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Members of the Family of IL-6-type Cytokines Activate Stat5a in Various Cell Types
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
236, 438–443 (1997)
RC976976
Members of the Family of IL-6-type Cytokines Activate Stat5a in Various Cell Types Roland P. Piekorz, Cordula Nemetz, and Gertrud M. Hocke1 Institute for Microbiology, Biochemistry and Genetics, University of Erlangen-Nu¨rnberg, Staudtstrasse 5, D-91058 Erlangen, Germany
Received June 6, 1997
Interleukin-6 (IL-6)-type cytokines activate transcription factors Stat1 and Stat3 (signal transducers and activators of transcription). Here we report that leukemia inhibitory factor (LIF) and IL-6 activate Stat5a in M1 myeloid leukemia cells in addition. In murine embryonal stem (ES) cells stably transfected with an expression vector for Stat5a treatment with LIF resulted in tyrosine phosphorylation and DNAbinding of this transcription factor. Transfection of an expression construct for Stat5a in human hepatoma cells caused a dose-dependent increase in LIF-triggered transcriptional activity. Our data demonstrate that Stat5a is activated by IL-6-type cytokines and can mediate transcriptional activity in addition to Stat1 and Stat3. q 1997 Academic Press
IL-6 and LIF belong to the family of IL-6-type cytokines which share several effects on various cell types by utilizing a common signal pathway (1). This so called Jak/Stat pathway involves tyrosine kinases of the family of Janus kinases (Jak) and transcription factors of the family of signal transducers and activators of transcription (Stat) (2, 3). Binding of the cytokine to specific receptor complexes leads to activation of receptor associated Jak kinases which phosphorylate cytoplasmic Stat factors at specific tyrosine residues. As a consequence, Stat factors dimerize and translocate to the nucleus to bind to specific Stat-binding sequences in the transcriptional control region of various target genes (2, 3). 1 Corresponding author. Fax. 0049-9131-858526. E-mail: ghocke@ biologie.uni-erlangen.de. Abbreviations: a2M, a2-macroglobulin; CHO, chinese hamster ovary; EMSA, electrophoretic mobility shift assay; EPO, erythropoietin; ES, embryonal stem; G-CSF, granulocyte-colony stimulating factor; GM-CSF, granulocyte macrophage-colony stimulating factor; IL6, interleukin-6; Jak, Janus kinase; LIF, leukemia inhibitory factor; MGF, mammary gland factor; RE, response element; Stat, signal transducer and activator of transcription.
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The promoter of the rat a2-macroglobulin (a2M) gene represents a bona fide IL-6-/LIF-response element (IL6-/LIF-RE) (4, 5). It confers transcriptional activity by IL-6-type cytokines not only in hepatic cells but also in embryonal stem (ES) cells (6). Its sequence represents a strong binding site for Stat3 and resembles the prolactin response element of the b-casein promoter, a binding site for Stat5, and the so called gamma-activating-site, a binding site for Stat1 (2, 7). A characteristic protein-DNA complex, referred to as complex II, was assembled between the IL-6/LIF-RE and nuclear proteins from various cell types after incubation of the cells with IL-6-type cytokines (4-6, 8). This complex contained activated Stat factors 1 and 3 in cytokine treated hepatic cells (9, 10) as well as in LIF-treated murine ES and embryonal carcinoma cells (6). In mice two highly related Stat5 genes exist encoding Stat5a and Stat5b. Stat5b was contained in nuclear extracts of rat liver cells additionally to Stat3 and was capable of binding to the IL-6-/LIF-RE (11). Stat5a/ MGF (mammary gland factor) was initially discovered as a prolactin-activated Stat factor in sheep mammary gland epithelial cells (12). Both forms can be activated in different cell types by various agents including cytokines of the IL-3-/IL-5/granulocyte macrophage-colony stimulating factor (GM-CSF)-family, thrombopoietin, erythropoietin (EPO), and growth hormone (13-16). In some cases preferential activation of one form of Stat5 was observed (16, 17). Activation of Stat5a was associated with differentiation processes in myeloid leukemic cells. Treatment of the human monoblastic leukemic cell line U-937 with GM-CSF caused activation of Stat5a and terminal differentiation (18). In addition, differentiation of U-937 cells was inducible by treatment with other agents such as interferons and phorbolester and in any case cor related with preferential activation of Stat5a (17). Recently a critical and causative role of the Jak/Stat signal pathway in differentiating M1 myeloid leukemic cells was shown. Induction of differentiation and growth arrest of M1 cells by treatment of the cells with
