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PDGF- and Insulin/IGF-1–Specific Distinct Modes of Class I_A PI 3-Kinase Activation in Normal Rat Retinas and RGC-5 Retinal Ganglion Cells

¹From the Penn State Heart and Vascular Institute, and the ²Departments of Medicine, ⁴Ophthalmology, ⁵Cellular and Molecular Physiology, and ⁶Pharmacology, Penn State College of Medicine, Hershey, Pennsylvania.

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- and -β immunoreactivity was observed in rat retinal Müller cells and in the RGC layer and blood vessels, respectively. In addition, PDGFR- and -β 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-/β tyrosine phosphorylation that induced the p85 regulatory subunit to activate p110/β-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.