Regulation of Transferrin Receptors in Human Hematopoietic Cell Lines*

Cells grown in the presence of ferric ammonium citrate or hemin exhibited a concentration and time-dependent decrease in 12SI-transferrin (Trf) binding. In contrast, cells grown in the presence of protoporphyrin IX or picolinic acid (an iron chelator) exhibited a marked increase in Trf binding. The decrease or increase in binding activity observed under these dif- ferent conditions of culture reflected, respectively, a reduction or increase in receptor number rather than an alteration in ligand receptor affinity. Growth of the cells in the presence of saturating concentrations of apotransferrin only induced a slight reduction in receptor number. Investigation of the Trf receptors’ turnover and biosynthesis clearly showed that iron and hemin decreased the synthesis of Trf receptors without any modification of the receptor turnover; in contrast, protoporphyrin IX and picolinic acid markedly in- creased the synthesis of Trf receptors. Our results suggest that hemin, iron, and protopor- phyrin IX may represent the main molecules involved in the regulation of Trf receptors.


Regulation of Transferrin Receptors in Human Hematopoietic
Creteil, France Cells grown in the presence of ferric ammonium citrate or hemin exhibited a concentration and timedependent decrease in 12SI-transferrin (Trf) binding. In contrast, cells grown in the presence of protoporphyrin IX or picolinic acid (an iron chelator) exhibited a marked increase in Trf binding. The decrease or increase in binding activity observed under these different conditions of culture reflected, respectively, a reduction or increase in receptor number rather than an alteration in ligand receptor affinity. Growth of the cells in the presence of saturating concentrations of apotransferrin only induced a slight reduction in receptor number. Investigation of the Trf receptors' turnover and biosynthesis clearly showed that iron and hemin decreased the synthesis of Trf receptors without any modification of the receptor turnover; in contrast, protoporphyrin IX and picolinic acid markedly increased the synthesis of Trf receptors.
Our results suggest that hemin, iron, and protoporphyrin IX may represent the main molecules involved in the regulation of Trf receptors.
It was recently suggested that polypeptide receptors on mammalian plasma membranes can be classified into two different categories on the basis of function (Kaplan, 1981). Such categorization is based on whether the major function of the receptor is to transmit information (class I receptors) or internalize the ligand (class I1 receptors) (Kaplan, 1981).
Ligand internalization by class I1 receptors provides the cell with a required factor, for example, cobalamin (Youngdahl-Turner et al., 1979) or cholesterol (Brown and Goldstein, 1975). On exposure to ligand, class I receptors may be downregulated (the number of surface receptors is reduced); in contrast, binding of ligand by class I1 receptors does not lead to regulation of receptor number. The best-studied class I1 receptor is the human fibroblast low-density lipoprotein receptor which is involved in the transport of cholesterol to the cells. Incubation of the cells with low-density lipoproteins results in a decrease in receptor number. Conversely, incubation of cells in the absence of low-density lipoproteins leads to an increase in surface receptor number (Brown et al., 1975). The regulation of low-density lipoprotein receptors results from modulation of receptor biosynthesis which is controlled by the concentration of free cholesterol (Brown et al., 1975).
Transferrin receptors appear to be another candidate for class I1 receptors. The plasma glycoprotein transferrin transports iron from the plasma to the cells (Finch and Huebers, 1982). The first step in the uptake of iron by the cells requires binding of Trf' to specific surface receptors. The interaction of Trf receptors in reticulocytes (Jandl and Katz, 1963;Kailis and Morgan, 1974), placenta (Wada et al., 1979;Galbraith et al., 1980a), activated lymphocytes (Galbraith et al., 1980b), hepatocytes (Young and Aisen, 1980), rat and human fibroblasts (Octave et al., 1981;Ward et al., 1982a), and a variety of neoplastic cell lines (Larrick and Cresswell, 1979;Hamilton et al., 1979;Sutherland et al., 1981;Trowbridge and Omary, 1981;Testa et al., 1982) has been reported. There is compelling evidence that Trf is required for the growth of cells in vitro (Hutchings and Sato, 1978). Recent studies have shown that iron salts down-regulate the Trf receptors (Ward et al., 1982b) and that the intracellular iron concentration may represent one of the most important factors in the control of the number of Trf receptors (Ward et al., 1982b).
