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Originally published In Press as doi:10.1074/jbc.M902864199 on April 4, 2000

J. Biol. Chem., Vol. 275, Issue 24, 18375-18381, June 16, 2000
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Proteasomes Regulate the Duration of Erythropoietin Receptor Activation by Controlling Down-regulation of Cell Surface Receptors*

Frédérique VerdierDagger §, Pierre WalrafenDagger , Nathalie HubertDagger , Stany Chrétien, Sylvie GisselbrechtDagger , Catherine LacombeDagger ||, and Patrick MayeuxDagger **

From the Dagger  Institut Cochin de Génétique Moléculaire, INSERM U363 and the || Service d'Hématologie, Hôpital Cochin, Université René Descartes, 27 Rue du Faubourg Saint Jacques, F75014 Paris, and the  Institut National de la Transfusion Sanguine, 6 Rue Alexandre Cabanel, F75015 Paris, France

Received for publication, April 14, 1999, and in revised form, April 3, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The binding of erythropoietin (Epo) to its receptor leads to the transient phosphorylation of the Epo receptor (EpoR) and the activation of intracellular signaling pathways. Inactivation mechanisms are simultaneously turned on, and Epo-induced signaling pathways return to nearly basal levels after 30-60 min of stimulation. We show that proteasomes control these inactivation mechanisms. In cells treated with the proteasome inhibitors N-Ac-Leu-Leu-norleucinal (LLnL) or lactacystin, EpoR tyrosine phosphorylation and activation of intracellular signaling pathways (Jak2, STAT5, phosphatidylinositol 3-kinase) were sustained for at least 2 h. We show that this effect was due to the continuous replenishment of the cell surface pool of EpoRs in cells treated with proteasome inhibitors. Proteasome inhibitors did not modify the internalization and degradation of Epo·EpoR complexes, but they allowed the continuous replacement of the internalized receptors by newly synthesized receptors. Proteasome inhibitors did not modify the synthesis of EpoRs, but they allowed their transport to the cell surface. N-Ac-Leu-Leu-norleucinal, but not lactacystin, also inhibited the degradation of internalized Epo·EpoR complexes, most probably through cathepsin inhibition. The internalized EpoRs were not tyrosine-phosphorylated, and they did not activate intracellular signaling pathways. Our results show that the proteasome controls the down-regulation of EpoRs in Epo-stimulated cells by inhibiting the cell surface replacement of internalized EpoRs.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Most receptors for hematopoietic growth factors, including erythropoietin (Epo),1 belong to the cytokine receptor family. Ligand binding to these receptors initiates a cascade of events leading to receptor dimerization or multimerization and activation of tyrosine kinases of the Jak family. According to this model, stimulation by Epo induces Epo receptor (EpoR) tyrosine phosphorylation by the receptor-associated Jak2 kinases (1). Phosphotyrosine residues of the receptor provide docking sites for intracellular signaling proteins with Src homology 2 (SH2) domains. Recruitment of these proteins to the EpoR induces the activation of several intracellular signaling pathways including STAT5 transcription factors (2-7), Ras/mitogen-activated protein kinases (8-10), and phosphatidylinositol 3-kinase (11-14). The activation of these signaling pathways is transient despite the presence of Epo in the culture medium. Indeed, negative regulatory pathways leading to the termination of intracellular signaling are simultaneously turned on by Epo. The phosphotyrosine phosphatase SHP1 has been shown to be involved in the termination of Epo-induced intracellular signaling (15). In agreement with this finding, truncated EpoRs unable to bind SHP1 are responsible for a benign erythrocytosis in human (16). Another mechanism for EpoR down-regulation is the internalization and degradation of the activated receptor (17-20). However, both the extent and the mechanism of this ligand-induced internalization and degradation process have not been carefully examined.

Although hormone-receptor complexes are most generally believed to be degraded through the endosome-lysosome pathway (see Ref. 21 for review), the involvement of the ubiquitin-proteasome proteolytic pathway in the down-regulation of the hepatocyte growth factor receptor (22) and the platelet-derived growth factor receptor (23) has been documented. In both cases, ligand binding appears to induce the polyubiquitination and the degradation of the receptor by a process involving the proteasome. Moreover, proteasome inhibitors have been shown to prolong the activation of Jak tyrosine kinases and of Stat5 transcription factors in cells stimulated by interleukin-2 (IL-2) (24), IL-3 (25), ciliary neurotrophic factor (26), and growth hormone (27). We have recently reported that these inhibitors also prolonged the duration of EpoR and STAT5 tyrosine phosphorylation in response to Epo (28). The mechanisms leading to these prolonged activations have not been determined; neither the Jak kinases nor the STAT5 molecules appear to be degraded during the deactivation process. It has been suggested that the proteasome could modulate a phosphatase activity (24, 27).

