| The new physiological function of reactive oxygen species (ROS) has been shown to mediating mitogen-stimulated cell signaling in several cell types. We have identified the mitogen-induced endogenous ROS generation and the range of exogenous H2O2 that initiate redox signaling and cell proliferation in human lens epithelial cells (HLE B3), using platelet-derived growth factor (PDGF) as a model. PDGF stimulated the lens cells to transiently generate ROS, which corroborated with activated MAPK pathways and increased cell proliferation, all of which were suppressed if the cells were pretreated with antioxidants or free radical scavengers. The ability of exogenous catalase to penetrate HLE B3 cells and to neutralize PDGF-induced ROS has been clarified and confirmed for the first time. ROS-mediated PDGF mitogenic action has been further established by our finding that H2O2 at 10-20 muM could mimic PDGF by stimulating signaling proteins and enhancing cell growth.; Specific inhibitors and mutant cells were used to examine the initial sites of PDGF-stimulated signaling that influence ROS production and cell growth. The results suggest that PDGF receptor kinase and its immediate downstream signaling components of Src family kinase, PI3K, and the small GTPase of Rac and Ras are essential for PDGF to induce ROS production, MAPK activation and cell proliferation. Patterns of MAPK activation further suggest a novel crosstalk among receptors, i.e., the transactivation between PDGF receptor (PDGFR) and other growth factor receptors, and the integrative receptor-signaling complex between PDGFR and G-protein coupled receptor.; Finally, microarray analysis of PDGF-stimulated HLE B3 cells displayed several groups of genes that were increased in expression. The first group is involved with protein synthesis for cell migration, structural maintenance, adhesion, and remodeling (e.g. ACTR2, FN1, ICAM1, and MMP7). A second group of genes is related to the regulation of cell cycle, proliferation, differentiation and transformation (e.g. PDGFRbeta, PIK3R2, FOS, IKBKB, IL-2, MAP2K7, ERK 1, JNK 2, STAT1/3/5A, Egr-1 and ATF2) while the third group of genes is associated with modulating ROS generation (e.g. PKCalpha, Akt1, IL-2, FAGL and PLCgamma). Interestingly, expression of EGFR was also modestly raised, likely the result of transactivation with PDGFR. |
