Osteocyte-derived sclerostin inhibits bone formation: its role in bone morphogenetic protein and Wnt signaling

Sclerosteosis and Van Buchem disease are rare, high-bone-mass disorders that have been linked to deficiency in the SOST gene, encoding sclerostin. Sclerostin belongs to the DAN family of glycoproteins, of which multiple family members have been shown to antagonize bone morphogenetic protein (BMP) and/or Wnt activity. Sclerostin is specifically expressed by osteocytes and inhibits BMP-induced osteoblast differentiation and ectopic bone formation. Sclerostin binds only weakly to BMPs and does not inhibit direct BMP-induced responses. Instead, sclerostin antagonizes canonical Wnt signaling by binding to Wnt coreceptors, low-density lipoprotein receptor-related protein 5 and 6. Several lipoprotein receptor-related protein-5 mutants that cause the high-bone-mass trait are defective in sclerostin binding. Thus, high bone mass in sclerosteosis and Van Buchem disease may result from increased Wnt signaling due to the absence of or insensitivity to sclerostin.     

Sclerostin promotes the apoptosis of human osteoblastic cells: a novel regulation of bone formation

A null mutation in the SOST gene is associated with sclerosteosis, an inherited disorder characterized by a high bone mass phenotype. The protein product of the SOST gene, sclerostin, is a bone morphogenetic protein (BMP) antagonist that decreases osteoblast activity and reduces the differentiation of osteoprogenitors. We sought to delineate the mechanism by which sclerostin modulated osteoblastic function by examining the effects of the protein on differentiating cultures of human mesenchymal stem cells (hMSC). Sclerostin significantly decreased alkaline phosphatase (ALP) activity and the proliferation of hMSC cells. In addition, hMSC cells treated with sclerostin displayed a marked increase in caspase activity. Elevated levels of fragmented histone-associated DNA in these cells were detected by ELISA and by TUNEL staining. Other BMP antagonists including noggin, Chordin, Gremlin, and Twisted gastrulation did not affect caspase activity. The sclerostin-mediated increase in caspase activity was blocked by caspase-1 and caspase-3 inhibitors. Sclerostin-induced changes in ALP activity and the survival of hMSC cells were partially restored by BMP-6, suggesting the involvement of additional growth factors. These findings show that sclerostin selectively controls the apoptosis of bone cells. The ability of sclerostin to interact with important growth factors such as BMPs likely serves as the basis by which it modulates the survival of osteoblasts. By making these growth factors unavailable for cell function, sclerostin promotes the apoptosis of bone cells, providing a novel level of control in the regulation of bone formation.     

Immunohistochemical localization of bone morphogenetic proteins (BMPs) 2, 4, 6, and 7 during induced heterotopic bone formation.

The distribution and staining intensity of bone morphogenetic proteins (BMPs) 2, 4, 6, and 7 were assessed by immunohistochemistry in ectopic bone induced in Nu/Nu mice by Saos-2 cell derived implants. Devitalized Saos-2 cells or their extracts can induce endochondral bone formation when implanted subcutaneously into Nu/Nu mice. BMP staining was mostly cytoplasmic. The most intense BMP staining was seen in hypertrophic and apoptotic chondrocytes, osteoprogenitor cells such as periosteal and perivascular cells, and osteoblasts. BMP staining in osteocytes and osteoclasts was variable, ranging from undetectable to intensely stained, and from minimal to moderately stained in megakaryocytes of the induced bone marrow. BMP-2, 4, 6, and 7 staining in Saos-2 implant-induced bone indicates the following: (1) Saos-2 cell products promote expression of BMPs by host osteoprogenitor cells, which in turn, leads to bone and marrow formation at ectopic sites; (2) strong BMP staining is seen in maturing chondrocytes, and thus may play a role in chondrocyte differentiation and/or apoptosis; (3) BMP expression in perivascular and periosteal cells indicates that osteoprogenitor cells also express BMP; (4) BMP release by osteoclasts may promote osteoblastic differentiation at sites of bone remodeling. These new data can be useful in understanding the role of BMPs in promoting clinical bone repair and in various pathologic conditions.     

