CHOP links endoplasmic reticulum stress to NF-κB activation in the pathogenesis of nonalcoholic steatohepatitis - PubMed (original) (raw)

Jeffrey A Willy et al. Mol Biol Cell. 2015.

Abstract

Free fatty acid induction of inflammation and cell death is an important feature of nonalcoholic steatohepatitis (NASH) and has been associated with disruption of the endoplasmic reticulum and activation of the unfolded protein response (UPR). After chronic UPR activation, the transcription factor CHOP (GADD153/DDIT3) triggers cell death; however, the mechanisms linking the UPR or CHOP to hepatoceullular injury and inflammation in the pathogenesis of NASH are not well understood. Using HepG2 and primary human hepatocytes, we found that CHOP induces cell death and inflammatory responses after saturated free fatty acid exposure by activating NF-κB through a pathway involving IRAK2 expression, resulting in secretion of cytokines IL-8 and TNFα directly from hepatocytes. TNFα facilitates hepatocyte death upon exposure to saturated free fatty acids, and secretion of both IL-8 and TNFα contribute to inflammation. Of interest, CHOP/NF-κB signaling is not conserved in primary rodent hepatocytes. Our studies suggest that CHOP plays a vital role in the pathophysiology of NASH by induction of secreted factors that trigger inflammation and hepatocellular death via a signaling pathway specific to human hepatocytes.

© 2015 Willy et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

PubMed Disclaimer

Figures

FIGURE 1:

FIGURE 1:

Saturated but not unsaturated free fatty acids induce the UPR before lipotoxicity in human hepatocytes. (A–C) Mouse primary hepatocytes (MPHs), rat primary hepatocytes (RPHs), HepG2 cells, and primary human hepatocytes (HPHs) were treated with the indicated concentrations of palmitate, stearate, or oleate for 24 h. Cell death was measured by LDH release. (D) _LC_50 values (micromoles) from LDH release assay were calculated in XLfit using four-parameter curve fits. (E) HepG2 cells were stained for neutral lipid accumulation using LipidTox Deep Red imaging for up to 24 h. Neutral lipid was quantified by counting total spot counts in PerkinElmer's Columbus software package and normalized to vehicle. (F–H) Cell death was measured by LDH release in HepG2 cells incubated with 600 μM palmitate, stearate, or oleate for the indicated times. (I, J) Immunoblot analysis of HepG2 lysates treated with 600 μM palmitate, 2 μM tunicamycin, 600 μM stearate, or 600 μM oleate for up to 24 h, as indicated. Measured proteins are indicated to the right. (K–N) Changes in gene transcripts from HepG2 cells incubated with 600 μM palmitate or oleate for up to 24 h, as indicated. The XBP1s/t is a ratio of the spliced XBP1 mRNA relative to total XBP1 transcript, and the measured transcript and length of FFA treatment are also shown.

FIGURE 2:

FIGURE 2:

Palmitate localizes to the ER and represses global initiation of translation. (A) Electron microscopy showing ultrastructures of HepG2 cells treated with vehicle, oleate, or palmitate for the indicated times. (B, C) Spinning disk confocal images of CLICK-IT–labeled palmitic acid, azide tagged with Alexa Fluor 488 alklyne, showing colocalization with the ER (calnexin) or PERK. The percentage colocalization of palmitate with calnexin and PERK is illustrated to the right. (D–F) Polysome profiles of lysates prepared from HepG2 cells treated with vehicle, palmitate, oleate, or tunicamycin for 12 h. (G) Polysome profile of PERK KO HepG2 cells treated with either vehicle or palmitate for 12 h. (H) Cell death of PERK KO HepG2 cells, as measured by LDH release, treated or not with palmitate. (I) Immunoblot analysis of control and PERK KO HepG2 cells after 12-h treatment with vehicle or palmitate.

FIGURE 3:

FIGURE 3:

CHOP induces secretion of factors involved in hepatocyte death. (A) Control (shCTRL) HepG2 cells or those knocked down for CHOP (shCHOP #1 and shCHOP #2) were treated with palmitate for 24 h, and cell death was measured by LDH release (top). The control and shCHOP cells were incubated for 12 h in the presence (+) or absence (–) of 600 μM palmitate, and the indicated proteins were measured by immunoblot (bottom). (B) shCTRL and shCHOP HepG2 cells were treated with palmitate for the indicated times, and cell death was measured by LDH release. (C) Control (shCTRL) HepG2 cells or those knocked down for ATF4 (shATF4 #1 and shATF4 #2) were treated with palmitate for 24 h, and cell death was measured by LDH release (top). Control and shATF4 cells were treated for 12 h in the presence (+) or absence (–) of 600 μM palmitate, and the indicated proteins were visualized by immunoblot (bottom). (D) Diagram depicting conditioned medium experiment. Donor shCTRL HepG2 cells were incubated with 600 μM palmitate or control vehicle for up to 24 h, as indicated. The conditioned medium was then transferred to recipient shCHOP HepG2 cells for 24 h, and cell death was measured by LDH release. (E) Conditioned medium was prepared from shCTRL HepG2 donor cells incubated for 3, 6, 12, or 24 h in the presence of palmitate. Conditioned medium was then transferred to recipient shCHOP HepG2, and after a 24-h incubation, cell death was measured by LDH release. (F, G) Conditioned medium prepared from donor shCTRL cells cultured with palmitate for 12 or 24 h was treated with RNase A, heat inactivation, or proteinase K, and the treated conditioned medium was then incubated with recipient shCHOP cells for 24 h. Cell death was measured by LDH release.

