ã Oncogene (2000) 19, 2296 ± 2304 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Epo regulates erythroid proliferation and dierentiation through distinct signaling pathways: implication for erythropoiesis and Friend virus-induced erythroleukemia Barry Zochodne1,4, Amandine HL Truong1,4, Kendra Stetler1, Rachel R Higgins1, Je Howard3, Dan Dumont1, Stuart A Berger2 and Yaacov Ben-David*,1 1 Department of Medical Biophysics, University of Toronto, Cancer Biology Research, Sunnybrook and Women's College Health Sciences Centre, 2075 Bayview Avenue, S-Wing, Toronto, Ontario, M4N 3M5 Canada; 2Department of Immunology, University of Toronto, Arthritis and Immune Disorder Network, University Health Network, 620 University Avenue, Suite 700, Toronto, Ontario M5G 2M9 Canada We have recently isolated the erythroleukemic cell line, HB60-5, that proliferates in the presence of erythropoietin (Epo) and stem cell factor (SCF), but undergoes terminal dierentiation in the presence of Epo alone. Ectopic expression of the ets related transcription factor Fli-1 in these cells resulted in the establishment of the Epo-dependent cell line HB60-ED that proliferates in the presence of Epo. In this study, we utilized these two cell lines to examine the signal transduction pathways that are activated in response to Epo and SCF stimulation. We demonstrate that Epo, but not SCF, phosphorylates STAT-5 in both HB60-5 and HB60-ED cells. Interestingly, SCF activates the Shc/ras pathway in HB60-5 cells while Epo does not. However, both Epo and SCF are capable of activating the Shc/ras pathway in HB60ED cells. Furthermore, enforced expression of gp55 in HB60-5 cells by means of infection with the Spleen Focus Forming virus-P (SFFV-P), confers Epo independent growth, which is associated with the up-regulation of Fli-1. Activation of the Shc/ras pathway is readily detected in gp55 expressing cells in response to both Epo and SCF, and is associated with a block in STAT-5B tyrosine phosphorylation. These results suggest that STAT-5 activation, in the absence of Shc/ras activation, plays a role in erythroid dierentiation. Moreover, Fli-1 is capable of switching Epo-induced dierentiation to Epo-induced proliferation, suggesting that this ets factor regulated genes whose products modulate the Epo-Epo-R signal transduction pathway. Oncogene (2000) 19, 2296 ± 2304. Keywords: erythropoiesis; Epo; Fli-1; signal transduction; Friend erythroleukemia Introduction In the past decade a number of important intracellular and extracellular factors have been indenti®ed that alter the fate of erythroid progenitor cells, namely whether to divide (self-renew), die (apoptosis), or *Correspondence: Y Ben-David 3 Current Address: HULC Cell & Molecular Biology Laboratory, Lawson Research Institute, St. Joseph's Health Centre, 268 Grosvenor St. London, Ontario N6A 4V2 Canada 4 Equal contribution made by ®rst two authors Received 20 October 1999; revised 20 January 2000; accepted 27 March 2000 dierentiate. Of these, perhaps hematopoietic growth factors (HGF) have been the most thoroughly characterized. Erythropoietin (Epo) and stem cell factor (SCF) are two HGFs that have been identi®ed as important regulators of erythropoiesis. Recognition of a role for SCF and its receptor, the tyrosine kinase c-Kit, in erythroid development stems largely from the study of W (White spotting) and Sl (Steel) mice that exhibit erythroid and other lineage-speci®c defects due to inherited mutations within the c-Kit and SCF genes (Pawson and Bernstein, 1990). Studies examining the pleiotropic eects of c-Kit signaling have identi®ed a number of important downstream eectors that are believed to be responsible for its potent mitogenic eects, including phosphatidylinositol 3'-kinase (PI3kinase), the SH2 adaptor protein (Shc), p21ras and mitogen-activated protein kinase (MAPK) (Tauchi et al., 1994; Timokhina et al., 1998; Sattler et al., 1997; Lev et al., 1992; Kinashi et al., 1995). Arguably the most important erythroid-speci®c HGF is Epo. The signaling events triggered by the binding of Epo to the Epo-receptor (Epo-R), like many other HGFs, is rather complex. However, it is clear that Epo possesses anti-apoptotic activity (Koury and Bondurant, 1990), a function that is somehow tightly coupled to its proliferative and dierentiation-inducing eects on erythroid progenitor cells (Youssou®an et al., 1993; Liboi et al., 1993). Although the exact molecular mechanisms underlying these contrasting cellular responses are not well understood, a number of downstream signaling eectors have been identi®ed, including Shc/ras, PI3-kinase, SHIP, as well as the JAK2/STAT5 pathways (Lecoq-Lafon et al., 1999; Damen et al., 1993, 1995a,b; He et al., 1995; Miura et al., 1994; Verdier et al., 1997; Gouilleux et al., 1995 ID: 1002; Pallard et al., 1995; Wakao et al., 1995). Epoinduced phosphorylation of the Epo-R at Tyrosine 343 promotes proliferation and an increase in STAT-5 activity (Damen et al., 1995b). However, recent studies link STAT-5 phosphorylation to Epo-induced dierentiation of erythroid