Proteasome inhibition promotes autophagy and protects from endoplasmic reticulum stress in rat alveolar macrophages exposed to hypoxia‐reoxygenation injury

Abstract

Alveolar macrophages play vital roles in acute lung injury, and macrophage response to hypoxia play relevant roles to disease mechanisms. There is growing evidence that cell death pathways play crucial roles in physiological and pathological settings and that the ubiquitin-proteasome system is involved in the regulation of these processes. However, the functional role of proteasome in alveolar macrophages exposed to hypoxia-reoxygenation (H/R) injury is unknown. We aimed to investigate the function of proteasome on alveolar macrophages exposed to H/R and the underlying mechanisms. NR8383 cells were pretreated with proteasome activator sulforaphane (SFN) or inhibitor MG-132 for 1 hr, and then submitted to 2/6 hr, 4/6 hr, and 6/6 hr H/R treatment. Cell viability was assessed with MTT assay. Autophagy was monitored using electron transmission microscope and flow cytometry and western blotting. The endoplasmic reticulum (ER) stress and unfolded protein response (UPR) pathways were equally analyzed by western blotting. Cell apoptosis was detected by immunohistochemistry, caspase3/7 activity, and western blotting. The viability of NR8383 cells exposed to H/R was affected by proteasome activity and proteasome inhibition significantly inhibited cell death. Treatment with MG-132 led to autophagy activation and induced the survival of NR8383 cells exposed to H/R. Pretreatment with SFN significantly decreased cell autophagy and induced cell death. ER stress was activated in H/R-treated NR8383 cells, and SFN further promoted ER stress whereas proteasome inhibition led to contrary results. Proteasome inhibtion hindered cell apoptosis as demonstrated by decreased caspase-3/7 activity, immunolabelling, and western blot results. Proteasome inhibition might be a promising approach for treating H/R injury-related lung diseases.

Acetylation modulates LC3 stability and cargo recognition

“…Following substrate ubiquitylation, p62 (or other adaptors) binds to poly-ubiquitinated proteins via the UBA domain. Increasing evidence revealed the crosstalk and reciprocal regulatory mechanisms between the two pathways, by which useless materials that accumulate following inhibition of one degradative system could be cleared by the other system [18][19][20]. We previously revealed starvation induced nuclear export of deacetylated LC3, which became the resource of membrane-associated LC3 [3,17].…”

“…Despite of the different mechanisms, ubiquitylation is the degradation signal by both systems. Increasing evidence revealed the crosstalk and reciprocal regulatory mechanisms between the two pathways, by which useless materials that accumulate following inhibition of one degradative system could be cleared by the other system [18][19][20]. The proteasome-mediated degradation of LC3 is one mechanism of autophagy regulation by proteasome.…”

Interferon regulatory factor 8 induces alveolar macrophage hypoxia/reoxygenation injury via the PINK1/Parkin mitophagy pathway

“…Although plenty of studies have employed different techniques to study the pathophysiology associated with lung ischemia-reperfusion injury, the speci c pathophysiological mechanisms of it remain incompletely understood. Studies from our laboratory have demonstrated the role of autophagy, endoplasmic reticulum stress and ubiquitin-proteasome system in HR injury or LIRI [11,16,17]. Other mechanisms include oxidative stress, innate immune responses and so on [3].…”

“…When the cells reached 80% con uence, they were digested with 0.25% trypsin. An HR model of NR8383 cells was established for mimicking lung ischemia-reperfusion injury as described previously [11]. Brie y, the cells were cultured in a humidi ed incubator with 0.5% oxygen for 6 h for hypoxia treatment, The cells were subsequently moved to a normoxic incubator and cultured for another 6 h as the reoxygenation treatment.…”

“…HR injury has been considered to cause inhibited cell viability and cell death [11]. Thus, in order to investigate the role of IRF8 in HR-mediated cell injury, CCK8 assay and LDH test were performed.…”