Key words

1 Introduction

Liver and biliary diseases can result from a wide variety of causes, including infectious agents, inherited defects, alcohol, toxins, and environmental insults. Common forms of liver and biliary diseases include viral hepatitis, alcoholic fatty liver disease, non-alcoholic fatty liver disease, and gallstones. Estimates suggest that hepatitis B and C virus are present in ~5 % and ~3 %, respectively, of the world’s population. Hepatocellular carcinoma is among the leading causes of cancer-related deaths and non-alcoholic fatty liver disease may affect 10–20 % of the population, largely due to the current worldwide obesity epidemic. Many liver and biliary diseases are characterized by both oxidative and endoplasmic reticulum stress, both of which can lead to inflammation, cell death, and global organ impairment. This chapter will discuss the endoplasmic reticulum (ER) and oxidative stress in the liver.

2 The Endoplasmic Reticulum

The ER is the largest continuous organelle in a eukaryotic cell and consists of an array of tubules (cisternae) that form a three-dimensional network (reticulum) stretching from the nuclear envelope to the cell surface. The smooth ER produces structural phospholipids and cholesterol, as well as significant amounts of triacylglycerol and cholesterol esters that have non-structural roles [1, 2]. The smooth ER is the main site of cholesterol synthesis, although much of this lipid is transported to other cellular organelles. Thus, the ER membrane is comprised of very low concentrations of cholesterol and complex sphingolipids [1]. It has been suggested that the loose packing of ER membrane lipids may provide an environment conducive to the insertion and transport of newly synthesized lipids and proteins [1]. This specialized lipid environment within the ER may have implications in diseases characterized by abnormal lipid accumulation, such as alcoholic and non-alcoholic fatty liver disease [3].

All eukaryotic cells contain a significant amount of rough ER, the site for protein folding and maturation. Proteins destined for secretion or insertion into membranes require modification, such as glycosylation and disulfide bond formation, which cannot be achieved in the cytosol [4]. The ER lumen provides a specialized environment for protein folding and maturation that is characterized by high concentrations of calcium, a low ratio (1:1–3:1) of reduced glutathione (GSH) to oxidized glutathione (GSSG), and a unique complement of molecular chaperones and folding enzymes [5]. The ER is also equipped with a quality control system that recognizes and degrades improperly folded proteins, termed ER-associated degradation (ERAD). ERAD can target and transport misfolded proteins from the ER lumen to the cytosolic proteasome machinery [6]. The unfolded protein response (UPR) monitors the ability of the ER lumen to match folding and degradation to the rate of entry of newly synthesized proteins and functions to restore ER homeostasis following periods of ER stress (i.e., accumulation of unfolded proteins within the ER lumen).

3 The Unfolded Protein Response

In mammalian cells, activation of the UPR (Fig. 5.1) generally involves three ER-localized proteins: inositol-requiring 1α (IRE1α), double-stranded RNA-dependent protein kinase-like ER kinase (PERK), and activating transcription factor-6 (ATF6) [7]. Each of these transmembrane proteins has an ER-luminal domain to sense unfolded proteins, a transmembrane domain for targeting to the ER membrane, and a cytosolic domain to transmit signals to the transcriptional and/or translational apparatus [8]. It is currently thought that in un-stressed cells all three proteins are maintained in an inactive state via their association with the ER protein chaperone glucose-regulated protein 78/immunoglobulin-heavy-chain-binding protein (GRP78). Subsequent to ER stress, GRP78 is released and sequestered on unfolded proteins, thereby allowing activation of PERK, IRE1α, and ATF6 [9]. PERK activation leads to phosphorylation of the α-subunit of the translation initiation factor eIF2 (p-eIF2α) and subsequent attenuation of translation initiation. Attenuation of translation also leads to selective translation of mRNAs containing open reading frames, such as activating transcription factor-4 (ATF4) [10, 11]. Increased expression of GADD34 (which also contains open reading frames), a member of the growth arrest and DNA damage family of proteins, is involved in dephosphorylation of eIF2α and therefore promotes reversal of translational attenuation [7]. IRE1α activation leads to splicing of X-box-binding protein-1 (XBP1s) mRNA and subsequent transcription of molecular chaperones (e.g., GRP78) and genes involved in ERAD (e.g., ER degradation-enhancing α—like protein (EDEM)) [11]. Activation of ATF6 leads to its release from the ER membrane, processing in the Golgi, and entry into the nucleus. Transcriptional targets of ATF6 include protein chaperones and XBP1 [8]. Thus, activation of the UPR initiates a spectrum of responses that include transient attenuation of global protein synthesis and an increased capacity for protein folding and degradation. This dual response may serve to not only minimize the increase in unfolded proteins in the ER lumen, but also the accumulation of chaperones and may be particularly relevant to cell types that produce a large amount of secreted proteins (e.g., β-cells) [12].

