Iron utilization in rabbit reticulocytes: A study using succinylacetone as an inhibitor of heme synthesis

We have investigated the effect of succinylacetone (4,6-dioxoheptanoic acid) on hemoglobin synthesis and iron metabolism in reticulocytes. Succinylacetone, 0.1 and 1 mM, inhibited [2-¹⁴C]glycine incorporation into heme by 91.2 and 96.4%, respectively, and into globin by 85 and 90.2%, respectively. 60 μM hemin completely prevented the inhibition of globin synthesis by succinylacetone, indicating that succinylacetone inhibits specifically the synthesis of heme. Added porphobilinogen, but not δ-aminolevulinic acid, partly overcame the inhibition of ⁵⁹Fe incorporation into heme caused by succinylacetone suggesting that the drug inhibits δ-aminolevulinic acid dehydratase in reticulocytes. Succinylacetone, 10 μM, 0.1 and 1 mM, inhibited ⁵⁹Fe incorporation into heme by 50, 90 and 93%, respectively, but stimulated reticulocyte ⁵⁹Fe uptake by about 25–30%. In succinylacetone-treated cells ⁵⁹Fe accumulates in a fraction containing plasma membranes and mitochondria as well as cytosol ferritin and an unidentified low molecular weight fraction obtained by Sephacryl S-200 chromatography. Reincubation of washed succinylacetone- and ⁵⁹Fe-transferrin-pretreated reticulocytes results in the transfer of ⁵⁹Fe from the particulate fraction (plasma membrane plus mitochondria) into hemoglobin and this process is considerably stimulated by added protoporphyrin. Although the nature of the iron accumulated in the membrane-mitochondria fraction in succinylacetone-treated cells is unknown some of it is utilizable for hemoglobin synthesis, while cytosolic ferritin iron would appear to be mostly unavailable for incorporation into heme.     

The onset of hemoglobin synthesis in spleens of irradiated mice after bone marrow transplantation.

Messenger RNA (mRNA) for globin was isolated from spleens of irradiated mice in which erythroid differentiation was induced by a bone marrow graft. The globin mRNA was isolated either by means of sucrose gradients of reticulocyte polysomal RNA or by affinity chromatography of total spleen RNA on poly (U)-sepharose. The globin mRNA was tested in a wheat embryo cell-free system. The appearance of mRNA in the spleen erythroid colonies was correlated with other parameters of erythroid differentiation such as globin synthesis, activity of delta-aminolevulinic acid synthetase and iron uptake. Poly(A) containing mRNA did appear already on the 3rd day after grafting. However, significant translational activity of globin mRNA could be demonstrated only one day later together with the increase in globin synthesis and delta-aminolevulinic acid synthetase and enhanced iron uptake. In the second part of this study mouse spleen cells rich in erythroid elements were incubated with a specific heme synthesis inhibitor (isonicotinic acid hydrazide, INH) and the synthesis of 9 S RNA was estimated. It was found that a 40-minute incubation with INH reduced uridine incorporation into 9 S RNA fraction by about 40%.     

Inhibition of heme synthesis decreases transferrin receptor expression in mouse erythroleukemia cells.

The coordination of transferrin receptor (TfR) expression and heme synthesis was investigated in mouse erythroleukemia (MEL) cells of line 707 treated with heme synthesis inhibitors or in a variant line Fw genetically deficient in heme synthesis. Cells of line 707 were induced for differentiation by 5 mM hexamethylene bisacetamide (HMBA). TfR expression increased in the course of induction, as judged by increased TfR mRNA synthesis, increased cytoplasmic TfR mRNA level, and by the increased number of cellular 125I-Tf binding sites. Addition of 0.1 mM succinylacetone (SA) decreased cellular TfR to the level comparable with the uninduced cells. The decrease was reverted by the iron chelator desferrioxamine (DFO) but not by exogenous hemin. In short-term (1-2 hours) incubation, SA inhibited 59Fe incorporation from transferrin into heme, whereas total cellular 59Fe uptake was increased. A decrease in TfR mRNA synthesis was apparent after 2 hours of SA treatment. Conversely, glutathione peroxidase mRNA synthesis, previously shown to be inducible by iron, was increased by SA treatment. Cells of heme deficient line Fw did not increase the number of Tf binding sites after the induction of differentiation by 5 mM sodium butyrate. SA had no effect on TfR expression in Fw cells. The results suggest that the depletion of cellular non-heme iron due to the increase in heme synthesis maintains a high level of transferrin receptor expression in differentiating erythroid cells even after the cessation of cell division.     