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IL-6-type cytokines was strictly dependent on activated Stat3, but not on Stat1 (19-21). So far a possible involvement of Stat5a was not investigated. Here we report that treatment of M1 cells with IL6-type cytokines caused activation and binding not only of Stats 1 and 3 but also of Stat5a to the IL-6-/LIF-RE, suggesting an additional role of Stat5a in induction of differentiation. Therefore the question arised whether activation of Stat5a is unique for M1 cells or a property of several cell types responsive to IL-6-type cytokines. MATERIALS AND METHODS Cell culture and reagents. M1 murine myelomonocytic leukemic cells (American Type Culture Collection) were grown in RPMI medium supplemented with 10% fetal calf serum (BioWhittaker, Boehringer Ingelheim, Heidelberg, Germany), 2 mM L-glutamine, 100 units/ml penicillin and 100 mg/ml streptomycin. ES cells and the human hepatoma cell line HepG2 were cultured as described (4, 6). The supernatant of a stably transfected CHO cell line secreting recombinant human LIF (Genetics Institute, Cambridge, MA) was used as a source of human LIF. Cultured cells were treated with this supernatant at a dilution of 1:100 for M1 and HepG2 cells and 1:500 for ES cells. Human recombinant IL-6 was used at a concentration of 50 units/ml (Genetics Institute, Cambridge, MA). Stat1a- and Stat5a-specific antibodies (15, 22) were kindly provided by Dr. J. Darnell (New York, NY) and by Dr. B. Groner (Freiburg, Germany) and Dr. J. Ihle (Memphis, TN), respectively. Antibodies against Stat3 (C-20), Stat5b (C-17) and control antibodies for EMSA-supershift were from Santa Cruz Biotechnology Inc. (Heidelberg, Germany). Antibodies recognizing both Stat5a and Stat5b were obtained from Transduction Laboratories (Lexington, KY). Anti-phospho tyrosine antibodies used were either pY20 (Santa Cruz Biotechnology Inc., Heidelberg, Germany) or 4G10 (UBI, Biotechnologies, Lake Placid, NY). Preparation of protein extracts. Nuclear protein and whole cell extracts were prepared according to published procedures (4, 23). M1 cells were starved in medium with 0.5% fetal calf serum for 14 h prior to stimulation with cytokines. ES cells were kept without LIF for 12 h prior to incubation with LIF for 15 min. Electrophoretic mobility shift assay (EMSA). EMSA was performed following published procedures with the radiolabeled oligonucleotide TB2 containing a dimer of a Stat-binding site (4). EMSAsupershift analysis was performed by preincubation of nuclear extracts with antibodies for 1 h prior to DNA-binding. Immunoprecipitation and Western blotting. Stat proteins were immunoprecipitated from whole cell extracts using specific antibodies and analysed by SDS-PAGE and Western blotting. Extracts from COS-1 cells transfected with the cDNAs for either Stat5a or Stat5b were provided by Dr. J. Ripperger (Erlangen, Germany). Plasmid construction, transfection and reporter assays. The cDNA encoding Stat5a/MGF was provided by Dr. B. Groner (Freiburg, Germany) (12) and cloned into the expression vector pSVSPORT-1 (Gibco BRL, Eggenstein, Germany). ES1 cells were cotransfected with the Stat5a expression construct (pSPORT-Stat5a) and pRSVneo in a 10:1 ratio by electroporation. Single cell clones were isolated by limiting dilution in 300 mg/ml G418 in the presence of LIF. For transient transfection assays, HepG2 hepatoma cells were transfected by the calcium phosphate procedure essentially as described (4). The construct p(Stat5-RE)6-TKLuc containing 6 copies of the Stat5 binding site of the b-casein promoter was used as reporter construct (24), and the galactosidase construct pBGeo (Gibco BRL, Eggenstein, Germany) as internal control. Each transfection was performed with 10 mg p(Stat5-RE)6-TKLuc, 2 mg pBGeo, and 5, 10 or
15 mg of pSport-Stat5a, equalizing the DNA quantities with salmon sperm DNA to 27 mg. Relative light units of luciferase and b-galactosidase activities were standardized to an equal volume of 1 ml. Transfection was performed in duplicate and repeated with similar results.
