Protective role of calreticulin in HFE hemochromatosis

HFE gene mutations are associated with over 80% of cases of hereditary hemochromatosis (HH), an iron-overload disease in which the liver is the most frequently affected organ. Research on HFE has traditionally focused on its interaction with the transferrin receptor. More recent studies have suggested a more complex function for this nonclassical MHC-I protein. The aim of this study was to examine how HFE and its two most common mutations affect the expression of selected genes in a hepatocyte-like cell line. Gene expression was analyzed in HepG2 cells overexpressing wild-type and mutant HFE. The effect of HFE in iron import and oxidative stress levels was assessed. Unfolded protein response (UPR)-activated gene expression was analyzed in peripheral blood mononuclear cells from characterized HH patients. C282Y HFE down-regulated hepcidin and enhanced calreticulin mRNA expression. Calreticulin levels correlated with intracellular iron increase and were associated with protection from oxidative stress. In C282Y(+/+) patients calreticulin levels correlated with the expression of the UPR marker BiP and showed a negative association with the number of hereditary hemochromatosis clinical manifestations. The data show that expression of C282Y HFE triggers a stress-protective response in HepG2 cells and suggest a role for calreticulin as a modifier of the clinical expression of HH.     

Hemochromatosis: As a conformational disorder

Hereditary hemochroamtosis (HH) refers to a unique clinicopathologic subset of iron overload syndromes that includes the disorder related to C282Y homozygous mutation of the hemochromatosis protein (HFE), the most common form of hereditary hemochromatosis. Recent reports have highlighted analogies with the class of disorders, known as the conformational diseases whereby HFE C282Y mutant protein forms aggregates and is subsequently retained in the endoplasmic reticulum (ER). In conformational disorders, accumulation of unfolded or misfolded proteins in the ER can activate a complex cascade linked to the regulation of diverse physiologic processes, disease onset and progression. To-date, reviews on HFE C282Y HH have largely dealt with the end-stage consequence of this disorder (iron overload). However, our review focuses on upstream molecular events resulting from the mislocalization of the aggregation-prone HFE C282Y protein leading to potential advances in treatment and diagnosis.     

Expression of hereditary hemochromatosis C282Y HFE protein in HEK293 cells activates specific endoplasmic reticulum stress responses

Background  

Hereditary Hemochromatosis (HH) is a genetic disease associated with iron overload, in which individuals homozygous for the mutant C282Y HFE associated allele are at risk for the development of a range of disorders particularly liver disease. Conformational diseases are a class of disorders associated with the expression of misfolded protein. HFE C282Y is a mutant protein that does not fold correctly and consequently is retained in the Endoplasmic Reticulum (ER). In this context, we sought to identify ER stress signals associated with mutant C282Y HFE protein expression, which may have a role in the molecular pathogenesis of HH.     

The unfolded protein response in hereditary haemochromatosis

To cope with the accumulation of unfolded or misfolded proteins the endoplasmic reticulum (ER) has evolved specific signalling pathways collectively called the unfolded protein response (UPR). Elucidation of the mechanisms governing ER stress signalling has linked this response to the regulation of diverse physiologic processes as well as to the progression of a number of diseases. Interest in hereditary haemochromatosis (HH) has focused on the study of proteins implicated in iron homeostasis and on the identification of new alleles related with the disease. HFE has been amongst the preferred targets of interest, since the discovery that its C282Y mutation was associated with HH. However, the discrepancies between the disease penetrance and the frequency of this mutation have raised the possibility that its contribution to disease progression might go beyond the mere involvement in regulation of cellular iron uptake. Recent findings revealed that activation of the UPR is a feature of HH and that this stress response may be involved in the genesis of immunological anomalies associated with the disease. This review addresses the connection of the UPR with HH, including its role in MHC-I antigen presentation pathway and possible implications for new clinical approaches to HH.     

Tauroursodeoxycholic acid reduces endoplasmic reticulum stress, acinar cell damage, and systemic inflammation in acute pancreatitis

