Developing an innovative SMAD reporter approach

Transforming growth factor-𝛽 (TGF-β) is a signaling molecule that has a regulatory role in many physiological processes such as cell differentiation, apoptosis, and regulation of immune response, especially wound healing. TGF-β, which is synthesized inactively, is stored inactively in the ECM by establishing covalent bonds with extracellular matrix (ECM) proteins through the Latent TGF-β Binding Protein (LTBP) molecule in its structure. After TGF-β is stimulated and activated by various stimuli, it binds to the receptor complex on the cell surface and stimulates the signaling pathway. The uncontrolled continuity of the activation process is associated with "fibrosis", which results in the irreversible accumulation of ECM elements. In addition, this pathway plays a role in the pathophysiology of cancer, autoimmune diseases and atherosclerosis. Therefore, it is important to monitör the TGF-β signaling pathway, especially whether the molecule triggers a response that regulates transcription in the cell, in order to develop new treatment methods targeting this pathway. Within the scope of this thesis study, a reporter fusion protein consisting of Secreted Alkaline Phosphatase (SEAP) and Green Fluorescent Protein (GFP) proteins was developed to enable in vitro monitoring of the TGF-β signal. In order to ensure the expression of this structure through the effect of TGF-β, the binding sequence of Suppressor of Mothers Against Decapentaplegic (SMAD) proteins that create a TGF-β-specific transcription response was used, and it was planned to develop an innovative dual reporter vector system. As a result of the studies, an increased GFP signal was shown by flow cytometry analysis in the TGF-β applied group, compared to the control group without TGF-β application, in the cells to which the developed reporter vector was transferred. In addition, an increase in the measurable GFP signal relative to the TGF-β concentration was observed. As a result of the study, it was observed that the resulting reporter vector structure did not exhibit the expected functional SEAP activity. In this regard, optimization studies are needed for SEAP functionality.