ko mice, p = 0.0029). No differences were observed in aortic calcium content between control mice and mice with renal failure. Conclusion In this study we managed to develop a mouse model of CKD-induced soft tissue calcification in the heart and kidneys of the animals, however no calcifications developed in the aorta. The higher calcium content that was observed in the heart of Sost ko mice compared to WT mice, might be an indication of a protective function of sclerostin during ectopic cardiac calcification

SOST Deficiency Aggravates Osteoarthritis in Mice by Promoting Sclerosis of Subchondral Bone.

As the initial part in the development of osteoarthritis (OA), subchondral bone sclerosis has been considered to be initiated by excess mechanical loading and proven to be correlated to other pathological changes. Sclerostin, which is an essential mechanical stress response protein, is encoded by the SOST gene. It is expressed in osteocytes and mature chondrocytes and has been proven to be closely correlated to OA. However, the relationship and mechanism between the SOST gene and the development of OA remain unclear. The aim of the present study was to investigate the role of the SOST gene in OA pathogenesis in the subchondral bone. A knee anterior cruciate ligament transection (ACLT) mouse osteoarthritis (OA) model on SOST-knockout (SOST KO) and wild-type (WT) mice was established. The pathogenic and phenotypic changes in the subchondral bone were investigated by histology, micro-CT, immunohistochemistry, TRAP staining, Masson staining, and Toluidine blue staining. It was found that sclerostin expression decreased in both the calcified cartilage and mineralized subchondral structures during the development of OA. Joint instability induced a severe cartilage degradation phenotype, with higher OARSI scores in SOST KO mice, when compared to WT mice. SOST KO mice with OA exhibited a higher BMD and BV/TV ratio, as well as a higher rate of bone remodeling and TRAP-positive cell number, when compared to the WT counterparts, but the difference was not significant between the sham-operation groups. It was concluded that loss of sclerostin aggravates knee OA in mice by promoting subchondral bone sclerosis and increasing catabolic activity of cartilage.

Sclerostin depletion may induce inflammation in the bone marrow

Abstract The interactions between long-term hematopoietic stem cells (LT-HSCs) and cells in the bone microenvironment result in changes in stem cell behavior that are incompletely understood. Our goal is to define the molecular mechanisms that guide the behavior of LT-HSCs after exposure to an irregular bone marrow (BM) niche. Sclerostin (Sost) is a secreted Wnt antagonist that is important for bone homeostasis. We hypothesized that that loss of Sost would influence LT-HSC differentiation and migration. To test this, we performed hematopoietic progenitor transplantation assays to track donor-derived hematopoiesis in wild-type (WT) and Sost-knockout (KO) recipients. We observed increased frequencies of donor-derived myelopoiesis as well as decreased lymphopoiesis in WT to KO chimeras possibly due to an inflammatory microenvironment. We are currently investigating LT-HSC inflammatory response genes, such as C/EBP-beta and PU.1, as well as BM serum inflammatory cytokine levels. As the SostKO mice age, we observe extramedullary hematopoiesis (EMH) in the spleen compared to age matched controls. We hypothesize that changes in the BM microenvironment as well as cytokine levels in the spleen induce LT-HSC migration to the spleen. We are characterizing the EMH LT-HSC splenic niche by investigating LT-HSC localization as well as examining cytokines, such as CXCL12, known to induce LT-HSC migration to the spleen. Taken together, our data indicates that depletion of Sost in the bone regulates LT-HSC differentiation and migration. Our results could have significant impact on the tracking of hematopoietic function in human patients treated with SOST-depleting antibodies for bone diseases such as osteoporosis.

Conditional Deletion of Sost in MSC-Derived Lineages Identifies Specific Cell-Type Contributions to Bone Mass and B-Cell Development