In this paper, we examine the involvement of the proteasome in the down-regulation of EpoRs following Epo stimulation. During Epo stimulation, the cell surface pool of EpoRs was strongly decreased due both to the internalization and degradation of the Epo·EpoR complexes and to the inhibition of insertion of new receptors in the plasma membrane. We show that proteasome activity was required for the inhibition of the replenishment of the plasma membrane receptor pool in Epo-stimulated cells. In cells treated by proteasome inhibitors, newly synthesized EpoRs were inserted in the plasma membrane where they immediately replaced the internalized receptors. These receptors were responsible for the prolonged activation of Epo signaling pathways in cells treated with proteasome inhibitors. Thus, proteasome controls the duration of Epo signaling by inhibiting the renewal of cell surface EpoRs in Epo-stimulated cells.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents and Cells-- Highly purified recombinant human Epo (specific activity 120,000 units/mg) used throughout this study was a generous gift from Dr. M. Brandt (Roche Molecular Biochemicals). The protease inhibitor N-Ac-Leu-Leu-norleucinal (LLnL) was obtained from Sigma. Lactacystin and the calpain inhibitor PD 150606 were obtained from Calbiochem. Anti-phosphotyrosine antibodies (4G10) were a generous gift from Dr. B. Drucker. Anti-EpoR antibodies and anti-Shc antibodies were produced by immunizing rabbits with recombinant proteins composed of the full intracellular domain of the human EpoR or the SH2 domain of the Shc protein fused to GST. Anti-GST antibodies were also prepared in our laboratory using the same protocol. Anti-EpoR antibodies from Santa Cruz Biotechnology (catalog number SC-695) were used for immunoblot analysis. Anti-PI 3-kinase antibodies (p85 subunit, catalog number 06-195) and anti-Jak2 antibodies (catalog number 06-255) were purchased from Upstate Biotechnologies Inc. We produced anti-STAT5A and anti-STAT5B antibodies by immunizing rabbits with peptides corresponding to the 12 last amino acids (AGLFTSARSSLS) of STAT5A or the 8 last amino acids of STAT5B (QWIPHAQS) coupled to keyhole limpet hemocyanin. A 1/1 mixture of anti-STAT5A/STAT5B was used for immunoprecipitation and Western blot experiments. Human UT-7 cells (29) were cultivated in alpha -minimum essential medium (MEM) containing 10% fetal calf serum complemented with 2 units/ml Epo. Before each experiment, the cells were serum- and growth factor-deprived by overnight incubation in Iscove's Dulbecco's modified Eagle's medium containing 0.1% deionized bovine serum albumin and 25 µg/ml iron-loaded human transferrin. The 606 murine erythroleukemia cell line (a generous gift of Dr. F. Moreau-Gachelin, Institut Curie, Paris), which expresses endogenous EpoRs, was derived from a Spi1 transgenic mouse. Wild-type EpoR-transfected BaF3 cells (5), TF-1 cells (30), and Mo7E cells were also used in these experiments. Cells were cultivated in alpha -MEM containing 10% fetal calf serum and complemented with 2.5 ng/ml granulocyte-macrophage colony-stimulating factor (Mo7E and TF-1 cells) or with 2.5% WEHI-conditioned medium as a source of IL-3 (BaF3 cells). WEHI-conditioned medium is the culture supernatant of WEHI 3B cells (ATCC TIB-68).

Whole Cell Extracts, Immunoprecipitation, and Western Blotting-- Whole cell extracts, immunoprecipitations and Western blots were done as described previously (31). Renaissance (NEN Life Science Products) was used for the Western blot development.

Epo Labeling, Epo Binding, and Epo Internalization Studies-- Epo labeling using IODO-GEN and Epo binding were performed as described previously (32, 33). A saturating 125I-Epo concentration (2 nM, 4 units/ml) was used for these experiments in order to suppress the effects of putative modifications of EpoR affinity induced by the different inhibitors used. Nonspecific binding determined using a 250-fold excess of unlabeled Epo was <5% in every case. Specific binding data are presented. In most experiments, an acidic wash was performed to separate cell surface-bound from internalized Epo. After incubation, cells were washed twice at 4 °C to remove unbound ligand. Cells were incubated in 0.5 ml of acidic buffer (150 mM NaCl, 50 mM sodium acetate, pH 3.5) for 3 min at 4 °C. The pH was then adjusted to 7.4 using 1 M Tris/HCl, pH 8.5, and the cell suspension was centrifuged. The radioactivities of the supernatant (cell surface-bound Epo) and of the cell pellet (internalized Epo) were determined. By using this method, more than 95% of cell-bound 125I-Epo was recovered in the acidic wash supernatant, when 125I-Epo was bound to the cells at 4 °C in order to inhibit Epo internalization. Each reported experiment was performed at least three times with similar results.

