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Thomas Gardner, Lijie Gong, Shirley He, Edith Arnold, Patrice Fort, Steven Abcouwer; Prevention of Caspase Activation and Apoptotic Death of R28 Retinal Neuronal Cells by IGF-1 Corresponds with AKT-Dependent Inhibition of FOXO1 Function. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3689.
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© ARVO (1962-2015); The Authors (2016-present)
Diabetic retinopathy is associated with diminished retinal AKT activity, chronic neurodegeneration and neuroinflammation. R28 retinal neuronal cells were used to examine the ability of IGF-1 to prevent apoptotic death during growth factor withdrawal and treatment with the inflammatory cytokine TNF-alpha. We hypothesized that the neurotrophic effects of IGF-1 are due to inhibition of the pro-apoptotic transcription factor FOXO1 downstream of AKT activation.
R28 cells were cultured on laminin in the presence of a cAMP analogue to induce neuronal differentiation and then were subjected to growth factor withdrawal through serum deprivation (SD). Apoptotic death was evaluated by caspase-3/7 activity, DNA fragmentation ELISA and lactate dehydrogenase (LDH) release. Activation of AKT and its substrates was evaluated by Western blotting with antibodies to phosphorylated and total proteins. Protein localization was assessed by subcellular fractionation followed by Western blotting and by microscopic visualization of FOXO1-EGFP-fusion protein. PI3K activity was inhibited with LY294002 and wortmannin. Messenger RNA levels were measured by qRT-PCR.
SD-induced cell death was significantly inhibited by IGF-1 in a concentration-dependent manner. Pretreatment with TNF-alpha amplified SD-induced death and diminished the sensitivity to low doses of IGF-1 (0.1-0.5 ng/ml). Protection of SD-induced death by IGF-1 coincided with phosphorylation of several protein substrates of AKT kinase, with FOXO1 being most prominent. SD resulted in dephosphorylation of FOXO1 at the threonine 24 activation site, leading to nuclear localization of the protein. IGF-1 caused marked FOXO1 Thr24 phosphorylation and reversed the effects of SD on FOXO1 nuclear accumulation. SD increased the contents of mRNAs for pro-apoptotic FOX01-target genes, including the BH3-only proteins BIM and NOXA, and this upregulation was reversed by IGF-1 treatment.
IGF-1 may provide neurotrophic support to retinal neurons by preventing the expression of pro-apoptotic FOXO1 target genes. Neuroinflammation in the diabetic retina may lessen neuroprotection by inhibiting the response to physiological levels of IGF-1 or other neurotrophins.
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