miR-433-3p Negatively Regulates Bone Formation and Targets Critical for Osteoblast Function

Osteoblastic differentiation is tightly controlled by multiple signaling pathways associated with key transcription factors, as well as a number of microRNAs (miRNAs). miRNAs are small noncoding RNAs that function in the post-transcriptional regulation of gene expression. While clinical strategies to combat the reduction in bone density have often centered on impeding further bone resorption, this strategy neglects to promote bone formation, a component which is particularly problematic in the aging population. miRNAs could serve to bridge that gap, as potential therapeutic candidates. Several studies have established that miRNAs can direct osteoprogenitors towards an osteoblastic lineage through modulation of Wnt, MAPK, TGFβ and BMP signaling. Thus, this thesis consists of 2 independent manuscripts. First, a primary research article details novel mechanisms by which one particular miRNA, miR-433-3p, regulates osteoblastic differentiation via modulation of Wnt, MAPK, PTH and glucocorticoid signaling. Second, a comprehensive review documents how miRNAs regulate TGFβ and BMP signaling to promote or repress osteoblast formation and function. Regarding miR-433-3p, this miRNA can specifically target Runx2 and is repressed by BMP2 signaling. Herein, we demonstrate that miR-433-3p is progressively decreased during osteoblastic differentiation of primary mouse bone marrow stromal cells in vitro, and we confirm its negative regulation of this process. Although repressors of osteoblastic differentiation often promote adipogenesis, inhibition of miR-433-3p did not affect adipocyte differentiation in vitro. Additionally, since multiple pathways regulate osteogenesis, using 3\u27 UTR-Luciferase reporter assays five novel miR-433 targets involved in PTH, MAPK, Wnt, and glucocorticoid signaling pathways were validated. In vivo, expression of a miR-433-3p inhibitor or tough decoy in the osteoblastic lineage increased trabecular bone volume. Mice expressing the miR-433 tough decoy displayed increased bone formation without alterations in osteoblast or osteoclast numbers or surface, indicating an impact on osteoblast activity. Overall, we demonstrated that miR-433 is a negative regulator of bone formation in vivo, targeting key bone-anabolic pathways including those involved in PTH signaling, MAPK, Wnt and endogenous glucocorticoids. Local delivery of miR-433 inhibitor could present a strategy for the management of bone loss disorders and bone defect repair. Having a wide understanding of the signaling pathways, differentiating factors, and the mechanisms necessary for the miRNA-mediated induction of multipotent cells along an osteoblastic lineage is key for the effective utilization of miRNAs as therapeutic candidates in the management of bone disorders. Thus, this thesis also contains a review that showcases miRNAs regulating TGFβ and BMP signaling pathways in bone forming osteoblasts, to illustrate their interaction with and impact on bone formation. BMP signaling is particularly relevant to bone defect repair, as localized delivery of osteogenic BMPs is presently used in the clinic