CTGF is increased in basal deposits and regulates matrix production through the ERK (p42/p44mapk) MAPK and the p38 MAPK signaling pathways.
OBJECTIVE
Matrix expansion is an early change in age-related maculopathy. The aim of this study was to determine whether connective tissue growth factor (CTGF) regulates the production of extracellular matrix components by retinal pigmented epithelial (RPE) cells.


METHODS
ARPE-19 cells were treated with CTGF and analyzed for fibronectin, laminin, and MMP-2 by RT-qPCR, Western blot analysis, or zymography. Cells were also pretreated with an MEK-1/2 inhibitor (PD98059) or a p38 inhibitor (SB203580) and an anti-CTGF antibody to analyze the signaling contributing to fibronectin, laminin, and MMP-2 production. Human maculas were analyzed for mRNA using laser capture microdissected RPE cells and by immunohistochemistry for the topographic distribution of CTGF.


RESULTS
CTGF induced fibronectin mRNA (P=0.006) and protein (P=0.006), and laminin mRNA (P=0.006) and protein (P=0.02) by ARPE-19 cells. CTGF also induced MMP-2 mRNA (P=0.002) and protein secretion (P=0.04). Using zymography, CTGF increased the latent and active forms of MMP-2 compared to controls (P=0.02). An anti-CTGF antibody inhibited fibronectin, laminin, and MMP-2 after CTGF stimulation. CTGF increased the phosphorylation of p38 and ERK1/2. Fibronectin and MMP-2 mRNA and protein were suppressed by a MEK-1/2 inhibitor, but not with a p38 inhibitor. Laminin expression was suppressed by both inhibitors. RT-qPCR analysis showed that macular RPE cells from human donors express CTGF. Immunohistochemistry of human maculas showed strong labeling of CTGF in Bruch membrane, including basal deposits and drusen.


CONCLUSIONS
CTGF is increased in basal deposits and drusen of AMD specimens, and it induces matrix protein production in ARPE-19 cells through the ERK (p42/p44(mapk)) and p38(mapk) signaling pathways.
Nagai
Norihiro
N
Wilmer Eye Institute, Johns Hopkins School of Medicine, Baltimore, Maryland 21287, USA.
Klimava
Alena
A
Lee
Wen-Hsiang
WH
Izumi-Nagai
Kanako
K
Handa
James T
JT
D001485:Basement Membrane / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor / Q000378:metabolism* / Q000494:pharmacology*; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis*; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007124:Immunoenzyme Techniques; D007797:Laminin / Q000235:genetics / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
C526317:CCN2 protein, human; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D005419:Flavonoids; D007093:Imidazoles; D007797:Laminin; D011725:Pyridines; D012333:RNA, Messenger; D011994:Recombinant Proteins; D055513:Connective Tissue Growth Factor; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D020778:Matrix Metalloproteinase 2; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one