Abstract
The two major intracellular protein degradation systems, the ubiquitin-proteasome system (UPS) and autophagy, work collaboratively in many biological processes including development, apoptosis, aging, and countering oxidative injuries. We report here that, in human retinal pigment epithelial cells (RPE), ARPE-19 cells, proteasome inhibitors, clasto-lactacystinβ-lactone (LA) or epoxomicin (Epo), at non-lethal doses, increased the protein levels of autophagy-specific genes Atg5 and Atg7 and enhanced the conversion of microtubule-associated protein light chain (LC3) from LC3-I to its lipidative form, LC3-II, which was enhanced by co-addition of the saturated concentration of Bafilomycin A1 (Baf). Detection of co-localization for LC3 staining and labeled-lysosome further confirmed autophagic flux induced by LA or Epo. LA or Epo reduced the phosphorylation of the protein kinase B (Akt), a downstream target of phosphatidylinositol-3-kinases (PI3K), and mammalian target of rapamycin (mTOR) in ARPE-19 cells; by contrast, the induced changes of autophagy substrate, p62, showed biphasic pattern. The autophagy inhibitor, Baf, attenuated the reduction in oxidative injury conferred by treatment with low doses of LA and Epo in ARPE-19 cells exposed to menadione (VK3) or 4-hydroxynonenal (4-HNE). Knockdown of Atg7 with siRNA in ARPE-19 cells reduced the protective effects of LA or Epo against VK3. Overall, our results suggest that treatment with low levels of proteasome inhibitors confers resistance to oxidative injury by a pathway involving inhibition of the PI3K-Akt-mTOR pathway and activation of autophagy.
Highlights
Autophagy allows cells to adapt to nutrient deficiency and cellular injuries
LA or Epo activated autophagy pathway in retinal pigment epithelial cells (RPE) To determine whether LA or Epo activate the autophagy pathway in RPE, we first examined the levels of Atg5 and Atg7 proteins, essential for autophagosome maturation, and measured the conversion of LC3 from LC3-I to LC3-II before and after LA or Epo treatment. 18–24 h treatment with LA (100,1000 nM) or
Our results demonstrate that the proteasome inhibitors LA or Epo activated the autophagy pathway, as measured by increased level of autophagosome proteins ATG5 and ATG7, increased conversion of LC3-I to LC3-II, and increased autophagic flux
Summary
Autophagy allows cells to adapt to nutrient deficiency and cellular injuries. It includes three main mechanisms: macroautophagy, microautophagy, and chaperone-mediated autophagy [1]. Macroautophagy (hereafter referred to as autophagy) begins with formation of autophagosome, which sequesters unused proteins and damaged cellular organelles. Autophagosome expansion, an early step in autophagy, involves insertion of LC3-II into vacuole membrane. This requires Atg (E1-like ubiquitin-activating enzyme), Atg (E2-like ubiquitin-conjugation enzymes), Atg5-Atg12-Atg complex (E3-like ubiquitin-ligase), and other Atgs to work in concert to conjugate phosphatidylethanolamine to LC3-I, forming LC3-II [2,3]. The delicate process of starvation-induced autophagy [4] is inversely regulated by mTOR which is activated by PI3K-Akt induced by insulin or other growth factor [5,6]
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