Biting the Iron Bullet: Endoplasmic Reticulum Stress Adds the Pain of Hepcidin to Chronic Liver Disease

Abstract

Hepcidin is a peptide hormone that is secreted by the liver and controls body iron homeostasis. Hepcidin overproduction causes anemia of inflammation, whereas its deficiency leads to hemochromatosis. Inflammation and iron are known extracellular stimuli for hepcidin expression. We found that endoplasmic reticulum (ER) stress also induces hepcidin expression and causes hypoferremia and spleen iron sequestration in mice. CREBH (cyclic AMP response element-binding protein H), an ER stress-activated transcription factor, binds to and transactivates the hepcidin promoter. Hepcidin induction in response to exogenously administered toxins or accumulation of unfolded protein in the ER is defective in CREBH knockout mice, indicating a role for CREBH in ER stress-regulated hepcidin expression. The regulation of hepcidin by ER stress links the intracellular response involved in protein quality control to innate immunity and iron homeostasis.

Comment

Vecchi et al.¹ describe a novel association between chemically-induced endoplasmic reticulum (ER) stress and alteration of iron homeostasis in mice. We review this finding and explore the broader implications for iron-related progression of chronic liver disease.

ER stress is a well-documented phenomenon in eukaryotic cells exposed to toxic chemicals, nutritional deprivation, and pathological agents (Fig. 1A). These varied insults all interfere with the normal folding and processing of newly synthesized proteins in the ER and elicit a coordinated set of responses in affected cells known collectively as the unfolded protein response (UPR).² Key aspects of the UPR include mechanisms for slowing the synthesis of new proteins in the ER, increased production of protein folding chaperones, activation of pathways for degrading misfolded proteins, and activation of self-destruct apoptotic pathways in severely damaged cells. Other arms of the UPR more directly address the cause(s) of ER stress, such as nuclear factor erythroid-2–related (Nrf2)-dependent induction of antioxidant enzymes.³ Together these responses maintain ER quality control and help the organism adapt to (and recover from) the stressful conditions that initiated the UPR.

Fig. 1.

The unfolded protein response (UPR) influences the course of liver disease and alters iron homeostasis. (A) Key facets of the hepatocyte UPR are shown that together either restore ER and cellular homeostasis or destroy severely afflicted cells. (B) Progression of many liver diseases follows a common course: pathogen-induced ROS, activation of ER stress, recruitment of immune cells, and accelerated liver damage. Induction of hepcidin by ER stress¹ represents a new mechanism linking liver disease to the regulation of iron homeostasis. As discussed in the text, this likely serves an antimicrobial defensive role when body iron stores are low or normal. Conversely, under conditions of elevated iron stores, hepcidin redistributes excess iron to macrophages. This may contribute to iron-related progression of liver diseases such as nonalcoholic fatty liver disease.

The UPR is increasingly postulated as a key homeostatic mechanism in hepatocytes that is capable of influencing the progression of chronic liver disease. It is likely highly relevant in hepatocytes given the high protein flux through the ER and the high rate at which reactive oxygen species (ROS) are generated, even in healthy cells. ROS and other sources of ER stress are increased in many chronic liver diseases, including hepatocellular carcinoma,⁴ viral hepatitis,^5,6 alcoholic liver disease,⁷ hereditary hemochromatosis,⁸ and nonalcoholic steatohepatitis (NASH).^9,10 Increased ER stress in liver disease is due partly to the effects of cytokines such as interleukin-6 secreted by the innate immune system.¹¹ Interleukin-6 triggers unique facets of the UPR in hepatocytes, including activation of the liver-specific transcription factor cyclic AMP–responsive element binding protein H (CREBH) and induction of an acute phase response.¹¹ Thus the UPR may be considered part of the liver's immune-mediated antimicrobial defense system.

Hepcidin, an antimicrobial peptide that regulates iron homeostasis, is emerging as an important systemic immune response mediator.¹² Inflammation and elevated iron stores are the two major stimuli for hepcidin secretion. Hepcidin acts by binding to ferroportin, an iron exporter enriched on the surface of cells active in iron transport, especially gut epithelial cells (enterocytes) and reticuloendothelial cells such as Kupffer cells, resulting in the internalization and degradation of ferroportin,¹³ and reduction of cellular iron export.

