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.
OBJECTIVE
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.
Biswas
Swarajit K
SK
Penn State Heart and Vascular Institute, Penn State College of Medicine, Hershey, Pennsylvania 17033, USA.
Zhao
Yan
Y
Nagalingam
Arumugam
A
Gardner
Thomas W
TW
Sandirasegarane
Lakshman
L
D000818:Animals; D000077214:Becaplermin; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D002867:Chromones / Q000494:pharmacology; D004789:Enzyme Activation; D005663:Furans / Q000494:pharmacology; D015151:Immunoblotting; D007150:Immunohistochemistry; D007328:Insulin / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D008297:Male; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D020574:Proto-Oncogene Proteins c-sis; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism; D020796:Receptor, Platelet-Derived Growth Factor alpha / Q000378:metabolism; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000378:metabolism; D012160:Retina / Q000187:drug effects* / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000187:drug effects* / Q000378:metabolism; D015398:Signal Transduction
D000077214:Becaplermin; D053148:Caspase 3; D002867:Chromones; D005663:Furans; D007328:Insulin; D007334:Insulin-Like Growth Factor I; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010982:Platelet-Derived Growth Factor; D011065:Poly(ADP-ribose) Polymerases; D051057:Proto-Oncogene Proteins c-akt; D020574:Proto-Oncogene Proteins c-sis; D011725:Pyridines; D011743:Pyrimidines; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin; D020796:Receptor, Platelet-Derived Growth Factor alpha; D020797:Receptor, Platelet-Derived Growth Factor beta; C522973:PI103; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one