Osteoblast-Specific Wnt Secretion Is Required for Skeletal Homeostasis and Loading-Induced Bone Formation in Adult Mice

Abstract

Wnt signaling is critical to many aspects of skeletal regulation, but the importance of Wnt ligands in the bone anabolic response to mechanical loading is not well established. Recent transcriptome profiling studies by our laboratory and others show that mechanical loading potently induces genes encoding Wnt ligands, including Wnt1 and Wnt7b. Based on these findings, we hypothesized that mechanical loading stimulates adult bone formation by inducing Wnt ligand expression. To test this hypothesis, we inhibited Wnt ligand secretion in adult (5 months old) mice using a systemic (drug) and a bone-targeted (conditional gene knockout) approach, and subjected them to axial tibial loading to induce lamellar bone formation. Mice treated with the Wnt secretion inhibitor WNT974 exhibited a decrease in bone formation in non-loaded bones as well as a 54% decline in the periosteal bone formation response to tibial loading. Next, osteoblast-specific Wnt secretion was inhibited by dosing 5-month-old Osx-CreERT2; Wls F/F mice with tamoxifen. Within 1 to 2 weeks of Wls deletion, skeletal homeostasis was altered with decreased bone formation and increased resorption, and the anabolic response to loading was reduced 65% compared to control (Wls F/F ). Together, these findings show that Wnt ligand secretion is required for adult bone homeostasis, and furthermore establish a role for osteoblast-derived Wnts in mediating the bone anabolic response to tibial loading.

Loading-Induced Bone Formation is Mediated by Wnt1 Induction in Osteoblast-Lineage Cells

“…OsxCreERT2; Wls F/F ; mice were tamoxifen-treated to inactivate Wntless – the Wnt-specific transport receptor responsible for shuttling Wnt ligands to the cell surface for exocytosis 8 – thereby blocking the secretion of all Wnts from osteoblasts and osteocytes. We showed that the periosteal response to tibial loading was 65% lower in Wls knockouts relative to WlsF/F controls, indicating that Wnts produced by osteoblast-lineage cells are important mediators of loading-induced bone formation 7 .…”

“…To achieve this, tamoxifen dissolved in corn oil was delivered by oral gavage for 5 days. We previously used a similar dosing strategy for Cre reporter analysis in 5-month old OsxCreERT2; Rosa-Ai9 mice, which showed robust TdTomato reporter expression on the bone surface (osteoblasts) and throughout the cortical bone (osteocytes) within days of tamoxifen treatment (Lawson, et al 2021) – indicating that OsxCreERT2 targets both osteoblasts and osteocytes in the bones of adult mice.…”

“…Bilateral tibias were collected and snap-frozen in liquid nitrogen as described previously 7, 12 . Briefly, tibias were exposed and soft tissues were removed from the bone surface using forceps, leaving the periosteum intact.…”

“…For example, loading was found to suppress expression of Wnt antagonist Sclerostin in the bone (Robling, et al 2008), and in an ulnar loading model, Tu et al subsequently showed that the downregulation of this antagonist was in fact necessary for the bone anabolic response to loading (Tu, et al 2012). Furthermore, mice lacking Wnt co-receptor Lrp5 exhibit a diminished bone anabolic response to loading (Zhao, et al, 2013), as do mice treated with a drug that blocks Wnt ligand secretion (Lawson, et al 2021).…”

“…Using OsxCreERT2 to target osteoblast lineage cells, 5-month old WlsF/F; OsxCreERT2 mice were tamoxifen-treated to inactivate Wntless – the Wnt-specific transport receptor responsible for shuttling Wnt ligands to the cell surface for exocytosis – to collectively block the secretion of all Wnt ligands. In a tibial loading model, we showed that the periosteal response to loading was 65% lower in Wls knockouts relative to OsxCreERT2-negative controls (Lawson, et al 2021). This study was congruent with a wider body of evidence implicating Wnt signaling in the skeletal response to loading, and furthermore showed that Wnt ligands produced by osteoblast-lineage cells were important mediators of loading-induced bone formation.…”