p38 MAPK mediates the expression of type I collagen induced by TGF-beta 2 in human retinal pigment epithelial cells ARPE-19.
OBJECTIVE
Transforming growth factor (TGF)-beta has been implicated as the key mediator of proliferative vitreoretinopathy, but the cellular mechanisms by which TGF-beta induces extracellular matrix protein (ECM) synthesis are not fully understood. The current study was conducted to examine whether the mitogen-activated protein kinase (MAPK) pathway is involved in TGF-beta 2-induced collagen expression in retinal pigment epithelial cells.


METHODS
Human retinal pigment epithelial cells ARPE-19 were cultured and stimulated with various concentrations of TGF-beta 2. The type I collagen gene (COL1A1, COL1A2) expression induced by TGF-beta 2 was evaluated by real-time RT-PCR. Synthesis of type I collagen was evaluated by the concentration of the C-terminal propeptide of type I (PICP) in the medium. The activation of MAPK pathways by TGF-beta 2 was assessed by immunoblot with anti-phospho-p38 and anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibodies. The role of MAPK was assessed using biochemical inhibitors. To examine the transcriptional activities of COL1A1 and COL1A2, luciferase reporter assays were also performed.


RESULTS
mRNA expression of COL1A1 and COL1A2 and type I collagen synthesis were activated by TGF-beta 2. Both ERK and p38 MAPK pathways were also activated by TGF-beta 2. The biochemical blockade of p38 MAPK activation, but not ERK activation, inhibited TGF-beta 2-induced type I collagen mRNA expression and type I collagen synthesis. Moreover, blockade of the p38 MAPK pathway inhibited the increase in both COL1A1 and COL1A2 promoter activities when induced by TGF-beta 2.


CONCLUSIONS
TGF-beta 2 activates p38 MAPK and p38 MAPK plays a role in relaying the TGF-beta 2 signal to type I collagen production in the retinal pigment epithelium.
Kimoto
Kenichi
K
Department of Ophthalmology, Faculty of Medicine, Oita University, Hasama-machi, Japan. kimotok@med.oita-u.ac.jp
Nakatsuka
Kazuo
K
Matsuo
Noritaka
N
Yoshioka
Hidekatsu
H
D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis / Q000235:genetics*; D024042:Collagen Type I / Q000096:biosynthesis / Q000235:genetics*; D000090602:Collagen Type I, alpha 1 Chain; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D010957:Plasmids; D011347:Procollagen / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053781:Transforming Growth Factor beta2; D048051:p38 Mitogen-Activated Protein Kinases
D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D011347:Procollagen; D012333:RNA, Messenger; C509041:TGFB2 protein, human; D016212:Transforming Growth Factor beta; D053781:Transforming Growth Factor beta2; C042813:procollagen type I carboxy terminal peptide; D003094:Collagen; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases