红细胞生成素受体3

The distinct erythropoietin functions that promote cell survival and proliferation are affected by aluminum exposure through mechanisms involving erythropoietin receptor Daniela Vittori , , Nicolás Pregi, Gladys Pérez, Graciela Garbossa and Alcira Nesse Laboratorio de Análisis Biológicos, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón II, Piso 4, Ciudad Universitaria, Ciudad de Buenos Aires (C1428EHA), Argentina Received 5 March 2004;  revised 23 July 2004;  accepted 6 August 2004.  Available online 21 August 2004. Abstract Erythropoietin ( Epo) promotes the development of erythroid progenitors by triggering intracellular signals through the binding to its specific receptor ( EpoR) . Previous results related to the action of aluminum (Al) on erythropoiesis let us suggest that the metal affects Epo interaction with its target cells. In order to investigate this effect on cell activation by the Epo–EpoR complex, two human cell lines with different dependence on Epo were subjected to Al exposure. In the Epo -independent K562 cells, Al inhibited Epo antiapoptotic action and triggered a simultaneous decrease in protein and mRNA EpoR levels. On the other hand, proliferation of the strongly Epo -dependent UT-7 cells was enhanced by long-term Al treatment, in agreement with the upregulation of EpoR expression during Epo starvation. Results provide some clues to the way by which Epo supports cell survival and growth, and demonstrate that not all the intracellular factors needed to guarantee the different signaling pathways of Epo -cell activation are available or activated in cells expressing EpoR. This study then suggests that at least one of the mechanisms by which Al interfere with erythropoiesis might involve EpoR modulation. Keywords: Erythropoietin; Aluminum; Erythropoietin receptor; K562 cell; UT-7 cell line; Apoptosis Article Outline 1. Introduction 2. Materials and methods 2.1. Materials 2.2. Cell lines and cultures 2.3. Aluminum cell loading 2.4. Fluorescent nuclear stain of apoptotic cells 2.5. Cell lysis 2.6. Immunoprecipitation 2.7. Western blotting 2.8. Reverse transcriptase–polymerase chain reaction (RT–PCR) 2.9. Statistics 3. Results 3.1. Response of cell lines to Epo 3.2. Effect of aluminum on Epo antiapoptotic activity 3.3. Aluminum effect on cell proliferation induced by Epo 3.4. Aluminum effect on EpoR expression 4. Discussion Acknowledgements References 1. Introduction Erythropoietin (Epo) is the main factor that promotes viability, proliferation, and differentiation of mammalian erythroid progenitor cells, functions that are transduced by the specific cell surface Epo receptor (EpoR) [1], [2] and [3]. In the course of our investigation focused on the mechanisms by which aluminum (Al) exposure could impair erythropoiesis, we found some clues to the possible interference of the metal with Epo activity. The main physiological target cells for Epo, late erythroid progenitors CFU-E (colony-forming units-erythroid), showed a remarkable inhibition of in vitro response to the growth factor, after being exposed to Al [4] and [5]. The steep reduction of CFU-E development due to this treatment only occurred under Epo stimulation, and Al affected cellular mechanisms of progenitor cells in such a way that the action of the metal could not be overcome by increasing Epo doses [4]. Furthermore, a similar negative effect upon bone marrow cells was reproduced in ex vivo assays after experimental Al exposure [6], [7], [8] and [9]. Since EpoR was detected in many different cells and tissues, evidence has been accumulated to show that Epo activity is not restricted to the erythroid compartment [10], [11], [12] and [13]. In addition, there is increasing knowledge supporting the idea that Epo signals that promote proliferation could be separated from the signals that promote protection against apoptosis [14]. Nevertheless, despite the available data on EpoR-activated