Proc. Natl. Acad. Sci. USAVol. 93, pp. 9600-9605, September 1996Cell Biology

Activation of Stat 5b in erythroid progenitors correlates with theability of ErbB to induce sustained cell proliferationGEORG MELLITZER*, OLIVER WESSELY*, THOMAS DECKERt, ANDREAS MEINKEt, MICHAEL J. HAYMANt,AND HARTMUT BEUG*§*Institute of Molecular Pathology, Dr. Bohrgasse 7, A 1030 Vienna, Austria; tVienna Biocenter, Institute of Microbiology and Genetics, University of Vienna, Dr.Bohrgasse 9, A 1030 Vienna, Austria; and tDepartment of Microbiology, Life Sciences Building, State University of New York, Stony Brook, NY 11794-8621

Communicated by Max L. Bimstiel, Research Institute of Molecular Pathology, Vienna, Austria, June 3, 1996 (received for review February 19,1996)

ABSTRACT Selfrenewal ofnormal erythroid progenitorsis induced by the receptor tyrosine kinase c-ErbB, whereasother receptors (c-Kit/Epo-R) regulate erythroid differenti-ation. To address possible mechanisms that could explain thisselective activity of c-ErbB, we analyzed the ability of thesereceptors to activate the different members of the Stat tran-scription factor family. Ligand activation of c-ErbB inducedthe tyrosine phosphorylation, DNA-binding, and reportergene transcription of Stat Sb in erythroblasts. In contrast,ligand activation of c-Kit was unable to induce any of theseeffects in the same cells. Activation of the erythropoietinreceptor caused specific DNA-binding of Stat 5b, but failed toinduce reporter gene transcription. These biochemical find-ings correlate perfectly with the selective ability of c-ErbB tocause sustained self renewal in erythroid progenitors.

The balance between proliferation and differentiation of he-matopoietic progenitors is of crucial importance for bothnormal and abnormal hematopoiesis (for review, see ref. 1).Recently, the ability to cultivate normal chicken erythroidprogenitors in vitro provided a system that allows a molecularanalysis of how growth factors regulate this balance. In thesecommitted erythroid progenitors, the erythropoietin receptor(EpoR) in cooperation with the insulin receptor regulateserythroid differentiation. In addition, these cells express thereceptor tyrosine kinases c-Kit and c-ErbB. Activation of thec-Kit receptor by its ligand stem cell factor (SCF) only inducesa transient proliferation of these progenitors. In contrast,activation of the c-ErbB/avian epidermal growth factor (EGF)receptor by its ligand type a transforming growth factor(TGF-a) induces sustained cell proliferation and inhibitsdifferentiation (referred to as "self-renewal"; refs. 2-4). Ac-tivation of a self-renewal program in committed progenitorsmay be a central mechanism that controls homeostasis inhematopoiesis and may be a likely target for leukemogenicmutations. Thus, our cell system may provide a unique op-portunity to analyzed the underlying mechanisms by whichprogenitor self-renewal can be controlled.

Recently, we observed that c-Kit and c-ErbB activate dis-tinct, but so far unidentified, downstream signaling pathways(O.W., G.M., M. Von Lindern, A. Levitzky, A. Gazit, J.Jschenko, M.J.H., and H.B., unpublished work). Thus, recep-tor specific signal transduction must contribute to the differ-ential ability of c-Kit and c-ErbB to induce self-renewal. Thepurpose of the studies reported here was to identify down-stream signaling molecules that could potentially be involvedin this differential regulation of committed progenitor self-renewal. One rapidly growing family of candidate signalingproteins that could be involved are the Stat (signal transducersand activators of transcription) proteins. These latent tran-

