Biomedical subjects

Martina Muckenthaler

Publications and source records attributed to Martina Muckenthaler.

9 recordsLinked to original sources

PubMed2005

Altered body iron distribution and microcytosis in mice deficient in iron regulatory protein 2 (IRP2).

Iron regulatory protein 2 (IRP2)-deficient mice have been reported to suffer from late-onset neurodegeneration by an unknown mechanism. We report that young adult Irp2-/- mice display signs of iron mismanagement within the central iron recycling pathway in the mammalian body, the liver-bone marrow-spleen axis, with altered body iron distribution and compromised hematopoiesis. In comparison with wild-type littermates, Irp2-/- mice are mildly microcytic with reduced serum hemoglobin levels and hematocrit. Serum iron and transferrin saturation are unchanged, and hence microcytosis is not due to an overt decrease in systemic iron availability. The liver and duodenum are iron loaded, while the spleen is iron deficient, associated with a reduced expression of the iron exporter ferroportin. A reduction in transferrin receptor 1 (TfR1) mRNA levels in the bone marrow of Irp2-/- mice can plausibly explain the microcytosis by an intrinsic defect in erythropoiesis due to a failure to adequately protect TfR1 mRNA against degradation. This study links a classic regulator of cellular iron metabolism to systemic iron homeostasis and erythropoietic TfR1 expression. Furthermore, this work uncovers aspects of mammalian iron metabolism that can or cannot be compensated for by the expression of IRP1.

Animals↗

PubMed2005

Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C Virus.

Clinical data suggest that iron is a negative factor in chronic hepatitis C; however, the molecular mechanisms by which iron modulates the infectious cycle of hepatitis C virus (HCV) remain elusive. To explore this, we utilized cells expressing a HCV replicon as a well-established model for viral replication. We demonstrate that iron administration dramatically inhibits the expression of viral proteins and RNA, without significantly affecting its translation or stability. Experiments with purified recombinant HCV RNA polymerase (NS5B) revealed that iron binds specifically and with high affinity (apparent Kd: 6 and 60 microM for Fe2+ and Fe3+, respectively) to the protein's Mg2+-binding pocket, thereby inhibiting its enzymatic activity. We propose that iron impairs HCV replication by inactivating NS5B and that its negative effects in chronic hepatitis C may be primarily due to attenuation of antiviral immune responses. Our data provide a direct molecular link between iron and HCV replication.

Carcinoma, Hepatocellular↗

PubMed2004

Impact of pre-analytical handling on bone marrow mRNA gene expression.

Large clinical trials on leukaemia, require the transport of bone marrow (BM) from participating clinics to central diagnostic laboratories. We have investigated the impact of RNA extraction protocols and time delays between sample aspiration and RNA extraction on RNA quality and gene expression profiles. Intact RNA can be extracted from BM samples stored at room temperature for up to 48 h. Gene expression analyses using Affymetrix U95Av2 GeneChips and a custom-designed cDNA array in parallel showed that even short-term storage of BM has dramatic effects on mRNA expression of individual transcripts. Many probe sets/genes showed either reproducible deregulation (18.8%, analysis of variance <0.05), or inconsistent expression that differed from patient to patient (38.4%). Moderate alterations were observed in 42.8% genes, with a maximum fold change <2.0 in all experiments and at all time points. These profound effects complicate the use of unstabilized, shipped BM samples for gene expression analyses. The comparison of a variety of RNA stabilization reagents (e.g. PAXgene) resulted in partial conservation of the mRNA expression patterns. Immediate density centrifugation or erythrocyte lysis and freezing at -80 degrees C represent simple procedures that reliably preserved mRNA gene expression patterns in BM.

Bone Marrow↗

PubMed2004

Iron-mediated degradation of IRP2, an unexpected pathway involving a 2-oxoglutarate-dependent oxygenase activity.

