Glia maturation factor-γ regulates murine macrophage iron metabolism and M2 polarization through mitochondrial ROS

Abstract In macrophages, cellular iron metabolism status is tightly integrated with macrophage phenotype and associated with mitochondrial function. However, how molecular events regulate mitochondrial activity to integrate regulation of iron metabolism and macrophage phenotype remains unclear. Here, we explored the important role of the actin-regulatory protein glia maturation factor-γ (GMFG) in the regulation of cellular iron metabolism and macrophage phenotype. We found that GMFG was downregulated in murine macrophages by exposure to iron and hydrogen peroxide. GMFG knockdown altered the expression of iron metabolism proteins and increased iron levels in murine macrophages and concomitantly promoted their polarization toward an anti-inflammatory M2 phenotype. GMFG-knockdown macrophages exhibited moderately increased levels of mitochondrial reactive oxygen species (mtROS), which were accompanied by decreased expression of some mitochondrial respiration chain components, including the iron-sulfur cluster assembly scaffold protein ISCU as well as the antioxidant enzymes SOD1 and SOD2. Importantly, treatment of GMFG-knockdown macrophages with the antioxidant N-acetylcysteine reversed the altered expression of iron metabolism proteins and significantly inhibited the enhanced gene expression of M2 macrophage markers, suggesting that mtROS is mechanistically linked to cellular iron metabolism and macrophage phenotype. Finally, GMFG interacted with the mitochondrial membrane ATPase ATAD3A, suggesting that GMFG knockdown–induced mtROS production might be attributed to alteration of mitochondrial function in macrophages. Our findings suggest that GMFG is an important regulator in cellular iron metabolism and macrophage phenotype and could be a novel therapeutic target for modulating macrophage function in immune and metabolic disorders.

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Glia Maturation Factor-Gamma Regulates the Intracellular Growth of Salmonella Via Modulation of Ferroportin in Murine Macrophages

Blood ◽

10.1182/blood.v126.23.2208.2208 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2208-2208 ◽

Cited By ~ 1

Author(s):

Wulin Aerbajinai ◽

Jungsoo Joo ◽

Ming Yan ◽

Chutima Kumkhaek ◽

Griffin P. Rodgers

Keyword(s):

Iron Metabolism ◽

Iron Content ◽

Glia Maturation Factor ◽

Intracellular Growth ◽

Intracellular Iron ◽

Iron Storage ◽

Murine Macrophages ◽

Protein Levels ◽

Raw264.7 Macrophages ◽

Maturation Factor

Abstract Salmonella is an intracellular bacterial pathogen that replicates within membrane-bound compartment and alters host iron metabolism for its own survival. Persistent survival and replication within phagocytes is central to the pathogenesis of Salmonella infections. Macrophages play a critical role in regulating iron metabolism for securing body iron sufficiency and controlling the availability of iron for intracellular proliferation of pathogens. However, the relationship of Salmonella-induced changes of macrophage iron metabolism to the survival and replication mechanism of this pathogen within macrophages remains poorly understood. Thus, it is critical to identify the host factors involved in the intracellular survival and replication of Salmonella in order to design more-efficient antimicrobial therapeutics. Glia maturation factor gamma (GMFG), a novel regulator of the actin-related protein-2/3 (Arp2/3) complex, is predominantly expressed in inflammatory cells. We have previously demonstrated that GMFG negatively regulate TLR4-induced proinflammatory signaling, but its function in macrophage response to intracellular bacteria infection remains unclear. In this study, we investigated the role of GMFG in Salmonella-infected murine macrophages by using small-interfering RNA (RNAi) techniques to knockdown GMFG. We found that knockdown of GMFG significantly enhanced the numbers of intracellular Salmonella growth (>3-fold, p< 0.008) at 24 hr postinfection compared with control siRNA transfected Raw264.7 macrophages. However, there was no significant difference in growth numbers of bacteria observed at 4 hr postinfection, indicating that GMFG does not influence bacterial phagocytosis. Immunofluorescence microscopy also revealed an accumulation of Salmonella, in GMFG knockdown macrophages at 24 hr postinfection. Knockdown of GMFG results in marked decreased the iron exporter ferroportin protein levels and increased iron storage ferritin-L protein levels in Raw264.7 macrophages. Further, the intracellular iron content was elevated in GMFG-knockdown macrophages compared with control macrophages (1.9-fold, p< 0.05). These observations indicate that regulatory impact of GMFG in Salmonella intracellular growth may be through modulation of macrophage iron metabolism. Moreover, consistent with previous studies, we found that Raw264.7 macrophage infections with Salmonella increase the expression of the iron transporter ferroportin and ferritin, indicating this is the host defense strategy against infection with intracellular microbes by limiting their access to iron. Although there was no marked altered in GMFG protein level after 24hr infection with Salmonella, GMFG knockdown macrophage infected with Salmonella displayed increased the intracellular iron content and iron storage protein ferritin compared with control macrophages. Further analysis of cytokines expression in Salmonella-infected GMFG-knockdown macrophage revealed enhanced the proinflammatory TNF-alpha mRNA (1.86-fold, p<0.05) and anti-inflammatory IL-10 mRNA (2.2-fold, p< 0.0) at 24 hr postinfection, which is paralleled with increased intracellular Salmonella replication. Our results suggest that GMFG modulation of crucial pathways of macrophage iron metabolism and immune function in murine macrophages infected with Salmonella. Disclosures No relevant conflicts of interest to declare.

