Effect of the interaction between Engrailed-1 and the TGFB/SMAD3 circuit in osteoblastic differentiation of mesenchymal stem cells

• Grant number: 25/01045-5
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: September 01, 2025
• End date: August 31, 2026
• Field of knowledge: Health Sciences - Dentistry - Oral and Maxillofacial Surgery
• Principal Investigator: Márcio Mateus Beloti
• Grantee: Robson Diego Calixto
• Supervisor: Vicki Rosen
• Host Institution: Faculdade de Odontologia de Ribeirão Preto (FORP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Institution abroad: Harvard University, Boston, United States
• Associated to the scholarship: 23/00538-2 - Effect of Engrailed-1 protein modulation in mesenchymal stem cells on osteoblastic differentiation and bone formation in defects created in mouse calvaria, BP.DR

Abstract

Engrailed-1 (EN1) is a homeobox transcription factor critical for various developmental processes, including bone and joint tissue formation. Its association with the TGF-B/SMAD signaling pathway has been demonstrated through its capacity to amplify the effects of TGF-B. However, the underlying mechanisms remain unclear, particularly in the context of bone repair and formation. Current evidence indicates that EN1 interacts directly with SMAD3, promoting the transcriptional activation of pro-fibrotic target genes, such as collagen and proteins associated with the differentiation of fibroblasts into myofibroblasts. Moreover, EN1 activation by TGF-B occurs in a SMAD3-dependent manner, suggesting the presence of a positive feedback loop in which TGF-B induces EN1 expression, which in turn interacts with SMAD3 to enhance the transcription of genes involved in tissue remodeling. This cooperative mechanism not only amplifies TGF-B signaling but also modulates the specificity of transcriptional targets involved in osteoblastic differentiation and other tissue functions. Building on these insights, the present study aims to evaluate the physical interaction and functional impact of the interplay between EN1 and the components of the TGF-B/SMAD3 signaling circuit in coordinating the osteoblastic differentiation of mesenchymal stem cells derived from mouse adipose tissue (AT-MSCs) with En1 silencing produced by the CRISPR-Cas9 technique. To achieve this, the following experiments will be conducted: 1) total RNA sequencing of AT-MSCs with En1 silencing, both with and without exogenous TGF-B, to analyze the gene expression profile of osteoblastic markers and signaling components involved in osteoblastic differentiation; 2) chromatin immunoprecipitation (ChIP) assays to assess the physical interactions between EN1 and TGF-B, as well as EN1 and SMAD3; and 3) luciferase assays to determine whether TGF-B and SMAD binding induces activity in the EN1 promoter region. Given that EN1 remains underexplored in the context of bone tissue, the findings of this study are expected to provide both scientifically novel insights and potential therapeutic applications.