Patricia Leroyer

A New Mouse Liver-specific Gene, Encoding a Protein Homologous to Human Antimicrobial Peptide Hepcidin, Is Overexpressed during Iron Overload

Considering that the development of hepatic lesions related to iron overload diseases might be a result of abnormally expressed hepatic genes, we searched for new genes up-regulated under the condition of iron excess. By suppressive subtractive hybridization performed between livers from carbonyl iron-overloaded and control mice, we isolated a 225-base pair cDNA. By Northern blot analysis, the corresponding mRNA was confirmed to be overexpressed in livers of experimentally (carbonyl iron and iron-dextran-treated mice) and spontaneously (β2-microglobulin knockout mice) iron-overloaded mice. In addition, β2-microglobulin knockout mice fed with a low iron content diet exhibited a decrease of hepatic mRNA expression. The murine full-length cDNA was isolated and was found to encode an 83-amino acid protein presenting a strong homology in its C-terminal region to the human antimicrobial peptide hepcidin. In addition, we cloned the corresponding rat and human orthologue cDNAs. Both mouse and human genes named HEPC are constituted of 3 exons and 2 introns and are located on chromosome 7 and 19, respectively, in close proximity toUSF2 gene. In mouse and human, HEPC mRNA was predominantly expressed in the liver. During both in vivo and in vitro studies, HEPC mRNA expression was enhanced in mouse hepatocytes under the effect of lipopolysaccharide. Finally, to analyze the intracellular localization of the predicted protein, we used the green fluorescent protein chimera expression vectors. The murine green fluorescent protein-prohepcidin protein was exclusively localized in the nucleus. When the putative nuclear localization signal was deleted, the resulting protein was addressed to the cytoplasm. Taken together, our data strongly suggest that the product of the new liver-specific gene HEPCmight play a specific role during iron overload and exhibit additional functions distinct from its antimicrobial activity.

Comparative analysis of mouse hepcidin 1 and 2 genes: evidence for different patterns of expression and co-inducibility during iron overload1

In contrast to the human genome, the mouse genome contains two HEPC genes encoding hepcidin, a key regulator of iron homeostasis. Here we report a comparative analysis of sequence, genomic structure, expression and iron regulation of mouse HEPC genes. The predicted processed 25 amino acid hepcidin 2 peptide share 68% identity with hepcidin 1 with perfect conservation of eight cysteine residues. Both HEPC1 and HEPC2 genes have similar genomic organization and have probably arisen from a recent duplication of chromosome 7 region, including the HEPC ancestral gene and a part of the adjacent USF2 gene. Insertion of a retroviral intracisternal A‐particle element was found upstream of the HEPC1 gene. Both genes are highly expressed in the liver and to a much lesser extent in the heart. In contrast to HEPC1, a high amount of HEPC2 transcripts was detected in the pancreas. Expression of both genes was increased in the liver during carbonyl–iron and iron–dextran overload. Overall our data suggest that both HEPC1 and HEPC2 genes are involved in iron metabolism regulation but could exhibit different activities and/or play distinct roles.

Metabolic imaging of tissues by infrared fiber-optic spectroscopy: an efficient tool for medical diagnosis

Infrared fingerprints of molecules in biology contain much information on cells metabolism allowing one to distinguish between healthy and altered tissues. Here, to collect infrared signatures, we used evanescent wave spectroscopy based on an original infrared transmitting tapered glass fiber. A strict control of the fiber diameter in the tapered sensing zone allows high sensitivity and wide spectral range exploration from 800 to 3000 cm(-1). Then, merely in depositing the mouse liver biopsies on the fiber, this device has enable us to differentiate between tumorous and healthy tissues.

Chalcogenide glass fibers used as biosensors

The development of a new generation of chalcogenide infrared glass fibers allows making an IR sensor that permits recording the fingerprints of biomolecules in the mid infrared (MIR) range. The measurements are based on the general concept of evanescent wave spectroscopy. To improve the detection, the diameter of the fiber is locally reduced. To test this optical sensor, we measured metabolic anomalies in relation with hepatic pathologies. Mouse liver tissues have been used and MIR spectra have been recorded by a mere contact between tissues and the surface of the fiber. Spectral differences reflect metabolic alterations, these can be identified and assigned. Furthermore, histologic studies confirm these results.

