The role of O-GlcNAcylation in the osteoclasts fusion and function

• Grant number: 19/15582-1
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: November 01, 2019
• End date: May 31, 2020
• Field of knowledge: Health Sciences - Dentistry
• Principal Investigator: Sandra Yasuyo Fukada Alves
• Grantee: Thaise Mayumi Taira
• Supervisor: Kent Soe
• Host Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Institution abroad: University of Southern Denmark (SDU), Denmark
• Associated to the scholarship: 17/23264-4 - Role of O-GlcNacylation in the bone loss in periapical lesion model, BP.DR

Abstract

The O-GlcNAcylation is a post-translational modification consisting in linking O-linked-N-Acetylglucosamine (O-GlcNAc) to serine and threonine residues of nuclear and cytoplasmic proteins by the action of enzyme OGT. O-GlcNAcylation, analogously to the phosphorylation process, control the function of several target proteins. Many cell types can be target of O-GlcNAcylation, including the osteoclasts that actively participate in the bone resorption process. Based on evidences from our research group, we believe that O-GlcNAcylation of proteins are important for receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis in vitro. Firstly, we observed that the stimulation of bone marrow macrophage with RANKL promoted increased level of O-GlcNAcylated proteins, which was sustained throughout the osteoclasts differentiation. Moreover, we tested two OGT inhibitors as pharmacological tools: 2-acetamido-2-deoxy-5-thio-D-glucopyranose (5SGlcNAc) uridine 5'-acetamido-2-deoxy-5-thiophosphoate and benzyl-2-acetamido-2-deoxy-±-dgalactopyronoside (BAD) and evaluated the impact of O-GlcNAc accumulation on osteoclasts differentiation. The pharmacological inhibition of OGT by 5SGlcNAc and BAD inhibited the differentiation of osteoclasts, evidenced by reduced number of TRAP+ cells. The expression of osteoclast markers such as NFATc1, ±V integrin, the enzyme cathepsin K, and the resorption capacity of the mature osteoclasts were all reduced after OGT inhibition. Moreover, we observed that addition of O-GlcNAc to the cell culture, a substrate of OGT, induced a significant increase in the size of TRAP-positive multinucleated cells, as well as the enzymatic activity of TRAP in the cell lysate. Although we still do not know exactly which proteins undergoes O-GlcNAcylation, our data together give us strong indications that this protein modification is important for osteoclastogenesis. It is important to mention that these conclusions are based on endpoint measurements such as counting the number of multinucleated OCs, number of nuclei per OC, and resorptive activity. In order to strengthen our analyses, we have set up a collaboration with Prof. Kent Søe from the Department of Pathology, Odense University Hospital, who is an expert in analyzing osteoclast fusion and function. They hold the knowledge of analyzing OC fusion in realtime making it possible to investigate novel details with regard to for example individual fusion partners. To explore the effect of O-GlcNAcylation on osteoclast fusion and function in real-time, we are applying for the BEPE fellowship program to spend 6 months in Prof. Kent Søe's laboratory to strengthen this part of the project with these new analytical approaches. (AU)