Menadione-induced oxidative stress leads to a rapid down-modulation of transferrin receptor recycling

Several human cell types have been demonstrated to possess high affinity receptors on their surface to which the transferrin glycoprotein binds and transports iron inside the cells (Testa, 1985; Testa et al., 1992). These receptors are dimeric glycoproteins composed of two identical subunits and their number can vary according to the growth rate of the cell type (Kuhn et al., 1984). After binding to their ligands, transferrin receptors cluster in coated pits and are internalized in the endocytic compartment in which the bound iron is released and the transferrin receptor-ligand complex remains intact (Huebers and Finch, 1987; Trowbridge, 1991). Receptor regulation clearly involves both plasma membrane function and the underlying cytoskeleton. Furthermore, cytoskeletal proteins have been suggested to be involved in antigen expression and receptor recycling (Schliwa, 1986). In particular, receptor internalization pathways seem to involve changes in plasma membrane properties together with (or consequent to) alterations of cytoskeletal element arrangement. This could be considered as an important mechanism by which the physiological expression of cell surface molecules can be modulated. It may occur both in vivo, in a variety of pathologic conditions (Hinshaw et al., 1986), and in vitro as a consequence of experimental treatment (Tripathi et al., 1991; Michelson et al., 1991).

Oxygen-derived free radical injury has been associated with several cytopathic conditions (Clark et al., 1985). This includes, or can be due to, a decrease in cellular redox capacity as observed in connection with cell aging and certain genetic diseases (Sies, 1991; Shinar et al., 1987; Niki et al., 1988). Oxidative stress, e.g. induced by various quinones, has also been suggested to determine membrane alterations, including both lipid peroxidation and modifications of membrane fluidity accompanied by a parallel increase in the intracellular calcium ion concentration (O’Brien, 1991). In particular, the naphthoquinone menadione was demonstrated to be reduced to a semiquinone radical, which forms superoxide anion free radicals that induce a progressive impairment of several cellular processes (Sies et al., 1991; Smith et al., 1984; Sies, 1985). Subcellular effects of such a compound were extensively described in several experimental models, such as isolated hepatocytes or cultured cells (Bellomo and Orrenius, 1985; Jewell et al., 1982; DiMonte et al., 1984). Recently, a role for specific cytoskeletal elements was hypothesized for menadione-induced cytotoxicity. In particular, surface morphological modifications ascribable to cytoskeletal oxidative damage were suggested (Bellomo et al., 1990a;

Mirabelli et al., 1988; Bellomo et al., 1990b). Thus, considering the close relationships between membrane and cytoskeletal functions, the effects of oxidative injury on specific surface molecules were investigated. The possible influence of redox imbalance on receptors exerting a physiological role by active recycling, e.g. transferrin receptors, was evaluated. The results obtained seem to demonstrate that oxidative modifications may have an important role in determining receptor expression and modulation.

K562 and HL-60 cells were grown in suspension culture in RPMI 1640 (Boehringer Mannheim, Germany) containing 10% fetal calf serum (Boehringer Mannheim) and antibiotics. The cells have a doubling time of about 24 hours. Cell viability was assessed by trypan blue dye exclusion test.

The culture medium was replaced with a buffer solution (phosphate buffered saline (PBS) supplemented with 1 mM CaCl₂ and MgCl₂, pH 7.3) and HL-60 and K562 cells were then treated with 200 μM menadione (Sigma) diluted in dimethyl sulphoxide (DMSO). Cells treated with equal amounts of vehicle alone were considered as controls.

Cells were also exposed to different chemicals as follows: cytochalasin B (CB) 5 μg/ml; phalloidin (PHA) 20 μg/ml; dithio- threitol (DTT) 2 mM; verapamil (VER) 150 μM for 30 minutes; nifedipine (NFD) 50 μM for 30 minutes; calcium ionophore A23187 25 μM for 30 minutes. All these agents were purchased from Sigma.

