revised 6 October1997
THE ROLE OF IRON IN CELL PROLIFERATION AND DIFFERENTIATION
Iron is indispensable for DNA synthesis and a host of metabolic processes (Cazzola et al., 1990). Iron starvation arrests proliferation, presumably because the metal is required by ribonucleotide reductase and other enzymes involved in cell division (Hoffbrand et al., 1976). Although transferrin receptors are expressed on all dividing cells in numbers roughly reflecting growth rate (Frazier et al., 1982), the erythron is the organ that relies most heavily on iron delivery by transferrin, as exemplified by atransferrinemic animals (Heilmeyer, 1966); (Goya et al., 1972); (Bernstein, 1987). But the transferrin cycle also appears to play a significant, if expendable, role in other cell types.
Iron and Lymphocyte Function
Studies of T-lymphocytes exemplify the general relationship between transferrin receptor expression and cell proliferation. Transferrin receptors, absent from resting T cells, have long been recognized as a marker of T cell activation. The initiation of cell division by a mitogen such as phytohemagglutinin dramatically increases both transferrin receptor surface expression and iron uptake (Larrick and Cresswell, 1979). Along the same lines, tumor cells upregulate transferrin receptor expression to optimize iron acquisition for proliferation.
When stimulated to proliferate, T-lymphocytes also synthesize and secrete transferrin (Bowman). The physiological role of this lymphocyte-produced transferrin is unknown. Some investigators have speculated that the transferrin could play an autocrine role. No evidence exists supporting this contention, however.
Blockade of transferrin receptor function can halt cell division. For instance, certain monoclonal antibodies against the transferrin receptor curb proliferation of tumor cells in vitro and in vivo (Lesley and Schulte, 1985); (White et al., 1990). Some of these antibodies actually prevent binding of transferrin to its receptor, while others suppress receptor recycling but do not abrogate ligand binding (reviewed by Trowbridge et al., 1987).)
Interestingly, there are reports suggesting that the transferrin receptor may have an additional role in activated T cells, apart from its iron uptake function. Anti-transferrin receptor monoclonal antibodies have been described that can trigger T cell activation and interleukin-2 secretion (Manger et al., 1987); (Cano et al., 1990); (Keyna et al., 1994). These antibodies presumably activate a signal transduction pathway beginning with the transferrin receptor, but independent of iron trafficking.
Iron Chelators and Cell Proliferation
The central role of iron in cell proliferation is further demonstrated by chelators that can cross the plasma membrane, bind the metal inside the cell, and limit its bioavailability. Agents such as desferrioxamine and desferrithiocin inhibit the growth of a variety of tumor cells in culture (Reddel et al., 1985) and greatly reduce T-cell proliferation (Chaudri et al., 1986); (Bierer and Nathan, 1990); (Polson et al., 1990); (Pattanapanyasat et al., 1992). The likely inhibitory mechanism is iron deprivation, with reduced ribonucleotide reductase activity and lower levels of deoxyribonucleotides. This in turn leads to mitotic arrest in S-phase (Lederman et al., 1984). The addition of iron to the medium reverses the growth inhibition. Chelators may also induce apoptosis or programmed cell death (Fukuchi et al., 1994).
Iron and Erythroid Precursors
Erythroid precursors need an extraordinary amount of iron to support hemoglobin synthesis and differentiation into mature red cells. The density of transferrin receptors on the cell surface changes during erythroid maturation. Transferrin receptors first appear in measurable numbers on CFU-Es, increasing to 300,000 per cell on pro-erythroblasts and as many as 800,000 per cell on basophilic erythroblasts, at the time of maximal iron uptake. Numbers then fall to 100,000 per cell on circulating reticulocytes, and negligible levels on mature red cells (Brittenham, 1994).
A strict correlation exists between iron requirement and transferrin receptor number, indicating that the abundance of transferrin receptors on the cell surface is a major determinant of erythroid iron uptake (Ponka, 1997). Because maturing red cells shed their transferrin receptors, the quantity of soluble transferrin receptor in plasma reasonably reflects erythropoiesis. In culture, a monoclonal antibody to the transferrin receptor that permits ligand binding but subsequently slows receptor recycling partially blocks erythroid burst cell iron uptake. The level of iron uptake is sufficient for cell division but not hemoglobin synthesis (Shannon et al., 1986).
