Sclerostin Stimulates Osteocyte Support of Osteoclast Activity by a RANKL-Dependent Pathway

Sclerostin is a product of mature osteocytes embedded in mineralised bone and is a negative regulator of bone mass and osteoblast differentiation. While evidence suggests that sclerostin has an anti-anabolic role, the possibility also exists that sclerostin has catabolic activity. To test this we treated human primary pre-osteocyte cultures, cells we have found are exquisitely sensitive to sclerostin, or mouse osteocyte-like MLO-Y4 cells, with recombinant human sclerostin (rhSCL) and measured effects on pro-catabolic gene expression. Sclerostin dose-dependently up-regulated the expression of receptor activator of nuclear factor kappa B (RANKL) mRNA and down-regulated that of osteoprotegerin (OPG) mRNA, causing an increase in the RANKL∶OPG mRNA ratio. To examine the effects of rhSCL on resulting osteoclastic activity, MLO-Y4 cells plated onto a bone-like substrate were primed with rhSCL for 3 days and then either mouse splenocytes or human peripheral blood mononuclear cells (PBMC) were added. This resulted in cultures with elevated osteoclastic resorption (approximately 7-fold) compared to untreated co-cultures. The increased resorption was abolished by co-addition of recombinant OPG. In co-cultures of MLO-Y4 cells with PBMC, SCL also increased the number and size of the TRAP-positive multinucleated cells formed. Importantly, rhSCL had no effect on TRAP-positive cell formation from monocultures of either splenocytes or PBMC. Further, rhSCL did not induce apoptosis of MLO-Y4 cells, as determined by caspase activity assays, demonstrating that the osteoclastic response was not driven by dying osteocytes. Together, these results suggest that sclerostin may have a catabolic action through promotion of osteoclast formation and activity by osteocytes, in a RANKL-dependent manner

Recombinant sclerostin antagonizes effects of ex vivo mechanical loading in trabecular bone and increases osteocyte lacunar size

Sclerostin has emerged as an important regulator of bone mass. We have shown that sclerostin can act by targeting late osteoblasts/osteocytes to inhibit bone mineralization and to upregulate osteocyte expression of catabolic factors, resulting in osteocytic osteolysis. Here we sought to examine the effect of exogenous sclerostin on osteocytes in trabecular bone mechanically loaded ex vivo. Bovine trabecular bone cores, with bone marrow removed, were inserted into individual chambers and subjected to daily episodes of dynamic loading. Cores were perfused with either osteogenic media alone or media containing human recombinant sclerostin (rhSCL) (50 ng/ml). Loaded control bone increased in apparent stiffness over time compared with unloaded bone, and this was abrogated in the presence of rhSCL. Loaded bone showed an increase in calcein uptake as a surrogate of mineral accretion, compared with unloaded bone, in which this was substantially inhibited by rhSCL treatment. Sclerostin treatment induced a significant increase in the ionized calcium concentration in the perfusate and the release of -CTX at several time points, an increased mean osteocyte lacunar size, indicative of osteocytic osteolysis, and the expression of catabolism-related genes. Human primary osteocyte-like cultures treated with rhSCL also released -CTX from their matrix. These results suggest that osteocytes contribute directly to bone mineral accretion, and to the mechanical properties of bone. Moreover, it appears that sclerostin, acting on osteocytes, can negate this effect by modulating the dimensions of the lacunocanalicular porosity and the composition of the periosteocyte matrix

The regulation of the sclerostin gene and the catabolic effects of sclerostin protein on bone.

Age and disease-related bone loss is a major health issue. Bone tissue is constantly remodelled throughout life in order to maintain a healthy skeleton and bone loss is caused by an imbalance in the remodelling process. Bone remodelling is a highly coordinated process between osteoclasts, osteoblasts and osteocytes, with bone targeted for renewal being resorbed by osteoclasts and the resorbed bone replaced by the activities of osteoblasts and osteocytes. During the synthesis of new bone organic matrix, osteoblasts become embedded and differentiate into osteocytes. Osteocytes were previously thought to be terminally differentiated, quiescent cells. However, a wealth of recent evidence suggests that osteocytes play important and dynamic roles. Recently, the osteocyte expressed protein, sclerostin, was identified to be a major regulator of bone formation. Various pharmaceutical companies are currently in the process of developing therapies to neutralise sclerostin, in order to reverse its antianabolic effects on bone. In pre-clinical and clinical studies to date, neutralising sclerostin had bone anabolic effects, and although anti-catabolic effects were also observed, these were usually reported as incidental events. Stemming from observations made by our group of pro-catabolic stimuli up-regulating sclerostin expression, it was hypothesised that sclerostin may have a catabolic action in addition to its anti-anabolic actions. Subsequent work identified the pre-osteocyte/osteocyte as cellular targets of sclerostin, and gene microarray analyses of osteocyte-like cells treated with recombinant sclerostin, led to the discovery of two novel mechanisms, by which sclerostin may act in a catabolic manner. As presented in Chapter 2, the work undertaken for this thesis demonstrated that sclerostin promotes osteocyte support of osteoclast formation and activity, consistent with recent reports by other groups that suggest osteocytes play a central role in regulating the formation and activity of osteoclasts. As presented in Chapter 3, sclerostin can also increase the expression by osteocytes of resorption-related molecules, in particular carbonic anhydrase 2. The importance of this observation is that acidification of the extracellular space by osteocytes could promote osteocytic release of mineral and increase of the osteocyte lacunar size, a process termed ‘osteocytic osteolysis’. The results presented in Chapter 3 provide the first mechanistic evidence for this process. As presented in Chapter 4, 1α,25-dihydroxyvitamin D (1,25D) was also identified as a regulator of SOST/sclerostin expression and a putative vitamin D response element (VDRE) was shown to be present in the proximal 6.3 kb SOST promoter. In summary, the novel work presented in this thesis expands our knowledge of the activity and regulation of sclerostin. Together, these findings suggest that a subset of pro-catabolic stimuli may induce sclerostin expression, which in turn may act to promote both osteoclastic and osteocytic removal of bone. This research has implications for the pharmacological inhibition of sclerostin, which is currently being pursued commercially. In embarking on such therapy, it is essential to understand the biology of sclerostin as completely as possible.Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 201

