Imatinib induces apoptosis by inhibiting PDGF- but not insulin-induced PI 3-kinase/Akt survival signaling in RGC-5 retinal ganglion cells.
OBJECTIVE
Platelet-derived growth factor (PDGF) and insulin promote the survival of neuronal cells, including retinal ganglion cells (RGCs), via activation of phosphoinositide 3-kinase (PI 3-kinase)/Akt signaling. Of importance, recent studies have shown that imatinib inhibition of PDGF receptors induces retinal toxicity in some patients. To date, the extent of activation and the functional significance of insulin-induced PI 3-kinase/Akt signaling remain unclear in the context of dysregulated PDGF receptor signaling in retinal cells. In the present study, we tested the hypothesis that the pro-survival effect of insulin-induced PI 3-kinase/Akt signaling is compromised by imatinib inhibition of PDGF receptor signaling in RGCs.


METHODS
RGC-5 cells were subjected to acute and long-term treatments with imatinib, a PDGF receptor tyrosine kinase inhibitor. Afterwards, the changes in RGC phenotype and apoptotic markers were assessed by fluorescence and phase contrast microscopy and caspase-3/poly(ADP-ribose) polymerase (PARP) cleavage, respectively. In addition, imatinib regulation of PDGF- and insulin-induced PI 3-kinase/Akt survival signaling was determined by immunoblot analyses, immunoprecipitation, and in vitro PI 3-kinase assays.


RESULTS
Treatment of RGC-5 cells with imatinib for up to 48 h resulted in apoptosis, which was not rescued by insulin supplementation. The apoptotic phenotype was associated with upregulation of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase. Time dependency experiments revealed that imatinib-mediated apoptosis was preceded by early and sustained abrogation of PDGF-induced increases in PDGF receptor tyrosine phosphorylation and phosphotyrosine-associated PI 3-kinase activity. In addition, imatinib inhibited PDGF-induced downstream phosphorylation of Akt, GSK-3beta, and p70S6kinase. However, imatinib exposure did not affect insulin-induced insulin receptor substrate (IRS)-associated PI 3-kinase activity and the downstream phosphorylation of Akt, GSK-3beta, and p70S6kinase.


