Antagonism of Canonical Beta-catenin Activity by Smad4 in Osteoprogenitors

Washington University, Saint Louis MO

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Abstract

[unreadable] DESCRIPTION (provided by applicant): In adult life, the recruitment and differentiation of osteoblast precursors to areas of active bone remodeling is orchestrated by molecular signals that induce undifferentiated cells in the bone marrow to proliferate and commit to the osteogenic lineage. Recent data have demonstrated that the Wnt and bone morphogenetic protein (BMP) signalig pathways are critical for osteogenic differentiation. Our laboratory has shown that ?- catenin, a transcription factor of the canonical Wnt pathway necessary for osteoblastogenesis, requires interaction with the BMP pathway, so that the two signals must act in tandem to manifest an osteogenic signal. Preliminary results strongly suggest that a critical interaction point between ? -catenin and BMP signals may involve Smad4, a transcription factor of the greater TGF-beta pathway. Specifically, Smad4 rectuits ? -catenin into Smad-dependent transcriptional complexes, at the expense of canonical Tcf/Lef activity. Based on these preliminary findings, I hypothesize that Smad4 interacts with ? -catenin in a manner that antagonizes Tcf/Lef signaling and proliferation in osteoprogenitors. I propose to test this hypothesis in two Specific Aims. In Aim I, C3H10T1/2 embryonic fibroblasts will be used to characterize in vitro how Smad4 interacts with ? -catenin to attenuate Tcf/Lef-activity, cyclin-D1 expression, and proliferation. This will be accomplished using a series of Tcf/Lef-dependent transcriptional reporters, gene expression analysis, and cell cycle assays. In Aim II, I will examine antagonism of canonical ? -catenin activity by Smad4 in osteoprogenitors in vivo. Here, a novel Cre-loxP system will be utilized to inducibly and conditionally ablate Smad4 in differentiating osteoblasts of young mice. I will phenotypically characterize these mice and apply cell biology studies to cells isolated from these mice to further characterize the role of Smad4 on ? -catenin and Tcf/Lef signaling and cell proliferation in vivo and in vitro. These studies will establish the biologic importance of Smad4/?? -catenin signaling cross-talk for osteogenesis. Bone metabolic disorders, such as osteoporosis, are due to becoming a major public health concern in the next decade with a large percentage of the population reaching senior age. Altered bone remodeling due to cellular dysfunctions in the bone forming and/or bone degrading cells of the skeleton has been demonstrated to play a role in such disorders. The proposed investigation will study a novel molecular mechanism involved in the generation of bone forming cells, osteoblasts, in the bone marrow. The results will facilitate identification of potential novel therapeutic targets for disease modification [unreadable] [unreadable] [unreadable]