The effects of inhibition of heme synthesis on the intracellular localization of iron in rat reticulocytes

These studies assessed the fate and localization of incoming iron in 6-8-day rat reticulocytes during inhibition of heme synthesis by succinylacetone. Succinylacetone inhibition of heme synthesis increased iron uptake by increasing the rate of receptor recycling without affecting receptor KD for transferrin, transferrin uptake, or total receptor number. Its net effect was to amplify the number of surface transferrin receptors by recruitment of receptors from an intracellular pool. Despite increased iron influx in inhibited cells, only 2-4% of total incoming iron was diverted into ferritin. The majority of incoming iron (65-80%) in succinylacetone-inhibited cells was recovered in the stroma, where ultrastructural and enzymic analyses revealed it to be accumulated mainly in mitochondria. Intramitochondrial iron (70-75%) was localized mainly in the inner membrane fraction. Removal of succinylacetone restored heme synthesis, utilizing iron accumulated within mitochondria for its support. Thus, inhibition of heme synthesis in rat reticulocytes results in accumulation of incoming iron in a functional mobile intramitochondrial precursor iron pool used directly for heme synthesis. Under normal conditions, there is no significant intracellular or intramitochondrial iron pool in reticulocytes, which are therefore dependent upon continuous delivery of transferrin-bound iron to maintain heme synthesis. Ferritin plays an insignificant role in iron metabolism of reticulocytes.     

Iron metabolism in K562 erythroleukemic cells

Iron delivery to K562 cells is enhanced by desferrioxamine through induction of transferrin receptors. Experiments were performed to further characterize this event with respect to iron metabolism and heme synthesis. In control cells, up to 85% of the iron taken up from iron-transferrin was incorporated into ferritin, 7% into heme, and the remainder into compartments not yet identified. In cells grown with desferrioxamine, net accumulation of intracellular desferrioxamine (14-fold) was observed and iron incorporation into ferritin and heme was inhibited by 86% and 75%, respectively. In contrast, complete inhibition of heme synthesis in cells grown with succinylacetone had no effect on transferrin binding or iron uptake. Exogenous hemin (30 microM) inhibited transferrin binding and iron uptake by 70% and heme synthesis by 90%. These effects were already evident after 2 h. Thus, although heme production could be reduced by desferrioxamine, succinylacetone, and hemin, cell iron uptake was enhanced only by the intracellular iron chelator. The effects of exogenous heme are probably unphysiologic and the greater inhibition of iron flow into heme can be explained by effects on early steps of heme synthesis. We conclude that in this cell model a chelatable intracellular iron pool rather than heme synthesis mediates regulation of iron uptake.     

Inhibition of heme biosynthesis in erythroid precursors (BFU-e) isolated from human peripheral blood: effects on globin synthesis

We studied the relationship between heme accumulation and globin synthesis in human erythroid precursors which were stimulated by 2 I.U. of erythropoietin in semi-solid cultures (1% methyl-cellulose, 20% fetal calf serum) and treated with 6-9 micrograms/ml of desferrioxamina (DF), a potent inhibitor of heme synthesis (6). Heme accumulation was detected by specific reaction with benzidine (4), globin synthesis by CM-cellulose column chromatography. Our results demonstrate that globin gene expression occurs in DF-treated erythroid cells which do not accumulate heme molecules. As heme does affect translation and stability of globin mRNA (10) our system might be suitable for studies focused on pathological alterations of erythropoiesis associated with the presence of unstable globin mRNAs and/or unstable globins.     

