Korean J Ophthalmol.  2007 Dec;21(4):244-250. 10.3341/kjo.2007.21.4.244.

Role of HGF/c-Met in Serum-Starved ARPE-19 Cells

• Jun EJ
• Kim HS
• Kim YH

• KMID: 754644
• DOI: http://doi.org/10.3341/kjo.2007.21.4.244

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 microgram/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.

Keyword

HGF/c-Met; PVR; RPE cells; Starvation

MeSH Terms

Blotting, Southern
Blotting, Western
Cell Movement/physiology
Cell Proliferation
Cells, Cultured
Culture Media, Serum-Free
*Gene Expression
Hepatocyte Growth Factor/biosynthesis/*genetics
Humans
Mitosis/physiology
Pigment Epithelium of Eye/cytology/*metabolism
Polymerase Chain Reaction
Proto-Oncogene Proteins c-met/biosynthesis/*genetics
RNA/*genetics

Figure

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  Fig. 1 ARPE-19 cell proliferation patterns and scattering during serum-starvation. Serum-starved ARPE-19 cells were counted using a hemocytometer (A). MTS assay was performed at 490 nm on a micro-enzyme-linked immunosorbent assay (ELISA) plate reader (B). ARPE-19 cells showed scattering pattern and spindle shape after serum starvation for 24 hours (C) and (D).

• 

  Fig. 2 Induction of HGF and HGF-R/c-met expression on serum-starved ARPE-19 cells, as determined by RT-PCR analysis (A) and Western blot (B) and (C). A. After serum starvation for 24 hours, RT-PCR using HGF and HGF-R primer was performed. We detected extremely high levels of c-met and HGF mRNA expression in serum-starved ARPE-19, but only a faint level of c-met mRNA expression in normal ARPE-19 cells was seen. B. To confirm the effect of stress, expression of c-Met proteins in serum-starved ARPE-19 cells was examined using Western blot. The results of western blot from stress-induced ARPE-19 are similar with those of PCR amplification of the cDNA (A). C. HGF expression was not detected from the total cell lysates, but it was detected from the concentrated media. The active form of HGF, mature HGF (69 kD), was detected after serum starvation for several days in concentrated media. *represents serum starvation periods. C; Normal ARPE-19 cell as a control, S; serum deprivation for 24 hours.

• 

  Fig. 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/ml concentrations 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-Met antibody (mAb) for 2 days.

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  Fig. 4 Expression pattern of the α-SMA by stress induction or neutralization against c-Met. RT-PCR and Southern blot analysis of α-SMA expression in ARPE-19 cells. C; normal ARPE-19 as a control, S; after serum starvation for 24 hours, normal mouse IgG 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-Met antibody (mAb) for 2 days.

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