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Sclerostin is a delayed secreted product of osteocytes that inhibits bone formation

Kenneth E. S. Poole^*,1, Rutger L. van Bezooijen^,, Nigel Loveridge^*, Herman Hamersma, Socrates E. Papapoulos, Clemens W. Löwik and Jonathan Reeve^*

^* MRC Bone Research Group, Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge, UK;
Department of Endocrinology and Metabolic Diseases and
Department of Molecular Cell Biology, Leiden University Medical Centre, Leiden, The Netherlands; and
Otolaryngologist, Weltevreden Park, South Africa

¹ Correspondence: Bone Research Group, Department of Medicine, Level 5, University of Cambridge, Addenbrooke’s Hospital, Box 157, Cambridge, England CB2 2QQ, UK. E-mail: kesp2{at}cam.ac.uk

SPECIFIC AIMS

Recent experiments have shown that sclerostin, the secreted protein product of the SOST gene is an osteocyte-derived inhibitor of cultured osteoblasts. Its absence results in the high bone mass clinical disorder sclerosteosis. The aim of this study was to determine which osteocytes express sclerostin in human bone and to test our hypothesis that sclerostin, as a secreted glycoprotein, is produced at or after primary mineralization by osteocytes in newly forming osteons to restrict bone formation by osteoblasts.

PRINCIPAL FINDINGS

1. Sclerostin was detected in a high proportion of osteocytes and was widespread in the lacuno-canalicular network; osteoblasts were consistently negative for staining in all biopsies
We investigated the distribution of sclerostin in bone cells using immunohistochemistry of fresh, frozen undecalcified sections of adult human bone. A large proportion of osteocytes in mineralized cortical and cancellous bones were positive for sclerostin with diffuse staining along the osteocyte canaliculi (Figs. 1 , 2 ). Osteoblasts, bone lining cells (retired osteoblasts that have ceased forming bone and cover most of the bone surface) and periosteal osteoblasts exhibited no sclerostin staining in iliac bone. Such osteocyte-specificity and canalicular staining was widespread throughout mineralized bone, but was absent in osteocytes located near to bone surfaces. A novel section mapping technique was employed using accurate montage images of whole cortices captured using an automated microscope stage and image analysis. With this method, we established the proportions of osteocytes staining for sclerostin, revealing a high percentage of sclerostin-positive cells in the cortex (median 84.5%, IQR 76.8, 89.4) and in cancellous bone (median 72.4%, IQR 67.7,83.3, P=0.08 compared with cortical bone).

View larger version (134K): [in this window] [in a new window]   Figure 1. Human iliac cortex bone stained with mouse monoclonal antisclerostin antibody (DAB brown chromagen). Toluidine blue counter stain. Most osteocytes (black arrow) stained for sclerostin but osteocytes close to forming surfaces (blue arrow) and osteoblasts (white arrow) did not. Polarized light. x63 magnification.

View larger version (141K): [in this window] [in a new window]   Figure 2. High power bright field image showing osteocytic sclerostin staining x252 (left) and a Z-stack composite montage (right) of two osteocytes and their canaliculi stained for sclerostin, oil immersion x1000.

2. Sclerostin-negative osteocytes were located significantly closer to bone surfaces than sclerostin positive osteocytes
We then used the section-mapping technique to explore the spatial relationships between sclerostin negative osteocytes and bone surfaces in cortical bone. The distance of each osteocyte from its closest surface (haversian canals, periosteal, and endosteal surfaces) was calculated automatically with a program that excluded the cut edge of the sections. This indicated that sclerostin-negative osteocytes were located significantly closer to bone surfaces (median distance 57 µm, IQR 33-96, n=1098) than the more numerous sclerostin-positive osteocytes (102 µm, IQR 68-144; n=5133, P<0.0001).

