©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mechanism of Transferrin Receptor Down-regulation in K562 Cells in Response to Protein Kinase C Activation (*)

(Received for publication, August 24, 1994; and in revised form, December 9, 1994)

Jeremy E. Schonhorn Thomas Akompong Marianne Wessling-Resnick (§)

From the Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Treatment with phorbol esters increases endocytosis of the transferrin receptor in K562 cells (Klausner, R. D., Harford, J., and van Renswoude, J.(1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3005-3009). In this report, we demonstrate that this effect is reversible within early times of protein kinase C activation (<2 h) but that prolonged exposure to phorbol esters results in a net loss of receptors. These effects are not due to the differentiation response of K562 cells to phorbol esters since bryostatin-1 also down-regulates the endocytosis of the transferrin receptor and shut downs receptor synthesis, but does not induce differentiation (Hocevar, B. A., Morrow, D. M., Tykocinski, M. L., and Fields, A. P.(1992) J. Cell Sci. 101, 671-679). We have characterized the early stages of receptor down-regulation which occur due to stimulation of receptor internalization from the cell surface. The fact that fluid-phase pinocytosis is also enhanced upon protein kinase C activation indicates that this effect is not specific for the transferrin receptor itself, but is a rather general cellular response to tumor-promoting phorbol esters. The fate of down-regulated transferrin receptors was followed in morphological and subcellular fractionation studies that demonstrate localization of this pool of receptors in early endocytic and recycling compartments. Our results exclude the possibility that transferrin receptor down-regulation results in trafficking of the receptor to lysosomal compartments for degradation. This idea is consistent with the observations that the time course of transferrin receptor degradation is not enhanced in stimulated K562 cells, while transferrin receptor synthesis is shut down. Our results rigorously demonstrate that activation of protein kinase C down-regulates the K562 cell transferrin receptor in two stages: acute regulation of early steps in endocytosis that results in an immediate reduction of 40% in cell surface number of receptors and a more chronic reduction in transferrin receptor synthesis upon prolonged exposure to phorbol esters (>15 h).


INTRODUCTION

It has been well-established that treatment of K562 cells with the phorbol ester 4beta-phorbol 12-myristate 13-acetate (PMA) (^1)perturbs endocytosis of the transferrin receptor, causing an apparent loss of cell surface receptors(1, 2) . This redistribution could be promoted by enhanced internalization, reduced externalization, and/or targeting of transferrin receptors to a compartment which does not participate in normal routes of receptor traffic. Early studies characterizing the parameters of transferrin receptor endocytosis in K562 cells suggested the existence of two receptor pools, one surface-bound and the other intracellular. Klausner et al. (2) proposed that transferrin binding initiates transferrin receptor internalization to intracellular compartments, which in turn stimulates the release of an intracellular transferrin receptor pool to the plasma membrane. PMA was thought to activate or enhance an endogenous endocytic signal of this pathway. In this model, contributions of two pools of receptors to recycling accounted for the down-regulation of the transferrin receptor in the absence of available ligand; PMA was considered to reproduce a ligand-induced endocytic initiation signal, thus altering the release of intracellular receptors, and presumably exerting this effect by the activation of protein kinase C.

In contrast to the ligand-induced endocytic transport mechanism, a mechanism of continuous transferrin receptor cycling in K562 cells was later identified. These conflicting results are attributed to the different methods utilized to determine cell surface receptors. Klausner et al.(2) assessed surface receptors with anti-transferrin receptor antibodies, a method subject to variabilities such as ligand-induced perturbations of antigenic determinants. In contrast, Watts (3) evaluated surface-bound receptors by following internalization of I-labeled transferrin receptors as measured by resistance to proteolytic digestion. Using this approach, it was observed that the transferrin receptor was internalized with identical kinetics in the presence or absence of available transferrin. These observations are consistent with the principle that it is the receptors, independent of available ligand, that are the true substrates for endocytosis(4) . Finally, although PMA treatment caused extensive phosphorylation of the receptor's cytoplasmic tail(5, 6) , no conclusive evidence for this modification as an endocytic signal has been obtained(7, 8, 9, 10) .

