Netrin-1 Protects Hepatocytes Against Cell Death Through Sustained Translation During the Unfolded Protein Response

Cell Mol Gastroenterol Hepatol. 2016 Jan 9;2(3):281-301.e9. doi: 10.1016/j.jcmgh.2015.12.011. eCollection 2016 May.


Background & aims: Netrin-1, a multifunctional secreted protein, is up-regulated in cancer and inflammation. Netrin-1 blocks apoptosis induced by the prototypical dependence receptors deleted in colorectal carcinoma and uncoordinated phenotype-5. Although the unfolded protein response (UPR) triggers apoptosis on exposure to stress, it first attempts to restore endoplasmic reticulum homeostasis to foster cell survival. Importantly, UPR is implicated in chronic liver conditions including hepatic oncogenesis. Netrin-1's implication in cell survival on UPR in this context is unknown.

Methods: Isolation of translational complexes, determination of RNA secondary structures by selective 2'-hydroxyl acylation and primer extension/dimethyl sulfate, bicistronic constructs, as well as conventional cell biology and biochemistry approaches were used on in vitro-grown hepatocytic cells, wild-type, and netrin-1 transgenic mice.

Results: HepaRG cells constitute a bona fide model for UPR studies in vitro through adequate activation of the 3 sensors of the UPR (protein kinase RNA-like endoplasmic reticulum kinase (PERK)), inositol requiring enzyme 1α (IRE1α), and activated transcription factor 6 (ATF6). The netrin-1 messenger RNA 5'-end was shown to fold into a complex double pseudoknot and bear E-loop motifs, both of which are representative hallmarks of related internal ribosome entry site regions. Cap-independent translation of netrin 5' untranslated region-driven luciferase was observed on UPR in vitro. Unlike several structurally related oncogenic transcripts (l-myc, c-myc, c-myb), netrin-1 messenger RNA was selected for translation during UPR both in human hepatocytes and in mice livers. Depletion of netrin-1 during UPR induces apoptosis, leading to cell death through an uncoordinated phenotype-5A/C-mediated involvement of protein phosphatase 2A and death-associated protein kinase 1 in vitro and in netrin transgenic mice.

Conclusions: UPR-resistant, internal ribosome entry site-driven netrin-1 translation leads to the inhibition of uncoordinated phenotype-5/death-associated protein kinase 1-mediated apoptosis in the hepatic context during UPR, a hallmark of chronic liver disease.

Keywords: ATF6, activated transcription factor 6; CMV, cytomegalovirus; DAPK, death-associated protein kinase; DMS, dimethyl sulfate; DR, dependence receptor; DTT, dithiothreitol; ER, endoplasmic reticulum; FLuc, Firefly luciferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; Hepatocyte; IRE1α, inositol requiring enzyme 1α; IRES, internal ribosome entry site; LSL, (Lox-Stop-Lox); NMIA, N-methyl-isatoic anhydride; Netrin; PBS, phosphate-buffered saline; PERK, protein kinase RNA (PKR)-like endoplasmic reticulum kinase; PP2A, protein phosphatase 2A; PR65β, erine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A beta isoform; RLuc, Renilla lucerifase; TUNEL, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling; Translation; Tu, tunicamycin; UNC5, uncoordinated phenotype-5; UPR; UPR, unfolded protein response; UTR, untranslated region; VR1, vanilloid receptor 1; eIF2α, Eukaryotic translation initiation factor 2A; mRNA, messenger RNA; pBic, Bicistronic plasmid; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; siRNA, small interfering RNA.