Hepatic fibrosis is driven by deposition of matrix proteins following liver injury. Hepatic stellate cells (HSCs) drive fibrogenesis, producing matrix proteins, including procollagen I, which matures into collagen I following secretion. Disrupting intracellular procollagen processing and trafficking causes endoplasmic reticulum stress and stress-induced HSC apoptosis and thus is an attractive antifibrotic strategy. We designed an immunofluorescence-based small interfering RNA (siRNA) screen to identify procollagen I trafficking regulators, hypothesizing that these proteins could serve as antifibrotic targets. A targeted siRNA screen was performed using immunofluorescence to detect changes in intracellular procollagen I. Tumor necrosis factor receptor associated factor 2 and noncatalytic region of tyrosine kinase-interacting kinase (TNIK) was identified and interrogated in vitro and in vivo using the TNIK kinase inhibitor NCB-0846 or RNA interference-mediated knockdown. Our siRNA screen identified nine genes whose knockdown promoted procollagen I retention, including the serine/threonine kinase TNIK. Genetic deletion or pharmacologic inhibition of TNIK through the small molecule inhibitor NCB-0846 disrupted procollagen I trafficking and secretion without impacting procollagen I expression. To investigate the role of TNIK in liver fibrogenesis, we analyzed human and murine livers, finding elevated TNIK expression in human cirrhotic livers and increased TNIK expression and kinase activity in both fibrotic mouse livers and activated primary human HSCs. Finally, we tested whether inhibition of TNIK kinase activity could limit fibrogenesis in vivo. Mice receiving NCB-0846 displayed reduced CCl 4 -induced fibrogenesis compared to CCl 4 alone, although α-smooth muscle actin levels were unaltered. Conclusions: Our siRNA screen effectively identified TNIK as a key kinase involved in procollagen I trafficking in vitro and hepatic fibrogenesis in vivo. (Hepatology Communications 2022;6:593-609).
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 throug...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.