scription factors reside in the cytoplasm, and their tyrosinephosphorylation, dimerization, and nuclear localization areinduced by various ligands of receptor tyrosine kinases (5-8).Stat 1 was first identified as a component of interferonactivated transcription factors (9). As a homodimer, the pro-tein binds to a DNA sequence designated interferon 'y-acti-vated site (GAS) (10). Stat 1 can also be activated by EGF andplatelet-derived growth factor to bind to a sis conditionedmedium-inducible (SIE) element (11-14). EGF- or platelet-derived growth factor-activated Stat 1 can bind to DNA as ahomodimer or form heterodimers with another Stat familymember, Stat 3, which is also phosphorylated on tyrosine inresponse to EGF or platelet-derived growth factor. Thesegrowth factors thus cause the formation of Stat 1 and Stat 3homodimers and of the Stat 1-Stat 3 heterodimers (15, 16).The third Stat recently implicated in a receptor tyrosine

kinase signaling pathway is Stat 5. This protein, originallydescribed as a prolactin responsive transcription factor inmammary gland epithelium (for review, see ref. 17), wasrecently found to be activated by a number of hematopoieticgrowth factor receptors (18-23). Injection of EGF into micealso caused the DNA-binding and phosphorylation of a Stat5-like protein in liver (7). In summary, this family of transcrip-tion factors is activated by numerous cytokines and receptortyrosine kinases. Thus, it represents a potential means by whichc-Kit and c-ErbB could regulate the expression of distincttarget genes, leading to the differential regulation of progen-itor self-renewal.

In this paper, we have examined the potential of variousmembers of the Stat family to transduce signals emanatingfrom c-ErbB and c-Kit as well as from the EpoR in erythro-blasts. We demonstrate that TGF-a, but not SCF, causesefficient phosphorylation and DNA-binding of Stat Sb, one oftwo closely related Stat 5 proteins (24). TGF-a also causestransactivation of reporter genes carrying high affinity Stat 5binding sites (IFP53-GAS) (25). In contrast, the EpoR causesspecific DNA-binding of Stat Sb, but fails to induce transac-tivation of respective reporter genes. These data implicate StatSb in playing a role in regulating self-renewal versus differen-tiation in erythroid progenitors.

MATERIALS AND METHODSCells. The generation, culture, and differentiation induction

of normal erythroid progenitors expressing endogenous c-ErbB and c-Kit as well as an exogenous c-ErbB have beendescribed previously (2, 4, 26). HD3E22, a variant of the HD3erythroblast line (27), will be described in detail elsewhere(O.W., G.M., and H.B., unpublished work). This cell line

Abbreviations: Epo, erythropoietin, EpoR, Epo receptor; SCF, stemcell factor; TGF-a, type a transforming growth factor; GAS, inter-feron -y-activated site; EGF, epidermal growth factor; SIE, sis condi-tioned medium inducible; CAT, chloramphenicol acetyltransferase.§To whom reprint requests should be addressed.

9600

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 93 (1996) 9601

expresses endogenous, bioactive c-Kit and c-ErbB, and an

exogenous murine EpoR (28). The ts-v-ErbB oncoproteinexpressed by this cell line is fully inactivated by the ErbB-specific tyrosine kinase inhibitor (PD 153035; ref. 29; see

below) or by shift to the nonpermissive temperature (42°C).V-ErbA, the second oncoprotein of HD3, is also inactive inpresence of PD 153035, since it requires the presence ofactivated receptor tyrosine kinases (A. Bauer, E. Ulrich, M.Andersson, H.B., and M. Von Lindern, unpublished work).Under these conditions, the HD3E22 cell line behaves similarto the normal erythroid progenitors.Growth Factor Treatment and Cell Lysis. For growth-factor

stimulation, HD3E22 cells were washed once with PBS andthen withdrawn from factors by cultivation for 12 hr indifferentiation medium (30), using 2.5,M PD153035 to inhibitv-ErbB/v-ErbA activity. PD 153035 abolishes ligand-inducedc-ErbB autophosphorylation and phosphorylation of c-ErbBsubstrates, but has no respective effects on many other recep-tors, including c-Kit, InsR, IGF-1R, Il-3R, and EpoR. Inaddition, PD 153035 specifically abolishes self-renewal induc-tion in erythroid progenitors by TGF-a activated c-ErbB, butdoes not affect proliferation/differentiation induction by li-gand-activated c-Kit or EpoR in the same cells (G.M. and H.B.,unpublished work).