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Effect of nitric oxide on expression of transferrin receptor and ferritin and on cellular iron metabolism in K562 human erythroleukemia cells

Blood ◽

10.1182/blood.v85.10.2962.bloodjournal85102962 ◽

1995 ◽

Vol 85 (10) ◽

pp. 2962-2966 ◽

Cited By ~ 47

Author(s):

R Oria ◽

L Sanchez ◽

T Houston ◽

MW Hentze ◽

FY Liew ◽

...

Keyword(s):

Nitric Oxide ◽

Iron Metabolism ◽

Transferrin Receptor ◽

Regulatory Protein ◽

Inhibitory Effect ◽

Receptor Expression ◽

Mrna Levels ◽

Intracellular Iron ◽

Cellular Iron ◽

Erythroleukemia Cells

Nitric oxide (NO) is known to increase the affinity of the intracellular iron-regulatory protein (IRP) for iron-response elements (IREs) in transferrin receptor and ferritin mRNAs, suggesting that it may act as a regulator of cellular iron metabolism. In this study, exogenous NO produced by adding the NO-generator S-nitroso-N-acetyl penicillamine gave a dose-dependent upregulation of transferrin receptor expression by K562 erythroleukemia cells and increased levels of transferrin receptor mRNA. NO did not affect the affinity of transferrin binding by the transferrin receptor. NO alone did not alter intracellular ferritin levels, but it did abrogate the inhibitory effect of the iron chelator desferrioxamine and potentiated the stimulatory effect of additional iron. NO also caused some increase in ferritin mRNA levels, which might mask any IRP-/IRE-mediated inhibitory effect of NO on ferritin translation. Although NO did not affect net iron uptake, it increased release of iron from K562 cells pulsed previously with 59Fe, and subcellular fractionation showed that it also increased the proportion of intracellular iron bound to ferritin. These findings provide direct evidence that NO can affect cellular iron metabolism and suggest that NO produced in vivo by activated bone marrow macrophages might affect erythropoiesis.

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Regulation of Cellular Iron Metabolism by Erythropoietin: Activation of Iron-Regulatory Protein and Upregulation of Transferrin Receptor Expression in Erythroid Cells

Blood ◽

10.1182/blood.v89.2.680 ◽

1997 ◽

Vol 89 (2) ◽

pp. 680-687 ◽

Cited By ~ 92

Author(s):

Günter Weiss ◽

Tracey Houston ◽

Stefan Kastner ◽

Karin Jöhrer ◽

Kurt Grünewald ◽

...

Keyword(s):