RESULTS Treatment of M1 cells with LIF or IL-6 activates DNA-binding of Stat factors 1, 3 and 5a. Treatment of M1 cells with LIF or IL-6 led to the formation of a characteristic protein-DNA complex, complex II (Fig. 1A, tracks 4 and 5). EMSA-supershift studies revealed the involvement of Stat proteins 1, 3 and 5a in the formation of complex II (Fig. 1B). However, Stat5b was not contained in this protein-DNA complex (Fig. 1B, last two tracks). No differences in the composition of complex II after treatment with LIF or IL-6 were detected (data not shown). Thus, Stat factors 1, 3 and 5a bound to the IL-6-/LIF-RE upon stimulation of M1 cells with IL-6-type cytokines. The amounts of tyrosine phosphorylated Stat factors increase after treatment of M1 cells with LIF or IL-6. Stat factors are activated by tyrosine phosphorylation prior to nuclear translocation and DNA-binding (2, 3). Therefore the status of tyrosine phosphorylation of Stat factors was investigated. Stimulated cells contained clearly increased amounts of tyrosine phosphorylated Stat3 and Stat5a compared to untreated cells (Fig. 2A). Interestingly, although Stat5b was detectable in M1 cells (Fig. 2B, lower panels, tracks 3-6), the level of tyrosine phosphorylation was very low and did not increase upon LIF- or IL-6-treatment of the cells (Fig. 2B, upper panel, tracks 4-6). These data confirmed that Stat factors capable of binding to DNA (Fig. 1B) were tyrosine phosphorylated and activated by treatment of M1 cells with LIF or IL-6. The lack of increase in tyrosine phosphorylation of Stat5b after cytokine treatment of M1 cells explains the missing detection of Stat5b in the assembly of the specific protein-DNA complex. The finding that treatment of M1 cells with LIF or IL-6 leads to activation of Stat5a is in contrast to observations of other authors. In their system activation of Stat5a was not inducible via the signal transducer chain gp130 (25). To prove that cytokine induced activation of Stat5a in M1 cells was not a property of this particular cell line other cell types responsive to IL-6-type cytokines were investigated. Exogenous Stat5a is activated by treatment of embryonal stem cells with LIF. ES cells are derived from the inner cell mass of preimplantation mouse embryos and can be cultured in an undifferentiated state in the presence of LIF. We have shown previously that ES cells proliferating by treatment with LIF contain only activated Stat1 and Stat3 (6). These cells contain no detectable amount of mRNA of Stat5 (C. Nemetz, unpublished results). Therefore ES cells were stably transfected with an expression construct coding for
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phosphorylated Stat5a was analysed by EMSA. Preincubation of nuclear extracts of LIF-treated stably transfected ES cells with specific Stat5a antibodies revealed a supershifted band similar to the one of LIFtreated M1 cells (Fig. 3B). Therefore upon stimulation with LIF exogenous Stat5a was tyrosine phosphorylated and was capable to bind DNA. In addition, Stat5a was able to confer transcriptional activity. Three independent experiments revealed at least a 1.4-fold higher LIF-inducible reporter activity in ZK21 cells compared to wildtype ES cells. A total of seven independent
FIG. 1. (A) The oligonucleotide TB2, representing an IL-6-/LIFRE, was used as a probe for EMSA-studies and incubated with buffer alone (1) or with nuclear extracts from: (2) ES cells, treated with LIF; (3) M1 cells, untreated; (4) M1 cells treated with LIF; (5) M1 cells treated with IL-6. II, complex II; U, unspecific complex; F, free probe. (B) Nuclear extracts of LIF-treated M1 cells either were used without antibodies (1) or were preincubated with control antibodies (2) or antibodies specific for the indicated Stat factors (3) and analyzed by EMSA. S, supershifted complexes.
Stat5a. Several clones expressing Stat5a protein were collected and analyzed. Stat5a was immunoprecipitated from clone ZK21 cells grown under various conditions (Fig. 3A). Tyrosine phosphorylation was strictly dependent on incubation of the cells with LIF. In untreated cells tyrosine phosphorylation was hardly detectable (Fig. 3A, track 2) but increased strongly after LIF-treatment (track 3), demonstrating a cytokine dependent phos phorylation process. Subsequently the DNA-binding capacity of tyrosine
FIG. 2. (A) Stat3 and Stat5a from untreated M1 cells (1) or cells stimulated with LIF (2) or IL-6 (3) for 15 min were immunoprecipitated (IP) and analyzed by Western blotting. (B) Stat5b from untreated M1 cells (4) and cells treated with LIF (5) or IL-6 (6) were immunoprecipitated with anti-Stat5b antibodies and Western blots were prepared. Tracks (1)–(2): Whole cell extracts of COS-1 cells transfected with the cDNAs for Stat5a (1) or Stat5b (2) as controls for the specificity of the antibodies. Track (3): Whole cell extract of M1 cells. The arrow in the upper panel indicates the signal corresponding to Stat5b. Membranes were reacted with anti-phosphotyrosine antibodies (pY20; P-Tyr) and with anti-Stat5 antibodies as indicated. IgH, Immunoglobulin heavy chains.
