From www.bloodjournal.org by guest on January 26, 2015. For personal use only. Inhibition of the Erythropoietin-Induced Erythroid Differentiation by Granulocyte-Macrophage Colony-Stimulating Factor in the Human UT-7 Cell Line Is Not Due To a Negative Regulation of the Erythropoietin Receptor By 0. Hermine, A. Dubart, F. Porteux, P. Mayeux, M. Titeux, D. Dumenil, and W. Vainchenker The human pluripotent UT-7 cell line is growth factor-dependent for proliferation and differentiation. We have previously shown that (11 granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Epo) induce a myeloid and erythroid pattern of differentiation, respectively; (2) GMCSF acts predominantly over Epo for cell differentiation; (3) GM-CSF induces a rapid downmodulation (4 hours) of Epo receptors (Epo-R) at the mRNA and binding site levels; and (4) in contrast, Epo has no effect on GM-CSF receptor (GMCSF-R) expression. These results suggested that UT-7 cell commitment or differentiation may be directed by a hierarchical action of growth factors through an early and rapid transmodulation of growth factor receptors. To test this hypothesis, we introduced and expressed the murine Epo-R (muEpo-R) in UT-7 cells using a retroviral strategy. Two retroviral vectors were constructed: one carrying the neomycin resistance gene, and another carrying a mouse Epo-R cDNA devoid of its regulatory untranslated 3’sequence placed under the transcriptional control of the viral long terminal repeat element (LTR) and the neomycin resistance gene. Three UT-7/Epo-R infected clones (12.6, I O ) and one UT-7lneomycin clone (Neo) were selected in medium containing G418. After growth factor deprivation (18 hours), Epo-Rs were expressed at the same level (approximately 6,000 receptors per cell) in all four clones 12, 6, IO, Neo, and in parental UT-7 cells, and exhibited similar affinity (0.1 t o 0.2 nmol/L). Cross-linking experiments showed that Epo is associated with three proteins of about 66, 85, and 100 kD in cells of parental UT-7, as well as in cells of clones 10 and 12. An inhibitory antibody directed specifically against the human Epo-R (huEpo-R Ab) abolished almost completely the crosslinking on parental UT-7 cells, but not on cells of clone 12, demonstrating that more than 90% cell surface Epo-Rs were of murine origin. The presence of GM-CSF significantly reduced the number of Epo-Rs expressed on parental UT-7 cells, but not on cells of clones 12, IO, and 6. HuEpo-R Ab inhibited Epo-induced parental UT-7 cell growth, but not that of cells of clone 12, suggesting that the muEpo-R is able t o induce human UT-7 cell proliferation. When cells of clone 12 were switched from a medium containing GM-CSFt o one with Epo, cell surface glycophorin A (GPA) was induced, as in parental UT-7 cells without inhibition by the huEpo-R Ab, demonstrating that the muEpo-R is also able t o transduce a differentiation signal in human cells. However, in cells of clones 12, 6, IO, and Neo, as well as in parental UT-7 cells, the induction of GPA by Epo was inhibited by GM-CSF. This finding demonstrates that, although GM-CSF does not downregulate muEpo-R binding sites on UT-7/muEpo-R infected clones, it still inhibits the effects of Epo on cell differentiation. Therefore, hierarchical regulation induced by growth factors for cell commitment or differentiation more likely acts downstream of cell surface receptors at either the signal transduction or transcriptional levels. 0 1996 by The American Society of Hematology. H differentiation of the target cells bearing the appropriate growth factor receptor. In contrast, their role as inducers of differentiation and commitment of a multipotent cell is still a matter of debate. On one hand, a stochastic model hypothesizes that pluripotent stem cells are intrinsically committed to lineage restricted progenitors without influence of external stimuli, growth factors simply permitting their survival, amplification, and further de~elopment.~.’ On the other hand, in some instructive models, it is postulated that growth factors interact with their receptors at the stem cell or multipotent cell levels to direct the commitment or restriction toward the different hematopoietic lineages.6 It has been reported that growth factors may act competitively by an internal autocrine mechanism'^' or hierarchically by triggering their own receptors on cell s~rface.’,’~ In the latter hypothesis, the fate of the pluripotent cell would depend on the concentration of the encountered growth factors and on the number and affinities of their receptors expressed on the cell surface. We have recently shown that the commitment and differentiation along the erythroid or myeloid pathways of the multipotent UT-7 cell line are also directed hierarchically by erythropoietin (Epo) or granulocyte-macrophage colony-stimulating factor (GM-CSF), respectively.” In this cell model, GM-CSF acts dominantly over Epo and rapidly downregulates the number of Epo receptors (Epo-R) on the cell surface. This downregulation is mediated by decreased mRNA levels. This model suggests that receptor transmodulation could be involved in the UT-7 cell commitment or differentiation along EMATOPOIESIS IS A continuous process leading to the production of mature blood cells of various lineages from a population of pluripotent stem cells that are capable of both extensive self-renewal and maturation. The mechanisms that regulate the commitment of stem cells or the lineage restriction of multipotential progenitors are poorly understood.’ However, several speculative models have been proposed based on studies of progenitor cell colony growth assays, in vitro differentiation of cell lines, and in vivo Most studies have focused on the role of growth factors. It is well established that hematopoietic growth factors are able to sustain proliferation and survival at all stages of From INSERM U 362, lnstitut Gustave Roussy, Villejuif; Hdpital Necker, Service d’Hdmatologie Clinique, Paris; and INSERM U 363, ICGM, Hopital Cochin, Paris, France. Submitted May I , 1995; accepted October 6, 1995. Supported by grants from the Association de la recherche sur le Cancer (ARC 6688) (Villejuij France) and from la Ligue Nationale contre le Cancer (Paris, France). Address reprint requests to 0. Hermine, MO, INSERM U 362, Institut Gustave Roussy, 94805 Villejuif Cedex, France. The publication costs of this article were defrayed in part by puge charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1996 by The American Society of Hematology. 0006-4971/96/8705-0032$3.00/0 1746 Blood, Vol 87, No 5 (March 1). 1996:pp 1746-1753 From www.bloodjournal.org by guest on January 26, 2015. For personal use only. DIFFERENTIATION AND GROWTH FACTOR 1747 MODULATION one restricted lineage. It could not, however, be ruled out that other mechanisms were involved in these processes. To define the role of Epo-R transmodulation by GM-CSF, we have retrovirally transduced the muEpo-R cDNA into UT-7 cells. The muEpo-R cDNA was placed under the transcriptional control of the retroviral LTR and its posttranscriptional regulatory sequences were removed to avoid a downregulation by GM-CSF. We show that muEpo-R is functional in transducing both cell proliferation and differentiation signals in UT-7 cells, and although Epo-Rs in transfected cells were not transmodulated, GM-CSF still acted as a dominant factor over Epo, strongly suggesting that cell commitment or differentiation is not a consequence of a receptor transmodulation. A pBT Zen mR-Epo SVNeo ATG * * 5.3 kb 4.9 kb 2 kb C MATERIALS AND METHODS Cell lines and culture. UT-7 cells, kindly provided by N. Komatsu (Tichi Medical School. Tochigi-Ken. Japan).” were grown at 37°C in Iscove‘s modified Dulbecco‘s medium (IMDM) (Jbio. Paris. France) containing 10% fetal calf serum (FCS) (Jbio) and the appropriate growth factors in a humidified atmosphere with 5% CO,. Human recombinant Epo (Cilag, France) and GM-CSF (Amgen. Thousand Oaks, CA) were used at 2 UlmL and 2.5 nglmL, respectively. The cell concentration was maintained between 2 and 5 X IO’ cellslmL by diluting the cells with fresh medium every 3 to 4 days. Inhibiton?anti-Epo ond CM-CSFreceptor monoclonal rintihodies. Monoclonal antibodies (MoAb) directed against the human Epo-R (16.5.1) (huEpo-R) and the huGM-CSF-R were obtained from Genetics Institute (Cambridge, MA) and lmmunex (Seattle, WA). respectively. Both MoAbs are IgGl and inhibit the binding of Epo and GM-CSF to their respective receptors. Cell pro1;feration and d;fferentiationa.rsa\:v. Cells ( I X 1 04/mL) were cultured in a 96-well microculture plate (Falcon no. 3072: Becton Dickinson, Mountain View, CA) with Epo (2 UlmL) or GMCSF (2.5 nglmL) in the presence or absence of inhibitory antibodies directed against the EPO-R or GM-CSF-R. Cell numbers and viability were then assessed by the trypan blue dye exclusion test. Cell differentiation was assessed by analysis of GPA expression by flow cytometry. Flow cvtometp analysis. Cell surface antigens were detected by indirect immunofluorescence staining with an anti-GPA MoAb (CLB-ery 1, CLB, Rotterdam, Netherland). Cells were incubated for 30 minutes at 4°C with the diluted MoAb. After washing twice in RPMl (Jbio). the cells were reincubated with fluorescent sheep Fab‘ antimouse Ig (Silenus, Hawthorn, Australia) for 30 minutes at 4°C. After two washes in RPMI, cells were analyzed according to their membrane antigenic density using a flow cytometer (FacSort, Becton Dickinson). The negative control was determined on cells indirectly labeled with an irrelevant lgGl antibody. Constructions and retroviral infections q f UT-7cells. The retroviral vector encoding Epo-R was constructed by inserting 1.500 bp of the muEpo-R cDNA (Sal I fragment) in the Xho I site of the pBTZen-SVNeo vector,” in which the neomycin resistance gene is expressed under transcriptional control of the SV40 promoter. The muEpo-R cDNA fragment starts 24 bases upstream of the ATG codon and ends at the stop codon, and is driven by the viral long terminal repeat (LTR). This construct (Fig I), or the Neo construct (not shown), were transfected into the ecotropic packaging cell line #2.“ Individual neomycin-resistant clones were derived, and their supernatants tested for viral production on NIH 3T3 cells. The EpoR virus-producing clone, which produces the largest number of viruses, was finally selected. and its viral integrated structure and TGA 23.1 kb 9.4 kb 6.6 kb 4.3 kb 2.3 kb 2kb - W r 0.5 kb 1 - ” 1 1- 2kb I Fig 1. (A) Retroviral construct. The Epo-R cDNA coding sequence was cloned at the Xho I site of the pBTZen-SVNeo vector. Splice donor (D) and acceptor (A) sites and ATG and TGA codons are indicated. The size of the different expected mRNAs are given. DNAs and RNAs were prepared from three clones of Epo-R virus infected UT-7 cells (clones 6,10, and 12). IBI Southern blot analysis. A 10-pg portion of genomic DNA of the different samples was digested with bglll and electrophoresed on a 0.8% agarose gel. (C) Northern blot analysis. A total of 5 p g RNA of the different samples were electrophoresed on a formaldehyde-agarosegel. After transfer both membranes were hybridized with a Neo probe. expression were tested. The titers of the clones ($2 Neo and $2 Epo-R) used for infection were 5 x IO‘ infectious viral particles per milliliter of supernatant. Supernatants of ecotropic $2 clones containing the vectors were used to infect the amphotropic helper cell line JJCRIP in the presence of 8 mglmL of polybrene (Aldrich. Steinheim, Germany); 48 hours after infection, the $CRIP cells supernatant was collected. UT-7 cells (2 x 10’ cells; 4 X IO‘lmL final concentration) were then infected by incubation for 48 hours in a mixture of 3 mL filtered (0.45 pmollL sterile filter (Nalgene. Rochester, NY)) viral supernatant, 2 mL of a cell culture medium containing GM-CSF (2.5 nglmL final concentration), and polybrene (4 mglmL final concentration). UT-7 cells (10, 50. or 100 X 10‘1 mL) were then plated in a semisolid medium containing 0.8% methylcellulose in IMDM supplemented with 10% FCS, 1% bovine serum albumin (BSA), 2.5 nglmL GM-CSF, and 0.8 mg/mL (3418. Individual clones were selected after 10 days, and expanded in liquid medium in presence of G418 (0.8 mglmL) and GM-CSF (2.5 nglmL). DNA and RNA analvsis. Total cellular RNA was isolated according to the method of Chomczynsky and Sacchi.” RNA ( 5 to 10 pg per lane) was size-fractionated by formaldehydelagarose gel electrophoresis and then transferred to a nylon membrane (Hybond N; Amersham, Les Ulis, France). The membrane was hybridized with a labeled Neo cDNA probe (Amersham Multiprime DNAlabeling system and a-”P-dCTP). From www.bloodjournal.org by guest on January 26, 2015. For personal use only. 1748 HERMINE ET AL Genomic DNA was extracted using the standard method," digested with BgnI restriction enzyme using conditions recommended by the supplier. After separation on 0.8% agarose gel, DNA fragments were transferred onto a nylon filter and hybridized to a 32Plabeled Neo cDNA probe (Amersham Multiprime DNA-labeling system and w3*P-dCTP). Binding experiments. Epo receptor characteristics were studied as reported earlier." Briefly, Epo was iodinated with a specific activity ranging from 500 to 2,000 Ci/mmol using lodogen and 0.5 to 2 X 10' cells (parental UT-7 cells, and cells of clones 10, 6 , and 12) were incubated for two hours at 25°C with various concentrations of '2'I-Ep~in 100 pL Iscove's modified Dulbecco medium containing 5% FCS and 0.1% sodium azide. After this time, the cells were centrifuged, and aliquots of the supernatants were used to determine the free hormone concentrations. The cells were then washed three times with ice cold PBS and the radioactivity bound to the cells was measured. Nonspecific binding was determined by incubating the cells with 100-fold excess of unlabeled ligand and the specific binding was analyzed using an unweighted least-square regression fitting