Progenitor Cells ◽

Iron Metabolism ◽

Transferrin Receptor ◽

Regulatory Protein ◽

Receptor Expression ◽

General Mechanism ◽

Iron Regulatory Protein ◽

Erythroid Progenitor ◽

Cellular Iron ◽

Erythroid Progenitor Cells

Abstract Erythropoietin (Epo) is the central regulator of red blood cell production and acts primarily by inducing proliferation and differentiation of erythroid progenitor cells. Because a sufficient supply of iron is a prerequisite for erythroid proliferation and hemoglobin synthesis, we have investigated whether Epo can regulate cellular iron metabolism. We present here a novel biologic function of Epo, namely as a potential modulator of cellular iron homeostasis. We show that, in human (K562) and murine erythroleukemic cells (MEL), Epo enhances the binding affinity of iron-regulatory protein (IRP)-1, the central regulator of cellular iron metabolism, to specific RNA stem-loop structures, known as iron-responsive elements (IREs). Activation of IRP-1 by Epo is associated with a marked increase in transferrin receptor (trf-rec) mRNA levels in K562 and MEL, enhanced cell surface expression of trf-recs, and increased uptake of iron into cells. These findings are in agreement with the well-established mechanism whereby high-affinity binding of IRPs to IREs stabilizes trf-rec mRNA by protecting it from degradation by a specific RNase. The effects of Epo on IRE-binding of IRPs were not observed in human myelomonocytic cells (THP-1), which indicates that this response to Epo is not a general mechanism observed in all cells but is likely to be erythroid-specific. Our results provide evidence for a direct functional connection between Epo biology and iron metabolism by which Epo increases iron uptake into erythroid progenitor cells via posttranscriptional induction of trf-rec expression. Our data suggest that sequential administration of Epo and iron might improve the response to Epo therapy in some anemias.

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Glia Maturation Factor-Gamma Regulates Alternative Activation of Macrophage By the Modulation of Iron Homeostasis through HO-1 in Ex Vivo

Blood ◽

10.1182/blood.v128.22.3677.3677 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3677-3677

Author(s):

Wulin Aerbajinai ◽

Chutima Kumkhaek ◽

Wenli Liu ◽

Griffin P. Rodgers

Keyword(s):

Iron Metabolism ◽

Iron Homeostasis ◽

Alternative Activation ◽

Raw 264.7 Cells ◽

Heme Oxygenase 1 ◽

Raw 264.7 ◽

Glia Maturation Factor ◽

Immunoblotting Analysis ◽

Protein Levels ◽

Maturation Factor

Abstract Macrophages play a key role at the crossroad of iron metabolism and immune function. They store and recycle iron derived from the phagocytosis of senescent erythrocytes. Macrophages iron homeostasis is coupled to their remarkable heterogeneity and functional plasticity. It is well known that the macrophage polarization process dictates expression profiles of genes involved in iron metabolism. M1 macrophages are characterized by increased iron retention, whereas, M2 macrophages showed increased iron recycling. However, the molecular mechanisms underlying iron metabolism link to regulation of macrophage-polarized phenotype are not fully understood. Glia maturation factor gamma (GMFG), a novel regulator of the actin-related protein-2/3 (Arp2/3) complex, is predominantly expressed in inflammatory cells. We have previously found that GMFG mediated macrophage resistance to Salmonella infection, but its function in iron metabolism and macrophage phenotype remains unclear. In this study, we explored the important role of GMFG in the regulation of iron metabolism and macrophages phenotypes interlinked. We found that GMFG expression was downregulated in a dose-dependent manner in murine bone marrow-derived macrophages (BMDM) and RAW-264.7 cells by treated with iron or heme. Immunoblotting analysis demonstrated that knockdown of GMFG in BMDM and RAW-264.7 cells lead to remarkable increased the protein levels of ferroportin (Fpn), transferrin receptor 1 (TfR1), as well as heme oxygenase 1 (HO-1), whereas decreased the ferritin light chain 1 (FtL1) compared with control siRNA transfected BMDM or Raw264.7 cells. Knockdown of GMFG display higher iron export capacity and elevated intracellular labile iron pool (LIP) compared with control macrophages. These results suggest that GMFG is the crucial regulator in macrophages iron metabolism because its downregulation caused an alteration in iron-handling proteins similar to IL-4 induced M2 polarization phenotype. Quantitative PCR analysis showed that M2 alternative activation markers Arg1, Mrc1, and Ym1 were noted to be induced in GMFG knockdown macrophages in the absence of M2-induceer cytokine treatment, confirming the skewing of these macrophages toward M2 alternative activation. Moreover, treatment of GMFG-knockdown BMDM or RAW-264.7 cells with Th2 cytokines IL-4 or IL-13 markedly enhanced the induction of several genes characteristic of M2 alternative activation, including Arg1, Mrc1, and Ym1 compared with control macrophages. Furthermore, M2 skewing was confirmed by the enhancement of the IL-4-induced Arg1 protein levels in GMFG knockdown macrophages relative to control macrophages by immunoblotting analysis. Interestingly, GMFG knockdown macrophages further markedly enhanced the IL-4-induced protein levels of HO-1 and TfR1 in M2 phenotypes, but there was no marked altered in protein level of Fpn or FtL1 compared with control macrophages. These results indicated that knockdown of GMFG might enhance the M2 phenotypes through modulation of HO-1. Finally, we observed that GMFG knockdown macrophages showed more accumulation of transcriptional factor Nrf2 in nuclear without alternation of its transcriptional expression levels compared with control macrophages, suggesting that downregulation of GMFG skewing macrophages toward a M2 phenotype might be through regulation of Nrf2-mediated HO-1 expression. Our results indicate that GMFG plays an important role in the regulation of M2 alternative activation through modulation of iron metabolism and act as a negative feedback loop in macrophages. Disclosures No relevant conflicts of interest to declare.