Cells were washed twice with PBS, suspended in differen-tiation medium, and treated either with or without 50 ng/mlTGF-a, 1 ,tg/ml SCF, and 20 units/ml human recombinantEpo for 15 min at 42°C. Control experiments showed that thisprocedure did not affect ligand-induced autophosphorylationof c-ErbB, c-Kit, or EpoR, but effectively abolished autophos-phorylation of the 68-74 kDa v-ErbB protein during thecourse of the experiment. Stimulation of primary SCF/TGF-aprogenitors was performed similar except that the cells did notreceive the tyrosine kinase inhibitor PD153035 during factorwithdrawal and were not shifted to 42°C.

After stimulation, 1 x 106 cells were lysed either in 10 ,ulelectrophoretic mobility-shift assay (EMSA) lysis buffer (20mM Hepes, pH 7.9/140 mM NaCl/1.5 mM MgCl2/1.0%Nonidet P-40/1 mM sodium orthovanadate/10 ,ug/ml aproti-nin/2 pugml leupeptin) or in 20,l immunoprecipitation buffer(1% Triton X-100/50 mM Tris-HCl, pH 8.0/100 mM NaCl/1mM sodium orthovanadate/10 ,tg/ml aprotinin/2 Ag/ml leu-peptin). Cell lysates were cleared by centrifugation for 15 minat 15,000 rpm before use.

Antibodies. Antibodies to mammalian ISGF3 and Stat 3cross-reacting with the respective chicken proteins were ob-tained from Signal Transduction Laboratories (anti-ISGF3,catalog no. G16920) and Santa Cruz Biotechnology (anti-Stat3; K-1SX, catalog no. sc-483X). Antibodies against the Stat 1C terminus were kindly provided by Chris Schindler (ColumbiaUniversity, New York). Antibodies raised in rabbits against a

glutathione S-transferase fusion protein with the huStat Sb Cterminus or a peptide corresponding to the huStat Sa C terminusare described in detail elsewhere (A.M., F. Barahmand-pour, S.Wohrl, D. Stoiber, and T.D., unpublished work).

Immunoprecipitation. Immunoprecipitation of Stat Sb pro-tein was done according to ref. 31 with some modifications.Cell lysate (100 gl) was incubated with 2 gl concentrated StatSb specific antibody for 12 hr at 4°C in 300 ,lI immunopre-cipitation buffer. Immune complexes were collected by proteinA-Sepharose, run on an 8% SDS/PAGE, and transferred tonitrocellulose membranes (DuPont). The Western blot was

probed first with an anti-phosphotyrosine antibody (4G10,Upstate Biotechnology, Lake Placid, NY) and then reprobedwith the Stat Sb specific antibody.

Oligonucleotides. For gel-shift experiments and competi-tions, the following double-stranded oligonucleotides were

used (the top strands are shown for all GAS elements): IFP 53,5'-GATCAAGTGTTTCTGAGAATCTGGGTGATT-3';Ly6A/E, 5'-GATCAATTTATGCATATTCCTGTAAGT-

GAC-3'; SIE m67, 5'-AATTCAGCATTTCCCGTAAATC-CCTCCG-3'; APRE, 5'-GCTGTACGGTAAAAGTGAG-CTCTTACGGGAATGGGAAT-3'.

Mobility-Shift and Super-Shift Analyses. To measure spe-cific complex formation of Stat proteins with DNA, thedouble-stranded oligonucleotides described above were la-beled with radio-labeled deoxynucleotides by filling in the endswith Klenow polymerase. The protein binding reaction wasperformed as described (32), using a buffer containing 10 mMHepes (pH 7.9), 50 mM NaCl, 1 mM EDTA, 10% (vol/vol)glycerol, 250 ng/ml poly(dIdC), and 1 mM sodium orthovana-date for 30 min. For super-shift experiments, the differentantibodies were added for the last 10 min of the bindingreaction (final dilution of 1:100 for the Stat Sa, Stat 5b, and Stat1C specific antisera, 1 ,tg of purified immunoglobulins in caseof the ISGF3 and Stat 3 antibodies)Transient Expression of Reporter Gene Constructs.

