The role of OB-targeted deletion of Rspo3 in adult bones skeletal homeostasis

• Grant number: 18/04795-1
• Support type: Scholarships abroad - Research Internship - Doctorate
• Effective date (Start): July 02, 2018
• Effective date (End): July 01, 2019
• Field of knowledge: Health Sciences - Dentistry
• Principal Investigator: Marcia Martins Marques
• Grantee: Ana Clara Fagundes Pedroni
• Supervisor abroad: Roland Baron
• Home Institution: Faculdade de Odontologia (FO). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Local de pesquisa : Harvard University, Boston, United States
• Associated to the scholarship: 17/00760-6 - Effect of cell sheet of human dental pulp stem cell, associated or not with photobiomodulation therapy, on the regeneration of critical size bone defects in calvaria of rats with type 2 diabetes mellitus, BP.DR

Abstract

Diabetes can lead to metabolic bone changes that promote an unbalanced state in bone mass, similar to osteoporosis. The WNT signaling pathway has already been described as involved in skeletal formation, bone regeneration and differentiation of mesenchymal stem cells in bone lineage - being an important pathway of interest in the treatment of bone volume disorders. Previous studies from Dr. Baron's research group found that genes RSPOs are Wnt signaling activators and that Wnt signaling has key roles in bone development and homeostasis suggest that RSPOs could have important functions in these processes. Preliminary results show: 1) that RSPO3 is expressed in osteoblasts and bone marrow stromal cells, 2) that RSPO3 activates canonical Wnt signaling and 3) that RSPO3 heterozygous mice display a bone phenotype characterized by increased bone formation and bone mass. The main goal of the studie proposed here is to explore the role of OB-targeted deletion of RSPO3 in adult bones. For this purpose, we will use the Col1a1-CreERT2 transgenic mice . These mice have a TM-inducible Cre-mediated recombination system driven by the 2.3 Kb mouse Col1a1 promoter. The Col1a1-CreERT2 fusion protein does not bind natural ligand (estrogen) at physiological concentrations, but will bind the synthetic ligand, 4-hydroxyTM (4-OHT) and gain access to the nuclear compartment to mediate recombination after exposure to exogenous TM. Methods: Transgenic mice (Col1a1-CreERT2/Rspo3f/f and Col1a1-CreERT2/Rspo3f/wt) and control 12-week-old mice will be injected IP with 1mg/10g body weight of TM or vehicle every other day for 5 days (3 injections) and sacrificed 4 weeks after the last injection, at 16 weeks of age. Deletion efficiency will be verified by PCR of genomic DNA extracted from long bones and calvariae. To insure that TM-induced gene recombination is achieved, Rspo3 RNA expression and protein levels will be evaluated by Q-RT-PCR and Western and immunohistochemistry analyses, respectively. Hindlimbs, vertebrae and calvariae will be collected for X-rays and BMD analysis, ¼CT imaging, dynamic histomorphometry, biomechanical properties and biochemical markers of bone formation and bone resorption will be measured. Research Relevance: Delineating the role of RSPO3 in postnatal bone mass homeostasis and the mechanisms by which this protein activates the Wnt signaling pathway has relevance to understanding of bone homeostasis and could have important implications for the generation of novel anabolic therapeutic approaches to treat osteoporosis and other diseases associated low bone mass and increased bone fragility, such as those found in diabetic patients.