Investigative ophthalmology & visual science · 2008
PDGF- and insulin/IGF-1-specific distinct modes of class IA PI 3-kinase activation in normal rat retinas and RGC-5 retinal ganglion cells.
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Biswas Swarajit K, Zhao Yan, Nagalingam Arumugam, Gardner Thomas W, Sandirasegarane Lakshman
Аннотация
PURPOSE: To compare PDGF- and insulin/IGF-1-induced class I(A) PI 3-kinase/Akt survival signaling in normal retinas and retinal ganglion cells (RGCs).
METHODS: Normal rat retinas and RGC-5 cells were used for (1) immunohistochemical and immunoblot studies to detect PDGF receptor (PDGFR) subtypes and (2) immunoprecipitation, immunoblot, and in vitro lipid kinase assays to determine basal and PDGF-induced class I(A) PI 3-kinase/Akt survival signaling, in comparison with insulin or IGF-1 responses. Furthermore, RGC-5 cells were exposed to broad-spectrum (LY294002) or p110 isoform-selective (PI-103) PI 3-kinase inhibitors (versus Akt inhibitor) to assess the consequent effects on Akt phosphorylation, caspase-3/PARP cleavage, apoptotic phenotype, and cell viability, as a function of serum trophic factors.
RESULTS: PDGFR-alpha and -beta immunoreactivity was observed in rat retinal Müller cells and in the RGC layer and blood vessels, respectively. In addition, PDGFR-alpha and -beta protein expression was observed in RGC-5 cells. Both retinas and RGC-5 cells exhibited a similar pattern of subunit-specific basal class I(A) PI 3-kinase activity, which was stimulated in a temporal and signal-specific manner by PDGF and insulin/IGF-1. Furthermore, RGC-5 cells showed PDGFR-alpha/beta tyrosine phosphorylation that induced the p85alpha regulatory subunit to activate p110alpha/beta-associated class I(A) PI 3-kinase, which in turn enhanced Akt phosphorylation. Exposure of serum-deprived RGC-5 cells to PI 3-kinase or Akt inhibitors increased susceptibility to apoptotic phenotype as revealed by caspase-3 and PARP cleavage.
CONCLUSIONS: The present findings provide direct evidence of two distinct modes of retinal class I(A) PI 3-kinase activation that occurs in response to PDGF receptor and insulin/IGF-1 receptor stimulation. PDGF-induced PI 3-kinase/PIP3/Akt axis may provide new therapeutic approaches to ameliorate cell death in diabetic retinopathy and other retinal neurodegenerations.
METHODS: Normal rat retinas and RGC-5 cells were used for (1) immunohistochemical and immunoblot studies to detect PDGF receptor (PDGFR) subtypes and (2) immunoprecipitation, immunoblot, and in vitro lipid kinase assays to determine basal and PDGF-induced class I(A) PI 3-kinase/Akt survival signaling, in comparison with insulin or IGF-1 responses. Furthermore, RGC-5 cells were exposed to broad-spectrum (LY294002) or p110 isoform-selective (PI-103) PI 3-kinase inhibitors (versus Akt inhibitor) to assess the consequent effects on Akt phosphorylation, caspase-3/PARP cleavage, apoptotic phenotype, and cell viability, as a function of serum trophic factors.
RESULTS: PDGFR-alpha and -beta immunoreactivity was observed in rat retinal Müller cells and in the RGC layer and blood vessels, respectively. In addition, PDGFR-alpha and -beta protein expression was observed in RGC-5 cells. Both retinas and RGC-5 cells exhibited a similar pattern of subunit-specific basal class I(A) PI 3-kinase activity, which was stimulated in a temporal and signal-specific manner by PDGF and insulin/IGF-1. Furthermore, RGC-5 cells showed PDGFR-alpha/beta tyrosine phosphorylation that induced the p85alpha regulatory subunit to activate p110alpha/beta-associated class I(A) PI 3-kinase, which in turn enhanced Akt phosphorylation. Exposure of serum-deprived RGC-5 cells to PI 3-kinase or Akt inhibitors increased susceptibility to apoptotic phenotype as revealed by caspase-3 and PARP cleavage.
CONCLUSIONS: The present findings provide direct evidence of two distinct modes of retinal class I(A) PI 3-kinase activation that occurs in response to PDGF receptor and insulin/IGF-1 receptor stimulation. PDGF-induced PI 3-kinase/PIP3/Akt axis may provide new therapeutic approaches to ameliorate cell death in diabetic retinopathy and other retinal neurodegenerations.
Предметные рубрики MeSH
AnimalsBecaplerminCaspase 3Cell LineCell SurvivalChromonesEnzyme ActivationFuransImmunoblottingImmunohistochemistryInsulinInsulin-Like Growth Factor IMaleMorpholinesPhosphatidylinositol 3-KinasesPhosphoinositide-3 Kinase InhibitorsPhosphorylationPlatelet-Derived Growth FactorPoly(ADP-ribose) PolymerasesProto-Oncogene Proteins c-aktProto-Oncogene Proteins c-sisPyridinesPyrimidinesRatsRats, Sprague-DawleyReceptor, IGF Type 1Receptor, InsulinReceptor, Platelet-Derived Growth Factor alphaReceptor, Platelet-Derived Growth Factor betaRetinaRetinal Ganglion CellsSignal Transduction