In acute pancreatitis, endoplasmic reticulum (ER) stress prompts an accumulation of malfolded proteins inside the ER, initiating the unfolded protein response (UPR). Because the ER chaperone tauroursodeoxycholic acid (TUDCA) is known to inhibit the UPR in vitro, this study examined the in vivo effects of TUDCA in an acute experimental pancreatitis model. Acute pancreatitis was induced in Wistar rats using caerulein, with or without prior TUDCA treatment. UPR components were analyzed, including chaperone binding protein (BiP), phosphorylated protein kinase-like ER kinase (pPERK), X-box binding protein (XBP)-1, phosphorylated c-Jun NH(2)-terminal kinase (pJNK), CCAAT/enhancer binding protein homologues protein, and caspase 12 and 3 activation. In addition, pancreatitis biomarkers were measured, such as serum amylase, trypsin activation, edema formation, histology, and the inflammatory reaction in pancreatic and lung tissue. TUDCA treatment reduced intracellular trypsin activation, edema formation, and cell damage, while leaving amylase levels unaltered. The activation of myeloperoxidase was clearly reduced in pancreas and lung. Furthermore, TUDCA prevented caerulein-induced BiP upregulation, reduced XBP-1 splicing, and caspase 12 and 3 activation. It accelerated the downregulation of pJNK. In controls without pancreatitis, TUDCA showed cytoprotective effects including pPERK signaling and activation of downstream targets. We concluded that ER stress responses activated in acute pancreatitis are grossly attenuated by TUDCA. The chaperone reduced the UPR and inhibited ER stress-associated proapoptotic pathways. TUDCA has a cytoprotective potential in the exocrine pancreas. These data hint at new perspectives for an employment of chemical chaperones, such as TUDCA, in prevention of acute pancreatitis.     

Over-expression of wild-type and mutant HFE in a human melanocytic cell line reveals an intracellular bridge between MHC class I pathway and transferrin iron uptake

Hereditary hemochromatosis (HH) is a frequent recessive disorder of iron metabolism characterised by systemic iron overload. In Northern Europe, more than 90% of HH patients are homozygous for a mis-sense mutation (C282Y) in the HFE1 gene product. The HFE protein is the heavy chain of a MHC class I-related molecule and associates with beta2 microglobulin and the transferrin receptor. Its precise roles in iron metabolism and in the pathophysiology of HH are still unclear. In order to identify the cellular processing of HFE, an important step towards the understanding of the function of the protein, we stably over-expressed the wild type and mutated forms fused to the Green Fluorescent Protein in a melanocytic MHC class I expressing cell line, the Mel Juso cell line. In wild type and mutant clones, the fusion proteins were not detected at the cell surface but only in the cytoplasm. Their sub-cellular localisation was determined by co-labelling of cells with organite-specific antibodies and confocal microscopy. HFE-GFP followed initially HLA class I intracellular processing but co-localised with transferrin in early endosomes without recycling at the cell surface. The C282Y-GFP fusion protein followed a different folding pathway to exit endoplasmic reticulum. Over-expression of the wild-type protein lead to a decrease in diferric transferrin uptake. Our model will be of use in the elucidation of the functional interaction between intracellular HFE and iron transporters transferrin/transferrin receptor complexes and Slc11A2 (also named N-Ramp2 or DMT1) in different endosomal compartments.     

Genetic hemochromatosis, a Celtic disease: is it now time for population screening?

In populations of northern European ancestry, hereditary hemochromatosis (HH) is tightly linked to mutations within the hemochromatosis gene (HFE gene). Over 93% of Irish HH patients are homozygous for the HFE gene C282Y mutation, providing a reliable diagnostic marker of the disease in this population. However, the prevalence of the C282Y mutation and that of the second HFE gene mutation, H63D, have yet to be determined within the Irish population. The objective of this study was to identify the true prevalence of the genetic form of HH in the Irish population. DNA was extracted from 1002 randomly selected newborn screening cards and analyzed for the C282Y and H63D mutations within the HFE gene. Complete results were obtained from 800 cards. Mutations were identified in 364 (46%) neonates. Eight (1%) neonates were homozygous for C282Y and 8 (1%) were homozygous for H63D. One hundred and fifty-five (19%) neonates were C282Y heterozygous and 226 (28%) were H63D heterozygous. Of these, 33 (4%) carried one copy of both C282Y and H63D mutations, i.e., compound heterozygous. Allele frequencies for C282Y and H63D were 11% and 15%, respectively. The high C282Y allele frequency in the Irish population together with its close linkage to HH indicate that C282Y genotyping is the preferred screening strategy for this disease in Ireland.     

Hereditary hemochromatosis in Spain

The C282Y mutation of the HFE gene has been reported as the main cause of hereditary hemochromatosis (HH). Another missense mutation (H63D) has also been detected at an increased frequency in a compound heterozygote state with the C282Y mutation in HH patients. However, these two mutations are not present in all of the HH patients, indicating that other mutations in the HFE gene, or in other loci, should exist. The present study reports the frequencies of the C282Y and H63D mutations in 74 Spanish HH patients and the results of the sequencing analysis of the HFE exons, intron-exon boundaries, and 588 bp of the 5' region in 5 patients negative for the C282Y mutation. We have detected a high frequency of the C282Y mutation (85.1%) in Spanish HH patients, indicating that this mutation is the most common defect associated with the disease in Spain. The screening of the HFE regions in our patients without the C282Y mutation has revealed the presence of five polymorphisms. However, no other pathological mutations have been found. Therefore, further efforts to characterize the unscreened part of the HFE gene or other loci should be taken to identify the potential genetic factors causing HH in the C282Y-negative patients.     