method (Enzfitter, Biosoft, Paris, France). In some experiments, the number of Epo binding sites was estimated from triplicate measurements realized using a saturating (2 nmol/L) '"7Epo concentration. Cross-linking experiments. Cell labeling with "'I-Epo and cross-linking with Disuccinimidyl suberate (DSS) (Pierce Chemical CO,Rockford, IL) were done as previously described." Briefly, the cells were labeled with '*'I-Epo for 30 minutes at 37°C in the presence or absence of inhibitory antibodies. Sodium azide (0.1%) was added to prevent internalization of the Epo-Epo-R complexes. The cells were washed twice with ice cold PBS and cross-linked for 30 minutes on ice with 0.2 mmol/L DSS. After two washes in PBS containing 0.1 molL ethanolamine pH 8.00, the cells were solubilized in 25 mmom HEPES pH 7.4 containing 150 mmoVL NaCI, 5 mmol/L EDTA, 1% Triton XIOO, 1 mmol/L phenylmethylsufonyl fluoride, and 1 pg/mL each of aprotinin, leupeptin, and pepstatin (all from Sigma, St Louis, MO). After centrifugation (27,OOOg 20 minutes), cellular extracts were immunoprecipitated with anti-Epo antibodies and protein A Sepharose and immunoprecipitated products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. RESULTS Transfected murine Epo-R is expressed on the cell surface of human UT-7 cells, and is not downregulated by GM-CSF. The human UT-7 cell line is strictly growth factor-dependent for survival, proliferation, and differentiation. We have previously shown that UT-7 cells express GM-CSF and EpoR, and that Epo-Rs are rapidly downregulated by GM-CSF during the first 4 hours of exposure. This effect occurred mainly at the transcriptional, and to a lesser extent, at the posttranscriptional levels (Herrnine et al, in preparation). To overcome this effect of GM-CSF on Epo-R expression, we introduced the murine cDNA Epo-R with a retroviral vector into UT-7 cells. This construct was devoid of the untranslated 3' regulatory elements" and was transcriptionally controlled by the viral LTR (Fig 1A). Three cell lines were derived from muEpo-R vector-infected UT-7 cells (clones 12, 6 , and 10) and expanded in liquid culture. To establish integration of the retrovirus in the individual clones, Southern blots were performed using BglII (Fig 1B). This restriction enzyme cuts twice within the proviral genome and is useful for comparisons of proviral Table 1. Binding of '251-Epoto UT-7 Cells and Cells of Clones 12, 10, and 6 in the Presence or Absence of GM-CSF minus GM-CSF plus GM-CSF UT-7 12 10 17,680 2 190 18,700 2 520 16,370 2 440 5,140 2 145 18,600 2 390 16,350 z 6 370 20,040 ? 420 18,200 i- 150 Cells were cultured in the presence of GM-CSF (2.5 ng/mL) or deprivated for 18 hours, and then were incubated with 2 nmol/L of '251-Epofor 2 hours at 25°C with 0.1% sodium azide. The data are presented in cpmlcell as the average of triplicate measurements ? SD. The results represent the average of three experiments. integration between different lines. Two bands were detected for each clone, a common one (2.1 kb) corresponding to the internal proviral fragment, and a second one corresponding to the 3' proviral junction fragment, which differed in the three different clones, demonstrating that they derived from independent infection events. A Neo vector infected clone (Neo) andor the parental cell line UT-7 were used as a control throughout this study. To check transcription of the proviral genome and expression of the muEpo-R, Northern blot analysis and Epo binding assays were performed. Using a Neo probe, Northem analysis identified three transcripts of the expected size, ie, 3.6, 3.2, and 2 kb for Neo infected cells (data not shown) and 5.3, 4.9, and 2 kb for Epo-R infected cells (Fig IC). In contrast, no signal was observed in the parental UT-7 cell line. Using binding experiments, we showed that surprisingly, and despite high expression of muEpo-R mRNA (hybridized with a Neo probe on Fig IC), muEpo-R transfected cells (clones 12, 6 , IO) had the same number of Epo-R on their cell surfaces as the UT-7 parental cell line and the Neo transfected cells (Neo) (Table 1 ). Furthermore, Scatchard analysis performed on clones 12 and 10 confirmed that they expressed a comparable number of Epo-R per cell with kD values close to parental UT-7 cells (Fig 2). However, because human recombinant '"I-Epo binds mu and hu Epo-R with roughly the same affinity," we could not determine from these results whether murine Epo-Rs were expressed on the surface of the cells from clones 6 , 10, 12. To answer this question, we performed cross-linking experiments with Epo on parental UT-7 cells and cells from clones 12 and 10 in the presence or