In the present paper, we define the role of iron salts, heme, and protoporphyrin IX in the control of the number of Trf receptors in human leukemic cell lines. The results showed the following. 1) Preincubation of the cells with ferric ammonium citrate markedly reduced the number of Trf receptors. 2) Addition of an iron chelator (such as picolinic acid) in the culture medium greatly increased the number of Trf receptors. 3) Hemin, as previously reported by us (Pelicci et al., 1982), exhibited an effect similar to that of iron salts. 4) Preincubation of the cells with protoporphyrin IX enhanced the number of Trf receptors. 5 ) Preincubation of the cells with apotransferrin only slightly modified the Trf-binding capacity. These studies clearly suggest that the biosynthesis of Trf receptors is modulated by iron salts, heme, and protoporphyrin IX, but not by previous exposure to the ligand.

EXPERIMENTAL PROCEDURES
Materials-Human transferrin was purchased from Sigma, and it was electrophoretically homogeneous. Ferric ammonium citrate and ferric ammonium sulfate were obtained, respectively, from Sigma and Prolabo (Paris, France). Bovine or human serum albumin were from Sigma. Carrier-free '*'I was bought from New England Nuclear. 69FeC13, 30 Ci/g of iron, was obtained from The Radiochemical Centre (Amersham, England).
Tissue culture flasks were from Costar (Cambridge, MA). Fetal and newborn calf serum were obtained from IBF or Eurobio (France).
The cells were washed three times in serum-free RPMI 1640 medium and then incubated in the presence of 125 pg/ml '$Fe-Trf in RPMI 1640 medium. To measure "nonspecific" iron uptake, cells were incubated with 125 pg/ml 59Fe-Trf in the presence of 10 mg/ml unlabeled transferrin. The incubation temperature was 37 "C. At the end of the incubation period, the cells (1 X lo6) were layered over a cushion of phthalate oil (1.02 density) and centrifuged 2 min at 10,000 X g to remove unbound "Fe-Trf. 59Fe content of the cell pellet was measured in a y counter. All data were averages of duplicate determinations (which were usually within 10% of each other) and were corrected for nonspecific binding (which did not exceed 5% of the total).
Transferrin Receptor Assay-Purified human transferrin was conjugated with ' ' ' I by the solid-phase lactoperoxidase method (New England Nuclear, radioiodination system) as previously described (Testa et al., 1982). The binding reaction was performed in polypropylene tubes (12 X 75 mm) in RPMI 1640 medium containing 0.1% bovine serum albumin (Sigma, Fraction V). Cell concentrations were 5 x IO6 cells/ml, labeled Trf was 200 ng/ml, and unlabeled Trf was 1 mg/ml. Unbound ligand was removed by passage of cells through a density cushion, as previously described (Testa et al., 1982). After incubation, 200-pl aliquots of the cell suspension were layered over 150 p1 of a mixture of dibutyl phthalate (Merck) and dinonyl phthalate (Merck) to a final density of 1.025 in 400-p1 plastic microfuge tubes and centrifuged in a Hettich microfuge (10,000 X g for 5 rnin). At the end of centrifugation, the supernatant and the majority of phthalate cushion were aspirated. The tips of the vials containing cell pellets were then severed with a scalpel, transferred to plastic vials, and the radioactivity was measured in a y counter. Total binding corresponded to the radioactivity in the cell pellet. Nonspecific binding was represented by the radioactivity hound to the cells in the presence of cold Trf (1 mg/ml) and was less than 5% of the total radioactivity bound per 106 cells. "Specific binding" was the difference between total and nonspecific binding.
Before binding, the cells were washed four times in 40 ml of Hanks' saline solution (Boehringer Mannheim, Germany). The number of washes did not modify the transferrin-binding capacity of the cells since they were grown in fetal calf serum, and bovine transferrin had a very low affinity for human transferrin receptors, as previously reported by other investigators (Ward et al., 198213) and by us (Titeux et al., 1984).