signal transduction molecules, little is known about the specific signals regulating this process. To further explore the common and/or unique signals triggered by Epo in human cells, we employed a model involving two Al-overloaded cell lines, K562 and UT-7, both expressing EpoR but showing different dependence on the hormone. Erythroleukemic K562 cells express low EpoR amounts [15] and are non-Epo-dependent. On the other hand, UT-7 cells, which express a large number of Epo binding sites, grow in response to Epo [16]. As it was stated before, Al is likely to affect erythroid cell activation by Epo [4], [5] and [8]. Therefore, since K562 and UT-7 cells show different Epo dependence, we assumed that the effect of the metal on the response of these cell lines to the growth factor would be a potentially useful model to investigate whether the multiple functions attributed to Epo are mediated by distinct intracellular signals. Moreover, this study may contribute to further understand the mechanisms by which Al affects erythroid cell response to Epo. 2. Materials and methods 2.1. Materials All chemicals used were of analytical grade. RPMI-1640 medium, bovine serum albumin (BSA), 2,7-diaminofluorene (DAF), Hoechst 33258 dye, sodium o-vanadate, phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, and pepstatin A were obtained from Sigma-Aldrich; Iscove's Modified Dulbecco's Medium (IMDM) and specific primers for EpoR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Invitrogen Life Technologies; polyclonal anti-EpoR antibody (SC-697) from Santa Cruz Biotechnology; monoclonal anti-phosphotyrosine (anti-PY) antibody and Protein A-agarose from BD Transduction Laboratories; Trizol Reagent from Gibco BRL; nitrocellulose membranes (Hybond), chemiluminiscent system kit (ECL) and Ready To Go T-Primed First-Strand Kit from Amersham Biosciences; agarose from Promega; ethidium bromide from Mallinckrodt; sodium dodecylsulfate (SDS), acrylamide, bis-acrylamide, aluminum chloride, Triton X-100, and Tween 20 from Merck; fetal bovine serum (FBS) (Bioser) and penicillin–streptomycin (PAA Laboratories) from GENSA and recombinant human erythropoietin (Epo, Hemax) from Biosidus (Argentina). 2.2. Cell lines and cultures Human erythroleukemic K562 cells, purchased from American Type Culture Collection (Manassas, VA), were grown in HEPES-buffered RPMI-1640 medium (pH 7.0±0.3), supplemented with 10% heat-inactivated FBS and 100 U/ml penicillin–100 μg/ml streptomycin [17]. UT-7 cell line, initially established from bone marrow cells obtained from a patient with acute megakarioblastic leukemia [16], was kindly provided by Dr. Patrick Mayeux (Cochin Hospital, Paris, France). Stock cultures were maintained in IMDM supplemented with 10% FBS and 1 U/ml Epo. Cell cultures were developed at 37 °C in an atmosphere containing 5% CO2 and 100% humidity, and one half of the medium was replaced every 3–4 days. Proliferation and cell viability were evaluated by the Trypan blue exclusion test, and cell differentiation was estimated by counting hemoglobin-positive cells after DAF–hydrogen peroxide reaction [18]. 2.3. Aluminum cell loading Al citrate was freshly prepared in 0.1 M Tris–HCl (pH 7.3) by mixing Al chloride and sodium citrate solutions (1:1.5 molar ratio), and added to culture media at 100 μM final concentration. This Al amount proved to produce in vitro toxic effects upon CFU-E cells similar to those attributed to in vivo Al overload [5] and [8]. 2.4. Fluorescent nuclear stain of apoptotic cells Cell cultured on slide covers were stained as follows: (a) addition of five drops of Carnoy solution (methanol/acetic acid, 3:1) for 2 min and complete removal of medium; (b) fixation with 1–2 ml Carnoy solution for 5 min (step repeated twice); (c) drying at 20 °C, after solution withdrawal; (d) addition of 1 ml of 1 μg/ml Hoechst 33258 dye prepared in Mc Ilvaine buffer (0.04 M citric acid, 0.12 M disodium phosphate, pH 5.5); (e) washing thrice with distilled water; (f) mounting by using Mc Ilvaine buffer. Fluorescent nucleus with apoptotic characteristics were detected by microscopy under UV light at 365 nm (Axioplan Fluorescent Microscope, Zeiss). Differential cell counting was performed by analyzing at least 400 cells. 