Two recent articles have now linked the hepatocyte UPR with hepcidin gene regulation. Oliveira et al. demonstrated that chemically-induced ER stress induced hepcidin gene expression in HepG2 cells via down-regulation of the C/EBPα inhibitor C/EBP homologous protein (CHOP),¹⁴ whereas Vecchi et al. used a distinct set of chemical and immunological stressors to activate the hepcidin gene in a CREBH-dependent fashion in HepG2 cells and in mice.¹

Vecchi et al. first investigated increased hepcidin expression in response to a diverse series of chemical stressors in HepG2 cells. One of these agents (tunicamycin) inhibits protein glycosylation in the ER, disrupting proper folding of nascent polypeptides and triggering the UPR. Increased hepcidin messenger RNA in response to tunicamycin resulted from transcriptional activation of hepcidin gene expression, the same basic mechanism used by other hepcidin regulators.¹² These findings were confirmed and extended in vivo; tunicamycin-treated mice showed increased liver hepcidin expression as well as decreased serum iron and elevated splenic iron.

Previous work had identified CREBH as a key mediator of the UPR in liver.¹¹ Vecchi et al. showed that CREBH knockdown with short interfering RNA decreased both basal and tunicamycin-induced levels of hepcidin messenger RNA in HepG2 cells. A CREBH transactivation site was identified in the hepcidin (HAMP) promoter and HAMP induction by tunicamycin was found to be impaired in CREBH knockout mice. Hepcidin gene activation by immunological challenge (lipopolysaccharide) was reduced and delayed but not eliminated in the CREBH knockout mice, consistent with the involvement of multiple transcriptional pathways in hepcidin regulation.

ER stress is thus the latest addition to a growing list of conditions that regulate hepcidin gene expression (Fig. 1B). Iron-related signals appear to have the major role. Iron increases production of bone morphogenetic protein-6,¹⁵ a transforming growth factor-β family member that binds to hemojuvelin and elicits smad4-mediated activation of hepcidin gene expression.¹⁶ Induction of hepcidin by iron may also depend on binding of diferric transferrin to the type II transferrin receptor.¹⁷ Immune-related signals are important activators of hepcidin expression, and these are mediated by the inflammatory response transcription factor C/EBPα¹⁸ and the jak/stat (Janus kinase/signal transducer and activator of transcription) pathway^19,20 in addition to ER stress.^1,14 Finally, inhibition of hepcidin secretion can occur in response to anemia, hypoxia, or increased erythropoiesis via a variety of transcriptional and posttranscriptional mechanisms.¹²

The study by Vecchi et al., is particularly intriguing because it establishes a direct connection between a cellular response common to essentially all chronic liver diseases (the UPR) and iron dysregulation. First, this is important because of hepcidin's central role in determining the total amount of body iron stores. Inappropriately low levels lead to iron loading and account for most forms of hereditary hemochromatosis.²¹ Thalassemias and transfusional iron overload are also associated with low hepcidin. Elevated liver iron in patients infected with hepatitis C virus has been linked to low hepcidin,²² and alteration in hepcidin expression may also contribute to increased liver iron associated with alcoholic fatty liver disease and nonalcoholic fatty liver disease (NAFLD).^23,24

Second, hepcidin levels influence the cellular distribution of excess iron, a potentially critical factor in its pathogenicity. Chronically low hepcidin (as can occur in hepatitis C and hemochromatosis) favors increased iron in hepatocyte stores and plasma. Conversely, high hepcidin (chronic inflammation and ER stress) favors depletion from blood but accumulation in reticuloendothelial cells. High hepcidin levels in diseases like obesity that are accompanied by chronic inflammation contribute to the anemia in these patients.²⁵ In further support of this idea, we recently found that presence of NASH was associated with a Kupffer cell pattern of iron staining (consistent with high hepcidin induced by immune-stimulated ER stress) in a cohort of patients with NAFLD (K.V. Kowdley, unpublished data). Progression of NASH to hepatocellular carcinoma has also been linked to elevated iron in Kupffer cells.²⁶

The UPR-induced hepcidin response seems counterproductive for most liver diseases. Initially, it may have been advantageous, for example in combating a microbial infection in the setting of a low to moderate body iron burden. However, for many modern-day liver diseases, the outcome may not always be as beneficial. The available data suggest that in the setting of abundant body iron, ER stress due to chronic diseases like NAFLD may result in a hepcidin-mediated redistribution of iron to reticuloendothelial cells, where it may further stimulate an immune response and exacerbate the disease. Additional research is needed to evaluate this extension of the exciting finding by Vecchi et al., but it is interesting to speculate that what may have once been a powerful weapon now looks more like a painful traitor in the liver's battle against chronic disease.