Investigation of the Binding of B3/25 Monoclonal Antibody to Transferrin Receptors on Whole Cells-In some experiments, the expression of transferrin receptors was evaluated by investigating the binding of B3/25 monoclonal antibody to transferrin receptors (Trowbridge and Omary, 1981). The cells were washed four times with Hanks' balanced saline solution containing 1 mg/ml bovine serum albumin (Sigma) and then 2 X lo6 cells for each point were incubated 90 min at 4 "C in the presence of increasing amounts of purified B3/ 25 monoclonal antibody diluted in the same incubation buffer. After three washes at 4 "C, the cells were then incubated 90 min at 4 "C in the presence of '251-goat IgG anti-mouse antibody (The Radiochemical Centre, Amersham, England; 10 pCi/pg). The cells were then layered on phthalate oil and processed as described above.
Dissolved Transferrin Receptor Assay-A simple assay was devised to measure solubilized transferrin receptor activity (Pelicci et al., 1982). It is based on a difference in solubility of free and transferrinbound receptors in polyethylene glycol. Cell samples were dissolved in phosphate-buffered saline solution containing 1% Triton X-100 (Sigma) and 1 mM phenylmethylsulfonyl fluoride (Sigma) and centrifuged 30 min at 20,000 X g. Dissolved receptors were incubated in a total volume of 0.2 ml for 30 min at 37 "C in a 0.1 M citrate/Tris buffer solution (pH 5.0) containing 0.1% bovine serum albumin, 0.1% Triton X-100, and 200 ng of lZ5I-transferrin. The receptor-transferrin complex was precipitated with 0.8 ml of polyethylene glycol solution (12% w/v) in 0.1 M citrate/Tris buffer (pH 5.0) containing the carrier human y-globulin (0.1%). The tubes were placed in a ice bath for 30 min and then centrifuged at 13,000 X g for 15 min at 4 "C. The supernatant and the precipitate were tested for radioactivity. Coprecipitation of free transferrin was measured by omitting the receptor from the tubes while nonspecific binding of transferrin was determined by preincubating the samples with 1 mg of nonradioactive transferrin before adding the radioactive transferrin.
Investigation of the Biosynthesis of Trf Receptors-10 X lo6 cells were washed three times in Dulbecco's methionine-free medium and then incubated in the same medium containing 10% fetal calf serum dialyzed overnight against Hanks' medium and 100 pCi of [35S] methionine (The Radiochemical Centre, Amersham, England). The cells were then washed five times in phosphate-buffered saline solution (0.15 M NaCI, 10 mM phosphate buffer (pH 7.40)) and then dissolved in the same solution containing 1% Triton X-100 and 2 mM phenylmethylsulfonyl fluoride (Sigma). After 30 min at 4 "C, the cells were centrifuged 30 min at 20,000 X g. The supernatant was added to 50 pl of Sepharose-transferrin, obtained by coupling purified human transferrin to CNBr-activated Sepharose 6B (Pharmacia, Uppsala, Sweden). The supernatant and the resin were first incubated 30 min at 20 "C and then overnight at 4 "C. The resin was then washed five or more times at 4 "C with phosphate-buffered saline containing 0.1% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride. 100 p1 of 2% SDS, 5% P-mercaptoethanol were added to the resin and boiled 5 min at 100 'C. After cooling, the supernatant was recovered and analyzed by SDS-polyacrylamide gel electrophoresis (Laemmli, 1970). After electrophoresis, the gels were stained with Coomassie Blue, destained, and treated for autoradiography as previously reported (Bonner and Laskey, 1974).
Investigation of Ferritin Concentration-The intracellular concentration of ferritin was evaluated by radioimmunoassay using a kit from Calbiochem-Behring Institute. Samples for ferritin determination were lysed in distilled water, freeze-thawed three times, and centrifuged at 20,000 X g for 30 min at 4 "C, and the supernatant was used for radioimmunodetermination of ferritin. Protein concentration was determined by the dye-binding method (Bio-Rad).