HD3E22 cells were transfected using the Lipofectamine re-agents (Life Technologies Grand Island, NY), according to themanufacturer's instructions. For transfection, 3.2 ,ug reporterDNA constructs (25) and 10 Al Lipofectamine reagents weremixed and preincubated, and the complex added to 1 x 107cells. After incubation for 4 hr, the cells were further cultivatedin differentiation medium containing TGF-a, SCF, or nofactor for 24 hr. After incubation, cytoplasmic extracts wereprepared by three cycles of freeze-thaw in 0.25 M Tris (pH 7.5)and chloramphenicol acetyltransferase (CAT) assays wereperformed as described (33). Transfection efficiency wasassessed by cotransfection of a reporter plasmid consisting ofa luciferase gene under the control of a Rous sarcoma viruspromoter or, in some experiments, by a plasmid containing aRous sarcoma virus promoter driven 03-galactosidase geneusing a chemiluminescence substrate (Tropix, Bedford, MA).CAT activities obtained were normalized to transfection effi-ciencies as well as cell numbers.

RESULTS AND DISCUSSIONSignaling from ErbB Causes Formation of Specific Stat/

DNA Complexes. Activation of Stat proteins gives rise to theformation of specific DNA-binding complexes that recognizea specific oligonucleotide sequence known as GAS element.Thus, as an initial step in identifying Stat proteins activated byc-ErbB and/or c-Kit after addition of ligand, extracts fromSCF- or TGF-a-treated HD3E22 erythroblasts (used heresince they tolerate growth factor withdrawal much better thanprimary erythroblasts, see Materials and Methods) were ana-lyzed in gel-shift assays (EMSA) for the formation of specificcomplexes with a number of different GAS-related DNAelements. These different GAS elements are selective forvarious Stat family members. As shown in Fig. 1A, cell extractsfrom TGF-a-treated HD3E22 cells contained proteins thatbound to the IFP-53 GAS element (25), but not to Ly6A/E. Aweak but TGF-a specific binding to SIE-M67 and APREsequences could be detected (sequences are defined in Mate-rials and Methods). In contrast to TGF-a, treatment with SCFor no growth factor failed to give rise to DNA-bindingcomplexes with IFP-53 GAS or the other elements in HD3E22cell extracts.

Next, we repeated these experiments in primary erythroidprogenitors, using cells expressing either endogenous c-Kitand c-ErbB (SCF/TGF-a progenitors; ref. 2) or high endog-enous c-Kit levels together with exogenous c-ErbB encoded byan avian retrovirus vector (26). Activation of c-ErbB caused asimilar, specific DNA-binding of Stat proteins as with theHD3E22 cell line (data not shown). In the SCF/TGF-aprogenitors, some background DNA complex formation wasobtained both without any factor and in the presence of SCF.In conclusion, this c-ErbB specific activation of a Stat/DNA

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9602 Cell Biology: Mellitzer et al.

WP53-GAS LY6-GAS APRE M67-SI:E oligoICs 11 Q 11 Is F-c u I 0 u I 0 u . v 'I growthfactor

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FIG. 1. Mobility-shift analysis, oligonucleotide competition assay, and antibody interference mobility-shift assay of TGF-a- or SCF-inducedHD3E22 cells. (A) Cell extracts prepared from HD3E22 treated for 15 min with TGF-a or SCF or left untreated (-) were analyzed by EMSAusing four different 32P-labeled oligonucleotides (IFP53-GAS, Ly6A/E-GAS, APRE, or M67-SIE; see text). (B) Extracts from TGF-a-induced cellswere assayed in the mobility-shift assay as above, using a labeled IFP53-GAS probe. The reaction was competed with a 20-, 100-, and 200-fold excessof the indicated nonradioactive oligonucleotides. The position of the specific Stat/DNA complexes formed is indicated by arrows. (C) HD3E22cells were treated with factors, extracted, and subjected to the mobility-shift assay using the IFP53-GAS probe as described for A. Incubation ofthe Stat/DNA complexes with various antibodies to different Stat proteins was done as described. Left arrow, position of the Stat/DNA complexes;right arrows, position of the complexes super-shifted by Stat Sb antibody.

complex has the desired properties of a signal transductionpathway involved in self-renewal induction.To directly demonstrate that the c-ErbB receptor tyrosine

kinase was responsible for Stat activation, we used a c-ErbB-specific tyrosine kinase inhibitor (PD 153035). PD 153035prevents ligand-induced autophosphorylation of c-ErbB andactivation of c-ErbB substrates, but has no effect on signaltransduction by all other receptors tested (see Materials andMethods; G.M. and H.B., unpublished work). When added toHD3E22 cells during c-ErbB activation by TGF-a, complexformation of Stats with the IFP-53 element was completelyinhibited (data not shown).To determine the specificity of the IFP-53 bound protein

complex, we tried to compete complex formation with anexcess of unlabeled DNA of all four GAS elements. Consistentwith the data shown in Fig. 1A, only the IFP-53 GAS elementeffectively competed complex formation of proteins fromTGF-a-stimulated erythroblast (Fig. 1B). The next best com-petitor was the APRE element. The two other elements wereeither unable to compete (such as the Ly6A/E, which is a Stat1-binding site), or competed only at high concentrations (like theSIE-M67 probe). These poorly competing sites are stronglyrecognized by Stat 3 and Stat 1 (12, 15, 34-36). Since the IFP-53GAS predominantly binds Stat 5 (32, 37, 38), these resultssuggested that a Stat 5-like protein is present in the specificDNA/protein complexes induced by TGF-a in erythroblasts.

Stat 5b Is Present in the DNA-Binding Complex. Havingdemonstrated the formation of a specific DNA-binding com-plex by activation of c-ErbB, we wanted to identify the Statproteins present in the DNA-binding complexes. This wasdone by testing various antibodies for their ability to cause asuper shift of the DNA bound protein complexes in the EMSAassay. We used antibodies that were able to specifically supershift Stat 1, Stat 3, Stat Sa, or Stat Sb complexes in suitableextracts from mammalian cells (A.M., F. Barahmand-pour, S.Wohrl, D. Stoiber, and T.D., unpublished work). These anti-bodies also detected proteins with the expected molecular

weights of Stat la, Stat 1,3, Stat 3, Stat Sa, and Stat Sb inWestern blots from both primary erythroblasts and from theHD3E22 cell line, demonstrating their ability to cross reactwith the respective chicken Stat proteins (data not shown).As shown in Fig. 1C, antibodies to Stat 1, Stat 3, or Stat Sa

were unable to super shift the TGF-a-induced DNA/proteincomplex. In contrast, the antibody directed against Stat Sbinduced a complete super shift of this complex, giving rise tothe formation of two distinct, larger complexes. It remainsunclear why the Stat Sb antibody induced super shift results intwo bands. One possibility is that the antibody cross-linked twoStat homodimer DNA complexes in the upper band. The factthat none of the antibodies to Stat 1 caused a super shift of theTGF-a induced DNA/protein complex is in line with theearlier finding that no TGF-a-induced complexes were ob-tained with high-affinity Stat 1-binding sites, e.g., the Ly6A/EGAS (Fig. 1A).c-ErbB is the chicken equivalent of the mammalian EGF

receptor (39). The mammalian receptor has been shown toactivate Stat 1 and Stat 3 in certain cell types (15, 16) and(directly or indirectly) causes Stat 5 activation in rat liver (7).As described above, chicken erythroblasts express all threeStat proteins (Stat 1, Stat 3, and Stat 5; data not shown).Therefore, it is surprising that Stat 5 seems to be selectivelyactivated by c-ErbB in this cell system. Except for potential celltype specific mechanisms, we have no explanation for thisselective activation seen in chicken cells.TGF-ot Induction of Stat 5b Allows Transcriptional Activa-

tion of an IFP53-GAS Promoter CAT Construct. The specificbinding of Stat Sb to DNA in response to TGF-a alreadyindicates that Stat Sb may be involved in the activation ofTGF-a-induced genes. To clarify if Stat Sb activation byc-ErbB would cause transcription of reporter genes containingStat 5-specific DNA elements, erythroid cells were transfectedwith a CAT reporter construct that contained either two orthree copies of the IFP53-GAS element, respectively. To beable to normalize for transfection efficiency, a construct

