The effect of the association of BMP-2 and Wnt-3a on temporal expression of Rad GTPase in osteoblastic cells

• Grant number: 17/26791-5
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: March 01, 2018
• End date: February 28, 2019
• Field of knowledge: Health Sciences - Dentistry - Oral and Maxillofacial Surgery
• Principal Investigator: Márcio Mateus Beloti
• Grantee: Bruna Scaf
• Host Institution: Faculdade de Odontologia de Ribeirão Preto (FORP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

Rad GTPase (Rad, Ras-related associated with diabetes) is a protein originally identified in the cardiac musculature of type II diabetes Mellitus patients and its expression and participation in different tissues and biological events has been investigated. Rad deficiency results in a significant decrease in bone mass, characterized by a decrease in osteoblastic activity and an increase in osteoclastogenesis. Furthermore, changes in Rad expression alter the mesenchymal stem cell fate by driving the osteoblastic or adipocytic differentiation. However, the information regarding the expression and function of Rad in osteoblastic differentiation and its interaction with well know osteogenic molecules are limited. Based on this and considering the participation of BMP and Wnt pathways in the osteogenesis process, the aim this study is to evaluate the effect of the association of BMP-2 and Wnt-3a on the temporal expression of Rad in osteoblastic cells. MC3T3-E1 cell line, subclone 14, will be cultured on polystyrene in the absence (control) or presence of BMP-2 (100 ng/mL) and / or Wnt-3a (10 ng/mL) recombinant proteins for periods up to 14 days. At 3, 5, 7, 10 and 14 days, the gene expression of the bone markers, alkaline phosphatase, bone sialoprotein, RUNX2, osterix, osteocalcin and osteopontin, and Rad and MGP (matrix GLA protein) will be evaluated by Real-time PCR. At 10 days, the in situ ALP activity will be evaluated by the Fast Red method. The numerical data will be submitted to the test of adherence to the normal distribution to determine the statistical analysis that should be used. The results of this study may achieve important data about the role of Rad in the bone tissue and may contribute to the development of new therapeutic strategies for bone regeneration. (AU)