Metabolic Labeling-- Growth factor-deprived UT-7 cells were washed with labeling medium (labeling medium: Met- and Cys-deficient MEM (Sigma M2289) containing 5% dialyzed fetal bovine serum, 25 mM HEPES, pH 7.4, and 1% complete MEM) and incubated for 15 min at 37 °C. Pilot experiments indicated that 125I-Epo binding and internalization, or proteasome inhibitors effects on 125I-Epo binding and internalization (see "Results"), were not modified when the cells were incubated in this medium. After incubation with or without proteasome inhibitor and/or Epo (see "Results"), the cells were pulse-labeled for 15 min with 0.25 mCi/ml of a mixture of [35S]Met and [35S]Cys (Trans-label, NEN Life Science Products). The cells were then quickly chilled, washed twice, and solubilized as described previously (31). Lysates were pre-cleared using preimmune serum and immunoprecipitated using either anti-EpoR or anti-GST control antibodies. Immunoprecipitates were separated by polyacrylamide gel electrophoresis, and labeled proteins were detected by fluorography. Radioactive bands were quantitated using a Molecular Dynamics PhosphorImager.

Northern Blot-- Total RNA extraction and Northern blot experiments were performed as described previously (34, 35). Quantitation of the radioactive bands was performed using a Molecular Dynamics PhosphorImager.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Proteasome Inhibitors Stabilize Intracellular Signaling Pathways Activated by Epo without Inhibiting a Phosphatase Activity Responsible for EpoR Dephosphorylation-- To determine the effect of proteasome inhibitors on Epo signaling, we examined the tyrosine phosphorylation of the EpoR in UT-7 cells incubated in the absence or presence of LLnL or lactacystin. In the absence of proteasome inhibitors, Epo-induced tyrosine phosphorylation of the EpoR was transient and was strongly decreased after 60 min of stimulation. In contrast, treatment of cells with 50 µM LLnL or lactacystin led to a sustained phosphorylation of the EpoR throughout the 60-min incubation period (Fig. 1A). Fig. 1B shows that cell treatment with LLnL also inhibited the dephosphorylation of other Epo-activated proteins such as the p55 and p48 forms of SHC, Jak2, and STAT5 and prolonged the association of PI 3-kinase with tyrosine-phosphorylated proteins. Probing total cell lysates with antibodies specific for these signaling proteins showed that their cellular levels were not modified during the 2-h incubation period with LLnL. Similar results were obtained using lactacystin (data not shown). Overall, these results show that the activation of all signaling pathways activated by Epo was prolonged in the presence of proteasome inhibitors.


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  		Fig. 1.   Proteasome inhibitors prolong the Epo-induced tyrosine phosphorylation of the EpoR and the activation of intracellular signaling pathways. A, growth factor-deprived UT-7 cells were preincubated for 15 min at 37 °C with or without 50 µM LLnL or 50 µM lactacystin. The cells were then stimulated with 10 units/ml Epo for the indicated times. Cellular extracts were immunoprecipitated (IP) with anti-EpoR antibodies, and immunoprecipitates were analyzed by Western blot (WB) with anti-phosphotyrosine (anti-PY) antibodies. ER indicates the EpoR. B, UT-7 cells were pretreated with or without 50 µM LLnL and stimulated with Epo as described in A. Cellular extracts corresponding to 107 cells were immunoprecipitated using anti-SHC, anti-Jak2, anti-STAT5, or anti-PI 3-kinase antibodies and analyzed by Western blot with anti-phosphotyrosine antibodies (left part of the figure). Simultaneously, total cell lysates corresponding to 5 × 105 cells were directly analyzed by Western blot using anti-SHC, anti-Jak2, anti-STAT5, anti-PI 3-kinase, or anti-SHP1 antibodies (right part of the figure). The identification of phosphotyrosine-containing proteins immunoprecipitated with anti-PI 3-kinase antibodies is reported elsewhere (49).

Prolonged EpoR tyrosine phosphorylation could be due either to the sustained activation of a tyrosine kinase or to inhibition of the tyrosine phosphatase activity responsible for EpoR dephosphorylation. We first tested whether proteasome inhibitors modified the cellular content of SHP1 that previously was shown to be involved in the termination of intracellular Epo signaling (15). As shown in Fig. 1B, neither proteasome inhibitors nor Epo modified the cellular level of SHP1. To test for the possible inhibition of a tyrosine phosphatase activity by proteasome inhibitors, we inhibited protein kinase activity. Prolonged EpoR tyrosine phosphorylation should not be suppressed when kinase activity was blocked, if this prolonged phosphorylation was due to the inhibition of a phosphatase activity. Attempts to inhibit Epo-induced tyrosine phosphorylation of the EpoR using the classical tyrosine kinase inhibitors genistein or herbimycin were unsuccessful for unknown reasons in UT-7. However, Epo-induced tyrosine phosphorylation of the EpoR was fully abolished by a 1-min preincubation with 500 nM staurosporine (data not shown). When LLnL-treated cells were stimulated by Epo, the tyrosine phosphorylation of the EpoR was stable for more than 60 min. However, when staurosporine was added after 30 min of Epo stimulation, the tyrosine phosphorylation of the EpoR quickly disappeared despite the presence of LLnL (Fig. 2, upper panel). This result indicates that the sustained tyrosine phosphorylation of the EpoR in proteasome inhibitor-treated cells required a continuous kinase activity and was not due to the inhibition of a phosphatase activity. Probing cellular extracts with anti-EpoR antibodies showed that staurosporine did not inhibit the expression of the mature form of the EpoR (Fig. 2, lower panel). This experiment also shows that the mature form of the EpoR disappeared during Epo stimulation but was maintained in LLnL-treated cells, suggesting that proteasome inhibitors could regulate the presence of EpoR at the cell surface during Epo stimulation.


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  		Fig. 2.   Prolonged tyrosine phosphorylation of the EpoR requires tyrosine kinase activity. Growth factor-deprived UT-7 cells were preincubated for 15 min at 37 °C with or without 50 µM LLnL. Cells were then stimulated with 10 units/ml Epo. After 30 min of Epo stimulation, LLnL-treated cells were separated in two parts, and one part was treated with staurosporine (final concentration, 500 nM). At the indicated times, samples corresponding to 107 cells were immunoprecipitated (IP) with anti-EpoR antibodies and analyzed by Western blot (WB) using anti-phosphotyrosine (anti-PY) antibodies (upper part of the figure). Total cell lysates (TCL) corresponding to 5 × 105 cells were also analyzed by Western blot using anti-EpoR antibodies (lower part of the figure). Deglycosylation studies showed that band 1 corresponds to the mature 66-kDa form of the EpoR, whereas band 2 corresponds to the 64-kDa EpoR previously observed in the endoplasmic reticulum of EpoR-transfected BaF3 cells (42).

Proteasome Inhibitors Inhibit the Down-regulation of EpoRs-- To determine whether proteasome inhibitors modified the cell surface expression of EpoRs, we studied the kinetics of 125I-Epo binding to UT-7 cells at 37 °C in the presence or absence of proteasome inhibitors. Internalized 125I-Epo·EpoR complexes were quantitated by removing the cell surface-bound 125I-Epo using an acidic buffer. In the absence of proteasome inhibitors, 125I-Epo transiently accumulated inside the cells, and both the cell surface and the intracellular pools of 125I-Epo strongly decreased after 30 min of incubation (Fig. 3). To verify that the disappearance of 125I-Epo corresponded to EpoR degradation, total cell extracts were probed with anti-EpoR antibodies. Fig. 4A shows that the levels of EpoR mature form (band 1) strongly decreased during Epo stimulation with kinetics similar to the disappearance of 125I-Epo binding, demonstrating that EpoR was degraded. In contrast, the maturing form of the receptor (band 2) was not significantly affected by Epo stimulation of these cells. Degradation of internalized 125I-Epo was measured by incubating the cells for 30 min with 2 nM 125I-Epo in order to allow the internalization of Epo·EpoR complexes. Then the cells were washed and incubated in 125I-Epo-free medium for various times. After 30 min of incubation, 80% of the internalized radioactivity was recovered in the incubation medium as trichloroacetic acid-soluble material (data not shown), demonstrating that most internalized 125I-Epo was degraded. Thus, after internalization both Epo and the EpoR were degraded with the same kinetics.


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  		Fig. 3.   Effects of LLnL and lactacystin on 125I-Epo binding and internalization. Growth factor-deprived UT-7 cells were preincubated for 15 min at 37 °C without inhibitors (circles), with 50 µM lactacystin (triangles), or with 50 µM LLnL (squares). After this time (corresponding to t = 0 on the graph) 125I-Epo (2 nM) was added with or without 500 nM unlabeled Epo. Aliquots corresponding to 2 × 106 cells were sampled at the indicated times. After washing to remove unbound 125I-Epo, the cells were incubated for 3 min in an acidic buffer to dissociate cell surface-bound 125I-Epo. The radioactivities removed by the acid wash (surface, solid lines, filled symbols) and resistant to the acid wash (internalized, broken lines, open symbols) were determined.


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  		Fig. 4.   EpoR protein levels in Epo-stimulated cells, effects of lactacystin and cycloheximide. Starved UT-7 cells were preincubated for 15 min with medium alone (A), with 50 µM lactacystin (B), with 50 µM lactacystin and 500 µM cycloheximide (C), with 50 µM LLnL (D), or with 50 µM LLnL and 500 µM cycloheximide (E). Then, Epo (final concentration, 2 units/ml) was added to the cells (time 0 of the figure). At the indicated times, whole cell extracts corresponding to 5 × 105 cells were analyzed by Western blot (WB) using anti-EpoR antibodies.

In the presence of lactacystin or LLnL, the cell surface pool of EpoRs did not decrease during the incubation with Epo (Fig. 3). Results from nine independent experiments showed that after 90 min of incubation with Epo, the number of Epo-binding sites was reduced to 19 ± 6% of the 10-min value in control cells, whereas it was not significantly modified in proteasome inhibitor-treated cells (91 ± 13%, mean ± S.D.). Probing total cell lysates with anti-EpoR antibodies shows that lactacystin and LLnL inhibited the disappearance of the mature form of the EpoR (Fig. 4, B and D, band 1). Lactacystin did not significantly modify the internalization of Epo·EpoR complexes and the intracellular pool of these complexes. In contrast, 125I-Epo accumulated inside cells treated with LLnL (Fig. 3). 125I-Epo accumulating inside LLnL-treated cells was immunoprecipitated by anti-EpoR antibodies (data not shown) showing that internalized Epo·EpoR complexes were protected by LLnL. In contrast to lactacystin, which appears to be highly specific for proteasome inhibition (36), LLnL also inhibits protease activity of calpains and cathepsin B (37). The specific calpain inhibitor PD150606 did not protect cell surface or internalized EpoRs (Fig. 5), showing that the protective effects of LLnL toward internalized Epo·EpoRs were not due to calpain inhibition. In contrast, lysosome inhibitors like NH4Cl and chloroquine, which do not inhibit proteasome activity, also inhibited the degradation of internalized Epo·EpoRs (Fig. 5). These results confirm that internalized EpoRs were ultimately degraded by lysosomes and strongly suggest that the protective effects of LLnL toward internalized complexes were due to its cathepsin inhibition properties. The effects of LLnL and lactacystin were also tested in four other cell lines as follows: the murine erythroleukemia cell line 606 that expresses endogenous EpoRs, and EpoR-transfected TF-1 cells, Mo7E cells, and BaF3 cells. In each cell line, both LLnL and lactacystin protected the cell surface pool of EpoRs during Epo stimulation, whereas LLnL, but not lactacystin, inhibited the degradation of internalized Epo·EpoR complexes.


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  		Fig. 5.   Effects of protease inhibitors and lysosome function inhibitors on Epo-induced EpoR regulation. Starved UT-7 cells were preincubated for 15 min with the indicated inhibitors (concentrations as follows: 50 µM for lactacystin, PD150606, and LLnL; 20 mM for NH4Cl, and 200 µM for chloroquine). The cells were then stimulated for 90 min with 2 nM 125I-Epo. Cell surface-bound Epo and internalized Epo were determined as described in Fig. 3.

Protein Synthesis Is Required to Stabilize the Plasma Membrane Pool of EpoRs, but Proteasomes Do Not Modify EpoR Synthesis-- Since internalized EpoRs accumulated inside the cells when receptor degradation was blocked by LLnL (Fig. 3 and immunoprecipitation studies), new receptors should be inserted in the plasma membrane to maintain the cell surface EpoR pool. We determined whether these receptors came from a preformed intracellular pool or whether protein synthesis was required to maintain the cell surface EpoR pool. For this purpose, cells were incubated with cycloheximide to inhibit protein synthesis. In the absence of proteasome inhibitors, cycloheximide did not significantly modify internalization and degradation of the EpoR (data not shown). When cycloheximide and LLnL were added together, the Epo-binding sites on the cell surface disappeared as they did in the absence of proteasome inhibitors (Fig. 6A). We confirmed this result using lactacystin (Fig. 6B). Probing total cell extracts with anti-EpoR antibodies showed that lactacystin did not protect the EpoRs in cells treated with Epo and cycloheximide (Fig. 4C). Under these conditions, the maturing form of the EpoR (band 2) first disappeared quickly followed by the disappearance of the mature form (band 1). Thus, renewal of the cell surface pool of EpoR required protein synthesis, and newly synthesized EpoRs were required to maintain the cell surface pool of receptors in proteasome inhibitor-treated cells. LLnL but not lactacystin protected internalized EpoRs from degradation even in the presence of cycloheximide (Fig. 4, C and E), confirming that the protective action of LLnL toward internalized EpoRs was not due to proteasome inhibition.


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  		Fig. 6.   Cycloheximide inhibits the replenishment of the EpoR cell surface pool. A, growth factor-deprived UT-7 cells were preincubated with 50 µM LLnL (squares), with 50 µM LLnL and 500 µM cycloheximide (CHX, diamonds) or with vehicle alone (circles) and labeled with 125I-Epo. At the indicated times, cell surface-bound (surface, solid lines, filled symbols) and internalized 125I-Epo (broken lines, open symbols) were determined as described in Fig. 3. B, growth factor-deprived UT-7 cells were preincubated for 15 min with or without 50 µM lactacystin and 500 µM cycloheximide and labeled for 90 min with 125I-Epo. At this time, cell surface-bound and internalized 125I-Epo was determined as described in Fig. 3.