FIGURE 4:

FIGURE 4:

CHOP directs hepatocyte secretion of TNFα and IL-8 upon exposure to palmitate. (A) RBM Myriad biomarker panel measuring the indicated secreted factors from HepG2 cells treated with either palmitate or oleate for 12 h. Values are normalized to vehicle treatment, and the number sign (#) indicates that biomarkers were statistically significant relative to the oleate treatment group. (B, C) Sandwich ELISAs measuring IL-8 and TNFα from conditioned media of shCTRL or shCHOP HepG2 cells treated with 600 μM palmitate or oleate for 12 h or vehicle control. (D) Control (shCTRL) HepG2 cells or those knocked down for IL-8 expression (shCXCL8 #1 and shCXCL8 #2) were treated with palmitate for 24 h, and cell death was measured by LDH release (top). The indicated proteins were measured by immunoblot using lysates prepared from control and shCXCL8 cells incubated for 12 h in the presence (+) or absence (–) of 600 μM palmitate (bottom). (E) Control HepG2 and those depleted for TNFα (shTNFA #1 and shTNFA #2) were treated with palmitate for 24 h, and cell death was measured by LDH release (top). Immunoblot analyses were also carried out to measure the indicated proteins in the shCTRL and shTNFA cells treated with palmitate for 12 h (+) or vehicle (–; bottom). (F, G) Sandwich ELISAs for shCXCL8 and shTNFA cells depicting repressed secreted polypeptides. (H, I) Cell migration assay for KG-1 cells using conditioned medium prepared from shCTRL, shCXCL8, or shTNFA cells treated with either vehicle or palmitate for 12 h. (J) Direct treatment indicates cell death of shCHOP cells incubated with palmitate for 24 h. Alternatively, donor shCTRL or shTNFA HepG2 cells were incubated with palmitate for 12 h. Conditioned medium was then applied to recipient shCHOP cells for 24 h, and cell death was measured by LDH release. (K) The shCHOP HepG2 cells were treated with vehicle, 600 μM palmitate, 10 ng/ml TNFα, 1000 ng/ml IL-8, or a combination of the recombinant proteins and palmitate for 24 h, as indicated. Cell death was measured by LDH release. (L) Conditioned medium was prepared from donor shCHOP HepG2 cells after 12-h treatment with vehicle or palmitate, and 10 ng/ml TNFα and/or 1000 ng/ml IL-8 was added to indicated conditioned medium before performing the KG1 cell migration assay.

FIGURE 5:

FIGURE 5:

CHOP is required for activation of NF-κB in hepatocytes treated with palmitate. (A) shCTRL or shCHOP HepG2 cells were treated with vehicle (–) or palmitate (+) for 12 h, followed by immunoblot analyses that measured the indicated proteins. (B) Nuclear localization of total and phospho-p65 was measured by immunofluorescence microscopy in HepG2 cells that were treated with palmitate or vehicle. In parallel, Hoescht staining was used to visualize nuclei, and the nuclear staining and p65 imaging was merged. (C) An NF-κB reporter plasmid was transiently transfected into shCTRL and shCHOP HepG2 cells, which were then treated with vehicle or palmitate for 12 h, and reporter firefly luciferase activity was measured and normalized to Renilla luciferase. (D–H) Levels of the indicated gene transcripts were measured by qPCR in HepG2 cells that were treated with palmitate for 12 h or vehicle. (I) Cell migration assays were carried out for 6 h using KG-1 cells incubated with conditioned medium prepared from HepG2 cells expressing shCTRL, shCHOP, or shRELA that were treated with palmitate for 12 h or vehicle. (J) The shCTRL and shRELA HepG2 cells were treated with vehicle (–) or palmitate (+) for 12 h, and the indicated proteins were measured by immunoblot analyses. (K) HepG2 cells expressing shCTRL, shCHOP, or shRELA were cultured in palmitate for 24 h, and cell death was measured by LDH release. (L) Conditioned medium was prepared from donor shCTRL cultured in palmitate for 12 h and applied to recipient shCTRL, shCHOP, or shRELA HepG2; after a 24-h incubation, cell death was measured by LDH release.

