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Stem Cells, Vol. 18, No. 5, 366-373, September 2000
© 2000 AlphaMed Press

Signaling Induced by Erythropoietin and Stem Cell Factor in UT-7/Epo Cells: Transient versus Sustained Proliferation

Connie L. Erickson-Miller, Louis M. Pelusa, Kenneth A. Lord

Department of Molecular Virology and Host Defense, SmithKline Beecham Pharmaceuticals, Collegeville, Pennsylvania, USA;
a Present Address: Indiana University School of Medicine, Department of Microbiology and Immunology and the Walther Oncology Center, and the Walther Cancer Institute, Indianapolis, Indiana, USA

Key Words. JAK • MAPK • STAT • Differentiation

Connie L. Erickson-Miller, Ph.D., Department of Molecular Virology and Host Defense, SmithKline Beecham Pharmaceuticals, 1250 South Collegeville Road, Collegeville, Pennsylvania 19406, USA. Telephone: 610-917-6790; Fax: 610-917-4181; e-mail: Connie_L_Erickson-Miller{at}SBPHRD.com


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
UT-7/Epo cells are human factor-dependent erythroleukemic cells, requiring erythropoietin (Epo) for long-term growth. Stem cell factor (SCF) stimulates proliferation of UT-7/Epo only transiently, for three to five days. An investigation of the signal transduction pathways activated by these cytokines in UT-7/Epo cells may identify those signals specifically required for sustained growth. Proliferation assays demonstrate that SCF generates a substantial growth response in UT-7/Epo cells; however, the cells do not multiply or survive past five to seven days. While Epo induces the activation of JAK2 and STAT5, SCF stimulation shows no activation of JAK2 or STATs 1, 3, or 5. The activation of MAPK (p42/44) by SCF was transient, lasting only 30 min, in contrast to Epo, which stimulated phosphorylation of p42/44 for up to 2 h. The expression of the early response genes c-fos, egr1, and cytokine-inducible SH2 protein (CIS) in response to SCF or Epo stimulation demonstrated that the transient expression of p42/44 correlated with the transient expression of c-fos and egr1. In addition, CIS was activated by Epo but not SCF. These data indicate that EpoR, JAK2, and STAT5 activation are not required for the initiation of proliferation of these erythroid cells, that the transient activation of p42/44 correlates with the transient gene expression of c-fos and egr1, and sustained expression of c-fos and egr1 as seen in UT-7/Epo cells continuously grown in Epo may be necessary for long-term proliferation.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The interaction of erythropoietin (Epo) and stem cell factor (SCF) with their respective receptors initiates the activation of multiple signaling cascades and changes in gene expression, resulting in proliferation and differentiation of hematopoietic progenitor cells. Understanding the common proliferative signals induced by these hematopoietic cytokines may elucidate pathways essential for proliferative control.

The binding of Epo to its receptor (EpoR), a member of the cytokine receptor superfamily, initiates a signal cascade which includes phosphorylation of EpoR, activation of JAK2 and STAT5 [1, 2], activation of PI3 kinase [3], and activation of the ras/raf/MAPK pathways [4, 5]. The receptor for SCF, c-kit, is a member of the tyrosine kinase family of receptors [6], and upon SCF binding undergoes autophosphorylation with secondary signals which include activation of the ras/raf/MAPK and PI3 kinase pathways [7] and JAK2 [4, 8, 9], depending on the cell type [10].

Early-response genes reported to be induced by Epo or associated with proliferation include c-fos [11-13], the transcription factor, egr1 (early growth response-1) [14], cytokine-inducible SH2 containing protein (CIS) [15], and c-myc [12]. The early response genes, c-fos and egr1 are predominantly associated with mitogenic stimuli, and function in proliferative responses in most cell contexts. C-myc and c-fos are also induced by SCF treatment [7, 16]. C-fos, c-myc and egr1 can be activated via the JAK/STAT and MAPK pathways [17, 18].