Twisted gastrulation and chordin inhibit differentiation and mineralization in MC3T3-E1 osteoblast-like cells

Bone morphogenetic proteins (BMPs) are potent inducers of osteoblast differentiation. The accessibility of BMP ligands for binding to their receptors is regulated by secreted proteins Twisted gastrulation (Tsg) and Chordin (Chd). Tsg antagonizes BMP signaling by forming ternary complexes with Chd and BMPs, thereby preventing BMPs from binding to their receptors. In addition to the anti-BMP function, Tsg also has pro-BMP activity, partly mediated by cleavage and degradation of Chd, which releases BMPs from ternary complexes. The roles of Tsg and Chd in osteoblast differentiation are not known. Therefore, in the present study, we investigated the effect of exogenous Tsg and Chd on osteoblast differentiation and mineralization using a well-characterized subclone of MC3T3-E1 osteoblast-like cells. Our results show that Tsg and Chd are expressed in MC3T3-E1 osteoblast-like cells. While Tsg mRNA levels decrease during osteoblast differentiation, Chd levels are found to increase. Tsg and Chd proteins accumulate in the cell culture media as the osteoblasts differentiate. Exogenous Tsg and Chd inhibit osteoblast differentiation and mineralization. Osteocalcin (OCN) mRNA levels decrease following both Tsg and Chd treatment. Tsg and Chd also inhibit alkaline phosphatase (ALP) activity in a dose-dependent manner. To provide insight into the mechanism of Tsg and Chd action, we investigated the effect of Tsg and Chd on BMP activity by determining phosphorylated Smad1 (pSmad1) levels. We show that both Tsg and Chd can independently and in combination reduce pSmad1 levels in MC3T3-E1 cells treated with BMP4. Further, BMP2 partially reverses the inhibitory effect of Tsg and Chd on ALP activity. Taken together, these results suggest that Tsg and Chd are involved in osteoblast differentiation and mineralization by regulating BMP signaling.     

Biphasic effects of transforming growth factor β on bone morphogenetic protein–induced osteoblast differentiation

Bone morphogenetic proteins (BMPs) exert an important role in skeletal development, adult bone homeostasis, and fracture healing and have demonstrated clinical utility for bone regeneration. However, BMPs fall short as regenerative agents because high doses need to be used to obtain therapeutic effects. Determining the molecular mechanisms controlling BMP‐induced bone formation may lead to the development of more effective BMP‐based therapies. To identify kinases mediating BMP‐induced osteoblast differentiation, we performed an siRNA screen to find kinases modulating BMP‐6‐induced alkaline phosphatase (ALP) activity. Surprisingly, although transforming growth factor β (TGF‐β) generally is considered to antagonize BMP‐induced osteoblast differentiation, C2C12 cells transfected with siRNAs targeting TGF‐β receptors displayed reduced BMP‐6‐induced ALP activity. Furthermore, pharmacologic inhibitors blocking the TGF‐β type I receptor impaired BMP‐induced ALP activity in KS483 and C2C12 cells and mineralization of KS483 cells. Consistently, costimulation with BMPs and TGF‐β further increased expression of osteoblast‐specific genes, ALP activity, and mineralization of KS483 cells and primary mesenchymal stem cells compared with BMPs alone. The stimulatory and inhibitory effects of TGF‐β were found to depend on timing and duration of the costimulation. TGF‐β inhibited BMP‐induced activation of a BMP‐Smad‐dependent luciferase reporter, suggesting that the stimulatory effect of TGF‐β is not due to increased BMP‐Smad activity. TGF‐β also inhibited the BMP‐induced expression of the BMP antagonist noggin and prolonged BMP activity. In conclusion, TGF‐β, besides acting as an inhibitor, also can, by dampening the noggin‐mediated negative‐feedback loop, enhance BMP‐induced osteoblast differentiation, which might be beneficial in fracture healing. © 2011 American Society for Bone and Mineral Research.     