Mechanical loading response in trabecular bone is abrogated by sclerostin - A direct demonstration

Sclerostin is expressed almost exclusively by mature osteocytes in bone. Recent findings from this lab indicate that sclerostin targets pre-osteocytes and osteocytes to regulate bone mineralisation(1), osteoclast activity(2), and, potentially, osteocytic osteolysis(3). Sclerostin expression in vivo is associated with the response of osteocytes to mechanical loading and unloading. The aim of this study was to examine the direct effects of sclerostin on loading-induced bone growth ex vivo. For this, 10x5mm bovine sternum trabecular bone cores were perfused with osteogenic media at 37°C for up to 3 weeks in individual bone culture chambers. The cores were divided into 3 groups; a) mechanically loaded (300 cycles, 4000 µstrain, 1 Hz/day), b) identical loading regime with continuous perfusion of 50 ng/ml recombinant human sclerostin and c) unloaded controls. Loading was accomplished using the Zetos™ long-term bone organ culture and piezo-electric bone loading system. Daily measurements of the bone core stiffness, media pH and ionic calcium concentrations were made. Histomorphometric assessment, including fluorochrome labelling analysis, was made at the end of the experiment. Bone stiffness increased greatly with mechanical loading but this was attenuated significantly with the addition of sclerostin. The pH of the media after 24 hours decreased and ionic calcium concentrations increased in the presence of sclerostin when compared to mechanical loading alone. Sclerostin also completely abrogated loading-induced calcium/calcein uptake by the bone cores. Together, the results suggest that an osteocyte/osteoclast response to sclerostin was responsible for these effects. Our results are the first direct evidence for a negative effect of sclerostin on the anabolic response of bone to mechanical loading

Direct effect of sclerostin on the mechanical loading response in bovine bone

Sclerostin is expressed exclusively by mature osteocytes in bone. Our recent findings indicate that sclerostin targets pre-osteocytes/osteocytes to regulate bone mineralisation(1), osteoclast activity(2), and, potentially, osteocytic osteolysis(3). Sclerostin expression in vivo is associated with the osteocyte response to mechanical loading/unloading. The aim of this study was to examine the effects of sclerostin on loading-induced bone growth ex vivo. For this, 10x5mm bovine sternum trabecular bone cores were perfused with osteogenic media at 37"C for up to 3 weeks in individual bone culture chambers. The cores were divided into 3 groups; a) mechanically loaded (300 cycles, 4000 microstrain, 1 Hz/day), b) identical loading regime with continuous perfusion of 50 ng/ml recombinant human sclerostin and c) unloaded controls. Loading was accomplished using a Zetos(TM) bone loading system. Daily measurements of bone stiffness, media pH and ionic calcium concentrations were made. Histomorphometric assessment, including fluorochrome labelling analysis, was made at the end of the experiment. Bone stiffness increased with mechanical loading but this was blocked by the addition of sclerostin. Media pH decreased and ionic calcium concentrations increased in the presence of sclerostin. Sclerostin also completely abrogated loading-induced calcium/calcein uptake, together suggesting that an osteocyte/osteoclast response to sclerostin was responsible for these effects. Our results are the first direct evidence for a negative effect of sclerostin on the anabolic response to mechanical loading. 1. Atkins et al. J Bone Miner Res. 201 1 26(7):1425-36. 2. Wijenayaka et al. PLoS One. 201 1 ;6(10):e25900. 3. Kogawa M. et al. Abstract at this meeting

Effects of rhSCL on TRAP⁺ multinucleated cell formation.