Several studies have suggested receptor crosstalk between EpoR and c-kit. In HCD57 cells which express endogenous EpoR and c-kit receptors and in 32D cells transfected with EpoR, initiation of EpoR signaling and proliferation following exposure to SCF in the absence of Epo is observed [19, 20]. Furthermore, the cytoplasmic box 1 domain of EpoR in part mediates mitogenic synergy with c-kit in FDCP1 cells transfected with EpoR [21] which occurs in the absence of JAK2 activation. However, while SCF induces cell proliferation and components of the EpoR signaling cascade, long-term cell proliferation is not maintained resulting from incomplete activation of the EpoR signaling cascade.

To further explore the common and/or unique signals initiated by Epo and SCF, we have utilized the human Epo-dependent UT-7/Epo cell line which expresses endogenous EpoR and c-kit receptors [22] and in which SCF produces a short-term unsustained proliferative response in the absence of Epo. Our results indicate that SCF-induced stimulation of cell proliferation occurs in the absence of activation of the JAK/STAT signaling pathway, and that while SCF activates the p42/44 MAPK pathway and the early response genes c-fos and egr1, activation is transient and substantially reduced in comparison to Epo. Transient and incomplete activation of the components of the EpoR signaling cascade by SCF likely is responsible for its inability to sustain long-term erythroid cell proliferation.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and Reagents
UT-7/Epo cells were obtained from Dr. Mark Showers, Boston, MA, and maintained in Iscove's modified Dulbecco's medium (IMDM) with 10% fetal calf serum (FCS) (Hyclone; Logan, UT; http://www.hyclone.com) and 0.4 U/ml recombinant human erythropoietin (rHuEpo) (Amgen; Thousand Oaks, CA; http://www.amgen.com). Carrier-free rHu interleukin 3 (IL-3) and rHu-SCF were purchased from R&D Systems (Minneapolis, MN; http://www.rndsystems.com) and rHu-GM-CSF from Immunex Corp. (Seattle, WA; http://www.immunex.com). Recombinant growth factors were resuspended in Dulbecco's phosphate-buffered saline (DPBS) containing 0.5% human serum albumin (HSA) and stored in frozen aliquots. Phenylmethylsulfonyl fluoride (PMSF), leupeptin, bestatin, and aprotinin were purchased from Sigma Chemicals (St. Louis, MO; http://www.sigma-aldrich.com). Poly(dIdC).poly(dIdC) was obtained from Pharmacia (Piscataway, NJ; http://www.pnu.com) and Pefabloc from Boehringer-Mannheim (Indianapolis, IN), antiphosphotyrosine monoclonal antibody, clone 4G10, and anti-JAK2-sepharose were purchased from Upstate Biotechnology (Lake Placid, NY; http://www.upstatebiotech.com).

Preparation of Cell Lysates
UT-7/Epo cells were grown in IMDM/10% FCS and deprived of Epo for 24 h. The cells were then treated with Epo and/or SCF for 10 min. After pelleting the cells, lysis buffer (0.05 M Tris-HCl, 1 mM sodium vanadate, 1 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1 mM Pefabloc, 10 µg /ml aprotinin, 10 µg /ml leupeptin) was added and the samples incubated on ice for 20 min with occasional vortexing. Following lysis, the samples were centrifuged at 1,800 rpm for 3 min at 4°C and the supernatants collected. Protein determinations were made with the BCA protein assay (Pierce; Rockford, IL).

JAK2 and p42/44 Activation
JAK2 activation was determined by immunoprecipitation with anti-JAK2 antibody and blotting with antiphosphotyrosine antibody. One hundred mg of each lysate were immunoprecipitated with 5 µg of JAK2-Protein A Sepharose for 1 h at 4°C, centrifuged and the pellet washed twice in cold lysis buffer. The pellet was resuspended in SDS Tris-glycine sample buffer with 2.5% 2-mercaptoethanol and 20 µl run on an 8% Tris glycine gel (Novex; San Diego, CA). The samples were transferred to polyvinyl difluoride (PVDF) membranes and blotted with antiphosphotyrosine for 1 h using 0.5% gelatin/PBS-Tween-20 as the blocking buffer, horse radish peroxidase-labeled sheep antimouse secondary antibody (Amersham; Arlington Hts., IL) for 1 h and developed using the enhanced chemiluminescence reagents (Amersham).