Control of heme synthesis during Friend cell differentiation: role of iron and transferrin

In many types of cells the synthesis of delta-aminolevulinic acid (ALA) limits the rate of heme formation. However, results from our laboratory with reticulocytes suggest that the rate of iron uptake from transferrin (Tf), rather than ALA synthase activity, limits the rate of heme synthesis in erythroid cells. To determine whether changes occur in iron metabolism and the control of heme synthesis during erythroid cell development Friend erythroleukemia cells induced to erythroid differentiation by dimethylsulfoxide (DMSO) were studied. While added ALA stimulated heme synthesis in uninduced Friend cells (suggesting ALA synthase is limiting) it did not do so in induced cells. Therefore the possibility was investigated that, in induced cells, iron uptake from Tf limits and controls heme synthesis. Several aspects of iron metabolism were investigated using the synthetic iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH). Both induced and uninduced Friend cells take up and utilize Fe for heme synthesis directly from Fe-SIH without the involvement of transferrin and transferrin receptors and to a much greater extent than from saturating levels of Fe-Tf (20 microM). Furthermore, in induced Friend cells 100 microM Fe-SIH stimulated 2-14C-glycine incorporation into heme up to 3.6-fold as compared to the incorporation observed with saturating concentrations of Fe-Tf. In contrast, Fe-SIH, even when added in high concentrations, did not stimulate heme synthesis in uninduced Friend cells but was able to do so as early as 24 to 48 h following induction. In addition, contrary to previous results with rabbit reticulocytes, Fe-SIH also stimulated globin synthesis in induced Friend cells above the level seen with saturating concentrations of transferrin. These results indicate that some step(s) in the pathway of iron from extracellular Tf to protoporphyrin, rather than the activity of ALA synthase, limits and controls the overall rate of heme and possibly hemoglobin synthesis in differentiating Friend erythroleukemia cells.     

Non-transferrin donors of iron for heme synthesis in immature erythroid cells

The mechanism of iron uptake from several iron-containing compounds by transferrin-depleted rabbit reticulocytes and mouse spleen erythroid cells was investigated. Iron complexes of DL-penicillamine, citrate and six different aroyl hydrazones may be utilized by immature erythroid cells for hemoglobin synthesis, although less efficiently than iron from transferrin. HTF-14, a monoclonal antibody against human transferrin, reacts with rabbit transferrin and inhibits iron uptake and heme synthesis by rabbit reticulocytes. HTF-14 had no significant effect on iron uptake and heme synthesis when non-transferrin donors of iron were examined. Ammonium chloride (NH4Cl) increases intracellular pH and blocks the release or utilization of iron from the internalized transferrin. NH4Cl only slightly affected iron incorporation and heme synthesis from non-transferrin donors of iron. Hemin inhibited transferrin iron uptake and heme synthesis, but had a much lesser effect on iron incorporation and heme synthesis from non-transferrin donors of iron. These results allow us to conclude that transferrin-depleted reticulocytes take up iron from all of the examined non-transferrin iron donors without the involvement of the transferrin/transferrin receptor pathway.     

Acquisition of iron from transferrin regulates reticulocyte heme synthesis

Fe-salicylaldehyde isonicotinoylhydrazone (SIH), which can donate iron to reticulocytes without transferrin as a mediator, has been utilized to test the hypothesis that the rate of iron uptake from transferrin limits the rate of heme synthesis in erythroid cells. Reticulocytes take up 59Fe from [59Fe]SIH and incorporate it into heme to a much greater extent than from saturating concentrations of [59Fe]transferrin. Also, Fe-SIH stimulates [2-14C]glycine into heme when compared to the incorporation observed with saturating levels of Fe-transferrin. In addition, delta-aminolevulinic acid does not stimulate 59Fe incorporation into heme from either [59Fe]transferrin or [59Fe]SIH but does reverse the inhibition of 59Fe incorporation into heme caused by isoniazid, an inhibitor of delta-aminolevulinic acid synthase. Taken together, these results suggest the hypothesis that some step(s) in the pathway of iron from extracellular transferrin to intracellular protoporphyrin limits the overall rate of heme synthesis in reticulocytes.     

