The unique regulation of Aedes aegypti larval cell ferritin by iron

https://doi.org/10.1016/j.ibmb.2007.01.003Get rights and content

Abstract

Mosquitoes must blood feed in order to complete their life cycle. The blood meal provides a high level of iron that is required for egg development. We are interested in developing control strategies that interfere with this process. We report the temporal effects of iron exposure on iron metabolism of Aedes aegypti larval cells. These cells take up iron in linear relationship to exposure time and distribute the iron primarily to the membranes. Iron uptake increases cytoplasmic, membrane and secreted ferritin. Membrane ferritin is abundant in cells treated with iron, increases in cells in the absence of iron exposure and is associated with the secretory pathway. Our data suggest that in contrast to mammals, mosquitoes control intracellular iron levels by producing membrane ferritin in anticipation of an iron load such as that provided by a blood meal and support the hypothesis that secreted ferritin is a primary iron storage protein for these animals.

Introduction

Mosquitoes transmit devastating diseases that kill millions of people each year (Jacob, 2001). Since disease transmission efficiency is in many cases low, rates reflect the great numbers of mosquito vectors. Female mosquitoes must blood feed in order to complete their life cycle. Among many nutrients, the blood meal provides a high level of iron that is required for development (Winzerling and Pham, 2004). Although sufficient iron is present in the blood meal to provoke the formation of toxic-free radicals, these animals have developed mechanisms that allow iron utilization while maintaining protection from iron-mediated oxidative stress (Nichol et al., 2002; Geiser et al., 2003). Similar to mammals, mosquitoes could be partially protected from oxidative stress by iron storage inside ferritin (Aisen et al., 2001; Nichol et al., 2002; Geiser et al., 2003; Hentze et al., 2004; Liu and Theil, 2005).

Vertebrate cytoplasmic ferritin generally consists of 24 subunits representing heavy (∼21 kDa) and light (∼19 kDa) chain polypeptides (Chasteen and Harrison, 1999). Recent data reveals that insect-secreted ferritin retains a similar 24-subunit structure (Hamburger et al., 2005), but the subunit composition differs. In the case of Aedes aegypti (yellow fever mosquito, Diptera), ferritin is composed of subunits of 24, 26, 28 and 30 kDa (Dunkov et al., 1995). The 24 and 26 kDa subunits are products of the same gene (heavy chain homologue—HCH) and retain the sites for ferroxidase catalytic activity that are present in the vertebrate ferritin heavy chain (Chasteen and Harrison, 1999; Pham, 2000). The 28 kDa subunit does not have a catalytic site and is a product of a different gene (light chain homologue—LCH, Geiser et al., 2003). The sequence of the 30 kDa subunit remains unknown.

In vertebrates, ferritin synthesis in response to iron is controlled primarily at the translational level, in part by the interaction of the iron regulatory protein 1 (IRP1) with an iron responsive element (IRE) in the 5′ untranslated region (UTR) of the ferritin subunit mRNA (Eisenstein, 2000; Pantopoulos, 2004). When intracellular iron is low, IRP1 IRE interaction prevents assembly of the ribosomal apparatus and blocks ferritin synthesis (Muckenthaler et al., 1998). When iron increases, IRP1 IRE interaction declines and synthesis increases. The change in IRP1 binding results from the formation of an iron sulfur cluster in the protein core (Beinert et al., 1996). When the cluster is present, the IRP1 cannot bind to the IRE. A functional IRE is found in the mRNAs for both vertebrate ferritin subunits. Although mosquitoes synthesize IRP1, only the mosquito HCH mRNA has a 5′UTR IRE (Dunkov et al., 1995; Zhang et al., 2002; Geiser et al., 2003). Studies support that HCH expression is subject to both transcriptional and translational control (Pham et al., 1999), whereas LCH synthesis reflects mRNA levels (Geiser et al., 2003; Pham and Chavez, 2005).

In A. aegypti, ferritin is expressed throughout the life cycle and increases in the ovaries, gut and hemolymph following a blood meal (Dunkov et al., 2002). HCH subunits predominate in hemolymph ferritin, are present in sugar-fed adults and increase dramatically with blood feeding. LCH is not found in sugar-fed adults, but is present following blood feeding. An increase in ferritin following iron exposure also has been reported for Manduca sexta (tobacco hornworm, Pham et al., 1996), Drosophila melanogaster (Georgieva et al., 2002) and Calpodes ethlius (Brazilian skipper butterfly, Nichol and Locke, 1990). In C. ethlius, apoferritin is observed in the secretory pathway of cells and holoferritin is observed in this pathway when iron is available (Nichol and Locke, 1990; Locke et al., 1991). Holoferritin also was observed in the endoplasmic reticulum of D. melanogaster (Nichol and Locke, 1990). Available evidence supports that insects secrete ferritin in response to an iron load.