MAPK activation was determined by Western blot with the New England Biolabs p42/44 kit (Beverly, MA; http://www.uk.neb.com/neb). Briefly, after running cell lysates on a 12% Tris-glycine gel and transferring to PVDF membrane, an antiphospho p42/44 antibody was used with 5% evaporated milk as the blocking reagent. CDP-Star was used to develop the bands. To control for equal loading, the same blots were stripped and reprobed with antibody to total immunoprecipitated p42/44 protein.

Proliferation Assays
UT-7/Epo cell proliferation in response to Epo or SCF was measured by 3H-thymidine incorporation. Fifty thousand cells in log phase growth were washed and plated in 100 µl IMDM/10% FCS per well of a 96-well microtiter plate with various concentrations of Epo or SCF. After three days incubation at 37°C, 3H-thymidine (1 uCi/well) (ICN; Irvine, CA; http://www.icnpharm.com) was added for 4 h and the plate harvested with 10% trichloroacetic acid and cold ethanol. Meltilex solid scintillant (Wallac; Gaithersburg, MD; http://www.wallac.com) was melted onto the filter containing samples and radioactivity measured on a Betaplate reader (Wallac). Data were reported as the mean of quadruplicate samples.

Northern Hybridizations
Procedures for probe preparation, Northern blotting and hybridizations were followed essentially as described [23]. RNA was extracted using the RNeasy kit (Qiagen; Valencia, CA), and blotted by electrophoresing total RNA (10 µg/lane) in denaturing formaldehyde/agarose gels, vacuum blotting to nylon, and ultraviolet crosslinking. cDNA probe fragments were labeled by Klenow fragment using random hexamers and {alpha}-32P-dATP at room temperature, and purified using Sephadex G-50 columns (Pharmacia). Northern hybridizations were carried out in 6X standard saline citrate, 1% SDS, 10% dextran sulfate and 100 µg/ml sheared salmon sperm DNA (Sigma Chemical Company) at 65°C overnight. High stringency washes consisted of 0.1X SSC, 0.1% SDS at 60°C for 1 h. Blots were exposed to phosphor storage screens and scanned for analysis.

Preparation of Nuclear Protein Extracts
Five million UT-7/Epo cells were harvested at 4°C after treatment with growth factor, washed 1x with DPBS and 1x in hypotonic buffer (20 mM Hepes, pH 7.9; 1 mM each EDTA, EGTA and sodium pyrophosphate; 10 mM sodium fluoride). The cell pellets were lysed in cold hypotonic buffer containing 0.05% NP-40 and protease inhibitors (0.5mM PMSF; 1 µg/ml each leupeptin, bestatin, aprotinin). Nuclei were pelleted in a microfuge pulsed at maximum and resuspended in high salt buffer (hypotonic lysis buffer containing 0.4M NaCl, 10% glycerol). Tubes were gently agitated at 4°C for 30 min, centrifuged at maximum and the supernatant containing nuclear proteins removed to a clean tube. Protein concentration was determined using the Bio-Rad micro assay (BioRad; Hercules, CA).

STAT Electrophoretic Mobility Shift Assays (EMSA)
DNA oligo probe sequences represent the STAT-binding elements from the IRF1 promoter (IRF1 probe) [24] and the Fc{gamma}R promoter (GRR probe) [25]. Oligodeoxynucleotide probes were prepared by annealing complementary oligos to generate a duplex with 5'-overhangs. The oligo sequences for the IRF1 probe were 5'-GAT CAT TTC GGG GAA ATC and 5'-GAT CGA TTT CCC CGA AAT. For the GRR probe the oligos used were 5'-CAT GTA TTT CCC AGA AAA G and 5'-GAT CTT TTC TGG GAA ATA C. Probes were labeled by filling-in with Klenow fragment using {alpha}-32P-dATP at 37°C, and purified using Sephadex G-50 columns (Pharmacia). DNA binding reactions contained 10-15 µg of nuclear protein, 1 µg poly(dIdC).poly(dIdC), 50 mM Tris, pH 7.5, 10% glycerol, 1 mM DTT, and 20-30 cpm of 32P labeled probe in 20 µl. Samples were incubated at ambient temperature for 20 min, then electrophoresed over 5% polyacrylamide in 0.25X TBE. Gels were dried and autoradiographed. In supershift experiments, nuclear protein extract was incubated with specific anti-STAT antibodies (Santa Cruz Biotechnology; Santa Cruz, CA) before addition of the oligo probe.


   RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Proliferation of UT-7/Epo Cells
UT-7/Epo cells exhibit a dose-dependent response to Epo reaching maximum at 0.2 U/ml Epo after three days, as measured by 3H-thymidine incorporation (Fig. 1Go). Recombinant SCF also induced thymidine incorporation in a dose-dependent manner, although at only ~70% of the level of Epo at the highest dose tested (200 ng/ml). There was no synergistic response in proliferation stimulated by SCF plus suboptimal levels of Epo (data not shown). In addition, there was no response of UT-7/Epo cells to IL-3 or GM-CSF under these conditions (data not shown). Direct viable cell counts confirmed the thymidine incorporation results, demonstrating an increase in the number of viable cells for up to three days in SCF, but no further proliferation and eventually cell death resulted if UT-7/Epo cells were maintained in SCF for extended time (Fig. 2Go). This was in contrast to Epo which continuously maintains cell proliferation.



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  		Figure 1. UT-7/Epo proliferation assay. Cells were incubated for 72 h in 0-0.8 U/ml Epo (circles) or 0-200 ng/ml rHuSCF (squares). The counts per minute of 3H-thymidine incorporated in the final 4 h are reported as the mean of quadruplicate samples. The maximum proliferation stimulated by SCF is 70% of that stimulated by 0.2 U/ml Epo.

 


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  		Figure 2. UT-7/Epo viable cell number following SCF and Epo treatment. Cells were initially plated at 1.5 x 105 cells/ml in IMDM/10% FCS and 0.4 U/ml Epo (top panel) or 100 ng/ml SCF (bottom panel). The viable cell number (x 105 cells/ml) was determined over 12 days culture by manual cell counts on a hemacytometer using trypan blue exclusion. Cells at >1 x 106 cells/ml at each time point were split to 1.5 x 105 cells/ml (*). Cells at <1 x 105 cells/ml were given 100 ng/ml fresh SCF (arrows).

 
Activation of EpoR, c-kit, JAK2, STAT5, and p42/44
It has been suggested that SCF, via its tyrosine kinase receptor c-kit, can cross phosphorylate the EpoR [20]. To further investigate this effect, lysates from SCF- or Epo-treated UT-7/Epo cells were immunoprecipitated with anti-c-kit or anti-EpoR antibodies and Western blotted with anti-phosphotyrosine. After a 10-min treatment of UT-7/Epo cells with SCF, the phosphorylation of c-kit was readily apparent; however, EpoR was not phosphorylated (Fig. 3Go, panel 1). There was no cross-phosphorylation of c-kit in Epo-treated cells; although, as expected, there was significant phosphorylation of EpoR (Fig. 3Go, panel 2). Following Epo binding to EpoR, activation of JAK2 kinase was readily detected (Fig. 3Go, panel 3 and 4). However, there was no activation of JAK2 following treatment of UT-7/Epo cells with concentrations of SCF which stimulate proliferation (100 ng/ml) at 10 min (Fig. 3Go). A time course in which samples were analyzed from various time points over 60 min indicates SCF does not have altered kinetics of JAK2 activation (data not shown). In general, there were very little effects demonstrated when SCF was added in combination with suboptimal amounts of Epo, but there was occasionally a smaller response than expected in both JAK2 activation and EpoR phosphorylation (Fig. 3Go).