Fig. 5.1
figure 1

Overview of the mammalian unfolded response. The presence of unfolded proteins in the ER lumen leads to dimerization and autophosphorylation of PERK and IRE1α, and the release and proteolytic cleavage of ATF6 in the Golgi. PERK-mediated phosphorylation of eIF2α leads to transient attenuation of translation, but selective translation of mRNAs containing upstream open reading frames, such as ATF4. Increased transcription and translation of GADD34 subsequently leads to dephosphorylation of eIF2α and resumption of translation. Activation of IRE1α leads to the splicing of XBP1. XBP1s, ATF4, and the cleaved form of ATF6 lead to transcriptional activation of a number of gene targets related to protein folding and ER-associated degradation (see text). Reprinted with permission from ANTIOXIDANT AND REDOX SIGNALING (2011, volume 15, issue 2), published by Mary Ann Liebert, Inc., New Rochelle, NY

Full size image

3.1 An Expanded View of the UPR

PERK is one of four protein kinases that can phosphorylate eIF2α; the other three are double-stranded RNA-activated protein kinase (PKR) which is activated in response to viral infection, general control non-derepressible 2 kinase (GCN2) which is activated in response to amino acid deprivation, and heme-regulated inhibitor kinase (HRI) which is primarily expressed in reticulocytes and appears to coordinate globin polypeptide synthesis with heme availability [13]. Protein kinase-mediated phosphorylation of eIF2α not only regulates translation, but also the activation of nuclear factor kappa-β (NFĸB), via reduction in the abundance of the NFĸβ inhibitor Iĸβ [11]. PERK can also phosphorylate nuclear erythroid 2 p45-related factor 2 (Nrf2), triggering the dissociation of Nrf2/Keap1 complexes and subsequent nuclear import of Nrf2 [14]. Thus, activation of this branch of the UPR links disruption of ER homeostasis to both inflammation, via NFκβ, and redox balance, via Nrf2 (see below).

IRE1α, in addition to catalyzing XBP1 splicing, has additional functions related to cellular signaling. Activated IRE1α can interact with the adaptor protein TNFR-associated factor 2 and lead to activation of c-Jun-NH2-terminal kinase and NFĸβ via apoptosis signaling-regulating kinase 1 [15]. IRE1α activation has also been linked to the activation of p38 mitogen-activated protein kinase and extracellular-regulated kinase [16–18]. These interactions suggest that the IRE1α branch of the UPR not only regulates adaptation to ER stress and cell survival via XBP1 splicing, but also activation of signaling pathways involved in inflammation, insulin action, and apoptosis. Regulated IRE1α-dependent decay of selected mRNAs can also reduce production of proteins destined for the ER lumen [19, 20].

