Chiara Vecchi

ER Stress Controls Iron Metabolism Through Induction of Hepcidin

Ironing Out Stress The peptide hormone, hepcidin, is secreted from the liver in response to extracellular factors, including inflammation, and regulates iron homeostasis by controlling transmembrane iron transport. Vecchi et al. (p. 877) showed that intracellular stress signals in the endoplasmic reticulum also control hepcidin expression and can thus modulate local or systemic iron traffic. This mechanism occurs through the transcription factor CREBH, which is a known mediator of the inflammatory response. Collectively, the results suggest a direct link between the intracellular stress response, innate immunity, and iron metabolism. Stress signals in the endoplasmic reticulum activate a transcription factor that induces the expression of an iron-regulatory hormone. 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.

Hepatitis C Virus–Induced Reactive Oxygen Species Raise Hepatic Iron Level in Mice by Reducing Hepcidin Transcription

BACKGROUND & AIMS Despite abundant clinical evidence, the mechanisms by which hepatic iron overload develops in patients with hepatitis C virus (HCV)-associated chronic liver disease remain unknown. The aim of this study was to investigate how hepatic iron overload develops in the presence of HCV proteins. METHODS Male transgenic mice expressing the HCV polyprotein and nontransgenic control mice (C57BL/6) were assessed for iron concentrations in the liver, spleen, and serum and iron regulatory molecules in vivo and ex vivo. RESULTS Transgenic mice had increased hepatic and serum iron concentrations, decreased splenic iron concentration, and lower hepcidin expression in the liver accompanied by higher expression of ferroportin in the duodenum, spleen, and liver. In response to hepatocellular iron excess, transferrin receptor 1 expression decreased and ferritin expression increased in the transgenic liver. Transgenic mice showed no inflammation in the liver but preserved the ability to induce hepcidin in response to proinflammatory cytokines induced by lipopolysaccharide. Hepcidin promoter activity and the DNA binding activity of CCAAT/enhancer-binding protein alpha (C/EBP) were down-regulated concomitant with increased expression of C/EBP homology protein, an inhibitor of C/EBP DNA binding activity, and with increased levels of reactive oxygen species in transgenic mice at the ages of 8 and 14 months. CONCLUSIONS HCV-induced reactive oxygen species may down-regulate hepcidin transcription through inhibition of C/EBPalpha DNA binding activity by C/EBP homology protein, which in turn leads to increased duodenal iron transport and macrophage iron release, causing hepatic iron accumulation.

Hypoxia induced downregulation of hepcidin is mediated by platelet derived growth factor BB

Objective Hypoxia affects body iron homeostasis; however, the underlying mechanisms are incompletely understood. Design Using a standardised hypoxia chamber, 23 healthy volunteers were subjected to hypoxic conditions, equivalent to an altitude of 5600 m, for 6 h. Subsequent experiments were performed in C57BL/6 mice, CREB-H knockout mice, primary hepatocytes and HepG2 cells. Results Exposure of subjects to hypoxia resulted in a significant decrease of serum levels of the master regulator of iron homeostasis hepcidin and elevated concentrations of platelet derived growth factor (PDGF)-BB. Using correlation analysis, we identified PDGF-BB to be associated with hypoxia mediated hepcidin repression in humans. We then exposed mice to hypoxia using a standardised chamber and observed downregulation of hepatic hepcidin mRNA expression that was paralleled by elevated serum PDGF-BB protein concentrations and higher serum iron levels as compared with mice housed under normoxic conditions. PDGF-BB treatment in vitro and in vivo resulted in suppression of both steady state and BMP6 inducible hepcidin expression. Mechanistically, PDGF-BB inhibits hepcidin transcription by downregulating the protein expression of the transcription factors CREB and CREB-H, and pharmacological blockade or genetic ablation of these pathways abrogated the effects of PDGF-BB toward hepcidin expression. Conclusions Hypoxia decreases hepatic hepcidin expression by a novel regulatory pathway exerted via PDGF-BB, leading to increased availability of circulating iron that can be used for erythropoiesis.