<p>The effect of rhSCL on osteoclastogenesis was tested in co-cultures of MLO-Y4 cells with A) mouse splenocytes and B) human PBMC. In both cases MLO-Y4 cells were seeded into type I collagen coated culture wells and cultured for 72 h in the absence or presence of rhSCL, as indicated, prior to the addition of either splenocytes or PBMC. All cultures received rhM-CSF at 25 ng/ml. RhOPG was added to some cultures at 100 ng/ml and rhRANKL (100 ng/ml) added to monocultures of either splenocytes or PBMC to confirm the osteoclast-forming potential of these populations (not shown). Media were replenished every 3 days. Cultures were fixed and stained for TRAP at day 6. TRAP-positive MNC, defined as cells with >3 nuclei, were counted from quadruplicate wells. Asterisks denote significant differences to the no SCL control (** <i>p</i><0.01, *** <i>p</i><0.001) and the effect of OPG relative to the corresponding SCL-only treatment is indicated by Φ (<i>p</i><0.001). C) In the case of MLO-Y4/PBMC co-cultures and cells formed in PBMC monocultures treated with rhRANKL, the relative size (in pixels) of TRAP⁺ MNC formed (in each case >50 cells) was measured by Image J analysis. Difference to the no SCL control is indicated by ** <i>p</i><0.01; † indicates difference of rhRANKL control to rhSCL at 100 ng/ml (<i>p</i><0.01). Data shown are representative of at least 3 independent experiments.</p

Effects of rhSCL on osteoclast resorptive activity.

<p>Co-cultures of MLO-Y4 cells and A) mouse splenocytes and B) human PBMC were established, in which MLO-Y4 cells were seeded onto bone-like Osteologic™ slides and cultured for 72 h in the absence or presence of rhSCL as indicated prior to the addition of either splenocytes or PBMC. All cultures received rhM-CSF at 25 ng/ml and some cultures were treated with rhOPG (100 ng/ml). Media were replenished every 3 days for 14 days. Cells were removed and slides developed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025900#s2" target="_blank">Materials and Methods</a> and resorption. The entire surface areas of the developed slides were imaged using an Olympus SZX10 dissecting microscope at high resolution, depicted for the PBMC co-cultures below the corresponding histograms in panel B) (red bars indicate 500 µm). Total resorbed area was then quantified from quadruplicate wells using ImageJ software. C) In the case of MLO-Y4/PBMC co-cultures, mean resorption pit size was determined and expressed as fold-change from the corresponding untreated co-culture. Differences to the ‘no SCL’ control in each panel are indicated by * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001. The effect of OPG relative to the corresponding SCL-only treatment is indicated by Φ (<i>p</i><0.001). Data shown are representative of 3 independent experiments.</p

Effects on gene expression of continuous exposure of mineralizing NHBC cultures to rhSCL.

<p>NHBC were cultured under mineralizing conditions for up to 35 days in the absence or presence of rhSCL at 50 ng/ml. Media were replenished every 3–4 days. At the time points indicated, total RNA was prepared and real-time RT-PCR performed to determine mRNA expression of A) <i>DMP1</i>, B) <i>SOST</i>, C) <i>OPG</i> and D) <i>RANKL</i>. Data shown are means of triplicate reactions ± SD normalized to expression of 18S mRNA. Significant differences to untreated control are indicated by * <i>p</i><0.05 and *** <i>p</i><0.001. Similar results were obtained from 3 independent experiments using NHBC from different donors.</p

Vitamin K promotes mineralization, osteoblast-to-osteocyte transition, and an anticatabolic phenotype by y-carboxylation-dependent and -independent mechanisms

Copyright © 2009 by the American Physiological Society.The vitamin K family members phylloquinone (vitamin K1) and the menaquinones (vitamin K2) are under study for their roles in bone metabolism and as potential therapeutic agents for skeletal diseases. We have investigated the effects of two naturally occurring homologs, phytonadione (vitamin K1) and menatetrenone (vitamin K2), and those of the synthetic vitamin K, menadione (vitamin K3), on human primary osteoblasts. All homologs promoted in vitro mineralization by these cells. Vitamin K1-induced mineralization was highly sensitive to warfarin, whereas that induced by vitamins K2 and K3 was less sensitive, implying that -carboxylation and other mechanisms, possibly genomic actions through activation of the steroid xenobiotic receptor, are involved in the effect. The positive effect on mineralization was associated with decreased matrix synthesis, evidenced by a decrease from control in expression of type I collagen mRNA, implying a maturational effect. Incubation in the presence of vitamin K2 or K3 in a three-dimensional type I collagen gel culture system resulted in increased numbers of cells with elongated cytoplasmic processes resembling osteocytes. This effect was not warfarin sensitive. Addition of calcein to vitamin K-treated cells revealed vitamin K-dependent deposition of mineral associated with cell processes. These effects are consistent with vitamin K promoting the osteoblast-to-osteocyte transition in humans. To test whether vitamin K may also act on mature osteocytes, we tested the effects of vitamin K on MLO-Y4 cells. Vitamin K reduced receptor activator of NF-B ligand expression relative to osteoprotegerin by MLO-Y4 cells, an effect also seen in human cultures. Together, our findings suggest that vitamin K promotes the osteoblast-to-osteocyte transition, at the same time decreasing the osteoclastogenic potential of these cells. These may be mechanisms by which vitamin K optimizes bone formation and integrity in vivo and may help explain the net positive effect of vitamin K on bone formationGerald J. Atkins, Katie J. Welldon, Asiri R. Wijenayaka, Lynda F. Bonewald and David M. Findla