Circulating Sclerostin and Dickkopf-1 (DKK1) in Predialysis Chronic Kidney Disease (CKD): Relationship with Bone Density and Arterial Stiffness

Abnormalities of bone metabolism and increased vascular calcification are common in chronic kidney disease (CKD) and important causes of morbidity and mortality. The Wnt signaling pathway may play a role in the bone and vascular disturbances seen in CKD, termed collectively "CKD-MBD." The aim of the study was to investigate the possible association of circulating concentrations of the secreted Wnt signaling inhibitors DKK1 and sclerostin with BMD and arterial stiffness in predialysis CKD. Seventy-seven patients (48 M, 29 F), mean age 57 (SD = 14) years with CKD stages 3B (n = 32) and 4 (n = 45) were studied. Sclerostin, DKK1, PTH, and 1,25(OH)(2)D were analyzed. BMD was measured at the lumbar spine (LS), femoral neck (FN), total hip (TH), and forearm (FARM). Arterial stiffness index was determined by contour analysis of digital volume pulse (SI(DVP)). There was a positive correlation between sclerostin and age (r = 0.47, p < 0.000). Sclerostin was higher in men than women (p = 0.013). Following correction for age and gender, there was a negative association between GFR and sclerostin (p = 0.002). We observed a positive association between sclerostin and BMD at the LS (p = 0.0001), FN (p = 0.004), and TH (p = 0.002). In contrast, DKK1 was negatively associated with BMD at the FN (p = 0.038). A negative association was seen between DKK1 and SI(DVP) (p = 0.027). Our data suggest that the Wnt pathway may play a role in CKD-MBD. Prospective studies are required to establish the clinical relevance of sclerostin and DKK1 as serological markers in CKD.     

Heparin affects human bone marrow stromal cell fate: Promoting osteogenic and reducing adipogenic differentiation and conversion

Heparins are broadly used for the prevention and treatment of thrombosis and embolism. Yet, osteoporosis is considered to be a severe side effect in up to one third of all patients on long-term treatment. However, the mechanisms underlying this clinical problem are only partially understood. To investigate if heparin affects differentiation of skeletal precursors, we examined the effects of heparin on the osteogenic and adipogenic lineage commitment and differentiation of primary human bone marrow stromal cells (hBMSCs). Due to the known inverse relationship between adipogenesis and osteogenesis and the capacity of pre-differentiated cells to convert into the respective other lineage, we also determined heparin effects on osteogenic conversion and adipogenic differentiation/conversion. Interestingly, heparin did not only significantly increase mRNA expression and enzyme activity of the osteogenic marker alkaline phosphatase (ALP), but it also promoted mineralization during osteogenic differentiation and conversion. Furthermore, the mRNA expression of the osteogenic marker bone morphogenic protein 4 (BMP4) was enhanced. In addition, heparin administration partly prevented adipogenic differentiation and conversion demonstrated by reduced lipid droplet formation along with a decreased expression of adipogenic markers. Moreover, luciferase reporter assays, inhibitor experiments and gene expression analyses revealed that heparin had putative permissive effects on osteogenic signaling via the BMP pathway and reduced the mRNA expression of the Wnt pathway inhibitors dickkopf 1 (DKK1) and sclerostin (SOST). Taken together, our data show a rather supportive than inhibitory effect of heparin on osteogenic hBMSC differentiation and conversion in vitro. Further studies will have to investigate the net effects of heparin administration on bone formation versus bone resorption in vivo to unravel the molecular mechanisms of heparin-associated osteoporosis and reconcile conflicting experimental data with clinical observations.     

Parathyroid hormone induces differentiation of mesenchymal stromal/stem cells by enhancing bone morphogenetic protein signaling