Thus, although the effects of PMA on transferrin receptor endocytosis are well documented, the precise mechanism of receptor down-regulation has yet to be elucidated. Recently, our laboratory re-examined the mechanism of PMA-induced transferrin receptor down-regulation in K562 cells using the In/Sur method of Wiley and Cunningham(4) . This approach was undertaken in order to exclude the possibility of ligand-induced endocytosis of the receptor or the internalization of a separate pool of unoccupied receptors that could be modulated in response to phorbol esters. These studies rigorously demonstrated that the rate of transferrin receptor endocytosis is significantly and specifically up-regulated in K562 cells exposed to phorbol esters: the endocytic rate constant for transferrin internalization is nearly doubled, 0.43 min compared to 0.28 min for control cells(11) . In contrast, there is little change in exocytic recycling of the receptor, arguing that the sole basis for the redistribution of receptors is due the kinetic alterations in endocytosis. Although this is consistent with the early observations of Klausner et al.(2) , additional changes in receptor traffic induced by phorbol esters cannot be excluded. For example, down-regulated receptors might be diverted to subcellular compartments that do not participate in the typical route of receptor trafficking, thus creating two populations of intracellular transferrin receptors in PMA-treated cells as initially proposed(2) . Consideration of this idea is prompted by the fact that treatment of K562 cells with phorbol esters also blocks growth and induces differentiation. Several investigations have demonstrated the multipotential nature of this cell line(12, 13) , and it has been established that PMA promotes stable megakaryocytic differentiation of the proerythroblastic K562 cells (14) . It is possible that during this response, transferrin receptors are simply cleared from the cell surface due to morphological changes effected by the cellular differentiation program. These changes might represent transport of the receptor to otherwise inaccessible compartments along a new membrane traffic pathway, for example, accelerated receptor degradation due to transport along the lysosomal pathway. In light of these arguments, this investigation was undertaken to specifically address the following questions: first, into what compartments are transferrin receptors redistributed upon exposure to PMA? Second, is down-regulation of the transferrin receptor a direct action of phorbol esters on regulation of the endocytic pathway or is this phenomenon simply a consequence of the differentiation program? Finally, are degradative processes accelerated under these conditions to account for the loss of receptors?


MATERIALS AND METHODS

Ligand Binding Studies

Cell culture and preparation of human erythroleukemia K562 cells for uptake and ligand binding studies was as described previously(11) . Receptor down-regulation was induced upon exposure 50 nM PDBu, 32.4 nM PMA (both from Sigma), or 50 nM bryostatin-1 (LC Laboratories, Woburn, MA) with incubation at 37 °C for times indicated in the legends. We thank the kind generosity of Dr. Katherine M. Call (Harvard School of Public Health) and Dr. W. S. May (Johns Hopkins Oncology Center) for the gifts of bryostatin-1 to initiate these studies.

To assess the extent of receptor down-regulation, binding of I-labeled transferrin to surface receptors was measured (11) . K562 cells were incubated with appropriate concentrations of I-transferrin (0.5-500 nM) in the presence or absence of unlabeled ligand (25 µM) for 1 h at 4 °C. Cells were collected by centrifugation, the cell pellet was rinsed with ice-cold PBS, and cell-associated radioactivity was measured. The amount of nonspecific I-transferrin associated with the cell pellet was determined in the presence of unlabeled ligand and substracted from each point. Scatchard analysis was employed to determine the number of transferrin-binding sites.

Soluble receptor binding studies were accomplished using the methods described by Klausner et al.(15) . Briefly, K562 cells were solubilized in TBS (10 mM Tris, pH 7.4, 150 mM NaCl) containing 0.1% Triton X-100. To 150-µl aliquots of the detergent-solublized cell extract, 50 µl of I-transferrin (0.5-500 nM) and 50 µl of buffer with or without 50 µg/ml unlabeled transferrin were added. After a 10-min incubation at room temperature, 250 µl of ice-cold 60% ammonium sulfate was added to the binding reactions to precipitate the ligand-receptor complexes. After 5 min on ice, the precipitate was collected by filtration through Whatman GF/C filters and washed three times with 30% ammonium sulfate containing 0.8% BSA. Filters were dried, counted, and specific I-transferrin binding was taken as the difference between radioactivity associated in the absence and presence of unlabeled transferrin.

Horseradish Peroxidase Uptake Experiments

K562 cells were incubated in the presence or absence of PMA for 2 h as described above except that after this time, cells were collected by centrifugation, washed, and resuspended in 25 mM HEPES, pH 7.4, 150 mM NaCl, 1 mg/ml glucose, 1 mg/ml BSA, with 1 mg/ml horseradish peroxidase (Sigma). After further incubation for 30 min at 37 °C, the cells were once again collected by centrifugation and extensively washed five times in ice-cold PBS. Cells were resuspended, lysed with 0.1% Triton X-100, and the clarified lysates were then assayed for horseradish peroxidase activity. Briefly, 80-µl samples of detergent lysate were incubated in a mixture containing 0.1 M imidazole, pH 7.0, and 4 mM peroxide with the addition of 20 µl of 11 mMo-dianisidine-HCl initiating the reaction (total volume 1.1 ml). The linear increase in absorbance at 460 nm over the first 1-2 min of the peroxidase reaction was measured to determine the DeltaA/min/mg K562 cell protein.

Confocal Fluorescence Microscopy

K562 cells were incubated at 37 °C in serum-free media containing 100 nM FITC-transferrin, then washed three times with ice-cold PBS, and fixed with 3.7% gluteraldehyde in PBS for 60 min at 4 °C. The fixed cells were subjected to five further washes in PBS and resuspended in 5-10 volumes of buffer/volume of cell pellet. Confocal fluorescence microscopy was performed at the Biomedical Imaging Laboratory at Harvard School of Public Health using a Molecular Dynamics Sarastro 2000 CLSM (Confocal Laser Scanning Microscope) fitted with a 25 milliwatt argon-ion laser. Optical sections (0.5 µm thick) were collected at magnification times 40 and were taken at the midplane of the cells. Excitation wavelenth was 488 nm and emission was >510 nm. Digital images presented are 1024 times 1024 in size with a pixel size of 0.25 µm.