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FIG. 3. (A) Stat5a from ES cells stably transfected with the cDNA for Stat5a was immunoprecipitated (IP) and analyzed by Western blotting. Cells were grown in the continuous presence of LIF (1) or deprived of LIF for 12 h and harvested without (2) or after restimulation with LIF for 15 min (3). The membrane was incubated with anti-phosphotyrosine antibodies (4G10; P-Tyr) and with antiStat5a antibodies as loading control. IgH, immunoglobulin heavy chains. (B) EMSA-supershift of nuclear extracts from LIF-treated M1 cells (1,2) and LIF-treated ES cells stably transfected with the cDNA for Stat5a (3,4). (1,3) no antibody treatment; (2,4) treatment with anti-Stat5a antibodies. II, complex II; S, supershifted complexes.
clones was screened and five of them showed similar transcriptional activities as clone ZK21 (C. Nemetz, manuscript in preparation). From these data we concluded that exogenous Stat5a was not only tyrosine phosphorylated after treatment of the cells with LIF but was also able to induce transcriptional activity. This effect was verified in a different cell system. Stat5a confers transcriptional activity in HepG2 human hepatoma cells. Human hepatoma cells contain only activated Stat1 and Stat3 after incubation with LIF or IL-6 ((10) and G. Hocke, unpublished data). In addition, although the cells display marginal amounts of both Stat5a and Stat5b, these factors were not activated upon cytokine treatment (C. Nemetz and G.
Hocke, unpublished observation). HepG2 cells were cotransfected with the expression construct pSPORTStat5a and the reporter construct p(Stat5-RE)6-TKLuc containing 6 copies of a preferentially binding site for Stat5 (24). A construct containing the b-galactosidase gene was used as internal control. HepG2 cells were transfected with increasing amounts of expression vector. Luciferase activities were measured in cells without and after treatment with LIF for 4 h and values were adjusted to b-galactosidase activity. The relative inductions of reporter activities in LIF-treated wildtype HepG2 cells and HepG2 cells transfected with cDNA for Stat5a were calculated (Table 1). Although the cells responded with an increase in transcriptional activity after treatment with LIF even in the absence of exogenous Stat5a, the response was significantly higher in cells cotransfected with the expression construct for Stat5a. The increase of transcriptional activities correlated in a dose-dependent manner with the amount of exogenous Stat5a (Table 1). Simultaneously galactosidase activity increased from 118 light units in HepG2 cells transfected without Stat5a cDNA to 4063 light units in cells transfected with 15 mg cDNA for Stat5a. We currently can not explain this remarkably transcriptional activitation. Despite of this general transcriptional activity the values of relative induction were clearly increased by treatment of the cells with LIF. The LIF-induced relative induction was 3.7 in cells transfected with 5 mg Stat5a cDNA and 6.1 in cells transfected with 15 mg of the expression construct (Table 1). Therefore, HepG2 cells transfected with 15 mg Stat5a cDNA expressed a 2.7-fold higher LIF-induced response than HepG2 cells missing exogenous Stat5a (Fig. 4). This finding demonstrated that Stat5a was also activated in hepatoma cells after treatment of the cells with LIF. In summary, Stat5a can be activated to bind DNA and to confer transcriptional activity in several cells types responsive to IL-6-types cytokines.
TABLE 1
Stat5a (mg)
0 5 10 15
LIF
Luc
Gal
Relative activity (Luc/Gal)
0 / 0 / 0 / 0 /
1587 4458 1473 5517 3009 15028 5659 39683
118 144 225 229 904 894 4063 4637
13.4 30.9 6.5 24.1 3.3 16.8 1.4 8.6
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Note. HepG2 cells were cotransfected with a luciferase reporter construct containing binding sites for Stat5, a b-galactosidase construct as internal control, and increasing amounts of an expression vector for Stat5a. Relative induction was defined as cytokine-induced relative activity divided by the activity of untreated cells.
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FIG. 4. Transfection of HepG2 cells with increasing amounts of Stat5a leads to a dose-dependent increase in LIF-induced transcriptional activity. HepG2 cells were transfected and transcriptional activity was measured as described in Table 1. Relative increases were defined as LIF-induced activity of HepG2 cells cotransfected with an expression construct for Stat5a divided by activity of HepG2 cells without exogenous Stat5a. The ordinate shows the relative increases in multiples of the values obtained for HepG2 cells without exogenous Stat5a.