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  		Figure 3. Phosphorylation of receptors, JAK2 and STAT5. UT-7/Epo cells were treated with either Epo or SCF for 10 min. Cell lysates were immunoprecipitated with either anti-c-kit (top panel) or anti-Epo R (second panel). SCF induced the phosphorylation of c-kit but no activation of EpoR, while Epo treatment resulted in EpoR phosphorylation, but had no effect on c-kit. JAK2 activation was determined by immunoprecipitation of lysates from cells treated for 10 min with Epo or SCF with anti-JAK2. Western blotting with anti-phosphotyrosine shows activated JAK2 in Epo-treated lysates (panel 3 and 4) (sixfold more at 1 U/ml than at 0.05 U/ml), but none in untreated or SCF-treated lysates. Suboptimal amounts of Epo with SCF gave no more activation than 0.05 U/ml Epo alone. Western blots for STAT5 activation used cells incubated for 10 min in Epo (0.05 or 1 U/ml) or SCF (10 or 100 ng/ml), immunoprecipitated with anti-STAT5 antibody and Western blotted using anti-phosphotyrosine. Reprobing with anti-STAT5 was used to determine equal loading (panel 5 and 6).

 
In the Epo signaling cascade, JAK2 activates STAT5, leading to homodimerization of STAT5 and its translocation into the nucleus. Immunoprecipitation of lysates from SCF- and Epo-treated UT-7/Epo cells with anti-STAT5 antibody, followed by blotting with antiphosphotyrosine, detected STAT5 activation by 0.05 and 1 U/ml Epo, but not by SCF (Fig. 3Go, panel 5 and 6). STAT1 and STAT3 were not phosphorylated on tyrosine by either Epo or SCF (data not shown). Activation of STAT5 by Epo but not SCF was confirmed by electrophoretic mobility shift experiments using nuclear extracts prepared from Epo-deprived UT-7/Epo cells stimulated for 30 min with various cytokines and analysis by gel shift using the IRF1 and GRR probes (Fig. 4Go). STAT protein/DNA complexes could only be observed after Epo stimulation. No STAT/DNA complexes were detected after stimulation with SCF. Supershift experiments with multiple anti-STAT antisera confirmed the presence of STAT5 in the Epo-induced complexes (Fig. 5Go). STATs 1 and 3 (Fig. 5Go) as well as STATs 4 and 6 (not shown) were not detected. With saturating antisera, the mobility of the entire complex was affected, implying that it was composed solely of STAT5 homodimers. Stimulation with a suboptimal amount of Epo (0.1 U/ml) generated a reduced level of STAT5 activation (approximately 50%). However, the inclusion of SCF in the treatments did not affect the kinetics or intensity of the STAT5-DNA complexes detected by gel shift, showing no evidence for alteration of STAT activation by SCF (data not shown).



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  		Figure 4. STAT activation in UT-7/Epo Cells is selective for Epo. Cells were stimulated with SCF (100 ng/ml) or Epo (4 U/ml) for 30 min. Nuclear protein extracts were analyzed by EMSA using the IRF1 and GRR probes.

 


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  		Figure 5. Epo activates only STAT5. Nuclear protein extracts were prepared from UT-7/Epo cells stimulated with 4 U/ml Epo for 30 min. Extracts (5 µg) were pretreated with antisera (1-3 µg) to STAT-1, -3, and -5 prior to analysis by EMSA using the IRF1 (STAT5 only) and GRR probes.

 
To determine if the proliferative response induced by SCF in UT-7/Epo cells could be correlated to particular signaling pathways, the activation of MAPK was analyzed by Western blotting with specific antibodies to phosphorylated p42/44. As expected, Epo induced activation of p42/44 within 10 min in a concentration-dependent manner (Fig. 6Go). SCF (100 ng/ml) also induced p42/44 activation, although at only 20% the level of 1 U/ml Epo. A time course of MAPK activation demonstrated that while both SCF and Epo activated p42/44 at 10 min, p42/44 was still activated 2 h following Epo treatment, whereas the level of activated p42/44 was more transient following exposure to SCF, being substantially reduced at 30 min and returned to baseline levels by 2 h.



