Original Full Length ArticleSost downregulation and local Wnt signaling are required for the osteogenic response to mechanical loading
Highlights
► The osteocyte network might detect variations in the level of mechanical strain and distribute signals leading to adaptive responses. ► Loading downregulates the bone formation inhibitor and Wnt antagonist Sost/sclerostin in osteocytes adjacent to high bone formation surfaces. ► We used DMP1-8 kb-SOST mice, in which human SOST is expressed in osteocytes and it is not decreased by loading. ► Maintaining high sclerostin expression in DMP1-8 kb-SOST mice prevented Wnt activation and the increase in bone formation induced by loading. ► Thus, mechanotransduction necessitates sclerostin reduction in osteocytes to direct Wnt signaling and osteogenesis to where bone is needed.
Introduction
The skeleton adapts to meet mechanical needs by changing its mass, shape, and microarchitecture [1], [2], [3]. Osteocytes (former osteoblasts buried in the bone matrix) are proposed to act as mechanosensors [4]. Whereas bone-forming osteoblasts and bone-resorbing osteoclasts are present on bone surfaces for relatively short periods of time and in low numbers, osteocytes are by far the most abundant resident cells and are present throughout the entire bone tissue. Osteocytes are also the core of a functional syncytium that extends from the mineralized bone matrix to the bone surface and the bone marrow, reaching the blood vessels. Their abundance and strategic location make osteocytes the most suitable candidates for detecting variations in the level of strain and for distributing signals leading to adaptive responses [5].
Regulation of the expression of sclerostin, a glycoprotein encoded by the Sost gene, has emerged as a compelling mechanism by which osteocytes control the activity of bone remodeling cells [6]. This protein is secreted by osteocytes and acts in a paracrine (and potentially autocrine) fashion to inhibit bone formation by antagonizing the pro-differentiating and survival actions of Wnts in osteoblasts. Genetic and pharmacologic evidence supports this mechanism. Loss of SOST expression in humans causes the high bone mass disorders Van Buchem's disease [7] and sclerosteosis [8]. Mice with targeted deletion of the Sost gene also display progressive high bone mass and increased bone strength [9], [10]; whereas, conversely, transgenic mice overexpressing SOST exhibit low bone mass [11], [12], [13]. Pharmacologic inhibition of sclerostin with neutralizing antibodies leads to marked anabolic effects in several preclinical osteopenic animal models and has been met with promising results in clinical settings [6], [14]. Sclerostin is also regulated by hormonal stimuli that affect the skeleton. In particular, elevation of parathyroid hormone (PTH), either in an intermittent or a continuous mode, downregulates sclerostin expression in osteocytes in mice and decreases the circulating levels of the protein in humans [15], [16], [17], [18], [19].
Mechanical forces are essential for the development, growth, and maintenance of the skeleton. Skeletal sites subjected to high mechanical strains exhibit high bone formation, whereas unloaded bones display reduced bone formation. These adaptive responses of the skeleton are thought to be mediated by osteocytes and to result from regulation of the Wnt signaling pathway [4]. Using the murine ulnar loading model [20], we have demonstrated that cortical bone areas exposed to high mechanical strain exhibit a reduction in sclerostin-positive osteocytes that is associated with higher bone formation on adjacent periosteal surfaces [21]. This evidence suggested that osteocytes coordinate the osteogenic response to mechanical force by downregulating sclerostin, thereby locally unleashing Wnt signaling. We now show that mice overexpressing a human SOST transgene in osteocytes, which is not downregulated by loading, failed to exhibit activation of the Wnt pathway and the anabolic response to mechanical stimulation. Thus, Sost downregulation is an obligatory step for mechanotransduction.
Section snippets
DMP1-SOST transgenic mice
DMP1-SOST transgenic mice were generated by inserting the human SOST cDNA (I.M.A.G.E. clone ID: 40009482, American Tissue Culture Collection, Manassas, VA) downstream from a DNA fragment containing 8 kb of the 5′-flanking region, the first exon, the first intron, and 17 bp of exon 2 of the murine dentin matrix protein 1 (DMP1) gene [22], and upstream from a 140 bp fragment containing the rabbit beta-globin polyadenylation sequence, as previously described [13]. Hemizygous DMP1-SOST mice or wild
Transgenic mice overexpressing human SOST in osteocytes (DMP1-SOST) exhibit low bone mass in the axial skeleton, but no changes in mass or geometry of long bones
In a previous study, we showed that DMP1-SOST mice exhibit a marked decrease in BMD in the spine with minimal if any changes in the long bones at 8 weeks of age [13]. Consistent with these earlier findings, longitudinal analysis of a cohort of DMP1-SOST mice and control littermates from 4 to 16 weeks of age demonstrated a progressive decrease in BMD in the lumbar spine and total body BMD in both female and male mice (Table 1). A transient decrease in femoral BMD was observed at 4 weeks of age in
Discussion
Although it has been long recognized that mechanical loading stimulates new bone formation on surfaces adjacent to areas to which strain is applied [20], [28], the cellular and molecular mechanism(s) responsible for this phenomenon still remains unclear. Earlier studies demonstrated that loading reduces the expression of sclerostin, the bone formation inhibitor encoded by the Sost gene, in osteocytes located close to high bone formation surfaces [21], and that activation of the Wnt pathway is
Acknowledgments
The authors thank R. Lee and J.D. Benson for technical assistance. This research was supported by the National Institutes of Health (R01DK076007 and an American Recovery and Reinvestment Act supplement).
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Current address: Department of Internal Medicine, College of Medicine, Yonsei University, Seoul, Republic of Korea.