Cells (10⁷) were washed 3 times in serum-free RPMI 1640, and then incubated in the presence of [⁵⁹Fe]transferrin (125 mM/ml) in RPMI 1640. To measure nonspecific iron uptake, cells were incubated with [⁵⁹Fe]transferrin (125 mM/ml) in the presence of unlabeled transferrin (10 mg/ml) at 37°C. At the end of the incubation period, the cells (10⁶) were layered over a cushion of phthalate oil (density, 1.02) and centrifuged for 2 minutes at 13,000 g to remove unbound [⁵⁹Fe]transferrin. The ⁵⁹Fe content of the cell pellet was measured in a gamma 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%). Iron-free human transferrin was obtained from Sigma and was radio-labeled with ⁵⁹Fe following a method described previously (Martinez-Medellin and Schulman, 1972). ⁵⁹Fe as ferric chloride, 30 Ci/g of iron, was obtained from Amer- sham.

Purified human transferrin (>99% pure) was conjugated with ¹²⁵I by the solid-phase lactoperoxidase system (New England Nuclear radio-iodination system), as reported previously (Testa et al., 1982). ¹²⁵I-transferrin-binding capacity was investigated both on intact cells and on cell samples dissolved in Triton X-100 (1%).

The binding reactions were performed in 12 mm × 75 mm polypropylene tubes in RPMI 1640 containing 0.1% human serum albumin (Sigma Fraction V). Cell concentrations were 5×10⁶ cells/ml. Unbound ligand was removed by passage of cells through a density cushion, as described previously (Testa et al., 1982). After incubation, 200 ml aliquots of the cell suspension were layered over 150 ml of a mixture of dibutyl phthalate and dinonyl phthalate (Merck) to a final density of 1.025 in 400 ml plastic microfuge tubes and centrifuged in a Hettich microfuge (10,000 g for 2 minutes). At the end of centrifugation, the supernatant and most of the dibutyl phthalate cushion were aspirated. The tips of the vials containing the cell pellet were then cut off with a scalpel and transferred to plastic vials, and the radioactivity was measured in a gamma counter. Total binding corresponded to the radioactivity in the cell pellet. ‘Nonspecific’ binding was represented by the radioactivity bound to the cells in the presence of cold transferrin (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. In order to make Scatchard plot analyses (Scatchard, 1949), binding was carried out for 90 minutes at 4°C with radiolabeled transferrin supplemented with increasing concentrations of unlabeled ligand.

Dissolved receptors (in 1% Triton X-100, 50 mM phenylmethyl- sulphonyl fluoride) were incubated in a total volume of 0.2 ml for 30 minutes 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 ¹²⁵I-transferrin complex was precipitated with 0.8 ml of polyethylene glycol (12% w/v) in 0.1 M citrate. Tris buffer (pH 5.0) contained the carrier human gamma-globulin (0.1%). The tubes were placed in an ice bath for 30 minutes and then centrifuged at 13000 g for 15 minutes 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 for nonspecific binding, the transferrin was determined by preincubating the samples with 1 mg of nonradioactive transferrin before adding the radioactive transferrin.

Transferrin internalization on intact cells was studied according to the method of Hopkins and Trowbridge (Hopkins and Trowbridge, 1983). Cells were incubated for 60 minutes at 4°C in RPMI-1640 medium with 1 μg/ml ¹²⁵I-Tf, rinsed three times at 4°C in PBS, and then incubated in RPMI-1640 medium at 37°C. At different time points aliquots of cells were removed and processed as follows: (i) the cells were first centrifuged (2 minutes at 3000 rpm), and the radioactivity present in the supernatant was counted; (ii) the cell pellet was incubated 2 minutes at 4°C with saline acetic acid, a procedure that allows the detachment of cell surface bound transferrin; (iii) the cells were then centrifuged and the radioactivity present in the supernatant and cell pellet was counted in a gamma-counter.