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Crucial function of vertebrate glutaredoxin 3 (PICOT) in iron homeostasis and hemoglobin maturation

Molecular Biology of the Cell ◽

10.1091/mbc.e12-09-0648 ◽

2013 ◽

Vol 24 (12) ◽

pp. 1895-1903 ◽

Cited By ~ 70

Author(s):

Petra Haunhorst ◽

Eva-Maria Hanschmann ◽

Lars Bräutigam ◽

Oliver Stehling ◽

Bastian Hoffmann ◽

...

Keyword(s):

Iron Metabolism ◽

Iron Homeostasis ◽

Regulatory Protein ◽

Iron Regulation ◽

Iron Starvation ◽

Intracellular Iron ◽

Cellular Iron ◽

Cellular Iron Uptake ◽

S Proteins

The mechanisms by which eukaryotic cells handle and distribute the essential micronutrient iron within the cytosol and other cellular compartments are only beginning to emerge. The yeast monothiol multidomain glutaredoxins (Grx) 3 and 4 are essential for both transcriptional iron regulation and intracellular iron distribution. Despite the fact that the mechanisms of iron metabolism differ drastically in fungi and higher eukaryotes, the glutaredoxins are conserved, yet their precise function in vertebrates has remained elusive. Here we demonstrate a crucial role of the vertebrate-specific monothiol multidomain Grx3 (PICOT) in cellular iron homeostasis. During zebrafish embryonic development, depletion of Grx3 severely impairs the maturation of hemoglobin, the major iron-consuming process. Silencing of human Grx3 expression in HeLa cells decreases the activities of several cytosolic Fe/S proteins, for example, iron-regulatory protein 1, a major component of posttranscriptional iron regulation. As a consequence, Grx3-depleted cells show decreased levels of ferritin and increased levels of transferrin receptor, features characteristic of cellular iron starvation. Apparently, Grx3-deficient cells are unable to efficiently use iron, despite unimpaired cellular iron uptake. These data suggest an evolutionarily conserved role of cytosolic monothiol multidomain glutaredoxins in cellular iron metabolism pathways, including the biogenesis of Fe/S proteins and hemoglobin maturation.

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Regulation of cellular iron metabolism

Biochemical Journal ◽

10.1042/bj20101825 ◽

2011 ◽

Vol 434 (3) ◽

pp. 365-381 ◽

Cited By ~ 505

Author(s):

Jian Wang ◽

Kostas Pantopoulos

Keyword(s):

Iron Metabolism ◽

Mammalian Cells ◽

Regulatory Protein ◽

Cell Types ◽

Excess Iron ◽

Cellular Iron ◽

System A ◽

Regulatory Circuit ◽

Transcriptional Regulatory ◽

Responsive Element

Iron is an essential but potentially hazardous biometal. Mammalian cells require sufficient amounts of iron to satisfy metabolic needs or to accomplish specialized functions. Iron is delivered to tissues by circulating transferrin, a transporter that captures iron released into the plasma mainly from intestinal enterocytes or reticuloendothelial macrophages. The binding of iron-laden transferrin to the cell-surface transferrin receptor 1 results in endocytosis and uptake of the metal cargo. Internalized iron is transported to mitochondria for the synthesis of haem or iron–sulfur clusters, which are integral parts of several metalloproteins, and excess iron is stored and detoxified in cytosolic ferritin. Iron metabolism is controlled at different levels and by diverse mechanisms. The present review summarizes basic concepts of iron transport, use and storage and focuses on the IRE (iron-responsive element)/IRP (iron-regulatory protein) system, a well known post-transcriptional regulatory circuit that not only maintains iron homoeostasis in various cell types, but also contributes to systemic iron balance.

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Superparamagnetic iron oxide nanoparticle uptake alters M2 macrophage phenotype, iron metabolism, migration and invasion

Nanomedicine Nanotechnology Biology and Medicine ◽

10.1016/j.nano.2015.11.020 ◽

2016 ◽

Vol 12 (4) ◽

pp. 1127-1138 ◽

Cited By ~ 37

Author(s):

José M. Rojas ◽

Laura Sanz-Ortega ◽

Vladimir Mulens-Arias ◽

Lucía Gutiérrez ◽

Sonia Pérez-Yagüe ◽

...