Subcellular Fractionation

Subcellular fractionation of K562 cells was accomplished on self-forming Percoll gradients as described previously(16) . K562 cells containing I-transferrin internalized under uptake conditions described above were washed with PBS and resuspended in TEA-sucrose (250 mM sucrose, 10 mM triethanolamine (TEA), pH 7.5, 1 mM EDTA). Cells were disrupted using a ball-bearing homogenizer, and a post-nuclear supernatant fraction was prepared. A 0.4-µl aliquot (1 mg of protein) was then mixed with 7.6 ml of 22% Percoll in breaking buffer and underlayed with a 0.5-ml 60% sucrose cushion. The samples were centrifuged in a Ti50 rotor (Beckman Instruments) for 90 min at 17,000 revolutions/min at 4 °C. Fractions (300 µl) were collected from the interface of the sucrose cushion and assayed for I-transferrin content and appropriate subcellular markers.

Cell Surface Iodination and Immunoprecipitation Experiments

K562 cells (5 times 10^6 cells/ml) were surface iodinated in PBS containing 15 mg/ml glucose, 2 units/ml lactoperoxidase (Calbiochem), 0.1 unit/ml glucose oxidase (Sigma), and 1 mCi of I (DuPont NEN), with incubation for 60 min at 4 °C. Surface-labeled K562 cells were washed extensively in PBS, resuspended in fresh media in the presence or absence of PMA, then incubated at 37 °C. At indicated times (0-24 h), 1 times 10^6 cells (2 ml) were collected, washed with PBS, and solubilized in 1 ml of 10 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100. I-Labeled transferrin receptors were immunoprecipitated using sheep anti-transferrin antiserum, a generous gift of Dr. Caroline Enns, Oregon Health Sciences University. Briefly, supernatant fractions from the solubilized samples were preabsorbed with 50 µl of Pansorbin (Calbiochem), collected by centrifugation, and 1.4 µl of antiserum was added along with 25 µl of fresh Pansorbin. After incubation for 1 h, Pansorbin-associated immune complexes were collected by centrifugation, washed with 100 µl of 10 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, then layered over 1 ml of RIPA (1% Triton X-100, 1% deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.5, 150 mM NaCl) containing 15% sucrose and centrifuged. Samples (25 µl) were analyzed on 7.5% SDS-polyacrylamide gels, subsequently dried, and placed on film for autoradiography. Molecular mass markers used in these experiments were: alpha(2)-macroglobulin (170 kDa), beta-galactosidase (116 kDa), fructose-6-phosphate kinase (85.2 kDa), glutamate dehydrogenase (55.5 kDa), and aldolase (39.2 kDa).

Northern Blot Analysis

K562 cell RNA was isolated using the method of Chomezynski and Sacchi(17) , and samples (20 µg) were electrophoresed on 1% agarose gels with 0.22 M formaldehyde, 20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA. RNAs were then transferred to nylon membranes (Nytran Plus, Schleicher and Schuell) and UV-cross-linked. Transfer membranes were prehybridized with 50% formamide, 1% SDS, 1 M NaCl, and 10% dextran sulfate at 42 °C. DNA fragments were radiolabeled with [P]dCTP by random priming (Pharmacia Oligolabeling Kit) in order to hybridize membranes using 13 times 10^6 counts/min of denatured P-labeled probe. After overnight hybridization, membranes were washed twice for 5 min at room temperature with 2 times SSC, then twice for 10 min at 65 °C in 2 times SSC, 1%SDS, and exposed to film for 2-5 h. The cDNA probes used in this study were a 5-kilobase BamHI insert for the human transferrin receptor isolated from pCDTR1 (18) and, as a control, the 0.7-kilobase PstI fragment of the human 36B4 ribosomal protein(19) .


RESULTS

Activation of Protein Kinase C Enhances Endocytosis in K562 Cells

Treatment of K562 cells with PMA has been shown to clear roughly 40-60% of the total cell surface transferrin receptors within 2 h of exposure to the protein kinase C activator(2) . Fig. 1confirms this result by presenting the I-transferrin binding curves for control (circle) and PMA-treated K562 cells (bullet). The number of cell surface binding sites (B(max)) was determined by Scatchard analysis to decrease from 3. 92 times 10^5 to 2.73 times 10^5 transferrin receptors/cell. Moreover, the down-regulation of transferrin receptors is fully reversible within this time frame. Table 1summarizes data from several experiments in which transferrin receptor down-regulation was induced by phorbol dibutyrate (PDBu). Unlike PMA, binding of PDBu to protein kinase C is fully reversible. Treatment with the phorbol ester for 2 h results in a net loss of 36% of cell surface receptors; further exposure up to 4 h leads to an even greater receptor redistribution (>40% loss from cell surface compared to control). If after 2 h the PDBu is removed by washing and the K562 cells are allowed to recover upon incubation for an additional 2 h, a significant increase in receptor number is observed relative to those cells wherein treatment with PDBu is continued (4 h total). These results support the idea that receptor down-regulation is fully reversible upon deactivation of protein kinase C. This is further demonstrated by the fact that if a specific inhibitor of protein kinase C, staurosporine, is added during the first 2 h of treatment, down-regulation of transferrin receptors is completely blocked. (^2)Finally, if staurosporine is added after 2 h of PDBu treatment and incubation in the presence of the phorbol ester is continued for an additional 2 h, then receptor down-regulation is completely reversed and the number of cell surface receptors resembles that of control (untreated) cells (Table 1).