FIGURE 6:

FIGURE 6:

CHOP regulates NF-κB through IRAK2 signaling. HepG2 cells expressing shCTRL or shCHOP were cultured in the presence (+) or absence (–) of palmitate. (A) The indicated proteins from these cells were then measured by immunoblot analyses. (B) Levels of the indicated IRAK isoform mRNAs expressed in these cells were measured by qPCR. (C) The shCTRL HepG2 cells or those expressing shIRAK2 were treated with vehicle (–) or palmitate (+) for 12 h, and the indicated proteins were measured by immunoblot. (D) Cell migration assay for KG-1 cells using conditioned medium prepared from shCTRL or shIRAK2 HepG2 cells that were treated with vehicle or palmitate for 12 h. (E, F) The mRNA levels of CXCL8 (IL-8) and TNFA (TNFα) were measured by qPCR in shCTRL and shIRAK2 HepG2 cells treated with palmitate for 12 h or vehicle. (G) HepG2 cells expressing shCTRL or shIRAK2 were cultured in the presence of palmitate for 24 h, and cell death was measured by LDH release. (H) Direct treatment indicates cell death as measured by LDH release of shCHOP cells incubated with palmitate for 24 h. Alternatively, donor shCTRL or shIRAK2 HepG2 cells were incubated with palmitate for 12 h. The conditioned medium was then applied to recipient shCHOP cells for 24 h, and cell death was measured by LDH release. (I) Polysome profiles of lysates prepared from HepG2 cells treated with palmitate or vehicle for 6 h. (J–L) Fractions were collected by the sucrose gradient analyses prepared from the HepG2 cells treated with palmitate or vehicle for 6 h, and the relative levels of the ATF4, CHOP, and IRAK2 mRNA were then determined by qPCR for each fraction. The percentage of the total levels for the indicated gene transcript in each of the seven fractions is illustrated. The percentage change in the indicated mRNA association with large polysomes (fractions 5–7) in response to palmitate is indicated above the polysome. For example, ATF4 showed a 38% increase in transcript levels into fractions 5–7 during palmitate treatment. (M) A similar analysis was carried out for changes in IRAK2 mRNA in polysome fractions in HepG2 cells treated with palmitate or vehicle for 12 h.

FIGURE 7:

FIGURE 7:

Human but not mouse primary hepatocytes activate NF-κB in response to palmitate. (A) Human HepG2 cells and mouse primary hepatocytes (MPHs) were treated with palmitate for 12 h or vehicle, and the indicated proteins were measured by immunoblot analyses (top). IRAK2 antibody from Abnova was used in the immunoblot analysis (see Supplemental Figure S3A). In parallel, IRAK2 mRNA was measured by qPCR in the HepG2 and mouse hepatocytes treated with palmitate for 12 h (bottom). (B) Levels of the indicated mRNAs were measured in mouse primary hepatocytes treated with palmitate for 12 h. (C) Human HepG2 cells and human primary hepatocytes (HPHs) were treated with palmitate for 12 h or vehicle, and the indicated proteins were measured by immunoblot (top). Furthermore, IRAK2 mRNA was measured by qPCR in the HepG2 and HPHs treated with palmitate for 12 h (bottom). (D) The indicated mRNA levels were measured in human primary hepatocytes treated with palmitate for 12 h. (E) Cell migration assay for human KG-1 and murine RAW cells using conditioned medium prepared from HepG2, HPHs, or MPHs treated with vehicle or palmitate for 12 h. (F) Direct treatment indicates cell death as measured by LDH release of shCTRL HepG2, shCHOP HepG2, or MPHs incubated with palmitate for 24 h. Alternatively, conditioned medium was prepared from donor shCTRL HepG2 or MPH cells that were incubated with palmitate for 24 h. The conditioned medium was then applied to recipient shCTRL HepG2, shCHOP HepG2, or MPHs for 24 h as indicated, and cell death was measured by LDH release. (G) Model for CHOP and UPR regulation of hepatocyte inflammation and death signaling during metabolic stress. CHOP regulation of key secreted factors, including TNFα and IL-8, occurs by signaling through NF-κB.

References

    1. Ariyama H, Kono N, Matsuda S, Inoue T, Arai H. Decrease in membrane phospholipid unsaturation induces unfolded protein response. J Biol Chem. 2010;285:22027–22035. - PMC - PubMed
    1. Bahcecioglu IH, Yalniz M, Ataseven H, Ilhan N, Ozercan IH, Seckin D, Sahin K. Levels of serum hyaluronic acid, TNF-alpha and IL-8 in patients with nonalcoholic steatohepatitis. Hepatogastroenterology. 2005;52:1549–1553. - PubMed
    1. Baird TD, Palam LR, Fusakio ME, Willy JA, Davis CM, McClintick JN, Anthony TG, Wek RC. Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKalpha. Mol Biol Cell. 2014;25:1686–1697. - PMC - PubMed
    1. Baird TD, Wek RC. Eukaryotic initiation factor 2 phosphorylation and translational control in metabolism. Adv Nutr. 2012;3:307–321. - PMC - PubMed
    1. Belfort R, Harrison SA, Brown K, Darland C, Finch J, Hardies J, Balas B, Gastaldelli A, Tio F, Pulcini J, et al. A placebo-controlled trial of pioglitazone in subjects with nonalcoholic steatohepatitis. N Engl J Med. 2006;355:2297–2307. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources