Review
Nuclear ferritin: A new role for ferritin in cell biology

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Abstract

Background

Ferritin has been traditionally considered a cytoplasmic iron storage protein. However, several studies over the last two decades have reported the nuclear localization of ferritin, specifically H-ferritin, in developing neurons, hepatocytes, corneal epithelial cells, and some cancer cells. These observations encouraged a new perspective on ferritin beyond iron storage, such as a role in the regulation of iron accessibility to nuclear components, DNA protection from iron-induced oxidative damage, and transcriptional regulation.

Scope of Review

This review will address the translocation and functional significance of nuclear ferritin in the context of human development and disease.

Major conclusions

The nuclear translocation of ferritin is a selective energy-dependent process that does not seem to require a consensus nuclear localization signal. It is still unclear what regulates the nuclear import/export of ferritin. Some reports have implicated the phosphorylation and O-glycosylation of the ferritin protein in nuclear transport; others suggested the existence of a specific nuclear chaperone for ferritin. The data argue strongly for nuclear ferritin as a factor in human development and disease. Ferritin can bind and protect DNA from oxidative damage. It also has the potential of playing a regulatory role in transcription.

General significance

Nuclear ferritin represents a novel new outlook on ferritin functionality beyond its classical role as an iron storage molecule.

Introduction

Iron (Fe) is an essential micronutrient in biological systems. Primarily, found in the active centers of enzymes and oxygen carrier proteins, iron plays a key role in several cellular processes, such as adenosine triphosphate (ATP), deoxyribonucleic acid (DNA), and neurotransmitter synthesis [1], [2], [3]. Despite its clear importance, unregulated levels of iron can have detrimental effects on the cell. To prevent iron-induced oxidative damage, iron storage and uptake are tightly regulated processes involving the cooperative effort of an array of uptake and storage molecules. Primary among the proteins capable of sequestering iron is a 24-mer protein complex known as ferritin. Ferritin has a hollow protein shell capable of sequestering free iron in its less toxic ferric form. Two functionally and genetically distinct ferritin subunits exist: L-ferritin and H-ferritin. In humans, their molecular masses are 19 and 21 kDa, respectively. Although the two subunits share approximately 55% of their sequence, as well as their multihelical three-dimensional structure, they are functionally distinct [4], [5]. The heavy subunit is primarily responsible for the ferroxidase activity of the ferritin complex, whereas the light subunit facilitates the storage of iron into the ferritin core [6], [7]. The efficient storage of iron (up to 4500 iron atoms per ferritin complex) requires the cooperativity of both ferritin subunits [7], [8]. For instance, the presence of a small proportion of L-ferritin within the ferritin complex can enhance iron hydrolysis and mineralization within the ferritin core [7]. On the other hand, the ferroxidase activity of H-ferritin is essential for uptake of free iron.

The expression of the ferritin subunits is under transcriptional and translational regulation [9]. Their mRNA contains iron response elements (IRE) that can increase the translation of ferritin when iron is abundant and vice versa [10], [11]. On a transcriptional level, several factors can enhance the expression of the ferritin subunits. For example, H-ferritin mRNA is affected by a range of different factors including TNF-α [12], IL-1β [13], IGF-1 [14], and heme [15]. Transcriptional regulation allows for a different distribution of the two subunits within cells enabling them to adapt to changes in iron bioavailability. Generally, organs with high iron turnover (i.e., heart) have been noted to have higher levels of H-ferritin, whereas organs involved in iron storage (i.e., liver) have higher levels of L-ferritin.

In 2001, a new ferritin type has been characterized [16]. This subunit is highly similar to H-ferritin and is specifically targeted to the Mitochondria. The precursor protein includes a mitochondrial leader sequence, which is cleaved upon entry to the mitochondria. The mature protein is capable of supramolecular assembly into ferritin shells and has an intact ferroxidase site [16]. The existence of a novel ferritin subunit in the mitochondria encourages the notion that ferritin may play specialized roles within organelles.

