Role of agrin receptors in adipose tissue-derived mesenchymal stem cell differentiation in cells of mesenchymal tissues

• Grant number: 24/00415-0
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Effective date (Start): June 01, 2024
• Effective date (End): May 31, 2025
• Field of knowledge: Health Sciences - Dentistry - Oral and Maxillofacial Surgery
• Principal Investigator: Márcio Mateus Beloti
• Grantee: Letícia Faustino Adolpho
• Supervisor: Suzanne Eldridge
• Host Institution: Faculdade de Odontologia de Ribeirão Preto (FORP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Research place: Queen Mary University of London, England
• Associated to the scholarship: 21/04824-4 - Effect of agrin overexpression in adipose tissue-derived mesenchymal stem cells on osteoblastic and adipocytic differentiation and on bone regeneration, BP.DR

Abstract

Agrin is an extracellular matrix protein with well-described functions in neuromuscular junctions, synaptic gaps of the central nervous system, and immunological synapses. Agrin can promote cardiac regeneration after myocardial infarction, maintenance of cartilage, and osteoblastic differentiation. Preliminary results demonstrated that the overexpression of agrin modulated the gene expression of its receptor low-density lipoprotein receptor-related protein 4 (Lrp4) and ±-dystroglycan (Dag1) during osteoblastic and adipogenic differentiation. Based on that and considering the relevance of agrin in tissues of mesenchymal origin such as bone, cartilage, fat and muscle, we hypothesized that differentiation in cells of these tissues can be modulated by changing the receptor stoichiometry in mesenchymal stem cells (MSCs) overexpressing agrin. To test our hypothesis, the aims of this study are to investigate the effects of: 1) DAG1 blockade in osteoblastic, chondrocyte, adipocytic and myocytic differentiation of MSCs overexpressing agrin, and 2) knockdown of Lrp4 in osteoblastic, chondrocyte, adipocytic and myocytic differentiation of MSCs overexpressing agrin, and 3) both knockdown of Lrp4 and DAG1 blockade during osteoblastic, chondrocyte, adipocytic and myocytic differentiation of MSCs overexpressing agrin. To evaluate the role of agrin receptors in cell differentiation, the DAG1 receptor will be blocked with specific antibodies and Lrp4 receptor will be knockdown by small interfering RNA (siRNA), and the cells will be induced to osteoblastic, chondrocyte, adipocytic and myocytic differentiation. Then, the gene expression of specific cell differentiation markers of each mesenchymal lineage will be evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and the protein expression will be evaluated by western blotting analysis. The osteoblastic, adipocytic, chondrocyte and myocytic phenotypes will be evaluated, respectively, by deposition of mineralized nodules, lipidic accumulation, proteoglycan deposition and skeletal muscle-specific myosin heavy chain (SkM-MHC) expression. The results of the present study will be of relevance to clarify the cellular and molecular basis of MSC differentiation in cells of multiple tissues from mesenchymal origin in the context of agrin signalling, which may contribute to the development of new therapies to treat diseases of mesenchymal tissues.