Figure 1: Down-regulation of K562 cell surface transferrin receptors by PMA. K562 human erythroleukemia cells were incubated in the absence (circle) or presence (bullet) of 32.4 nM PMA for 2 h at 37 °C. The cells were then washed three times in PBS at 4 °C, and cell surface I-transferrin binding was measured as described under ``Materials and Methods.'' Briefly, K562 cells (0.5-1 times 10^6 cells) were resuspended in 25 mM HEPES, pH 7.4, 150 mM NaCl, 1 mg/ml BSA with indicated concentrations of I-transferrin (0.5-50 nM), with or without 25 µM unlabeled transferrin (final volume 250 µl). The binding reactions were incubated at 4 °C with constant mixing for 60 min and terminated upon centrifugation. The cell pellets were washed briefly one time in ice-cold PBS and associated counts/minute was measured by gamma counting. Shown is the specific amount of bound I-transferrin determined from the difference in cell-associated counts/minute in the absence and presence of unlabeled transferrin. Data are from a single experiment representative of similar experiments repeated on numerous occasions.





All of the data described above are consistent with previous work characterizing the down-regulation of transferrin receptors in K562 cells treated with phorbol esters(2, 11, 20) . The action of PMA on receptor down-regulation has been shown to enhance endocytosis of transferrin(2) , and work from our laboratory recently confirmed that the endocytic rate constant for receptor internalization is increased about 2-fold in stimulated cells(11) . The results presented here extend these previous findings by further documenting the reversible nature of this phenomenon and demonstrating that it is the biological action of phorbol esters to activate protein kinase C directly that alters endocytosis of the K562 cell transferrin receptors.

Does the activation of protein kinase C lead to other changes in K562 cell endocytosis or is this effect specific for transferrin receptors? The fact that the transferrin receptor is a substrate for phosphorylation by protein kinase C led to the original suggestion that perhaps this event triggered endocytosis of the receptor(2, 15) . However, there is little data to substantiate a role for transferrin receptor phosphorylation in internalization, particularly since mutagenesis studies have demonstrated that key serine residues phosphorylated by protein kinase C are not essential for receptor internalization(7, 8, 9, 10) . To specifically address this issue, the fluid-phase uptake of horseradish peroxidase by K562 cells was also examined under these conditions. Fig. 2compares endocytic uptake of horseradish peroxidase by control and PMA-treated cells. Exposure of K562 cells to the phorbol ester stimulates fluid-phase internalization of the probe about 2.5-fold, consistent with the idea that activation of protein kinase C results in a general cellular response to increase endocytosis. The finding that fluid-phase endocytosis is also enhanced by PMA treatment strongly argues that receptor down-regulation is not due to phosphorylation of transferrin receptor itself nor that this effect is specific for the transferrin receptor alone. Thus, the effects of phorbol esters are most likely exerted through protein kinase C regulation of elements involved in the endocytic pathway of K562 cells.


Figure 2: PMA treatment stimulates fluid-phase pinocytosis. The effects of phorbol esters on the fluid-phase internalization of horseradish peroxidase by K562 cells were measured as described under ``Materials and Methods.'' K562 cells were first incubated in the presence or absence of 32.4 nM PMA for 2 h, then washed in PBS, and incubated for 30 min in 25 mM HEPES, pH 7.4, 150 mM NaCl, 1 mg/ml glucose, 1 mg/ml BSA containing 1 mg/ml horseradish peroxidase. After fluid-phase uptake was complete, cells were extensively washed in PBS, then lysed with 0.1% Triton X-100, and cell-associated horseradish peroxidase activity was assayed using o-dianisidine as substrate. Fluid-phase uptake measurements (DeltaA/min/mg cell protein) is given in the mean ± S.D. from duplicate measurements from a single experiment. Similar results were obtained on two other occasions.



Down-regulation of Transferrin Receptors to Endocytic Compartments

In order to investigate the cytolocalization of down-regulated transferrin receptors, K562 cells were treated with PMA in the continuous presence of FITC-transferrin. Internalization of the fluorescently labeled ligand thus marks the intracellular path of redistributed receptors. Fig. 3presents results of confocal fluorescence microscopy studies examining the distribution of FITC-transferrin in control (left panels) and PMA-treated cells (right panels). As described previously(12, 13, 21) , treatment with the phorbol ester even for short times (<2 h) results in pronounced surface blebbing compared to control cells. However, it should be noted that these surface blebs are relatively devoid of the fluorescent ligand. Both control and PMA-treated cells reveal a punctate distribution of FITC-transferrin in endosomal domains immediately adjacent to the plasmalemma and in recycling endosomes located in the perinuclear region of the cells(22) . Although the PMA-treated cells contain increased fluorescence intensity relative to control cells, corresponding to increased numbers of internalized receptors during the time of exposure to ligand, there is otherwise little difference in the general pattern of distribution of intracellular FITC-transferrin. From these results we conclude that upon stimulation with PMA the pattern of transferrin receptor traffic is unchanged; that is, in association with its receptor, transferrin is internalized to endosomal compartments and is then recycled back to the cell surface.