2.5. Cell lysis Cells were washed with ice-cold phosphate-buffered saline (PBS) solution containing 1 mM sodium o-vanadate, and lysed with hypotonic buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100) containing protease inhibitors (1 mM PMSF, 4 μM leupeptin, 2 μM pepstatin A, 1 μg/ml aprotinin) and 1 mM sodium o-vanadate, in a ratio of 200 μl/107 cells. After 30 min of incubation on ice, insoluble material was removed by centrifugation at 15,000×g for 15 min. 2.6. Immunoprecipitation Cell extracts were incubated with 3 μg/ml polyclonal anti-EpoR antibody during 1 h at 4 °C. Then, Protein A-agarose was added and, after overnight incubation at 4 °C in a rotating shaker, immunoprecipitates were collected by centrifugation at 15,000×g during 15 min and washed twice with the lysis buffer, containing the mentioned protease inhibitors and o-vanadate. 2.7. Western blotting Immunoprecipitates were boiled for 3 min in the Laemmli buffer [19], resolved by SDS–polyacrylamide gel electrophoresis (T=8%) and then, electroblotted onto a Hybond nitrocellulose membrane during 1.5 h (transfer buffer: 25 mM Tris, 195 mM glycine, 0.05% SDS, pH 8.3, and 20% (v/v) methanol). Residual binding sites on the membrane were blocked with 5% ECL membrane blocking agent in Tris-buffered saline (25 mM Tris, 137 mM NaCl, 3 mM KCl, pH 7.4) containing 0.1% Tween 20 (TBS–Tween) for 1 h at room temperature. The blots were then incubated with the appropriate concentration of monoclonal anti-PY antibody during 1 h at 4 °C, washed three times for 10 min each with TBS–Tween, and probed with a 1:1000 dilution of anti-mouse horseradish peroxidase-conjugated antibody for 1 h at 20 °C. After washing, the blots were incubated with the enhanced chemiluminiscence substrate (ECL kit) and the bands detected by using a Fujifilm Intelligent Dark Box II (Fuji) equipment coupled to a LAS-1000 digital camera. To visualize the bands, the Image Reader LAS-1000 and LProcess V1.Z2 programs were employed. The blots were then stripped with 62.5 mM Tris–HCl (pH 6.8), 2% SDS, 100 mM 2-mercaptoethanol at 50 °C for 30 min, washed, blocked, and reprobed with anti-EpoR antibody. 2.8. Reverse transcriptase–polymerase chain reaction (RT–PCR) Total RNA was isolated by means of Trizol Reagent and its concentration estimated by measuring the optical density at 260 nm [20]. cDNA was synthesized by reverse transcription using Ready To Go T-Primed First-Strand Kit, starting from a sample of total RNA (2.5 μg). An aliquot of cDNA was amplified by 25 PCR amplification cycles for UT-7 cells and 30 cycles for K562 cells (94 °C for 20 s, primer annealing at 64 °C for 30 s, extension at 72 °C for 40 s) and a final incubation at 60 °C for 7 min. Specific primers were employed for EpoR [21] and for the internal standard GAPDH [22]. The PCR products were examined by electrophoresis on 1.5% agarose gel containing ethidium bromide. Gels were photographed and analyzed through the ArrayGauge and ImageGauge software. 2.9. Statistics Results are expressed as mean±S.E.M. When corresponding, the non-parametric Mann–Whitney U-Test or the Kruskal–Wallis One-Way Analysis of Variance Test was employed. At least differences with P<0.05 were considered the criterion of statistical significance. Correlation between variables was described by the Pearson r coefficient. 