progenitor cells. Iwatsuki et al. (1997), showed that tyrosine phosphorylation of the Epo-R at position Y343 and Y401 is required for STAT-5 activation and erythroid dierentiation. These con¯icting results underscore the need to clarify the exact role of STAT-5 phosphorylation during Epoinduced signaling events. Friend erythroleukemia is a well known multi-staged tumor model that is aptly suited for studying the Epo signaling in erythropoiesis and Friend leukemia B Zochodne et al mechanisms governing the proliferation and maturation of erythroid progenitor cells (Ben-David and Bernstein, 1991). In the past decade alone researchers have discovered that the signaling pathway de®ned by Epo-Epo-R is a common target of the transforming strains of Friend virus. The genomic integrity of both Epo and the Epo-R are often speci®cally disrupted during the course of F-MuLV and FV-P-induced erythroleukemias (Li et al., 1990; McDonald et al., 1987; Hankins et al., 1989; Howard et al., 1996). Moreover, gp55p, the glycoprotein of the env of the polycythemia strain of spleen focus-forming virus (SFFVp), has been shown to bind to and activate the Epo-R (Li et al., 1990). The Epo mimicry of gp55p is thought to be responsible for the early polyclonal proliferation of erythroid progenitor cells during the preleukemic stage of FV-P induced disease (Kabat, 1990). Overall, Friend virus has also been found to suppress the dierentiation signals controlled by the Epo/Epo-R pathway, through retroviral insertional mutagenesis (Ben-David and Bernstein, 1991). Genes speci®cally targeted by retroviral insertional mutagenesis have been identi®ed for each strain of Friend virus. These genes include the p53 tumor suppressor gene and two members of the ets family of transcription factors, Spi-1 and Fli-1 (Ben-David et al., 1988, 1991; Hicks and Mowat, 1988; MoreauGachelin et al., 1988). Activation of Fli-1 has been identi®ed as an early transformation event associated with F-MuLV induced erythroleukemias (Ben-David et al., 1990). Examination of its role during the transformation of erythroid progenitor cells suggest that constitutive expression of Fli-1 dramatically increases the self-renewal potential of proerythroblasts. This block in erythroid dierentiation appears to stem, in part, from the ability of Fli-1 to transcriptionally repress the retinoblastoma (Rb) tumor suppressor gene, a central component of the cell cycle machinery (Tamir et al., 1999). Disruption of the cell cycle in this manner would be advantageous to the virus since exit from the cell cycle is a prerequisite for terminal erythroid dierentiation but detrimental to further viral replication. In this study, we have investigated the role of Epo and SCF in regulating the self-renewal and dierentiation potential of erythroid progenitor cells. Toward this goal, we used a novel erythroleukemia cell line, designated HB60-5, that proliferates in the presence of SCF and Epo, but undergoes terminal erythroid dierentiation in response to Epo alone (Tamir et al., 1999). We previously demonstrated that a transient but dramatic decrease in the expression levels of Fli-1 is associated with cell cycle arrest and terminal dierentiation of this cell line (Tamir et al., 1999). Ectopic expression of Fli-1 in HB60-5 or expression as a result of insertional mutagenesis conferred Epo-dependent proliferation, suggesting that constitutive high levels of Fli-1 expression constitutes an important molecular `switch' that disables the dierentiation signaling of Epo. By using HB60-5 and its Epodependent derivative HB60-ED cells, we provide evidence that the proliferation of HB60-5 cells is speci®cally associated with the activation of the PI3K and Shc/ras pathways, while Epo-induced terminal dierentiation appears to be mainly associated with STAT-5 activation. Interestingly, expression of gp55 in HB60-5 cells resulted in Epo and SCF independent growth. The block in Epo-induced dierentiation elicited by gp55p was found to be associated with a block in STAT-5 phosphorylation and activation of the Shc/ras pathway, which correlated with high levels of Fli-1 expression. 2297 Results Epo-induced differentiation of HB60-5 cells is associated with the phosphorylation of STAT-5 Previous studies have demonstrated that Epo stimulates the phosphorylation of both Shc and STAT-5b protein whereas SCF activates Shc/ras signaling (Penta and Sawyer, 1995; Damen et al., 1993, 1995b; Gouilleux et al., 1995; Wakao et al., 1995). To verify the status of these phosphorylation events in HB60-5 cells, we brie¯y starved and then incubated these cells with Epo, SCF or both growth factors (10 min) and then immunoprecipitated with STAT-5b and Shc antibodies. Hybridization of these blots with anti-Shc and anti-STAT-5b antibodies clearly showed the presence of Shc (52 and 46 kDa) and STAT-5b (93 kDa) in these IPs. Western blot analysis with anti-PY antibodies also revealed that STAT-5b became phosphorylated in response to Epo or Epo and SCF together, but not in response to SCF alone in HB60-5 cells (Figure 1B). However, Shc