Control of PDGF-induced reactive oxygen species (ROS) generationand signal transduction in human lens epithelial cells

[摘要] Purpose: The mitogenic action of PDGF has been shown to associatewith reactive oxygen species (ROS) generation, but the mechanism leadingto ROS production and subsequent cell proliferation is not clear. Weinvestigated the upstream membrane-bound target proteins involved inPDGF-stimulated signal transduction in human lens epithelial cell (HLEB3), using specific inhibitors and transfected cells.Methods: PDGF (1 ng/ml)-stimulated ROS generation was measured usingfluorescent reaction of DCFDA by confocal microscope in live HLE B3cells. Western blot analysis was used to determine the activated MAPkinases in cell lysates. Specific inhibitors used in this study were:AG1296 for PDGF receptor (PDGFR); AG1517 for EGF receptor (EGFR);pertussis toxin for cytokine-binding G protein coupled receptor (GPCR);PP1 for Src-family kinases; LY294002 for phosphatidylinositol-3 kinase(PI3K). Small GTP-binding proteins Rac and Ras were studied usingtransfectants of dominant negative Rac (Rac N17), Ras (Ras N17) orconstitutively active Rac (Rac V12). Cell proliferation was quantifiedusing BrdU incorporation method.Results: Inhibitions of PDGF receptor kinase, the docking proteincomponent Src-family kinases, and the survival element PI3K alleradicated PDGF-stimulated ROS production and corroborated with thesuppressed cell growth. These inhibitions also attenuated the activatedERK1/2, JNK, and Akt, all downstream targets of the above factors.Interestingly, inhibiting GPCR or EGFR also showed the same effect butto a lesser degree. Co-inhibiting receptors to PDGF and EGF with orwithout co-inhibiting GPCR eradicated the PDGF signaling systemcompletely. Transiently transfected cells with plasmid from smallGTP-binding proteins Rac N17 or Ras N17 diminished PDGF action in ROSgeneration, cell proliferation and MAP kinase activation, while cellswith Rac V12 enhanced the PDGF effect.Conclusions: Our data clarified the potential mechanism of PDGFsignaling in the lens epithelial cells, in which concerted efforts ofthe upstream components of PDGF receptor kinase, Src-family kinases,PI3K, Rac, and Ras proteins are required. This report also providednovel findings that GPCR and EGF receptors may control PDGF signaling inthe lens epithelial cells via integrative signaling and transactivationmechanisms, respectively.

[学科分类] 生物化学/生物物理