Inhibition of mRNA synthesis in rabbit bone marrow nuclei in vitro by quinone metabolites of benzene

mRNA synthesis by rabbit bone marrow nuclei has been shown to be inhibited by the quinone metabolites of benzene, hydroquinone and p-benzoquinone, in a concentration-dependent manner with 50% inhibitory concentration (IC50[M]) for both compounds of 6 X 10(-6) M. Catechol and 1,2,4-benzenetriol also showed a concentration-dependent inhibition of synthesis, however, 50% inhibition was not reached by 10(-4) M. Phenol did not inhibit mRNA synthesis even at 10(-3) M. It is possible that myelotoxicity from benzene might result from such an inhibition of mRNA synthesis by quinone metabolites in pluripotent and/or committed bone marrow stem cells.     

Evidence supporting a physiological role for the hemin-controlled translational repressor of globin synthesis in reticulocytes.

The synthesis of heme and globin in rabbit reticulocytes was compared at 35 and 25 degrees C. The lower temperature decreased heme synthesis significantly more than globin synthesis and resulted in a much greater accumulation of globin dimers. After 16 h of incubation in the absence of iron, globin synthesis in reticulocytes which were at 35 degrees C could not be stimulated by iron, whereas cells which were at 25 degrees C responded with nearly control levels of globin synthesis. Since the formation of the hemin-controlled translational repressor in reticulocyte lysates is also decreased much more than protein synthesis at reduced temperature the results provide evidence for a physiological role for the translational repressor in controlling globin synthesis in reticulocytes.     

Heterogeneity of globin protein synthesis in bone marrow cells of patients with homozygous beta-thalassemia from Tadzhikistan

The synthesis of globin proteins in blood reticulocytes of homozygous beta-thalassemic patients from Tadzhikistan has been previously studied. beta-thalassemia with sharp repression of beta-globin protein synthesis (alpha/beta greater than 10) has been shown to be most representative for the region. In this work, the synthesis of globin proteins has been studied in bone marrow cells of homozygous beta-thalassemic patients. Comparison of data on globin synthesis in bone marrow cells and in blood reticulocytes of the patients has revealed that in some cases the disbalance of chain synthesis in both cell types is equal. In other cases the disbalance in bone marrow cells is less than in blood cells, indicating the instability of beta-globin mRNA that is partially degrading in the process of cell maturation. Homozygous beta-thalassemic cases with low content of Hb F in blood cells (5-10%), with substantial disbalance of alpha and beta-globin synthesis and marked production of gamma-globins in bone marrow cells and in blood reticulocytes are of special interest. It has been assumed that parallel to beta-thalassemia some instability of gamma-globin proteins takes place in these patients.     

Inhibition of heme synthesis induces apoptosis in human erythroid progenitor cells

Heme synthesis by erythroid progenitor cells is maintained by erythropoietin (EP), insulin-like growth factor-I (IGF-I), and stem cell factor (SCF), and without these growth factors apoptosis (programmed cell death) occurs. To clarify the possible interaction between heme synthesis and programmed cell death of human erythroid progenitor cells, the effect of specific inhibition of heme synthesis on apoptosis of highly purified human erythroid colony forming cells (ECFC) was studied. When the amount of uncleaved DNA was determined as a measure of apoptosis, the heme synthesis inhibitors, succinylacetone (SA) (0.1 mmol/L) or isonicotinic acid hydrazide (INH) (10 mmol/L), significantly decreased the amount of uncleaved DNA (P < 0.01) in the presence of erythropoietin (EP). Addition of recombinant heavy-chain ferritin (rHF) (10 nmol/L), or deprivation of transferrin from the culture medium, which decreased heme synthesis, also reduced the amount of uncleaved DNA (P < 0.01). The production of apoptosis by diverse inhibitors of heme synthesis was in each case reversed by the addition of hemin (0.1 mmol/L) and did not occur with HL-60 cells. When the colony-forming capacity of ECFC was determined by plasma clot assay, SA, INH, or rHF reduced the number of CFU-E (P < 0.01), and the effect of SA was reversed by hemin. The addition of SA did not alter the c-myc response of ECFC to EP. These data indicate that inhibition of heme synthesis induces apoptosis of human erythroid progenitor cells, in a manner independent of an early c-myc response, and suggest that the presence of apoptosis in ineffective erythropoiesis may be secondary to impaired heme synthesis. © 1995 Wiley-Liss, Inc.     

Succinylacetone, a potent inhibitor of heme biosynthesis: effect on cell growth, heme content and delta-aminolevulinic acid dehydratase activity of malignant murine erythroleukemia cells.