To elucidate further the mechanism of action of the proteasome, we tested whether proteasomes were involved in the regulation of the EpoR synthesis. During Epo stimulation, the levels of mRNA measured by Northern blot analysis decreased in both control and LLnL-treated cells. The level of EpoR mRNA was more reduced in LLnL-treated cells than in control cells, possibly because the duration of Epo signaling was increased in LLnL-treated cells (Fig. 7A). Indeed, it has been shown that Epo down-regulates the expression of its receptor gene (38). EpoR synthesis was assessed by a 15-min pulse labeling with 35S-labeled amino acids (Fig. 7B) in cells incubated for 1 h with Epo and/or lactacystin. Similar results were obtained using LLnL. These experiments show that proteasome inhibitors did not modify the synthesis of EpoRs, thus suggesting that proteasomes control the transport of EpoRs to the cell surface.


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  		Fig. 7.   Proteasome inhibitors do not modify EpoR synthesis. A, growth factor-deprived UT-7 cells were preincubated for 15 min with or without 50 µM LLnL and stimulated with 10 units/ml Epo. At the indicated times, total RNA was extracted from cells and analyzed by Northern blot using an EpoR probe. The blot was exposed to radiography film (inset), and radioactivity was quantified using a Molecular Dynamics PhosphorImager. The blot was then stripped and hybridized with an actin probe. The main graph shows the ratio EpoR probe signal/actin probe signal determined using the PhosphorImager. B, growth factor-deprived UT-7 cells were preincubated for 15 min with or without 50 µM lactacystin and incubated in the presence or absence of 10 units/ml Epo for 60 min in metabolic labeling medium as described under "Experimental Procedures." [35S]Met and [35S]Cys were added during the last 15 min of incubation. After washing, the cells were solubilized and cell extracts were immunoprecipitated (IP) with anti-GST control antibodies (G) or with anti-EpoR antibodies (ER). Immunoprecipitates were separated by electrophoresis and revealed by fluorography.

The Internalized Receptors Are Not Tyrosine-phosphorylated and Do Not Activate Intracellular Signaling-- Since internalized 125I-Epo·EpoR complexes were protected from degradation in cells treated with LLnL, we determined whether they were able to activate intracellular signaling. We first tested whether the internalized EpoRs were tyrosine-phosphorylated. To that end, Epo was removed from the cell surface receptor by incubating the cells with an acidic buffer. The cells were then lysed, and the Epo·EpoR complexes were immunoprecipitated using anti-Epo antibodies. As shown in Fig. 8A, most tyrosine-phosphorylated EpoRs were lost when the cells were acid-washed before solubilization, indicating that tyrosine-phosphorylated EpoRs were located at the cell surface. To confirm this result, UT-7 cells were preincubated for 15 min with LLnL and stimulated for 60 min with Epo in the presence or absence of cycloheximide. Such a protocol allowed the accumulation of similar amounts of Epo·EpoR complexes inside the cells but did not allow us to maintain the plasma membrane pool of receptors in cycloheximide-treated cells (see Fig. 6). Fig. 8B shows that the tyrosine phosphorylation of the EpoR returned to basal levels in cells treated with cycloheximide and LLnL exactly as in control cells. Similarly, the activation of Jak2 and STAT5 was not maintained in the presence of cycloheximide (Fig. 8B), although the levels of Jak2 and STAT5 proteins did not significantly decrease during the incubation with cycloheximide (data not shown). Finally, when Epo was removed from LLnL-treated cells after 45 min of incubation, the tyrosine phosphorylation of the receptors quickly decreased (Fig. 8C), despite the continuous presence of LLnL that maintained the pool of internalized Epo·EpoR complexes. These results demonstrate that internalized EpoRs were not tyrosine-phosphorylated and that they probably did not transduce any intracellular signal. Thus, only cell surface EpoRs were responsible for prolonged EpoR signaling in proteasome inhibitor-treated cells.


