New TARGETs for osteoporosis: from mesenchymal stem cells to the regulation of osteogenesis and BONE mass – TARGETBONE

Osteoporosis, the major cause of fractures, results from an imbalance between bone formation and bone resorption. While accelerated bone resorption characterizes postmenopausal bone loss related to estrogen failure, reduced bone formation is responsible for idiopathic and age-related bone loss. The objective of TARGETBONE is to provide an integrated understanding of the cellular and molecular mechanisms that underlay the pathophysiology of osteoporosis focusing on the differentiation process of Bone Marrow Mesenchymal Stem Cells (BM-MSC) and bone marrow progenitors towards the osteoblastic lineage. The transcription factors Dlx5 and Dlx6, which play an important role in osteogenesis, are expressed by BM-MSC and are upstream regulators of Osterix (Osx) and Runx2, two master genes involved in the induction of osteoblast differentiation. Our aim is to characterize the cellular and molecular factors that promote BM-MSCs differentiation modifying directly the function of Dlx5 and Dlx6 in BM-MSCs or in more differentiated progenitors in vivo and in vitro. To this end, we will generate the conditional deletion of Dlx5/6 in mouse osteoblast progenitors by crossing our recently generated Dlx5/6Hboxlox/lox with Osx-creERT2 mice to obtain Dlx5/6Osx-creERT2 mice in which Tamoxifen injection will permit to delete both gene in Osx-expressing precursors. The bone phenotype of these mice will be studied, and BM-MSCs and more differentiated osteoblasts will be cultured and analysed. Complementary genetic, viral and siRNA strategies will be used to analyze the effect of Dlx5/6 on the differentiation potential of cultured BM-MSC from mice and from humans. The capacity of normal and ablated cells to differentiate towards the osteoblastic or adipocytic lineage will be tested using standardized molecular approaches.
The interaction of Dlx5 with the adaptator protein MAGED1 is required to determine the cellular and/or transactivating activity of Dlx5 during osteoblast differentiation. We will characterize the protein complexes which include Dlx5 by analyzing through mass-spectroscopy the proteins that are coprecipitated with Dlx5 and/or MAGED1. The conformational interaction of Dlx5 and MAGED1 will be investigated through crystallography and in silico analysis with the objective to identify new protein-preotein interactions which could become targets for drug discovery. The identification of new molecular targets will prompt the design of innovative molecular tools to promote bone anabolic response. To further translate the impact of our findings to osteoporosis, we will analyze the osteogenic functions exerted by DLX5/6 in human bone and human BM-MSCs from different ages. The strength of TARGETBONE resides in the multidisciplinary nature of the consortium. The combined expertise of three complementary teams expert in molecular biology, drug discovery and osteoporosis provides a unique opportunity to transfer this new knowledge from basic science to clinical applications.