Abstract

Background: Liver injury activates hepatic stellate cells (HSCs) which drive fibrosis through secreting extracellular matrix proteins. Proteins destined for secretion are cotranslationally translocated into the endoplasmic reticulum (ER), folded, and exported for secretion. Activated HSCs exhibit increased protein translation leading to ER stress, which is sensed by ER membrane proteins Activating transcription factor 6 (ATF6α), Inositol-requiring enzyme 1 (IRE1α), and Protein kinase R-like ER kinase (PERK). These sensors initiate the Unfolded Protein Response (UPR) to relieve stress through processes including ER-associated degradation (ERAD): where misfolded proteins are targeted for proteasome degradation. The UPR is critical for HSC activation and fibrogenesis, but whether ERAD plays a key pro-fibrotic role is unknown. Publicly available RNAseq data revealed upregulation of ERAD components in cirrhotic livers, thus we hypothesized that ERAD is crucial for HSC activation and fibrogenesis.

Methods: Primary human HSCs (hHSCs) were activated with TGFβ in combination with UPR inhibitors (Ceapin A7, 4µ8C, or GSK2656157 to inhibit ATF6α, IRE1α, or PERK respectively), or ERAD inhibitor Eeyarestatin 1 (EER1). ERAD components and HSC activation were analyzed by Western blot, qPCR, and microscopy. Immortalized hHSC (LX-2) cells were transfected with an siRNA targeting ERAD component SEL1L and analyzed for HSC activation. In vivo, C57BL/6J mice were fed a high fat diet for 8 weeks (D12102Ci) or control diet with biweekly injections of 2mg/kg EER1 or vehicle. Liver sections were analyzed for fibrosis by Western blot and Sirius red staining.

Results: TGFβ increased ERAD components ERdj4, ERdj5, HERPUD1, and HRD1, while decreasing protein levels of ERAD substrate OS-9, indicative of increased ERAD activity (p < 0.05). IRE1α inhibition limited TGFβ induction of ERdj4 and collagen deposition (p < 0.05), while PERK inhibition increased HRD1, ERdj4, and ERdj5 levels, and TGFβ-induction of collagen deposition (p < 0.05), linking ERAD and HSC activation. ERAD inhibition decreased αSMA and Collagen 1 protein levels in hHSCs treated with TGFβ, and knockdown of SEL1L decreased collagen I levels at 24h. Unexpectedly, disrupting ERAD increased collagen deposition at 48h in both hHSCs and LX-2 cells. Finally, in vivo studies revealed that ERAD inhibition limited diet induced fibrogenesis and HSC activation.

Conclusion: ERAD components increased in activated HSCs, and ERAD inhibition limited fibrogenesis in vivo. In vitro, ERAD disruption limited HSC activation at earlier time points (24h), but increased fibrogenesis at later time points (48h). We hypothesize that prolonged ERAD inhibition induces protein trafficking and secretion to remove misfolded proteins from the ER in vitro, but that long term ERAD inhibition limits fibrogenesis in vivo. This work identifies ERAD as a possible therapeutic target for fibrosis and fibrogenesis.