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Role of HGF/c-Met in serum-starved ARPE-19 cells.

Jun EJ, Kim HS, Kim YH - Korean J Ophthalmol (2007)

Bottom Line: ARPE-19 cell proliferation was also enhanced with recombinant HGF treatment.Neutralization against c-Met inhibited the proliferation of serum-deprived ARPE-19 by 64.5% (n=9, S.D. 5.5%).In conclusion, c-Met induced under non-physiologic conditions has significant effects on the activation of RPE cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, College of Medicine, The Catholic University of Korea, Seoul, Korea.

ABSTRACT

Purpose: Hepatocyte growth factor (HGF) and its receptor (HGFR/c-Met) regulate motility, mitogenesis, and morphogenesis in a cell type-dependent fashion. We report the role of HGF and c-Met on stress-induced ARPE-19 human retinal pigment epithelial (RPE) cells in this study.

Methods: The cells were cultured either with or without serum. Southern and Western blot analyses were done to determine the expression patterns of HGF/c-Met in serum-starved ARPE-19 cells. The cell proliferation pattern in serum-starved condition was analyzed using MTS assay. Inhibition level of cell proliferation was analyzed using a neutralizing monoclonal antibody against c-Met (2 microg/ml).

Results: Abnormal cell proliferation and scattering of ARPE-19 cells was observed under serum starvation. HGF/c-Met were expressed in serum-starved ARPE-19 cells. ARPE-19 cell proliferation was also enhanced with recombinant HGF treatment. Neutralization against c-Met inhibited the proliferation of serum-deprived ARPE-19 by 64.5% (n=9, S.D. 5.5%). Serum starvation appears to induce epithelial-mesenchymal transition of ARPE-19 cells, resulting in scatter, and the expression of alpha-smooth muscle actin (alpha-SMA), a marker for fibrosis.

Conclusions: In conclusion, c-Met induced under non-physiologic conditions has significant effects on the activation of RPE cells.

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Related in: MedlinePlus

A. Effect of HGF on proliferation of ARPE-19. After serum starvation for 24 hours, HGF at 0.01, 0.1, 1, 10, 50 ng/mlconcentrations were added and incubation was done for 48 hours under serum-free conditions. HGF stimulated ARPE-19 cell proliferation in a dose-dependent manner. (n=9, S.D. 3.7%, p<0.01) B. Inhibition of ARPE-19 cell proliferation with neutralizing the HGF receptor/c-Met. To investigate the relationship between the induction of c-met, HGF expression and serum-starved ARPE-19 proliferation, we performed a colorimetric procedure to the known MTS proliferation assay. Neutralized ARPE-19 cells with anti-c-met antibody (2 µg/ml) inhibited cell proliferation up to 55%. A and B data are presented as means±S.D, n=9. C and D. Down regulation of HGF/c-Met in ARPE-19 cells with neutralizing c-Met antibody. After neutralization, HGF was down-regulated at the transcriptional level (C), but c-Met was at translational level (D). C; normal ARPE-19 as a control, S; after serum starvation for 24 hours, PBS was added for 2 days as a control of mAb, S+mAb; after serum starvation for 24 hours, neutralization was performed using monoclonal anti-mouse c-Metantibody (mAb) for 2 days.
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Figure 3: A. Effect of HGF on proliferation of ARPE-19. After serum starvation for 24 hours, HGF at 0.01, 0.1, 1, 10, 50 ng/mlconcentrations were added and incubation was done for 48 hours under serum-free conditions. HGF stimulated ARPE-19 cell proliferation in a dose-dependent manner. (n=9, S.D. 3.7%, p<0.01) B. Inhibition of ARPE-19 cell proliferation with neutralizing the HGF receptor/c-Met. To investigate the relationship between the induction of c-met, HGF expression and serum-starved ARPE-19 proliferation, we performed a colorimetric procedure to the known MTS proliferation assay. Neutralized ARPE-19 cells with anti-c-met antibody (2 µg/ml) inhibited cell proliferation up to 55%. A and B data are presented as means±S.D, n=9. C and D. Down regulation of HGF/c-Met in ARPE-19 cells with neutralizing c-Met antibody. After neutralization, HGF was down-regulated at the transcriptional level (C), but c-Met was at translational level (D). C; normal ARPE-19 as a control, S; after serum starvation for 24 hours, PBS was added for 2 days as a control of mAb, S+mAb; after serum starvation for 24 hours, neutralization was performed using monoclonal anti-mouse c-Metantibody (mAb) for 2 days.

Mentions: In normal healthy eyes, retinal pigment epithelial (RPE) cells form a polarized monolayer adjacent to the photoreceptors and are involved in various activities that are essential to retinal homeostasis and visual function. RPE cells are commonly activated by failure of retinal adhesion that causes proliferative vitreoretinopathy (PVR). Scar tissue develops as epiretinal and subretinal membranes, and this causes a pathological condition in which these cells become activated and mobilized.1 RPE cells are a key component of epiretinal and subretinal membranes, and in those eyes that develop PVR, the RPE cells beneath the detached neural retina undergo an epithelial-to-mesenchymal transition that involves a wide range of changes. The epithelium loses its hexagonal shape and resembles fibroblasts. The cells undergo a change from being a static non-dividing cell becoming a migratory and proliferating variant.2 Transition of RPE cells is modulated by soluble growth factors and cytokines such as tumor necrosis factor-α (TNF-α), interlukin-1 (IL-1) and platelet-derived growth factor (PDGF).3 The basic pathomolecular mechanism by which the sedentary RPE cells become activated is still poorly understood. Among the involved factors, hepatocyte growth factor (HGF) or scatter factor (SF) is associated with increasing the mobility of various types of epithelium.2,4


Role of HGF/c-Met in serum-starved ARPE-19 cells.