absence of an inhibitory anti-huEpo-R antibody. This antibody specifically inhibits Epo binding to the huEpo-R, but not to the muEpo-R (data not shown). As reported earlier, '251-Ep0cross-linking showed the presence, in UT-7 cells, of three proteins with molecular masses of 66 kD (corresponding to the Epo-R binding chain), 85 M), and 100 kD (corresponding to the putative Epo-R accessory proteins).'"'" In the parental UT-7 cells, addition of the inhibitory antihuman Epo-R MoAb inhibited the cross-linking of '251-Ep~. In contrast, although clones 12 and 10 exhibited the same pattern of Epo cross-linked proteins as parental UT-7 cells, the inhibitory anti-huEpo-R MoAb had little or no effect (Fig 3), demonstrating that virtually all (>90%) cell surface Epo-Rs were of murine origin. In agreement with this finding, GM-CSF (2.5 ng/mL) had no effect on the number of Epo-R or on their affinity with Epo as assessed by '251-Ep0binding assay (Table 1) and From www.bloodjournal.org by guest on January 26, 2015. For personal use only. DIFFERENTIATION AND GROWTH FACTOR MODULATION 1749 P ANTI-hEPO-R t kDa UT-7 0 -GM-CSF 5000 R Kd=390pM 0 +GM-CSF 1990 R Kd=320pM 0.2 CllO Cl12 nnn -+ - +- + 205P 116D 97r (P100 -p85 (p66 U \ 66, m 0.1 0 I I 50 100 b I B: pM Clonel 0 0 -GM-CSF 6900 R ~ d = 4 7 pM 1 0 +GM-CSF 6300 R Kd-580pM \ m 0.1 50 0 100 6: pM A Clonel 2 0.2 * U \ m -GM-CSF 7200 R Kd-517pM +GM-CSF 6900 R Kd=580pM 0.1 0 I I 50 100 B: pM b Fig 3. Murine Epo receptors are expressed on cell surface of clones 12 and 10. '251-Epoprotein was cross-linked t o parental UT-7 cells (PI, and cells of clones 10 (CI 10) and 12 (CI 12). Cells were labeled with 500 pmol/L of lZ51-Epoin the absence (-1 or in the presence (+I of antihuman Epo-R MoAb. Epo and Epo-R were cross-linked with DSS and complexes were analyzed as described in Materials and Methods. Arrows on right side indicate the molecular weights of proteins cross-linked with Epo. Scatchard analysis (Fig 2). In contrast, and as reported earlier," GM-CSF decreased significantly the Epo-R number of the UT-7 parental cell line from 5,000 to 2.000 (receptors/ cell) with no affinity change (kD = 390 pmol/L v 310 pmol/ L) (Fig 2). Taken together, these results demonstrate that the transduced cell lines express a high number of muEpo-R on their cell surface. These receptors are associated with the same putative accessory proteins as the huEpo-R but are not downregulated by GM-CSF. Tronsdiicecl mirEpo-R is able to induce proliferotion of humori UT-7 cells in the presence of Epo. We then examined whether the muEpo-R is capable of transducing a proliferative signal when triggered by Epo. The growth response t Fig 2. Number and affinity of Epo receptors on parental UT-7 cells and cells of clones 10 and 12. Scatchard analysis of Epo binding sites on UT-7 cells, clones 10 and 12 in the presence ( 0 )or absence (0) of GM-CSF (after 18 hours of growth factor deprivation). The number of Epo receptors and their affinities are indicated for each condition. Cells were grown in presence of 2.5 n g h L GM-CSF, and then washed twice and resuspended in presence or not of GM-CSF (2.5 ng/mL) for 18 hours. '251-Epo binding was then determined by Scatchard analysis using 0.5 x 10'cells/100 FL per point as described in Materials and Methods. B (bound) or F (Free) '251-Epo. From www.bloodjournal.org by guest on January 26, 2015. For personal use only. 1750 HERMINE ET AL Table 2. Growth of Parental UT-7 Cells and Transduced UT-7 Cells in the Presence of Epo or GM-CSF Table 3. Transduced muEpo-R is Able to Induce Proliferationin the Human UT-7 Cells ~ UT-7 10 12 6 Neo EPO GM-CSF 6,300 t 200 6,100 t 200 6,600 f 400 4,200 f 200 4,700 f 400 80 f 20 ~ Epo 3,200 2 80 GM-CSF 2.600 2 80 3,400 2 100 2,900 2 80 2,580 2 60 2,600 _i 80 1,800 80 1.500 _t 60 3,100 t 120 2,700 _t 80 UT-7 cells and cells from clones 12, 10,6, and Neo were grown in the presence of either Epo (2 U/mL) or GM-CSF (2.5 ng/mL) in 96-well microplates (lo3cells/ 100 pLI. The number of viable cells per 100 pL was determined by a trypan blue exclusion on day 4. The data represent the mean t SD of a representative experiment made in quadruplicate. was assessed by counting the viable cells after 96 hours of culture (Table 2). As reported earlier, parental UT-7 cells responded to GM-CSF (2.5 n g k ) and Epo ( 2 U k ) . Clones 12 and 10 grew in the presence of GM-CSF and Epo, as well as the parental cell line, suggesting that muEpo-R is able to transduce a growth signal in human cell lines (Table 2). Clone 6 grew slower than the others in the presence of Epo. However, the same growth alteration was observed in the presence of GM-CSF. Moreover, transfected clones could be maintained in the presence of Epo or GM-CSF for '/ GM-CSF I500 I ,, Q..,. n k- .,-0 -............ ,,e ...........0 zsw swo 2w 5w 7sw 750 low0 1254) Anti-hGM-CSF-R Anti-hEpo-R lm Antibody dilutions P ai 3 s 0 50 2500 swo 75w lwoo 2w 5w 750 1wo 12: iw Anti-hGM-CSF-R Anti-hEpo-R Antibody Dilutions Fig 4. Inhibitory concentration of antihuman Epo-R and antihuman GM-CSF-R antibodies. Cells ([email protected]/lOOpLJ were cultured for 4 days in a 96-well mimcutture plate with Epo (2 UlmLJ or GM-CSF (2.5 ng/mLJ in the presence of various dilutions of inhibitory antihuman Epo-R or antihuman GM-CSF-R MoAbs. Cell numbers per 100 p L and v i a b i l i were then assessed by the trypan blue dye exclusion test. Control Epo-R moAb GM-CSF-R moAb ~ Cells from clone 12 (103/100pLJwere cultured with Epo or GM-CSF i n the presence or absence of inhibitory concentration of anti-huEpoR or anti-huGM-CSF-R MoAbs. The number of viable cells per 100 fiL was determined by a trypan blue exclusion on day 4. The results represent the data of one experiment made in quadruplicate. a long period of time, and deprivation of Epo from the culture medium resulted in a rapid cell death in 2 or 3 days as for the parental UT-7 (data not shown). Although the number of residual human Epo-R on cell surface of clones 12, 6, and 10 was extremely low, we could not rule out that they were responsible for the entire growth signal. Indeed, I O 0 to 300 Epo-R per cell are sufficient to induce a biological effect in normal erythroid progenitors or in some Epo-responsive cell lines.*'-'' Therefore, we repeated the same experiments in the presence of the inhibiting antibody directed against the huEpo-R (16.1.5). We first determined the inhibitory concentration of antibodies directed against huEpo-R and huGM-CSF-R used as a control, which inhibit parental UT-7 cell growth in the presence of Epo ( 2 U/mL) or GMCSF (2.5 ng/mL), respectively (Fig 4). We next grew clone 12 in the presence of Epo (2 U/mL) or GM-CSF (2.5 ng/ mL) with an inhibitory concentration of antibody directed against huEpo-R (dilution was 1/50 vol/vol) or huGM-CSFR (dilution was 1/2,000 vol/vol). The antibody directed against the hu Epo-R did not inhibit the growth of clone 12 in presence of either Epo or GM-CSF, whereas the antibody directed against the huGM-CSF-R did so in the presence of GM-CSF, but not of Epo (Table 3). Similar results were found in the two other clones. These results demonstrate that muEpo-R can transduce a proliferative signal in the human UT-7 cell line. MuEpo-R is able to induce erythroid differentiation of the human UT-7 cells in the presence of Epo. We have previously shown that UT-7 is a multipotent cell line." Epo or GM-CSF can induce erythroid or myeloid programs of differentiation, which are well assessed by GPA or CD33 expression, respectively. In the presence of GM-CSF, the level of GPA expression is low and increases on exposure to Epo, increasing to a maximum in about 10 days of culture. Therefore, to assess the ability of muEpo-R to induce cell differentiation, cells from clone 12 grown in a GM-CSFcontaining medium were switched to an Epo-containing medium with the presence of an inhibitory concentration of antibody directed against huEpo-R (16.1.5), or an antihuGM-CSF-R (as control) for 10 days. After this period, GPA expression was determined by flow cytometry analysis. As shown in Fig 5, Epo markedly increased GPA expression in clone 12, while the inhibitory antibody directed against huEpo-R did not prevent this induction, demonstrating that the muEpo-R is also able to induce an erythroid program of From www.bloodjournal.org by guest on January 26, 2015. For personal use only. 1751 DIFFERENTIATION AND GROWTH FACTOR MODULATION J 1 I Epo+ anti-hGM-CSF-R I I 0 I. f- ;L'+ ;I i I Epo + Clone 10 lgGl . . I. i j; .'! anti-hEpo-R GM-CSF + Epo GPA Fig 5. Differentiation of muEpo-R transduced cells in the presence of inhibitory concentration of anti-huEpo-R and anti-huGM-CSF-R antibodies. Cells from clone 12 were grown in presence of GM-CSF 12.5 ng/mL) for at least five passages, washed, and thereafter reincubated in the presence of either GM-CSF (2.5 nglmLl or Epo (2 UlmL) and with inhibitory concentration of anti-huEpo-R or anti-huGMCSF-R for 10 days. Cells were then analyzed for GPA expression as described in Materials and Methods. An lgGl irrelevant antibody was used as the negative control (IgG1). I" GFA 1 GM-CSF differentiation in a human cell line. Similar results were found in the two other clones. Although GM-CSF does not downregulate Epo-R number on the cell sugace of transfected clones, it still acts dominantly