DISCUSSION The main conclusions drawn from the presented data were: 1) Treatment of M1 myeloid leukemic cells with LIF or IL-6 led to activation and DNA-binding of Stat factors 1, 3 and 5a. 2) LIF-treatment of embryonal stem cells stably transfected with a cDNA for Stat5a caused tyrosine phosphorylation and DNA-binding of this factor. 3) LIF-treatment of hepa toma cells transfected with a cDNA for Stat5a led to a dose-dependent increase of transcriptional activity. Various cell types respond to treatment with IL-6type cytokines with activation of transcription factors Stat1 and Stat3 (6, 10). DNA-binding of Stat5b to the IL-6/LIF-RE occured in rat liver cells after induction of an acute phase response which is mainly triggered by IL-6 (11). In accordance with this finding exogenous Stat5b confered transcriptional activation in COS-1 cells using chimeric receptors consisting of the extracellular domain of the receptor for granulocyte-colony stimulating factor (G-CSF) and the intracellular domain of gp130 (26). Contradictory to these results transfection studies in COS-7 cells with chimeric receptors containing modulations of the intracellular domain of gp130 led to the conclusion that the common receptor chain gp130 is not able to transduce the cytokine signal to DNA-binding of Stat5a (25). However, the authors observed a weak Stat5a activation after exchange of
the box 3 motif of gp130 by a tyrosine module of the ligand binding chain of the LIF-receptor. We studied activation of Stat5 in the myelomonocytic cell line M1, which expresses high levels of most of the components of the Jak/Stat pathway (27). Treatment of these cells with LIF or IL-6 caused tyrosine phosphorylation and DNA-binding not only of Stat1 and Stat3, but in addition of Stat5a (Fig. 1B and 2). Interestingly, the amount of tyrosine phosphorylated Stat5b was not increased (Fig. 2B), similar to treatment of M1 cells with G-CSF (28). The G-CSF-receptor displays homology to gp130 (29) and should activate the Jak/ Stat pathway in a similar way. Mutated forms of this receptor were analyzed in M1 cells. Even in the absence of any activated tyrosine modules in the intracellular part Stat5 was activated to bind DNA (28). Therefore activation of Stat5 may not only be triggered by tyrosine phosphorylation directly via gp130, but may depend alternatively on the presence of adaptor protein(s), which ‘‘crosstalk’’ between gp130, Jak-kinase(s) and Stat5. In line with this assumption it was recently demonstrated that a direct interaction of Jak kinases and Stat5 resulted in activation of Stat5 (30). Activation of Stat5a can therefore be reached by diverse mechanisms ((15, 30), this work). In two other cell types which contain a complete Jak/ Stat cascade but no activated Stat5a the signaling pathway under study was capable to activate exogenous Stat5a. Treatment of the cells with LIF led to tyrosine phosphorylation and DNA-binding of exogenous Stat5a causing modulation of transcriptional activity (Tab. 1). These findings suggest that cells containing the important components of the Jak/Stat signal cascade at sufficient levels are able to activate Stat5a after cytokine treatment. In summary, despite of the critical role of the Jak/ Stat signal pathway in general, alterations in the spectrum of activated Stat factors may not only be correlated with, but responsible for some of the events triggered by treatment of various cells with IL-6-type cytokines. ACKNOWLEDGMENTS The expert technical assistance of Brigitte Go¨pfert is gratefully acknowledged. This work was supported by Research Grants Deutsche Forschungsgemeinschaft (DFG) Hi 291/5-2 TP6 and Thyssen Stiftung (awarded to G.M.H.). R.P.P. and C.N. were recipients of graduate student fellowships from the Graduiertenkolleg ‘‘RNASynthese’’ supported by the DFG.
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Members of the Family of IL-6-type Cytokines Activate Stat5a in Various Cell Types Roland P. Piekorz, Cordula Nemetz, and Gertrud M. Hocke1 Institute for Microbiology, Biochemistry and Genetics, University of Erlangen-Nu¨rnberg, Staudtstrasse 5, D-91058 Erlangen, Germany
Received June 6, 1997
Interleukin-6 (IL-6)-type cytokines activate transcription factors Stat1 and Stat3 (signal transducers and activators of transcription). Here we report that leukemia inhibitory factor (LIF) and IL-6 activate Stat5a in M1 myeloid leukemia cells in addition. In murine embryonal stem (ES) cells stably transfected with an expression vector for Stat5a treatment with LIF resulted in tyrosine phosphorylation and DNAbinding of this transcription factor. Transfection of an expression construct for Stat5a in human hepatoma cells caused a dose-dependent increase in LIF-triggered transcriptional activity. Our data demonstrate that Stat5a is activated by IL-6-type cytokines and can mediate transcriptional activity in addition to Stat1 and Stat3. q 1997 Academic Press
IL-6 and LIF belong to the family of IL-6-type cytokines which share several effects on various cell types by utilizing a common signal pathway (1). This so called Jak/Stat pathway involves tyrosine kinases of the family of Janus kinases (Jak) and transcription factors of the family of signal transducers and activators of transcription (Stat) (2, 3). Binding of the cytokine to specific receptor complexes leads to activation of receptor associated Jak kinases which phosphorylate cytoplasmic Stat factors at specific tyrosine residues. As a consequence, Stat factors dimerize and translocate to the nucleus to bind to specific Stat-binding sequences in the transcriptional control region of various target genes (2, 3). 1 Corresponding author. Fax. 0049-9131-858526. E-mail: ghocke@ biologie.uni-erlangen.de. Abbreviations: a2M, a2-macroglobulin; CHO, chinese hamster ovary; EMSA, electrophoretic mobility shift assay; EPO, erythropoietin; ES, embryonal stem; G-CSF, granulocyte-colony stimulating factor; GM-CSF, granulocyte macrophage-colony stimulating factor; IL6, interleukin-6; Jak, Janus kinase; LIF, leukemia inhibitory factor; MGF, mammary gland factor; RE, response element; Stat, signal transducer and activator of transcription.
