The hypoferremic response to acute inflammation is maintained in thalassemia mice even under parenteral iron loading

Background Hepcidin controls iron homeostasis by inducing the degradation of the iron efflux protein, ferroportin (FPN1), and subsequently reducing serum iron levels. Hepcidin expression is influenced by multiple factors, including iron stores, ineffective erythropoiesis, and inflammation. However, the interactions between these factors under thalassemic condition remain unclear. This study aimed to determine the hypoferremic and transcriptional responses of iron homeostasis to acute inflammatory induction by lipopolysaccharide (LPS) in thalassemic (Hbbth3/+) mice with/without parenteral iron loading with iron dextran. Methods Wild type and Hbbth3/+ mice were intramuscularly injected with 5 mg of iron dextran once daily for two consecutive days. After a 2-week equilibration, acute inflammation was induced by an intraperitoneal injection of a single dose of 1 µg/g body weight of LPS. Control groups for both iron loading and acute inflammation received equal volume(s) of saline solution. Blood and tissue samples were collected at 6 hours after LPS (or saline) injection. Iron parameters and mRNA expression of hepcidin as well as genes involved in iron transport and metabolism in wild type and Hbbth3/+ mice were analyzed and compared by Kruskal–Wallis test with pairwise Mann–Whitney U test. Results We found the inductive effects of LPS on liver IL-6 mRNA expression to be more pronounced under parenteral iron loading. Upon LPS administration, splenic erythroferrone (ERFE) mRNA levels were reduced only in iron-treated mice, whereas, liver bone morphogenetic protein 6 (BMP6) mRNA levels were decreased under both control and parenteral iron loading conditions. Despite the altered expression of the aforementioned hepcidin regulators, the stimulatory effect of LPS on hepcidin mRNA expression was blunt in iron-treated Hbbth3/+ mice. Contrary to the blunted hepcidin response, LPS treatment suppressed FPN1 mRNA expression in the liver, spleen, and duodenum, as well as reduced serum iron levels of Hbbth3/+ mice with parenteral iron loading. Conclusion Our study suggests that a hypoferremic response to LPS-induced acute inflammation is maintained in thalassemic mice with parenteral iron loading in a hepcidin-independent manner.

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Tmprss6, An Inhibitor of Hepatic Bmp/Smad Signaling, Is Required for Hepcidin Suppression and Iron Loading In a Mouse Model of β-Thalassemia

Blood ◽

10.1182/blood.v116.21.164.164 ◽

2010 ◽

Vol 116 (21) ◽

pp. 164-164

Author(s):

Karin E. Finberg ◽

Rebecca L. Whittlesey ◽

Stefano Rivella ◽

Nancy C. Andrews

Keyword(s):

Iron Deficiency ◽

Iron Homeostasis ◽

Heme Iron ◽

Mrna Levels ◽

Iron Loading ◽

Wild Type ◽

Smad Signaling ◽

Hepcidin Expression ◽

Non Heme Iron ◽

Hepcidin Mrna

Abstract Abstract 164 TMPRSS6, a transmembrane protease produced by the liver, is an essential regulator of mammalian iron homeostasis. TMPRSS6 inhibits the expression of hepcidin, a circulating peptide that decreases intestinal iron absorption and macrophage iron release, by down-regulating hepatic BMP/SMAD signaling for hepcidin production. Accordingly, TMPRSS6 mutations result in elevated hepcidin levels, impaired absorption of dietary iron, and systemic iron deficiency. Interestingly, in congenital iron loading anemias such as β-thalassemia, hepcidin levels are inappropriately low relative to body iron stores, a finding that has been postulated to result from the production of a hepcidin-repressing factor in the setting of ineffective erythropoiesis. Here we asked if Tmprss6 is required to achieve the hepcidin suppression present in Hbbth3/+ mice, a model of β-thalassemia intermedia. To test this, we bred Hbbth3/+ mice to mice harboring a targeted disruption of the Tmprss6 serine protease domain. We generated mice of various Hbb-Tmprss6 genotype combinations and compared parameters of systemic iron homeostasis at 8 weeks of age. Consistent with prior studies of Hbbth3/+ mice, Hbbth3/+ mice harboring 2 wild-type Tmprss6 alleles (Hbbth3/+Tmprss6+/+ mice) showed non-heme iron concentrations of liver, spleen, and kidney that were significantly elevated compared to wild-type controls. Homozygosity for Tmprss6 mutation, however, ameliorated the iron overload phenotype of Hbbth3/+ mice and led to systemic iron deficiency. Tissue non-heme iron concentrations were markedly reduced in Hbbth3/+Tmprss6−/− mice as compared to Hbbth3/+Tmprss6+/+ mice and were similar to levels observed in Tmprss6−/− mice harboring 2 wild-type Hbb alleles. Hbbth3/+Tmprss6−/− mice had hemoglobin levels similar to the thalassemic levels present in Hbbth3/+Tmprss6+/+ mice. However, compared to Hbbth3/+Tmprss6+/+ mice, Hbbth3/+Tmprss6−/− mice showed markedly reduced erythrocyte mean corpuscular volume and serum transferrin saturation, as well as increased red blood cell count. Interestingly, homozygous loss of Tmprss6 in Hbbth3/+ mice also led to marked reduction in splenomegaly and improvement in peripheral red blood cell morphology. Consistent with prior studies of Hbbth3/+ mice, Hbbth3/+Tmprss6+/+ mice displayed hepatic hepcidin mRNA levels that were similar to wild-type and were, therefore, inappropriately decreased relative to their increased hepatic iron stores. Hepatic mRNA levels of Bmp6, encoding a Bmp ligand that is transcriptionally regulated by iron and acts as a key regulator of hepcidin expression in vivo, were significantly elevated in Hbbth3/+Tmprss6+/+ mice, suggesting that their relative hepcidin deficiency does not result from impaired Bmp6 transcription. While Hbbth3/+Tmprss6+/+ mice showed suppressed hepcidin levels, homozygous loss of Tmprss6 alleviated their hepcidin suppression and led to elevated hepcidin mRNA levels similar to Tmprss6−/− mice harboring 2 wild-type Hbb alleles. Hbbth3/+Tmprss6−/− mice also showed elevated hepatic mRNA encoding Id1, another transcriptional target of Bmp/Smad signaling. These findings indicate that Tmprss6 is required to achieve the suppression of hepatic hepcidin expression that underlies systemic iron overload in Hbbth3/+ mice. Furthermore, our results demonstrate that, by up-regulating hepatic Bmp/Smad signaling for hepcidin production, genetic loss of Tmprss6 induces profound changes in systemic iron homeostasis in this thalassemia model. Disclosures: No relevant conflicts of interest to declare.

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Hepcidin inhibition improves iron homeostasis in ferrous sulfate and LPS treatment model in mice

Drug Research ◽

10.1055/a-1542-8531 ◽

2021 ◽

Author(s):

Vishal Patel ◽

Amit Joharapurkar ◽

Samadhan Kshirsagar ◽

Maulik Patel ◽

Hiren Patel ◽

...

Keyword(s):

Ferrous Sulfate ◽

Serum Iron ◽

Iron Homeostasis ◽

The Body ◽

Rapid Screening ◽

Iron Dextran ◽

Liver Iron ◽

Hepcidin Expression ◽

Serum Hepcidin ◽

Lps Treatment

Abstract Background Hepcidin, a liver-derived peptide, regulates the absorption, distribution, and circulation of iron in the body. Inflammation or iron overload stimulates hepcidin release, which causes the accumulation of iron in tissues. The inadequate levels of iron in circulation impair erythropoiesis. Inhibition of hepcidin may increase iron in circulation and improve efficient erythropoiesis. Activin-like kinase (ALK) inhibitors decrease hepcidin. Methods In this work, we have investigated an ALK inhibitor LDN193189 for its efficacy in iron homeostasis. The effect of LDN193189 treatment was assessed in C57BL6/J mice, in which hepcidin was induced by either ferrous sulfate or lipopolysaccharide (LPS) injection. Results After two hours of treatment, ferrous sulfate increased serum and liver iron, serum hepcidin, and liver hepcidin expression. On the other hand, LPS reduced serum iron in a dose-related manner after six hours of treatment. LDN193189 treatment increased serum iron, decreased spleen and liver iron, decreased serum hepcidin and liver hepcidin expression in ferrous sulfate-treated mice, and increased serum iron in LPS-induced hypoferremia. We observed that ferrous sulfate caused a significantly higher increase in liver iron, serum hepcidin, and liver hepcidin than turpentine oil or LPS in mice. Iron dextran (intraperitoneal or intravenous) increased serum iron, but LDN193189 did not show hyperferremia with iron dextran stimulus. Conclusion Ferrous sulfate-induced hyperferremia can be a valuable and rapid screening model for assessing the efficacy of hepcidin inhibitors.

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Expression of Ara-C Metabolizing Enzymes Mediates in Vitro Sensitivity to Ara-C in Primary AML Cells From Patients with Denovo AML,

Blood ◽

10.1182/blood.v118.21.3481.3481 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3481-3481

Author(s):

Ajay Abraham ◽

Savitha Varatharajan ◽

Ashok kumar Jayavelu ◽

Shaji R Velayudhan ◽

Rayaz Ahmed ◽

...

Keyword(s):

Mrna Expression ◽

Candidate Genes ◽

P Value ◽

Mrna Levels ◽

Wild Type ◽

Mutation Status ◽

Expression Levels ◽

In Vitro Sensitivity ◽

Mrna Expression Levels

Abstract Abstract 3481 Wide inter-individual variation in terms of treatment outcome and toxic side effects of treatment exist among patients with AML receiving chemotherapy with cytarabine (ara-C) and daunorubicin. The pre-requisite for the cytotoxic action of pro-drug Ara-C is the enzymatic conversion to its active tri-phosphorylated form ara-CTP. Many drug activating (Deoxycytidine kinase (dCK) and human Equilibrative Nucleoside Transporter 1 (hENT1) and deactivating (Cytidine deaminase (CDA), 5'nucleotidase (NT5C2) genes and ribonucleoside reductase (RRM1), which are involved in transport and biotransformation of cytarabine contribute to the variation in ara-C sensitivity in AML patients. FLT3-ITD and NPM1 mutations act as major poor and good prognostic markers respectively in cytogenetically normal AML. The effect of these mutations in ara-C metabolism remains to be elucidated. The present study aims to determine independent as well as the combined effect of ara-C metabolizing genes mRNA expression on in-vitro ara-C cytotoxicity and the role of FLT3-ITD and NPM mutations on mRNA expression of these genes. Diagnostic bone marrow sample (median blasts 65%; range 21 – 98%) from 98 adult patients with de novo AML (other than AML-M3) were included in this study. mRNA expression levels for each target gene relative to housekeeping gene GAPDH was analyzed using Taqman based gene expression assays. In vitro cytotoxicity was assessed using MTT cell viability assay and IC-50 was calculated. In vitro sensitivity or resistance was classified on the basis of the IC-50 values <6uM and >6uM ara-C respectively. FLT3 ITD and NPM mutation status at diagnosis were determined through PCR followed by Genescan analysis using genomic DNA samples. Type of NPM mutation was identified by sequencing. When ara-C IC-50 values were compared with the mRNA expression levels of these candidate genes, Ara-C sensitive samples (n= 30; IC-50 < 6uM) showed significantly higher mRNA expression of dCK and hENT1 compared to those with Ara-C resistance (n=51) IC50 >6uM (median 314 (61.56 – 1232) vs. 180 (31.87 – 749.2); p = 0.0004 and median 172.1 (44.12 – 657.6) vs. 96.19 (37.49 – 432.4), p= 0.0008 respectively. RRM1 and NT5C2 did not show any association with in vitro Ara-C cytotoxicity, while CDA showed a trend towards association with lower CDA expression in ara-C sensitive samples. Based on these findings we put forward Ara-C resistance index (RI). RI is calculated by the formula RI = ΔCT (dCK X ENT1)/ ΔCT CDA. (Smaller ΔCT value= higher mRNA expression). RI values were significantly higher in resistant (IC50 >6uM) compared to sensitive cells (median: 6.084; range 1.89–11.82) vs. 3.702 (1.89–9.80); p=<0.0001). This association should now be validated in an independent cohort. Effects of NPM and FLT3 mutation status on Ara-C metabolizing genes were then evaluated. No significant association was found between FLT3-ITD status and the mRNA expression of these candidate genes. Interestingly, dCK mRNA levels were significantly higher in samples with NPM mutation (n=39) compared to NPM wild type (n=59); median 272.3 (41.64–1232) vs. 188.6 (31.87–1030); p value= 0.01. When analysed separately, patients with NPM type A mutation (n=27) showed significantly higher dCK expression (median 347.4 (41.64–1232) vs. 188.6 (31.87–1030); p value= 0.003 compared to those with wild type NPM1. This first report showing an association between expression profiles of ara-C metabolizing genes and NPM mutation should form the basis for evaluating their clinical correlations. Disclosures: No relevant conflicts of interest to declare.

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SRC-homology 2 domain-containing phosphatase 2 (SHP2) in epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma (LUAD).

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.9540 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. 9540-9540

Author(s):

Rafael Rosell ◽

Masaoki Ito ◽

Jordi Codony-Servat ◽

Ana Giménez-Capitán ◽

Mireia Serra-Mitjans ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Mrna Expression ◽

Mrna Levels ◽

Wild Type ◽

Src Homology 2 ◽

Src Homology ◽

Epidermal Growth ◽

Src Homology 2 Domain ◽

Egfr Mutant

9540 Background: Epidermal growth factor (EGFR)-mutant lung adenocarcinomas (LUADs) display impaired phosphorylation of extracellular signal-regulated kinase (ERK) and SRC-homology 2 domain-containing phosphatase 2 (SHP2) in comparison with EGFR wild-type LUADs. However, the function of SHP2 in early EGFR-mutant LUADs and EGFR wild-type LUADs has not been reported. We posit that SHP2 mRNA expression could be a predictive marker in resected EGFR-mutant LUADs versus EGFR wild-type patients (pts). Methods: We examined 267 resected LUADs from Japan and Spain. mRNA expression levels of AXL, MET, CDCP1, STAT3, YAP1 and SHP2 were analyzed by quantitative reverse transcriptase polymerase chain reaction (PCR). EGFR mutant cell lines were investigated for their activity of SHP2. Results: Among the 267 enrolled pts, 100 (37.3%) were EGFR-mutant LUADs. Five-year recurrence-free survival (RFS) and overall survival (OS) were lower for EGFR-mutant LUADs with high SHP2 mRNA levels (hazard ratio = 1.83 and 2.28, respectively. p = 0.03 and p = 0.04). However, SHP2 was not associated with RFS nor OS in the 167 wild-type EGFR LUADs. In EGFR-mutant cells, RMC-4550 (SHP2 inhibitor) plus erlotinib showed synergism via inhibition of AKT (S473) and ERK1/2 (T202/Y204). While erlotinib translocates SHP2 (Y542) into the nucleus, either RMC-4550 alone, or in combination with erlotinib, relocalizes SHP2 into the cytoplasm membrane, limiting AKT and ERK activation. Conclusions: High SHP2 mRNA is related to shorter RFS and OS in EGFR-mutant LUADs, but not in EGFR wild-type LUADs. The findings indicate that the addition of SHP2 inhibitors could improve adjuvant therapy in EGFR-mutant LUADs.

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C-Abl Is Required for the Development of Hyperoxia-Induced Retinopathy

Journal of Experimental Medicine ◽

10.1084/jem.193.12.1383 ◽

2001 ◽

Vol 193 (12) ◽

pp. 1383-1392 ◽

Cited By ~ 10

Author(s):

Irene Nunes ◽

Rosemary D. Higgins ◽

Lucia Zanetta ◽

Peter Shamamian ◽

Stephen P. Goff

Keyword(s):

Mrna Expression ◽

Retinal Neovascularization ◽

Mrna Levels ◽

Vascular Endothelial ◽

Wild Type ◽

Vegf Mrna ◽

Nonreceptor Tyrosine Kinase ◽

Kinase C ◽

Endothelial Growth Factor ◽

Inspired Oxygen

The requirement for the nonreceptor tyrosine kinase c-abl in the pathogenesis of retinopathy of prematurity (ROP) was examined using the mouse model for ROP and c-abl–deficient mice. Hyperoxia-induced retinal neovascularization was observed in wild-type and heterozygous mice but animals that were homozygous null for c-abl did not develop a vasoproliferative retinopathy in response to hyperoxia. Two gene products, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF), have been implicated in the pathogenesis of ROP. The mRNA expression of ET-1 and VEGF was assessed in mice maintained in normoxia and in hyperoxia-exposed mice. ET-1 mRNA levels were unchanged in wild-type mice throughout the hyperoxia treatment, suggesting that ET-1 mRNA expression is not regulated by the increase in inspired oxygen. In wild-type mice maintained in room air, VEGF mRNA levels rose threefold from postnatal day 6 (P6) to P17. When wild-type mice were treated with the hyperoxia regimen, a fivefold decrease in VEGF mRNA expression was observed from P7 to P16. However, retinal VEGF expression in hyperoxia-treated homozygous null mice did not decrease and remained at control levels. These data suggest that c-abl is required for the hyperoxia-induced retinal neovascularization and hyperoxia-induced decrease in VEGF mRNA levels.

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Blunted hepcidin response to inflammation in the absence of Hfe and transferrin receptor 2

Blood ◽

10.1182/blood-2010-08-303859 ◽

2011 ◽

Vol 117 (10) ◽

pp. 2960-2966 ◽

Cited By ~ 18

Author(s):

Daniel F. Wallace ◽

Cameron J. McDonald ◽

Lesa Ostini ◽

V. Nathan Subramaniam

Keyword(s):

Transferrin Receptor ◽

Serum Iron ◽

Iron Homeostasis ◽

Hereditary Hemochromatosis ◽

Homeostatic Regulation ◽

Wild Type ◽

Regulatory Peptide ◽

Basal Expression ◽

Low Levels ◽

Transferrin Receptor 2

AbstractThe induction of the iron-regulatory peptide hepcidin by proinflammatory cytokines is thought to result in the withholding of iron from invading pathogens. Hfe and transferrin receptor 2 (Tfr2) are involved in the homeostatic regulation of hepcidin and their disruption causes hereditary hemochromatosis (HH). To determine whether either Hfe or Tfr2 is involved in the inflammatory pathway regulating hepcidin, we analyzed the effect of inflammation in 3 mouse models of HH. The inflammatory response and indicators of iron homeostasis were measured in wild-type, Hfe−/−, Tfr2−/−, and Hfe−/−/Tfr2−/− mice injected with lipopolysaccharide (LPS). The administration of LPS significantly reduced serum iron in wild-type and Hfe−/− mice, with smaller reductions in Tfr2−/− and Hfe−/−/Tfr2−/− mice. Low basal levels of hepcidin in the Hfe−/−/Tfr2−/− mice were increased in response to LPS, but remained significantly lower than in the other strains of mice. These results suggest that despite the absence of Hfe and Tfr2, hepcidin is responsive to inflammation; however, the low basal expression and subsequent low levels of circulating hepcidin are insufficient to reduce serum iron effectively. This suggests that in HH, the iron-withholding response to invading pathogens may be inadequate, and this is especially the case in the absence of both Hfe and Tfr2.

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Multiple mechanisms are responsible for altered expression of ornithine decarboxylase in overproducing variant cells

Molecular and Cellular Biology ◽

10.1128/mcb.6.8.2865-2871.1986 ◽

1986 ◽

Vol 6 (8) ◽

pp. 2865-2871

Author(s):

L McConlogue ◽

S L Dana ◽

P Coffino

Keyword(s):

Steady State ◽

Gene Amplification ◽

Ornithine Decarboxylase ◽

Mrna Levels ◽

Wild Type ◽

Altered Expression ◽

Cells Of Origin ◽

Multistep Process ◽

Steady State Levels ◽

Synthesis And Degradation

We selected and characterized a series of mouse S49 cell variants that overproduce ornithine decarboxylase (ODC). Previously, we described variants that have an amplified ODC gene and produce about 500-fold more ODC than the wild-type cells of origin (L. McConlogue and P. Coffino, J. Biol. Chem. 258:12083-12086, 1983). We examined a series of independent variants that overproduce ODC to a lesser degree and found that a number of mechanisms other than gene amplification are responsible for the increased ODC activity. Variants were selected for resistance to 0.1 mM difluoromethylornithine, an inhibitor of ODC, by either a single or a multistep process. All showed increased ODC activity and increased ODC mRNA steady-state levels. The half-life of the enzyme was not increased in any of the variants. In one class of variant the increase of ODC mRNA was sufficient to account for ODC overproduction. In a second class, the rate of synthesis of ODC polypeptide per ODC mRNA was at least four- to eightfold higher than that in wild-type cells. Therefore, these variants were altered in the translatability of ODC mRNA. Southern analysis showed that gene amplification does not account for the increased ODC mRNA levels in any of the variants. In both variant and wild-type cells, ODC activity was responsive to changes in polyamine pools; activity was reduced following augmentation of pool size. This change in activity was associated with modification of the rate of synthesis and degradation of ODC but no change in the level of ODC mRNA.

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The defect in Fas mRNA expression in MRL/lpr mice is associated with insertion of the retrotransposon, ETn.

Journal of Experimental Medicine ◽

10.1084/jem.178.2.723 ◽

1993 ◽

Vol 178 (2) ◽

pp. 723-730 ◽

Cited By ~ 171

Author(s):

J L Chu ◽

J Drappa ◽

A Parnassa ◽

K B Elkon

Keyword(s):

Mrna Expression ◽

Tumor Necrosis Factor Receptor ◽

Growth Factor Receptor ◽

Surface Protein ◽

Northern Blotting ◽

Nucleotide Sequencing ◽

Mrna Levels ◽

Wild Type ◽

Fas Mrna ◽

A Cell

Fas is a cell surface protein of the tumor necrosis factor receptor, nerve growth factor receptor, CD40 family, and is involved in the control of lymphocyte apoptosis. A mutation in the Fas gene in MRL/lpr mice results in massive lymphoproliferation (lpr) and accelerated autoimmunity. To further study the nature of this defect, Fas mRNA expression was evaluated by reverse transcriptase polymerase chain reaction as well as by Northern blotting. These studies revealed that the wild-type Fas message was produced at approximately 10-fold lower levels in the lpr compared with the ++ substrain of MRL mice. In addition to the wild-type transcript, lpr mice also synthesized chimeric transcripts containing an insertion of the early retrotransposon (ETn). Molecular cloning and nucleotide sequencing of a Fas-ETn chimeric cDNA suggested that the striking reduction in wild-type Fas mRNA levels and the presence of aberrant transcripts in MRL/lpr mice are most likely explained by the insertion of the ETn retrotransposon into an intron of the Fas gene and induction of alternative splicing involving the 5' ETn long terminal repeat.

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Iron-controlled infection with Neisseria meningitidis in mice

Infection and Immunity ◽

10.1128/iai.29.3.886-891.1980 ◽

1980 ◽

Vol 29 (3) ◽

pp. 886-891 ◽

Cited By ~ 2

Author(s):

B E Holbein

Keyword(s):

Neisseria Meningitidis ◽

Serum Iron ◽

Reticuloendothelial System ◽

High Serum ◽

Iron Dextran ◽

Working Hypothesis ◽

Intraperitoneal Infection ◽

Parenteral Iron

An iron-controlled infection was obtained after the intraperitoneal infection of Neisseria meningitidis strain M1011 into normal mice. The infection progressed rapidly but then disappeared in concert with the disappearance of plasma transferrin iron. Parenteral iron dextran enhanced and prolonged the infection in mice at dosages above 15 mg of Fe per kg. Studies on the distribution of iron dextran within the physiological iron pools and the importance of timing with the iron dextran addition indicated that high serum iron, available early during infection, was necessary to promote infection. High levels of iron in the reticuloendothelial system did not stimulate infection. A working hypothesis to explain the roles of iron in infection was developed: N. meningitidis obtains iron for growth from the transferrin pool, and iron dextran maintains transferrin iron levels during infection.

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