The Unfolded Protein Response Regulates ER-phagy in Fibrogenic Hepatic Stellate Cells

Zachary Hanquier, Yvonne Thomason, Jessica L. Maiers

Research output: Contribution to journalArticlepeer-review


INTRODUCTION: Liver injury activates Hepatic Stellate Cells (HSCs) which secrete fibrogenic proteins such as collagen I to promote scarring. Increased translation of the collagen I precursor procollagen I by HSCs causes ER stress due to 30% of nascent procollagen I failing to fold correctly, placing a burden on the ER; however, it is unclear how HSCs adapt to this stress. ER stress activates the Unfolded Protein Response (UPR) which signals through pathways mediated by Activating Transcription Factor 6α (ATF6α), Inositol Requiring Enzyme 1 (IRE1α), or Protein Kinase R-like ER kinase (PERK). ATF6α or IRE1α inhibition limits HSC activation and promotes apoptosis in vitro, while deletion of ATF6α or IRE1α limits fibrogenesis and reduces HSC number in vivo. While it is clear that the UPR plays a crucial role in HSC activation and survival, the mechanisms that facilitate this role are unknown. Recent work shows that misfolded procollagen I can undergo ER-phagy, where receptors on the ER recruit autophagic membranes to engulf portions of the ER containing misfolded proteins and target them for degradation. ER-phagy can be activated by ER stress, but the fibrogenic role and regulation of ER-phagy in HSCs is unknown. We hypothesized that UPR induction of ER-phagy targets misfolded procollagen I for degradation, thus promoting HSC survival and fibrogenesis. METHODS: Expression of ER-phagy receptors (Cell-cycle progression gene 1 (CCPG1), Family with sequence similarity 134B (FAM134B), and Atlastin 3 (ATL3)) was assessed in 1) livers from patients with advanced fibrosis or controls (GSE25097), 2) murine livers harvested from age- and sex-matched mice following bile-duct ligation (3 weeks) or sham controls; and 3) primary hHSCs or mHSCs, or immortalized hHSCs (LX-2) following TGFβ treatment (2ng/mL, 24h). ER-phagic flux was measured in LX-2 cells expressing a fluorescent ER-phagy reporter (RAMP4-GFP-mCherry), with RAMP4 as a known ER-phagic cargo. UPR signaling was disrupted using inhibitors targeting ATF6α (6µM Ceapin-A7) or IRE1α (0.5µM 4µ8C), or RNAi targeting PERK. CCPG1 or FAM134B were knocked out from LX-2 cells using CRISPR-Cas9. HSC activation and UPR signaling were measured by qPCR and Western blot. RESULTS: Expression of CCPG1 and ATL3 increased in fibrotic human livers compared to controls, while CCPG1 mRNA, and FAM134B protein and mRNA levels increased in fibrotic mouse livers compared to controls. In vitro activation of primary hHSCs or mHSCs also increased CCPG1, FAM134B, and ATL3 protein and mRNA levels, as well as increased ER-phagic flux in LX-2 cells. Regulation of ER-phagic flux and expression of ER-phagy receptors was UPR-dependent, with inhibition of ATF6α or IRE1α blocking TGFβ-induced ER-phagic flux, while PERK knockdown increased ER-phagic flux. Interestingly, CCPG1 or FAM134B loss did not impact HSC activation, or induce the pro-apoptotic UPR. CONCLUSIONS: ER-phagy receptors increased in fibrotic human and murine livers, and TGFβ upregulated ER-phagy receptors and increased ER-phagic flux in HSCs through UPR-dependent mechanisms. Deletion of CCPG1 or FAM134B did not impact HSC activation, suggesting redundancy between ER-phagic receptors. Future studies will focus on understanding the fibrogenic role of ER-phagy in HSCs.

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics


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