Figure 1.
Growth factors induced cell migration of ARPE19 and RPE50.
(A) ARPE 19 was cultivated with a wound healing culture insert and serum-starved for 48 h. After removal of culture insert (0 h), cells were treated with 50 nM of HGF (H50), EGF (E50), TGFβ1 (T50) or PDGF (P50), 50 ng/ml HB-EGF (HB-E50), 25 nM HGF coupled with 25 nM EGF (H25/E25) or 25 nM HGF coupled with 25 ng/ml EGF (H25/HB-E25) in serum free medium for 18 h and photographed. The cells migrated into the blanking area between the indicated orange lines representing the boundary between the blanking area and cell culture at 0 h. The results were representatives of four reproducible experiments. (B) Quantitative analysis of relative migration of ARPE19 (gray) and RPE 50 (black) treated with growth factors as indicated in (A) and supplemental Figure 1, respectively. The number of cells migrated into the wound area were counted under phase contrast microscope (at 200-fold magnification). For each treatment, total cell numbers in four microscopic fields were counted. Relative migration of the cells were calculated as the ratio of cell numbers of each treatment vs. that of untreated cells, taken the ratio of untreated cells as 1.0. The results were averages of three experiments with coefficient of variation (C.V.) of 5.5%. (*) and (**) represent statistical significance (P<0.05 and P<0.005, respectively) for comparison of the growth factor-treated with untreated group. (##) represent statistical significance (P<0.005) for comparison of the combined growth factors vs. single growth factors group as described in the text. (C) and (D) RPE 50 (black) and ARPE19 (gray) were untreated (-), treated with 50 nM HGF (C) and EGF (D), or HGF and EGF coupled with DMSO (as solvent control) or various inhibitors as indicated. Wound healing assay were performed and relative migrations were quantitated as described in (B). JNJ in (C): JNJ38877605. The results were averages of 3 experiments with coefficient of variation (C.V.) of 5.5%. (**) represent statistical significance (P<0.005) for comparison of the inhibitor- vs. DMSO-treated group.
Figure 2.
Growth factors induced ERK phosphorylation in RPE50 and ARPE19.
(A) RPE50 cells were untreated (-), treated with 50 nM of HGF, EGF, TGFβ and PDGF for the time indicated. (B) RPE50 cells were untreated (-), treated with 50 nM of HGF or EGF or 50 ng/ml HB-EGF coupled with DMSO or various inhibitors as indicated for 0.5 h. (C) RPE50 (upper panel) and ARPE 19 (lower panel) cells were untreated (a and b), treated with 50 nM of HGF (H50) (a and b) and EGF (E50) (a), 50 ng/ml HB-EGF (HB-E 50) (b), 25 nM of EGF and HGF in combination (E25/H25) (a) or 25 ng/ml HB-EGF and 25 nM HGF in combination (HB-E25/H25) (b) for 0.5 h. Western blot of p-ERK were performed using ERK as internal control. The relative ratios of the intensity for p-ERK/ERK (indicated below each lane) were calculated, taking the ratio of the untreated group as 1.0. The results were averages of 2 experiments with coefficient of variation (C.V.) of 6.0–7.0%.
Figure 3.
Growth factors induced PKC activation in RPE50 and ARPE19.
(A) RPE50 cells were untreated (-), treated with 50 nM of HGF or EGF, or 50 ng/ml HB-EGF for the time indicated. (B) and (E) RPE50 cells were untreated (-), treated with 50 nM EGF or EGF coupled with DMSO or various inhibitor as indicated for 0.5 h. (C) and (D) RPE50 cells were untreated, treated with 50 nM of HGF (H50) or EGF (E50), 50 ng/ml HB-EGF (HB-E50), 25 nM of EGF and HGF in combination (E25/H25) or 25 ng/ml HB-EGF and 25 nM HGF in combination (HB-E25/H25) for 0.5 h. PKC activity assay were performed. Relative PKC activity was obtained as the ratio of calculated PKC activity of each treatment vs. that of untreated cell, taking the ratio of untreated cell as 1.0. The results were averages of 3 experiments with coefficient of variation (C.V.) of 5.5%. In (A), (*) and (**) represent statistical significance (P<0.05 and P<0.005, respectively) for comparison of the growth factor-treated vs. untreated group. In (B) and (E), (*) and (**) represent statistical significance (P<0.05 and P<0.005, respectively) for comparison of the inhibitor- vs. DMSO-treated group. In (C) and (D), (**) represent statistical significance (P<0.005) for comparison of the single growth factor-treated vs. untreated group. (##) represent statistical significance (P<0.005) for comparison of the combined growth factors-treated vs. single growth factor-treated group as described in the text.
