The PI3K/Akt pathway mediates the expression of type I collagen induced by TGF-β 2 in human retinal pigment epithelial cells

Abstract

Purpose Transforming growth factor (TGF)-β is a key mediator of proliferative vitreoretinopathy, but the cellular mechanisms by which TGF-β induces extracellular matrix protein (ECM) synthesis are not fully understood. This study examined whether the PI3K/Akt pathway is involved in TGF-β 2-induced collagen expression in human retinal pigment epithelial cells. Methods Human retinal pigment epithelial cells ARPE-19 were cultured and stimulated with TGF-β 2. The role of the PI3K/Akt pathway was evaluated using the biochemical inhibitor, wortmannin. The effect of wortmannin on the expression of type I collagen mRNA (COL1A1, COL1A2) induced by TGF-β 2 was evaluated by real-time RT-PCR. The effect of wortmannin on the synthesis of type I collagen induced by TGF-β 2 was assessed by an immunocytochemical analysis with anti-type I collagen antibody. Luciferase reporter assays were performed to examine the effect of wortmannin on the transcriptional activities of COL1A2. A luciferase assay using a mutation construct of the Smad binding site in COL1A2 promoter (Smad-mut/Luc) was also performed to examine the crosstalk between the Smad pathway and the PI3K/Akt pathway. The effects of wortmannin on the transcriptional activity of Smad3 were also examined using CAGA12-Luc. Moreover, the effect of wortmannin on TGF-β 2-induced Smad7 mRNA expression was evaluated. Results The biochemical blockade of PI3K/Akt activation inhibited TGF-β 2- induced type I collagen mRNA expression and type I collagen synthesis. The blockade of PI3K/Akt pathway inhibited the increase in COL1A2 promoter activities when induced by TGF-β 2 and reduced TGF-β 2 induction of Smad-mut/Luc promoter activity and CAGA12-Luc activity. Moreover, wortmannin increased the TGF-β 2- induced Smad7 mRNA expression levels. Conclusions The PI3K/Akt pathway plays a role in relaying the TGF-β 2 signal to induce type I collagen synthesis in the retinal pigment epithelium through Smaddependent and Smad-independent pathways. © Springer-Verlag 2011.