Parathyroid hormone (PTH) stimulates bone remodeling and induces differentiation of bone marrow mesenchymal stromal/stem cells (MSCs) by orchestrating activities of local factors such as bone morphogenetic proteins (BMPs). The activity and specificity of different BMP ligands are controlled by various extracellular antagonists that prevent binding of BMPs to their receptors. Low-density lipoprotein receptor-related protein 6 (LRP6) has been shown to interact with both the PTH and BMP extracellular signaling pathways by forming a complex with parathyroid hormone 1 receptor (PTH1R) and sharing common antagonists with BMPs. We hypothesized that PTH-enhanced differentiation of MSCs into the osteoblast lineage through enhancement of BMP signaling occurs by modifying the extracellular antagonist network via LRP6. In vitro studies using multiple cell lines, including Sca-1⁺CD45^–CD11b^–MSCs, showed that a single injection of PTH enhanced phosphorylation of Smad1 and could also antagonize the inhibitory effect of noggin. PTH treatment induced endocytosis of a PTH1R/LRP6 complex and resulted in enhancement of phosphorylation of Smad1 that was abrogated by deletion of PTH1R, β-arrestin, or chlorpromazine. Deletion of LRP6 alone led to enhancement of pSmad1 levels that could not be further increased with PTH treatment. Finally, knockdown of LRP6 increased the exposure of endogenous cell-surface BMP receptor type II (BMPRII) significantly in C2C12 cells, and PTH treatment significantly enhanced cell-surface binding of ¹²⁵I-BMP2 in a dose- and time-dependent manner, implying that LRP6 organizes an extracellular network of BMP antagonists that prevent access of BMPs to BMP receptors. In vivo studies in C57BL/6J mice and of transplanted green fluorescent protein (GFP)-labeled Sca-1⁺CD45^–CD11b^–MSCs into the bone marrow cavity of Rag2^−/− immunodeficient mice showed that PTH enhanced phosphorylation of Smad1 and increased commitment of MSCs to osteoblast lineage, respectively. These data demonstrate that PTH enhancement of MSC differentiation to the osteoblast lineage occurs through a PTH- and LRP6-dependent pathway by endocytosis of the PTH1R/LRp6 complex, allowing enhancement of BMP signaling. © 2012 American Society for Bone and Mineral Research.     

Bone morphogenetic proteins 2, 5, and 6 in combination stimulate osteoblasts but not osteoclasts in vitro

Bone regeneration is required for fracture healing. Various procedures have been used to promote osteogenesis with bone morphogenetic proteins (BMPs). We assessed the effects of BMP‐2, BMP‐5, and BMP‐6 in isolated and combined use on the generation of osteoblasts and osteoclasts by comparing the osteoclastic potency of each on osteoclasts of primary murine bone marrow cells. Subsequently, cells were stained for tartrate‐resistant acid phosphatase, and real time PCR analysis of receptor activator of NKκB ligand and osteoprotegerin was conducted. The same combination of BMPs was used to assess their potential to enhance osteoblasts, employing a mineralization assay and real‐time PCR analysis of collagen type‐1, runx2, and osterix. While BMP‐2 alone and the combination of BMP‐2 and BMP‐5 significantly enhanced osteoclastogenesis, BMP‐2, BMP‐5, and BMP‐6 in combination did not have additional effects. However, the combined use of BMP‐2, BMP‐5, and BMP‐6 had an additive effect on matrix mineralization and osterix expression in osteoblasts. Our study shows that the combination of BMP‐2, BMP‐5, and BMP‐6 stimulates osteoblasts but not osteoclastogenesis. Thus, the synergistic use of various BMPs might improve effective bone regeneration in the clinical setting. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res     

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.     

New bone formation in axial spondyloarthritis

New bone formation of the entheses with possible progression to ankylosis is among the hallmarks of axial spondyloarthritis. Radiographic scores are available for evaluating axial new bone formation. The complex systems that underlie new bone formation involve Wnt bone-formation signaling and the natural Wnt inhibitors DKK-1 and sclerostin, as well as growth factors such as the bone morphogenetic proteins (BMPs); these systems are modulated by proinflammatory cytokines, most notably TNF. Factors that predict progressive new bone formation include the presence of syndesmophytes, various biological markers (CRP, MMP-3, sclerostin, DKK-1, and BMP), smoking, and prior vertebral corner abnormalities (inflammation and, above all, resolved inflammation). Nonsteroidal antiinflammatory agents seem able to slow the pace of radiographic progression, an effect not documented to date with TNF antagonists in patients with axial spondyloarthritis.