Histone deacetylases (Hdacs) are crucial regulators of gene expression and are therefore targets for new medical therapies. Hdac3 is expressed in osteoblasts and binds to Runx2 to regulate osteoblastic gene expression, thus playing an important role in bone development and maintenance. Conditional knockout of Hdac3 in osteochondral progenitor cells (Hdac3-CKO) leads to decreased osteoblast activity and a low bone mass phenotype. This bone loss may be due to disregulation of Wnt/?-catenin signaling, a key regulatory pathway for bone remodeling. Importantly, levels of Axin2 (a negative feedback inhibitor of Wnt signaling) are increased in Hdac3-CKO mice. However, it is not clear whether Axin2 levels are increased as a result of upregulated canonical Wnt signaling, leading to a concomitant increase in Axin2 (as part of a negative feedback loop) or if the increased Axin2 levels in Hdac3-CKO mice result from losing a direct repressive effect of Hdac3 on the Axin2 promoter. The objective of the proposed research is to determine how Hdac3 suppression affects Wnt/2-catenin signaling in osteoblast lineage cells. The central hypothesis is that Hdac3 is actively recruited to the Axin2 promoter by Runx2 to repress Axin2's inhibition of the Wnt pathway, thus ?-catenin-dependent bone formation. The first specific aim to test this hypothesis is to determine the consequences of Hdac3-depletion on canonical Wnt signaling in vivo. This will be accomplished by crossing Hdac3-CKO mice with two mouse strains in which Wnt signaling can be directly quantified (i.e., Wnt signaling reporter strains). Quantification of 2-galactosidase staining on bones from these mice will reveal the effects of Hdac3 deficiency on an artificial reporter of canonical Wnt signaling (TOPGAL) and on a natural target of canonical Wnt signaling (Axin2). The second specific aim is to determine relationships between Hdac3 and Axin2 on bone mass and bone formation in vivo. Femurs and tibias will be analyzed in Hdac3- CKO:Axin2-KO mutant mice (and littermate controls) with micro-computed tomography, histomorphometry, and mechanical testing to determine if deficiencies in both Hdac3 and Axin2 result in a normal (i.e., wild-type) skeletal phenotype. The final specific aim is to define the molecular mechanisms by which Hdac3 regulates Axin2. Mechanistic in vitro experiments including transcription and chromatin immunoprecipitation assays will explain how Hdac3 associates with and represses the Axin2 promoter. These studies are novel and important because they will be the first to mechanistically link Hdac3 with Axin2 and the Wnt pathway in vivo and in vitro. The results will be important for understanding how Hdacs interact with Runx2 and canonical Wnt signaling to obtain and maintain optimal bone mass, thereby increasing knowledge of osteoblast maturation, skeletal development and bone regeneration/repair. Understanding this relationship is important because Axin2 is an intracellular and auto-feedback regulator of Wnt/?-catenin signaling, a pathway that is currently being targeted in the development of new anabolic skeletal therapies.
We propose to study novel interactions between histone deacetylases 3 (Hdac3), Axin2, and the canonical Wnt signaling pathway in mice derived from an Hdac3-deficient model. The proposed project is significant because the Wnt signaling pathway is a target for new osteogenic therapies, and our studies will investigate factors that may modulate its effects. Furthermore, the work will have collective impact because studies of Hdac depletion will enhance understanding of how Hdac inhibitors (e.g., ZolinzaTM, Valproate, used clinically as anti-cancer and anti-epileptic therapies) affect the skeleton.
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