Kupffer cells and macrophages are not required for hepatic hepcidin activation during iron overload

Hepcidin, the iron hormone, is produced by the liver in response to iron and inflammation. Its synthesis during inflammation is triggered by cytokines, but the details of iron activation are obscure. We tested the role of Kupffer cells and macrophages by studying iron‐loaded or inflamed mice with selective inactivation of Kupffer cells or the in vitro effect of conditioned human macrophages on hepcidin expression. Hepcidin messenger RNA (mRNA) expression was studied by Northern blot and reverse transcriptase polymerase chain reaction analysis in mice that were treated with 40 mg/kg gadolinium (III) chloride (GdCl3) as a Kupffer cell inactivating agent and subjected to inflammatory challenges with either lipopolysaccharide (LPS) and turpentine or iron overload by iron‐dextran administration. Similar analyses were performed in human hepatoma cells (HepG2) cultured with medium from LPS‐ or iron‐conditioned macrophages from blood donors or patients with HFE‐linked hereditary hemochromatosis (HH). In vivo, LPS and particularly turpentine stimulated hepcidin mRNA expression, and this effect was prevented by the inactivation of Kupffer cells. Also, iron overload markedly upregulated hepatic hepcidin mRNA, but this activity persisted in spite of Kupffer cell blockade. In vitro, the medium of LPS‐treated normal or hemocromatotic macrophages turned on hepcidin expression. On the contrary, medium of iron‐manipulated macrophages, regardless of their HFE status, did not affect hepcidin mRNA steady‐state levels. In conclusion, Kupffer cells are required for the activation of hepcidin synthesis during inflammation, and HH inflamed macrophages are capable of mounting a normal response, eventually leading to hepcidin stimulation. However, both Kupffer cells and human macrophages are dispensable for the regulatory activity exerted by iron on hepatic hepcidin. (HEPATOLOGY 2005;41:545–552.)

Gluconeogenic Signals Regulate Iron Homeostasis via Hepcidin in Mice

Background & Aims Hepatic gluconeogenesis provides fuel during starvation, and is abnormally induced in obese individuals or those with diabetes. Common metabolic disorders associated with active gluconeogenesis and insulin resistance (obesity, metabolic syndrome, diabetes, and nonalcoholic fatty liver disease) have been associated with alterations in iron homeostasis that disrupt insulin sensitivity and promote disease progression. We investigated whether gluconeogenic signals directly control Hepcidin, an important regulator of iron homeostasis, in starving mice (a model of persistently activated gluconeogenesis and insulin resistance). Methods We investigated hepatic regulation of Hepcidin expression in C57BL/6Crl, 129S2/SvPas, BALB/c, and Creb3l3–/– null mice. Mice were fed a standard, iron-balanced chow diet or an iron-deficient diet for 9 days before death, or for 7 days before a 24- to 48-hour starvation period; liver and spleen tissues then were collected and analyzed by quantitative reverse-transcription polymerase chain reaction and immunoblot analyses. Serum levels of iron, hemoglobin, Hepcidin, and glucose also were measured. We analyzed human hepatoma (HepG2) cells and mouse primary hepatocytes to study transcriptional control of Hamp (the gene that encodes Hepcidin) in response to gluconeogenic stimuli using small interfering RNA, luciferase promoter, and chromatin immunoprecipitation analyses. Results Starvation led to increased transcription of the gene that encodes phosphoenolpyruvate carboxykinase 1 (a protein involved in gluconeogenesis) in livers of mice, increased levels of Hepcidin, and degradation of Ferroportin, compared with nonstarved mice. These changes resulted in hypoferremia and iron retention in liver tissue. Livers of starved mice also had increased levels of Ppargc1a mRNA and Creb3l3 mRNA, which encode a transcriptional co-activator involved in energy metabolism and a liver-specific transcription factor, respectively. Glucagon and a cyclic adenosine monophosphate analog increased promoter activity and transcription of Hamp in cultured liver cells; levels of Hamp were reduced after administration of small interfering RNAs against Ppargc1a and Creb3l3. PPARGC1A and CREB3L3 bound the Hamp promoter to activate its transcription in response to a cyclic adenosine monophosphate analog. Creb3l3–/– mice did not up-regulate Hamp or become hypoferremic during starvation. Conclusions We identified a link between glucose and iron homeostasis, showing that Hepcidin is a gluconeogenic sensor in mice during starvation. This response is involved in hepatic metabolic adaptation to increased energy demands; it preserves tissue iron for vital activities during food withdrawal, but can cause excessive iron retention and hypoferremia in disorders with persistently activated gluconeogenesis and insulin resistance.

Huh‐7: A human “hemochromatotic” cell line

Hereditary hemochromatosis (HC) is commonly associated with homozygosity for the cysteine‐to‐tyrosine substitution at position 282 (C282Y) of the HFE protein. This mutation prevents HFE from binding beta2‐microglobulin (beta2M) and reaching the cell surface. We have discovered that a widely used hepatoma cell line, Huh‐7, carries a HFE mutation similar to that associated with human HC. By HFE gene sequencing of Huh‐7 genomic DNA, we found a TAC nucleotide deletion (c. 691_693del) responsible for loss of a tyrosine at position 231 (p. Y231del) of the HFE protein. This mutation affects a conserved hydrophobic region in a loop connecting two beta strands that make up the alpha3 domain of HFE, not far from the 282 site. HFE was detected by western blot in HepG2 but not in Huh‐7 cell membrane fractions. In WRL‐68 cells expressing wild‐type HFE, the HFE protein was largely found at the plasma membrane where it colocalizes with beta2M. On the contrary, the HFE‐Y231del mutant, similarly to an exogenously expressed HFE‐C282Y mutant, failed to reach the plasma membrane and did not colocalize with membrane‐expressed beta2M. C282Y mutant HFE in HC is associated with inadequate hepcidin expression. We found that Huh‐7 cells display lower hepcidin messenger RNA levels as compared to HepG2 cells, which carry a wild‐type HFE. Interestingly, hepcidin messenger RNA levels increased significantly in Huh‐7 cells stably expressing exogenous wild‐type HFE at the plasma membrane. Conclusion: Huh‐7 cells may represent a novel and valuable tool to investigate the role of altered HFE traffic in iron metabolism and pathogenesis of human HFE HC. (HEPATOLOGY 2010.)

HCV RNA in serum of asymptomatic blood donors involved in post-transfusion hepatitis (PTH)

A group of blood donors involved in post-transfusion hepatitis was investigated for the presence of the anti-HCV antibody and of HCV RNA as a more direct infection marker. RNA was extracted from serum, reverse transcribed and amplified using primers which belonged to the non structural region. The amplified product of the PCR reaction was 582 base pairs. Seven (25.9%) of the 27 blood donors examined were found anti-HCV-positive by ELISA; five (71.4%) of these were HCV RNA positive. Among the 20 anti-HCV-negative blood donors, four (20.0%) were HCV RNA positive. ALT levels were below 45 UI/l in 18 donors, while the other nine had ALTs over the limit accepted for transfusion. The anti-HCV-negative HCV RNA-positive blood donors had normal ALTs. Our study offers a direct explanation for the substantial proportion of residual cases of anti-HCV-positive post-transfusion hepatitis and suggests the necessity of creating a register of blood donors who have at some time presented blood enzyme abnormalities and for whom second level investigations such as HCV RNA should be used.

Behavior of Antibody Profile against Hepatitis C Virus in Patients on Maintenance Hemodialysis

The prevalence of anti-hepatitis C virus (HCV) in dialysis setting is still a nonstandard datum. In particular, it is not known of the phenomenon is stable or increasing or decreasing, even in a given geographical area. We studied the behavior of anti-HCV prevalence during a 12-month follow-up in 415 hemodialysis patients treated at a single institution and belonging to a limited geographical area with standard HCV endemic. Point prevalence of anti-HCV has shown a tendency to growth linked in part of the incidence of infection, in part to new positivities in patients already on dialysis treatment. More than 50% of the new HCV-positive patients, had no history of classical parenteral transmission of the virus. These findings suggest that HCV infection is a phenomenon on the increase in dialysis units and that dialysis treatment emerges as an independent risk factor in contracting infection.

Presence of HCV RNA in serum of asymptomatic blood donors involved in post-transfusion hepatitis (PTH).

To study the causes of residual posttransfusion hepatitis, serum from implicated donors was tested by PCR for the presence of HCV RNA. Of 20 anti HCV negative donors, 4 were HCV RNA positive and thus, infective. The results suggest that higher-level investigations are necessary for prospective donors who present blood enzyme abnormalities or other questionable characteristics.