Erythroid differentiation and protoporphyrin IX down-regulate frataxin expression in Friend cells: characterization of frataxin expression compared to molecules involved in iron metabolism and hemoglobinization

Abstract

Friedreich ataxia (FA) is caused by decreased frataxin expression that results in mitochondrial iron (Fe) overload. However, the role of frataxin in mammalian Fe metabolism remains unclear. In this investigation we examined the function of frataxin in Fe metabolism by implementing a well-characterized model of erythroid differentiation, namely, Friend cells induced using dimethyl sulfoxide (DMSO). We have characterized the changes in frataxin expression compared to molecules that play key roles in Fe metabolism (the transferrin receptor [TfR] and the Fe transporter Nramp2) and hemoglobinization (␤-globin). DMSO induction of hemoglobinization results in a marked decrease in frataxin gene (Frda) expression and protein levels. To a lesser extent, Nramp2 messenger RNA (mRNA) levels were also decreased on erythroid differentiation, whereas TfR and ␤-globin mRNA levels increased. Intracellular Fe depletion using desferrioxamine or pyridoxal isonicotinoyl hydrazone, which chelate cytoplasmic or cytoplasmic and mitochondrial Fe pools, respectively, have no effect on frataxin expression. Furthermore, cytoplasmic or mitochondrial Fe loading of induced Friend cells with ferric ammonium citrate, or the heme synthesis inhibitor, succinylacetone, respectively, also had no effect on frataxin expression. Although frataxin has been suggested by others to be a mitochondrial ferritin, the lack of effect of intracellular Fe levels on frataxin expression is not consistent with an Fe storage role. Significantly, protoporphyrin IX down-regulates frataxin protein levels, suggesting a regulatory role of frataxin in Fe or heme metabolism. Because decreased frataxin expression leads to mitochondrial Fe loading in FA, our data suggest that reduced frataxin expression during erythroid differentiation results in mitochondrial Fe sequestration for heme biosynthesis. IntroductionFriedreich ataxia (FA) is an inherited neurodegenerative condition 1,2 with an incidence of 1:30 000 in the European population. 3 The gene FRDA that is defective in FA encodes a mitochondrial protein known as frataxin. 2,3 In 97% of individuals with FA the defect in this gene is due to a GAA triplet repeat expansion in intron 1 (chromosome 9q13) that results in a marked decrease in gene expression. 2,3 Over the last few years evidence has accumulated to suggest that frataxin plays a role in mitochondrial iron (Fe) metabolism. However, the function of mammalian frataxin remains unclear. 24 Experiments using the yeast, Saccharomyces cerevisiae, led to a model that may explain the pathogenesis of FA. 10,12 The yeast gene YFH1 is homologous to the human gene FRDA, 12 and encodes a mitochondrial protein (Yfh1p) involved in Fe homeostasis and respiration. 5,9,10,12 When the YFH1 gene was deleted, a marked accumulation of mitochondrial Fe occurred, resulting in loss of mitochondrial DNA and impaired respiration. 6,7,12,13 Thus, this led to a hypothesis that the accumulated Fe resulted in the production of free radicals that damage biologic targets. When YFH1 was reintroduc...

Elucidation of the mechanism of mitochondrial iron loading in Friedreich's ataxia by analysis of a mouse mutant

“…Supporting our findings, a direct interaction between purified frataxin and Fech has been reported, suggesting a role for frataxin in heme synthesis (9,20). Indeed, we have suggested that frataxin acts as a metabolic switch to regulate mitochondrial iron utilization between heme and ISC biosynthesis (8). Moreover, an investigation in frataxin knockout mice showed decreased Isu1, Cpox, and Fech mRNA transcripts (not protein levels) (21), which is validated by our findings.…”

“…The precise function of frataxin remains unclear, but it has been implicated in mitochondrial iron metabolism, particularly iron-sulfur cluster (ISC) synthesis (3)(4)(5). Frataxin depletion results in mitochondrial DNA damage, mitochondrial iron accumulation, ISC deficiency, perturbed heme synthesis, and oxidative damage (6)(7)(8)(9). In addition, recent studies have suggested cytosolic iron deficiency in Friedreich's ataxia models, as indicated by increased RNA-binding activity of iron regulatory protein 2, increased expression of transferrin receptor 1 (Tfr1), and decreased cytosolic ferritin (10,11).…”

Mitochondria in hematopoiesis and hematological diseases

“…In this latter disease, mutations in the gene encoding frataxin, a protein associated with the mitochondrial inner membrane and involved in (Fe-S) cluster formation, induces Fe accumulation in the mitochondria of non-erythroid cells such as neurons and cardiomyocytes (Campuzano et al, 1996;Pandolfo, 2003). However, no anemia is observed in Friedreich ataxia and Fe does not accumulate in mitochondria of erythroid cells because frataxin levels are low in these cells to favor Fe utilization for heme production (Becker et al, 2002;Napier et al, 2005).…”

The Evolution of Iron Chelators for the Treatment of Iron Overload Disease and Cancer

“…This organelle is also responsible for heme and [Fe-S] cluster biosynthesis, and hence, any mitochondrial damage has a large impact on the function of a cell (Cooper and Schapira, 2003). Although the exact function of frataxin has not been determined, it is thought to take part in mitochondrial iron metabolism and [Fe-S] cluster synthesis (Puccio et al, 2001;Becker et al, 2002;Muhlenhoff et al, 2002; for reviews, see Pandolfo, 2002;Alper and Narayanan, 2003;Napier et al, 2005). This correlates with iron accumulation seen in the liver, heart, and spleen of FA patients in a pattern consistent with mitochondria (Sachez-Casis et al, 1976;Lamarche et al, 1993), whereas a decrease in respiratory chain activities of the [Fe-S] cluster-containing complexes I, II, and III is also observed (Rotig et al, 1997;Puccio et al, 2001; for reviews, see Becker and Richardson, 2001;Lodi et al, 2002;Cooper and Schapira, 2003).…”