RESULTS
Effects on Cell Growth-The addition of hemin (from 1 to 100 @M) or protoporphyrin IX (from 0.1 to 10 PM) to the culture medium did not significantly affect the rate of cell growth. In contrast, ferric ammonium citrate or sulfate increased the rate of growth of the cells, especially the K 562 cells (Fig. 1). Picolinic acid at concentrations between 1-2 mM inhibited cell growth slightly; at higher concentrations (3  picolinic acid, apotransferrin, and iron-saturated transferrin on Trf receptor number and affinity K 562 cells were grown with ferric ammonium citrate (10 Fg/ml), heme (0.1 mM), picolinic acid (2 mM), apotransferrin (50 Fg/ml), iron-saturated transferrin (50 gg/ml), or protoporphyrin IX (10 pM) for 3 days before assay of Trf-binding activity. The results represent the range of values obtained in five different experiments.  3. Effect of ferric ammonium citrate, hemin, and protoporphyrin IX on the growth rate, iron uptake, and lZ6I-Trfbinding capacity of K 562 cells. K 562 cells were incubated with increasing concentrations of ferric ammonium citrate, hemin, or protoporphyrin IX. Growth rate was estimated by counting the cells every day. For the determination of '*'I-Trf binding and iron uptake, the cells were washed and then incubated 30 min at 20 "C in the presence of "'I-Trf (500 ng/ml) and 2 h at 37 "C in the presence of 59Fe-Trf (125 pg/ml). and in the presence of ferric ammonium citrate, apotransferrin, or a combination of apotransferrin and ferric ammonium citrate. The cells were then washed and incubated with a fixed nonsaturating concentration of 12sI-Trf (500 ng/ml) and increasing concentrations of cold Trf, ranging from 500 ng/ml to 1 mg/ml, for 30 min at 20 "C. The data were plotted according to the method of Scatchard (1949). mM or more), picolinic acid completely inhibited the cell growth.

ImI-Trf bound
Effects of Iron, Heme, Protoporphyrin IX, Picolinic Acid, Apotransferrin, and Iron-saturated Transferrin on the Erpression of Trf Receptors-The addition of iron (ferric ammonium citrate or sulfate) and heme to the culture medium markedly decreased Trf-binding activity (Table I) (Table I). Scatchard analysis revealed that the decrease or increase in binding activity observed under these different conditions were due, respectively, t o a decrease or increase in receptor number rather than an alteration in ligand-receptor affinity (Fig. 2). As shown in Fig. 3, incubation of cells with ferric ammonium citrate, hemin, or protoporphyrin IX led to a concentration-dependent decrease or increase in Trf-binding activity.
Comparable results were obtained when the Trf-binding capacity was estimated by investigating the binding of B3/25 or 42/6 monoclonal antibodies which specifically recognize Trf receptors (Trowbridge and Omary, 1981;Trowbridge and Lopez, 1982) (Fig. 4) presence of protoporphyrin IX bound 2.5 times more B3/25 antibody then control cells. Experiments with Trf-59Fe2 showed that a decrease or increase in iron uptake occurred, respectively, proportional to the decrease or increase in receptor number (Fig. 5). However, the changes in iron uptake were less marked than those observed for Trf binding. The modulation of Trf receptors observed in the cells grown in the presence of these different compounds was not dependent upon (a) a change in the kinetics of association of lZ5I-Trf to the cells a t 20 or 37 "C, or (b) a change in the dissociation of Trf from the cells (data not shown).
The decrease in Trf receptor number produced by ferric

FIG. 7. Recovery of Trf-binding capacity following removal of cells from ferric ammonium citrate-or hemin-supplemented media.