phosphorylation was only detected in cells after stimulation with SCF or SCF and Epo, but not Epo alone (Figure 1A). These results demonstrate that Epo, but not SCF, can activate STAT-5b in HB60-5 and that Shc phosphorylation correlates with proliferation. Since phosphorylated STAT-5b enters the nucleus (Mui et al., 1995), we next examined its localization in HB60-5 cells following growth factor stimulation. After 10 minutes of stimulation with Epo or Epo and SCF, the majority of the phosphorylated STAT-5b band was found in the nucleus (Figure 1C). As expected, SCF alone did not alter the phosphorylation status of STAT-5b in either cytoplasmic or nuclear fractions. It has recently been reported that the Src homology 2containing inositol phosphate (SHIP) is associated with Shc and is phosphorylated in response to Epo and SCF (Damen et al., 1996). Indeed, we also detected the 140 kDa phosphorylated SHIP proteins in IPs with Shc antibody in extracts from SCF or SCF+Epo stimulated HB60-5 cells, but not with Epo alone (Figure 1A). These experiments, therefore, demonstrated that in HB60-5 cells Epo but not SCF activated STAT-5 while the Shc/ras pathway was activated by SCF. Activation of Shc/ras pathway by Epo in HB60-ED cells Unlike HB60-5 cells, stimulation of the Fli-1 overexpressed HB60-ED cells with Epo resulted in the phosphorylation and activation of Shc (Figure 2A). A similar phosphorylation pattern was also detected in the Epo-dependent cell line HB9.1-ED, a F-MuLV induced erythroleukemic cell line which also expresses high levels of Fli-1 due to retroviral insertional activation of this gene (Figure 2B). Activation of Shc was also detected in HB60-ED cells following SCF stimulation, although the extent of phosphorylation was signi®cantly reduced in these cells. Similar to HB60-5 cells, stimulation of Oncogene Epo signaling in erythropoiesis and Friend leukemia B Zochodne et al 2298 Figure 1 SCF and Epo activate Shc and STAT-5 in HB60-5 cells, respectively. Lysates (100 mg) from HB60-5 cells, stimulated with the indicated growth factors were immunoprecipitated with either Shc (A) of STAT-5b (B) antibodies and subjected to Western blot analysis using the indicated antibodies. (C) Cytoplasmic and nuclear proteins were prepared from HB60-5 cells after stimulation with the indicated growth factor. 100 mg of proteins from each preparation was immunoprecipitated with STAT-5b antibody, subjected to SDS ± PAGE and Western blotted with the STAT-5b antibody Figure 2 Mitogenic activation of Shc/ras pathway by Epo and SCF correlates with proliferation. Lysates prepared from HB60-ED cells (A) or the Epo-dependent HB9.1-ED cells (B) after stimulation with the indicated growth factors, were subjected to IP-Western blot analysis with the indicated antibodies (left panel), as described in Figure 2. Arrows (right panel) show the positions of the detected proteins. The symbol (*) indicates the position of a phosphorylated protein that co-immunoprecipitated with the antiSTAT-5b antibody but whose identity is yet to be determined HB60-ED and HB9.1-ED with Epo also activated STAT-5b phosphorylation (Figure 2A,B). Interestingly, a 180 kDa protein that was also phosphorylated in response to Epo, co-immunoprecipitated with STAT-5b Oncogene in HB60-ED cells (Figure 2A). This band was not detected in the STAT-5b IP of HB60-5 cells (Figure 1B). Together these results suggest that dierentiation of HB60-5 cells is associated with STAT-5 phosphoryla- Epo signaling in erythropoiesis and Friend leukemia B Zochodne et al tion, whereas their proliferation may require Shc/ras pathway activation by Epo. SCF activated the Epo-R through a direct interaction of cKit with the Epo-R Previously we showed that HB60-5 cells proliferate in response to SCF, and that the addition of Epo dramatically increases this proliferative response (Tamir et al., 1999). The activation of Epo-R through direct interaction with the phosphorylated c-Kit receptor has been suggested to be responsible for the synergistic eect of Epo and SCF during erythroid proliferation (Wu et al., 1995). However, a recent con¯icting study showed that in erythroid cells c-Kit, although critical to the phosphorylation of Epo-R by SCF, does not directly interact with the Epo-R (Jacobs-Helber et al., 1997). To verify the role of SCF in erythroid proliferation, HB60-5 cells were starved for 12 h and subsequently stimulated with either Epo or SCF. SCF stimulation followed by immunoprecipitation with the Epo-R antibody revealed a 145 kDa phosphorylated band, as well as three additional phosphorylated bands of 68, 66 and 95 kDa (Figure 3A). Phosphorylation of the latter three bands was also stimulated by Epo with their precipitation being blocked by the addition of competitor Epo-R peptide (Figure 3A). A similar pattern of cytokine mediated-phosphorylation was also seen in HB60-ED cells (Figure 3B) and the Epo-dependent HB9.1-ED cell line (Figure 3C), which was derived from a FMuLV induced-erythroleukemia (Howard et al., 1993). Based on their molecular weight and interaction