Figure 3: FITC-transferrin is internalized to endosomal domains in PMA-treated K562 cells. Confocal fluorescence microscopy was performed to identify the cytolocalization of endocytosed FITC-transferrin. K562 cells were treated with or without 32.4 nM PMA for 2 h at 37 °C, during which time 100 nM FITC-labeled transferrin was included in the incubation medium to be internalized. The cells were then washed extensively with PBS, fixed with 3.7% gluteraldehyde, and prepared for microscopy. Fluorescence confocal microscopy was carried out as described under ``Materials and Methods'' with optical sections (0.5 µm thick) taken at the mid-plane of the cells at magnification times 40. Images obtained from two different experiments are shown. A 5-µm scale bar is shown.



To verify the results of these morphological studies, we also examined the profile of I-transferrin bearing compartments in control and PMA-treated cells by subcellular fractionation on self-forming Percoll density gradients(16) ; this method is routinely employed to separate intracellular compartments and in fact, the presence of radiolabeled transferrin is commonly used as a marker to denote endocytic vesicles(23) . The results presented in Fig. 4demonstrate that the fractionation profiles of control and PMA-treated cells are identical: I-transferrin appears in fractions containing membrane vesicles of light buoyant density as described previously(16) . Most importantly, the radiolabeled ligand does not appear in dense compartments separated on this gradient, in particular, those fractions containing lysosomes as marked by beta-hexosaminidase activity (panel B). The observations made in these subcellular fractionation experiments support the conclusion that the endocytic pathway of the transferrin receptor is not altered by activation of protein kinase C and that down-regulated receptors reside in endosomal compartments of the K562 cells.


Figure 4: Sub-cellular fractionation profiles of PMA-treated and control K562 cells. PMA-treated and control K562 cells were incubated at 37 °C for 1 h in the presence of 5 nMI-transferrin. The cells were then collected, washed with PBS, and disrupted in TEA-sucrose using a ball-bearing homogenizer in order to obtain a post-nuclear supernatant. PNS fractions (400 µl) were mixed with 7.6 ml of 22% Percoll in TEA-sucrose and the entire mixture was then underlayed with a 0.5-ml 60% sucrose cushion. The samples were centrifuged in a Ti50 rotor for 90 min at 17,000 revolutions/min at 4 °C and fractionated from the interface of the sucrose cushion (fractions from bottom of the tube are on the left). 100 µl of each fraction was taken to measure I-transferrin content (panel A). Aliquots (10 µl) were also assayed for beta-hexosaminidase activity with methylumbelliferyl-beta-N-acetylglucosaminide (1 mM) in MacIlvaine buffer (pH 4.5) containing 0.1% Nonidet P-40 and 0.1% gelatin. After 30 min at 37 °C, trichloroacetic acid was added to the reaction mixture (5% final concentration), and clarified supernatants were assayed for released methylumbelliferone (excitation 365 nm, emission 450 nm). Panel B shows activity profiles of lysosomal beta-hexosaminidase (in fluorescence units). There was no significant difference between the subcellular fractionation profiles for control (circle) and PMA-treated K562 cells (bullet).



Transferrin Receptors Are Down-regulated in K562 Cells Exposed to Bryostatin-1

In addition to the down-regulation of K562 cell transferrin receptors outlined above, phorbol esters are also known to induce the stable differentiation of this cell line. This well-characterized phenomenon includes the induction of surface expression of the platelet-specific antigen, gpIIIa, the disappearance of erythrocyte-specific lineage antigen, glycophorin A, the increased presence of platelet peroxidase, and enhanced secretion of granulocyte/macrophage-colony-stimulating factor and interleukin-6 (14) . While PMA exerts these effects to promote megakaryoblastic differentiation, the synthesis of DNA and growth of the K562 cells completely ceases. Although temporally these rather gross changes occur over a 1-4-day time span, an immediate response of initiation of the differentiation program could be marked by the rapid down-regulation of transferrin receptors.

Recently, Hocevar et al.(24) demonstrated that exposure of K562 cells to the macrocyclic lactone, bryostatin-1, activates protein kinase C but does not induce differentiation. Although structurally unrelated to phorbol esters(25) , bryostatin-1 appears to activate protein kinase C in an isotype-specific manner and, in K562 cells, bryostatin-1 promotes differential translocation of the beta isotype of protein kinase C to the nucleus(24) . We therefore took advantage of the specificity of bryostatin-1's action to investigate whether transferrin receptor down-regulation would occur in the absence of differentiation.