K 562 cells were grown in media supplemented with 25 pg/ml of Fe3+ in the form of ferric ammonium citrate (0) or with 100 p~ hemin (0). After 72 h, cells were removed from ferric ammonium citrate-or hemin-containing media, washed, and then allowed to grow in standard media. At indicated times, IZ6I-Trf-binding studies were performed following the conditions reported above. Zero time represents the time at which cells were transferred to standard media. The data are expressed as the percentage of specific binding with respect to that observed in control cells. ammonium citrate or hemin was time-dependent and was maximal by 24 h (Fig. 6). The decrease in receptor number occurred regardless of growth state. Similarly, the increase in Trf-binding capacity produced by protoporphyrin IX was time-dependent and was maximal by 48 h. When cells grown in media supplemented with hemin, ferric ammonium citrate, or protoporphyrin IX were removed to nonsupplemented media, the Trf-binding capacity returned to control levels by 24-48 h (Fig. 7).
The binding experiments on whole cells a t 20 "C permits the evaluation of surface receptors. Since several recent studies have afforded evidence that most of transferrin receptors are on intracellular membranes, we evaluated lZ5I-Trf binding also on cell samples dissolved in 1% Triton X-100. This technique allows the investigation of both surface-bound and intracellular localized transferrin receptors. Using this technique, we confirmed the results obtained by quantifing the transferrin-binding capacity on intact cells. Thus, we showed that cells grown in the presence of ferric ammonium citrate and hemin, respectively, exhibited a marked decrease in their Trf-binding capacity (Table 11). In contrast, cells grown in the presence of picolinic acid and protoporphyrin IX, respec-  After 72 h of incubation, actinomycin D (1 pg/ml) was added. At indicated times, '9-Trf-binding activity was determined under the conditions mentioned above. Actinomycin D completely inhibited cell growth, but until 24 h of incubation, did not modify the cell viability as estimated by the trypan blue exclusion test.

FIG. 9. Reappearance of Trf receptors after trypsin treatment of K 562 cells grown in media with and without iron or hemin supplementation. K 562 cells were grown in standard media
supplemented with Fe3+ (25 pg/ml) in the form of ferric ammonium citrate, or with hemin (100 p~) . Cells were exposed to trypsin (0.1 mg/ml) in serum-free RPMI 1640 medium for 30 min at 37 "C. The trypsin was neutralized by addition of fetal calf serum (20%); the cells were washed three times and then grown in media with or without iron or hemin supplementation. At specific times, cells were removed, and the Trf-binding capacity was measured under the conditions mentioned above. Cells in standard (O), in iron-supplemented (A), or hemin-supplemented (0) media. tively, exhibited a marked enhancement of their transferrinbinding capacity (Table 11).
Mechanism of Receptor Regulation-Changes in receptor number could result from alterations in the rate of receptor biosynthesis and/or degradation. In order to determine whether hemin, ferric ammonium citrate, and protoporphyrin IX affect the rate of receptor degradation, actinomycin D was added to control and hemin, protoporphyrin or ferric ammonium citrate-incubated cells. As demonstrated in Fig. 8, apparently none of these compounds significantly modified the rate of Trf receptor degradation.
In other experiments, cycloheximide was used instead of actinomycin D. Using cycloheximide, the rate of degradation of transferrin receptors was very similar to that observed with actinomycin D.
To investigate whether preincubation of cells with these compounds modifies receptor half-life, we followed an approach previously described (Ward et al., 1982b). Control cells and cells preincubated with ferric ammonium citrate, hemin, or protoporphyrin IX were first treated with trypsin. The rate of receptor reappearance was measured following transfer of the cells to their respective trypsin-free media. Trypsinized control cells and trypsinized iron-, hemin-, and protoporphyrin IX-supplemented cells resynthesized receptors to the levels present before trypsin treatment. The rate of recovery of Trf binding was greater in protoporphyrin IX-treated cells than in controls; in contrast, iron-or hemin-treated cells exhibited a rate of receptor reappearance about 50% that of control cells (Fig. 9). However, when the percentage of recov-  Effect of iron (ferric ammonium citrate), heme, protoporphyrin IX, and picolinic acid on the intracellular concentration of ferritin K 562 cells were grown with ferric ammonium citrate (10 pglml), heme (0.1 mM), picolinic acid (2 mM), or protoporphyrin IX (10 p M ) for 3 days before assay of ferritin. The results represent the range of values obtained in five different experiments. ery of Trf binding is plotted against time, the reappearance of receptors reached half of the maximal level at 6-8 h for control cells, at 6-8 h for iron-supplemented cells, at 7-9 h for hemin-treated cells, and at 8-10 h for protoporphyrin IXtreated cells. These results indicate similar half-lives for Trf receptors under these different conditions of culture. Thus, our data suggest that incubationof the cells with iron, hemin, or protoporphyrin IX affects receptor biosynthesis without affecting receptor degradation. T o directly investigate this point, we studied the biosynthesis of Trf receptors by incubation of the cells in the presence of [%]methionine followed by purification of Trf receptors on Sepharose-transferrin (Fig.  10). In control cells, the synthesis of Trf receptors corresponded to 0.2% of the total protein synthesis of K 562 cells.
