In this study, a HEK293 cell line model system transiently expressing WT HFE or its C282Y mutant counterpart was used to determine the ER stress mechanisms associated with C282Y HFE production and retention. We demonstrated that the C282Y mutant protein co-localized with the ER resident protein calnexin and undergoes marked degradation compared to WT HFE, providing a suitable model of study for the C282Y HFE protein. C282Y HFE mutant protein accumulates in the ER and middle Golgi compartment, and consequently undergoes degradation [5, 6, 38–40]. Mutant proteins with such impaired egress have been shown to cause ER stress in a variety of disorders, which in turn may give rise to a variety of stress pathways such as EOR, UPR activation and cellular death [41–43].
Upregulation of the EOR pathway leads to activation of the transcription factor NF-κB . In this study we found that C282Y HFE protein induced NF-κB, which consequently resulted in a marked increase in protein production of both interleukin-8 (IL-8) and monocyte chemotatic protein-1 (MCP-1), along with increased transcriptional activation of IL-8 in C282Y HFE-expressing cell line. IL-8 has been shown to be an important mediator in various diseases [44, 45]. MCP-1 is secreted by a variety of cells as a response to several inflammatory stimuli and activates and attracts monocytes/macrophages . Furthermore, MCP-1 concentrations are deregulated in patients with alcoholic hepatitis or cirrhosis [46, 47] and in patients with hepatitis C . These findings suggest that the pathology of HH in C282Y/C282Y HFE patients may involve an aberrant inflammatory action [49, 50].
Indeed, in this regards Lee et al found that HFE expression was increased during ER stress, induced by serum deprivation, menadione and β-amyloid. This increase in HFE expression was independent of transferrin receptor and ferritin. Furthermore, the labile iron pool was consistently decreased when HFE expression was increased, suggesting that the observed induction of HFE has a protective function by limiting cellular iron exposure during stress [51, 52].
UPR pathway involves up-regulation of the secretory pathway's capacity to process proteins and entails the transcriptional up-regulation of a co-ordinately expressed set of genes encoding ER chaperones, enzymes, and structural components of the ER. The UPR pathway culminates in the expression of glucose-responsive genes (grp) such as, grp78. Expression of the ER charpone grp78/BiP is a classical marker for UPR activation in mammalian cells. The grp78 promoter contains a consensus binding site called the ER stress response element; recognised by ATF6, a transcription factor specifically activated by ER stress. Several lines of evidence support the essential role of ATF6 in the ER stress response and have revealed it to be a proximal transducer of GRP78/BiP . Recent studies of proteomic analysis of hepatic iron overload in mice have shown increased levels of GRP78/Bip protein . This upregulation in response to iron excess has been demonstrated previously in human cells and it has been suggested that up-regulation of GRP78/Bip may indicate increased demand for re-folding or retention of proteins in the ER of iron-overloaded cells . The UPR is also known to up-regulate CHOP, which is generally linked to ER stress. CHOP protein belongs to the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors and is thought to play a critical role in cell survival or cell death during ER stress . Prolonged activation of the UPR responses is known to result in cell death. Studies by Hacki et al, suggested that perturbing ER functions induces a specific crosstalk between the ER and mitochondria . It is also worth noting that constitutive activation of NF-κB promotes survival of a range of cells, including B cells, hepatic cells and cancer cells. However, whereas NF-κB is most commonly involved in suppressing apoptosis by transactivating the expression of anti apoptotic genes, it is associated with promoting programmed cell death in response to ER stress via the EOR pathway by calcium release from the ER, resulting in mitochondrial dysfunction and apoptosis [57–59]. In neuronal degenerating disease, ER stress results in activation of NF-κB, up-regulation of GRP78 protein levels, and ensuing apoptotic cell death due to the expression of mutant protein . The data in this study reveals a potential role for NF-κB in C282Y HFE mediated cellular death.
TUDCA is a non-toxic compound which is known to be effective in preventing cytotoxic processes. It has been demonstrated to inhibit activation of GRP78 and caspase-3, along with cytochrome c release. TUDCA stabilizes the lipid and protein structure of mitochondrial outer membranes, thus inhibiting Bax binding to the outer membrane . Indeed, mitochondrial-derived reactive oxygen species may be involved [61, 62] as suggested by recent studies which report that TUDCA prevents the generation of ROS . The potential of TUDCA to prevent apoptosis and caspase activation may prove beneficial in protecting cells predisposed to disruptions in the ER. Cytochrome c release and caspase-3 activation along with decreased bcl-2 promoter activity suggest the damaging action of mutant C282Y protein in cellular stress and therefore TUDCA may protect cells during C282Y HFE protein induced stress.
A variable clinical presentation among C282Y homozygous individuals suggests an important contribution of disease modifiers acting on the same signalling pathways. Indeed, while the C282Y mutation results in an altered disease phenotype, mice studies have shown that the C282Y mutation does not completely disrupt the function of HFE, emphasising the importance of additional insults. Furthermore, activation of the UPR pathway can have a protective role . Therefore, it may be suggested that activation of this pathway by C282Y HFE protein expression as shown in this report might explain the milder phenotype of C282Y HFE mice compared to a similar but more severe phenotype of the HFE knockout mice . Z A1AT deficiency represents a possible genetic insult which is known to act on the EOR and UPR pathways and serves as an excellent model for conformational disease [64, 65]. Recent work has revealed the lack of UPR activation by mutant Z A1AT, suggesting it is unlikely that mutant Z A1AT is "sensed" by the machinery of the UPR. Indeed, the signal appears not to be transmitted as a result of a block in the afferent or efferent components of the response, demonstrated by a lack of increased GRP78 activity. However, these studies have shown that Z A1AT UPR activation is found as a result of a secondary stimulus, revealing its activation in the presence of known chemical stress inducers . We wanted to examine the co-existence of both C282Y HFE and Z A1AT in our in vitro cell model system, whereby the C282Y HFE could act as a genetic insult in the context of secondary stimuli acting on the Z A1AT UPR pathway. Our findings indicate the potential for C282Y HFE to activate the Z A1AT UPR pathway (Fig. 3A and 3D), thus implicating C282Y HFE as a factor which may 'trigger' and/or explain part of the clinical expression of Z A1AT deficiency and vice versa. Recent reports, have suggested that the heterozygous carrier state for the mutant Z gene, found in 1.5 % to 3% of the population, is not itself a common cause of liver injury but may be a modifier gene for other liver diseases . Indeed, a large patient population study recently indicated that the Z A1AT heterozygous state may have a role in worsening liver disease .