Fig. 5demonstrates that similar to the effects of phorbol esters, bryostatin-1 induces 50% loss of cell surface receptors in K562 cells. Panel A presents the Scatchard plots of I-transferrin binding to cells exposed to 50 nM bryostatin-1 compared to control (untreated cells). Although in this particular experiment K562 cells were treated with bryostatin-1 overnight, preliminary experiments determined that down-regulation of transferrin receptors was maximal within 4 h of exposure to the protein kinase C activator (data not shown). For comparison, panel B shows Scatchard analysis of transferrin binding to K562 cells after overnight exposure to PDBu. For both treatments, activation of protein kinase C provoked greater than 50% loss of binding sites. Within this time period of exposure to phorbol esters, other studies have established that K562 cells initiate expression of the megakaryocytic phenotype(14) , consistent with the cessation of cell growth. In contrast, K562 cells treated with bryostatin-1 continue to grow and fail to differentiate as noted by the lack of gpIIIa expression or induction of granulocyte/macrophage-colony-stimulating factor and interleukin-6 secretion(24) . In these experiments, the growth of the K562 cells was monitored to confirm that PDBu-treated cells ceased to divide and that bryostatin-1-treated cells were similar to control (untreated) K562 cells in their rate of growth (data not shown). Therefore, the observation that both bryostatin-1 and phorbol esters down-regulate the transferrin receptor indicates that activation of protein kinase C regulates endocytosis of K562 cells through pathways independent of those involved in cellular differentiation.


Figure 5: Bryostatin-1 down-regulates K562 cell surface transferrin receptors. K562 cells were incubated with 50 nM bryostatin-1 overnight, then prepared for I-transferrin binding studies as described for Fig. 1and Table 1. Panel A presents the results of Scatchard analysis for treated (bullet) and control () cells. I-Transferrin binding measurements were also performed with K562 cells treated with bryostatin-1 overnight then incubated for an additional 2 h with 100 nM staurosporine (circle) comparison, Panel B shows Scatchard analysis of I-transferrin binding to K562 cells treated overnight with 50 nM PDBu () and for an additional 2 h with 100 nM staurosporine (box). Shown are data obtained from an individual experiment with both PDBu and bryostatin-1 treatments performed in parallel. The experiment was repeated three times, with data summarized in Table 2.





Prolonged Exposure to Protein Kinase C Activators Decreases Synthesis of Transferrin Receptors

As shown in Table 1, the protein kinase C inhibitor staurosporine is capable of reversing receptor down-regulation after short times of exposure to phorbol esters (2-4 h). In contrast, the subsequent addition of staurosporine after overnight treatment with PDBu fails to redistribute receptors back to the cell surface (Fig. 5). Down-regulation induced by either PDBu or bryostatin-1 could not be reversed when the inhibitor was added >15 h after the induction of transferrin receptor down-regulation. The results from several experiments are summarized in Table 2, which clearly document that while K562 cells treated overnight with both protein kinase C activators display a decreased number of cell surface receptors, subsequent exposure to staurosporine does not reverse this effect. At first glance, the fact that the protein kinase C inhibitor is without effect is not surprising, since prolonged treatment with both activators results in a net loss of cellular protein kinase C activity(26, 27) . Together with the data of Table 1, our observations suggest that activation of protein kinase C results in an acute stimulation of endocytosis to redistribute cell surface receptors to intracellular compartments. However, the results of Table 2and Fig. 5also indicate that, in addition, prolonged exposure to the protein kinase C activators may lead to a persistent, irreversible loss of transferrin receptors.

To investigate this phenomenon, the total number of transferrin receptors in K562 cells treated overnight with phorbol esters was measured in a soluble receptor binding assay. As shown by the results of Fig. 6, K562 cells exposed to PDBu overnight display about a 50% loss in detectable I-transferrin binding activity relative to control (panel A). In contrast, no decrease in binding activity is observed after 2 h of treatment. In a similar manner, overnight treatment with bryostatin-1 was also observed to induce a loss in I-transferrin-binding sites (panel B). Thus, while short times of protein kinase C alters only the number of cell surface transferrin receptors, prolonged activation leads to a reduction in both cell surface and intracellular receptors.


Figure 6: Transferrin binding to total solubilized receptors. -Transferrin binding to detergent-solublized receptors was assayed using ammonium sulfate to precipitate receptor-ligand complexes(3) . K562 cells were treated for 16 h in the absence (circle) or presence of either 50 nM PDBu (bullet) or 50 nM bryostatin-1 (). As a control, K562 cells were incubated for only 2 h with 100 nM PDBu (). Briefly, the cells were then washed in PBS, and detergent extracts were prepared in TBS containing 0.1% Triton X-100. Binding of I-transferrin to detergent-solublized receptors (cell surface and intracellular) was performed as described under ``Materials and Methods'' with nonspecific binding measured in the presence of excess unlabeled transferrin. Shown is measured amount of transferrin bound (pmol/10^6 cells) to total soluble receptors as a function of [I-transferrin]. Results from one of three separate experiments is shown, with 50% loss of total transferrin receptors after overnight treatment with either PDBu or bryostatin-1 demonstrated on each occasion.