Iron may induce both DNA synthesis and repair in rat hepatocytes stimulated by EGF/pyruvate

BACKGROUND/AIMS Hepatocellular carcinoma develops frequently in the course of genetic hemochromatosis, and a role of iron overload in hepatic carcinogenesis is strongly suggested. METHODS The aim of our study was to investigate the effect of iron exposure on DNA synthesis of adult rat hepatocytes maintained in primary culture stimulated or not by EGF/pyruvate and exposed to iron-citrate complex. RESULTS In EGF/pyruvate-stimulated cultures, the level of [3H] methyl thymidine incorporation was strongly increased as compared to unstimulated cultures. The addition of iron to stimulated cultures increased [3H] methyl thymidine incorporation. The mitotic index was also significantly higher at 72 h. However, the number of cells found in the cell layer was not significantly different from iron-citrate free culture. By flow cytometry, no difference in cell ploidy was found between iron-treated and untreated EGF/pyruvate-stimulated cultures. A significant increase in LDH leakage reflecting a toxic effect of iron was found in the cell medium 48 h after cell seeding. In addition, [3H] methyl thymidine incorporation in the presence of hydroxyurea was increased in iron-treated compared to untreated cultures. CONCLUSIONS Our results show that DNA synthesis is increased in the presence of iron in rat hepatocyte cultures stimulated by EGF/pyruvate, and they suggest that DNA synthesis is likely to be related both to cell proliferation and to DNA repair. These observations may allow better understanding of the role of iron overload in the development of hepatocellular carcinoma.

Regulation of ferritin and transferrin receptor expression by iron in human hepatocyte cultures.

HepG2 cell cultures and human hepatocyte primary cultures were used to develop appropriate hepatocytic in vitro models of iron load in order to further understand the pathophysiological mechanisms occurring in the liver of patients with hemochromatosis. The first step of this study was to obtain an efficient iron supply in conditions of minimal toxicity. It was demonstrated that iron complexed to citrate entered efficiently into HepG2 cells and human hepatocytes. This iron load was obtained with minimal toxicity in both culture models as evaluated by the intracellular LDH activity and the total protein content. The second step was to study the effect of iron on ferritin and transferrin receptor expression. In HepG2 cell cultures, intracellular and extracellular ferritin concentrations were strikingly increased by iron in dose- and time-dependent manners. However, the relative amounts of H and L ferritin mRNAs were not significantly affected by iron, suggesting that ferritin regulation occurred at a translational level. On the other hand, in human hepatocyte cultures, the increase of intracellular and extracellular ferritin concentrations was accompanied by an increase in the amounts of H and L ferritin mRNAs. In this model, iron-induced ferritin biosynthesis seemed to be more complex than in HepG2 cells and to be governed by transcriptional and/or post-transcriptional regulatory mechanisms. However, an additional translational level of regulation could not be excluded. In contrast, transferrin receptor expression was decreased by iron in HepG2 cells as well as in human hepatocyte cultures. This decrease was associated with a decrease in the mRNA steady-state level. In both culture models, transferrin receptor regulation seemed to occur at a transcriptional or post-transcriptional level. These results demonstrate that normal human hepatocytes in primary culture respond to iron in a manner close to that observed in vivo and thereby provide a promising experimental model for further understanding pathophysiological mechanisms involved in human hemochromatotic liver.

Iron excess limits HHIPL-2 gene expression and decreases osteoblastic activity in human MG-63 cells

SummaryIn order to understand mechanisms involved in osteoporosis observed during iron overload diseases, we analyzed the impact of iron on a human osteoblast-like cell line. Iron exposure decreases osteoblast phenotype. HHIPL-2 is an iron-modulated gene which could contribute to these alterations. Our results suggest osteoblast impairment in iron-related osteoporosis.IntroductionIron overload may cause osteoporosis. An iron-related decrease in osteoblast activity has been suggested.MethodsWe investigated the effect of iron exposure on human osteoblast cells (MG-63) by analyzing the impact of ferric ammonium citrate (FAC) and iron citrate (FeCi) on the expression of genes involved in iron metabolism or associated with osteoblast phenotype. A transcriptomic analysis was performed to identify iron-modulated genes.ResultsFAC and FeCi exposure modulated cellular iron status with a decrease in TFRC mRNA level and an increase in intracellular ferritin level. FAC increased ROS level and caspase 3 activity. Ferroportin, HFE and TFR2 mRNAs were expressed in MG-63 cells under basal conditions. The level of ferroportin mRNA was increased by iron, whereas HFE mRNA level was decreased. The level of mRNA alpha 1 collagen type I chain, osteocalcin and the transcriptional factor RUNX2 were decreased by iron. Transcriptomic analysis revealed that the mRNA level of HedgeHog Interacting Protein Like-2 (HHIPL-2) gene, encoding an inhibitor of the hedgehog signaling pathway, was decreased in the presence of FAC. Specific inhibition of HHIPL-2 expression decreased osteoblast marker mRNA levels. Purmorphamine, hedgehog pathway activator, increased the mRNA level of GLI1, a target gene for the hedgehog pathway, and decreased osteoblast marker levels. GLI1 mRNA level was increased under iron exposure.ConclusionWe showed that in human MG-63 cells, iron exposure impacts iron metabolism and osteoblast gene expression. HHIPL-2 gene expression modulation may contribute to these alterations. Our results support a role of osteoblast impairment in iron-related osteoporosis.

