Transferrin receptor regulation is coupled to intracellular ferritin in proliferating and differentiating HL60 leukemia cells

Cultured myeloid leukemia cells display transferrin receptors but decrease receptor display after differentiation induction or accumulation of intracellular iron. To determine whether regulation of transferrin receptors and ferritin were linked under these disparate conditions, serum-free and fetal bovine serum (FBS) cultures of HL60 promyelocytic leukemia cells were used to investigate relationships between transferrin receptor display and intracellular ferritin. Using 125I-transferrin binding and immunofluorescence staining for transferrin receptors, HL60 cells cultured in serum-free, transferrin-free medium expressed fewer transferrin receptors and contained increased ferritin when compared to cells cultured with FBS or transferrin supplemented, serum-free medium. When placed in medium containing transferrin, cells previously grown in transferrin-free medium rapidly re-expressed transferrin receptors and decreased their ferritin content. HL60 cells induced to differentiate into granulocytes or macrophages also decreased transferrin receptor display and increased their ferritin content. Transferrin receptor display and ferritin content in both proliferating and differentiating myeloid leukemia cells are inversely related and their regulation is closely linked. Regulation of transferrin receptor display and ferritin synthesis may be important events regulating myeloid cell growth and differentiation.     

Cell cycle effects of iron depletion on T-47D human breast cancer cells

T-47D human breast cancer cells grown in culture medium containing low concentrations of fetal calf serum (FCS) proliferated very slowly, with an accumulation of cells in the G2 phase of the cell cycle, increased polyploid cells, and increased expression of transferrin receptors. Cell proliferation was stimulated by the addition of human transferrin or ammonium ferric citrate to the medium. Growth inhibition and accumulation of G2-phase cells could also be produced in T-47D cells grown in medium containing 10% FCS by the addition of the iron chelator, desferrioxamine. It is concluded that cellular deprivation of iron and/or transferrin is the major cause of reduced proliferation rates and G2-phase arrest which accompany the culture of these cells in medium supplemented with low concentrations of FCS.     

Transferrin receptor 2-alpha supports cell growth both in iron-chelated cultured cells and in vivo

In most cells, transferrin receptor (TfR1)-mediated endocytosis is a major pathway for cellular iron uptake. We recently cloned the human transferrin receptor 2 (TfR2) gene, which encodes a second receptor for transferrin (Kawabata, H., Yang, R., Hirama, T., Vuong, P. T., Kawano, S., Gombart, A. F., and Koeffler, H. P. (1999) J. Biol. Chem. 274, 20826-20832). In the present study, the regulation of TfR2 expression and function was investigated. A select Chinese hamster ovary (CHO)-TRVb cell line that does not express either TfR1 or TfR2 was stably transfected with either TfR1 or TfR2-alpha cDNA. TfR2-alpha-expressing cells had considerably lower affinity for holotransferrin when compared with TfR1-expressing CHO cells. Interestingly, in contrast to TfR1, expression of TfR2 mRNA in K562 cells was not up-regulated by desferrioxamine (DFO), a cell membrane-permeable iron chelator. In MG63 cells, expression of TfR2 mRNA was regulated in the cell cycle with the highest expression in late G(1) phase and no expression in G(0)/G(1). DFO reduced cell proliferation and DNA synthesis of CHO-TRVb control cells, whereas it had little effect on TfR2-alpha-expressing CHO cells when measured by clonogenic and cell cycle analysis. In addition, CHO cells that express TfR2-alpha developed into tumors in nude mice whereas CHO control cells did not. In conclusion, TfR2 expression may be regulated by the cell cycle rather than cellular iron status and may support cell growth both in vitro and in vivo.     

