Abstract
Purpose :
Elovanoids (ELVs), a novel class of lipid mediators are derived from 32:6n3 and 34:6n3. We established structure/stereochemistry of ELVs and showed ELV mediate cell death protection from uncompensated oxidative stress (UOS) in human retinal pigment epithelium (RPE) and in brain (Jun B, et al. Sci Rep 2017; Bhattacharjee S, et al. Sci Adv. 2017). This study investigates the role of ELVs on telomerase activity and telomere-independent function under UOS. Non-telomeric functions for the telomerase proteins TERT and TTI1 (TELO2-interacting protein 1 homolog) influence different key cellular processes, e.g., cell survival and oxidative stress resistance. Emerging data shows the interaction of TERT with the intracellular signaling pathways PI3K-Akt, mTOR, NF-κB and WNT/β-catenin. These telomere-independent functions and signaling pathways are critical for the molecular logic of cell survival. Thus, we investigated whether ELVs function in modulating telomerase activity.
Methods :
Primary cultures of human RPE (48h) were serum starved for 8h, oxidative-stress was introduced by H2O2 (1200µM) +TNFα (10ng/ml) and then treated with ELVs (200nM) for 16h. Western-blot analysis using 20-25μg protein was performed on 4-12% Bis-Tris gel for 2h at 125 volts using 6h treated cell extracts. Proteins transferred on a nitrocellulose membrane, blocked 1h with 3% BSA solution, were probed with primary antibodies of anti-TERT and anti-TTI1. Proteins were detected by chemiluminescence using an ECL kit.
Results :
Induction of TERT and TTI1 occurred upon UOS, and was attenuated by treatment with ELVs. Since the physico-chemical properties of these new lipid mediators differ from those known for other mediators (18-22 C), we explore here the bioactivity of different ELV structural analogs, including methyl esters and the acetylenic derivatives ELV-N-32-Me, ELV-N34-Me, and ELV-N34-Me-A.
Conclusions :
Our data suggest that ELV reduction of UOS, neuroprotection, and cell survival targets TERT and TTI1. We are currently defining ELV mechanisms in RPE on non-telomeric functions of these genes. The TTI1 complex is involved in double-stranded DNA damage repair, and we know DNA repair enzymes are activated in photoreceptor inner segments by photo-oxidative stress. This novel signaling may be fundamental to sustain photoreceptor integrity.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.