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The promoter of the rat a2-macroglobulin (a2M) gene represents a bona fide IL-6-/LIF-response element (IL6-/LIF-RE) (4, 5). It confers transcriptional activity by IL-6-type cytokines not only in hepatic cells but also in embryonal stem (ES) cells (6). Its sequence represents a strong binding site for Stat3 and resembles the prolactin response element of the b-casein promoter, a binding site for Stat5, and the so called gamma-activating-site, a binding site for Stat1 (2, 7). A characteristic protein-DNA complex, referred to as complex II, was assembled between the IL-6/LIF-RE and nuclear proteins from various cell types after incubation of the cells with IL-6-type cytokines (4-6, 8). This complex contained activated Stat factors 1 and 3 in cytokine treated hepatic cells (9, 10) as well as in LIF-treated murine ES and embryonal carcinoma cells (6). In mice two highly related Stat5 genes exist encoding Stat5a and Stat5b. Stat5b was contained in nuclear extracts of rat liver cells additionally to Stat3 and was capable of binding to the IL-6-/LIF-RE (11). Stat5a/ MGF (mammary gland factor) was initially discovered as a prolactin-activated Stat factor in sheep mammary gland epithelial cells (12). Both forms can be activated in different cell types by various agents including cytokines of the IL-3-/IL-5/granulocyte macrophage-colony stimulating factor (GM-CSF)-family, thrombopoietin, erythropoietin (EPO), and growth hormone (13-16). In some cases preferential activation of one form of Stat5 was observed (16, 17). Activation of Stat5a was associated with differentiation processes in myeloid leukemic cells. Treatment of the human monoblastic leukemic cell line U-937 with GM-CSF caused activation of Stat5a and terminal differentiation (18). In addition, differentiation of U-937 cells was inducible by treatment with other agents such as interferons and phorbolester and in any case cor related with preferential activation of Stat5a (17). Recently a critical and causative role of the Jak/Stat signal pathway in differentiating M1 myeloid leukemic cells was shown. Induction of differentiation and growth arrest of M1 cells by treatment of the cells with
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IL-6-type cytokines was strictly dependent on activated Stat3, but not on Stat1 (19-21). So far a possible involvement of Stat5a was not investigated. Here we report that treatment of M1 cells with IL6-type cytokines caused activation and binding not only of Stats 1 and 3 but also of Stat5a to the IL-6-/LIF-RE, suggesting an additional role of Stat5a in induction of differentiation. Therefore the question arised whether activation of Stat5a is unique for M1 cells or a property of several cell types responsive to IL-6-type cytokines. MATERIALS AND METHODS Cell culture and reagents. M1 murine myelomonocytic leukemic cells (American Type Culture Collection) were grown in RPMI medium supplemented with 10% fetal calf serum (BioWhittaker, Boehringer Ingelheim, Heidelberg, Germany), 2 mM L-glutamine, 100 units/ml penicillin and 100 mg/ml streptomycin. ES cells and the human hepatoma cell line HepG2 were cultured as described (4, 6). The supernatant of a stably transfected CHO cell line secreting recombinant human LIF (Genetics Institute, Cambridge, MA) was used as a source of human LIF. Cultured cells were treated with this supernatant at a dilution of 1:100 for M1 and HepG2 cells and 1:500 for ES cells. Human recombinant IL-6 was used at a concentration of 50 units/ml (Genetics Institute, Cambridge, MA). Stat1a- and Stat5a-specific antibodies (15, 22) were kindly provided by Dr. J. Darnell (New York, NY) and by Dr. B. Groner (Freiburg, Germany) and Dr. J. Ihle (Memphis, TN), respectively. Antibodies against Stat3 (C-20), Stat5b (C-17) and control antibodies for EMSA-supershift were from Santa Cruz Biotechnology Inc. (Heidelberg, Germany). Antibodies recognizing both Stat5a and Stat5b were obtained from Transduction Laboratories (Lexington, KY). Anti-phospho tyrosine antibodies used were either pY20 (Santa Cruz Biotechnology Inc., Heidelberg, Germany) or 4G10 (UBI, Biotechnologies, Lake Placid, NY). Preparation of protein extracts. Nuclear protein and whole cell extracts were prepared according to published procedures (4, 23). M1 cells were starved in medium with 0.5% fetal calf serum for 14 h prior to stimulation with cytokines. ES cells