Death Receptor 5 Signaling Promotes Hepatocyte Lipoapoptosis

Sophie C Cazanave,Justin L Mott,Steven F Bronk,Nathan W Werneburg,Christian D Fingas,X Wei Meng,Niklas Finnberg,Wafik S El-Deiry,Scott H Kaufmann,Gregory J Gores

doi:10.1074/jbc.m111.280420

Abstract

Nonalcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA), endoplasmic reticulum (ER) stress, and hepatocyte lipoapoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5) is significantly elevated in patients with nonalcoholic steatohepatitis, and steatotic hepatocytes demonstrate increased sensitivity to TRAIL-mediated cell death. Nonetheless, a role for TRAIL and/or DR5 in mediating lipoapoptotic pathways is unexplored. Here, we examined the contribution of DR5 death signaling to lipoapoptosis by free fatty acids. The toxic saturated free fatty acid palmitate induces an increase in DR5 mRNA and protein expression in Huh-7 human hepatoma cells leading to DR5 localization into lipid rafts, cell surface receptor clustering with subsequent recruitment of the initiator caspase-8, and ultimately cellular demise. Lipoapoptosis by palmitate was not inhibited by a soluble human recombinant DR5-Fc chimera protein suggesting that DR5 cytotoxic signaling is ligand-independent. Hepatocytes from murine TRAIL receptor knock-out mice (DR(-/-)) displayed reduced palmitate-mediated lipotoxicity. Likewise, knockdown of DR5 or caspase-8 expression by shRNA technology attenuated palmitate-induced Bax activation and apoptosis in Huh-7 cells, without altering induction of ER stress markers. Similar observations were verified in other cell models. Finally, knockdown of CHOP, an ER stress-mediated transcription factor, reduced DR5 up-regulation and DR5-mediated caspase-8 activation upon palmitate treatment. Collectively, these results suggest that ER stress-induced CHOP activation by palmitate transcriptionally up-regulates DR5, likely resulting in ligand-independent cytotoxic signaling by this death receptor.

Highlights

death receptor 5 (DR5) Expression Is Increased by Palmitate Treatment and Contributes to Hepatocyte Lipoapoptosis—Consistent with prior observations [16], treatment of Huh-7 cells for 8 h with the saturated free fatty acids (FFA) palmitate resulted in a 2–3-fold increase in DR5 mRNA expression as compared with vehicle-treated cells (Fig. 1A)
Further evidence for TNF-related apoptosis-inducing ligand (TRAIL) death receptors (DR) contribution to saturated FFA-mediated toxicity was obtained by demonstrating that hepatocytes from DRϪ/Ϫ mice (Fig. 2D) were more resistant to lipoapoptosis by palmitate than wild-type (WT) hepatocytes (Fig. 2E); these hepatocytes genetically deficient in murine DR remained sensitive to apoptosis induced by TNF␣ (28 ng/ml) plus actinomycin D (0.2 ␮g/ml) for 24 h (37 Ϯ 2 apoptotic nuclei) as compared with WT hepatocytes (43 Ϯ 3 apoptotic nuclei)
Death receptor specificity for these observations was confirmed as hepatocytes from lpr mice, which contain a Fas receptor that is unable to signal for death, and readily underwent lipoapoptosis by palmitate (Fig. 2E)

Summary

Introduction

The results implicate TRAIL death signaling by DR5 as a mediator of palmitate-induced hepatocyte lipoapoptosis downstream of ER stress-mediated CHOP induction of DR5 expression. Consistent with these observations, incubation of Huh-7 cells with a soluble human recombinant DR5-Fc chimera protein, which inhibits TRAIL-mediated death by competing with cell surface DR4 and DR5 for TRAIL, did not inhibit palmitate-induced apoptosis (Fig. 3B).

Results

Conclusion