Iron-dependent trafficking of 5-lipoxygenase and impact on human macrophage activation

Beatrice Dufrusine, Andrea Di Francesco, Sergio Oddi, Lucia Scipioni, Clotilde Beatrice Angelucci, Claudio D'Addario, Mauro Serafini, Ann Kathrin Häfner, Dieter Steinhilber, Mauro Maccarrone, Enrico Dainese

Research output: Contribution to journalArticle

Abstract

5-lipoxygenase (5-LOX) is a non-heme iron-containing dioxygenase expressed in immune cells that catalyzes the two initial steps in the biosynthesis of leukotrienes. It is well known that 5-LOX activation in innate immunity cells is related to different iron-associated proinflammatory disorders, including cancer, neurodegenerative diseases, and atherosclerosis. However, the molecular and cellular mechanism(s) underlying the interplay between iron and 5-LOX activation are largely unexplored. In this study, we investigated whether iron (in the form of Fe3+ and hemin) might modulate 5-LOX influencing its membrane binding, subcellular distribution, and functional activity. We proved by fluorescence resonance energy transfer approach that metal removal from the recombinant human 5-LOX, not only altered the catalytic activity of the enzyme, but also impaired its membrane-binding. To ascertain whether iron can modulate the subcellular distribution of 5-LOX in immune cells, we exposed THP-1 macrophages and human primary macrophages to exogenous iron. Cells exposed to increasing amounts of Fe3+ showed a redistribution (ranging from ~45 to 75%) of the cytosolic 5-LOX to the nuclear fraction. Accordingly, confocal microscopy revealed that acute exposure to extracellular Fe3+, as well as hemin, caused an overt increase in the nuclear fluorescence of 5-LOX, accompanied by a co-localization with the 5-LOX activating protein (FLAP) both in THP-1 macrophages and human macrophages. The functional relevance of iron overloading was demonstrated by a marked induction of the expression of interleukin-6 in iron-treated macrophages. Importantly, pre-treatment of cells with the iron-chelating agent deferoxamine completely abolished the hemin-dependent translocation of 5-LOX to the nuclear fraction, and significantly reverted its effect on interleukin-6 overexpression. These results suggest that exogenous iron modulates the biological activity of 5-LOX in macrophages by increasing its ability to bind to nuclear membranes, further supporting a role for iron in inflammation-based diseases where its homeostasis is altered and suggesting further evidence of risks related to iron overload.

Original languageEnglish
Article number1347
JournalFrontiers in Immunology
Volume10
Issue numberJUN
DOIs
Publication statusPublished - Jan 1 2019

Fingerprint

Arachidonate 5-Lipoxygenase
Macrophage Activation
Iron
Macrophages
Hemin
Interleukin-6
5-Lipoxygenase-Activating Proteins
Iron Chelating Agents
Dioxygenases
Fluorescence Resonance Energy Transfer
Deferoxamine
Iron Overload
Membranes
Leukotrienes
Nuclear Envelope
Innate Immunity
Confocal Microscopy
Neurodegenerative Diseases
Atherosclerosis
Homeostasis

Keywords

  • 5-lipoxygenase
  • Enzyme activation
  • Iron
  • Macrophage activation
  • Macrophages
  • Nuclear translocation

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

Iron-dependent trafficking of 5-lipoxygenase and impact on human macrophage activation. / Dufrusine, Beatrice; Francesco, Andrea Di; Oddi, Sergio; Scipioni, Lucia; Angelucci, Clotilde Beatrice; D'Addario, Claudio; Serafini, Mauro; Häfner, Ann Kathrin; Steinhilber, Dieter; Maccarrone, Mauro; Dainese, Enrico.

In: Frontiers in Immunology, Vol. 10, No. JUN, 1347, 01.01.2019.