For cell surface transferrin receptor labeling, cells washed three times in Hanks’ balanced salt solution were incubated for 30 minutes at 4°C with 100 ml of a 1:40 dilution of the anti-transferrin receptor monoclonal antibody (mAb); Sigma and Peel-Freeze antibodies in parallel experiments were used. The cells were then incubated with FITC-conjugated anti-mouse IgG at 4°C for 30 minutes. Cells were then fixed with 1% paraformaldehyde in PBS. For actin staining and intracytoplasmic tranferrin receptor detection, after adhesion to glass surfaces, cells were fixed with 3.7% formaldehyde and permeabilized with 0.5% Triton X-100 (Sigma). Actin was stained with fluorescein-phalloidin (Sigma) after a 30 minute incubation at 37°C. For transferrin receptor detection, the specific monoclonal antibodies (Sigma or Peel Freeze) were incubated for 60 minutes at 37°C. Cells were then incubated with antimouse IgG-fluorescein-linked whole antibody (Amersham International). Finally, all the samples were mounted with glycerol-PBS (1:1) and observed with a Nikon Microphot fluorescence microscope.

Control and treated cells were seeded on glass coverslips coated with polylysine. After adhesion to the glass surface, cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at room temperature for 20 minutes. Following post-fixation in 1% osmium tetroxide for 30 minutes, cells were dehydrated through a graded ethanol series, critical point dried in CO₂ and gold-coated by sputtering. The samples were examined with a Cambridge 360 scanning electron microscope.

First, menadione-induced oxidative stress was evaluated by a special set of experiments carried out using scanning and transmission electron microscopy. Membrane surface bleb- bing, which is often observed in stressed cells, was detected in both the HL-60 and K562 cell lines studied here after 30 minutes incubation with menadione.

Subsequently, binding studies on transferrin (Tf) were carried out. Incubation of both HL-60 and K562 cells at 37°C in the presence of menadione induced a rapid and marked reduction of membrane-associated transferrin-binding capacity. This phenomenon appeared 2-5 minutes after the addition of menadione to the cells, while 5-10 minutes of incubation resulted in a >50% loss of the Tf-binding capacity (Fig. 1, left). As a control of the specific activity of menadione, a series of parallel experiments performed in the same experimental conditions was also carried out. In particular, GM-CSF-binding ability was examined and no significant changes in the binding capacity of this molecule were detected (Fig. 1, right). In addition, Scatchard analysis of ¹²⁵I-Tf binding to the cells (Fig. 2a) showed that menadione elicited a very marked reduction of the number of Tf-binding sites without significantly affecting the affinity of Tf receptors for Tf, as well as a marked reduction of Tf-mediated intracellular Fe²⁺ release (Fig. 2b). The reduced Tf-binding capacity induced by oxidative stress seems to be energy-dependent since, when the cells were incubated at 4°C with menadione, no reduction of Tf-bind- ing capacity was detected (Fig. 3). Furthermore, in order to evaluate whether the reduction of Tf-binding ability was related either to an internalization or masking of Tf receptors, Tf-binding capacity was also investigated in cell samples dissolved in Triton X-100, a technique which allows the evaluation of both the membrane-associated and the intracellular pools of the Tf receptor. Using such an assay, it appeared that no significant differences in the total Tf- binding capacity existed in menadione-treated cells (Fig. 3). On the basis of these findings, it is suggested that menadione can modify membrane-associated Tf receptor processing. As a result of this modification, these receptors are not capable of binding Tf, but remain physically present in the cell. This conclusion was directly supported by immunofluorescence experiments on detergent-permeated cells. In fact, cells treated with menadione showed a remarkable decrease of surface expression of Tf receptors five minutes after menadione-exposure (Fig. 4a-c). This finding was also confirmed by analysis of membrane fluorescence using a fluorescence-activated cell sorter (data not shown). In contrast, intracellular staining with anti-Tf receptor mAbs was much higher in menadione-exposed than in control cells (Fig. 4d-f). Additional experiments were specifically carried out in the attempt to evaluate the possible impairment of the internalization process by menadione and its possible modulation of the recycling of Tf receptors on the cell surface. In order to perform these studies, cells were first incubated at 4°C in the presence of ¹²⁵I-Tf, washed to remove unbound labeled Tf and then incubated at 37°C in the absence or in the presence of menadione. The time-course of the percentage of Tf released into the medium, of membrane-associated Tf and of intracellular Tf was determined (Fig. 5). The analysis of these three parameters both in control and menadione-treated cells provided evidence that: (i) the extent of the decrease of membrane-associated Tf following the shift at 37°C was similar both in control and menadione-treated cells (Fig. 5a); (ii) the amount of intracellular Tf was higher in menadione-treated cells when compared to the level observed in control cells (Fig. 5b); (iii) the amount of Tf released into the medium (Tf recycled back to the cell surface) was significantly higher for control cells when compared to mena- dione-incubated cells (Fig. 5c). These results indicate that menadione could inhibit the recycling of Tf. Thus, with this in mind, several attempts were made to identify the possible mechanisms responsible for the effects of menadione on Tf binding. Experiments were carried out in order to demonstrate a possible role of microtubules or microfilaments in mediating the effects of menadione on Tf binding. In fact, the pre-treatment of the cells with agents causing depolymerization of actin filament bundles, such as cytochalasin B, did not protect against the reduction of Tf- binding capacity induced by menadione. In contrast, compounds eliciting a stabilizing effect on actin filaments, such as the toxin phalloidin, partially protect the cells against the effect of menadione on Tf-binding capacity (Fig. 6). Accordingly, a marked rearrangement of actin filaments accompanied by a partial depolymerization of the microtubular network were observed in both cell lines after exposure to menadione (data not shown).

