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Hepatocyte Growth Factor Protects RPE Cells from Apoptosis Induced by Glutathione Depletion

¹From the Arnold and Mabel Beckman Macular Research Center; the ²Departments of Pathology and ⁴Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, California; and the ⁵Doheny Eye Institute, Los Angeles, California.

PURPOSE. To study the mechanism of the protective effect of hepatocyte growth factor (HGF) in oxidative injury to RPE cells induced by glutathione (GSH) depletion.

METHODS. RPE cells were treated with HGF for 24 hours (20 ng/mL) and then were treated with DL-buthionine-(S,R)-sulfoximine (BSO) for an additional 24 hours. Cell death, apoptosis, and GSH levels were measured. Levels of intracellular reactive oxygen species (ROS) and their cellular localization were assessed by confocal microscopy. Expression of Bcl-2 and release of cytochrome c from mitochondria were quantified. The effect of BSO on caspase-3 activation and expression was determined. Gene expression of key enzymes of GSH metabolism by real-time PCR and regulation and translocation of the transcription factor NF-E2–related factor (Nrf2) by BSO were examined.

RESULTS. Treatment with BSO-induced apoptosis in RPE caused a significant decrease in intracellular GSH and in GSH/GSSG ratios. Marked increases in lipid peroxidase (LPO), ROS, and mitochondrial cytochrome c release and a decrease in Bcl-2 expression were observed. Elevated GSH/GSSG ratio (especially in mitochondria), decreased LPO and ROS, attenuation of apoptosis, and partial restoration of Bcl-2 expression were found in the HGF-pretreated cells. BSO activated caspase-3, and this effect was significantly blocked by HGF. Both HGF and BSO induced anti-oxidant gene expression. Nrf2 translocated to the nuclear region after treatment with BSO, whereas HGF did not induce such translocation.

CONCLUSIONS. The protective effect of HGF may be attributed in part to the elevation of mitochondrial GSH. BSO and HGF act in concert to enhance GSH-related gene expression in stressed RPE cells.

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