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  		Figure 6. Activated MAPK (p42/44) in response to Epo or SCF. UT-7/Epo cells were treated with either Epo (1U/ml) or SCF (100 ng/ml) for 0-60 min and cell lysates prepared at 0, 10, 30, and 120 min. Western blotting using antibody to phosphorylated p42/44 shows that MAPK is activated by Epo at 10 min and remains activated for at least 2 h. SCF activated MAPK at 10 min but was significantly diminished by 30 min and barely detectable at 2 h.

 
Expression of Epo Early-Response Genes Induced by Epo and/or SCF
To examine the role of the Epo early-response genes in transient or sustained proliferation of UT-7/Epo cells, total RNA was extracted from both Epo- and SCF-treated UT-7/Epo cells, blotted and hybridized to cDNA probes for c-fos, egr1 and CIS (Fig. 7Go). Maximal stimulation was obtained with 4 U/ml Epo or 100 ng/ml SCF. The messages for c-fos and egr1 were rapidly induced by both Epo and SCF at 30 min. The message for c-fos could still be detected at 2 h in both SCF- and Epo-treated cells and was undetectable at 6 h. Message for egr1 was reduced but still prominent at 2 h in Epo-treated cells but was substantially reduced in SCF-treated cells. Epo-induced CIS message expression was slower, with peak expression levels at 2 h, and more prolonged, with CIS message still detected at 6 h after treatment. CIS message was not detected following SCF treatments. Pretreatment with cycloheximide did not block induction of these genes, and in fact, led to super-induction, characteristic of primary response genes.



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  		Figure 7. Expression of egr1, c-fos, and CIS in treated UT-7/Epo cells. RNA was prepared from UT-7/Epo cells that were treated for 0.5, 2 or 6 h with 100 ng/ml SCF or 4 U/ml Epo. Northern blots (10 µg RNA/lane) were hybridized to cDNA probes for c-fos, egr1, and CIS.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Epo activation of its receptor leads to stimulation of the JAK/STAT and ras/raf/MAPK pathways, both of which regulate changes in early erythroid cell proliferation, maturation and gene expression [1, 2, 4, 11, 12, 14, 15]. Binding of SCF to its tyrosine kinase receptor, c-kit, activates ras/raf/MAPK in multiple cell types including erythroid cells [7]. Previous reports have shown that SCF can activate or cross-phosphorylate EpoR in erythroid precursor cells [26] and erythroid cells [19-21], suggesting a mechanism whereby SCF enhances the proliferation and maturation of erythroid progenitor cells. However, although SCF activates EpoR in these systems, this effect is not sufficient for sustained cell proliferation and survival [20, 21] and is not mediated by activation of the JAK/STAT pathway [20, 21], but rather may be an effect mediated by activation of MAPK [20].

In this study using an Epo-dependent human erythroid cell line UT-7/Epo, we have confirmed the inability of SCF to sustain erythroid cell proliferation and activate the JAK/STAT pathway. Furthermore, we have confirmed the activation of MAPK by SCF and extended these studies by demonstrating that in comparison to Epo, SCF activation of MAPK is reduced and transient. In addition, SCF induces more transient expression of the early response genes c-fos and egr1 than Epo and unlike Epo, does not induce CIS. The transient effects of SCF on these signaling pathways may explain the inability of SCF to sustain erythroid cell proliferation.

The ability of SCF to activate JAK2 may be a cell-specific phenomenon and variable. SCF has been reported to activate JAK2 in M-07e, VTF-1, and FDCP cells [8, 9, 27], and not JAK2 in M-07e or HCD57 cells [10, 20]. Our results in UT-7/Epo cells are consistent with others who have failed to demonstrate JAK2 activation in erythroid cells or cell lines [20, 21]. Examinations of both nuclear and cytoplasmic extracts of UT-7/Epo cells show no STAT activation by SCF. In addition, as reported in other factor-dependent cells [28], there were no synergistic effects on STAT activation and translocation, even when SCF was added to suboptimal amounts of Epo. Thus, while JAK2 and STAT5 activation may be required for sustained proliferation in response to Epo, they appear to be unnecessary for the initiation of DNA synthesis (i.e., thymidine incorporation) for up to three days. It should be noted that cell lines, even factor-independent lines, have intrinsic changes which may affect their signaling pathways and early gene responses. The results may elucidate pathways of key importance in proliferation and differentiation, but the responses may be different from cell line to cell line and may not be identical to those that occur in primary cells.

