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N. Nagai, M. Fujihara, T. Wu, J. Handa; Effect of Connective Tissue Growth Factor on ECM Modulation in Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5541. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelial (RPE) cells play a pivotal role in the pathogenesis of choroidal neovascularization (CNV). Connective tissue growth factor (CTGF), a 38KDa cysteine-rich polypeptide, induces extracellular matrix (ECM) production and causes fibrosis. Modulation of ECM is important for potently related with the CNV development including promoting angiogenesis or promotion the fibrosis withintic component of CNV membranes. Matrix metalloproteinases (MMPs) are a family of calcium and zinc dependent enzymes associated with remodeling of the ECM. We investigated the role of CTGF on ECM modulation in RPE Cells.
ARPE-19 cells were seeded in 6 well plates and maintained in MEM plus 10% heat-inactivated FBS. When cultures achieved confluence, the medium was removed and replaced with serum-free MEM containing 1 % bovine albumin. Cells were stimulated with recombinant CTGF (1, 10 or 100 ng/ml) or vehicle in serum-starved MEM. After a 36-hour incubation, the supernatant was collected, concentrated tenfold, and processed for western blot analyses for fibronectin and MMP2, and gelatin zymography for MMP-2. After a 15-minute incubation, the cell lysates were processed for western blot analyses for p38 and ERK (extracellular signal-regulated kinase) MAP (mitogen-activated protein) kinases. Additionally, cells were pretreated with a MEK-1 inhibitor, PD98059, or a p38 MAPK inhibitor, SB203580 for 2 hours and stimulated with CTGF to analyze the signaling contributing fibronectin and MMP-2 production.
Fibronectin levels in the supernatant were significantly increased by the treatment with CTGF. MMP-2 production analyzed by western blotting and MMP-2 activation analyzed by zymography were also stimulated by the addition of CTGF. CTGF induced activation of both p38 and ERK MAP kinases. Inhibition of ERK MAP kinase signaling with a MEK-1 inhibitor, PD98059, suppressed production of fibronectin and MMP-2 in RPE cells.
The present data reveal that CTGF can positively and negatively regulate ECM in RPE cells by stimulating fibronectin production and MMP-2 activation via ERK MAP kinase signaling, showing the potent role of CTGF in regulating the extracellular matrix.
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