In our previous studies, we evaluated the effects of iron dose on ferritin expression in A. aegypti CCL-125 larval cells (Geiser et al., 2006). We found that larval cells synthesize and secrete ferritin in response to iron exposure. Cytoplasmic ferritin is maximal following exposure to low levels of iron and consists of both subunits. Secreted ferritin increases in direct linear relationship to iron dose and is composed primarily of HCH subunits. Although exposure of larval cells to low iron concentrations increases cytoplasmic iron levels, higher levels of exposure result in a decline in cytoplasmic iron indicating that iron is removed from the cells. In keeping with the lower cytoplasmic iron levels, IRP1 IRE binding activity is minimally reduced.

We now report that A. aegypti larval cells take up iron in direct, linear relationship to exposure time. Although iron uptake rapidly expands a labile pool, total cytoplasmic iron levels do not change. In contrast, membrane iron levels increase with iron exposure time. In keeping with these changes, cytoplasmic, membrane and secreted ferritin show different temporal relationships.

Section snippets

Cell culture and experimental protocol

A. aegypti larval cells (CCL-125) were obtained from the American Type Culture Collection (Manassas, VA). The cells were maintained in Medium A: 75% DMEM high glucose (Invitrogen Corporation, Carlsbad, CA) and 25% Sf-900 II SFM (Invitrogen) supplemented with 15% heat-inactivated fetal bovine serum (Gemini Bio-Products, Calabasas, CA) and 0.15% antibiotics/antimycotics (Invitrogen), as stock cultures in a water-jacketed incubator with 10% humidity and a 95% air–5% CO2 atmosphere at 28 °C.

Iron uptake significantly increases the cytoplasmic labile iron pool

We evaluated cell numbers and viability to assure that iron treatment did not alter these parameters. As shown in Fig. 1(A), cell numbers increase significantly (p<0.001) with time relative to 0 h for all treatment groups due to cell division, but do not vary significantly as a result of treatment conditions (Fig. 1(A)). Cell viability also does not change significantly with time of iron exposure or with treatment conditions (Fig. 1(B)). Calcein is a chemosensor that detects iron to 0.02 μM in

Discussion

We are interested in the mechanisms that allow mosquitoes to adapt to the iron load of a blood meal and to utilize iron for development. Our data indicate that A. aegypti larval epithelial cells take up iron in direct, linear relationship to exposure time. Iron uptake results in the rapid transfer of iron to the membranes as well as a shift in cytoplasmic iron into a labile pool. To our knowledge, this is the first report in insect cells documenting levels of iron distributed to various cell

Acknowledgements

This work was supported by funds from the National Institutes of Health, National Institute of General Medical Sciences (GM056812), the Agricultural Experiment Station and the College of Agriculture and Life Sciences and the Center for Insect Science of the Arizona Research Laboratories at the University of Arizona. The authors thank M. K. Amistadi, M-C. Shen and E. Kohlhepp for their technical support.

References (60)

  • T. Georgieva et al.

    Drosophila melanogaster ferritin: cDNA encoding a light chain homologue, temporal and tissue specific expression of both subunit types

    Insect Biochem. Mol. Biol.

    (2002)
  • S. Ghosh et al.

    Regulated secretion of glycosylated human ferritin from hepatocytes

    Blood

    (2004)
  • A.E. Hamburger et al.

    Crystal structure of a secreted insect ferritin reveals a symmetrical arrangement of heavy and light chains

    J. Mol. Biol.

    (2005)
  • M.W. Hentze et al.

    Balancing acts: molecular control of mammalian iron metabolism

    Cell

    (2004)
  • O. Kakhlon et al.

    Repression of ferritin expression increases the labile iron pool, oxidative stress and short-term growth of human erythroleukemia cells

    Blood

    (2001)
  • S.A. Kohler et al.

    Succinate dehydrogenase b mRNA of Drosophila melanogaster has a functional iron-responsive element in its 5′-untranslated region

    J. Biol. Chem.

    (1995)
  • A.M. Konijn et al.

    The cellular labile iron pool and intracellular ferritin in K562 cells

    Blood

    (1999)
  • M. Locke et al.