Finally, experiments were also carried out in order to evaluate the role of calcium ions on menadione-mediated down-modulation of membrane Tf receptors. Thus, the effect of drugs inhibiting calcium entry into the cells was considered. Pre-treatment of the cells with verapamil or nifedipine, agents blocking extracellular calcium entry, completely abrogated the inhibitory effect of menadione on Tf binding. In line with this observation, menadione was unable to down-modulate membrane Tf receptors if the cells were incubated in medium containing no calcium. Finally, experiments were performed in order to evaluate the effect of the calcium ionophore A23187 on Tf receptor traffic. In fact, the calcium ionophore as well as menadione was effective in inducing a rapid and marked reduction of Tf binding (Fig. 7). Additional experiments (immunofluorescence on detergent-permeated cells, and studies of ¹²⁵I- Tf internalization) provided evidence that the acute downmodulation of surface Tf receptors elicited by the calcium ionophore can be ascribed to an intracellular block of receptor recycling (data not shown).

Our results provide evidence that the human hematopoietic cell lines K562 and HL-60 exhibit a rapid and marked reduction in iron uptake following menadione treatment. To investigate the possible mechanisms responsible for this phenomenon, several different analyses were performed with various approaches. First, general cytopathic effects, i.e. surface blebbing, and receptor-specific alterations were studied in the attempt to evaluate possible links among these two events. Second, different experimental conditions were used in order to assess possible intracellular mechanisms responsible for the oxidative stress-induced downmodulation of Tf membrane receptors.

On the basis of our findings, possible non-specific membrane effects exerted by menadione exposure and leading to Tf receptor changes, can be ruled out. Specifically, menadione induced a remarkable reduction of the maximal amount of ⁵⁹Fe taken up by the cells, without affecting the KM of iron uptake. The affinity of the Tf receptor for its ligand was apparently unchanged by oxidative stress exposure, while the ligand and receptor recycling is specifically impaired. These data provide evidence for a substantial integrity of the Tf receptor, excluding primary functional alterations of the molecule induced by menadione.

