ERN1 blocking peptide

Autophagy dysfunction is a potential toxic effect of nanoparticles. Previous studies have indicated that silica nanoparticles (SiNPs) induce macroautophagy/autophagy dysfunction, while the precise mechanisms remain uncertain. Hence, the present study investigated the molecular mechanisms by which SiNPs enhanced autophagosome synthesis, which then contributed to autophagy dysfunction. First, the effects of SiNPs on autophagy and autophagic flux were verified using transmission electron microscopy, laser scanning confocal microscopy, and western blot assays. Then, the activation of endoplasmic reticular (ER) stress was validated to be through the EIF2AK3 and ATF6 UPR pathways but not the ERN1-XBP1 pathway, along with the upregulation of downstream ATF4 and DDIT3. Thereafter, the ER stress inhibitor 4-phenylbutyrate (4-PBA) was used to verify that SiNP-induced autophagy could be influenced by ER stress. Furthermore, specialized lentiviral shRNA were employed to determine that autophagy was induced via specific activation of the EIF2AK3 and ATF6 UPR pathways. Finally, the 2 autophagic genes LC3B and ATG12 were found to be transcriptionally upregulated by downstream ATF4 and DDIT3 in ER stress, which contributed to the SiNP-enhanced autophagosome synthesis. Taken together, these data suggest that SiNPs induced autophagosome accumulation via the activation of the EIF2AK3 and ATF6 UPR pathways in hepatocytes, which offers a new insight into detailed molecular mechanisms underlying SiNP-induced autophagy dysfunction, and specifically how UPR pathways regulate key autophagic genes. This work provides novel evidence for the study of toxic effects and risk assessment of SiNPs.

BACKGROUND & AIMS: Endoplasmic reticulum to nucleus signaling 1 (ERN1, also called IRE1A) is a sensor of the unfolded protein response that is activated in the livers of patients with nonalcoholic steatohepatitis (NASH). Hepatocytes release ceramide-enriched inflammatory extracellular vesicles (EVs) after activation of IRE1A. We studied the effects of inhibiting IRE1A on release of inflammatory EVs in mice with diet-induced steatohepatitis. METHODS: C57BL/6J mice and mice with hepatocyte-specific disruption of Ire1a (IRE1 alpha(Delta hep)) were fed a diet high in fat, fructose, and cholesterol to induce development of steatohepatitis or a standard chow diet (controls). Some mice were given intraperitoneal injections of the IRE1A inhibitor 4 mu 8C. Mouse liver and primary hepatocytes were transduced with adenovirus or adeno-associated virus that expressed IRE1A. Livers were collected from mice and analyzed by quantitative polymerase chain reaction and chromatin immunoprecipitation assays; plasma samples were analyzed by enzyme-linked immunosorbent assay. EVs were derived from hepatocytes and injected intravenously into mice. Plasma EVs were characterized by nanoparticle-tracking analysis, electron microscopy, immunoblots, and nanoscale flow cytometry; we used a membrane-tagged reporter mouse to detect hepatocyte-derived EVs. Plasma and liver tissues from patients with NASH and without NASH (controls) were analyzed for EV concentration and by RNAscope and gene expression analyses. RESULTS: Disruption of Ire1a in hepatocytes or inhibition of IRE1A reduced the release of EVs and liver injury, inflammation, and accumulation of macrophages in mice on the diet high in fat, fructose, and cholesterol. Activation of IRE1A, in the livers of mice, stimulated release of hepatocyte-derived EVs, and also from cultured primary hepatocytes. Mice given intravenous injections of IRE1A-stimulated, hepatocyte-derived EVs accumulated monocyte-derived macrophages in the liver. IRE1A-stimulated EVs were enriched in ceramides. Chromatin immunoprecipitation showed that IRE1A activated X-box binding protein 1 (XBP1) to increase transcription of serine palmitoyltransferase genes, which encode the rate-limiting enzyme for ceramide biosynthesis. Administration of a pharmacologic inhibitor of serine palmitoyltransferase to mice reduced the release of EVs. Levels of XBP1 and serine palmitoyltransferase were increased in liver tissues, and numbers of EVs were increased in plasma, from patients with NASH compared with control samples and correlated with the histologic features of inflammation. CONCLUSIONS: In mouse hepatocytes, activated IRE1A promotes transcription of serine palmitoyltransferase genes via XBP1, resulting in ceramide biosynthesis and release of EVs. The EVs recruit monocyte-derived macrophages to the liver, resulting in inflammation and injury in mice with diet-induced steatohepatitis. Levels of XBP1, serine palmitoyltransferase, and EVs are all increased in liver tissues from patients with NASH. Strategies to block this pathway might be developed to reduce liver inflammation in patients with NASH.