Treatment of K562 cells with desferrioxamine, a permeable iron chelator, led to an increase in the number of transferrin receptors. Increasing intracellular iron levels by treatment of cells with either human diferric transferrin or hemin lowered the level of the transferrin receptors. By using a cDNA clone of the human transferrin receptor, we showed that the changes in the levels of the receptor by iron were accompanied by alterations in the levels of the mRNA for the receptor. The rapidity of these changes indicated that the mRNA had a very short half-life. By using an in vitro transcriptional assay with isolated nuclei, we obtained evidence that this regulation occurred at the transcriptional level.All proliferating cells acquire iron via transferrin (Tf), a serum glycoprotein that binds to a specific receptor on the cell surface. The receptor-Tf complex is internalized by a process of receptor-mediated endocytosis in which the iron is dissociated from the Tf (3). Investigators have previously shown that the levels of intracellular iron regulate the level of expression of the Tf receptor (6, 8,9,14). Raising the levels of intracellular iron by treatment of K562 cells (a human erythroleukemic cell line) with either human diferric Tf or ferric ammonium citrate results in decreased levels of the receptor. Conversely, treatment with desferrioxamine, a permeable iron chelator, leads to marked elevations in receptor levels. These changes in cellular content of the Tf receptor through manipulation of intracellular iron levels are caused by altered rates of biosynthesis of the receptor. Corresponding changes in the levels of translatable mRNA for the receptor were measured by in vitro translation (9).Recently, cDNA clones for the human Tf receptor have been isolated (4,12). By using one such clone, we showed that the alterations of the biosynthetic rate of the receptor produced by manipulations of iron levels were caused by changes in the levels of the mRNA for the receptor. The modulation of the expression of the Tf receptor gene provided an important example of the specific feedback regulation of a eucaryotic gene. In this regard, the regulation of Tf receptor expression produced by changes in iron levels is analogous to the regulation of low-density lipoprotein receptors produced by cholesterol (11). We showed that cellular control of iron uptake was accomplished, at least in part, through transcriptional control of the synthesis of the Tf receptor message. The rapid adjustment of specific mRNA levels was possible because of the short half-life of this message. We expect that the mechanisms underlying the regulation of this gene will be further elucidated when the genetic regulatory sequences conferring iron sensitivity are determined.

Abstract. When the human erythroleukemia cell line K562 is treated with OKT9, a monoclonal antibody against the transferrin receptor, effects on receptor dynamics and degradation ensue. The apparent half-life of the receptor is decreased by >50% as a result of OKT9 treatment. The transferrin receptor is also rapidly redistributed in response to OKT9 such that a lower percentage of the cellular receptors are displayed on the cell surface. OKT9 treatment also leads to a decrease in the total number of receptors participating in the transferrin cycle for cellular iron uptake. The reduction in iron uptake that results from the loss of receptors from the cycle leads to enhanced biosynthesis of the receptor. Receptors with bound OKT9 continue to participate in multiple cycles of iron uptake. However, OKT9 treatment appears to result in a relatively small increase per cycle in the departure of receptors from participation in iron uptake to a pathway leading to receptor degradation. Radiolabeled OKT9 is itself degraded by K562 cells and this degradation is inhibitable by leupeptin or chloroquine. In the presence of leupeptin, OKT9 treatment results in the enhanced intracellular accumulation of transferfin. Because the time involved in the transferrin cycle is shorter 02.5 min) than the normal half-life of the receptor (8 h), a small change in recycling efficiency caused by OKT9 treatment could account for the marked decrease in receptor half-life. In this paper the implications of these findings are discussed as they relate to systems in which receptor number is regulated by ligand. p ROLIFERATING cells require exogenous iron and acquire it via receptor-mediated endocytosis of the ironcarrier protein transferrin (Tf) 1 (for review see reference 6). Iron uptake via the Tf cycle involves binding of diferric Tf to a specific, high-affinity surface receptor followed by internalization of the receptor ligand complex. Within the cell, the complex encounters an acidic (~pH 5) environment that is instrumental in iron unloading (22, 31). Apo-Tf is then released intact from the cell, and the transferrin receptors (TtR) are reused (2, 9). The Tf cycle shares certain aspects with other systems of receptor-mediated endocytosis but also exhibits distinctive features, including the return of undegraded ligand and receptor to the cell exterior.In the present study, we describe perturbations of the pathway traversed by the TfR that result from exposure of K562 cells to a monoclonal anti-receptor antibody (OKT9). The monoclonal antibody OKT9 recognizes the human TfR at a site distinct from the ligand binding site and has been used to examine the structural features of the receptor as well as in the chromosomal localization and cloning of the TfR gene (3,15,25 Materials and Methods Cells and ChemicalsK562, a human erythroleukemia cell line, was grown in RPMI 1640 with 25 mM Hepes containing 10% fetal bovine serum, penicillin, and streptomycin (growth medium). Ceils were maintained at densities of 2-6 × 105/ml at 37"C in 5% CO2 incubator....