with speci®c antibodies, the 68 and 95 kDa proteins appear to be identical to the Epo-R and STAT-5, respectively (data not shown here). Interestingly, the phosphorylated 145 kDa band that immunoprecipitated with the Epo-R also weakly hybridized with the c-Kit antibody (Figure 3A,B). To con®rm that the 145 kDa band was indeed c-Kit, SCF stimulated cell extracts were ®rst immunoprecipitated with Epo-R, separated from the beads, re-immunoprecipitated with the c-Kit antibody and Western blotted with anti-phospho-tyrosine (PY) antibody. As shown in Figure 3D, a 145 kDa band migrates with the phosphorylated c-Kit band was precipitated by the double IP procedure. These data show that the phosphorylated c-Kit and the Epo-R are found in the same complex. The identity of the 66 kDa band that was also precipitated with the Epo-R from HB60-5 and HB60-ED cell extracts is yet to be determined. Phosphatidylinositol 3-kinase (PI3K) has been previously shown to be phosphorylated in response to Epo and SCF stimulation and has been shown to bind to both receptors (Verdier et al., 1997; Damen et al., 1995a; Sattler et al., 1997; Lev et al., 1992). Using HB60-5 and HB60-ED cells, we also examined the binding of the p85 subunit of PI3K to the Epo-R and c-Kit. Western blot analysis revealed the PI3K was associated with both the Epo-R and c-Kit in IP with extracts from SCF stimulated HB60-5 and HB60-ED cells (Figure 3A,B). However, the Epo-induced association of p85 with the Epo-R was only seen in the IP with extracts from HB60-ED cells, and not with extracts from the parental HB60-5 cells, demonstrating that PI3K activation is connected with the Epodependent proliferation observed in this cell line. 2299 gp55 induces Epo and SCF independent growth HB60-5 cells The cell line HB60-5 is similar to FV-P inducederythroleukemia cell lines in that it has undergone insertional activation of the Spi-1 gene and a p53 mutation (Johnson et al., 1993; Tamir et al., 1999). However, since this cell line was derived from a F-MuLV infected tumor, it is negative for gp55 expression (Figure 4). Given that gp55p mimics the proliferative eects of Epo in erythroblasts, we examined the eect of gp55p expression in HB60-5 cells. The resulting SFFV-P Figure 3 Epo-R is phosphorylated by SCF stimulation through a direct interaction with the activated c-Kit. Cell extracts (100 mg) prepared from either SCF or Epo stimulated HB60-ED (A), HB60-5 (B) and HB9.1-ED (C) were immunoprecipitated with either an Epo-R antibody (with or without a competitive Epo-peptide), or a c-Kit antibody, as indicated. The precipitates were then subjected to SDS ± PAGE, transferred to PVDF membrane, and immunoblotted with the anti-phosphotyrosine antibody (anti-PY). These same blots were then sequentially hybridized with the anti-c-Kit antibody and an anti-p85 antibody. (D) Extracts (2 mg) prepared from HB60-5 cells, that were stimulated with SCF (lane 4) or without SCF (lane 3), were immunoprecipitated with an anti-Epo-R antibody and then re-immunoprecipitated with an anti-c-Kit antibody, as described in the methods. The blotted membrane was then immunoblotted with the anti-phosphotyrosine antibody (a-PY). Extracts (200 mg) from the starved HB60-5 cells (lane 1) or SCF stimulated cells (lane 2) that were only immunoprecipitated with the c-Kit antibody, were used as controls Oncogene Epo signaling in erythropoiesis and Friend leukemia B Zochodne et al 2300 infected HB60-5 cell line, HB60-SSFV, was similar to the FV-P induced-erythroleukemia cell line DP28-9 (BenDavid et al., 1988) in expressing gp70 (encoded by FMuLV) and gp55 (encoded by SFFV) (Figure 4). In contrast to HB60-5 cells that undergo cell growth arrest when cultured in the presence of Epo, expression of gp55 in these cells conferred SCF and Epo independent growth in culture (Figure 5A,B). We have previously shown that the expression of globin genes (dierentiation marker for erythroid cells) is elevated in response to Epo in HB60-5 cell (Tamir et al., 1999). Since the expression of globin in HB60-SSFV cells was not aected by the addition of Epo (Figure 5C), this data suggests that the expression of gp55 in HB60-5 cells blocks dierentiation. Moreover, gp55 appears to stabilize the levels of Fli-1 expression in HB60-SSFV cells, since previously we have shown that Fli-1 levels undergo a transient but dramatic decrease in response to Epo (Figure 5c) (Tamir et al., 1999). These results suggest that gp55p prevents the transient but dramatic decrease in Fli-1 expression upon Epo stimulation. (Figure 6A). Moreover, Shc activation by these growth factors was also associated with SHIP phosphorylation which co-immunoprecipitated with the anti-Shc antibody. Discussion We have shown here, using the unique erythroleukemia cell lines HB60-5 and HB60-ED, that Epo-induced dierentiation correlates with distinct signaling events. STAT-5 phosphorylation correlates with the dieren- Expression of gp55 in HB60-5 cells overrides STAT-5b phosphorylation by Epo Since gp55 expression confers SCF and Epo independent growth to HB60-5 