In contrast, the synthesis of Trf receptors was lower in cells grown in the presence of ferric ammonium citrate (0.05-0.196 of total protein synthesis). Picolinic acid strongly increased the synthesis of Trf receptors to levels 3-5 times higher than in control cells (0.5-1% of total protein synthesis).
Effect of Ferric Ammonium Citrate, Hemin, Protoporphyrin IX, and Picolinic Acid on the Intracellular Content of Ferritin-In order to investigate the effect of ferric ammonium citrate, hemin, protoporphyrin IX, and picolinic acid on the level of iron stored into the cells, we evaluated the intracellular concentration of ferritin. In fact, it was well established that the level of ferritin present in the cell was dependent upon the intracellular concentration of iron (Aisen and Lis-towsky, 1980). These experiments showed that (a) ferric ammonium citrate induced a marked increase in the level of ferritin; (b) hemin elicited moderate enhancement in the concentration of ferritin; and (c) both protoporphyrin IX and picolinic acid elicited a very marked decrease in the concentration of ferritin (see Table 111).

DISCUSSION
Our results clearly show that several compounds related to iron or heme metabolism can modulate the transferrin-binding capacity of human leukemic cell lines. More particularly, we showed that incubation with iron salts or Trf-Fez resulted in time and concentration-dependent reduction in Trf-binding capacity of the cells. Analysis of the binding data by the method of Scatchard (Scatchard, 1949) revealed that such reduction was the result of decreased receptor number rather than alteration in ligand-receptor affinity. Incubation of cells with apo-Trf did not alter receptor number. These results indicate that an increased accumulation of iron in the cells leads to a decrease in Trf binding. Similar results were previously reported in Hela cells (Ward et al., 1982b).
This conclusion is further supported by the observation that an iron chelator, picolinic acid, in the culture medium greatly increased Trf-binding capacity. As previously reported (Fernandez-Pol, 1977;Fernandez-Pol, 1978;Gusley and Jett, 1981), picolinic acid at low concentrations (0.1 to 2 mM) reduces iron uptake; a t higher concentrations (3 mM or higher), this compound completely inhibits iron uptake.
Furthermore, this interpretation is further supported by the experiments of ferritin quantification. These experiments clearly showed that cells exhibiting a higher level of intracellular ferritin (cells grown in the presence of ferric ammonium citrate and hemin) have the lower number of Trf receptors; in contrast, cells possessing the lower level of ferritin exhibit the higher level of Trf-binding capacity.
Thus, our data clearly indicate that the expression of Trf receptors depends on the amount of iron accumulated into the cells. This system exhibits a simple physiological principle. When cells accumulate large amounts of iron, they reduce the number of Trf receptors in order to prevent further accumulation of iron; in contrast, when the intracellular concentration of iron is low and the cells need more iron, they induce an increase in Trf receptors to permit rapid accumulation of iron. This mechanism may be especially relevant for cellular physiology since iron is required for both cell growth and for synthesis of heme-containing molecules.
