Graefe s Archive for Clinical and Experimental Ophthalmology · 2011 · 74 citations · 31 references
Life SciencesMolecular BiologyOphthalmologyCell SignalingSignal TransductionTranscriptional RegulationCollagen DepositionExtracellular MatrixExtracellular Matrix DepositionExtracellular Matrix RemodelingGrowth Factor SignalingRetinal Pigment EpitheliumLuciferase Reporter AssaysLuciferase ReportersPhosphatidylinositol 3-Kinase-akt PathwayPhosphoinositide 3-Kinase-akt ActivationPi3 Kinase-akt SignalingPi3k-akt Signaling PathwayRetinal Pigment Epithelium Cell CultureSmad Signaling Pathway
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-β2-induced collagen expression in human retinal pigment epithelial cells. METHODS: Human retinal pigment epithelial cells ARPE-19 were cultured and stimulated with TGF-β2. 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-β2 was evaluated by real-time RT-PCR. The effect of wortmannin on the synthesis of type I collagen induced by TGF-β2 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-β2-induced Smad7 mRNA expression was evaluated. RESULTS: The biochemical blockade of PI3K/Akt activation inhibited TGF-β2-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-β2 and reduced TGF-β2 induction of Smad-mut/Luc promoter activity and CAGA12-Luc activity. Moreover, wortmannin increased the TGF-β2-induced Smad7 mRNA expression levels. CONCLUSIONS: The PI3K/Akt pathway plays a role in relaying the TGF-β2 signal to induce type I collagen synthesis in the retinal pigment epithelium through Smad-dependent and Smad-independent pathways.
31
Identification of Smad7, a TGFβ-inducible antagonist of TGF-β signalling
Atsuhito Nakao, Mozhgan Afrakhte, Anita Morn et al. · Nature · 1997 · 1.8K citations