Treatment of K562 cells, a human erythroleukemia cell line, with desferrioxamine raised the levels of the receptor for transferrin (Tf) two-to threefold over that of the control cells. The levels of receptor were reduced by at least 50 and 35% of that of the control in cells treated with diferric Tf and ferric ammonium citrate, respectively. These changes were of total cellular receptors with no alteration in the proportion of receptors found on the cell surface. The half-lives of the receptor were identical in cells treated with desferrioxamine, diferric Tf, or ferric ammonium citrate. Cells metabolically labeled with [35S]methionine showed a 2.5-fold increase in the rate of receptor synthesis when treated with desferrioxamine and a 35 and 65% decrease when treated with ferric ammonium citrate and diferric Tf, respectively. In vitro translations of. polyadenylated mRNA isolated from cells incubated with desferrioxamine showed a 2.5-fold increase in translatable mRNA for the receptor, whereas treatment of cells with ferric ammonium citrate and diferric Tf resulted in a 25 and 50% reduction, respectively, in translatable mRNA for this receptor.Iron is an essential constituent of all cells. It is required for the normal functioning of a wide array of cellular enzymes, including those of the respiratory pathway (2, 4, 6). Cells receive iron via the receptor-mediated endocytosis of irontransferrin (Tf). Large amounts of iron can be stored in cells in ferritin and as hemosiderin. The iron needs of cells vary and are high in fetal, erythropoietic, and rapidly proliferating cells. Concomitant with these needs is a high level of expression of Tf receptors in placental cells, reticulocytes, and proliferating cells (1,8,10). It is not known exactly how cells regulate their iron uptake, but clearly the modulation of the number of Tf receptors can play a role in such regulation. Recently, Bridges et al. (Fed. Proc. 42:2193 and Mattia et al. (9) showed that K562 cells can alter Tf receptor numbers in response to intracellular iron chelation by desferrioxamine. The locus of this regulation was shown to be at the level of mRNA for the receptor (9). Ward et al. (12) reported a down regulation of Tf receptors in HeLa cells that were treated with iron salts. In this paper we report the extension of our studies on the regulation of Tf receptor biosynthesis in K562 cells. We show that delivering iron to cells results in a twofold decrease in specific synthesis of the Tf receptor. A decrease in the levels of mRNA is responsible for this drop. Furthermore, we compare the efficacy of Tf with that of ferric ammonium citrate in lowering receptor biosynthesis and establish the range of receptor regulation achievable with iron depletion or iron introduction without causing observable cellular toxicity.

Iron regulation of the human transferrin receptor gene was examined in murine cells transformed with chimeric constructs containing the human transferrin receptor gene's promoter and either the structural gene for bacterial chloramphenicol acetyltransferase or the human transferrin receptor cDNA. The activity of the transferrin receptor gene's promoter with the heterologous indicator gene was found to be 3-fold higher in cells treated with the iron chelator desferrioxamine than in cells treated with the iron source, hemin. A higher degree of iron regulation was seen in the expression of the human transferrin receptor cDNA driven by its own promoter. The receptor cDNA under the control of the simian virus 40 early promoter was also iron-regulated. Several human transferrin receptor transcripts differing in their 3' end were produced in the murine cells regardless of the promoter used, with the shorter transcripts being relatively unregulated by iron. Deletion of cDNA corresponding to most of the 3' untranslated portion of the mRNA for the receptor ablated the iron regulation. We conclude that at least two genetic elements exist for the regulation of the transferrin receptor gene by iron. One has its locus in the DNA upstream of the transferrin receptor gene's transcription start site, and the other is dependent upon the integrity of the sequences in the 3' end of the gene.

A Chinese hamster ovary cell mutant defective in the receptor-mediated endocytosis of several unrelated ligands (Robbins, A. R., S. S. Peng, and J. L. Marshall, 1983, J. Cell Biol., 96:1064-1071) failed to accumulate iron provided in the form of diferric transferrin. Analysis of the steps of the transferrin cycle indicated that binding and internalization of transferrin proceeded normally in mutant cells. However, the mutant appeared unable to dissociate iron from transferrin, as evidenced by release of diferric transferrin from the mutant versus apotransferrin from the parent. Uptake of ferric ions from the growth medium was enhanced in the mutant.