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Proc. Natl. Acad. Sci. USA 93 (1996)

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Proc. Natl. Acad. Sci. USA 93 (1996) 9603

expressing the luciferase gene under the control of a Roussarcoma virus promoter was cotransfected. The transfectedcells were treated with TGF-a or, as controls, with SCF or nogrowth factor. Extracts were prepared and the CAT activity ofrespective cell extracts was then determined. TGF-a treatmentresulted in a 3- to 4-fold induction of CAT activity, whileSCF-treated cells expressed CAT levels that were even lowerthan in the untreated control cells (Fig. 2). Similar results wereobtained in four other experiments, yielding up to 20-foldstimulation ofCAT activity by ligand activated c-ErbB when animproved method for determining transfection efficiency wasused (data not shown). Thus, activation of c-ErbB is sufficientto cause transactivation of IFP53-GAS-regulated reportergenes, indicating that signals originating from this receptortyrosine kinase produce a transcriptionally active Stat 5 dimer.The resulting Stat Sb-specific gene expression could play animportant role in the induction of self-renewal by c-ErbB.Ligand Activation of ErbB Causes Tyrosine Phosphoryla-

tion of a 92 kDa Stat 5b Protein. The binding of Stat proteinsto DNA is dependent on their tyrosine phosphorylation (6). Toanalyze whether Stat 5 proteins were tyrosine phosphorylatedupon activation of c-ErbB, HD3E22 cells were treated withTGF-a or SCF or left untreated. Extracts were subjected toimmunoprecipitation with a Stat 5b antibody, followed byWestern blot analysis using an anti-phosphotyrosine antibody.As shown in Fig. 3, TGF-a activated c-ErbB induced thetyrosine phosphorylation of a protein doublet with molecularmasses of 91-92 kDa that was specifically immunoprecipitatedby a Stat 5b antibody. No such phosphorylated proteins weredetected in the SCF-treated cells or in the controls. Reprobingof the Western blot with the Stat 5b specific antibody showedthat equal amounts of Stat 5b protein were immunoprecipi-tated under all conditions (Fig. 3). Again, these results couldbe reproduced in normal c-Kit/c-ErbB expressing erythroidprogenitors (data not shown). The two bands detected by ourantibody may represent full-length and short versions of Stat5 recently found in mammalian myeloid cells (18).

Trials to demonstrate tyrosine phosphorylation of other Statproteins by c-ErbB or c-Kit in either HD3E22 cells or primaryerythroblasts met with limited success. Using a Stat la/,3antibody (anti ISGF-3 antibody) that cross-reacts with severalStat proteins, a 91 kDa protein was found to be phosphorylatedin response to TGF-a, whereas a 84 kDa protein was similarlyphosphorylated in response to TGF-a and SCF (data not

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FIG. 2. Selective activation of IFP53-GAS-containing reporter genesby TGF-a. HD3E22 cells were transfected with CAT-reporter geneconstructs containing two (IFP-2) or three (IFP-3) IFP53-GAS elementsand grown for 24 hr in the presence of the indicated growth factors(TGF-a, SCF, or no growth factor). CAT activity in cell extracts was

plotted after normalization for transfection efficiency and cell number.

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FIG. 3. TGF-a selectively induces tyrosine phosphorylation of Stat5b. HD3E22 cells treated with TGF-a, SCF, or left untreated weresubjected to immunoprecipitation Western blot analysis using ananti-Stat 5b antibody. Immunoprecipitates were resolved by SDS/PAGE and then immunoblotted with anti-phosphotyrosine antibody(Upper, a-p-tyr) or with anti-Stat 5b-specific antibody (Lower, a-Stat5b), respectively. Size markers at the left are in kilodaltons (kDa). Thetyrosine phosphorylated Stat 5 protein induced by TGF-a (Upper)corresponds to the top protein band in the Western blot (Lower). Theimmunoglobulin heavy chain (Ig-hc) is indicated.