4 The UPR and Antioxidant Defense

Oxidative stress is thought to be an important pathogenic event in many liver diseases. The ER provides a unique oxidizing environment for protein folding and disulfide bond formation. Each disulfide bond formed during oxidative protein folding produces a single reactive oxygen species. It has been estimated that secretory cells produce 3–6 million disulfide bonds per minute, thus protein folding in the ER is intimately linked to the generation of reaction oxygen species and potentially oxidative stress [21, 22]. Conversely, cellular oxidative stress can disrupt ER homeostasis and induce ER stress [23–25]. Therefore, it is not surprising that the UPR engages the antioxidant program via the transcription factor Nrf2 [26]. Nrf2 belongs to the Cap “n” Collar family of basic leucine zipper transcription factors and regulates the expression of antioxidant response element (ARE)-containing genes [26]. Nrf2 is highly expressed in the liver and kidney and is a substrate of the proximal UPR sensor PERK [27]. Importantly, Nrf2 deletion results in rapid onset and progression of steatohepatitis in mice provided a methionine-choline-deficient diet, often used to model components of non-alcoholic fatty liver disease [28]. In addition, Nrf2-deficient mice were characterized by increased mortality in response to endotoxin- and cecal ligation and puncture-induced septic shock [29]. As noted above, PERK-mediated phosphorylation of eIF2α also leads to the selective translation and upregulation of ATF4. Along with Nrf2, this transcription factor has been linked to the maintenance of cellular glutathione [14]. Thus, the PERK arm of the UPR appears to play a critical role in the defense against oxidative stress and the downstream substrate Nrf2 has been directly linked to steatohepatitis.

In addition to the PERK arm of the UPR, recent evidence has also linked the IRE1α-XBP1 branch of the UPR to the regulation of antioxidant defenses [30]. In this study, hydrogen peroxide-mediated cell death occurred more extensively in mouse embryonic fibroblast cells deficient in XBP1. XBP1 deficiency resulted in reduced catalase expression, and overexpression of XBP1 restored catalase expression in XBP1-deficient cells. Thus, XBP1 may provide protection from oxidative stress; however, whether this regulation occurs in hepatocytes is presently unknown.

5 The ER Lumen as a Source of Oxidative Stress

The ER lumen is an oxidizing environment characterized by a GSH:GSSG ratio of 1:1–3:1, much lower than the cytosolic ratio of 30:1–100:1 [31]. This environment is, in part, maintained by ER oxidase 1 (Ero1) and GSSG, and disulfide bond formation in the ER lumen appears to primarily result from electron transfer reactions involving Ero1, protein disulfide isomerase, and molecular oxygen. Hydrogen peroxide is a product of these transfer reactions and therefore disulfide bond formation and protein folding in the ER lumen are associated with the formation of reactive oxygen species [21, 32]. In addition, the luminal NADPH concentration may play an important antioxidant defense role in liver cells in a manner that appears to be independent of the thiol/disulfide redox system [33].

Very few studies have directly examined whether and how protein folding/misfolding influences oxidative stress. In one study, HIP-deficient cells that lack the ability to eliminate misfolded proteins from the ER were employed. Introduction of low levels of a mutant misfolded form of the vacuolar protein carboxypeptidase Y induced ER stress, accumulation of reactive oxygen species, and cell death [34]. Malhotra et al. utilized hydrodynamic delivery of FVIII (coagulation factor VIII, prone to misfolding in the ER lumen) DNA expression vectors into the tail vein of mice [35]. Accumulation of FVIII resulted in oxidative stress (monitored by dihydroethidine staining, malondialdehyde, GSH) and activation of the UPR in the liver. Treatment with butylated hydroxyanisole reduced accumulation of FVIII and attenuated oxidative stress and UPR activation. Taken together, these data are consistent with the notion that protein misfolding in the ER lumen can produce ROS and that ROS and accumulation of misfolded proteins induce ER stress and activate the UPR. In this context, ROS may be generated as a consequence of disulfide bond formation, depletion of cellular GSH, and/or mitochondrial oxidative phosphorylation.

6 Oxidative Stress as an Activator of the UPR

ROS can originate from exposure to irradiation and environmental pollutants and enzymatic reactions involving the mitochondrial respiratory chain, arachidonic acid pathway, cytochrome P450 family, glucose, amino acid, xanthine and NADP/NADPH oxidases, and nitric oxide synthase [36]. In cultured liver cells the combination of hydrogen peroxide generation using glucose oxidase and proteasome inhibition resulted in activation of the UPR and formation of inclusion bodies that was reduced by either pretreatment with N-acetyl-cysteine or the chemical chaperone, 4-phenylbutyrate [23].