One explanation for the apparent loss of receptors is that it may reflect an increase in transferrin receptor degradation. However, as demonstrated by the results of Fig. 3and Fig. 4, PMA does not appear to provoke altered routes of endocytic traffic such that the transferrin receptor becomes retargeted to lysosomal compartments for degradation. To directly ascertain the fate of down-regulated receptors, we performed cell surface iodination to label K562 cell surface transferrin receptors, then induced down-regulation of these receptors upon exposure to PMA. After the extended times of incubation indicated in Fig. 7, K562 cells were solubilized, and the I-labeled receptors were immunoprecipitated to be analyzed by SDS-PAGE and subsequent autoradiography. As shown by the results of Fig. 7, the time course of cell surface transferrin receptor degradation is unaffected in the PMA-treated K562 cells. For both control and PMA-treated cells, the receptor half-life appears to be on the order of about 8 h. The fact that the time course of receptor degradation is unaffected in PMA-treated cells rigorously excludes the possibility that in addition to its immediate effects on endocytosis, activation of protein kinase C also redirects membrane traffic to degradative lysosomal compartments, consistent with the observations discussed above ( Fig. 3and Fig. 4).


Figure 7: Time course of cell surface transferrin receptor degradation. K562 cells were surface iodinated by the lactoperoxidase-glucose oxidase coupled method described under ``Materials and Methods.'' After surface labeling, the cells were resuspended in alpha-minimal essential medium and incubated in the presence or absence of 32.4 nM PMA for 0-16 h, at which time the cells were collected to be processed for immunoprecipitation of I-labeled transferrin receptors. Immunoprecipitates were analyzed by SDS-PAGE and the results of autoradiography of the gel (4 day exposure) are presented. The experiment was repeated four times with similar results.



Since the half-life of cell surface transferrin receptors is unchanged in K562 cells exposed to PMA, the simplest explanation for the results of Fig. 6is that in response to protein kinase C, transferrin receptor synthesis is completely shut down resulting in a net loss of receptors. This interpretation is consistent with the results of Ho et al.(28) which demonstrate that activation of protein kinase C in HL60 cells dramatically eliminates transferrin receptor mRNA after 12 h of exposure to PMA. To examine whether phorbol esters promote a similar effect in K562 cells, RNA was prepared from cells treated with 50 nM PMA for various times of incubation up to 16 h. The results of the Northern blot analysis shown in Fig. 8indicate that activation of protein kinase C leads to a rapid loss of transferrin receptor message, with a significant decrease in mRNA levels observed within 4 h of exposure to the phorbol ester. We therefore propose that for K562 cells, phorbol esters induce down-regulation of transferrin receptors in two stages: an acute phase during which the rate of endocytosis is stimulated, although the pattern of transferrin receptor traffic is unchanged, followed by a more chronic down-regulation of transferrin receptor synthesis resulting in a net loss of receptors.


Figure 8: Northern blot analysis of K562 cell transferrin receptor mRNA after treatment with PMA. Total RNA was isolated from K562 cells incubated for the indicated times with 50 nM PMA, and 20-µg samples were electrophoresed on 1% formaldehyde agarose gels and blotted to nylon membranes to be UV-cross-linked. The Northern blot was hybridized with radiolabeled probes for transferrin receptor mRNA and 36B4 mRNA (control) as indicated. Identical results were obtained in a similar experiment performed on a separate occasion.




DISCUSSION

The effects of phorbol esters on the receptor-mediated endocytosis of transferrin have been studied in many different cell lines, including A431 cells(6) , mouse 3T3 and Ltk cells(7, 8) , and Chinese hamster ovary cells(9, 10) . In each of these studies, activation of protein kinase C was documented to increase transferrin receptor phosphorylation, but either little change in surface number of receptors was observed(7) , or increased numbers of transferrin receptors appearing on the cell surface were reported(8, 9, 10) . It was determined that the latter effect is promoted by increased rates of exocytosis such that receptor recycling is stimulated and transferrin receptors become redistributed to the cell surface(9) . Despite the fact that receptor phosphorylation was initially implicated to play a role in the membrane trafficking of the transferrin receptor(1, 5, 15) , site-directed mutagenesis studies that altered or deleted the key serine residue phosphorylated by protein kinase C (serine 24) revealed little change in the uptake of ligand or kinetics of transferrin receptor internalization-recycling(7, 8, 9) . These observations have led to the current view that receptor phosphorylation does not play a role in the receptor-mediated endocytosis and recycling of transferrin.

In contrast to observations made for these other cell lines, endocytosis of the transferrin receptor by erythroid or leukemic cell lines is stimulated by PMA(2, 5, 20, 29) . As shown by the work presented here, as well as in many past investigations(2, 11, 29) , PMA-stimulated K562 cells display a pattern of increased internalization resulting in a decreased number of cell surface transferrin receptors. Since none of these cell lines have been utilized in site-directed mutagenesis studies examining receptor phosphorylation, the role for serine 24 in PMA-stimulated internalization has not been directly assessed. The fact that phorbol esters will provoke differentiation of these cell lines further complicates the situation since differentiation-dependent changes in transferrin receptor endocytosis and the state of receptor phosphorylation have also been reported(30) . Finally, although down-regulation of the transferrin receptor was determined to result from enhanced rates of endocytosis(2, 11) , the fate of these redistributed receptors remained poorly defined. All of these uncertainties prompted this investigation of the pattern of transferrin receptor traffic in K562 cells in order to gain a better understanding of the factors involved in PMA-stimulated internalization.