Highly reactive hydroxyl radicals may be important contributors to rapid radical-mediated alterations of membrane fluidity via lipid peroxidation phenomena, while the superoxide anions, the main products of menadione-induced oxidative stress, could affect protein thiol group redox status (O’Brien, 1991; DiMonti et al., 1984; Rice-Evans and Hochstein, 1981; Thor et al., 1982). Different lipid-protein interactions or protein changes might cause, as previously hypothesized (Gaffney, 1975; Sefton and Gaffney, 1974), the appearance (or disappearance) of specific surface antigens (receptors), which may be responsible, in turn, for changes in membrane order (Santini et al., 1990). Finally, the specificity of the effect exerted by menadione on the transferrin receptor was also supported by investigating the binding of other molecules, e.g. granulocyte-macrophage colony stimulating factor (GM-CSF), which demonstrated a lack of direct alteration induced by the drug on the number of membrane GM-CSF receptors.

As far as the second point is concerned, i.e. the possible relationships of receptor function alterations with general biological and morphological effects of menadione, e.g. surface blebbing, our findings seem to indicate that these are separate phenomena sharing some common mechanisms. In fact, two main features have to be taken into account. First, the induced modification of Tf receptors is a very early phenomenon (few minutes), while structural changes are not. For instance, this was demonstrated by fluorescence analysis showing that a remarkable decrease in the number of membrane Tf receptors, associated with an increase in the number of intracellular Tf receptors was noticeable five minutes after induction of oxidative stress, and by the observation that surface blebbing can occur after only 30 minutes of stress. Second, it is conceivable that calcium ions and the cytoskeleton play a major role in both phenomena. An induced imbalance of calcium homeostasis, as well as an impairment of cytoskeletal element function by a specific action on thiol groups was in fact previously hypothesized for menadione (Bellomo et al., 1990a; Malorni et al., 1991; Mirabelli et al., 1989). In nucleated cells, these alterations may lead to typical cell surface alterations such as surface blebbing, a phenomenon previously described in several other experimental conditions (Bignold and Ferrante, 1988; Coakley, 1987; Malorni et al., 1990). In particular, an oxidation of sulphydryl groups was demonstrated to occur in actin cytoskeletal filaments of several cell types (Bellomo et al., 1990a; Mirabelli et al., 1988; Mirabelli et al., 1989). Furthermore, some actin-binding proteins were shown to regulate the function of the microfilament system in a calcium-dependent manner (Howard et al., 1990). Finally, microtubule integrity was demonstrated to be necessary for intracellular transfer of the ligand (Schliwa, 1986; Robert et al., 1985). Thus, the previously demonstrated depolymerizing effects of menadione on microtubules and its thiol group-mediated cross-linking on actin microfilaments in nucleated cells (Bellomo et al., 1990a; Mirabelli et al., 1989) could be in some way responsible for the impairment of Tf receptor trafficking reported here. Hence, in view of the role of cytoskeletal elements (i.e. F-actin, some actin- binding proteins, tubulin) in receptor trafficking, specific experiments were also performed by using the thiol reductant dithiothreitol. Unfortunately, this compound, which was able to ‘protect’ cells from surface blebbing, was not able to modify the receptor-cycling impairment in both cell lines examined here. In conclusion, on the basis of these results, we hypothesize that Tf receptor function impairment is a specific effect of menadione.

The mechanisms leading to the target effect of oxidative stress on Tf receptor cycling was thus investigated. It is well known that transferrin receptor expression is essential for cell growth and its regulatory process has been extensively studied (Cazzola et al., 1990). For instance, in T lymphocytes, the growth promoting effects of interleukin-2 are partially mediated by transferrin receptor induction (Neck- ers and Cossman, 1983). In addition, B-cell proliferation also requires the expression of Tf receptors and their blockade inhibits DNA synthesis (Seligman et al., 1991). Other mechanisms can be responsible for Tf receptor regulation or function. The re-distribution of transferrin receptors from the external membrane to the cytoplasmic pool can be modulated by phorbol esters, possibly as a consequence of an increased receptor phosphorylation (May et al., 1983; Mattia et al., 1984). Finally, the de-regulation of transferrin receptor expression was also considered as a general characteristic of neoplastic cells (Neckers and Cossman, 1983). Thus, the role of the Tf receptor in several physiological and pathological processes, as well as in cell proliferation, is well established. With this in mind, we can consider the findings reported here as further proof of the specific link between oxidative imbalance, functional impairment of important molecules on the cell surface and cell damage. In fact, several cellular pathologies, as well as neoplastic transformation, can be due to, or have been associated with, free radical formation (Clark et al., 1985; Kappus, 1987; Allen, 1987; Boobis et al., 1989).

