Probing the iron pool. Focus on "Detection of intracellular iron by its regulatory effect".

ALL MAMMALIAN CELLS BENEFIT from a healthy dose of iron. In addition to its prominent role as the oxygen-carrying moiety in hemoglobin, iron is a cofactor for cytochromes and a variety of enzymes. During the past decade, a great deal has been learned about cellular iron transport and its regulation (1), but the understanding of iron balance within cells remains limited. Li et al. (Ref. 3; see p. C1547 in this issue) report the development of very useful probes with which to study intracellular iron content. Free iron is dangerous, because it catalyzes the formation of reactive oxygen species. For that reason, most biological iron is bound to proteins (e.g., transferrin, ferritin) to keep it from causing trouble. However, a very small amount of iron appears to be, at least functionally, free within the cytoplasm. This labile iron pool represents 5% of the total cellular iron. It is chelatable, composed of both Fe and Fe , and associated with a variety of small molecules, including organic anions, polypeptides, and phospholipids (reviewed in Ref. 2). It is a dynamic compartment that can change rapidly and must be managed by intracellular homeostatic mechanisms. Various attempts have been made to quantify the labile iron pool. Cell fractionation, although helpful for other purposes, is notoriously unreliable for measuring free iron. Fluorescent dyes such as calcein and phen-green are both quenched by metal binding, allowing measurement of changes in free iron over seconds to minutes. However, these dyes have a few drawbacks. First, the chelation of iron may by itself perturb rapidly changing iron pools. Second, calcein and phen-green cannot be used for long-term experiments, because the dyes bleach and leak over time. Third, they are not specific for iron: both can interact with other metals. Li et al. (3) have used a different approach with a biological reporter that exploits the most studied homeostatic mechanism for intracellular iron. Iron-regulatory elements (IRE) are encoded in untranslated portions of mRNA (reviewed in Ref. 1). These IRE are hydrogen-bonded RNA stem-loop structures that can interact with at least two cellular proteins, termed “iron-regulatory proteins” (IRP1 and IRP2). Both IRP1 and IRP2 are modulated by the cellular iron endowment. This regulatory mechanism is particularly elegant because IRE have different functions, depending on their location in the mRNA. When an IRE is located in the 5 untranslated region (5 UTR), as in the mRNA encoding ferritins, IRP binding interrupts protein synthesis. When IRE are located in the 3 UTR, however, as in the transferrin receptor mRNA, IRP binding facilitates protein synthesis by conferring mRNA stability. Li et al. (3) have placed cDNA encoding derivatives of green fluorescent protein under the regulatory control of the IRE-IRP system. They have used 5 and 3 IRE to regulate distinguishable fluorophores, allowing two complementary measurements from each cell. When iron is scarce, translation of cyan fluorescent protein, linked to a 5 IRE, is inhibited by IRP binding, while translation of yellow fluorescent protein, linked to a 3 IRE, is enhanced. In the presence of free intracellular iron, this pattern is reversed. This allows for ratiometric analysis of iron-responsive gene expression. This new method is at least as sensitive as dye-based methods, and it complements the other approaches nicely. IRP are abundant in most cell types, and the introduction of the IRE-containing reporter constructs does not seem to perturb intracellular iron pools or alter their regulation of endogenous IRE-containing mRNA. Individual cells can be examined for responses on time scales of hours, rather than seconds or minutes, because of ongoing, regulated synthesis of the fluorescent proteins, which themselves have relatively short halflives. The reversibility afforded by protein degradation allows the same cells to be monitored under changing conditions, e.g., as they differentiate. IRP are highly specific for regulation by iron; other metals have little or no effect on IRP activities. This method is superior to simply assessing the expression of endogenous IRE-regulated proteins (e.g., ferritin, with a 5 IRE, or transferrin receptor, with a 3 IRE) because each of those molecules modulates intracellular iron homeostasis and each is subject to other modes of regulation in addition to IRP regulation. There are, however, some disadvantages. IRE-regulated fluorescent probes are not helpful for shorter time frames, because it takes several hours for their biosynthesis. Furthermore, the reporter constructs must be introduced deliberately into cells by transfection or by viral infection. Finally, although it appears that IRP are controlled by the labile iron pool, there may be important intracellular compartments that are not probed by their activities. Nonetheless, these fluorescent IRE-based reporters are likely to be useful. For example, the assay should easily be adaptable to a multiwell plate format, allowing automated screening of chemical compound libraries to identify potential new chelators. This could be valuable in the development of new drugs to treat iron overload conditions. Li et al. (3) have already begun efforts to introduce IREregulated fluorescent reporter transgenes into mice. Development of reporter mice will facilitate analysis of cellular iron content within complex tissues and during development. They will allow, for example, direct examination of iron content of important cell types (e.g., enterocytes, macrophages) in mouse models of human hemochromatosis. In vivo information obAddress for reprint requests and other correspondence: N. C. Andrews, Karp Family Research Laboratories 8-125, Children’s Hospital Boston, 300 Longwood Ave., Boston, MA 02115-5737 (E-mail: [email protected]. harvard.edu). Am J Physiol Cell Physiol 287: C1537–C1538, 2004; doi:10.1152/ajpcell.00435.2004.