Beug and co-workers demonstrated that an anti-transferrin receptor monoclonal antibody to chick erythroid cells blocked erythroid differentiation at the erythroblast or early reticulocyte stage, and promoted premature, pyknotic cell death (Schmidt et al., 1986). The antibody apparently prevented normal cycling of transferrin receptors, and inhibited efficient iron uptake. The effect was specific to differentiation. The antibody did not inhibit proliferation of a variety of other cell lines.
Ferric salicylaldehyde-isonicotinyl-hydrazone (Fe-SIH) was added to antibody-treated cells to determine whether direct delivery of iron by this compound could rescue the normal erythroid program. Interestingly, the Fe-SIH only partially restored maturation of antibody-treated avian cells. The investigators postulated that insufficient levels of heme or hemoglobin might shut off production of proteins required for differentiation.
These data are in concord with Ponka and co-workers, who have shown that the rate of heme synthesis is influenced by the efficiency of an unknown step in iron uptake. They have localized the critical step distal to the interaction of ferric transferrin with the transferrin receptor but proximal to insertion of iron into heme by ferrochelatase (Ponka and Schulman, 1993).
Hemin Promotes Cell Differentiation
A wealth of literature demonstrates that oxidized heme (hemin) promotes differentiation of erythroleukemia cell lines in tissue culture (Ross and Sautner, 1976); (Rutherford et al., 1979); (Mager and Bernstein, 1979); (Bonanou-Tzedaki et al., 1981). Conversely, deficient heme biosynthesis abrogates chemical induction of differentiation in an erythroleukemia cell line subclone (Rutherford et al., 1979). Hemin's differentiation capacity extends to other types of cells, such as preadipocytes, suggesting a general effect on differentiation that extends beyond the erythroid lineage (ref).
A hemin-inducible transcriptional regulatory element exists in the enhancer-like locus control region (LCR) upstream of the human beta globin genes (Ney et al., 1990). Although far from the structural genes, the LCR is critical for high level globin expression. The region activates transcription, at least in part, through binding of a factor termed NF-E2 to this hemin-inducible DNA sequence element. NF-E2 DNA-binding activity has been purified from murine and human erythroleukemia cell lines (Andrews et al., 1993a); (Andrews et al., 1993b); (Ney et al., 1993).
NF-E2 activity correlates with the expression of a heterodimeric complex of a tissue-specific 45 kDa polypeptide (p45 NF-E2) and a widely expressed 18 kDa polypeptide (p18) that heterodimerize to form a basic-leucine zipper transcriptional regulatory protein. NF-E2 recognition elements are also found in the promoters for red cell-specific forms of the heme biosynthetic enzymes porphobilinogen deaminase and ferrochelatase (Mignotte et al., 1989); (Taketani et al., 1992).
Interestingly, forced expression of p45 NF-E2 in non-erythroid cells stimulates iron uptake (Chang and Andrews, in preparation). The functional data and the fact that NF-E2 sites are found in association with this constellation of genes involved in red cell development, suggests that NF-E2 coordinates hemoglobin production by regulating the expression of globin proteins, heme biosynthesis and iron uptake (Andrews, 1994).
Other reports indicate that heme biosynthesis indirectly regulates globin transcription, as well as expression of the genes encoding the transferrin receptor and ferritin (Battistini et al., 1991); (Battistini et al., 1991); (Coccia et al., 1992). Heme also regulates globin mRNA translation. While the onset of globin protein synthesis precedes that of heme in developing erythroblasts (Nathan et al., 1961), the intracellular concentration of heme directly regulates globin synthesis (London et al., 1987). Although the precise mechanisms remain to be elucidated, iron uptake, heme biosynthesis and globin protein production clearly are coordinately regulated. Interrelated regulatory networks apparently allow red cell precursors to maximize hemoglobin formation without accumulating excess globin proteins, unbound iron or toxic protoporphyrin intermediates.
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