Changes in nutrient flux, particularly fatty acid flux, may influence the functional capacity of the ER, in part, via effects on redox balance. Elevated free fatty acids, in particular saturated fatty acids, have been linked to activation of the UPR in a number of cell types, including hepatocytes [37–40]. Antioxidants, such as taurine, effectively reduce saturated fatty acid-mediated oxidative stress and UPR activation in both H4IIE liver cells and primary hepatocytes [41].

Ethanol impairs protein secretion in hepatocytes and the serum protein deficiency that can lead to clotting disorders, edema, and impaired iron delivery [42]. ER stress and activation of the UPR by ethanol is conserved across vertebrates and reductions in ER stress reduce alcohol-induced liver injury [42, 43]. Using a zebrafish larvae model, Tsedensodnom et al. demonstrated that ethanol exposure induced oxidative stress and that oxidative stress and low doses of ethanol synergize to induce the UPR in the liver [44]. In alcoholic liver disease, ROS is generated by ER-localized cytochrome P450’s and, perhaps, interactions between the ER and mitochondria (discussed below) [45]. Regardless of the source of ROS, these data are consistent with the notion that ROS can impair the secretory pathway in hepatocytes and lead to ER stress and UPR activation.

7 ER–Mitochondrial Interactions and Oxidative Stress

Protein folding in and clearance of aggregated proteins from the ER lumen requires energy, thus ER homeostasis is linked to mitochondrial bioenergetics and adenosine triphosphate (ATP) supply. Physical interactions between the ER and mitochondria have been documented and involve specific tethering proteins and elements of the cytoskeleton [46, 47]. One function of these physical interactions is to provide efficient calcium transfer from the ER to mitochondria, thereby regulating the activity of matrix dehydrogenases required for mitochondrial respiration and ATP production. In turn, ATP can be supplied to the ER lumen to support the energy requirements for protein folding and clearance [48, 49].

Several proteins have been identified as components of the ER–mitochondria tethering mechanism including mitofusin-1 and -2 [50]. In particular, mitofusin-2 appears to be enriched at ER–mitochondria contact sites, can influence ER morphology, and can directly tether ER and mitochondria via homo- and hetero-typic interactions [50]. Importantly, the absence of mitofusin-2 increased the distance between the ER and mitochondria and impaired mitochondrial calcium uptake [50]. Liver-specific ablation of mitofusin-2 in mice resulted in ER and oxidative stress, increased stress pathway signaling (c-Jun NH2 Terminal Kinase), and impaired insulin signaling [51]. Chemical chaperones or the antioxidant N-acetylcysteine improved insulin signaling and glucose homeostasis in liver-specific mitofusin-2 knockout mice [51]. It should be noted that hydrogen peroxide increased the expression of mitofusin-2 in cardiomyocytes and selective upregulation of mitofusin-2 was sufficient to induce myocyte apoptosis [52]. One interpretation of these data is that both reductions and increases in mitofusin-2 may interfere with mitochondrial fusion/fission, physical interactions between the ER and mitochondria, and potentially calcium and ATP transfer.

8 Summary

ER and oxidative stress are typically present in metabolic diseases, such as obesity and diabetes, neurodegenerative diseases, such as Alzheimer and Parkinson disease, and atherosclerosis. Protein folding in the ER lumen generates ROS and in turn, ROS can influence the fidelity of protein folding. The quality control system that responds to ER stress, the UPR, can regulate multiple pathways, including antioxidant defense. Thus, the UPR can be viewed as an adaptive mechanism that ultimately attempts to maintain cell survival and function. The ER is also structurally and functionally linked to mitochondria, and thus mitochondrial ATP and ROS may influence or interact with the ER lumen. Liver diseases are often characterized by both ER and oxidative stress and thus understanding how these two processes interact is critical to understanding the pathogenesis of these diseases.