SQSTM1/p62 depletion leads to the Rab7 accumulation and inflammatory response in ARPE-19 cells
Abstract
Purpose
SQSTM1/p62 is a central player in the selective autophagy directing ubiquitinated protein aggregates, mitochondria, and bacterial pathogens to autophagosomes. Autophagy culminates into the fusion of matured autophagosomes with lysosomes that contain degradative enzymes. One regulator of the fusion is Rab7, which also has a role in the movement of autophagosomes along microtubules. In this study, we have investigated the significance of SQSTM1/p62 in the functionality of human retinal pigment epithelial (RPE) cells and its possible role in the regulation of Rab7.
Methods
SQSTM1/p62 was blocked in ARPE-19 cells using spesific siRNA. Proteasomes were inhibited with MG-132, which has been shown to induce autophagy flux by providing more substrate to the autophagic machinery. Additionally, AMPK-activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) was used to induce autophagy. Rab7 protein levels were determined with western blot and the secretion of inflammatory cytokines using the ELISA method.
Results
According to our results, interference of autophagy by SQSTM1/p62 depletion, especially in the presence of other stimuli, such as MG-132 or AICAR, leads to the accumulation of Rab7. Along with higher amounts of Rab7, increased inflammatory response was observed.
Conclusions
Our results emphasize the importance of SQSTM1/p62 in the regulation of autophagy and inflammation in RPE cells that play the principal role in the pathogenesis of age-related macular degeneration (AMD).