Sclerostin (Sost) is a negative regulator of bone formation and blocking its function via antibodies has shown great therapeutic promise by increasing both bone mass in humans and animal models. Sclerostin deletion in Sost KO mice (Sost-/- ) causes high bone mass (HBM) similar to sclerosteosis patients. Sost-/- mice have been shown to display an up to 300% increase in bone volume/total volume (BV/TV), relative to age-matched controls. It has been postulated that the main source of skeletal sclerostin is the osteocyte. To understand the cell-type specific contributions to the HBM phenotype described in Sost-/- mice, as well as to address the endocrine and paracrine mode of action of sclerostin, we examined the skeletal phenotypes of conditional Sost loss-of-function (SostiCOIN/iCOIN ) mice with specific deletions in (1) the limb mesenchyme (Prx1-Cre; targets osteoprogenitors and their progeny); (2) midstage osteoblasts and their progenitors (Col1-Cre); (3) mature osteocytes (Dmp1-Cre); and (4) hypertrophic chondrocytes and their progenitors (ColX-Cre). All conditional alleles resulted in significant increases in bone mass in trabecular bone in both the femur and lumbar vertebrae, but only Prx1-Cre deletion fully recapitulated the amplitude of the HBM phenotype in the appendicular skeleton and the B-cell defect described in the global KO. Despite WT expression of Sost in the axial skeleton of Prx1-Cre deleted mice, these mice also had a significant increase in bone mass in the vertebrae, but the sclerostin released in circulation by the axial skeleton did not affect bone parameters in the appendicular skeleton. Also, both Col1 and Dmp1 deletion resulted in a similar 80% significant increase in trabecular bone mass, but only Col1 and Prx1 deletion resulted in a significant increase in cortical thickness. We conclude that several cell types within the Prx1-osteoprogenitor-derived lineages contribute significant amounts of sclerostin protein to the paracrine pool of Sost in bone. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.

Sclerostin expression in distinct osteolineage cell types differentially regulates hematopoietic stem cell and B lymphocyte development

Abstract Investigation of the reciprocal effects of hematopoietic cell types and osteolineage cells on each other’s cell fates have clinical applications to improve bone fracture healing, prevent age-related bone loss and immune deficiencies. A significant knowledge gap exists as to the contribution of different osteolineage cells to the differentiation, proliferation, and long term survival of B cells in the BM. In global sclerostin knockout (SostKO) mice, we previously uncovered a cell-extrinsic requirement for sclerostin on B lymphocyte development. It is widely accepted that sclerostin is transcribed primarily in osteocytes (OCYs), but whether is it expressed and/or active in other bone progenitors has been controversial. In order to identify the Sost-expressing osteolineage cell type that is responsible for B cell support, we analyzed hematopoietic differentiation in mice lacking Sost in mesenchymal stem cells (MSCs) [Prx1-Cre], osteoblasts (OBs) [Col1a-Cre] and OCYs [Dmp1-Cre] through the use of conditional Sost-KO (cKO) mice. Loss of Sost expression in MSCs resulted in reduced BM cellularity, whereas this was unaffected by Sost-deficiency in OB and OCYs. Furthermore, MSC-specific depletion of Sost caused an accumulation of early B cell progenitors. In contrast, loss of Sost in OBs and OCYs resulted in a reduction of mature “recirculating” B cells in the BM. Remarkably, the percentage of long-term CD150+ CD48− HSCs was significantly reduced in MSC-cKO mice, but was not affected in OB- and OCY-cKOs. The data described herein suggest that Sost deficiency in MSCs, OBs, and OCYs differentially regulates hematopoietic stem cell retention, survival, maintenance and B cell differentiation in the bone microenvironment.

Genetic Sost Deletion and Pharmacological Inhibition of Sclerostin Prevent Multiple Myeloma-Induced Bone Loss without Affecting Tumor Growth

