ELAVL1/HuR-mediated accumulation of SQSTM1/p62 during proteasomal inhibition in human ARPE-19 cells

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Abstract

Purpose

Impaired protein degradation in retinal pigment epithelial (RPE) cells contributes to age-related macular degeneration (AMD) pathogenesis. SQSTM1/p62 protein is involved in autophagy and proteasome-mediated proteolysis, and expressed strongly under toxic stimuli. ELAVL1/HuR is a RNA-binding protein which, in response to various stressors, regulates gene expression, finally affecting key cellular functions. Main aims of our study were to investigate whether p62 mRNA would be a target of HuR protein, and whether a link between these two proteins exists in RPE cells. Finally, the effects of proteasome inhibition and autophagy induction on p62 and HuR levels in RPE cells were also evaluated.

Methods

AlphaScreen technology and immunoprecipitation coupled with real-time qPCR were used to study the binding between HuR protein and p62 mRNA. ARPE-19 cells were treated with MG-132 (proteasome inhibitor; 5μM) and/or AICAR (AICA ribonucleotide, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside; 2mM). P62 and HuR levels were analyzed by real-time qPCR and Western blotting. HuR silenced ARPE-19 cells and negative control cells were also employed.

Results

MG-132 treatment up-regulates HuR, which binds p62 mRNA thus contributing, at post-transcriptional level, to the increase of p62 protein level in ARPE-19 cells. The addition of AICAR promoted cleansing by autophagy of p62, but not HuR.

Conclusion

p62, whose mRNA represents a novel target of HuR, is positively regulated as a cellular response to proteasome inhibition. P62 is degraded by autophagy-mediated pathway, while HuR through proteasome. These findings may be relevant for AMD.

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