Regulation of K562 cell transferrin receptors by exogenous iron

Single-cell analysis of K562 human erythroleukemia cells by flow cytometry was used to demonstrate the specific role of iron in regulating transferrin receptors (TfRs) and to establish that TfR expression does not necessarily correlate with growth rate. Exogenous iron concentration in culture was manipulated by supplementing the medium with sera having different iron concentrations over the range 0.6 to 5.4 micrograms/ml, by the addition of iron in the form of FeCl3, iron-saturated serum, or diferric transferrin, and by the addition of the iron chelator Desferal (desferrioxamine). TfR expression was negatively correlated with exogenous iron content: any treatment that reduced exogenous iron supply by at least 15% resulted in as much as a 1.8-fold increase in external receptors, detected as binding by both transferrin and monoclonal anti-TfR antibodies, and a 1.5-fold increase in the pool of internal receptors, as detected by anti-TfR antibody binding. None of these treatments altered growth rate, total cellular protein content, protein synthetic rate, cell cycle distribution or cell size. The rapid (12 hr) and reversible induction of internal and external receptors by Desferal was inhibited by cycloheximide and therefore may have resulted from de novo synthesis and not just mobilization of internal receptor pool to the cell surface. The correlation between growth rate and TfR expression previously observed in these and other cells must be secondary to cellular mechanisms that maintain intracellular iron pools by regulating synthesis, recycling, and cell surface expression of TfRs.     

Relationships between the cell cycle and the expression of c-myc and transferrin receptor genes during induced myeloid differentiation

We examined the relationship of cellular oncogene c-myc and transferrin receptor (TfR) gene expression to cell proliferation and cell cycle progression during myeloid differentiation in the HL-60 myeloid leukemia cell line. In order to determine levels of mRNA for these genes in HL-60 cells induced to differentiate along the myeloid pathway, RNA was isolated from HL-60 cells incubated with retinoic acid for 24 h and Northern blots were probed with labeled cDNAs for c-myc and TfR. c-myc mRNA decreased within 3 h of retinoic acid addition, and TfR mRNA decreased after 9 h; both mRNAs continued to decrease over 24 h. RNA was also isolated from HL-60 cells separated by centrifugal elutriation into cell cycle phases. TfR and c-myc cDNA probes hybridized equally to RNA from uninduced cells in all phases of the cell cycle. However, after 24 h incubation with the differentiation inducer retinoic acid, TfR mRNA was expressed substantially less in the G1 stage, whereas c-myc mRNA was still expressed equally in all cell cycle phases. These data indicate that, although TfR and c-myc expression are both associated with cell proliferation in the HL-60 line, TfR is down-regulated specifically in G1 upon induction of terminal differentiation whereas c-myc expression is disassociated from cell cycle control in these cells.     

Enhancement of cytotoxicity of artemisinins toward cancer cells by ferrous iron

Iron(II) heme-mediated activation of the peroxide bond of artemisinins is thought to generate the radical oxygen species responsible for their antimalarial activity. We analyzed the role of ferrous iron in the cytotoxicity of artemisinins toward tumor cells. Iron(II)-glycine sulfate (Ferrosanol) and transferrin increased the cytotoxicity of free artesunate, artesunate microencapsulated in maltosyl-beta-cyclodextrin, and artemisinin toward CCRF-CEM leukemia and U373 astrocytoma cells 1.5- to 10.3-fold compared with that of artemisinins applied without iron. Growth inhibition by artesunate and ferrous iron correlated with induction of apoptosis. Cell cycle perturbations by artesunate and ferrous iron were not observed. Treatment of p53 wild-type TK6 and p53 mutated WTK1 lymphoblastic cells showed that mutational status of the tumor suppressor p53 did not influence sensitivity to artesunate. The effect of ferrous iron and transferrin was reversed by monoclonal antibody RVS10 against the transferrin receptor (TfR), which competes with transferrin for binding to TfR. CCRF-CEM and U373 cells expressed TfR in 95 and 48% of the cell population, respectively, whereas TfR expression in peripheral mononuclear blood cells of four healthy donors was confined to 0.4-1.3%. This indicates that artemisinins plus ferrous iron may affect tumor cells more than normal cells. The IC(50) values for a series of eight different artemisinin derivatives in 60 cell lines of the U.S. National Cancer Institute were correlated with the microarray mRNA expression of 12 genes involved in iron uptake and metabolism by Kendall's tau test to identify iron-responsive cellular factors enhancing the activity of artemisinins. This pointed to mitochondrial aconitase and ceruloplasmin (ferroxidase).     