3. Results 3.1. Response of cell lines to Epo To study the degree of Epo dependence of K562 and UT-7 cell lines, erythroid differentiation, cell growth, and survival were analyzed. Three-day cultures were used to determine the response to Epo regarding cell viability and proliferation in dose–effect assays, in the range between 0.1 and 10.0 U Epo/ml (Fig. 1). UT-7 cultures showed a close relationship between Epo dose (between 0 and 1.0 U Epo/ml) and viable cell number. The Epo dependence of these cells was clearly described by the Pearson coefficient (r=0.83, P<0.001). No further linear increase of cell growth was obtained with Epo concentration of 10.0 U Epo/ml. Full-size image (18K) Fig. 1. Proliferative response of K562 and UT-7 cells to Epo. Cells were initially plated at 2×105 cells/ml in the presence of increasing Epo concentrations. The viable cell number was determined after 3 days by the Trypan blue exclusion test. Results are expressed as mean±S.E.M. of five independent experiments. Pearson coefficient between UT-7 viable cell number and Epo concentration in the range between 0 and 1.0 U/ml was r=0.83 (P<0.001). View Within Article On the contrary, K562 cells grew independently of the Epo concentration present in the culture medium (Fig. 1). The effect of Epo on cell differentiation was measured by the development of cells containing hemoglobin after three days of treatment. In doses up to 10 U/ml, the hormone did not play any role in erythroid differentiation of both cell lines, since percentages of hemoglobinized cells showed no significant differences with respect to spontaneous differentiation (without Epo). Results of cell differentiation in cultures stimulated with 10 U/ml Epo were: 10.3±1.8% vs. 8.5±0.8% (n=9) for K562 cells, and 4.3±1.7% vs. 5.5±1.2% (n=5) for UT-7 cells. 3.2. Effect of aluminum on Epo antiapoptotic activity From the experimental evidence described above, it is clear that K562 cells are Epo-independent to grow and differentiate. Thus, we assumed that EpoR expression in these cells would be related to the prevention of programmed cell death. To analyze this hypothesis, we examined the ability of Epo to inhibit hemin-induced apoptosis. Apoptotic cells were detected by fluorescence microscopy after Hoechst staining in cultures developed in the presence of hemin and Epo (Fig. 2). Whereas the mean value of spontaneous apoptosis was low (2.4±0.3%), it increased almost 10 times under hemin induction (24.6±3.6%), and the latter effect decreased 45% due to Epo influence, being the percentage of apoptotic cells 13.9±2.4%. Full-size image (23K) Fig. 2. Effect of aluminum on the Epo antiapoptotic activity. Apoptosis was measured in K562 cells cultured during 5 days in the presence of 50 μM hemin; 50 μM hemin+10 U/ml Epo or 50 μM hemin+10 U/ml Epo+100 μM Al citrate, as well as in control cells incubated without any treatment or in the presence of 100 μM Al citrate (upper panel). In UT-7 cell cultures, apoptosis was achieved by a 3-day Epo deprivation while the hormone antiapoptotic effect was demonstrated in 1 U/ml Epo stimulated cultures, regardless of whether 100 μM Al citrate was present or absent (lower panel). Apoptotic cells were detected by fluorescence microscopy after Hoechst staining. Each bar represents percentage value (mean±S.E.M.) of apoptotic cells with respect to total cell number. *Significant differences with respect to both (Hemin) and (Hemin+Epo+Al) (P<0.05, n=5). **Significant increase with respect to cultures performed in the presence of Epo (P<0.01, n=3). View Within Article As we already mentioned, one of the purposes of this work was to analyze whether the model of cells exposed to Al might be useful to study different mechanisms of Epo activation. Therefore, we evaluated Al ability to modulate Epo antiapoptotic action. When K562 cells induced to apoptosis by hemin were cultured with the simultaneous presence of a high Epo dose and Al, the protective effect of the hormone was counteracted, being the amount of apoptotic cells 25.5±3.3% (Fig. 2). It is worth mentioning that Al per se did not affect cell death (3.0±1.0%). Since UT-7 cells are Epo-dependent to survive, apoptosis can be achieved by Epo deprivation. As expected, cells deprived from the hormone for three days suffered a high degree of apoptosis, which was almost prevented by 1 U/ml Epo (76.8±3.8% vs. 12.2±1.6%, P<0.01) (Fig. 2). On the other hand, no Al effect was observed upon the Epo protective action. The apoptosis originated by Epo starvation was prevented by the hormone in such a way that minimal changes introduced by Al could be concealed. 