cells, we examined the phosphorylation status of Shc and STAT-5b in HB60-SFFV cells. In contrast to HB60-5, incubation of HB60-SSFVcells with Epo (1 units/ml), was not able to activate STAT-5b phosphorylation (Figure 6B). As shown in Figure 6C, regardless of the concentration of Epo used for cell stimulation, STAT-5b remained unphosphorylated in HB60-SFFV cells, whereas maximum STAT-5b phosphorylation levels were readily detected in HB60-5 cells stimulated with only 0.5 units of Epo. A similar block in STAT-5b activation by Epo was also seen in the FV-P induced erythroleukemia cell line DP28-9 (Figure 6C). However, a low but constitutive level of STAT-5b phosphorylation was seen in these cells. With regard to the status of Shc phosphorylation, the addition of both Epo and SCF resulted in an increase in Shc phosphorylation in HB60-SFFV cells Figure 4 Expression of gp55 in HB60-5 cells. Total protein extracts (20 mg) prepared from the indicated cells were resolved on a 12% SDS-polyacrylamide gel, transferred to a PVDF membrane and hybridized with a gp55 speci®c antibody. Equal loading was determined by hybridizing the same membrane to Erk-2 antibody. The locations of gp70 and gp55 bands are shown by arrows Oncogene Figure 5 Expression of gp55 in HB60-5 cells confers growth factor independence. Triplicate culture (56104) of HB60-5 (A) and HB60-SSFV (B) cells were grown in absence of no growth factor, Epo (0.5 units/ml), SCF (100 ng/ml) or both Epo and SCF, and the number of viable cells was determined at days 1 ± 4. (C) Total RNA was isolated from HB60-SSFV cells after 12 and 24 h growth with or without Epo and separated on a 1% agarose gel, transferred to the nitrocellulose membrane and sequentially hybridized with Fli-1, a-globin and GAPDH probes Epo signaling in erythropoiesis and Friend leukemia B Zochodne et al 2301 Figure 6 Expression of gp55 in HB60-5 cells promotes the activation of Shc while inhibiting STAT-5b phosphorylation. HB60SSFV, HB60-5 and DP28-9 erythroleukemic cells were starved in a-MEM+0.5% FBS, stimulated with Epo (1 units/ml for A and 0.5 units/ml for C) or SCF (100 ng/ml for B) for 10 min, washed with PBS and lysed. The lysates (100 mg) were immunoprecipitated with either Shc (A) or STAT-5b antibodies (B and C), separated on SDS-polyacrylamide gels and subjected to Western blot analysis using the indicated antibodies tiation of HB60-5 cells but not with the proliferation of these cells by Epo. Conversely, activation of the Shc/ ras pathway by Epo was only detected under proliferative conditions but not during dierentiation of these cells. As well, since constitutive expression of Fli-1 in these cells can switch dierentiation to proliferation in response to Epo stimulation, these results demonstrate the importance of this transcription factor in the regulation of Epo/Epo-R signaling and erythroid transformation. Analysis of Shc activation by Epo had revealed that this SH2-domain containing adaptor was mainly phosphorylated in the proliferative HB60-ED cells. Moreover, activation of SHIP and p85/PI3K was also detected in proliferating HB60-ED cells. Our results are consistent with published studies that have shown increased phosphorylation of Shc and other SH2 containing protein such as P85/PI3K, SHIP and Grb2 during Epo-induced proliferation of erythroblasts (Damen et al., 1993, 1995a, 1996; He et al., 1995; Verdier et al., 1997). Interestingly, Epo-induced dierentiation of the parental HB60-5 cells requires very distinct signaling events, namely the tyrosine phosphorylation of STAT-5b. The ability of gp55p to block STAT-5b tyrosine phosphorylation in response to Epo supports the notion that it may be an important dierentiation signaling event. This is further supported by the lack of STAT-5b phosphorylation by SCF, which is a known mitogenic factor for erythroid cells. Several reports (Sui et al., 1998; Gregory et al., 1998; Pallard et al., 1995), with the exception of one (Brizzi et al., 1999), also suggest that SCF does not activate STAT-5b. However, SCF phosphorylated Shc and its associated protein SHIP in both HB60-5 and HB60-ED cells resulting in proliferation. Interestingly, during proliferation of HB60-5 cells with Epo and SCF, phosphorylation of STAT5 is also detected in these cells. Based on the results presented here, it is likely that activation of Shc/ ras pathway by SCF may block STAT-5 mediated dierentiation signal which can also be induced by Epo in HB60-5 cells. Further support for the involvement of STAT-5b in erythroid dierentiation comes from recent knock-out studies. Double knock-out STAT-5a/5b mice are viable with no apparent defect in erythropoiesis (Teglund et al., 1998). However, these mice eventually developed splenomegaly which is associated with a dramatic increase in the number of Ter 119 erythroid positive spleen cells (Moriggl et al., 1999). While the authors attributed this altered morphology to spleen hyperplasia, there is a reasonable possibility that the lack of STAT-5 in the erythroid progenitor cells of the double knockout mice may