The results presented here define that activation of protein kinase C down-regulates the K562 cell transferrin receptor in two distinct phases. The first acute phase of phorbol ester treatment results in enhanced rates of endocytosis(2, 11) ; this effect is reversible within the first few hours of exposure and is the direct result of protein kinase C activation since the kinase-specific inhibitor staurosporine restores cell surface transferrin receptor numbers to basal levels. The fact that fluid-phase uptake of horseradish peroxidase is also stimulated under these conditions indicates that the effect of protein kinase C activation is not specific for the transferrin receptor itself. Although this does not exclude a role for serine 24 phosphorylation in the process of down-regulation, this observation does suggest that the target of protein kinase C action most likely resides in elements of the endocytic apparatus. This idea is consistent with proposals first made by Backer and King (31) concerning the PMA-triggered internalization of receptors independent of their phosphorylation states.

A second point to be made concerning this initial stage of receptor down-regulation is that it does not occur as a consequence of the induction of cellular differentiation. Our results demonstrate that bryostatin-1, a protein kinase C activator that does not provoke differentiation of K562 cells(24) , displays all of the properties of phorbol esters; that is, exposure to bryostatin-1 accelerates endocytosis and down-regulates transferrin receptors from the surface of K562 cells. This response clearly distinguishes receptor down-regulation from pathways of cellular differentiation and further substantiates a role for protein kinase C in the regulation of endocytosis. Finally, the pattern of transferrin receptor membrane traffic is unaltered in PMA-stimulated K562 cells, excluding the possibilities that down-regulated receptors become rerouted to new intracellular compartments or internalized into lysosomal, degradative pathways. Our combined results support the hypothesis that phosphorylation of factor(s) involved in early stages of receptor-mediated endocytosis is responsible for the PMA-stimulated down-regulation of transferrin receptors in K562 cells.

In addition to these early and immediate effects of protein kinase C activation, prolonged treatment of K562 cells with phorbol esters or bryostatin-1 results in decreased synthesis of transferrin receptors. The second chronic phase of receptor down-regulation promotes not only a decrease in the number of transferrin receptors appearing on the cell surface, but a general loss of both cell surface and intracellular receptors as well. While one explanation for this overall loss of receptors during this chronic phase of down-regulation is that PMA-stimulated internalization results in transferrin receptor traffic to the lysosome, the results of our morphological and subcellular fractionation experiments do not support this idea. Instead, based on the results of our Northern blot analysis we conclude that prolonged activation of protein kinase C leads to a marked reduction in the level of transferrin receptor mRNA. This effect is identical to the regulation of transferrin receptor synthesis observed in HL-60 leukemia cells, which also display transferrin receptor down-regulation in response to PMA, exhibiting decreased receptor mRNA, and a pattern of monocytic differentiation(28) . Since the level of receptor message observed in their studies temporally correlated with the cessation of HL-60 cell growth, Ho et al.(28) concluded that the reduction in transferrin receptor levels was not a consequence of cellular differentiation. Rather, these investigators proposed that transferrin receptor down-regulation may signal programmed inhibition of cell growth, which is necessary for terminal differentiation. Our results do not support this idea since bryostatin-1 also reduces transferrin receptor levels; thus, receptor synthesis may be regulated by protein kinase C independent of the acquisition of the differentiated phenotype. This suggests that the phenomenon of receptor down-regulation is not responsible for the inhibition of cell growth associated with terminal differentiation, and in fact the bryostatin-1-treated K562 cells continue to divide.

Our results clearly support the model that activation of protein kinase C down-regulates the K562 cell receptor in the two stages outlined above: 1) immediate stimulation of early steps in endocytosis, and 2) a secondary decline of transferrin receptor synthesis. Of particular interest is the immediate and reversible action of protein kinase C to clear transferrin receptors from the cell surface. Limited evidence for a role of phosphorylation in the endocytic pathway exists(31) ; however, in vitro studies have documented that inhibitors of protein phosphatases, okadaic acid and microcystin-LR, block endocytic vesicle fusion(32) . Although the latter studies by Woodman et al.(32) determined that this effect was not exerted via cdc2 kinase, the actual kinase involved in in vitro endosome fusion remains unknown. It should be noted that in vivo studies have also indicated that okadaic acid inhibits endocytosis in HeLa cells(33) , consistent with the notion that elements of the endocytic pathway can be modulated by phosphorylation. Our study demonstrates that the down-regulation of K562 cell transferrin receptors provides an excellent paradigm to study the role of phosphorylation and protein kinase C in endocytosis, and future work in this area encompassing in vitro studies may provide further insights into the regulation of vesicle traffic.


FOOTNOTES

*
This work was supported by Grant CB15 from the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Junior Faculty Award from the American Cancer Society. To whom correspondence should be addressed.

(^1)
The abbreviations used are: PMA, 4beta-phorbol 12-myristate 13-acetate; TEA, triethanolamine; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; PAGE, polyacrylamide gel electrophoresis; PDBu, phorbol dibutyrate; BSA, bovine serum albumin; MOPS, 4-morpholinepropanesulfonic acid.

(^2)
J. E. Schonhorn and M. Wessling-Resnick, personal observations.


ACKNOWLEDGEMENTS

We thank Drs. Kathy Call and Dale Goad for their helpful advice and expert guidance in performing Northern blot analysis experiments.


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