Abstract
Purpose :
Recently, we demonstrated that TGF-β induced cell-cell junction formation when added to confluent corneal endothelial cells (CECs) in vitro. The purpose of this study was to investigate the signalling pathways associated with this enhanced intercellular junction formation.
Methods :
Human CECs (N = 8 populations) at passage 3 were cultured using a dual media approach containing TGF-β2 (5 ng/ml) or not (control condition) during the maturation phase. RNA was collected for gene profiling microarray (SurePrint G3 Human GE; 60 000 probes, Agilent Technologies) after the third TGF-β2 dose. Proteins were also collected for phosphorylated proteins screening (TGF-β phospho antibody array, 176 antibodies against phosphorylated and total forms, Fullmoon biosystems; Phospho kinase array, 45 antibodies against phosphorylated forms, R&D systems). Proteome profiling results were confirmed by western blot analysis.
Results :
Microarray analysis revealed that the presence of TGF-β2 upregulated 105 genes (2-fold change) and downregulated 50 genes. Functional analysis showed that 20% of the overexpressed genes encoded for cell-extracellular matrix adhesion proteins. Among the 129 phosphorylated proteins studied, 18 had a 2-fold difference between TGF-β2 condition over the control condition, including SMAD3, AKT, FAK, STAT5 and SRC kinases family. Western blot analysis confirmed SMAD3 (2.2X, p = 0.03) and AKT (1.8X, p = 0.07) activation by TGF-β2.
Conclusions :
This study suggests a role for cell-extracellular matrix adhesion and it identifies kinases (AKT, SMAD3, FAK, STAT5 and SRC family) potentially associated with TGF-β induced cell-cell junctions. Understanding intercellular junctions formation sheds insight into the role of TGF-β in regulating the corneal endothelium’s integrity in vivo.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.