The recent genetic evidence that LRP5 plays a key role in supporting bone formation provides strong presumptive evidence that the canonical wnt signaling pathway is a primary regulator of osteoblastic bone formation. The present proposal constitutes an effort to address the mechanism and scope of this regulation. This will be accomplished by developing an in vivo system of controlled gene knockout in adult mice, and applying this to the targeted knockout of the beta-catenin gene in osteoblast lineage cells. We hypothesize that canonical wnt signaling in osteoblasts produces a positive effect on bone formation via different mechanisms at different stages of osteoblast differentiation, increasing proliferation in early osteoblasts and inhibiting apoptosis in fully differentiated osteoblasts. We also hypothesize that the anabolic action of parathyroid hormone (PTH) is exerted via the ability of PTH receptor signaling to potentiate the anabolic action of canonical wnt signaling. To test these hypotheses, we propose to first determine the differential role of the canonical wnt/LRP pathway in preosteoblasts and in mature osteoblasts by regulated knockout of beta-catenin expression. The tetracycline regulatory system will be used to allow for controlled expression of the cre recombinase in osteoblasts in vivo. The use of the 3.6 kb and 2.3 kb type I collagen promoters will allow us to ablate beta-catenin in pre-osteoblasts and mature osteoblasts (3.6 kb promoter) or only in mature osteoblasts (2.3 kb promoter). We will determine the effect of induced knockout of beta-catenin on skeletal homeostasis by microCT and on the number of osteoblasts undergoing proliferation and apoptosis. In preliminary studies, we have found that PTH receptor signaling synergizes with beta-catenin in the activation of LEF/TCF transcription factors, the downstream target of canonical wnt signaling. We will therefore use the beta-catenin knockout model to determine whether this convergent signaling is an essential pathway for the anabolic effect of exogenous PTH. Successful completion of these studies will help to define the role of canonical wnt signaling in skeletal function, and will provide novel insights into the mechanism underlying the anabolic effects of PTH.