Figure 4.
Prevention of growth factors-induced cell migration and ERK phosphorylation of RPE by inhibitors of PKC isozymes.
RPE50 (black) (A, B) and ARPE 19 (gray) cells (A) were cultivated with a wound healing culture insert and depleted of serum for 24 h. After removing the culture insert, cells were untreated (-), treated with 25 nM HGF/EGF in combination (A) or 50 nM EGF (B), or those coupled with DMSO, PD98059 or various PKC isozyme inhibitors as indicated for 18 h. Wound healing assay were performed and quantitated as described in Fig. 1B. (*) and (**) represent statistical significance (P<0.05 and P<0.005, respectively) for comparison of the inhibitors-treated vs. DMSO-treated group. The results were averages of 5 experiments with coefficient of variation (C.V.) of 5.5%. (C) RPE50 cells were untreated (-), treated with 50 nM of EGF or EGF coupled with DMSO or various inhibitors as indicated for 0.5 h. Western blot of p-ERK were performed using ERK as internal control. The relative ratios of the intensity for p-ERK/ERK (indicated below each lane) were calculated, taking the ratio of the untreated group as 1.0. The results were averages of 2 experiments with coefficient of variation (C.V.) of 6.0–7.0%.
Figure 5.
Prevention of growth factors-induced cell migration and ERK phosphorylation of RPE50 and ARPE19 by depletion of PKC isozymes.
(A) RPE50 (black) and ARPE 19 (gray) cells were cultivated with a wound healing culture insert for 24 h, transfected with shRNA of GFP or PKC α, β or δ for 36 h followed by treatment with 50 nM EGF in serum free medium for 18 h. Wound healing assay were performed and quantitated as in Figure 1B. (*) and (**) represent statistical significance (P<0.05 and P<0.005, respectively) for comparison of the PKC isozyme shRNA vs. GFP shRNA group. (B) and (C) RPE50 was transfected with shRNA of GFP or PKCα, β or δ for 36 h followed by treatment with 50 nM EGF for 0.5 h (B) or none (C). Western blot of p-ERK (B) and PKCα, β or δ(C) were performed using ERK as internal control. The relative ratios of the intensity for p-ERK/ERK (B) and PKC isozyme/ERK (C) were calculated, taking the ratio of the untreated group as 1.0. The results were averages of 2 experiments with coefficient of variation of 6.0–7.0%.
Figure 6.
Inhibitors of the of EGF and HGF receptors prevented migration and ERK phosphorylation reciprocally induced by HGF and EGF.
RPE50 cells were untreated, treated with 50 nM of HGF or EGF coupled with the indicated inhibitors (AG: AG1478, JNJ: JNJ38877605) or DMSO (as solvent control) for 24 h (A) or 30 min (B and C). Wound healing cell migration assay (A), PKC assay (B) and Western blot of p-ERK (C) were performed. In (A) and (B), (*) and (**) represent statistical significance (P<0.05 and P<0.005, respectively) for comparison of the inhibitors-treated vs. DMSO-treated group. Western blot of ERK served as internal control for (C). The relative ratios of the intensity for p-ERK/ERK (indicated below each lane) were calculated, taking the ratio of the untreated group as 1.0. The results were averages of 2 experiments with coefficient of variation (C.V.) of 6.0–7.0%.
Figure 7.
Prevention of cell migration of RPE50 induced by vitreous fluid by inhibitors of PKC and ERK.
RPE50 cells were cultivated with a wound healing culture insert for 24 h. Cells were then untreated (-), treated with vitreous fluids PVR-vitreo, PDR-vitreo and RD-vitreo obtained from patients of PVR, PDR and RD, respectively, for 18 h. Each vitreous fluid was normalized with equal amount of protein (10–20 µg). Wound healing assay were performed and quantitated as described in Figure 1 B. The results were averages of 3 experiments with coefficient of variation of 7.5%. (*) and (**) represent statistical significance (P<0.05 and P<0.005, respectively) for comparison of the vitreous fluid treated-sample vs. untreated sample. In each vitreous fluid–treated group, (#) and (##) represent statistical significance (P<0.05 and P<0.005, respectively) for comparison of the inhibitor- vs. DMSO-treated group.