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C. Chen, J.Y. Zhou, K. Xing, M.F. Lou; The Physiological Function of Reactive Oxygen Species, the Redox Signaling, in the Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):312.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Low level of reactive oxygen species (ROS) has been shown to play an important role in host defense and stimulation of cell proliferation in several cell types. This study is to identify the mitogen-induced endogenous ROS generating system and the range of exogenous H2O2 that initiate redox signaling and cell proliferation in human lens epithelial cells (HLE B3). Methods: Confluent HLE cells (1.4 millions) were waned from serum by first culturing overnight in 20% fetal bovine serum (FBS) , then in 2% FBS. The cells were starved for FBS for 30 min before loaded with fluorescent dye (2’,7’-dichlorofluorescein diacetate (DCFDA)) for 5 min in the dark. A bolus of H2O2 (0.02-0.1 mM) or platelet-derived growth factor (PDGF) at 1 ng/ml was added into the medium. The generation of fluorescence was immediately measured by confocal microscope (λεΧ = 488 nm, λem = 522 nm, laser power = 10%) in the presence or absence of ROS inhibitors: N-acetyl-L-cysteine (NAC, 30 mM for 30 min), catalase (1 mg/ml, overnight), and L-mannitol (100 mM for 30 min) at 5 min time intervals. For determining the activation of mitogen activated protein kinases (MAPKs), the cells were treated as described above without loading of the dye. Cells were lyzed with lysis buffer on plate at the end of 5, 10, 15, 20, 40, 60 min. The cell lysates were analyzed by western blot for the activation of MAPK pathways (ERK, JNK, and p38). Results: The endogenous ROS was produced by PDGF stimulation in a time- and concentration-dependent manner. The fluorescence was completely eliminated in cells preloaded with NAC, mannitol or catalase. This pattern of ROS generation coincided with the transient stimulation patterns of Raf-MEK-ERK cascade as well as JNK, but not p38. The diffusion and transient accumulation of exogenous H2O2 (0.04-0.1 mM) in cells during 60 min exposure could also be quantified by DCF-DA dye. The same range of H2O2 showed a transient stimulation of JNK and ERK, similar to that of PDGF. Conclusions: Growth factors can stimulate ROS generation and further activate MAPKs in HLE cells. Exogenous H2O2 at low level mimics growth factor and may play a physiological role for cell proliferation. Supported by NIH grant EY 10590. None
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