were kept without LIF for 12 h prior to incubation with LIF for 15 min. Electrophoretic mobility shift assay (EMSA). EMSA was performed following published procedures with the radiolabeled oligonucleotide TB2 containing a dimer of a Stat-binding site (4). EMSAsupershift analysis was performed by preincubation of nuclear extracts with antibodies for 1 h prior to DNA-binding. Immunoprecipitation and Western blotting. Stat proteins were immunoprecipitated from whole cell extracts using specific antibodies and analysed by SDS-PAGE and Western blotting. Extracts from COS-1 cells transfected with the cDNAs for either Stat5a or Stat5b were provided by Dr. J. Ripperger (Erlangen, Germany). Plasmid construction, transfection and reporter assays. The cDNA encoding Stat5a/MGF was provided by Dr. B. Groner (Freiburg, Germany) (12) and cloned into the expression vector pSVSPORT-1 (Gibco BRL, Eggenstein, Germany). ES1 cells were cotransfected with the Stat5a expression construct (pSPORT-Stat5a) and pRSVneo in a 10:1 ratio by electroporation. Single cell clones were isolated by limiting dilution in 300 mg/ml G418 in the presence of LIF. For transient transfection assays, HepG2 hepatoma cells were transfected by the calcium phosphate procedure essentially as described (4). The construct p(Stat5-RE)6-TKLuc containing 6 copies of the Stat5 binding site of the b-casein promoter was used as reporter construct (24), and the galactosidase construct pBGeo (Gibco BRL, Eggenstein, Germany) as internal control. Each transfection was performed with 10 mg p(Stat5-RE)6-TKLuc, 2 mg pBGeo, and 5, 10 or
15 mg of pSport-Stat5a, equalizing the DNA quantities with salmon sperm DNA to 27 mg. Relative light units of luciferase and b-galactosidase activities were standardized to an equal volume of 1 ml. Transfection was performed in duplicate and repeated with similar results.
RESULTS Treatment of M1 cells with LIF or IL-6 activates DNA-binding of Stat factors 1, 3 and 5a. Treatment of M1 cells with LIF or IL-6 led to the formation of a characteristic protein-DNA complex, complex II (Fig. 1A, tracks 4 and 5). EMSA-supershift studies revealed the involvement of Stat proteins 1, 3 and 5a in the formation of complex II (Fig. 1B). However, Stat5b was not contained in this protein-DNA complex (Fig. 1B, last two tracks). No differences in the composition of complex II after treatment with LIF or IL-6 were detected (data not shown). Thus, Stat factors 1, 3 and 5a bound to the IL-6-/LIF-RE upon stimulation of M1 cells with IL-6-type cytokines. The amounts of tyrosine phosphorylated Stat factors increase after treatment of M1 cells with LIF or IL-6. Stat factors are activated by tyrosine phosphorylation prior to nuclear translocation and DNA-binding (2, 3). Therefore the status of tyrosine phosphorylation of Stat factors was investigated. Stimulated cells contained clearly increased amounts of tyrosine phosphorylated Stat3 and Stat5a compared to untreated cells (Fig. 2A). Interestingly, although Stat5b was detectable in M1 cells (Fig. 2B, lower panels, tracks 3-6), the level of tyrosine phosphorylation was very low and did not increase upon LIF- or IL-6-treatment of the cells (Fig. 2B, upper panel, tracks 4-6). These data confirmed that Stat factors capable of binding to DNA (Fig. 1B) were tyrosine phosphorylated and activated by treatment of M1 cells with LIF or IL-6. The lack of increase in tyrosine phosphorylation of Stat5b after cytokine treatment of M1 cells explains the missing detection of Stat5b in the assembly of the specific protein-DNA complex. The finding that treatment of M1 cells with LIF or IL-6 leads to activation of Stat5a is in contrast to observations of other authors. In their system activation of Stat5a was not inducible via the signal transducer chain gp130 (25). To prove that cytokine induced activation of Stat5a in M1 cells was not a property of this particular cell line other cell types responsive to IL-6-type cytokines were investigated. Exogenous Stat5a is activated by treatment of embryonal stem cells with LIF. ES cells are derived from the inner cell mass of preimplantation mouse embryos and can be cultured in an undifferentiated state in the presence of LIF. We have shown previously that ES cells proliferating by treatment with LIF contain only activated Stat1 and Stat3 (6). These cells contain no detectable amount of mRNA of Stat5 (C. Nemetz, unpublished results). Therefore ES cells were stably transfected with an expression construct coding for
