Biochemical Mechanisms For Ironfolate Interactions

Iron Metabolism Influences Folate Status

Regulation of intracellular folate concentrations is complex and is influenced by dietary folate intake, intestinal and cellular transport systems, polyglutamylation of the cofactor, and folate turnover, including folate catabolism (19,24,38). Folate catabolism is defined as the oxidative and irreversible scission of the p-aminobenzoyl(poly)glutamate moiety of the cofactor from the quinazoline ring system, a reaction that destroys folate as a metabolic cofactor (24,39,40). Certain physiological states are associated with increased rates of folate catabolism and simultaneous frank folate deficiency despite adequate dietary folate intake. These states of increased folate catabolism include cancer (41,42), antiepiletic drug therapy, and pregnancy in rodents (43,44) and perhaps humans (45-48), as evidenced by elevated concentrations of p-aminobenzoylglutamate in urine (38).

The in vivo catabolism of folate cofactors has been assumed to result from the nonenzymatic, oxidative degradation of labile folate cofactors, including dihydrofolate (44) and 10-formyltetrahydrofolate (24). Each of these cofactors is readily oxidized in vitro (39,40). However, observations that increased rates of folate catabolism occur during defined physiological states indicate that folate catabolism may be a regulated, enzyme-mediated process (24). Recently, a protein was purified from crude rat liver homogenates that effectively generated p-aminobenzoylglutamate from 5-formyltetrahydrofolate (49). The protein was identified as ferritin, a multisubunit protein that sequesters and stores intracellular iron (50). Ferritin is a 24-mer composed of heavy-chain (HCF) and light-chain (LCF) subunits. Expression of the rat HCF cDNA in Chinese hamster ovary cells results in increased rates of folate turnover and decreased intracellular folate concentrations, even when cells are cultured in the presence of pharmacological levels of folic acid (49). Therefore, increased rates of ferritin-mediated folate catabolism can affect intracellular folate concentrations in cell cultures, indicating that changes in iron metabolism may influence cellular folate concentrations in vivo.

Iron Status Influences Folate Metabolism

Clinical manifestations of folate deficiency during iron deficiency can occur in the absence of depleted tissue folate (17). This observation indicates that catabolism-mediated folate deficiency cannot, in itself, fully account for the iron-folate relationship. Iron status is influenced by three pools of cellular iron: the functional pool that is bound by iron-requiring proteins, the storage pool that is ferritin-bound, and the labile, or regulatory, pool that exists free in solution. Elevated expression of HCF in the absence of increased iron availability lowers intracellular regulatory iron concentrations and triggers the cellular iron-deficiency response, which includes increased expression of the transferrin receptor and increased rates of iron uptake (51). A recent study demonstrated that increased expression of HCF, but not increased LCF, in cell cultures alters the relative distribution of folate one-carbon-substituted cofactors, indicating that HCF expression alters the flux of one-carbon units through folate-requiring anabolic pathways (52). HCF contains a ferrioxidase activity that catalyzes the oxidation of cyto-plasmic Fe2+ to Fe3+, a reaction that is associated with cellular iron chelation and storage within the ferritin polymer, whereas LCF does not contain this activity and does not function as an active iron chelator (52). Therefore, decreases in the cellular regulatory iron pool may mediate the changes in folate-dependent pathways observed with increased HCF expression in cell culture. The alterations in folate metabolism associated with increased HCF expression are the result, at least in part, of increased activity of the folate-dependent enzyme cytoplasmic serine hydroxymethyltransferase (cSHMT). The increases in cSHMT activity occur without alterations in cSHMT mRNA levels, but rather are caused by elevated cSHMT protein levels resulting from increased rates of cSHMT mRNA translation. Increased expression of the LCF cDNA does not affect cSHMT protein levels, indicating that increases in cSHMT expression respond either directly to increases in HCF protein or are responsive to HCF-induced decreases in the regulatory iron pool (52).

The role of cSHMT in folate metabolism has been elusive. The serine hydroxymethyltransferase (SHMT) enzyme catalyzes the reversible interconversion of serine and tetrahydrofolate to glycine and methylene tetrahydrofolate:

serine + tetrahydrofolate <—> glycine + methylenetetrahydrofolate

Serine is the primary source of the one-carbon units that are required for folate-dependent biosynthetic reactions, indicating that SHMT plays an important role in folate metabolism (13-15). There are two cellular isozymes of SHMT that are encoded by separate genes; one isozyme resides in the cytoplasm (cSHMT) and the other resides in the mitochondria (mSHMT) (53,54). Loss of mSHMT activity in Chinese hamster ovary cells results in a glycine auxotrophy and cellular deficits in one-carbon units, indicating that mSHMT is responsible for the catabolism of serine to glycine and a single carbon and that cSHMT cannot compensate for loss of mSHMT function (55). Several studies have indicated that SHMT activity in the cytoplasm favors serine synthesis, and cSHMT enzyme is expressed at high levels in the liver and kidney (53,56,57). These observations may reflect glycine's role as an important gluconeogenic amino acid in these tissues. The definitive metabolic function of cSHMT in other cell types is less certain.