CONCLUSIONS
Together, these data indicate that disruption of PDGF receptor signaling compromises the pro-survival effect of insulin-induced IRS-dependent PI 3-kinase/Akt signaling in RGCs, and that the maintenance of PDGF-induced PI 3-kinase/Akt signaling is critical for the survival of retinal neuronal cells.
Biswas
Swarajit K
SK
Department of Medicine, Penn State Hershey Heart & Vascular Institute, Penn State College of Medicine, Hershey, PA 17033, USA.
Zhao
Yan
Y
Sandirasegarane
Lakshman
L
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001549:Benzamides; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D000068877:Imatinib Mesylate; D007328:Insulin / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D010879:Piperazines / Q000494:pharmacology*; D010982:Platelet-Derived Growth Factor / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology*; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*
D001549:Benzamides; D007328:Insulin; D010879:Piperazines; D010982:Platelet-Derived Growth Factor; D011743:Pyrimidines; D019000:Phosphotyrosine; D000068877:Imatinib Mesylate; D011065:Poly(ADP-ribose) Polymerases; D019869:Phosphatidylinositol 3-Kinases; D011972:Receptor, Insulin; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D051057:Proto-Oncogene Proteins c-akt; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D038362:Glycogen Synthase Kinase 3; D053148:Caspase 3
J Biol Chem. 2007 Sep 7;282(36):26077-88
17620332
Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):8159-63
1654560
Science. 2005 Mar 18;307(5716):1711
15774738
Neurosci Lett. 1995 Oct 13;199(1):37-40
8584221
Invest Ophthalmol Vis Sci. 2005 Apr;46(4):1330-8
15790899
Invest Ophthalmol Vis Sci. 2008 Aug;49(8):3687-98
18421086
J Clin Invest. 1998 Aug 15;102(4):783-91
9710447
Mol Cell Neurosci. 2008 Mar;37(3):507-18
18243733
J Neurochem. 2004 Apr;89(1):109-18
15030394
Curr Biol. 2004 Jul 13;14(13):R497-500
15242626
Lancet. 2002 May 18;359(9319):1751-2
12049868
Jpn J Ophthalmol. 2008 Jul-Aug;52(4):331-333
18773274
Development. 2000 Apr;127(8):1641-9
10725240
J Neurosci. 1995 Nov;15(11):7095-104
7472464
J Biol Chem. 2001 Aug 31;276(35):32814-21
11443130
Leukemia. 2004 Jun;18(6):1138-9
15085156
J Cell Sci. 2000 Jan;113 Pt 2:207-14
10633072
Brain Res. 2001 Mar 23;895(1-2):111-8
11259767
J Biol Chem. 2007 Apr 13;282(15):10922-34
17303574
Invest Ophthalmol Vis Sci. 2006 Jan;47(1):427-38
16384993
J Cell Sci. 1998 Apr;111 ( Pt 7):907-15
9490635
J Biol Chem. 1997 Jun 27;272(26):16089-92
9195902
Cell. 2003 Jan 10;112(1):63-75
12526794
Mol Biol Cell. 2005 May;16(5):2207-17
15728722
J Neurochem. 2006 Oct;99(2):524-36
17029604
FASEB J. 2003 Sep;17(12):1709-11
12958184
Invest Ophthalmol Vis Sci. 2009 Mar;50(3):1033-40
19029027
Prog Neurobiol. 2006 Jul;79(4):205-21
16916571
Annu Rev Biochem. 1998;67:481-507
9759495
J Neurochem. 2006 Jul;98(2):588-600
16805849
J Biol Chem. 1994 Jul 22;269(29):18961-7
8034653
J Neurochem. 2004 Nov;91(4):920-35
15525346
J Neurosci. 2005 Feb 2;25(5):1240-8
15689562
Mol Pharmacol. 2002 Aug;62(2):225-33
12130673
FEBS Lett. 2003 Jan 30;535(1-3):17-22
12560071
Mol Cell Neurosci. 2005 Aug;29(4):569-79
15936213
Biochem J. 2001 Mar 1;354(Pt 2):359-68
11171115
Trends Immunol. 2007 Apr;28(4):161-8
17336157
Cancer Res. 2005 Mar 15;65(6):2047-53
15781610
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):3100-5
14981233
J Neurosci. 2000 Jan 15;20(2):2-8
10632601
J Biol Chem. 2000 Feb 18;275(7):5228-37
10671571
J Biol Chem. 2008 Jul 11;283(28):19781-92
18480052
J Clin Invest. 2002 Jan;109(1):141-9
11781359
Eur J Neurosci. 2008 Oct;28(7):1255-64
18973553
Diabetes. 2006 Apr;55(4):1148-56
16567541
Vision Res. 1998 Oct;38(20):3031-9
9893812
J Ocul Pharmacol Ther. 2003 Aug;19(4):371-5
12964961
J Cataract Refract Surg. 2005 Dec;31(12):2427-8
16473243
J Clin Oncol. 2004 Nov 15;22(22):4653-5
15542819
Nat Biotechnol. 2001 May;19(5):475-9
11329020
Biochem Soc Trans. 2006 Dec;34(Pt 6):1287-90
17073803
Brain Res Mol Brain Res. 2001 Jan 31;86(1-2):1-12
11165366
J Neurobiol. 1999 Jun 15;39(4):536-46
10380075
Int Ophthalmol. 2007 Feb;27(1):43-4
17410337
J Biol Chem. 1996 Jul 5;271(27):16151-9
8663218
Mol Pharmacol. 2006 May;69(5):1527-33
16436588