However, compounds possessing a tetrapyrrolic ring can also modulate the Trf-binding capacity of the cells. Thus, hemin, as previously reported by us (Pelicci et al., 1982), significantly reduces the number of Trf-binding sites in a manner similar to iron. In contrast, protoporphyrin IX which is identical to heme but is devoid of iron markedly increases the number of Trf receptors. The effect of heme could be related to the presence of iron in its molecule: in fact, accumulation of heme by the cells corresponds equally to an accumulation of iron. In contrast, it is difficult to understand the mechanism by which protoporphyrin IX increases the number of Trf receptors. This effect does not derive from reduced iron accumulation since protoporphyrin IX does not inhibit iron uptake by the cells.
However, experiments of ferritin quantification showed that cells grown in the presence of protoporphyrin IX exhibited a marked reduction of their ferritin concentration. It is interesting that in other biological systems, hemin and protoporphyrin IX exhibit antagonistic effects: thus, protoporphyrin IX is a potent stimulator of guanylate cyclase which is inhibited by heme (Ignarro et al., 1982); heme acts as a mitogen for lymphocytes, whereas protoporphyrin IX is unable to stimulate the proliferation of the cells (Stenzel et al., 1981). A possible role for cyclic nucleotides in the regulation of Trf receptors is now under investigation in our laboratory.
The modulation of Trf receptors by all the compounds mentioned above implicates a modification of the number of receptors without change in the affinity of the receptor for the ligand. Modifications in the kinetics of association or dissociation were not observed. Furthermore, differences in the proportion of membrane-bound and intracellular receptors were not found, as suggested by experiments of lZ5I-Trf binding on cell samples dissolved in 1% Triton X-100.
The modulation by these compounds of the number of Trf receptors became apparent only several hours after their addition to the cells. Such changes in receptor number could have represented a modification of the degradation of Trf receptors and/or a modulation of the synthesis of receptors. When new receptor synthesis was blocked with actinomycin D or cycloheximide, both control cells and cells exposed to iron, heme, or protoporphyrin IX demonstrated the same apparent rate of receptor degradation. However, investigation of the half-life of a receptor by using protein or mRNA synthesis inhibitors may cause artifactual changes in the rate of receptor degradation. Therefore, we investigated receptor half-life by measuring the reappearance of Trf-binding activity following trypsinization. The time required for both trypsinized control cells and cells incubated with ferric ammonium citrate, hemin, or protoporphyrin IX to resynthesize half of their maximal level of receptors was similar. This result strongly suggests that hemin, protoporphyrin IX, and ferric ammonium citrate did not alter the rate of receptor degradation.
To more directly investigate the mechanism of regulation of Trf receptors, we studied the effect of hemin, ferric ammonium citrate, protoporphyrin IX, and picolinic acid on the biosynthesis of Trf receptors. Under standard conditions of culture, the biosynthesis of Trf receptors in K 562 cells represents about 0.1-0.2% of the total protein synthesis. Cells grown 3 days in the presence of ferric ammonium citrate or hemin exhibited a marked reduction of Trf receptor biosynthesis. In contrast, cells grown in the presence of protoporphyrin IX or picolinic acid exhibited a strong increase in the synthesis of Trf receptors. It is interesting that cells grown in the presence of picolinic acid synthesize on the average five times more Trf receptors than do control cells. Thus, picolinic acid-treated cells may represent an interesting source of mRNA specific for Trf receptors with respect to gene cloning.
Short incubation (2 h) of the cells in the presence of ferric ammonium citrate, hemin, protoporphyrin IX, or picolinic acid did not modify the rate of receptor biosynthesis with respect to the control. These results suggest that Trf receptors are probably modulated by a transcriptional mechanism.
This study affords evidence that Trf receptors can be modulated. The pattern of regulation appears to be analogous to that previously described for lipoprotein receptors (Brown et al., 1975). Receptor number is regulated not by binding of low-density lipoproteins, but by the intracellular concentration of cholesterol metabolites. Receptor number is also regulated by an alteration in the rate of receptor biosynthesis (Brown and Goldstein, 1975). Thus, great analogies exist between Trf receptors and lipoprotein receptors.