Research output: Contribution to journalArticle

Dufrusine, B, Francesco, AD, Oddi, S, Scipioni, L, Angelucci, CB, D'Addario, C, Serafini, M, Häfner, AK, Steinhilber, D, Maccarrone, M & Dainese, E 2019, 'Iron-dependent trafficking of 5-lipoxygenase and impact on human macrophage activation', Frontiers in Immunology, vol. 10, no. JUN, 1347. https://doi.org/10.3389/fimmu.2019.01347
Dufrusine, Beatrice ; Francesco, Andrea Di ; Oddi, Sergio ; Scipioni, Lucia ; Angelucci, Clotilde Beatrice ; D'Addario, Claudio ; Serafini, Mauro ; Häfner, Ann Kathrin ; Steinhilber, Dieter ; Maccarrone, Mauro ; Dainese, Enrico. / Iron-dependent trafficking of 5-lipoxygenase and impact on human macrophage activation. In: Frontiers in Immunology. 2019 ; Vol. 10, No. JUN.
@article{6593dc744eb6428892ebb674c7a31845,
title = "Iron-dependent trafficking of 5-lipoxygenase and impact on human macrophage activation",
abstract = "5-lipoxygenase (5-LOX) is a non-heme iron-containing dioxygenase expressed in immune cells that catalyzes the two initial steps in the biosynthesis of leukotrienes. It is well known that 5-LOX activation in innate immunity cells is related to different iron-associated proinflammatory disorders, including cancer, neurodegenerative diseases, and atherosclerosis. However, the molecular and cellular mechanism(s) underlying the interplay between iron and 5-LOX activation are largely unexplored. In this study, we investigated whether iron (in the form of Fe3+ and hemin) might modulate 5-LOX influencing its membrane binding, subcellular distribution, and functional activity. We proved by fluorescence resonance energy transfer approach that metal removal from the recombinant human 5-LOX, not only altered the catalytic activity of the enzyme, but also impaired its membrane-binding. To ascertain whether iron can modulate the subcellular distribution of 5-LOX in immune cells, we exposed THP-1 macrophages and human primary macrophages to exogenous iron. Cells exposed to increasing amounts of Fe3+ showed a redistribution (ranging from ~45 to 75{\%}) of the cytosolic 5-LOX to the nuclear fraction. Accordingly, confocal microscopy revealed that acute exposure to extracellular Fe3+, as well as hemin, caused an overt increase in the nuclear fluorescence of 5-LOX, accompanied by a co-localization with the 5-LOX activating protein (FLAP) both in THP-1 macrophages and human macrophages. The functional relevance of iron overloading was demonstrated by a marked induction of the expression of interleukin-6 in iron-treated macrophages. Importantly, pre-treatment of cells with the iron-chelating agent deferoxamine completely abolished the hemin-dependent translocation of 5-LOX to the nuclear fraction, and significantly reverted its effect on interleukin-6 overexpression. These results suggest that exogenous iron modulates the biological activity of 5-LOX in macrophages by increasing its ability to bind to nuclear membranes, further supporting a role for iron in inflammation-based diseases where its homeostasis is altered and suggesting further evidence of risks related to iron overload.",
keywords = "5-lipoxygenase, Enzyme activation, Iron, Macrophage activation, Macrophages, Nuclear translocation",
author = "Beatrice Dufrusine and Francesco, {Andrea Di} and Sergio Oddi and Lucia Scipioni and Angelucci, {Clotilde Beatrice} and Claudio D'Addario and Mauro Serafini and H{\"a}fner, {Ann Kathrin} and Dieter Steinhilber and Mauro Maccarrone and Enrico Dainese",
year = "2019",
month = "1",
day = "1",
doi = "10.3389/fimmu.2019.01347",
language = "English",
volume = "10",
journal = "Frontiers in Immunology",
issn = "1664-3224",
publisher = "Frontiers Media S.A.",
number = "JUN",

}

TY - JOUR

T1 - Iron-dependent trafficking of 5-lipoxygenase and impact on human macrophage activation