Endoplasmic reticulum stress inhibition protects steatotic and non-steatotic livers in partial hepatectomy under ischemia�Creperfusion

During partial hepatectomy, ischemia–reperfusion (I/R) is commonly applied in clinical practice to reduce blood flow. Steatotic livers show impaired regenerative response and reduced tolerance to hepatic injury. We examined the effects of tauroursodeoxycholic acid (TUDCA) and 4-phenyl butyric acid (PBA) in steatotic and non-steatotic livers during partial hepatectomy under I/R (PH+I/R). Their effects on the induction of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress were also evaluated. We report that PBA, and especially TUDCA, reduced inflammation, apoptosis and necrosis, and improved liver regeneration in both liver types. Both compounds, especially TUDCA, protected both liver types against ER damage, as they reduced the activation of two of the three pathways of UPR (namely inositol-requiring enzyme and PKR-like ER kinase) and their target molecules caspase 12, c-Jun N-terminal kinase and C/EBP homologous protein-10. Only TUDCA, possibly mediated by extracellular signal-regulated kinase upregulation, inactivated glycogen synthase kinase-3β. This is turn, inactivated mitochondrial voltage-dependent anion channel, reduced cytochrome c release from the mitochondria and caspase 9 activation and protected both liver types against mitochondrial damage. These findings indicate that chemical chaperones, especially TUDCA, could protect steatotic and non-steatotic livers against injury and regeneration failure after PH+I/R.     

Chaperones Ameliorate Beta Cell Dysfunction Associated with Human Islet Amyloid Polypeptide Overexpression

In type 2 diabetes, beta-cell dysfunction is thought to be due to several causes, one being the formation of toxic protein aggregates called islet amyloid, formed by accumulations of misfolded human islet amyloid polypeptide (hIAPP). The process of hIAPP misfolding and aggregation is one of the factors that may activate the unfolded protein response (UPR), perturbing endoplasmic reticulum (ER) homeostasis. Molecular chaperones have been described to be important in regulating ER response to ER stress. In the present work, we evaluate the role of chaperones in a stressed cellular model of hIAPP overexpression. A rat pancreatic beta-cell line expressing hIAPP exposed to thapsigargin or treated with high glucose and palmitic acid, both of which are known ER stress inducers, showed an increase in ER stress genes when compared to INS1E cells expressing rat IAPP or INS1E control cells. Treatment with molecular chaperone glucose-regulated protein 78 kDa (GRP78, also known as BiP) or protein disulfite isomerase (PDI), and chemical chaperones taurine-conjugated ursodeoxycholic acid (TUDCA) or 4-phenylbutyrate (PBA), alleviated ER stress and increased insulin secretion in hIAPP-expressing cells. Our results suggest that the overexpression of hIAPP induces a stronger response of ER stress markers. Moreover, endogenous and chemical chaperones are able to ameliorate induced ER stress and increase insulin secretion, suggesting that improving chaperone capacity can play an important role in improving beta-cell function in type 2 diabetes.     

The effects of wild-type and mutant HFE expression upon cellular iron uptake in transfected human embryonic kidney cells

In order to measure the effects of HFE (haemochromatosis) upon iron uptake, stable expression of wild-type and C282Y, H63D and S65C mutant HFE cDNA was established in HEK 293 cells. Control cells were transfected with empty vector. Expression of HFE mRNA and protein was detected in the cell lines transfected with HFE cDNA, but not in the control cell line. The ferritin concentration in wild-type cells cultured in 40 microM ferric ammonium citrate was 69% of that in control cells and 81% of that in C282Y cells. The ferritin concentration in H63D cells was intermediate between wild-type and C282Y and the ferritin concentration in S65C cells was similar to wild-type cells. Uptake of transferrin-iron in wild-type, C282Y and control cells was measured over 45 min. The Hill coefficients for transferrin-iron uptake were similar. The V(max) for transferrin-iron uptake in wild-type cells was 59.5% of control cells and 69.5% of C282Y cells. Estimates of K(m) were 232 nM for wild-type cells, 338 nM for C282Y cells and 570 nM for controls. Transferrin receptor levels were lowered, but not significantly, in the HFE transfected cells. The results show that HFE reduces transferrin-iron uptake, probably as an uncompetitive inhibitor.     