    Vacuolar apoferritin synthesis by the fat body of an insect

    J. Insect Physiol.

    (1991)
  • M.A. Markwell et al.

    A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples

    Anal. Biochem.

    (1978)
  • O. Melefors

    Translational regulation in vivo of the Drosophila Melanogaster mRNA encoding succinate dehydrogenase iron protein via iron responsive elements

    Biochem. Biophys. Res. Commun.

    (1996)
  • M. Muckenthaler et al.

    IRP-1 binding to ferritin mRNA prevents the recruitment of the small ribosomal subunit by the cap-binding complex eIF4F

    Mol. Cell

    (1998)
  • H. Nichol et al.

    Structured RNA upstream of insect cap distal iron responsive elements enhances iron regulatory protein-mediated control of translation

    Insect Biochem. Mol. Biol.

    (2002)
  • H.K. Nichol et al.

    The localization of ferritin in insects

    Tissue Cell

    (1990)
  • D.Q. Pham et al.

    Manduca sexta hemolymph ferritin: cDNA sequence and mRNA expression

    Gene

    (1996)
  • D.Q.-D. Pham et al.

    Identification and mapping of the promoter for the gene encoding the ferritin heavy-chain homologue of the yellow fever mosquito Aedes aegypti

    Insect Biochem. Mol. Biol.

    (2003)
  • D.R. Richardson et al.

    The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells

    Biochim. Biophys. Acta

    (1997)
  • H.R. Sanders et al.

    Blood meal induces global changes in midgut gene expression in the disease vector, Aedes aegypti

    Insect Biochem. Mol. Biol.

    (2003)
  • K.L. Schalinske et al.

    Phosphorylation and activation of both iron regulatory proteins 1 and 2 in HL-60 cells

    J. Biol. Chem.

    (1996)
  • T.N. Tran et al.

    Secretion of ferritin by rat hepatoma cells and its regulation by inflammatory cytokines and iron

    Blood

    (1997)
  • D. Zhang et al.

    Cloning and molecular characterization of two mosquito iron regulatory proteins

    Insect Biochem. Mol. Biol.

    (2002)
  • Cited by (19)

    • Ovarian morphological features and proteome reveal fecundity fitness disadvantages in β-cypermethrin-resistant strains of Blattella germanica (L.) (Blattodea: Blattellidae)

      2020, Pesticide Biochemistry and Physiology
      Citation Excerpt :

      Iron is a vital metal involved in oxygen delivery, cell proliferation and drug metabolism, while it is also a highly toxic metal producing reactive oxygen species (Harigae, 2010). To avoid shortage or toxicity of iron, the two properties of ionic iron, an essential nutrient and a potent toxin, must be balanced (Geiser et al., 2007). Ferritin regulates the concentration of ionic iron in cells so that it is sufficient to catalyze oxidative metabolism without causing oxidative damage, but the mechanisms of ferritin are different in different insects and are poorly understood (Tan et al., 2012).

    • Identification and characterization of a Macrobrachium nipponense ferritin subunit regulated by iron ion and pathogen challenge

      2014, Fish and Shellfish Immunology
      Citation Excerpt :

      In plants, however, iron regulates ferritin expression at the transcriptional level [41]. In previous studies, iron loading has been shown to stimulate ferritin gene expression in fruit flies and mosquitos [37,42]. In our study, the expression of the ferritin transcript increased significantly by approximately seven-fold in prawn muscle and four-fold in prawn gill 3 h after iron injection.

    • Transcriptional up-regulation of a novel ferritin homolog in abalone Haliotis discus hannai Ino by dietary iron

      2010, Comparative Biochemistry and Physiology - C Toxicology and Pharmacology
      Citation Excerpt :

      Mammalian ferritin levels increased in order to allow iron storage and availability for use when needed, as well as protection from iron-mediated oxidative stress (Hentze et al., 2004; Liu and Theil, 2005). It has been shown that FT levels markedly increased with high levels of dietary iron in fruit fly (Georgieva et al., 2002) and mosquito (Geiser et al., 2007). These results are consistent with the findings in the present study, and expression levels of HdhNFT mRNA in hepatopancreas and haemocyte of abalone significantly increased with dietary Fe (Fig. 6).

    • Insect ferritins: Typical or atypical?

      2010, Biochimica et Biophysica Acta - General Subjects
    • Expression profile of the iron-binding proteins transferrin and ferritin heavy chain subunit in the bumblebee Bombus ignitus

      2009, Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology
    View all citing articles on Scopus
    View full text