As a consequence of its ability to sequester iron, ferritin plays a key protective role against oxidative stress. Unbound intracellular ferrous iron is capable of generating free radicals and reactive oxidative species (ROS) through Fenton chemistry causing lipid peroxidation, DNA breaks, and other forms of cellular damage [17], [18], [19]. Several overexpression and deletion studies have demonstrated the effect of ferritin, specifically H-ferritin, on survival under conditions of oxidative stress. In Caenorhabditis elegans, deletion of a mammalian H-ferritin homolog reduced lifespan under conditions of iron overload [20]. In HeLa and human murine erthroleukemia cells, overexpression of wild-type H-ferritin induced an iron-deficient phenotype—with increased IRP activity, transferrin receptor levels, and a reduction of L-ferritin levels [21], [22]. Moreover, these cells displayed increased resistance to hydrogen peroxidase cytotoxicity [22]. Interestingly, these effects were not observed when overexpressing L-ferritin or a ferroxidase -deficient mutant of H-ferritin, suggesting a major role for H-ferritin in controlling intracellular iron levels [22]. Deletions of H-ferritin in mice are embryonic lethal, signifying its protective role during the vulnerable early stages of blastocyst development [23]. But, in the context of this review, it may have roles in the developing cells at the nuclear level that have yet to be discovered. Mice heterozygous for the H-ferritin deletion lacked any apparent abnormalities but had elevated levels of L-ferritin in serum and tissue [24]. A closer look at the brains of heterozygous mice revealed an increase in oxidative stress, indicating a possible connection between iron metabolic dysfunction in the brain and neuronal diseases [25].

Aside from its role as an antioxidant, H-ferritin seems to have the ability to bind signaling elements involved in survival pathways. Recently, H-ferritin has been reported as a negative intracellular regulator for the CXC chemokine receptor 4 (CXCR4) [26]. CXCR4 is a coreceptor for T-tropic HIV [27] and is expressed on a wide range of human malignancy [28], [29], [30]. Induction by its ligand, CXC ligand 12 (CXCL12), activates several signaling cascades including MAPK–ERK1/2 [31], Jak/STAT [31], and AKT [32]. H-ferritin overexpression in HeLa cells led to the inhibition of CXCL12-mediated signaling through MARK–ERK [26]. Knockdown of H-ferritin reversed these effects and prolonged the activation of downstream pathways. These results suggest a significant role for ferritin in modulating the activity of CXCR4. This study also generated a novel insight into nuclear ferritin translocation. Exposure to CXCL12 was associated with a time-dependent phosphorylation and nuclear translocation of ferritin. The potential functional implications for this movement will be discussed in a later section.

DNA-binding protein from starved cells (Dps) was first characterized in 3-day starved Escherichia coli cultures [33]. Since then, hundreds of Dps-like proteins have been identified in bacteria and archaea. This prokaryotic protein family is primarily involved in iron homeostasis and the protection against oxidative stress [34], [35]. Some Dps-like proteins have evolved the ability to bind DNA. The Dps–DNA interaction shows a high degree of stability and is independent of sequence [33]. Furthermore, this interaction is believed to stabilize DNA and protect it during periods of metabolic and oxidative stress.

The Dps-like family shows a striking resemblance to ferritin both structurally and functionally, suggesting a common ancestor. The Dps monomer is composed of a four-helix bundle and is capable of forming multimers similar to the ferritin subunits [36], [37]. Moreover, the assembled Dps shell is capable of iron oxidation, uptake, and storage [37], [38]. Thus, the similarity between Dps-like proteins and the ferritins is consistent with extending the function of ferritin beyond iron homeostasis and into DNA binding.

Section snippets

Nuclear ferritin

Over the last two decades, several studies have reported the presence of ferritin in cell nuclei. The earliest observations of nuclear ferritin were in cells under pathological conditions, such as hepatocytes of mice following iron overload [39]. The first report of nuclear ferritin in cells under normal physiological conditions came from the study of corneal epithelial cells in chicken [40] followed by the presence of nuclear ferritin in neurons [41]. Tissue sections of the corneal epithelium

Translocation mechanism of nuclear ferritin

In eukaryotic cells, proteins are selectively segregated to the nucleus through the action of the nuclear pore complex. It is believed that small water soluble molecules (< 40 kDa) can enter the nucleus through passive diffusion [47]. However, efficient transport of proteins, large or small, requires the involvement of several factors through signal- and energy-dependent pathways. A well-understood nuclear translocation mechanism involves the ‘basic’ nuclear localization signal (NLS) first

Functional significance of nuclear ferritin

Ferritin has been reported to be unevenly distributed within the nucleoplasm, raising the possibility of specific interactions with nuclear components [40], [52]. Nuclear fractionation studies in the astrocytoma cell line SW1088 demonstrated that the bulk of nuclear ferritin is within the soluble nuclear fraction, whereas intermediate levels of ferritin are associated with the nuclear matrix [52]. In corneal epithelial cells, nuclear ferritin was reported to be found throughout the nucleus,

Future direction

The data argue strongly for nuclear ferritin as a factor in human disease and development. Of particular interest is the role of nuclear ferritin in cancer. The movement of ferritin to the nucleus might exert a protective effect on DNA, especially during periods of metabolic or oxidative stress. Such function could increase resistance to chemotherapeutic agents that target DNA.

A recent report has linked nuclear ferritin to triple A syndrome—a rare and poorly understood neurological disorder [64]

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