Growth of human tumor cell lines in transferrin-free,low-iron medium

Summary Iron is essential for tumor cell growth. Previous studies have demonstrated that apart from transferrin-bound iron uptake, mammalian cells also possess a transport system capable of efficiently obtaining iron from small molecular weight iron chelates (Sturrock et al., 1990). In the present study, we have examined the ability of tumor cells to grow in the presence of low molecular weight iron chelates of citrate. In chemically defined serum-free medium, most human tumor cell lines required either transferrin (5 μg/ml) or a higher concentration of ferric citrate (500 μM) as an iron source. However, we have also found that from 13 human cell lines tested, 4 were capable of long-term growth in transferrin-free medium with a substantially lower concentration of ferric citrate (5 μM). When grown in medium containing transferrin, both regular and low-iron dependent cell lines use transferrin-bound iron. Growth of both cell types in transferrin medium was inhibited to a certain degree by monoclonal antibody 42/6, which specifically blocks the binding of transferrin to the transferrin receptor. On the contrary, growth of low-iron dependent cell lines in transferrin-free, low-iron medium (5 μM ferric citrate) could not be inhibited by monoclonal antibody 42/6. Furthermore, no autocrine production of transferrin was observed. Low-iron dependent cell lines still remain sensitive to iron depletion as the iron(III) chelator, desferrioxamine, inhibited their growth. We conclude that low-iron dependent tumor cells in transferrin-free, low-iron medium may employ a previously unknown mechanism for uptake of non-transferrin-bound iron that allows them to efficiently use low concentrations of ferric citrate as an iron source. The results are discussed in the context of alternative iron uptake mechanisms to the well-characterized receptor-mediated endocytosis process.     

Release of the soluble transferrin receptor is directly regulated by binding of its ligand ferritransferrin

The human transferrin receptor (TfR) is shed by an integral metalloprotease releasing a soluble form (sTfR) into serum. The sTfR reflects the iron demand of the body and is postulated as a regulator of iron homeostasis via binding to the hereditary hemochromatosis protein HFE. To study the role of transferrin in this process, we investigated TfR shedding in HL60 cells and TfR-deficient Chinese hamster ovary cells transfected with human TfR. Independent of TfR expression, sTfR release decreases with increasing ferritransferrin concentrations, whereas apo-transferrin exhibits no inhibitory effect. To investigate the underlying mechanism, we generated several TfR mutants with different binding affinities for transferrin. Shedding of TfR mutants in transfected cells correlates exactly with their binding affinity, implying that the effect of ferritransferrin on TfR shedding is mediated by a direct molecular interaction. Analysis of sTfR release from purified microsomal membranes revealed that the regulation is independent from intracellular trafficking or cellular signaling events. Our results clearly demonstrated that sTfR does not only reflect the iron demand of the cells but also the iron availability in the bloodstream, mirrored by iron saturation of transferrin, corroborating the important potential function of sTfR as a regulator of iron homeostasis.     

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.     

Iron chelation in cell cultures by two conjugates of 2, 3 - dihydroxybenzoic acid (2, 3 - DHB)

A cell culture system has been used for screening iron chelating compounds for therapeutic use in patients with iron overload. Two conjugates of 2, 3 - dihydroxybenzoic acid (2, 3 - DHB) with spermidine are more effective than desferrioxamine in removing iron from cells and, in addition, are capable of removing iron from transferrin.     