Iron regulatory protein 2 (IRP2), a central posttranscriptional regulator of cellular and systemic iron metabolism, undergoes proteasomal degradation in iron-replete cells. The prevailing model postulates that the mechanism involves site-specific oxidation of 3 cysteine residues (C168, C174, and C178) within a 73-amino-acid (73-aa) degradation domain. By expressing wild-type and mutated versions of IRP2 in H1299 cells, we find that a C168S C174S C178S triple mutant, or a deletion mutant lacking the entire "73-aa domain," is sensitive to iron-mediated degradation, like wild-type IRP2. The antioxidants N-acetylcysteine, ascorbate, and alpha-tocopherol not only fail to stabilize IRP2 but, furthermore, promote its proteasomal degradation. The pathway for IRP2 degradation is saturable, which may explain earlier data supporting the "cysteine oxidation model," and shows remarkable similarities with the degradation of the hypoxia-inducible factor 1 alpha (HIF-1 alpha): dimethyl-oxalylglycine, a specific inhibitor of 2-oxoglutarate-dependent oxygenases, stabilizes IRP2 following the administration of iron to iron-deficient cells. Our results challenge the current model for IRP2 regulation and provide direct pharmacological evidence for the involvement of 2-oxoglutarate-dependent oxygenases in a pathway for IRP2 degradation.

Animals↗

PubMed2003

Iron overload in adult Hfe-deficient mice independent of changes in the steady-state expression of the duodenal iron transporters DMT1 and Ireg1/ferroportin.

Patients suffering from hereditary hemochromatosis (HH) show progressive iron overload as a consequence of increased duodenal iron absorption. It has been hypothesized that mutations in the HH gene HFE cause misprogramming of the duodenal enterocytes towards a paradoxical iron-deficient state, resulting in increased iron transporter expression. Previous reports concerning gene expression levels of the duodenal iron transporters DMT1 and IREG1 in HH patients and animal models are controversial, however, and in many cases only mRNA expression levels were investigated. To analyze the duodenal expression of DMT1, Ireg1, Dcytb, and hephaestin and the association with iron overload in adult Hfe(-/-) mice, an Hfe(-/-) mouse line was generated. Duodenal DMT1 and Ireg1 protein levels, duodenal DMT1, Ireg1, Dcytb, hephaestin, and TfR1 mRNA levels, and hepatic hepcidin mRNA levels were quantified and the correlation to liver iron contents was calculated. We report that duodenal DMT1 and Ireg1 mRNA levels and DMT1 and Ireg1 protein levels remained unaffected by the Hfe deletion. Furthermore, duodenal hephaestin and TfR1 mRNA expression and hepatic hepcidin mRNA expression remained unaltered, while the duodenal mRNA expression of the brush border ferric reductase Dcytb was significantly increased in Hfe(-/-) mice. We found no correlation between the expression level of any of the analyzed transcripts and the liver iron content. In conclusion, the lack of correlation between DMT1 and Ireg1 protein expression and the liver iron content suggests that elevated duodenal iron transporter expression is not required for high liver iron overload. Hfe(-/-) mice do not necessarily display features of iron deficiency in the duodenum, indicated by an increase in mRNA and protein levels of DMT1 and Ireg1. Rather, the duodenal ferric reductase Dcytb may act as a possible mediator of iron overload in Hfe deficiency.

Animals↗

PubMed2003

Mouse brains deficient in H-ferritin have normal iron concentration but a protein profile of iron deficiency and increased evidence of oxidative stress.

Several neurodegenerative disorders such as Parkinson's Disease (PD) and Alzheimer's Disease (AD) are associated with elevated brain iron accumulation relative to the amount of ferritin, the intracellular iron storage protein. The accumulation of more iron than can be adequately stored in ferritin creates an environment of oxidative stress. We developed a heavy chain (H) ferritin null mutant in an attempt to mimic the iron milieu of the brain in AD and PD. Animals homozygous for the mutation die in utero but the heterozygotes (+/-) are viable. We examined heterozygous and wild-type (wt) mice between 6 and 8 months of age. Macroscopically, the brains of +/- mice were well formed and did not differ from control brains. There was no evidence of histopathology in the brains of the heterozygous mice. Iron levels in the brain of the +/- and wild-type (+/+) mice were similar, but +/- mice had less than half the levels of H-ferritin. The other iron management proteins transferrin, transferrin receptor, light chain ferritin, Divalent Metal Transporter 1, ceruloplasmin, were increased in the +/- mice compared to +/+ mice. The relative amounts of these proteins in relation to the iron concentration are similar to that found in AD and PD. Thus, we hypothesized that the brains of the heterozygote mice should have an increase in indices of oxidative stress. In support of this hypothesis, there was a decrease in total superoxide dismutase (SOD) activity in the heterozygotes coupled with an increase in oxidatively modified proteins. In addition, apoptotic markers Bax and caspase-3 were detected in neurons of the +/- mice but not in the wt. Thus, we have developed a mouse model that mimics the protein profile for iron management seen in AD and PD that also shows evidence of oxidative stress. These results suggest that this mouse may be a model to determine the role of iron mismanagement in neurodegenerative disorders and for testing antioxidant therapeutic strategies.

