The role of Syndecan 4 in the cellular traffic and extracellular matrix remodeling

• Grant number: 24/20473-5
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: April 01, 2025
• End date: March 31, 2027
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Helena Bonciani Nader
• Grantee: Naiara Climas Pereira
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated research grant: 23/07464-4 - Glycosaminoglycans and the cross-talk cell and extracellular matrix: a translational approach, AP.TEM

Abstract

Syndecan-4 (Syn4) is a heparan sulfate proteoglycan extensively involved in cell-extracellular matrix (ECM) communication and ECM remodeling. This study aims to investigate the role of Syn4 in cellular trafficking and molecular regulation of ECM, utilizing genetically modified cell models. The research will address mechanisms influencing cell adhesion, migration, and associated signaling pathways.We will use endothelial cell models, including wild-type cells, Syn4 knockout cells (shRNA-Syn4-EC), and cells overexpressing Syn4. Knockout cells exhibit a disorganized cytoskeleton and increased cell migration due to the uncoupling of vinculin from F-actin filaments, whereas Syn4-overexpressing cells show resistance to anoikis. The organization and functionality of the endoplasmic reticulum (ER), Golgi complex, mitochondria, lysosomes, and endocytic pathways will be investigated in these models. Preliminary data suggest that shRNA-Syn4-EC cells show increased expression of the transcription factor C-MYC and reduced microRNA204 expression, indicating Syn4's regulatory role in gene expression and cell signaling.The experimental strategy includes analyzing endocytosis and exocytosis through cell transfection using fluorescent markers such as caveolin1 (Cav1)-mCherry and clathrin light chain (CLC)-mRFP. Additionally, ER arrangement will be monitored using vectors containing the KDEL sequence fused to the cerulean fluorescent protein. Confocal microscopy and FRET techniques will be employed to analyze the localization and interaction of macromolecular complexes. In parallel, the activation of MAP kinases (ERK, JNK, p38) and the involvement of RhoGTPases in cytoskeleton remodeling and adhesion regulation will be investigated.Gene expression analysis of molecules associated with cellular trafficking and ECM remodeling will be conducted via qPCR, while gene profiling will be performed using digital PCR and nanopore technology. Furthermore, Syn4 gene silencing through the CRISPR-Cas9 technique will allow an evaluation of its influence on adhesion regulation, endocytic trafficking, and associated signaling pathways.Expected results include the identification of mechanisms through which Syn4 modulates cell adhesion, endocytosis, and intracellular homeostasis. Moreover, understanding the relationship between Syn4, C-MYC, and microRNA204 in gene expression regulation and cell signaling is anticipated. These findings may contribute to the development of novel therapeutic approaches targeting ECM modulation and cell-ECM communication control.This study will provide crucial insights into the role of Syn4 in cellular trafficking and ECM remodeling, contributing to advances in cell biology and potential therapeutic applications.