A 41 kDa transferrin related molecule acts as an autocrine growth factor for HL-60 cells

HL-60 cells produce an autostimulatory growth factor. Since the stimulatory effect of HL-60 conditioned medium is only observed in the absence of exogenous transferrin we have assayed HL-60 cells for the production of transferrin and found that they produce polypeptides which react with transferrin antibodies. 35S-methionine labelling, immunoprecipitation and subsequent separation by SDS-gel electrophoresis reveals the presence of a major transferrin related 41 +/- 2 kDa species released by HL-60 cells. Physiological levels of iron salts completely abolish the requirement of exogenous transferrin which indicates that the endogenous transferrin related polypeptides in the presence of exogenous inorganic iron salts are sufficient for the proliferation of HL-60 cells provided insulin or related growth factors are present. The addition of transferrin receptor antibodies inhibits the stimulatory action of the endogenous transferrin related activity.     

The influence of cell growth media on the stability and antitumour activity of methionine enkephalin.

Studies with cultured tumour cell lines are widely used in vitro to evaluate peptide-induced cytotoxicity as well as molecular and biochemical interactions. The objectives of this study were to investigate the influence of the cell culture medium on peptide metabolic stability and in vitro antitumour activity. The degradation kinetics of the model peptide methionine enkephalin (Met-E, Tyr-Gly-Gly-Phe-Met), demonstrated recently to play an important role in the rate of proliferation of tumour cells in vitro and in vivo, were investigated in cell culture systems containing different amounts of fetal bovine serum (FBS). The influence of enzyme inhibitors (bestatin, captopril, thiorphan) on the Met-E degradation was also investigated. The results obtained in the Dulbecco's modified Eagle medium containing 10% FBS indicated a rapid degradation of Met-E (t(1/2) = 2.8 h). Preincubation of the medium with a mixture of peptidase inhibitors reduced the hydrolysis of Met-E, as shown by the increased half-life to 10 h. The in vitro activity of Met-E against poorly differentiated cells from lymph node metastasis of colon carcinoma (SW620) and human larynx carcinoma (HEp-2) cells was determined. Tumour cells were grown for 3 weeks prior to the experiment in a medium supplemented with 10%, 5% or 2% FBS. Statistically significant to mild or no suppression of cell proliferation was observed in all cultures. In both cell lines, a significant suppression of cell growth by a combination of peptidase inhibitors and Met-E, compared with cells exposed to the peptide alone and cells grown in the absence of Met-E, was observed. This study indicated that caution must be exercised in interpreting the antiproliferative effects of peptide compounds in conventional drug-response assays.     

Replacement of serum by insulin and transferrin supports growth and differentiation of the human promyelocytic cell line, HL-60

The human promyelocytic leukemia cell line HL-60 can be grown in serum-free synthetic medium supplemented with insulin and transferrin alone. Growth of HL-60 in this defined medium is at a rate approx. 80% of that in medium containing serum. Moreover, the distinct morphological and histochemical myeloid characteristics of HL-60 are maintained in such serum-free medium. The HL-60 promyelocytes are induced by DMSO to differentiate to mature granulocytes equally well in both serum-supplemented and serum-free medium. However, this defined medium does not support colony growth of HL-60 in semi-solid medium such as methylcellulose.     

Sodium ascorbate (vitamin C) induces apoptosis in melanoma cells via the down-regulation of transferrin receptor dependent iron uptake

Sodium ascorbate (vitamin C) has a reputation for inconsistent effects upon malignant tumor cells, which vary from growth stimulation to apoptosis induction. Melanoma cells were found to be more susceptible to vitamin C toxicity than any other tumor cells. The present study has shown that sodium ascorbate decreases cellular iron uptake by melanoma cells in a dose- and time-dependent fashion, indicating that intracellular iron levels may be a critical factor in sodium ascorbate-induced apoptosis. Indeed, sodium ascorbate-induced apoptosis is enhanced by the iron chelator, desferrioxamine (DFO) while it is inhibited by the iron donor, ferric ammonium citrate (FAC). Moreover, the inhibitory effects of sodium ascorbate on intracellular iron levels are blocked by addition of transferrin, suggesting that transferrin receptor (TfR) dependent pathway of iron uptake may be regulated by sodium ascorbate. Cells exposed to sodium ascorbate demonstrated down-regulation of TfR expression and this precedes sodium ascorbate-induced apoptosis. Taken together, sodium ascorbate-mediated apoptosis appears to be initiated by a reduction of TfR expression, resulting in a down-regulation of iron uptake followed by an induction of apoptosis. This study demonstrates the specific mechanism of sodium ascorbate-induced apoptosis and these findings support future clinical trial of sodium ascorbate in the prevention of human melanoma relapse.     