In Multiple myeloma (MM) plasma cells grow in the bone marrow and induce localized lytic lesions due to increased bone resorption and suppressed bone formation. High levels of Sclerostin, a potent inhibitor of bone formation, are found in sera of MM patients. The expression of Sost/Scleorstin by osteocytes is also increased in mice bearing MM tumors, suggesting that Sclerostin might play a role in MM-induced bone disease. The goal of this study was to examine the impact of Sost/Sclersotin on tumor growth and MM-induced bone disease.We first examined the effect of genetic deletion of Sost. Therefore, we generated global Sost KO mice in an immunodeficient background (SCID), which exhibited the expected high bone mass phenotype associated with Sost deficiency. 6-wk-old SostKO or WT control littermates mice were injected intratibially with 105 JJN3 human MM (hMM) cells or saline (n=7-10/group), and sacrificed after 4wks. Sost KO and WT mice injected with hMM had equivalent tumor engraftment as demonstrated by serum human kappa light chain levels. hMM-injected WT mice exhibited ~50% decrease in tibia cancellous bone volume (BV/TV) and trabecular number (Tb.N), and increased trabecular separation (Tb.Sp). In contrast, hMM-injected Sost KO mice displayed no changes in BV/TV or bone architecture. Importantly, X-ray analysis revealed that the number and area of osteolytic lesions was reduced in Sost KO by 60% and 74%, respectively, compared to WT mice.We next examined the effect of pharmacological inhibition of Sclerostin in an immunocompetent preclinical model of established MM. 6-wk-old C57BLKalwRij mice were injected intratibially with 105 5TGM1 murine MM cells (mMM) or saline. After 4wks mMM-injected mice had a 2-fold increase in the serum tumor engraftment marker IgG2b. Saline or mMM-injected mice were then treated with a Sclerostin neutralizing antibody (Scl-Ab; 15mg/kg/wk) or control antibody (IgG; n=6-10/group). After 4wks of treatment, serum IgG2b levels were similar in mMM-injected mice receiving Scl-Ab or IgG. mMM-injected mice receiving IgG injections had ~35% decreased BV/TV, Tb.N, and increased Tb.Sp. In contrast, mMM-injected mice receiving Scl-Ab displayed increased trabecular BV/TV (52%), Tb.N (22%), Tb.Th (33%) and decreased Tb.Sp (14%), results that did not differ from saline-injected mice treated with Scl-Ab. Moreover, the number of osteolytic lesions was reduced by 46% in Scl-Ab treated mice when compared to the IgG-treated group. Further, mMM-injected mice treated with IgG or Scl-Ab showed similar increases in the bone resorption markerCTX in the circulation, whereas mMM-injected mice treated with Scl-Ab had a smaller decrease in the bone formation marker P1NP in sera compared to IgG-treated mice (22 vs 45%).Consistent with the lack of effect of the Scl-Ab on MM tumor growth in vivo, Scl-Ab did not affect the proliferation or viability of MM cells in vitro. We then examined next if Scl-Ab alters the anti-myeloma activity of dexamethasone (DEX), bortezomib (BOR) and the Notch inhibitor GSIXX. As expected, DEX, BOR and GSIXX increased the number of dead mMM and hMM cells in a time- and dose-dependent manner. Importantly, the increase in the number of dead hMM and mMM cells induced by DEX, BOR and GSIXX remained unchanged when Scl-Ab was co-administered.Taken together, these results demonstrate that increased Scl production by osteocytes inhibits bone formation and contributes to MM-induced bone loss. Further, our data shows that pharmacological inhibition of Scl does not alter tumor growth or the activity of anti-myeloma drugs. These findings provide the rationale for combining Scl-Ab with anti-tumor drugs to simultaneously prevent tumor growth and the bone diseases in MM patients. DisclosuresRoodman:Amgen: Consultancy.

Mice with sclerostin gene deletion are resistant to the severe sublesional bone loss induced by spinal cord injury.

The bone loss secondary to spinal cord injury (SCI) is associated with several unique pathological features, including the permanent immobilization, neurological dysfunction, and systemic hormonal alternations. It remains unclear how these complex pathophysiological changes are linked to molecular alterations that influence bone metabolism in SCI. Sclerostin is a key negative regulator of bone formation and bone mass. We hypothesized that sclerostin could function as a major mediator of bone loss following SCI. To test this hypothesis, 10-week-old female sclerostin knockout (SOST KO) and wild type (WT) mice underwent complete spinal cord transection or laminectomy (Sham). At 8weeks after SCI, substantial loss of bone mineral density was observed at the distal femur and proximal tibia in WT mice but not in SOST KO mice. By μCT, trabecular bone volume of the distal femur was markedly decreased by 64% in WT mice after SCI. In striking contrast, there was no significant reduction of bone volume in SOST KO/SCI mice compared with SOST KO/sham. Histomorphometric analysis of trabecular bone revealed that the significant reduction in bone formation rate following SCI was observed in WT mice but not in SOST KO mice. Moreover, SCI did not alter osteoblastogenesis of marrow stromal cells in SOST KO mice. Our findings demonstrate that SOST KO mice were protected from the major sublesional bone loss that invariably follows SCI. The evidence indicates that sclerostin is an important mediator of the marked sublesional bone loss after SCI, and that pharmacological inhibition of sclerostin may represent a promising novel approach to this challenging clinical problem.

Effect of sclerostin removal in vivo on experimental periodontitis in mice.