Keyword(s):

Iron Oxide ◽

Iron Metabolism ◽

Superparamagnetic Iron Oxide ◽

Migration And Invasion ◽

Iron Oxide Nanoparticle ◽

M2 Macrophage ◽

Macrophage Phenotype ◽

Nanoparticle Uptake ◽

Oxide Nanoparticle ◽

Superparamagnetic Iron Oxide Nanoparticle

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Iron regulatory protein-1 and -2: transcriptome-wide definition of binding mRNAs and shaping of the cellular proteome by iron regulatory proteins

Blood ◽

10.1182/blood-2011-04-343541 ◽

2011 ◽

Vol 118 (22) ◽

pp. e168-e179 ◽

Cited By ~ 69

Author(s):

Mayka Sanchez ◽

Bruno Galy ◽

Bjoern Schwanhaeusser ◽

Jonathon Blake ◽

Tomi Bähr-Ivacevic ◽

...

Keyword(s):

Iron Metabolism ◽

Rna Binding ◽

Isotope Labeling ◽

Rna Binding Proteins ◽

Regulatory Protein ◽

Regulatory Proteins ◽

Iron Regulatory Proteins ◽

Ribonucleoprotein Complexes ◽

Cellular Iron ◽

Irp1 And Irp2

Abstract Iron regulatory proteins (IRPs) 1 and 2 are RNA-binding proteins that control cellular iron metabolism by binding to conserved RNA motifs called iron-responsive elements (IREs). The currently known IRP-binding mRNAs encode proteins involved in iron uptake, storage, and release as well as heme synthesis. To systematically define the IRE/IRP regulatory network on a transcriptome-wide scale, IRP1/IRE and IRP2/IRE messenger ribonucleoprotein complexes were immunoselected, and the mRNA composition was determined using microarrays. We identify 35 novel mRNAs that bind both IRP1 and IRP2, and we also report for the first time cellular mRNAs with exclusive specificity for IRP1 or IRP2. To further explore cellular iron metabolism at a system-wide level, we undertook proteomic analysis by pulsed stable isotope labeling by amino acids in cell culture in an iron-modulated mouse hepatic cell line and in bone marrow-derived macrophages from IRP1- and IRP2-deficient mice. This work investigates cellular iron metabolism in unprecedented depth and defines a wide network of mRNAs and proteins with iron-dependent regulation, IRP-dependent regulation, or both.

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Glia Maturation Factor-Gamma Regulates Autophagy and Scavenger Receptor-Mediated Phagocytosis in Murine Macrophages

10.21203/rs.3.rs-147914/v1 ◽

2021 ◽

Author(s):

Wulin Aerbajinai ◽

Jianqiong Zhu ◽

Kyung Chin ◽

Griffin Rodgers

Keyword(s):

Phagocytic Activity ◽

Scavenger Receptor ◽

Receptor Expression ◽

Related Factor ◽

Glia Maturation Factor ◽

Autophagosome Formation ◽

Ampk Signaling ◽

Murine Macrophages ◽

Maturation Factor ◽

Liver Kinase B1

Abstract Autophagy and phagocytosis are critical processes involved in maintaining macrophage homeostasis and cellular immunity. Because dysfunction of autophagy is observed in many human pathologies, it is important to understand the regulatory mechanisms governing crosstalk between autophagy and phagocytosis. Glia maturation factor-gamma (GMFg) is a novel regulator of the Arp2/3 complex, its role in modulating autophagy and phagocytosis remains unknown. Here, we show that knockdown of GMFg in murine macrophages inhibited autophagosome formation and compromised lysosomal function. GMFg knockdown suppressed phosphorylation of liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signaling pathway components, suggesting a role for this pathway in GMFg regulation of autophagy. Moreover, GMFg-knockdown macrophages displayed increased the expression of scavenger-receptor MSR1 and CD36, which was dependent on activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor, and exhibited increased phagocytic activity. In contrast, overexpression of GMFg in murine macrophages increased autophagosome abundance and suppressed both scavenger-receptor expression and phagocytic activity. These findings suggest that GMFg regulates autophagy through AMPKregulated control of autophagosome formation, while mediating phagocytosis through modulation of scavenger-receptor abundance in macrophages, and may provide insight into therapeutic approaches to autophagy-related diseases and autophagy-regulated phagocytosis in immune and metabolic disorders.

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