Ferroportin Diseases: Functional Studies, a Link Between Genetic and Clinical Phenotype

Ferroportin (FPN) mediates iron export from cells and this function is modulated by serum hepcidin. Mutations in the FPN gene (SLC40A1) lead to autosomal dominant iron overload diseases related either to loss or to gain of function, and usually characterized by normal or low transferrin saturation versus elevated transferrin saturation, respectively. However, for the same mutation, the phenotypic expression may vary from one patient to another. Using in vitro overexpression of wild‐type or mutant FPN proteins, we characterized the functional impact of five recently identified FPN gene mutations regarding FPN localization, cell iron status, and hepcidin sensitivity. Our aim was to integrate functional results and biological findings in probands and relatives. We show that while the p.Arg371Gln (R371Q) mutation had no impact on studied parameters, the p.Trp158Leu (W158L), p.Arg88Gly (R88G), and p.Asn185Asp (N185D) mutations caused an iron export defect and were classified as loss‐of‐function mutations. The p.Gly204Ser (G204S) mutation induced a gain of FPN function. Functional studies are useful to determine whether or not a FPN gene mutation found in an iron overloaded patient is deleterious and to characterize its biological impact, especially when family studies are not fully informative and/or additional confounding factors may affect bio‐clinical expression.

Stearoyl coenzyme A desaturase 1 expression and activity are increased in the liver during iron overload

In humans, hepatic iron overload can lead to hepatocellular carcinoma development. Iron related dysregulation of hepatic genes could play a role in this phenomenon. We previously found that the carbonyl-iron overloaded mouse was a useful model to study the mechanisms involved in the development of hepatic lesions related to iron excess. The aim of the present study was to identify hepatic genes overexpressed in conditions of iron overload by using this model. A suppressive subtractive hybridization was performed between hepatic mRNAs extracted from control and 3% carbonyl-iron overloaded mice during 8 months. This methodology allowed us to identify stearoyl coenzyme A desaturase 1 (SCD1) mRNA overexpression in the liver of iron loaded mice. The corresponding enzymatic activity was also found to be significantly increased. In addition, we demonstrated that both SCD1 mRNA expression and activity were increased in another iron overload model in mice obtained by a single iron-dextran subcutaneous injection. Moreover, we found, in both models, that SCD1 mRNA was not only influenced by the quantity of iron in the liver but also by the duration of iron overload since SCD1 mRNA upregulation was not detected in earlier stages of iron overload. In addition, we found that cellular repartition likely influenced SCD1 mRNA expression. In conclusion, we demonstrated that iron excess in the liver induced both the expression of SCD1 mRNA and its corresponding enzymatic activity. The level and duration of iron overload, as well as cellular repartition of iron excess in the liver likely play a role in this induction. The fact that the expression and activity of SCD1, an enzyme adding a double bound into saturated fatty acids, are induced in two models of iron overload in mice leads to the conclusion that iron excess in the liver may enhance the biosynthesis of unsaturated fatty acids.

Regulation of ferritin expression by alcohol in a human hepatoblastoma cell line and in rat hepatocyte cultures

Serum ferritin increases in chronic alcoholism, without clear explanation. We have previously shown that alcohol increases ferritin levels in a human hepatoblastoma cell line (HepG2). The aims of the present work were: 1) To extend our results in normal rat hepatocyte cultures, and 2) To determine the mechanism by which alcohol enhances ferritin levels. In HepG2 cells, high alcohol concentrations (300 mM) during long exposure (4 days) increased the synthesis of H and L ferritin subunits, in association with increased levels of ferritin mRNAs. In rat hepatocyte cultures, the synthesis of L ferritin increased after 24 h of exposure to lower alcohol concentrations (10 mM); alcohol had no effect on ferritin mRNAs levels. In both cell types, the alcohol effect was not related to an increase in iron intracellular incorporation. In HepG2 cells, desferrioxamine (Df), a potent iron chelator, abolished ferritin synthesis in the presence or absence of alcohol, and abolished the alcohol induction of ferritin mRNAs. In rat hepatocytes, Df decreased ferritin synthesis to a similar level in the presence or absence of alcohol. Alcohol increased ferritin synthesis differently in HepG2 cells and in normal rat hepatocyte cultures. In the latter case, the alcohol effect was observed at low concentration. Despite a striking inhibiting effect of Df on ferritin synthesis, in both cellular models a mechanism accounting for increased ferritin synthesis independently of iron is suggested. Globally, these data strongly suggest that hyperferritinemia in chronic alcoholism could be related to the induction of ferritin by alcohol.