We explored the effects of sclerostin removal in vivo on experimental periodontitis in mice. A ligature of Porphyromonas gingivalis-saturated collagen silk was applied to the cervical region of the first molar tooth in 10 wild-type (WT) mice and 10 sclerostin-knockout (SOST-KO) mice, and the animals were fed 10% sucrose for 2 months. Another 10 WT mice and 10 SOST-KO mice were similarly treated, but then fed a normal diet for 2 months. The maxillae were then harvested for morphological and molecular examinations. The mice with periodontitis showed significantly more severe alveolar bone loss than control mice, the most significant absorption being observed in WT mice. Immunohistochemical staining demonstrated upregulation of RANKL and ERK1/2-MAPK expression and downregulation of OPG expression in mice with periodontitis, especially WT mice. Therefore, removal of sclerostin appears to modestly protect the alveolar bone from resorption in this experimental setting. (J Oral Sci 58, 271-276, 2016).

Effect of SOST gene deletion on the progression of renal interstitial fibrosis in obstructive kidney injury

Objectives: The role of SOST/sclerostin in mediating tissue fibrogenic response to injury/inflammation remains largely unknown. Thus, we conducted this study to determine whether SOST/sclerostin plays a role in renal interstitial fibrosis (RIF) for the first time. Methods: Unilateral ureteral obstruction (UUO) was performed to create obstructive kidney injury model. Twelvemale SOST knockout (SOST KO) mice and 12 age-matched wild-type (WT) mice were divided into three groups: sham surgery, UUO 3 d and UUO 7 d. The mice were sacrificed at each time point and kidney tissues were collected. Histopathological changes were evaluated by hematoxylin and eosin and Masson staining, while α-smooth muscle actin (α-SMA), type I collagen (Col-I) and fibronectin (FN) expression levels were detected by RT-PCR and western-blot. Results: In sham control group, neither WT nor SOST KO exhibited fibrotic change. On 3 days after UUO, total renal histopathological score and fibrotic area were aggravated and α-SMA, Col-I and FN expressions were upregulated, but no difference was observed between WT and SOST KO. On 7 days after UUO, compared with WT, SOST KO mice showed higher total renal histopathological score and fibrotic area percentage, as well as a higher level of fibrogenic marker mRNA/protein expression (except for α-SMA mRNA and FN mRNA). Conclusion: It is supposed that SOST gene is involved in the regulation of RIF progression. In obstructive kidney injury, SOST gene deletion would probably enhance renal fibrogenic response and promote the progression of RIF. But more evidences are needed to further identify the role of SOST/sclerostin in mediating RIF progression.

Loss of sclerostin promotes osteoarthritis in mice via β-catenin-dependent and -independent Wnt pathways.

IntroductionSclerostin is a Wnt inhibitor produced by osteocytes that regulates bone formation. Because bone tissue contributes to the development of osteoarthritis (OA), we investigated the role of sclerostin in bone and cartilage in a joint instability model in mice.MethodsTen-week-old SOST-knockout (SOST-KO) and wild-type (WT) mice underwent destabilization of the medial meniscus (DMM). We measured bone volume at the medial femoral condyle and osteophyte volume and determined the OA score and expression of matrix proteins. Primary murine chondrocytes were cultured with Wnt3a and sclerostin to assess the expression of matrix proteins, proteoglycan release and glycosaminoglycan accumulation.ResultsSclerostin was expressed in calcified cartilage of WT mice with OA. In SOST-KO mice, cartilage was preserved despite high bone volume. However, SOST-KO mice with DMM had a high OA score, with increased expression of aggrecanases and type X collagen. Moreover, SOST-KO mice with OA showed disrupted anabolic–catabolic balance and cartilage damage. In primary chondrocytes, sclerostin addition abolished Wnt3a-increased expression of a disintegrin and metalloproteinase with thrombospondin motifs, matrix metalloproteinases and type X collagen by inhibiting the canonical Wnt pathway. Moreover, sclerostin inhibited Wnt-phosphorylated c-Jun N-terminal kinase (JNK) and rescued the expression of anabolic genes. Furthermore, sclerostin treatment inhibited both Wnt canonical and non-canonical JNK pathways in chondrocytes, thus preserving metabolism.ConclusionSclerostin may play an important role in maintaining cartilage integrity in OA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0540-6) contains supplementary material, which is available to authorized users.

Sclerostin deficiency is linked to altered bone composition.

