title: Lipopolysaccharide stimulates p62-dependent autophagy-like aggregate clearance in hepatocytes.
abstract: Impairment of autophagy has been associated with liver injury. TLR4-stimulation by LPS upregulates autophagy in hepatocytes, although the signaling pathways involved remain elusive. The objective of this study was to determine the signaling pathway leading to LPS-stimulated autophagy in hepatocytes. Cell lysates from livers of wild type (WT; C57BL/6) mice given LPS (5 mg/kg-IP) and hepatocytes from WT, TLR4ko, and MyD88ko mice treated with LPS (100 ng/mL) up to 24 h were collected. LC3II, p62/SQSTM1, Nrf2, and beclin1 levels were determined by immunoblot, immunofluorescence, and qPCR. Autophagy-like activation was measured by GFP-LC3-puncta formation and LC3II-expression. Beclin1, Nrf2, p62, MyD88, and TIRAP were knocked-down using siRNA. LC3II-expression increased in both liver and hepatocytes after LPS and was dependent on TLR4. Beclin1 expression did not increase after LPS in hepatocytes and beclin1-knockdown did not affect LC3II levels. In hepatocytes given LPS, expression of p62 increased and p62 colocalized with LC3. p62-knockdown prevented LC3II puncta formation. LPS-induced LC3II/p62-puncta also required MyD88/TIRAP signaling and localization of both Nrf2 and NF κ B transcription factors to the nucleus to upregulate p62-expression. Therefore, TLR4-activation by LPS in hepatocytes induces a p62-mediated, not beclin1-mediated, autophagy-like clearance pathway that is hepatoprotective by clearing aggregate-prone or misfolded proteins from the cytosol and preserving energy homeostasis under stress.
authors.affiliation: Department of Surgery, University of Pittsburgh, NW607 MUH, 3459 Fifth Avenue, Pittsburgh, PA 15213, USA.
authors.forename: Christine
authors.identifier: 0000-0003-2617-7835
authors.initials: C
authors.lastname: Chen
authors.affiliation: Department of Surgery, University of Pittsburgh, NW607 MUH, 3459 Fifth Avenue, Pittsburgh, PA 15213, USA.
authors.forename: Meihong
authors.initials: M
authors.lastname: Deng
authors.affiliation: Department of Surgery, University of Pittsburgh, NW607 MUH, 3459 Fifth Avenue, Pittsburgh, PA 15213, USA ; Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA.
authors.forename: Qian
authors.initials: Q
authors.lastname: Sun
authors.affiliation: Department of Surgery, University of Pittsburgh, NW607 MUH, 3459 Fifth Avenue, Pittsburgh, PA 15213, USA ; Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, PA 15213, USA.
authors.forename: Patricia
authors.initials: P
authors.lastname: Loughran
authors.affiliation: Department of Surgery, University of Pittsburgh, NW607 MUH, 3459 Fifth Avenue, Pittsburgh, PA 15213, USA.
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Billiar
authors.affiliation: Department of Surgery, University of Pittsburgh, NW607 MUH, 3459 Fifth Avenue, Pittsburgh, PA 15213, USA.
authors.forename: Melanie J
authors.initials: MJ
authors.lastname: Scott
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects*; D000071186:Beclin-1; D006360:Heat-Shock Proteins / Q000378:metabolism*; D022781:Hepatocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008810:Mice, Inbred C57BL; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism; D051267:NF-E2-Related Factor 2 / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D066329:Protein Aggregates / Q000187:drug effects*; D017472:Receptors, Interleukin-1 / Q000378:metabolism; D000071456:Sequestosome-1 Protein; D015398:Signal Transduction / Q000187:drug effects; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D000071186:Beclin-1; C115010:Becn1 protein, mouse; D006360:Heat-Shock Proteins; D008070:Lipopolysaccharides; C480860:Map1lc3b protein, mouse; D008562:Membrane Glycoproteins; D008869:Microtubule-Associated Proteins; D053594:Myeloid Differentiation Factor 88; D051267:NF-E2-Related Factor 2; D016328:NF-kappa B; D066329:Protein Aggregates; D017472:Receptors, Interleukin-1; D000071456:Sequestosome-1 Protein; C101343:Sqstm1 protein, mouse; C499045:TIRAP protein, mouse; D051197:Toll-Like Receptor 4