Changes in cSHMT expression influence both thymidylate synthesis and homocysteine remethylation in cell cultures, but in a reciprocal manner (52,58) (Fig. 1). cSHMT activity has been shown to be rate limiting in thymidine biosynthesis in MCF-7 cells, and elevated cSHMT expression in these cells increases the flux of one-carbon units through the thymidylate synthesis pathway (52). Simultaneously, increased cSHMT expression inhibits the homocysteine remethylation pathway by two distinct mechanisms (58) (Fig. 1). Evidence suggests that the cSHMT enzyme competes with methylenetetrahydrofolate reductase (MTHFR) for one-carbon units in the form of methylenetetrahydrofolate; that is, cSHMT-catalyzed serine synthesis competes with 5-methyltetrahydrofolate synthesis and ultimately homocysteine remethylation (58). The cSHMT enzyme can also inhibit homocysteine remethylation by a second mechanism. The cSHMT enzyme is a high-affinity 5-methyltetrahydrofolate-binding protein (59), and increased expression of the cSHMT cDNA or induction of endogenous cSHMT expression by increasing HCF expression greatly increases intracellular 5-methyltetrahydrofolate concentrations through sequestration of the cofactor (58). This decrease in intracellular 5-methyltetrahydrofolate availability impairs homocysteine remethylation and results in markedly decreased cellular concentrations of S-adenosylmethionine. Collectively, these studies suggest that the cytoplasm contains two pools of cSHMT, one that interacts with and perhaps channels one-carbon units to the enzyme thymidylate synthase and a second "free" pool that competes with MTHFR for methylenetetrahydrofolate (58). Furthermore, although previous studies have indicated that homocysteine remethylation has a higher priority compared to deoxyribonucleotide biosynthesis, increased expression of cSHMT reverses that priority by accelerating folate-dependent thymidylate synthesis while simultaneously inhibiting homocysteine remethylation. This "switch" is influenced by HCF expression (52), retinoic acid, and developmental stage (60).

Effect of Chemical Iron Chelators on cSHMT Expression

Chemical iron chelators inhibit cell proliferation in some but not all tumor cell lines (61-63). The iron chelators mimosine and deferoxamine have been demonstrated to alter and deplete intracellular deoxyribonucleotide pools, presumably by inhibiting the iron-dependent enzyme ribonucleotide reductase (64-66). It has been suggested that iron chelators inhibit the cell cycle by this mechanism. However, this suggestion has remained controversial because depletion of cellular deoxyribonucleotide concentrations would be expected to inhibit the elongation phase of DNA replication, whereas mimosine inhibits the formation of replication bubbles, or the initiation phase of DNA replication (67,68). Additionally, neither mimosine nor de-feroxamine effectively inhibits cell cycle progression in cells of embryonic origin; therefore, other mechanisms must be considered. Mimosine alters folate metabolism in human MCF-7 cells, a cell line known to be growth arrested by iron chelators, but mimosine does not influence folate metabolism or cell proliferation in human neuroblastoma (62). Mimosine inhibits cSHMT expression in MCF-7 cells at the level of transcription, presumably by activating a transcriptional silencing factor whose activity is increased by chemical iron chelators (62). This factor appears to be tissue-specific, as it has only been found in MCF-7 cells. However, it is unlikely that inhibition of cSHMT expression is the primary mechanism whereby iron chelators inhibit cell cycle progression because characterization of MCF-7 mutants that are resistant to mimosine has revealed that they also fail to express cSHMT. Therefore, it has been proposed that activation of this silencing factor may regulate other proteins necessary for cell cycle progression.

At first glance, the transcriptional silencing effect of chemical iron chelators on cSHMT expression appears to contradict the effect of HCF-induced activation of cSHMT expression (52). However, these two chelators have very different effects on cellular iron stores. HCF is only capable of influencing the regulatory iron pool, whereas chemical iron chelators are capable of depleting both the free and functional iron pools. Therefore, the effect of chemical iron chelators on cSHMT transcription, an effect not observed when HCF expression is increased, likely results from chelation of the functional iron pool.

Indicators of Impaired Folate Metabolism

Impairment of folate metabolism is made manifest by several clinical and biochemical outcomes that serve as indicators, or proxies, of whole-body folate status (17,69). As indicated in Fig. 2, short-term folate deficiency lowers serum folate concentrations; therefore, this measurement is a good indicator of recent folate intake. Longer-term folate depletion results in increased segmentation of peripheral blood neutrophils. Peripheral neutrophils isolated from folate-replete subjects normally contain no more than three or four lobes (average = 3.2 lobes), but during folate deficiency, they can become hypersegmented and contain as many as six lobes or average greater than 3.5 lobes. This hypersegmentation is believed to result from impaired DNA synthesis. Longer-duration deficiency results in increased excretion of formiminoglutamate (FIGLU), an intermediate in the folate-dependent catabolism of histidine. Finally, severe depletion of whole-body folate stores is evidenced by low red blood cell (RBC) folate and megaloblastic bone marrow and anemia. RBC folate is a good indicator of long-term folate status because the RBC accumulates folate during erythropoiesis, and these folate levels are retained throughout the RBC life span. RBC folate correlates well with liver folate in the absence of pernicious anemia. Impairment of folate metabolism is also quantified by the deoxyuridine (dU) suppression assay, which measures the efficiency of de novo thymidine biosynthesis (69). Thymidine can be synthesized de novo by a folate-dependent

Weeks following folate restriction

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