Nrf2 and HuR/ELAV1 as determinant factors for VEGF expression and secretion in ARPE‐19 cells under stress stimuli

Abstract

Aims/Purpose: The transcription nuclear factor E2‐related factor 2 (Nrf2) pathway is a key antioxidant system in the RPE, protecting it from oxidative stress and inflammation. Its dysfunction is a renowned aspect of Age‐related Macular Degeneration (AMD). As previously documented, RNA‐binding protein HuR/ELAVL1 interacts with Nrf2 mRNA, as well as many of its targets at the post‐transcriptional level. Among them, vascular endothelial growth factor (VEGF) is a crucial one, being involved in both physiological maintenance of vasculature and pathological angiogenesis. Purposes of our study were disclosing the crosstalk between Nrf2 and HuR in RPE cells subjected to stressful stimuli and understanding their regulatory effect on VEGF.Methods: Wild type, Nrf2‐ or HuR‐deficient ARPE‐19 cells were used. As stressful stimuli, MG132 (5 μM) and Bafilomycin (50 nM) for 24 h, or 4‐Hydroxynonenal (4‐HNE; 50 or 100 μM) for 6 h, were used. Cell viability was assayed by enzymatic fluorescence analysis; VEGF expression and extracellular secretion were evaluated by RT‐qPCR and ELISA, respectively.Results: When subjected to intense toxic stimuli, HuR‐deficient cells display impaired viability, although to a lesser extent compared to Nrf2‐deficient cells. In these silenced cell models, 4‐HNE seems to be more harmful than the autophagy inhibitors. Nrf2 or HuR deficiency markedly affects VEGF levels and secretion, even more than stressful conditions. Furthermore, distinctive effects of silencing either Nrf2 or HuR on the level of the other factor have been disclosed.Conclusions: Both HuR and Nrf2 are involved in the constitutive regulation of VEGF levels; contexts characterized by oxidative stress or impaired clearance induce more significant changes in RPE featured by an impairment of Nrf2 than of HuR levels. Further elucidations in how the crosstalk between these two factors regulate common targets (e.g., VEGF) may improve our understanding of RPE homeostasis and alteration, and consequently AMD pathophysiology.