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.     

The macrophage cell surface glyceraldehyde-3-phosphate dehydrogenase is a novel transferrin receptor

The reticuloendothelial system plays a major role in iron metabolism. Despite this, the manner in which macrophages handle iron remains poorly understood. Mammalian cells utilize transferrin-dependent mechanisms to acquire iron via transferrin receptors 1 and 2 (TfR1 and TfR2) by receptor-mediated endocytosis. Here, we show for the first time that the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is localized on human and murine macrophage cell surface. The expression of this surface GAPDH is regulated by the availability of iron in the medium. We further demonstrate that this GAPDH interacts with transferrin and the GAPDH-transferrin complex is subsequently internalized into the early endosomes. Our work sheds new light on the mechanisms involved in regulation of iron, vital for controlling numerous diseases and maintaining normal immune function. Thus, we propose an entirely new avenue for investigation with respect to transferrin uptake and regulation mechanisms in macrophages.     

Transferrin receptor expression and the regulation of placental iron uptake

Placental transferrin receptors, located at the apical side of syncytiotrophoblast, mediate placental iron uptake. Regulation of transferrin receptors on the fetal-maternal exchange area could be a major determinant in the regulation of trans-placental iron transport.Transferrin receptor expression in cultured human term cytotrophoblasts is on a much lower level than in choriocarcinoma cells, with a higher proportion of receptors located on the cell surface. Differentiation of cells, either due to longer culture periods or to 8-bromo-cAMP treatment does not lead to an increase of transferrin receptor expression. In vitro, the level of expression is largely regulated by the cellular density in the culture dishes. Low cellular occupancy of the dish leads to a high level of transferrin receptors. Treatment with iron-sources results in a down regulation of transferrin receptors.Thus, though the level of transferrin receptors in cultured normal trophoblast is at a constant level, unaffected by differentiation, high levels of maternal transferrin-iron availability can lead to a decrease in placental iron uptake. This feed-back mechanism makes placental iron uptake independent of maternal iron stores.Abbreviations hCG human Chorionic Gonadotrophin - TfR Transferrin Receptor     

Differential regulation of iron regulatory element-binding protein(s) in cell extracts of activated lymphocytes versus monocytes-macrophages.

The intracellular iron level exerts a negative feedback on transferrin receptor (TfR) expression in cells requiring iron for their proliferation, in contrast to the positive feedback observed in monocytes-macrophages. It has been suggested recently that modulation of TfR and ferritin synthesis by iron is mediated through a cytoplasmic protein(s) (iron regulatory element-binding protein(s) (IRE-BP)), which interacts with ferritin and TfR mRNA at the level of hairpin structures (IRE), thus leading to inhibition of transferrin mRNA degradation and repression of ferritin mRNA translation. In the present study we have evaluated in parallel the level of TfR expression, ferritin, and IRE-BP in cultures of: (i) circulating human lymphocytes stimulated to proliferate by phytohemagglutinin (PHA) and (ii) circulating human monocytes maturing in vitro to macrophages. The cells were grown in either standard or iron-supplemented culture. TfR and ferritin expression was evaluated at both the protein and mRNA level. IRE-BP activity was measured by gel retardation assay in the absence or presence of beta-mercaptoethanol (spontaneous or total IRE-BP activity, respectively). Spontaneous IRE-BP activity, already present at low level in quiescent T lymphocytes, shows a gradual and marked increase in PHA-stimulated T cells from day 1 of culture onward. This increase is directly and strictly correlated with the initiation and gradual rise of TfR expression, which is in turn associated with a decrease of ferritin content. Both the rise of TfR and spontaneous IRE-BP activity are completely inhibited in iron-supplemented T cell cultures. In contrast, the total IRE-BP level is similar in both quiescent and PHA-stimulated lymphocytes, grown in cultures supplemented or not with iron salts. Monocytes maturing in vitro to macrophages show a sharp increase of spontaneous and, to a lesser extent, total IRE-BP; the addition of iron moderately stimulates the spontaneous IRE-BP activity but not the total one. Here again, the rise of spontaneous IRE-BP from very low to high activity is strictly related to the parallel increase of TfR expression and, suprisingly, also with a very pronounced rise of ferritin expression observed at both the mRNA and protein level. It is noteworthy the effect of beta-mercaptoethanol is cell specific, i.e. the ratio of total versus spontaneous IRE-BP activity is different in activated lymphocytes and maturing monocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of transferrin receptor expression by inhibitors of nucleic acid synthesis