AU - Dufrusine, Beatrice

AU - Francesco, Andrea Di

AU - Oddi, Sergio

AU - Scipioni, Lucia

AU - Angelucci, Clotilde Beatrice

AU - D'Addario, Claudio

AU - Serafini, Mauro

AU - Häfner, Ann Kathrin

AU - Steinhilber, Dieter

AU - Maccarrone, Mauro

AU - Dainese, Enrico

PY - 2019/1/1

Y1 - 2019/1/1

N2 - 5-lipoxygenase (5-LOX) is a non-heme iron-containing dioxygenase expressed in immune cells that catalyzes the two initial steps in the biosynthesis of leukotrienes. It is well known that 5-LOX activation in innate immunity cells is related to different iron-associated proinflammatory disorders, including cancer, neurodegenerative diseases, and atherosclerosis. However, the molecular and cellular mechanism(s) underlying the interplay between iron and 5-LOX activation are largely unexplored. In this study, we investigated whether iron (in the form of Fe3+ and hemin) might modulate 5-LOX influencing its membrane binding, subcellular distribution, and functional activity. We proved by fluorescence resonance energy transfer approach that metal removal from the recombinant human 5-LOX, not only altered the catalytic activity of the enzyme, but also impaired its membrane-binding. To ascertain whether iron can modulate the subcellular distribution of 5-LOX in immune cells, we exposed THP-1 macrophages and human primary macrophages to exogenous iron. Cells exposed to increasing amounts of Fe3+ showed a redistribution (ranging from ~45 to 75%) of the cytosolic 5-LOX to the nuclear fraction. Accordingly, confocal microscopy revealed that acute exposure to extracellular Fe3+, as well as hemin, caused an overt increase in the nuclear fluorescence of 5-LOX, accompanied by a co-localization with the 5-LOX activating protein (FLAP) both in THP-1 macrophages and human macrophages. The functional relevance of iron overloading was demonstrated by a marked induction of the expression of interleukin-6 in iron-treated macrophages. Importantly, pre-treatment of cells with the iron-chelating agent deferoxamine completely abolished the hemin-dependent translocation of 5-LOX to the nuclear fraction, and significantly reverted its effect on interleukin-6 overexpression. These results suggest that exogenous iron modulates the biological activity of 5-LOX in macrophages by increasing its ability to bind to nuclear membranes, further supporting a role for iron in inflammation-based diseases where its homeostasis is altered and suggesting further evidence of risks related to iron overload.

AB - 5-lipoxygenase (5-LOX) is a non-heme iron-containing dioxygenase expressed in immune cells that catalyzes the two initial steps in the biosynthesis of leukotrienes. It is well known that 5-LOX activation in innate immunity cells is related to different iron-associated proinflammatory disorders, including cancer, neurodegenerative diseases, and atherosclerosis. However, the molecular and cellular mechanism(s) underlying the interplay between iron and 5-LOX activation are largely unexplored. In this study, we investigated whether iron (in the form of Fe3+ and hemin) might modulate 5-LOX influencing its membrane binding, subcellular distribution, and functional activity. We proved by fluorescence resonance energy transfer approach that metal removal from the recombinant human 5-LOX, not only altered the catalytic activity of the enzyme, but also impaired its membrane-binding. To ascertain whether iron can modulate the subcellular distribution of 5-LOX in immune cells, we exposed THP-1 macrophages and human primary macrophages to exogenous iron. Cells exposed to increasing amounts of Fe3+ showed a redistribution (ranging from ~45 to 75%) of the cytosolic 5-LOX to the nuclear fraction. Accordingly, confocal microscopy revealed that acute exposure to extracellular Fe3+, as well as hemin, caused an overt increase in the nuclear fluorescence of 5-LOX, accompanied by a co-localization with the 5-LOX activating protein (FLAP) both in THP-1 macrophages and human macrophages. The functional relevance of iron overloading was demonstrated by a marked induction of the expression of interleukin-6 in iron-treated macrophages. Importantly, pre-treatment of cells with the iron-chelating agent deferoxamine completely abolished the hemin-dependent translocation of 5-LOX to the nuclear fraction, and significantly reverted its effect on interleukin-6 overexpression. These results suggest that exogenous iron modulates the biological activity of 5-LOX in macrophages by increasing its ability to bind to nuclear membranes, further supporting a role for iron in inflammation-based diseases where its homeostasis is altered and suggesting further evidence of risks related to iron overload.

KW - 5-lipoxygenase

KW - Enzyme activation

KW - Iron

KW - Macrophage activation

KW - Macrophages

KW - Nuclear translocation

UR - http://www.scopus.com/inward/record.url?scp=85069051059&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85069051059&partnerID=8YFLogxK

U2 - 10.3389/fimmu.2019.01347

DO - 10.3389/fimmu.2019.01347

M3 - Article

AN - SCOPUS:85069051059

VL - 10

JO - Frontiers in Immunology

JF - Frontiers in Immunology

SN - 1664-3224

IS - JUN

M1 - 1347

ER -