enhance their proliferation. Interestingly, Socolovsky et al. (1999) have recently shown that the STAT-5 double knockout embryos are anemic. The low number of erythroid progenitor cells seen in these mice was shown to be associated with a higher apoptotic rate due to lower Bcl-xl expression. These data strongly suggest that STAT-5 is essential for high erythropoietic rate during fetal development. Additional support for the role of STAT-5 in erythroid development has emerged from a study using a dominant negative mutant of STAT-5 that inhibited the dierentiation of erythroblasts and erythroleukemic cells (Iwatsuki et al., 1997). It will therefore be intriguing to determine whether STAT-57/7 mice are more susceptible to Friend virus induced-erythroleukemias. Perhaps the most striking result that supports the above observation is the ability of gp55 to block Epo-induced phosphorylation in HB60-5 cells. In addition, these gp55 expressing cells are Epo and SCF independent with an intact Epo-activated Shc/ ras response. Interestingly, Yamamura et al. (1998) have recently reported that normal erythroblasts and erythroleukemic cells expressing gp55 have constitutive STAT-5 phosphorylation. However, they reported that the phosphorylated STAT-5 was unable to bind to a speci®c STAT-5 consensus DNA sequence and did not translocate to the nucleus. Indeed, we also see constitutive phosphorylation of STAT-5b in a FV-P induced erythroleukemia cell line, but at very low levels. Nevertheless, their results are consistent with ours in that gp55/Epo-R signaling in erythroblasts is distinct from Epo-R signaling. Although our results also suggest that gp55/Epo-R signaling could also be identical to Epo/ Oncogene Epo signaling in erythropoiesis and Friend leukemia B Zochodne et al 2302 Oncogene Epo-R only in transformed erythroblasts that proliferate in response to Epo. Our previous studies have demonstrated that in HB60-5 cells Fli-1 ¯uctuates during Epo-induced dierentiation while constitutive expression of this transcription factor results in Epo-induced proliferation (Tamir et al., 1999). Recently, a similar result was also reported in chicken erythroblasts transfected with Fli-1 (Pereira et al., 1999). Similarly, gp55 expression in HB60-5 cells conferred a growth advantage that was associated with elevated Fli-1 expression levels. Given that gp55p activates the Shc/ras pathway in HB60-5 cells, it may be that Fli-1 is a downstream component of this pathway. In support of this notion we have recently identi®ed Fli-1 as a downstream target of the SCF/c-Kit signaling which also activates the Shc/ras pathway (Tamir et al., 1999). Fli-1 may therefore belong to a growing list of ets related transcription factors whose expression is thought to be regulated by the ras pathway (Janknecht, 1996; O'Hagan et al., 1996; Wasylyk et al., 1998; Fowles et al., 1998). Our data demonstrates that Fli-1 over-expression in HB60-5 cells converts Epo-induced dierentiation to Epo-induced proliferation. Furthermore, this dierentiation is correlated with the activation of Shc/ras through the Epo-R. While the mechanism by which Fli-1 alters Epo signaling has not yet been identi®ed, these results suggest that some of the downstream target genes of this transcription factor may be involved in the modulation of the Epo/Epo-R signal transduction pathway. Hence, identi®cation of these target genes should provide important insights into the molecular events that regulate normal erythropoiesis and malignant transformation by Fli-1. The nature of the synergy between Epo and SCF with respect to the proliferation of HB60-5 cells appears to be closely linked to the ability of c-Kit to phosphorylate the Epo-R. Previously, Wu et al. (1995) demonstrated that c-Kit was capable of interacting with and rapidly phosphorylating the Epo-R. Although a recent report (Jacobs-Helber et al., 1997) suggested an indirect mechanism of Epo-R phosphorylation by cKit, our data certainly provides evidence that c-Kit binds to the Epo-R and induces its phosphorylation. Since the phosphorylation of either c-Kit or Epo-R activates the Shc/ras pathway in HB60-ED cells, both SCF and Epo likely promote the growth of these cells through their accumulative eects on multiple overlapping and distinct pathways that are also undoubtedly targeted by gp55. In summary, by utilizing the cell lines HB60-5 and HB60-ED, we have shown that activation of Shc/ras pathway was strongly associated with SCF and Epoinduced cell growth. In contrast, Epo-induced activation of the STAT-5 pathway was associated with terminal dierentiation of HB60-5 cells. Constitutive expression of Fli-1 dramatically modi®ed the eect of Epo on these cells by blocking their dierentiation and promoting their proliferation, which was associated with Shc activation. These same responses to Epo were produced in cells ectopically expressing gp55p. The viral glycoprotein activated Fli-1 expression and blocked STAT-5 activation by Epo. Together, these results shed further light on the signaling events that discriminate between