Jun EJ, Kim HS, Kim YH - Korean J Ophthalmol (2007)

A. Effect of HGF on proliferation of ARPE-19. After serum starvation for 24 hours, HGF at 0.01, 0.1, 1, 10, 50 ng/mlconcentrations were added and incubation was done for 48 hours under serum-free conditions. HGF stimulated ARPE-19 cell proliferation in a dose-dependent manner. (n=9, S.D. 3.7%, p<0.01) B. Inhibition of ARPE-19 cell proliferation with neutralizing the HGF receptor/c-Met. To investigate the relationship between the induction of c-met, HGF expression and serum-starved ARPE-19 proliferation, we performed a colorimetric procedure to the known MTS proliferation assay. Neutralized ARPE-19 cells with anti-c-met antibody (2 µg/ml) inhibited cell proliferation up to 55%. A and B data are presented as means±S.D, n=9. C and D. Down regulation of HGF/c-Met in ARPE-19 cells with neutralizing c-Met antibody. After neutralization, HGF was down-regulated at the transcriptional level (C), but c-Met was at translational level (D). C; normal ARPE-19 as a control, S; after serum starvation for 24 hours, PBS was added for 2 days as a control of mAb, S+mAb; after serum starvation for 24 hours, neutralization was performed using monoclonal anti-mouse c-Metantibody (mAb) for 2 days.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2629891&req=5

Figure 3: A. Effect of HGF on proliferation of ARPE-19. After serum starvation for 24 hours, HGF at 0.01, 0.1, 1, 10, 50 ng/mlconcentrations were added and incubation was done for 48 hours under serum-free conditions. HGF stimulated ARPE-19 cell proliferation in a dose-dependent manner. (n=9, S.D. 3.7%, p<0.01) B. Inhibition of ARPE-19 cell proliferation with neutralizing the HGF receptor/c-Met. To investigate the relationship between the induction of c-met, HGF expression and serum-starved ARPE-19 proliferation, we performed a colorimetric procedure to the known MTS proliferation assay. Neutralized ARPE-19 cells with anti-c-met antibody (2 µg/ml) inhibited cell proliferation up to 55%. A and B data are presented as means±S.D, n=9. C and D. Down regulation of HGF/c-Met in ARPE-19 cells with neutralizing c-Met antibody. After neutralization, HGF was down-regulated at the transcriptional level (C), but c-Met was at translational level (D). C; normal ARPE-19 as a control, S; after serum starvation for 24 hours, PBS was added for 2 days as a control of mAb, S+mAb; after serum starvation for 24 hours, neutralization was performed using monoclonal anti-mouse c-Metantibody (mAb) for 2 days.
Mentions: In normal healthy eyes, retinal pigment epithelial (RPE) cells form a polarized monolayer adjacent to the photoreceptors and are involved in various activities that are essential to retinal homeostasis and visual function. RPE cells are commonly activated by failure of retinal adhesion that causes proliferative vitreoretinopathy (PVR). Scar tissue develops as epiretinal and subretinal membranes, and this causes a pathological condition in which these cells become activated and mobilized.1 RPE cells are a key component of epiretinal and subretinal membranes, and in those eyes that develop PVR, the RPE cells beneath the detached neural retina undergo an epithelial-to-mesenchymal transition that involves a wide range of changes. The epithelium loses its hexagonal shape and resembles fibroblasts. The cells undergo a change from being a static non-dividing cell becoming a migratory and proliferating variant.2 Transition of RPE cells is modulated by soluble growth factors and cytokines such as tumor necrosis factor-α (TNF-α), interlukin-1 (IL-1) and platelet-derived growth factor (PDGF).3 The basic pathomolecular mechanism by which the sedentary RPE cells become activated is still poorly understood. Among the involved factors, hepatocyte growth factor (HGF) or scatter factor (SF) is associated with increasing the mobility of various types of epithelium.2,4

Bottom Line: ARPE-19 cell proliferation was also enhanced with recombinant HGF treatment.Neutralization against c-Met inhibited the proliferation of serum-deprived ARPE-19 by 64.5% (n=9, S.D. 5.5%).In conclusion, c-Met induced under non-physiologic conditions has significant effects on the activation of RPE cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, College of Medicine, The Catholic University of Korea, Seoul, Korea.

ABSTRACT

Purpose: Hepatocyte growth factor (HGF) and its receptor (HGFR/c-Met) regulate motility, mitogenesis, and morphogenesis in a cell type-dependent fashion. We report the role of HGF and c-Met on stress-induced ARPE-19 human retinal pigment epithelial (RPE) cells in this study.

Methods: The cells were cultured either with or without serum. Southern and Western blot analyses were done to determine the expression patterns of HGF/c-Met in serum-starved ARPE-19 cells. The cell proliferation pattern in serum-starved condition was analyzed using MTS assay. Inhibition level of cell proliferation was analyzed using a neutralizing monoclonal antibody against c-Met (2 microg/ml).

Results: Abnormal cell proliferation and scattering of ARPE-19 cells was observed under serum starvation. HGF/c-Met were expressed in serum-starved ARPE-19 cells. ARPE-19 cell proliferation was also enhanced with recombinant HGF treatment. Neutralization against c-Met inhibited the proliferation of serum-deprived ARPE-19 by 64.5% (n=9, S.D. 5.5%). Serum starvation appears to induce epithelial-mesenchymal transition of ARPE-19 cells, resulting in scatter, and the expression of alpha-smooth muscle actin (alpha-SMA), a marker for fibrosis.

Conclusions: In conclusion, c-Met induced under non-physiologic conditions has significant effects on the activation of RPE cells.

Show MeSH
Related in: MedlinePlus