3.3. Aluminum effect on cell proliferation induced by Epo Since Al seemed to alter Epo action and UT-7 cells proved to be Epo-dependent (Fig. 1), this cell line was used to investigate whether the metal might alter cell proliferation. Figure 3 shows the results of cell growth and viability determined in three-day cultures carried out under different conditions: (a) cells incubated without Epo (−Epo), and (b) cells incubated for 3 days with 1 U/ml Epo, with or without the addition of Al (Epo 1U+Al 3d and Epo 1U). Since this acute Al exposure proved not to affect cell proliferation, this 3-day assay was repeated with cells previously treated with Al for 30 days (Epo 1U+Al 30d). The interest to investigate cell behavior after such long-term exposure was based on the slow and silent effects on different tissues reported for the non-essential metal. Unlike the short-term Al treatment, chronic exposure to the metal induced a significant increase in the proliferative UT-7 cell response to Epo (Fig. 3: Epo 1U+Al 30d vs. Epo 1U, P<0.001). This assay demonstrated that in cells chronically exposed to Al the mean period necessary to duplicate cell population was 26±0.6 h, whereas in cells cultured without the metal it was 37±0.9 h, thus showing statistically significant differences between them (P<0.005). In order to determine whether this unexpected response could be attributed to the behavior of Al as a proliferative factor, an assay without Epo was developed in the presence of the metal (Fig. 3: Al 3d). Results showed that Al did not act as a proliferative inducer (Al 3d vs. −Epo, NS) in UT-7 cells. Full-size image (25K) Fig. 3. UT-7 cell proliferation under the effect of Epo and aluminum. UT-7 cell cultures were performed under different conditions. Cells without any previous treatment were incubated for 3 days without Epo (−Epo) and with 0.1 or 1 U/ml Epo (Epo 0.1U and Epo 1U). Similar assays were also made in the presence of 100 μM Al citrate (Al 3d, Epo 0.1U+Al 3d and Epo 1U+Al 3d). Cells previously exposed to 100 μM Al citrate during 30 days were stimulated with 0.1 U/ml (Epo 0.1 U+Al 30d) or 1 U/ml Epo (Epo 1 U+Al 30d) for 3 days. *Significant differences with respect to Epo 1U (P<0.001, n=5). **Significant differences with respect to Epo 0.1U (P<0.005, n=5). View Within Article To further investigate possible changes in sensitivity to Epo of cells after long-term Al treatment, cultures were stimulated with lower Epo amounts (0.1 U/ml). Figure 3 shows that chronically Al-overloaded cells cultured in the presence of 0.1 U/ml Epo reached the proliferation rate of Al-untreated cells stimulated with 1 U/ml Epo (Epo 0.1U+Al 30d vs. Epo 1U, NS). In contrast, the lack of effect due to a 3-day Al exposure (Epo 0.1U+Al 3d) emphasized the chronic effect of Al upon these UT-7 cells. 3.4. Aluminum effect on EpoR expression In order to determine whether the inhibition of the Epo antiapoptotic effect upon K562 cells was related to the interference of Al with EpoR activation signals, we examined receptor expression and phosphorylation. Cells were cultured for 5 days in the presence of Al. Then, treated and untreated cells were activated adding 10 U/ml Epo for 10 min. Cell lysates were immunoprecipitated with anti-EpoR antibody and Western blots were revealed using anti-PY antibody to detect tyrosine-phosphorylated proteins associated to the activated EpoR. This association was further confirmed through immune reaction with anti-EpoR antibody in the stripped blots. As can be seen in Figure 4, several proteins are re