over Epo to inhibit erythroid differentiation. We have previously shown that the GM-CSF effect on UT-7 differentiation predominates over that of Epo. In the presence of GM-CSF plus Epo, the expression of GPA was identical to that obtained with GM-CSF alone." To assess whether GM-CSF still inhibited Epo-induced cell differentiation in muEpo-R expressing cells (clones 12, 6, lo), and in Neotransfected cells (Neo), we grew them in the presence of Epo, washed and then switched them to an Epo, GM-CSF, or GM-CSF plus Epo-containing medium for 10 days. GPA expression was then assessed by flow cytometry analysis. In clones 12, 10 (Fig 6), and 6 (data not shown), GPA expression was downmodulated by GM-CSF and by the combination of Epo plus GM-CSF with the same magnitude, as observed with the cells of Neo clone (Fig 6) and UT-7 parental cells (data not shown). Using the same conditions of culture, Clone Ne0 GM-CSF * 0 c. C O 3*) 0 0 0 > Fig 6. Expression of GPA on transduced UT-7 cells in the presence of GM-CSF, Epo, and a combination of Epo plus GM-CSF. Cells from clones 12,10, and Neo were grown in the presence of Epo (2 UlmL) for at least five passages, washed, and thereafter reincubated in the presence of Epo (2 UlmLl. GM-CSF (2.5 nglmL), or a combination of Epo (2 UlmLl plus GM-CSF (2.5 nglmL1 for 10 days. Cells were then analyzed for GPA expression as described in Materials and Methods. lgGl irrelevant antibody was used as the negative control (IgG1). (Y 0 10" . .... 10' 10 GPA 10 From www.bloodjournal.org by guest on January 26, 2015. For personal use only. 1752 HERMINE ET AL Table 4. Binding of '251-Epoto UT-7 Cells and Cells of Clones 12 in the Presence or Absence of GM-CSF at Day 10 UT-7 GM UT-7 EPO 12 GM 12 Epo 12 GM +GM-CSF 2,270 ? 115 2,840 i- 270 4,975 t 540 7,960 t- 720 6,090 -GM-CSF 6,935 ? 30 6,030 i 120 5,170 ? 50 6,935 2 615 6,150 + Epo z 290 115 Cells were cultured in the presence of Epo (2 U/mL). washed, and then switched to an Epo (2 UlmL), GM-CSF (2.5 ng/mLI, or GM-CSF (2.5 ng/mL) + Epo ( 2 UlmL) medium for 10 days. For binding analysis cells were washed and resuspended in the presence or not of GM-CSF for 18 hours and were then incubated with 2 nmollL of '''I-Epo for 2 hours at 25°C with 0.1% sodium azide. The data are presented in number of receptorslcell as the average of triplicate measurements t SD. The results represent the average of three experiments. the number of Epo-R was also examined at day IO. As shown in Table 4, on clone 12 in the presence of GM-CSF, downregulation of Epo-R did not occur. In contrast, in UT7 parental cells the Epo-R number was still downmodulated by GM-CSF with the same magnitude as at day 1 (Fig 2). Taken together these findings demonstrate that although GM-CSF does not downregulate Epo-R, it still acts dominantly over Epo to inhibit erythroid differentiation. DISCUSSION Understanding of the mechanisms involved in stem cell commitment has been hampered by the difficulties of purifying, identifying, and maintaining pluripotent cells in sufficient number. An alternative is provided by the study of permanent pluripotent hematopoietic cell lines dependent on the presence of growth factors for survival, proliferation, commitment, and differentiation. Studies using cell lines, and to a lesser extent, normal bone marrow cells, have established a hierarchical modulation of growth factor effects for cell differentiation. For example, interleukin (1L)-3 or GMCSF may inhibit macrophagic," erythr~id,'~ or granulo~ytic'~ differentiation induced by M-CSF, Epo, and G-CSF, respectively. On one hand, it has been suggested, but not demonstrated, that transmodulation of receptors may be one mechanism involved in this hierarchy.'."' On the other hand, in some murine growth factor receptor transduced cell lines, this phenomenon may occur even without negative regulation of receptor^.^^.^^ We have previously shown that, although of leukemic origin, the human UT-7 cell line provides a good model to study lineage restriction hierarchically induced by growth factors." Indeed, UT-7 cells may display features of megakaryocytic, eosinophilic, basophilic, and erythroid lineages. In this cell line, the presence of Epo favors differentiation toward the erythroid lineage (demonstrated by the presence of erythroblasts that are hemoglobinized and express high levels of glycophorin A), whereas GM-CSF, as well as IL3, even in the presence of Epo, inhibits this Epo-induced erythroid differentiation. This effect was associated with a rapid downmodulation of Epo-R within the first 4 hours of GM-CSF exposure. In this report, we wondered whether this GM-CSF- induced Epo-R downmodulation