High bone mass in animals and humans with sclerostin deficiency is associated with increased bone strength, which is not the case for all disorders with high bone mineral density, some of which are even associated with fragility fractures owing to unfavorable bone composition. In the current study we investigated whether alterations in bone composition may contribute to the bone strength characteristics associated with lack of sclerostin. We examined cortical bone of Sost-knockout (KO) mice (n = 9, 16 weeks old) and sclerosteosis patients (young [4 to 14 years], n = 4 and adults [24 and 43 years], n = 2) by quantitative backscattered electron imaging and Raman microspectroscopy and compared it to bone from wild-type mice and healthy subjects, respectively. In Sost-KO mice endocortical bone exhibited altered bone composition, whereas subperiosteal bone was unchanged. When comparing endocortical bone tissue of identical tissue age as defined by sequential dual fluorochrome labeling the average bone matrix mineralization was reduced -1.9% (p < 0.0001, younger tissue age) and -1.5% (p < 0.05, older tissue age), and the relative proteoglycan content was significantly increased. Similarly, bone matrix mineralization density distribution was also shifted toward lower matrix mineralization in surgical samples of compact bone of sclerosteosis patients. This was associated with an increase in mineralization heterogeneity in the young population. In addition, and consistently, the relative proteoglycan content was increased. In conclusion, we observed decreased matrix mineralization and increased relative proteoglycan content in bone subcompartments of Sost-KO mice-a finding that translated into sclerosteosis patients. We hypothesize that the altered bone composition contributes to the increased bone strength of patients with sclerostin deficiency.

Lack of sclerostin promotes osteoarthritis by activating canonical and non-canonical WNT pathways

Lack of sclerostin promotes osteoarthritis by activating canonical and non-canonical WNT pathways

Sclerostin deficient mice rapidly heal bone defects by activating β-catenin and increasing intramembranous ossification

We investigated the influence of the osteocyte protein, sclerostin, on fracture healing by examining the dynamics and mechanisms of repair of single-cortex, stabilized femoral defects in sclerostin knockout (Sost−/−; KO) and sclerostin wild-type (Sost+/+; WT) mice. Fourteen days following generation of bone defects, Sost KO mice had significantly more bone in the healing defect than WT mice. The increase in regenerating bone was due to an increase in the thickness of trabecularized spicules, osteoblast numbers and surfaces within the defect. Enhanced healing of bone defects in Sost KO mice was associated with significantly more activated β-catenin expression than observed in WT mice. The findings were similar to those observed in Axin2−/− mice, in which β-catenin signaling is known to be enhanced to facilitate bone regeneration. Taken together, these data indicate that enhanced β-catenin signaling is present in Sost−/− mice that demonstrate accelerated healing of bone defects, suggesting that modulation of β-catenin signaling in bone could be used to promote fracture repair.

Increased heterogeneity of bone matrix mineralization in a patient with familial hypercalcaemic hypocalciuria due to the loss of function of the calcium sensing receptor

Increased heterogeneity of bone matrix mineralization in a patient with familial hypercalcaemic hypocalciuria due to the loss of function of the calcium sensing receptor

Lower bone matrix mineralization in cases of sclerostin deficiency

Lower bone matrix mineralization in cases of sclerostin deficiency

Increased callus mass and enhanced strength during fracture healing in mice lacking the sclerostin gene

Humans with inherited sclerostin deficiency have high bone mass. Targeted deletion of the sclerostin gene in mice (SOST-KO) causes increases in bone formation, bone mass and bone strength. Inhibition of sclerostin by a monoclonal antibody increases bone formation and enhances fracture healing in rodent and primate models. In this study, we describe the temporal progression of femoral fracture healing in SOST-KO mice compared with wild type (WT) control mice to further characterize the role of sclerostin in fracture healing. Sixty-seven male 9-10 week-old SOST-KO (N=37) and WT (N=30) mice underwent a closed femoral fracture. Weekly radiography was used to monitor the progress of healing. Histologic sections were used to characterize callus composition, evaluate callus bridging, and quantify lamellar bone formation on days 14 and 28. Densitometry and biomechanical testing were utilized to characterize bone mass and strength at the fractured and contralateral femurs on day 45. A significant improvement in time to radiographic healing (no discernible fracture line) was observed in SOST-KO mice, which corresponded to an increase in histologic bony bridging at 14 days (38% versus 0% in WT). Both genotypes appeared to be nearly fully bridged at 28 days post-fracture. The increased bridging at 14 days was associated with 97% greater bone area and 40% lower cartilage area in the callus of SOST-KO mice as compared to WT mice. Bone formation-related endpoints were higher in SOST-KO mice at both 14 and 28 days. At 45 days post-fracture, peak load and bone mass were significantly greater in the fractured femurs of SOST-KO mice as compared to WT mice. In conclusion, fractures in mice lacking sclerostin showed accelerated bridging, greater callus maturation, and increased bone formation and strength in the callus.