Bone marrow mesenchymal stem cell donors with a high body mass index display elevated endoplasmic reticulum stress and are functionally impaired

Bone marrow mesenchymal stem cells (BM‐MSCs) are promising candidates for regenerative medicine purposes. The effect of obesity on the function of BM‐MSCs is currently unknown. Here, we assessed how obesity affects the function of BM‐MSCs and the role of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) therein. BM‐MSCs were obtained from healthy donors with a normal (<25) or high (>30) body mass index (BMI). High‐BMI BM‐MSCs displayed severely impaired osteogenic and diminished adipogenic differentiation, decreased proliferation rates, increased senescence, and elevated expression of ER stress–related genes ATF4 and CHOP. Suppression of ER stress using tauroursodeoxycholic acid (TUDCA) and 4‐phenylbutyrate (4‐PBA) resulted in partial recovery of osteogenic differentiation capacity, with a significant increase in the expression of ALPL and improvement in the UPR. These data indicate that BMI is important during the selection of BM‐MSC donors for regenerative medicine purposes and that application of high‐BMI BM‐MSCs with TUDCA or 4‐PBA may improve stem cell function. However, whether this improvement can be translated into an in vivo clinical advantage remains to be assessed.     

Mutant HFE H63D protein is associated with prolonged endoplasmic reticulum stress and increased neuronal vulnerability

A specific polymorphism in the hemochromatosis (HFE) gene, H63D, is over-represented in neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer disease. Mutations of HFE are best known as being associated with cellular iron overload, but the mechanism by which HFE H63D might increase the risk of neuron degeneration is unclear. Here, using an inducible expression cell model developed from a human neuronal cell line SH-SY5Y, we reported that the presence of the HFE H63D protein activated the unfolded protein response (UPR). This response was followed by a persistent endoplasmic reticulum (ER) stress, as the signals of UPR sensors attenuated and followed by up-regulation of caspase-3 cleavage and activity. Our in vitro findings were recapitulated in a transgenic mouse model carrying Hfe H67D, the mouse equivalent of the human H63D mutation. In this model, UPR activation was detected in the lumbar spinal cord at 6 months then declined at 12 months in association with increased caspase-3 cleavage. Moreover, upon the prolonged ER stress, the number of cells expressing HFE H63D in early apoptosis was increased moderately. Cell proliferation was decreased without increased cell death. Additionally, despite increased iron level in cells carrying HFE H63D, it appeared that ER stress was not responsive to the change of cellular iron status. Overall, our studies indicate that the HFE H63D mutant protein is associated with prolonged ER stress and chronically increased neuronal vulnerability.     

Hereditary hemochromatosis in a Brazilian university hospital in São Paulo State (1990-2000)

Hereditary hemochromatosis (HH) is the most common genetic disease among individuals of European descent. Two mutations (845G-->A, C282Y and 187C-->G, H63D) in the hemochromatosis gene (HFE gene) are associated with HH. About 85-90% of patients of northern European descent with HH are C282Y homozygous. The prevalence of HH in the Brazilian population, which has a very high level of racial admixture, is unknown. The aims of the present study were to identify individuals with diagnostic criteria for HH among patients with a body iron overload attended at the university hospital of the Faculty of Medicine of Ribeirao Preto from 1990 to 2000, and to evaluate the prevalence of HFE mutations. We screened first-degree relatives for HFE mutations. Four of 72 patients (three men and one woman, mean age 47 years) fulfilled the criteria for HH. HFE mutations were studied in three patients [two C282Y homozygotes (patients 1 and 2) and one H63D heterozygote]. Patient 1 had four children (all C282Y heterozygotes with no iron overload) and seven brothers and sisters: two sisters (66 and 76 years old) were C282Y homozygotes and both had an iron overload (a liver biopsy in one showed severe iron deposits), one sister (79 years old) was a compound heterozygote with no iron overload, one brother (78 years old) was a C282Y heterozygote with no iron overload, two individuals were H63D heterozygotes (one brother, 49 years old, obese, with a body iron overload and abnormal liver enzymes - a biopsy showed non-alcoholic steatohepatitis, and one 70-year-old sister with no iron overload). Patient 2 had two children (22 and 24 years old who were C282Y heterozygotes with no iron overload) but no brothers or sisters. These results showed that HH was uncommon among individuals attended at our hospital, although HFE mutations were found in all patients. Familial screening is valuable for the early diagnosis of individuals at risk since it allows treatment to be initiated before the onset of the clinical manifestations of organ damage associated with HH.     