was responsible for the inhibition of erythroid differentiation. Thus, to address this question, we have transduced and highly expressed a muEpo-R species that cannot be downregulated in the UT- 7 cells to study the effects of a combination of Epo plus GM-CSF. As expected from the high homology between human and murine Epo-Rs," we first demonstrated that muEpo-R is able to transduce both proliferative and differentiative signals in a human cell line. Furthermore, in UT-7, the murine binding Epo-R chain was able to associate with two human 85 and 105 kD proteins, thus suggesting a strong homology between these putative accessory chains in different species. Although clones 6, 10, and 12 expressed variable amounts of the endogenous huEpo-R mRNA, the number of Epo-R expressed were constant and roughly identical to that o f parental cells. Moreover, virtually all cell surface receptors were of murine origin. This finding strongly suggests that the Epo-R number expressed at the cell surface level may be limited by the requirement of an accessory chain for its membrane transportation. Further work is needed to understand the mechanisms operating at the molecular level to translocate Epo-R to the cell surface in these infected cells. Although muEpo-R was not downregulated by GM-CSF, GM-CSF still acted dominantly over Epo to inhibit erythroid differentiation. This result shows that the dominant effect of GM-CSF over Epo is not a consequence of an Epo-R downmodulation. Alternatively, because Epo-R expression can also be considered as an early erythroid differentiation marker, GM-CSF inhibition of Epo-R expression may reflect a rapid evidence of the GM-CSF driven inhibition of the erythroid differentiation program. Thus, Epo-R inhibition is more likely a consequence and not the cause of the GMCSF-induced inhibition of the erythroid differentiation. Taken together these findings strongly suggest that the hierarchy between growth factors for cell commitment or differentiation is probably not directed by transmodulation of receptors. The levels at which GM-CSF can inhibit the differentiation signal transduced by the Epo-R are numerous and are under investigation. Activated Epo-R and GM-CSFR may compete for various intracellular proteins that transduce differentiation signals to the nucleu~.'~ However, both receptors used the same known transduction signalization such as the Ras and the JAK2/STATS pathways." Therefore, inhibition of erythroid differentiation by GM-CSF may be related to another mechanism. At the nucleus level, differential activation by Epo or GM-CSF of DNA binding proteins, or proto-oncogenes expression such as csuch as GATA myc, or c-myb)"," may also contribute to the inhibition of erythroid commitment or differentiation. Alternatively. GMCSF may reduce the length of the G I phase of the cell cycle and thus inhibits erythroid differentiation as suggested by Krosl et in Epo-R-transfected B d F 3 cell line treated with IL-3. In conclusion, our data suggest that the dominant effect of GM-CSF over Epo is not a consequence of a receptor transmodulation, but more likely occurs at the signal transduction or transcriptional level. Therefore, the modulation of receptors would be considered more as an early marker of differentiation.'2,33However, this conclusion is based on the use of a leukemic cell line, and it would be important, but rather difficult, to reproduce such results on normal multipotent progenitors. From www.bloodjournal.org by guest on January 26, 2015. For personal use only. DIFFERENTIATION AND GROWTH FACTOR MODULATION ACKNOWLEDGMENT The authors are indebted to J.-L. Villeval and I. Dusanter for helpful discussion and critical review of the manuscript, to Genetics Institute (Cambridge, MA), to Immunex (Seattle, WA), and Amgen (Thousand Oaks, CA) for providing the antihuman Epo-R MoAb, the anti-GM-CSF-R MoAb, and the human rhGM-CSF, respectively. REFERENCES I . Metcalf D: The molecular control of cell division, differentiation commitment and maturation in haemopoietic cells. Nature 339:27, 1989 2. 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For personal use only. 1996 87: 1746-1753 Inhibition of the erythropoietin-induced erythroid differentiation by granulocyte-macrophage colony-stimulating factor in the human UT-7 cell line is not due to a negative regulation of the erythropoietin receptor O Hermine, A Dubart, F Porteux, P Mayeux, M Titeux, D Dumenil and W Vainchenker Updated information and services can be found at: http://www.bloodjournal.org/content/87/5/1746.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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