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  		Fig. 8.   Internalized EpoRs do not stimulate intracellular signaling. A, growth factor-deprived UT-7 cells were preincubated for 15 min with 50 µM LLnL and stimulated with 10 units/ml Epo for 10 and 90 min. Cells were then recovered and incubated for 3 min in ice-cold phosphate-buffered saline (-) or acidic buffer (+) to remove the cell surface-bound Epo. The cells were then solubilized; the Epo·EpoR complexes were immunoprecipitated (IP) with anti-Epo antibodies and analyzed by Western blot (WB) using anti-phosphotyrosine antibodies (anti-PY) or anti-EpoR antibodies (anti-ER). B, growth factor-deprived UT-7 cells were preincubated for 15 min with or without 50 µM LLnL and 500 µM cycloheximide. The cells were then stimulated with 10 units/ml Epo for the indicated times and solubilized. EpoRs, Jak2, and STAT5 were immunoprecipitated with specific antibodies and analyzed by Western blot using anti-phosphotyrosine antibodies. C, growth factor-deprived UT-7 cells were preincubated for 15 min with or without 50 µM LLnL and stimulated with 10 units/ml Epo for the indicated times (first incubation). After 45 min, LLnL-treated cells were washed with ice-cold culture medium containing 50 µM LLnL to remove Epo. Washed cells were then incubated at 37 °C in culture medium with (+) or without (-) 10 units/ml Epo. The cells were then solubilized; EpoRs were immunoprecipitated, and immunoprecipitates were analyzed by Western blot using anti-phosphotyrosine antibodies.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The mechanisms leading to the arrest of the intracellular signaling process activated by the EpoR are poorly understood. The phosphotyrosine phosphatase SHP1 is involved in these inactivation mechanisms by dephosphorylating and inactivating the EpoR-associated Jak2 kinase. EpoRs unable to bind SHP1 exhibit a prolonged activation of Jak2 (15). In addition, Epo·EpoR complexes are internalized, and Epo is degraded by a process sensitive to inhibitors of lysosomal function (Ref. 17 and our results). However, the importance of this process in the termination of intracellular Epo signal transduction has not been reported. Our results show that, during Epo stimulation, the cell surface pool of EpoRs was strongly decreased due to the degradation of the EpoR after internalization (Figs. 3 and 4) and to the lack of replacement of the internalized EpoRs. This down-regulation of the cell surface EpoRs was reversible; Epo removal from the culture medium led to the recovery of the full cell surface EpoR pool after 6 h of incubation (data not shown). It has been recently shown that proteasome inhibitors prolonged the activation of the EpoR (28) and of several other cytokine receptors such as those of IL-2 (24), IL-3 (25), growth hormone (27), ciliary neurotrophic factor (26), and possibly interferon-gamma (39). Several hypotheses have been advanced to explain these results. It has been suggested that proteasome inhibitors could inhibit a phosphatase activity responsible for cytokine receptor inactivation (25, 27) or that they could protect cytokine receptors from degradation by a mechanism involving the ubiquitinated protein CIS (28). Our results show that proteasome inhibition did not modify the dephosphorylation (Fig. 2) nor the internalization and degradation of the EpoRs. Although the degradation of Epo and EpoR was inhibited by LLnL, it was not modified by the specific proteasome inhibitor lactacystin. It seems most likely that the inhibition of internalized EpoR degradation was due to the inhibition of cathepsins that have been shown to be inhibited by LLnL (37). In agreement with this hypothesis, lysosome inhibitors such as NH4Cl and chloroquine inhibited the degradation of internalized Epo·EpoR complexes confirming that these complexes were ultimately degraded by the lysosomes (Fig. 5). Internalized EpoRs that accumulated inside LLnL-treated cells were not tyrosine-phosphorylated and appeared unable to transduce intracellular signals (Fig. 8). Thus, they did not contribute to the prolonged signaling observed in cytokine-stimulated cells treated with LLnL. The inability of these receptors to transduce signals is surprising. Indeed, EpoRs should be continuously activated since they remained associated with Epo as demonstrated by the co-immunoprecipitation of 125I-Epo with the EpoRs. Accordingly, we have observed that the tyrosine kinase activity of internalized SCF-Kit complexes whose degradation was also blocked by LLnL remained stimulated and that internalized c-Kit was tyrosine-phosphorylated (data not shown). A plausible explanation would be that the internalized EpoRs did not remain associated with the Jak2 tyrosine kinases that are required for signal transduction. Moreover, these results suggest that internalization of the Epo·EpoR complexes could require the dephosphorylation of the receptor. Growth hormone receptors also seem to be dephosphorylated before internalization (27, 40) suggesting a common deactivation mechanism preceding internalization for cytokine receptors. We are currently testing this hypothesis.