Consistent with reports that Epo phosphorylation of EpoR results in MAPK activation [4, 5, 7], p42/44 activation in UT-7/Epo cells is readily detected following either low or high concentrations of Epo. While Miura et al. used mutant EpoR constructs that demonstrated that the ability to proliferate was not correlated with the activity of MAPK [4], there are reports of other cell types, such as G-CSF-stimulated NFS-60 cells or SCF-treated B6M cells, in which MAPK activity has either correlated with, or been required for, proliferation [7, 29, 30]. In UT-7/Epo cells, like primary erythroid progenitor cells [26], MAPK activation appears to be one component common to both the SCF- and Epo-stimulated signaling pathway, contributing to proliferation. However, we now demonstrate that the effects of SCF on p42/44 activation in UT-7/Epo are transient in comparison to Epo, which may explain the inability of SCF to sustain erythroid cell proliferation.

Following binding of SCF to c-kit, association with EpoR occurs in the Box 2 region of EpoR, believed to be involved in mitogenesis [19]. Thus, SCF activation of c-kit in transduced cells indirectly activates p42/44 through activation of EpoR resulting in GRB2 and/or SHC binding and subsequent activation of the components leading to p42/44 phosphorylation [31]. Phosphorylation of EpoR by SCF also occurs in the HCD57 cell line and primary erythroid progenitor cells [20, 26]. However, in contrast, phosphorylation of EpoR was not induced by SCF in UT-7/Epo cells. Therefore, in UT-7/Epo cells it is likely that SCF activates MAPK directly through Syp and SHC association with c-kit [20, 32].

The c-fos and egr1 promoters can be activated through activation of the ras/MAPK cascade [17, 18]. The observation that c-fos and egr1 messages were only transiently induced in UT-7/Epo cells by SCF stimulation is consistent with the short-term activation of MAPK following this treatment. In situations where there is sustained MAPK activation, such as that induced by Epo, there is continued c-fos and egr1 expression and long-term proliferation is maintained. This is supported by data that log phase UT-7/Epo cells grown continuously in the presence of 0.4 U/ml Epo have a basal level of c-fos and egr1 messages, which becomes undetectable in the absence of Epo (data not shown).

In summary, we conclude that while p42/44 activation is a common path in the stimulation of UT-7/Epo proliferation, it is the transient nature of this kinase activation and the subsequent transient expression of early response genes (i.e., c-fos and egr1) that are responsible for the short-term proliferative effects of SCF. Differential effects of transient regulation of MAPK have also been described in the PC12 system [33]. Nerve growth factor (NGF) and epidermal growth factor (EGF) stimulation of PC12 utilized similar signals, but there was a quantitative difference in p21ras activation responsible for NGF induction of differentiation. EGF only transiently activated p21ras and only affected proliferation [33]. In addition, activation of JAK2 and STAT5 is not required to initiate proliferation; however, activation of these components, along with the full signal cascade induced by Epo binding to EpoR, provides the additional signals, including prolonged activation of p42/44, that provides the sustained signals necessary for a continued proliferative response.


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The authors would like to thank Maria-Nicole Sandoli and Caroline King for expert technical assistance. The authors and this work were supported by SmithKline Beecham Pharmaceuticals.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  8. Brizzi MF, Zini MG, Aronica MG et al. Convergence of signaling by interleukin-3, granulocyte-macrophage colony-stimulating factor, and mast cell growth factor on JAK2 tyrosine kinase. J Biol Chem 1994;269:31680-31684.[Abstract/Free Full Text]

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Received June 8, 2000; accepted for publication June 8, 2000.



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