CTGF is increased in basal deposits and regulates matrix production through the ERK (p42/p44mapk) MAPK and the p38 MAPK signaling pathways

Abstract

Purpose: 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.

Figure 1

Stimulation of fibronectin and laminin expression in ARPE-19 cells by CTGF. (A, B) RT-qPCR analyses show significant mRNA increase of fibronectin and laminin expression in CTGF-treated ARPE-19 cells. Statistically significant differences compared with the control are indicated. (C, top) Representative Western blot showing CTGF significantly increased protein levels of fibronectin in the supernatant of ARPE-19 cells. Fibronectin signal was normalized to cell lysate protein. (D) Representative Western blot showing increased laminin in cell lysates of ARPE-19 cells treated with CTGF. Production was normalized to α-tubulin. Statistically significant differences from controls are indicated.

Figure 2

Blockade of CTGF by FG-3019 inhibited CTGF-induced mRNA expression (A, B) and protein production (C, D) of fibronectin (A, C) and laminin (B, D). Statistically significant differences compared with the control (IgG1) are indicated.

Figure 3

Stimulation of MMP-2 expression in ARPE-19 cells by CTGF. (A) RT-qPCR analyses show significant increase of MMP-2 expression in CTGF-treated ARPE-19 cells. Statistically significant differences, compared with the control. (B, top) Western blot from a representative experiment. CTGF significantly increased protein levels of MMP-2 in the supernatants of ARPE-19 cells. (C, D) Blockade of CTGF by FG-3019 inhibited CTGF-induced mRNA expression (C) and protein production (D) of MMP-2.

Figure 4

Stimulation of MMP-2 activation in ARPE-19 cells by CTGF. MMP-2 protein activity was evaluated by zymography in presence of 10 or 40 ng/mL CTGF for 36 hours. Top: gelatin zymogram from a representative experiment. Bottom: statistically significant differences (P = 0.02), compared with the control.

Figure 5

Activation of p38 (A) and ERK1/2 (B) MAP kinases by CTGF in ARPE-19 cells. Top: Western blot for phosphorylated and total levels of p38 and ERK1/2 in the ARPE-19 cells. Bottom: the graphs show the ratios of phosphorylated to total p38 (A) and ERK1/2 (B). p38 and ERK1/2 were significantly activated by CTGF. Statistically significant differences (P = 0.02) are seen, compared with controls.

Figure 6

Representative Western blot showing specific inhibition by (A) p38 MAPK (SB203580; 20 μM, designated SB) of phosphorylated p38 (p-p38), and (B) ERK1/2 (PD098059; 20 μM, designated PD) inhibition of phosphorylated ERK1/2 (p-ERK1/2) of ARPE-19 cells stimulated with CTGF.

Figure 7

Inhibition of fibronectin (A, D), laminin (B, E), and MMP-2 (C, F) expression by an ERK1/2 inhibitor. (A–C) RT-qPCR analyses show significant suppression of fibronectin (A), laminin (B), and MMP-2 (C) expression by the MEK-1 inhibitor, PD98059. (D–F) Top: Western blot from a representative experiment. Bottom: graphical representation of differences compared with the vehicle control. Statistically significant differences are indicated. PD, ERK1/2 inhibitor PD098059; SB, p38 MAPK inhibitor SB203580.

Figure 8

Immunohistochemistry of CTGF in human maculas. (A, B) A 78-year-old female with AMD shows strong labeling for CTGF throughout Bruch membrane (BrM). Strong immu-nolabeling is seen in the RPE basement membrane (long arrows), basal deposits (short arrows), a druse (*), and the choriocapillaris basement membrane. The choroid also shows staining for CTGF. (C) A 42-year-old female with no history of AMD shows light immunostaining of Bruch membrane and the choroid. (D) IgG control of the 78-year-old female shown in (A, B). Scale bar, 15 μm.