We investigated the effects of the iron chelator desferrioxamine on the expression of transferrin receptors (TfR) by CCRF-CEM human T-cell leukaemia and B16 mouse melanoma cells growing in tissue culture. Desferrioxamine (DFOA) enhanced TfR expression when added in the dose range of 10(-5)-10(-4) to CCRF-CEM cells, but was toxic to these cells, the lower concentrations producing a slowing of cell growth with a build up in S-phase, while higher concentrations caused cell death with a block at the G1/S-phase interface. These toxic effects are compatible with its previously reported inhibition of the non-haem iron containing (M2) subunit of ribonucleotide reductase. In marked contrast, DFOA caused the growth of B16 melanoma cells to arrest in G1, without loss of cloning efficiency, and resulted in a fall in TfR expression to approximately 50% of control values. These results suggested that the effects of DFOA on TfR expression were linked to DNA synthesis rather than to a more generalised inhibition of iron-dependent cellular processes. It was subsequently found that inhibition of the M2 subunit of ribonucleotide reductase in CCRF-CEM cells with 5 X 10(-5) M hydroxyurea, which is not an iron chelator, also enhanced TfR expression, as did thymidine and cytosine arabinoside, which have different enzyme targets. By measuring cellular DNA and RNA content simultaneously it was shown that all of these agents caused unbalanced growth, i.e., inhibited DNA synthesis more than RNA synthesis. In contrast, 6-thioguanine was more inhibitory to RNA synthesis, and treatment with this drug caused a fall in TfR expression. Thus, although CCRF-CEM cells treated with DFOA show enhanced TfR expression, similar effects are also seen with other inhibitors of DNA synthesis, provided that RNA synthesis is allowed to continue. These results provide further evidence that the regulation of TfR expression by proliferating cells is specifically linked to DNA synthesis rather than to the iron requirements of other cellular processes.     

Characterization of glyceraldehyde-3-phosphate dehydrogenase as a novel transferrin receptor

A majority of cells obtain of transferrin (Tf) bound iron via transferrin receptor 1 (TfR1) or by transferrin receptor 2 (TfR2) in hepatocytes. Our study establishes that cells are capable of acquiring transferrin iron by an alternate pathway via GAPDH.These findings demonstrate that upon iron depletion, GAPDH functions as a preferred receptor for transferrin rather than TfR1 in some but not all cell types. We utilized CHO-TRVb cells that do not express TfR1 or TfR2 as a model system. A knockdown of GAPDH in these cells resulted in a decrease of not only transferrin binding but also associated iron uptake. The current study also demonstrates that, unlike TfR1 and TfR2 which are localized to a specific membrane fraction, GAPDH is located in both the detergent soluble and lipid raft fractions of the cell membrane. Further, transferrin uptake by GAPDH occurs by more than one mechanism namely clathrin mediated endocytosis, lipid raft endocytosis and macropinocytosis. By determining the kinetics of this pathway it appears that GAPDH-Tf uptake is a low affinity, high capacity, recycling pathway wherein transferrin is catabolised. Our findings provide an explanation for the detailed role of GAPDH mediated transferrin uptake as an alternate route by which cells acquire iron.