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Use of specific modulator of SRC - PP1 - as a strategy to differentiate osteoblast

Grant number: 14/04305-3
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): May 01, 2014
Effective date (End): April 30, 2016
Field of knowledge:Health Sciences - Dentistry - Oral and Maxillofacial Surgery
Principal Investigator:Willian Fernando Zambuzzi
Grantee:Ana Clara Denadai
Home Institution: Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil

Abstract

Our group has worked with the signal transduction mechanisms involved in the differentiation of osteoblasts and found that modulation of the activity of Src by LMWPTP (Low Molecular Weight Protein Tyrosine Phosphatase) is a necessary condition for this event. Based on these findings, We hypothesized that exogenous Src Inhibition stimulate osteoblast differentiation. Thus, in this design we decided to propose the use of a specific inhibitor of Src (PP1) as the differentiation of pre- osteoblasts (MC3T3 -E1) strategy. Initially, we will have a dose - response curve, evaluating its cytotoxic effect in order to choose one subtoxic concentration. In studies of cell differentiation, pre- osteoblasts will be cultured in 6-well plates and maintained in the presence of the inhibitor for 3, 7 and 14 days. Use as an experimental control group without treatment and one where the cells are treated with classical cell differentiation molecules : ascorbic acid (50 mu.g / ml) and b- glycerophosphate (10 mM). The alkaline phosphatase activity will be monitored over periods so as to check the mechanisms of cell differentiation. Furthermore, we will monitor the phosphorylation of Src in both residues ( Y416 and Y527 ) profile in order to predict its activity by using western blotting methodology. The results will be properly addressed with statistical methods.