Epo-induced proliferation and dierentiation. Materials and methods Tumors and cell lines The establishment of the erythroleukemic cell line HB60-5 cells was previously described (Tamir et al., 1999). HB60-ED cells are polyclonal derivative of HB60-5 cells transfected with a Fli-1 expression vector (Tamir et al., 1999). The Epodependent erythroleukemia cell line HB9.1-ED, was derived from a culture of greatly enlarged spleens of BALB/c mice injected at birth with F-MuLV (Howard et al., 1993). The erythroleukemia cell lines DP28-9 and CB7 were induced by FV-P and F-MuLV, respectively as described elsewhere (BenDavid et al., 1988). Cells were maintained in alpha minimum essential medium (a-MEM) with 10% fetal bovine serum (FBS). HB60-5 and HB60-ED cells were cultured in a-MEM medium supplemented with 15% heat inactivated FBS, 0.5 unit/ml of Epo (Boehringer Mannheim) and 100 ng/ml of SCF. To induce dierentiation, HB60-5 cells were washed twice with PBS and incubated in the presence of 15% FBS and 1 unit/ml of Epo. Viral infection HB60-5 cells were infected with SFFV-P virus by co-culturing these cells with an SSFV producing amphotrophic GP+ envAM12 helper free packaging cell line (Markowitz et al., 1988) for 48 h in the presence of Epo and SCF. The unattached HB60-5 cells were separated from the viral producer packaging cell lines, washed with PBS and grown in the presence of Epo and SCF for two additional days. The polyclonal HB60-SFFV cell line was then established by growing these cells in the absence of growth factors. RNA extraction and Northern blotting Total cellular RNA from erythroleukemia cell lines was isolated by using TRIzol reagent as described by the supplier (Gibco BRL). Twenty mg of total RNA was dissolved in 2.2 M formaldehyde, denatured at 658C for 5 min and electrophoresed in a 1% agarose gel containing 0.66 M formaldehyde. After transfer to Zetaprobe membranes, the ®lters were hybridized with 26106 c.p.m./ml of a-32P-dCTP labeled probe. Western blotting and immunoprecipitations Erythroleukemia cell lines were lysed with RIPA buer (0.5% NP-40, 50 mM Tris HCl, pH 8.0, 120 mM NaCl, 50 mM NaF, 10 mg/ml aprotinin, 100 mg/ml leupeptin and 10 mM PMSF). For Western blotting, 50 mg of cell lysates were resolved on a 12% SDS-polyacrylamid gel and immunoblotted as described (Howard et al., 1996). Antibody to gp55 was obtained from Quality Biotech Inc. (New Jersey) and antibody against Erk-2 from Santa Cruz, CA, USA. For immunoprecipitations (IPs), cells were starved for 9 h in a a-MEM+0.5% FBS. Subsequent to induction with Epo (1 units/ml) or SCF (100 ng/ml) for 10 min at 378C, the cells were collected by centrifugation, washed with cold PBS and lysed in RIPA buer as previously described (Tamir et al., 1999). Protein concentration was determined using the BioRad protein assay (Bio-Rad Laboratories, Munchen, Germany). After pre-clearing with Protein-G and -A, 200 mg of lysates were used in IPs and subjected to Western blotting, as described (Tamir et al., 1999). For double IP procedure, the lysates (2 mg) from starved or SCF stimulated cells were ®rst immunoprecipitated with antibody to Epo-R, bound to sepharose-A beads and the bound proteins were washed three times with the IP lysis buer. The proteins were released by boiling the beads in the same lysis buer containing 1% SDS for 1 min. Supernatant was then diluted 10-fold with the IP buer and immunoprecipitated for c-Kit Epo signaling in erythropoiesis and Friend leukemia B Zochodne et al using the c-Kit antibody. The Shc antibody was a gift from Dr Jane McGlade (University of Toronto). STAT-5b, p85 subunit of PI3K and cKit antibodies were purchases from Santa-Cruz, CA, USA. After a brief vortexing and a further incubation on ice for 5 min, the suspension was spun at 14000 r.p.m. for 5 min at 48C. Supernatants, containing the nuclear extract, were aliquoted and stored at 7808C. 2303 Preparation of cytoplasmic and nuclear extracts Approximately 16106 cells, cultured as described above, were pelleted, washed once with PBS and once with hypotonic buer (10 mM HEPES, pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT). Cell pellets were resuspended in 400 uL of hypotonic buer and incubated on ice for 10 min. After a brief vortexing (10 s) and further incubation on ice for 5 min, cells were pelleted at 8000 r.p.m. for 10 min. The supernatant, which contains the cytoplasmic material, was aliquoted and stored at 7808C. Pellets, which contain nuclei, were resuspended in 200 uL buer C (20 mM HEPES, pH 7.5 1.5 mM, MgCl2, 0.5 mM DTT 0.5 mM PMSF 25% glycerol) containing 420 mM NaCl and incubated on ice for 20 min. Acknowledgments We thank Dr Jane McGlade for the generous gift of Shc antibody. We also thank Drs Kirill Rosen for his comment on the manuscript and Lynda Woodcock for help in preparation of the manuscript. This work was supported by grants from the National Cancer Institute of Canada to Y Ben-David and SA Berger and Medical Research Council of Canada to D Dumont. RR Higgins was supported by a fellowship from the Leukemia Research Fund