title: The PI3K/Akt pathway mediates the expression of type I collagen induced by TGF-β2 in human retinal pigment epithelial cells.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Ophthalmology, Faculty of Medicine, Oita University, Hasama-machi, Yufu-shi, Oita 879-5593, Japan.
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Yokoyama
authors.forename: Kenichi
authors.initials: K
authors.lastname: Kimoto
authors.forename: Yuji
authors.initials: Y
authors.lastname: Itoh
authors.forename: Kazuo
authors.initials: K
authors.lastname: Nakatsuka
authors.forename: Noritaka
authors.initials: N
authors.lastname: Matsuo
authors.forename: Hidekatsu
authors.initials: H
authors.lastname: Yoshioka
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Kubota
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D006801:Humans; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010957:Plasmids; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D015398:Signal Transduction; D051900:Smad3 Protein / Q000235:genetics; D051906:Smad7 Protein / Q000235:genetics; D014162:Transfection; D053781:Transforming Growth Factor beta2 / Q000494:pharmacology*; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D024042:Collagen Type I; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; C497790:SMAD3 protein, human; C497806:SMAD7 protein, human; D051900:Smad3 Protein; D051906:Smad7 Protein; D053781:Transforming Growth Factor beta2; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D000077191:Wortmannin