Several in vitro and in vivo studies have demonstrated that oxidative stress can modify cell physiology by altering redox status of proteins or phospholipids involved in numerous cellular functions (Sun, 1990; Hyslop et al., 1988; Trump et al., 1989; Wolff and Dean, 1986). The results reported here seem to suggest that earlier modifications occurring to key molecule physiology, e.g. transferrin receptors, can be of great importance for the cascade of events leading to cytotoxicity processes previously demonstrated for oxidative stress (Trump et al., 1989; Orrenius et al., 1989). The inhibition of iron uptake induced by menadione is associated with a parallel and marked inhibition of transferrin-binding capacity. As discussed above, this last phenomenon was very rapid, occurring within a few minutes of menadione addition, and appears evident when investigated on whole cells. However, experiments on transferrin binding carried out in cell samples dissolved in 1% Triton X-100 showed that menadione-treated cells exhibited a transferrin-binding capacity similar to that of control cells. These findings strongly suggest that menadione modified a large part of the transferrin receptors present in the cells. As a result of this modification, these receptors became unavailable for binding transferrin, but they remained physically present in the cells. Furthermore, transferrin internalization experiments also showed that menadione-treated cells were able to internalize the trans- ferrin-transferrin receptor complex like the control cells, but exhibited a significantly lower capacity to recycle transferrin and transferrin receptors back to the cell surface. Taken together these findings suggest a down-modulation process similar to the one observed in the down-modulation of membrane transferrin receptors induced by phorbol esters (May et al., 1983; Testa et al., 1984). It was initially suggested that the phorbol ester-induced down-modulation of transferrin receptor could be related to a receptor hyperphosphorylation at the level of the serin 24 residue (May et al., 1985). However, site-directed mutagenesis studies have shown that phosphorylation does not affect endocyto- sis of the transferrin receptor-ligand complex (Rothenberger et al., 1987; Zerial et al., 1987). Furthermore, no evidence is available to show that menadione is able to induce, as phorbol esters do, protein kinase C activation.

Finally, the investigation of the biochemical mechanism responsible for the down-modulation of transferrin receptor elicited by menadione converged on calcium homeostasis. Calcium ions play a role in receptor regulation (Carpentier et al., 1992) and in the oxidative stress-induced cascade of intracellular events (Orrenius et al., 1989; Bel- lomo et al., 1982; Nicotera et al., 1988; Richter and Kass, 1991). Our studies provide evidence that calcium ions could play a major role in the blocking of receptor cycling. Two major pieces of evidence seem to emerge: (i) agents capable of inhibiting Ca²⁺ uptake into the cell, such as verapamil and nifedipine, completely abolished the menadione- induced down-modulation of transferrin receptors; (ii) calcium ionophores mimick, at least partially, the effect of menadione by inducing a rapid and marked transferrin receptor down regulation. However, it must be pointed out that in other cell types calcium ionophores may have a different effect on Tf receptor regulation as compared to those observed in the present study. In conclusion, changes in the regulation of the expression of important molecules on the cell surface, such as receptors for growth factors, might also contribute to the pathogenesis of oxidation-induced morbid states (Snyder et al., 1985).

This work was supported by grants from the Italian Ministry of Health and from CNR, ACRO Project, grant no. 92.0363.PF39.