RT Journal Article
A1 Kong, Dejuan
A1 Shanmugam, Sumathi
A1 Gong, Lijie
A1 Fort, Patrice E
A1 Gardner, Thomas W
A1 Abcouwer, Steven F
T1 Insulin-like growth factor 1 rescues R28 retinal neurons from apoptotic death through ERK-mediated BimEL phosphorylation independent of Akt
JF Investigative Ophthalmology & Visual Science
JO Invest. Ophthalmol. Vis. Sci.
YR 2016
VO 57
IS 12
SP 2741
OP 2741
SN 1552-5783
AB    IGF-1 is thought to promote neuronal cell survival largely through activation of Akt and deactivation of FoxO proteins. We employed serum deprivation (SD) of R28 cells to explore the mechanisms by which insulin-like growth factor 1 (IGF-1) protects retinal neurons from apoptotic death and to determine the contribution of the Akt/FoxO and MEK/ERK/BimEL pathways.    R28 cells were induced to undergo apoptosis by SD for up to 48 h. IGF-1 was added at concentrations of 10 to 100 ng/mL. Apoptotic cell death was evaluated by western blotting for caspase-9 and caspase-3 cleavage, caspase-3/7 activity, and lactate dehydrogenase (LDH) activity release. Cell signaling was evaluated by western blotting with phospho-specific antibodies to Akt, GSK3, FoxO1, FoxO3, ERK1/2, and Bim isoforms. FoxO protein translocation was assayed by immunofluorescence and subcellular fractionation. Transfection with siRNA was used to silence expression of Rictor, FoxO1, FoxO3 and Bim.    SD caused a progressive increase in cell death. IGF-1 as low as 10 ng/mL afforded significant protection. IGF-1 caused a rapid activation of Akt, as indicated by phosphorylation of Akt-T308, Akt-S473, PRAS40-T246, and FoxO1/3-T32/T24. This led to prevention of FoxO1/3 nuclear translocation and FoxO-mediated Bim mRNA upregulation in response to SD. IGF-1 also caused MAPK/MEK pathway activation as indicated by ERK1/2-T202/Y204 and Bim-S65 phosphorylation. Surprisingly, inhibition of Akt activation with LY294002 or by Rictor knockdown did not block the protective effect of IGF-1. In contrast, inhibition of MEK activity with PD98059 prevented Bim phosphorylation and blocked IGF-1 protection. In addition, knockdown of Bim expression was protective while co-silencing of FoxO1/3 expression had no effect.    Although IGF-1 may provide long-term neurotrophic support by activation of Akt, inhibition of FoxO activity and prevention of Bim gene transcription, this pathway was not essential for protection from SD-induced apoptosis by IGF-1. Instead, IGF-1 protection was dependent on activation of the MEK/ERK pathway leading to BimEL phosphorylation, which is known to prevent Bax/Bak oligomerization and activation of the intrinsic mitochondrial apoptosis pathway. Targeting this pathway may lead to novel neuroprotective strategies to prevent the progression of sight-threatening diseases.  This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016. 
RD 4/12/2021