shown). The identity of the latter protein and the reason for itsinability to produce a gel shift with the Stat 1 DNA elementsused remains unclear.Our results indicate that ligand-activated c-ErbB that causes

sustained self-renewal in erythroid progenitors specificallyactivates Stat 5b proteins by causing their tyrosine phosphor-ylation, specific binding to DNA, and activation of reportergene transcription. In contrast, activation of c-Kit does not leadto the formation of Stat/DNA complexes and also does notinduce sustained self-renewal. However, it remains unclearwhether c-Kit is incapable of activating the Stat pathway, sinceit could induce tyrosine phosphorylation of Stat-like targets,but was unable to cause binding of such proteins to DNA.The Activated EpoR Causes DNA Binding but Not Trans-

activation by Stat 5b. Our results raise the obvious question,how cytokine receptors known to activate Stat 5b in other celltypes (see Introduction) would behave in our cells with respectto Stat 5b activation and induction of self-renewal. Thisquestion is difficult to address in chicken cells, since mostknown cytokines or their receptors are not available as re-combinant proteins. Recently, however, we could show that themurine EpoR is able to trigger normal, terminal erythroiddifferentiation if expressed in primary or established chickenerythroblasts (28). The EpoR activates Stat 5 proteins inmammalian nonerythroid cell lines (20, 22). We thereforeanalyzed if activation of the murine EpoR expressed in theHD3E22 cell line was able to cause DNA-binding of andtransactivation by Stat 5b. Extracts from HD3E22 cells treatedwith human recombinant Epo or left untreated were analyzedfor complex formation with the Stat 5-specific IFP-53 GASelement. In addition, antibodies to different Stat proteins weretested for their ability to super shift the Epo-induced com-plexes. Fig. 4A clearly demonstrates that the ligand-activated

Cell Biology: Mellitzer et al.

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9604 Cell Biology: Mellitzer et al.

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FIG. 4. The activated EpoR induces Stat 5b binding to DNA butfails to cause transactivation. (A) Cell extracts were prepared fromHD3E22 cells after a 15-min treatment with Epo or no growth factor(-), and examined by mobility-shift assay using a labeled IFP53-GASprobe. Incubation of the Stat/DNA complexes with antibodies todifferent Stat proteins was done as described for Fig. 1C. Lower arrow,Stat Sb/DNA complex; upper arrow, Stat Sb DNA complex super shiftedby anti-Stat Sb antibody. (B) HD3E22 cells were transfected with a CATreporter gene construct containing three (IFP-3) IFP53-GAS elementsand grown for 24 hr in the presence of the indicated growth factors (Epo,TGF-a, or no growth factor). CAT activity in cell extracts was plottedafter normalization for transfection efficiency and cell number. Theresults of two independent experiments are indicated (1, 2).

EpoR induced the formation of a specific DNA/proteincomplex that could be super shifted by the Stat 5b antibody.Thus, similar to c-ErbB, the EpoR can induce DNA-binding ofStat Sb. Interestingly, however, activation of the EpoR failedto induce transactivation of a IFP53-GAS regulated reportergene, whereas activated c-ErbB used as a positive controlresulted in a 10-fold stimulation of CAT activity (Fig. 4B).Thus, the activity of Stat proteins is probably regulated atmultiple levels, not only by tyrosine phosphorylation causingDNA-binding, but also by other events (e.g., serine phosphor-ylation; ref. 40) necessary for transactivation.

In conclusion, ligand activation of c-ErbB specifically acti-vates DNA-binding and transcriptional activation by Stat Sband thus presumably gives rise to the activation of gene

expression by this protein. The activation of Stat 5b by c-ErbBclearly correlated with its ability to activate self-renewal, sincetwo other receptors, c-Kit and EpoR, failed to cause transac-tivation by Stat 5b and were unable to induce sustainedproliferation. Future studies will be necessary to determine ifthis activation is necessary and/or sufficient to activate self-renewal, to identify the gends that are activated by this Statcomplex and to determine their role in self-renewal induction.

We thank Dr. Chris Schindler for his kind gift of the anti-Stat 1antibody and Evi Deiner for expert technical assistance.

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