Hereditary hemochromatosis: the clinical significance of the S65C mutation

Hereditary hemochromatosis (HH) is a common genetic disease with iron overload in certain organs, especially the liver. Most cases are homozygous for the C282Y mutation in the HFE gene; a few are C282Y heterozygous, compound C282Y/H63D heterozygous, or have no known mutation. A third mutation, S65C, has been associated with HH, but this finding is disputed. We have studied the clinical significance of various genotypes with the S65C mutation. In a population-based screening for HH in 65,238 persons, 613 had high serum transferrin saturation in two blood samples and were invited for HFE genotyping. In 556 persons with complete data sets, we studied the serum ferritin concentration and the risk of being diagnosed with phenotypic HH in the various genotypic groups. The phenotypic diagnosis was given without knowing the genotypic result. Except for the C282Y homozygotes, no differences in median serum ferritin concentrations were found between the various genotypic groups. However, the C282Y/S65C compound heterozygous group had a higher risk of being diagnosed with phenotypic HH than the wild-type group, as did the C282Y homozygous and the C282Y/H63D compound heterozygous groups. When combined with the C282Y mutation, the S65C mutation is associated with an increased risk of being diagnosed with phenotypic HH.     

Reducing endoplasmic reticulum stress does not improve steatohepatitis in mice fed a methionine- and choline-deficient diet

Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of nonalcoholic steatohepatitis. The ER stress response is activated in the livers of mice fed a methionine- and choline-deficient (MCD) diet, yet the role of ER stress in the pathogenesis of MCD diet-induced steatohepatitis is unknown. Using chemical chaperones on hepatic steatosis and markers of inflammation and fibrosis in mice fed a MCD diet, we aim to determine the effects of reducing ER stress. C57BL/6J mice were fed a MCD diet with or without the ER chemical chaperones 4-phenylbutyric acid (PBA) and tauroursodeoxycholic acid (TUDCA) for 2 wk. TUDCA and PBA effectively attenuated the ER stress response in MCD diet-fed mice, as evidenced by reduced protein levels of phosphorylated eukaryotic initiation factor 2α and phosphorylated JNK and suppression of mRNA levels of CCAAT/enhancer binding protein homologous protein, glucose-regulated protein 78 kDa, and X-box binding protein 1. However, PBA and TUDCA did not decrease MCD diet-induced hepatic steatosis. MCD diet-induced hepatic inflammation, as evidenced by increased plasma alanine aminotransferase and induction of hepatic TNFα expression, was also not reduced by PBA or TUDCA. PBA and TUDCA did not attenuate MCD diet-induced upregulation of the fibrosis-associated genes tissue inhibitor of metalloproteinase-1 and matrix metalloproteinase-9. ER chemical chaperones reduce MCD diet-induced ER stress, yet they do not improve MCD diet-induced hepatic steatosis, inflammation, or activation of genes associated with fibrosis. These data suggest that although the ER stress response is activated by the MCD diet, it does not have a primary role in the pathogenesis of MCD diet-induced steatohepatitis.     

Phenylbutyric acid induces the cellular senescence through an Akt/p21(WAF1) signaling pathway

It has been well known that three sentinel proteins - PERK, ATF6 and IRE1 - initiate the unfolded protein response (UPR) in the presence of misfolded or unfolded proteins in the ER. Recent studies have demonstrated that upregulation of UPR in cancer cells is required to survive and proliferate. Here, we showed that long exposure to 4-phenylbutyric acid (PBA), a chemical chaperone that can reduce retention of unfolded and misfolded proteins in ER, induced cellular senescence in cancer cells such as MCF7 and HT1080. In addition, we found that treatment with PBA activates Akt, which results in p21(WAF1) induction. Interestingly, the depletion of PERK but not ATF6 and IRE1 also induces cellular senescence, which was rescued by additional depletion of Akt. This suggests that Akt pathway is downstream of PERK in PBA induced cellular senescence. Taken together, these results show that PBA induces cellular senescence via activation of the Akt/p21(WAF1) pathway by PERK inhibition.     

Frequencies in the Japanese Population of HFE Gene Mutations

We studied the frequencies of C282Y and H63Dmutations in the HFE gene, thought to be responsible forhereditary hemochromatosis (HH), in 504 chromosomesobtained from 252 unrelated Japanese. Allele-specific PCR and PCRrestriction fragment lengthpolymorphism methods revealed that the C282Y mutationwas not found and the H63D mutation was low in frequency(at only 0.99%) compared with data from European people. Since most HH is thought to be associated withthe HFE gene mutation, the low incidence of thesemutations is a likely reason for the rarity of thisdisease in the Japanese population.