3. Osteons in the process of bone formation contained more sclerostin-negative osteocytes
To test whether the closer proximity of the sclerostin negative osteocytes to surfaces was because they were recently embedded osteocytes at sites of bone formation, serial sections of sclerostin-immunostained, alkaline phosphatase and double tetracycline-labeled bone were analyzed. By using polarized light to capture the serial sections (Fig. 1) , interstitial regions could be excluded to focus on either forming (alkaline phosphatase, ALP positive) or quiescent (ALP negative) osteons. After categorizing osteons based on their sclerostin status, contingency analysis confirmed that quiescent (ALP negative) osteons were more likely to contain all sclerostin positive osteocytes whereas forming (ALP positive) osteons were more likely to contain sclerostin negative or mixed osteocytes (²=33.5, P < 0.0001).

4. Recently embedded osteocytes, including those within unmineralized osteoid were almost all negative for sclerostin; sclerostin secretion by new osteocytes is therefore delayed until the cells have matured and are surrounded by a mineralized matrix
Analysis of high power images confirmed that 27 of 28 (96.4%) recently embedded osteocytes (located between the first fluorescent label and the bone surface) were negative for sclerostin. Since the first of the two demeclocycline treatments finished 16 days before the bone biopsy was performed, the majority of new osteocytes were negative for sclerostin staining for at least 16 days. Our findings are in keeping with experiments using cultures of differentiating osteoblasts, since SOST mRNA expression was not detected in undifferentiated cells but occurred after the onset of in vitro mineralization. The majority of nonremodeling cortical osteons (66%) contained exclusively sclerostin positive osteocytes indicating that after mineralization, production of this inhibitory signal is prevalent in bone.

5. On approaching the periosteum, the proportion of sclerostin negative cells increased logarithmically
Examining the cortex by starting from 150 microns parallel to the periosteal surface and moving in 10 micron wide bands toward the periosteal surface there was a logarithmic increase in the proportion of sclerostin negative osteocytes.

6. Absence of sclerostin did not appear to influence osteocyte lacunar density
We found no reduction in the recruitment of osteocytes (as inferred from osteonal lacunar density) in the osteons of bone specimens removed at operation from 3 cases of sclerosteosis and 3 controls. This finding suggests that sclerostin primarily influences the later stage of bone formation rather than earlier events such as osteocyte recruitment.

CONCLUSIONS AND SIGNIFICANCE

We conclude that sclerostin secretion by newly embedded osteocytes is delayed so that the cells must mature or receive a later signal that triggers sclerostin expression. These findings are consistent with the concept that newly embedded osteocytes secrete sclerostin only after the local onset of mineralization to inhibit cortical bone formation and osteon infilling by cells of the osteoblast lineage (Fig. 3 ). In this way, a sufficient but not excessive amount of cortical bone may be formed to fill in osteonal and other canals without compressing their contents. Likewise it is envisaged that trabeculae are maintained at appropriate thickness.

View larger version (41K): [in this window] [in a new window]   Figure 3. Schematic diagram of the proposed regulation of a remodeling cortical osteon by osteocytic sclerostin expression.

Osteocytes, the most abundant cells in adult human bone are thought to sense loading stimuli and regulate remodeling and bone turnover processes. Our findings suggest that osteocytes, which have access to surface osteoblasts and bone lining cells through their dendritic connections, may provide the key inhibitory signal sclerostin to inactive bone surfaces. In contrast, active osteoblasts within forming osteons are protected from inhibition by sclerostin by a layer of sclerostin negative osteocytes, permitting bone formation to continue at specific sites (Fig. 3) including the periosteum.

This study adds to the growing body of knowledge concerning the osteocytes’ regulatory role in maintaining bone structure and strength. Our findings are the first demonstration of how live osteocytes might fine-tune bone formation by the timely secretion of an inhibitory signal. This observation shifts the emphasis away from sclerostin as a possible regulator of preosteoblast proliferation (e.g., through interaction with bone morphogenetic proteins) toward a role in determining bone’s microarchitectural development through its very precise geographical and temporal expression as a local modulator of mature osteoblast function. These experiments strengthen the supposition that sclerostin is a key inhibitor, determining the normal extent of bone formation and consequently protecting against the deleterious effects of uncontrolled bone growth (sclerosteosis). Our findings help us understand the functional role of osteocytes in maintaining bone tissue and by showing how restricted sclerostin expression is in bone, making the SOST gene product a highly promising target for developing new pharmaceutical approaches to managing osteoporosis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4221fje;

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