Alzheimer Disease↗

PubMed2003

Standardization of protocols in cDNA microarray analysis.

Systematic variations can occur at various steps of a cDNA microarray experiment and affect the measurement of gene expression levels. Accepted standards integrated into every cDNA microarray analysis can assess these variabilities and aid the interpretation of cDNA microarray experiments from different sources. A universally applicable approach to evaluate parameters such as input and output ratios, signal linearity, hybridization specificity and consistency across an array, as well as normalization strategies, is the utilization of exogenous control genes as spike-in and negative controls. We suggest that the use of such control sets, together with a sufficient number of experimental repeats, in-depth statistical analysis and thorough data validation should be made mandatory for the publication of cDNA microarray data.

DNA, Complementary↗

PubMed2003

Regulatory defects in liver and intestine implicate abnormal hepcidin and Cybrd1 expression in mouse hemochromatosis.

Individuals with hereditary hemochromatosis suffer from systemic iron overload due to duodenal hyperabsorption. Most cases arise from a founder mutation in HFE (845G-->A; ref. 2) that results in the amino-acid substitution C282Y and prevents the association of HFE with beta2-microglobulin. Mice homozygous with respect to a null allele of Hfe (Hfe-/-) or homozygous with respect to the orthologous 882G-->A mutation (Hfe(845A/845A)) develop iron overload that recapitulates hereditary hemochromatosis in humans, confirming that hereditary hemochromatosis arises from loss of HFE function. Much work has focused on an exclusive role for the intestine in hereditary hemochromatosis. HFE deficiency in intestinal crypt cells is thought to cause intestinal iron deficiency and greater expression of iron transporters such as SLC11A2 (also called DMT1, DCT1 and NRAMP2) and SLC11A3 (also called IREG1, ferroportin and MTP1; ref. 3). Published data on the expression of these transporters in the duodenum of HFE-deficient mice and humans are contradictory. In this report, we used a custom microarray to assay changes in duodenal and hepatic gene expression in Hfe-deficient mice. We found unexpected alterations in the expression of Slc39a1 (mouse ortholog of SLC11A3) and Cybrd1, which encode key iron transport proteins, and Hamp (hepcidin antimicrobial peptide), a hepatic regulator of iron transport. We propose that inappropriate regulatory cues from the liver underlie greater duodenal iron absorption, possibly involving the ferric reductase Cybrd1.

Animals↗

PubMed2002

Relationships and distinctions in iron-regulatory networks responding to interrelated signals.

Specialized cDNA-based microarrays (IronChips) were developed to investigate complex physiological gene-regulatory patterns in iron metabolism. Approximately 115 human cDNAs were strategically selected to represent genes involved either in iron metabolism or in interlinked pathways (eg, oxidative stress, nitric oxide [NO] metabolism, or copper metabolism), and were immobilized on glass slides. HeLa cells were treated with iron donors or iron chelators, or were subjected to oxidative stress (H(2)O(2)) or NO (sodium nitroprusside). In addition, we generated a stable transgenic HeLa cell line expressing the HFE gene under an inducible promoter. Gene-response patterns were recorded for all of these interrelated experimental stimuli, and analyzed for common and distinct responses that define signal-specific regulatory patterns. The resulting regulatory patterns reveal and define degrees of relationship between distinct signals. Remarkably, the gene responses elicited by the altered expression of the hemochromatosis protein HFE and by pharmacological iron chelation exhibit the highest degree of relatedness, both for iron-regulatory protein (IRP) and non-IRP target genes. This finding suggests that HFE expression directly affects the intracellular chelatable iron pool in the transgenic cell line. Furthermore, cells treated with the iron donors hemin or ferric ammonium citrate display response patterns that permit the identification of the iron-loaded state in both cases, and the discrimination between the sources of iron loading. These findings also demonstrate the broad utility of gene-expression profiling with the IronChip to study iron metabolism and related human diseases.

DNA, Complementary↗