4,6-Dioxoheptanoic acid (succinylacetone, SA) was examined with regard to its ability to a) inhibit the second enzyme of the heme pathway, δ-aminolevulinic acid (ALA) dehydratase, b) lower the heme concentration, and c) inhibit cell growth of murine erythroleukemia (MEL) cells in culture. SA profoundly inhibited ALA dehydratase in broken cell preparations at concentrations as low as 10^?7 M. The stimulation of hemoglobin production by DMSO and butyrate in MEL cells was inhibited by the addition of SA to the cell medium. When 1 mM SA was added to the medium, there was a profound inhibition of ALA dehydratase activity, and the heme concentration of cells declined progressively with each cell division. Cell growth was markedly inhibited after two cell divisions.     

The effect of heme on intracellular iron pools during differentiation of friend erythroleukemia cells

The inhibition of cellular iron uptake by hemin described previously in reticulocytes was studied in murine erythroleukemia (Friend) cells that can be induced to differentiate in culture by dimethyl sulfoxide (DMSO). Hemin had no effect on iron uptake into noninduced cells. After the induction by DMSO, hemin inhibited iron uptake into Friend cells and this effect of hemin became more pronounced with the further progress of differentiation. The reduction of cellular iron accumulation was caused mainly by inhibition of iron incorporation into heme, iron uptake into the non-heme pool was influenced by hemin treatment. Inhibition of heme synthesis by isonicotinic acid hydrazide (INH) caused an accumulation of iron in mitochondria in DMSO-induced cells but not in uninduced cells. On the basis of these results, a specific system transporting iron to mitochondria induced by DMSO treatment is suggested as a target for the inhibitory action of hemin. In Friend cells of the Fw line which are deficient in ferrochelatase, heme has no effect on iron uptake. The addition of INH to the Fw cells does not enhance the iron accumulatoni in mitochondria.     

The effect of cysteine and 2,4-dinitrophenol on heme and nonheme absorption in a rat intestinal model

Previous studies have showed that purified heme iron forms insoluble polymers that are poorly absorbed. The presence of peptides and of amino acids maintaining heme iron in a soluble form could improve its bioavailability. The digestive uptake and transfer of a concentrated hydrolysate of heme peptides (HPH) and of iron gluconate (Gluc) at 100 μM were compared in vitro in a Ussing chamber. The effects of an enhancing amino acid (L-cysteine) on the uptake and transfer of both forms were assessed. An inhibitor of the oxidative phosphorylation (2,4-dinitrophenol; DNP) was used to differentiate the active and passive mechanisms of the absorption. The mucosal uptake (%Tot) and enterocyte transfer (%S) of the two sources of iron did not differ. DNP significantly reduced %Tot and %S of both forms. Cysteine significantly enhanced %Tot and %S of HPH and Gluc, partly corrected the inhibition exerted by DNP on %Tot of HPH and %S of both forms, and fully restored %Tot of Gluc. In presence of peptides produced by globin hydrolysis, the absorption of hemoglobin iron was efficient; it was mostly energy dependent and, therefore, should have occurred by a regulated transcellular pathway. Cysteine enhanced the passive uptake of iron and the passive processes involved in the enterocyte transfer of the common pool made of both sources (heme and nonheme) of iron. These results showed that heme iron can be purified and concentrated without impairing its digestive absorption, provided it remains in presence of peptides or amino acids.     

Increase in globin chains and globin mRNA in erythroleukemia cells in response to hemin.

Addition of 50 μm hemin to mouse erythroleukemia cells cultured in 0.5% dimethyl-sulfoxide (DMSO) resulted in >10-fold stimulation of globin chain synthesis as a percentage of acid precipitable protein. In cultures fully induced with 1.5% DMSO, addition of 15 mm 3-amino-1,2,4-triazole (AT), an inhibitor of heme synthesis, reduced globin chain synthesis to uninduced levels and reduced globin mRNA levels to less than 20% of induced values. The inhibition of AT was prevented by simultaneous addition of 25 μm hemin to the cultures. Using RNA-DNA hybridization analysis, the amount of globin mRNA sequences as a fraction of total cytoplasmic RNA was also increased by addition of 50 μm hemin to cultures with 0.5% DMSO. The results suggest that exogenous hemin can promote globin chain synthesis, that endogenously synthesized heme can be required for globin chain synthesis, and that hemin directly or indirectly also alters the appearance or degradation of globin mRNA sequences in the cytoplasm.     

