TOPGAL Mice Show That the Canonical Wnt Signaling Pathway Is Active During Bone Development and Growth and Is Activated by Mechanical Loading In Vitro

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Abstract
We identified cellular targets of canonical Wnt signaling within the skeleton, which included chondrocytes, osteoblasts, and osteocytes in growing bone, but only osteocytes and chondrocytes in the mature skeleton. Mechanical deformation induced Wnt signaling in osteoblasts in vitro. Genetic evidence in mice and humans has implicated the canonical Wnt signaling pathway in the control of skeletal development and bone mass. However, little is known of the details of Wnt signaling in the skeleton in vivo. We used Wnt indicator TOPGAL mice to identify which cells activated this pathway during bone development and in the mature skeleton. We examined canonical Wnt signaling during embryonic and neonatal bone development in TOPGAL mice. The TOPGAL transgene consists of a beta-galactosidase gene driven by a T cell factor (TCF)beta-catenin responsive promoter so that canonical Wnt activity can be detected by X-gal staining. Expression of Wnt signaling components was examined in primary calvarial cell cultures by RT-PCR. The effect of mechanical deformation on Wnt signaling was examined in primary calvarial cells grown on collagen I and stretched using Flexercell Tension Plus System FX-4000T. Immunohistochemistry was used to examine the localization of beta-catenin in cartilage, bone, and cultured calvarial cells exposed to physical deformation. Canonical Wnt signaling was active in several cell types in the fetal and neonatal skeleton, including chondrocytes, osteoblasts, and osteocytes. With age, activation of Wnt signaling became less prominent but persisted in chondrocytes and osteocytes. Although osteoblasts in culture expressed many different individual Wnt's and Wnt receptors, the TOPGAL transgene was not active in these cells at baseline. However, Wnt signaling was activated in these cells by physical deformation. Together with the activation of canonical Wnt signaling in osteocytes seen in vivo, these data suggest that Wnt signaling may be involved in the coupling of mechanical force to anabolic activity in the skeleton.