Proteomic changes in extracellular vesicles from hepatocytes after cytotoxic ethanol injury

Abstract

AimsExtracellular vesicles (EVs) play an important role in maintaining normal homeostasis or driving pathogenesis in the liver. For example, various disease‐associated processes (e.g. steatosis, inflammation, fibrosis) are, on the one hand, promoted by EVs produced by hepatocytes after cell stress, injury, or infection yet, on the other hand, inhibited by EVs produced by normal hepatocytes. To better understand these functional differences, proteomic analysis was performed on EVs produced by AML12 mouse hepatocytes maintained in vitro under normal conditions or after exposure to ethanol and TNFα.MethodsAML12 cells were cultured under serum‐free conditions either alone or in the presence of 50mM ethanol for 48 hours plus 30ng/ml TNF‐α over the latter 24 hrs. EVs were purified from conditioned medium by sequential steps of low‐, high‐ and ultra‐ centrifugation. Proteins from two separate EV preparations collected under control (EVNorm) or ethanol/TNFα (EVETOH/TNF) culture conditions were extracted, digested with trypsin, and protein identification was performed using nano‐liquid chromatography‐nanospray tandem mass spectrometry (LC/MS/MS). Sequence data were searched against the Uniprot Mouse database using Mascot Daemon by Matrix Science version 2.6.0 (Boston, MA) Proteins with less than 1% FDR as well as a minimal of 2 significant peptides detected were considered as valid proteins.Results293 proteins were present in duplicate EVNorm samples and 186 proteins were present in duplicate EVETOH/TNF samples. 152 proteins were shared by both EVNorm and EVETOH/TNF and were present at relatively higher levels (quantitative value 10–1000) than the 141 EVNorm‐specific proteins (quantitative value 5–50) or the 34 EVETOH/TNF ‐specific proteins (quantitative values 1–10). Among the most abundant shared proteins, FN, CLUS, ACTG, KPYM and TERA showed diminished levels in EVETOH/TNF versus EVNorm while LG3BP, CFAB, H2AY, RS27A, and IGSF8 showed increased levels in EVETOH/TNF versus EVNorm. GO analysis showed that of the proteins that were unique or enriched in EVNorm or EV ETOH/TNF, some components were shared (exosome, membrane, cytoplasm, nucleus, focal adhesion, extracellular components, cell‐cell adherins, nucleolus, ribonucleoproteins) while other components were unique to either EVNorm (cytoskeleton, mitochondria, myelin sheath, protein complex) or EVETOH/TNF (ribosome, endocytosis, cytosol, Golgi). KEGG analysis showed that the principal pathways for EVNorm‐specific/enriched proteins related to antibiotic synthesis, carbon metabolism, proteasome, actin cytoskeleton, virus infection, focal adhesion, PI3K‐AKT, amino acid metabolism, and glycolysis whereas EVETOH/TNF‐specific/enriched proteins related to ribosome, endocytosis, proteasome, system lupus erythematosus, alcoholism and mineral absorption.ConclusionsEVs from normal or ethanol/TNFα ‐treated hepatocytes show dynamic differences in their respective proteomes. The underlying changes in EV protein composition, both quantitatively and qualitatively, likely contribute to the role that these EV populations play in regulating hepatic homeostasis and the pathogenic outcomes following cytotoxic ethanolic injury in the liver.Support or Funding InformationNIH grants R21 AA025974 and R21 AA023626