of Canada. References Ben-David Y and Bernstein A. (1991). Cell, 66, 831 ± 834. Ben-David Y, Giddens EB and Bernstein A. (1990). Proc. Natl. Acad. Sci. USA, 87, 1332 ± 1336. Ben-David Y, Giddens EG, Letwin K and Bernstein A. (1991). Genes Devel., 5, 908 ± 918. Ben-David Y, Prideaux VR, Chow V, Benchimol S and Bernstein A. (1988). Oncogene, 3, 179 ± 185. Brizzi MF, Dentelli P. Rosso A, Yarden Y and Pegoraro L. (1999). J. Biol. Chem., 274, 16965 ± 16972. Damen JE, Cutler RL, Jiao H, Yi T and Krystal G. (1995a). J. Biol. Chem., 270, 23402 ± 23408. Damen JE, Liu L, Cutler RL and Krystal G. (1993). Blood, 82, 2296 ± 2303. Damen JE, Liu L, Rosten P, Humphries RK, Jeerson, AB, Majerus PW and Krystal G. (1996). Proc. Natl. Acad. Sci. USA, 93, 1689 ± 1693. Damen JE, Wakao H, Miyajima A, Krosl J, Humphries RK, Cutler RL and Krystal G. (1995b). EMBO J., 14, 5557 ± 5568. Fowles LF, Martin ML, Nelsen L, Stacey KJ, Redd D, Clark YM, Nagamine Y, McMahon M, Hume DA and Ostrowski MC. (1998). Mol. Cell. Biol., 18, 5148 ± 5156. Gouilleux F, Pallard C, Dusanter-Fourt I, Wakao H, Haldosen LA, Norstedt G, Levy D and Groner B. (1995). EMBO J., 14, 2005 ± 2013. Gregory RC, Jiang N, Todokoro K, Crouse J, Paci®c RE and Wojchowski DM. (1998). Blood, 92, 1104 ± 1118. Hankins Wd, Chin DL, Dons R and Sigounas G. (1989). Ann. N.Y. Acad. Sci., 554, 21 ± 28. He TC, Jiang N, Zhuang H and Wojchowski DM. (1995). J. Biol. Chem., 270, 11055 ± 11061. Hicks GG and Mowat M. (1988). J. Virol., 62, 4752 ± 4755. Howard JC, Berger L, Bani MR, Hawley R and Ben-David Y. (1996). Oncogene, 12, 1405 ± 1415. Howard JC, Youse® S, Cheong G, Bernstein A and BenDavid Y. (1993). Oncogene, 8, 2721 ± 2729. Iwatsuki K, Endo T, Misawa H, Yokouchi M, Matsumoto A, Ohtsubo M, Mori KJ and Yoshimura A. (1997). J. Biol. Chem., 272, 8149 ± 8152. Jacobs-Helber SM, Penta K, Sun Z, Lawson A and Sawyer ST. (1997). J. Biol. Chem., 272, 6850 ± 6853. Janknecht R. (1996). Mol. Cell. Biol., 16, 1550 ± 1556. Johnson P, Chung S and Benchimol S. (1993). Mol. Cell. Biol., 13, 1456 ± 1463. Kabat D. (1990). Curr. Top. Microbiol. Immunol., 148, 1 ± 42. Kinashi T, Escobedo JA, Williams LT, Takatsu K and Springer TA. (1995). Blood, 82, 2086 ± 2090. Koury MJ and Bondurant MC. (1990). Science, 248, 378 ± 381. Lecoq-Lafon C, Verdier F, Fichelson S, Chretien S, Gisselbrecht S, Lacombe C and Mayeux P. (1999). Blood, 93, 2578 ± 2585. Lev S, Givol D and Yarden Y. (1992). Proc. Natl. Acad. Sci. USA, 89, 678 ± 682. Li JP, D'Andrea AD, Lodish HF and Baltimore D. (1990). Nature, 343, 762 ± 764. Liboi E, Carroll M, D'Andrea AD and Mathey-Prevot B. (1993). Proc. Natl. Acad. Sci. USA, 90, 11351 ± 11355. Markowitz D, Go S and Bank A. (1988). J. Virol., 62, 1120. McDonald J, Beru N and Goldwasser E. (1987). Mol. Cell. Biol., 7, 365 ± 370. Miura Y, Miura O, Ihle JN and Aoki N. (1994). J. Biol. Chem., 269, 29962 ± 29969. Moreau-Gachelin F, Tavitian A and Tambourin P. (1988). Nature, 331, 277 ± 280. Moriggl R, Topham DJ, Teglund S, Sexl V, McKay C, Wang D, Homeyer A, van Deursen J, Sangster MY, Bunting KD, Grosveld GC and Ihle JN. (1999). Immunity, 10, 249 ± 259. Mui AL, Wakao H, O'Farrell AM, Harada N and Miyajima A. (1995). EMBO J., 14, 1166 ± 1175. O'Hagan RC, Tozer RG, Symons M, McCormick F and Hassell JA. (1996). Oncogene, 13, 1323 ± 1333. Pallard C, Gouilleux F, Benit L, Cocault L, Souyri M, Levy D, Groner B, Gisselbrecht S and Dusanter-Fourt I. (1995). EMBO J., 14, 2847 ± 2856. Pawson T and Bernstein A. (1990). Trends Genet., 6, 350 ± 356. Penta K and Sawyer ST. (1995). J. Biol. Chem., 270, 31282 ± 31287. Pereira R, Quang CT, Lesault I, Dolznig H, Beug H and Ghysdael J. (1999). Oncogene, 18, 1597 ± 1608. Sattler M, Salgia R, Shrikhande G, Verma S, Pisick E, Prasad KV and Grin JD. (1997). J. Biol. Chem., 272, 10248 ± 10253. Socolovsky M, Fallon AE, Wang S, Brugnara C and Lodish HF. (1999). Cell, 93, 181 ± 191. Sui X, Krantz SB, You M and Zhao Z. (1998). Blood, 92, 1142 ± 1149. Tamir A, Howard J, Higgins RR, Li YJ, Berger L, Zacksenhaus E, Reis M and Ben-David Y. (1999). Mol. Cell. Biol., 19, 4452 ± 4464. Tauchi T, Boswell HS, Leibowitz D and Broxmeyer HE. (1994). J. Exp. Med., 179, 167 ± 175. Teglund S, McKay C, Schuetz E, van Deursen JM, Stravopodis D, Wang D, Brown M, Bodner S, Grosveld G and Ihle JN. (1998). Cell, 93, 841 ± 850. Oncogene Epo signaling in erythropoiesis and Friend leukemia B Zochodne et al 2304 Oncogene Timokhina I, Kissel H, Stella G and Besmer P. (1998). EMBO J., 17, 6250 ± 6262. Verdier F, Chretien S, Billar C, Gisselbrecht S, Lacombe C and Mayeux P. (1997). J. Biol. Chem., 272, 26173 ± 26178. Wakao H, Harada N, Kitamura T, Mui AL and Miyajima A. (1995). EMBO J., 14, 2527 ± 2535. Wasylyk B, Hagman J and Gutierrez-Hartmann A. (1998). Trends Biochem. Sci., 23, 213 ± 216. Wu H, Klingmuller U, Besmer P and Lodish HF. (1995). Nature, 377, 242 ± 243. Yamamura Y, Senda H, Kageyama Y, Matsuzaki T, Noda M and Ikawa Y. (1998). Mol. Cell. Biol., 18, 1172 ± 1180. Youssou®an H, Longmore G, Neumann D, Yoshimura A and Lodish HF. (1993). Blood, 81, 2223 ± 2236.
© Copyright 2025 Paperzz