The role of heme in the regulation of the late program of Friend cell erythroid differentiation.

The addition of a chemical inducer, such as dimethylsulfoxide (DMSO), to cultures of mouse Friend erythroleukemic cells results in the induction of a number of late erythroid events, including the accumulation of globin mRNA, the inducation of hemoglobin synthesis, the appearance of erythrocyte membrane antigens (EMA), and the cessation of cell division. The experiments presented in this study demonstrate that heme is necessary but not sufficient for the loss of proliferative capacity associated with DMSO-induced Friend cell differentiation, whereas the accumulation of globin mRNA and EMA can occur in the absence of heme synthesis or heme itself. These conclusions were reached by selectively inhibiting heme synthesis in DMSO-treated cells in two independent ways: (i) Inducible cells were treated with 3-amino-1,2,4-triazole (AT), a drug which inhibits the induction of heme synthesis in Friend cells in a dose-dependent manner. Treatment of inducible Friend cells with 1.5% DMSO for five days caused the plating efficiency in methyl cellulose to decrease to 1% of that in untreated cultures. However, treatment of the cells with DMSO plus AT almost totally prevented this decrease in plating efficiency. The addition of exogenous hemin, which alone had no significant effect on plating efficiency, largely reversed the effect of AT in DMSO-treated cells, reducing the plating efficiency to below 5%. In contrast to the marked effects of AT on the proliferative capacity of differentiating Friend cells, the levels of globin mRNA and EMA were only partially decreased in cells treated with DMSO plus AT, compared to cells treated with DMSO alone. (ii) The relationship between heme synthesis, terminal cell division, and the induction of globin mRNA was investigated further through the use of non-inducible Friend cell variant clones. One such non-inducible clone, M18, appears to be a phenotypic analog of inducible cells treated with DMSO plus AT. Clone M18 did not accumulate heme or hemoglobin, as detected by benzidine staining, nor lose its proliferative capacity in response to DMSO. However, globin mRNA was induced by DMSO in this clone. Treatment of clone M18 with DMSO plus hemin overcame the block in hemoglobin accumulation suggesting that M18 has a defect in the induction of heme biosynthesis. In addition, exposure of M18 cells to DMSO plus hemin caused a gradual decrease in plating efficiency which was not due to non-specific toxicity. Prior incubation of M18 cells in DMSO for three to five days was necessary before hemin caused a rapid loss of proliferative capacity. Thus, these results, in agreement with the AT studies on inducible Friend cells and previous studies on the induction of EMA in clone M18, indicate that there may be both heme-dependent and heme-independent events in the program of Friend cell differentiation.     

Characterization of a human plasma membrane heme transporter in intestinal and hepatocyte cell lines

Heme is the most bioavailable form of dietary iron and a component of many cellular proteins. Controversy exists as to whether heme uptake occurs via specific transport mechanisms or passive diffusion. The aims of this study were to quantify cellular heme uptake with a fluorescent heme analog and to determine whether heme uptake is mediated by a heme transporter in intestinal and hepatic cell lines. A zinc-substituted porphyrin, zinc mesoporphyrin (ZnMP), was validated as a heme homolog in uptake studies of intestinal (Caco-2, I-407) and hepatic (HepG2) cell lines. Uptake experiments to determine time dependence, heme inhibition, concentration dependence, temperature dependence, and response to the heme synthesis inhibitor succinylacetone were performed. Fluorescence microscope images were used to quantify uptake and determine the cellular localization of ZnMP; ZnMP uptake was seen in intestinal and hepatic cell lines, with cytoplasmic uptake and nuclear sparing. Uptake was dose- and temperature dependent, inhibited by heme competition, and saturated over time. Preincubation with succinylacetone augmented uptake, with an increased initial uptake rate. These findings establish a new method for quantifying heme uptake in individual cells and provide strong evidence that this uptake is a regulated, carrier-mediated process.