Platelet derived growth factor (PDGF)-induced reactive oxygen species in the lens epithelial cells: the redox signaling

Abstract

Low level of reactive oxygen species (ROS) has been shown to play an important role in host defense and mediating mitogen-stimulated cell signaling in several cell types. This study is to identify the mitogen-induced endogenous ROS generation and the range of exogenous H 2O 2 that initiate redox signaling and cell proliferation in human lens epithelial cells (HLE B3), using platelet-derived growth factor (PDGF) as a model. To detect ROS generation, serum starved HLE cells (1·6 million) were loaded with fluorescent dye, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), before exposing to PDGF (1 ng ml −1). The fluorescence generated from the oxidant-sensitive DCFH, the intracellular product of DCFH-DA hydrolysate, was immediately measured in live cells by confocal laser light microscopy ( λ Ex =488 nm, λ Em =522 nm, laser power=10%). PDGF-stimulated cells showed strong transient fluorescence during the 60 min while no fluorescence could be seen in the unstimulated cells. The PDGF-induced fluorescence could be suppressed with cells preloaded with N-acetyl- l-cysteine (NAC, 30 m m), catalase (1 mg ml −1), or D-mannitol (100 m m). The ability of catalase to penetrate and function in HLE cells was confirmed by western blot, enzyme activity and immunofluorescence microscopic analyses. PDGF induced DNA synthesis within one hour as measured by 3H-thymidine incorporation, and transiently activated the mitogen-activated protein kinases (MAPKs) of ERK1/2 and JNK. PDGF-stimulated DNA synthesis and MAPK activation were eliminated in the presence of catalase or mannitol. Low levels of H 2O 2 (10–20 μ m) mimicked PDGF in both MAPK stimulation and cell proliferation. In conclusion, the mitogenic stimulus function of PDGF in HLE cells appears to be mediated via ROS to activate MAPKs and cell proliferation, which can be mimicked by low levels of H 2O 2. It is proposed that the physiological function of ROS, the redox signaling, is present in the HLE cells and may play an important role in the development and maintenance of the lens.