Lipopolysaccharide Stimulates p62-Dependent Autophagy-Like Aggregate Clearance in Hepatocytes

Abstract EN

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 NFκB 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.