Our results show that prolonged Epo signaling induced by proteasome inhibitors was due to the continuous replacement of internalized EpoRs by new receptors in the plasma membrane of Epo-stimulated cells. These receptors were quickly activated by Epo present in the incubation medium. When Epo was removed from the incubation medium or when membrane insertion of new receptors was blocked by protein synthesis inhibitors, intracellular signaling quickly stopped despite the presence of proteasome inhibitors (see Fig. 8). Thus, proteasomes appear to control the down-regulation of EpoR and the duration of Epo signaling by inhibiting the replacement of internalized EpoRs. The control of EpoR expression by the proteasomes appears to be essentially effective during Epo stimulation. Indeed, proteasomes did not significantly modify the expression of EpoRs in resting cells, and preincubation of cells for 90 min with 50 µM lactacystin only increased the number of cell surface EpoRs of 24 ± 11% (data not shown). The same result was previously reported for cells treated with LLnL (41). Therefore, pretreatment of cells with proteasome inhibitors did not significantly modify the level of activation of the EpoR and of the subsequently activated signaling pathways in the initial times of Epo stimulation (see Fig. 1). In contrast, during Epo stimulation the number of cell surface EpoRs remained roughly constant in proteasome inhibitor-treated cells because new receptors were continuously inserted in the plasma membrane, whereas the number of cell surface EpoRs quickly decreased in control cells. Newly synthesized EpoRs are involved in this process since EpoRs disappeared during incubations with cycloheximide despite the presence of proteasome inhibitors. However, we cannot exclude that synthesis of other proteins was also needed for the cell surface renewal of EpoRs. Neither the synthesis of the EpoRs nor the accumulation of maturing forms were modified by proteasome inhibitors (Figs. 4 and 7), suggesting that proteasomes could inhibit the transport of the synthesized receptors to the cell surface. The expression of the EpoRs at the cell surface of hematopoietic cells is tightly controlled by unidentified mechanisms. Cellular EpoR protein level does not seem to be the limiting factor for cell surface expression since overexpression of exogenous EpoRs into hematopoietic cells leads to the expression of a low number of cell surface receptors, whereas most receptors remain inside the cells (42). Moreover, infection of UT-7 cells with an amphotropic virus encoding the murine EpoR does not increase the number of EpoRs expressed at the cell surface but leads to the complete replacement of the endogenous human EpoRs by a similar number of murine EpoRs (43). These results strongly suggest that an unidentified factor that associates with the EpoR controls the cell surface expression of the EpoR. An attractive hypothesis would be that this limiting factor is sensitive to the proteasome. When proteasome activity is blocked, this factor could recycle and allow the transport of newly synthesized receptors to the cell surface. In agreement with this hypothesis, it has been demonstrated that components of a multimeric complex can be sorted separately along the endocytic pathway. Indeed, the alpha  chain of the IL-2 receptor has been shown to recycle to the cell surface after ligand-induced internalization of the IL-2 receptor complex, whereas the beta  and gamma  chains are degraded (44). Alternatively, the Jak2 molecule could be the limiting factor controlling the level of cell surface expression of the EpoR. After Epo stimulation, these kinases could be degraded by a mechanism involving the proteasomes and could be recycled when proteasome activity is blocked. Although the intracellular levels of Jak2 were not significantly modified during incubation with Epo and/or proteasome inhibitors (Fig. 1), we cannot exclude the presence of separate intracellular pools of Jak2 with only a restricted fraction of Jak2 being available to the EpoR. It has been recently shown that Tyk2 was required for sustained expression of the interferon-alpha receptor 1 protein (45). This mechanism would also explain why proteasome inhibitors also prolong the activation of other cytokine receptors that associate to Jak kinases. In contrast, lactacystin did not maintain the cell surface pool of c-Kit during SCF stimulation (data not shown), suggesting that the proteasome action could be restricted to receptors of the cytokine family.

Our results provide an explanation for the extended activation of the EpoR in cells treated with proteasome inhibitors. Prolonged activation of Epo signaling in proteasome inhibitor-treated cells is not restricted to leukemic cell lines. We have recently shown (46) that it also occurs in primary erythroid progenitors. Whether this mechanism could be responsible for increased responsiveness to growth factors observed in hematological disorders such as polycythemia vera remains to be determined. Interestingly, it has been recently shown that mutations in the granulocyte colony-stimulating factor receptor that inhibit ligand-induced internalization and lead to extended receptor activation frequently occurred in acute myeloid leukemia (47, 48), suggesting that elimination of the down-regulation mechanisms of cytokine receptors could lead to severe hematological disorders in human.

    ACKNOWLEDGEMENTS

We thank Dr. Joelle Finidori and Dr. Alice Dautry (Paris) for helpful discussions and Dr. Lucia Rothman-Denes (University of Chicago) for critical reading of the manuscript.

    FOOTNOTES

* This work was supported in part by a grant from the Comité de Paris of the Ligue Nationale Contre le Cancer (Associate Laboratory 8).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by Glaxo Wellcome and the French Society of Hematology (SFH).

** To whom correspondence should be addressed: ICGM, INSERM U363, Hôpital Cochin, 27 rue du Faubourg Saint Jacques, F75014 Paris, France. Tel.: 33 1 46 33 14 09; Fax: 33 1 46 33 92 97; E-mail: mayeux@cochin.inserm.fr.

Published, JBC Papers in Press, April 4, 2000, DOI 10.1074/jbc.M902864199

    ABBREVIATIONS

The abbreviations used are: Epo, erythropoietin; EpoR, erythropoietin receptor; IL, interleukin; LLnL, N-Ac-Leu-Leu-norleucinal; MEM, minimum essential medium; PI 3-kinase, phosphatidylinositol 3-kinase; GST, glutathione S-transferase; STAT, signal transducers and activators of transcription.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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