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phosphorylated Stat5a was analysed by EMSA. Preincubation of nuclear extracts of LIF-treated stably transfected ES cells with specific Stat5a antibodies revealed a supershifted band similar to the one of LIFtreated M1 cells (Fig. 3B). Therefore upon stimulation with LIF exogenous Stat5a was tyrosine phosphorylated and was capable to bind DNA. In addition, Stat5a was able to confer transcriptional activity. Three independent experiments revealed at least a 1.4-fold higher LIF-inducible reporter activity in ZK21 cells compared to wildtype ES cells. A total of seven independent
FIG. 1. (A) The oligonucleotide TB2, representing an IL-6-/LIFRE, was used as a probe for EMSA-studies and incubated with buffer alone (1) or with nuclear extracts from: (2) ES cells, treated with LIF; (3) M1 cells, untreated; (4) M1 cells treated with LIF; (5) M1 cells treated with IL-6. II, complex II; U, unspecific complex; F, free probe. (B) Nuclear extracts of LIF-treated M1 cells either were used without antibodies (1) or were preincubated with control antibodies (2) or antibodies specific for the indicated Stat factors (3) and analyzed by EMSA. S, supershifted complexes.
Stat5a. Several clones expressing Stat5a protein were collected and analyzed. Stat5a was immunoprecipitated from clone ZK21 cells grown under various conditions (Fig. 3A). Tyrosine phosphorylation was strictly dependent on incubation of the cells with LIF. In untreated cells tyrosine phosphorylation was hardly detectable (Fig. 3A, track 2) but increased strongly after LIF-treatment (track 3), demonstrating a cytokine dependent phos phorylation process. Subsequently the DNA-binding capacity of tyrosine
FIG. 2. (A) Stat3 and Stat5a from untreated M1 cells (1) or cells stimulated with LIF (2) or IL-6 (3) for 15 min were immunoprecipitated (IP) and analyzed by Western blotting. (B) Stat5b from untreated M1 cells (4) and cells treated with LIF (5) or IL-6 (6) were immunoprecipitated with anti-Stat5b antibodies and Western blots were prepared. Tracks (1)–(2): Whole cell extracts of COS-1 cells transfected with the cDNAs for Stat5a (1) or Stat5b (2) as controls for the specificity of the antibodies. Track (3): Whole cell extract of M1 cells. The arrow in the upper panel indicates the signal corresponding to Stat5b. Membranes were reacted with anti-phosphotyrosine antibodies (pY20; P-Tyr) and with anti-Stat5 antibodies as indicated. IgH, Immunoglobulin heavy chains.
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FIG. 3. (A) Stat5a from ES cells stably transfected with the cDNA for Stat5a was immunoprecipitated (IP) and analyzed by Western blotting. Cells were grown in the continuous presence of LIF (1) or deprived of LIF for 12 h and harvested without (2) or after restimulation with LIF for 15 min (3). The membrane was incubated with anti-phosphotyrosine antibodies (4G10; P-Tyr) and with antiStat5a antibodies as loading control. IgH, immunoglobulin heavy chains. (B) EMSA-supershift of nuclear extracts from LIF-treated M1 cells (1,2) and LIF-treated ES cells stably transfected with the cDNA for Stat5a (3,4). (1,3) no antibody treatment; (2,4) treatment with anti-Stat5a antibodies. II, complex II; S, supershifted complexes.
clones was screened and five of them showed similar transcriptional activities as clone ZK21 (C. Nemetz, manuscript in preparation). From these data we concluded that exogenous Stat5a was not only tyrosine phosphorylated after treatment of the cells with LIF but was also able to induce transcriptional activity. This effect was verified in a different cell system. Stat5a confers transcriptional activity in HepG2 human hepatoma cells. Human hepatoma cells contain only activated Stat1 and Stat3 after incubation with LIF or IL-6 ((10) and G. Hocke, unpublished data). In addition, although the cells display marginal amounts of both Stat5a and Stat5b, these factors were not activated upon cytokine treatment (C. Nemetz and G.
Hocke, unpublished observation). HepG2 cells were cotransfected with the expression construct pSPORTStat5a and the reporter construct p(Stat5-RE)6-TKLuc containing 6 copies of a preferentially binding site for Stat5 (24). A construct containing the b-galactosidase gene was used as internal control. HepG2 cells were transfected with increasing amounts of expression vector. Luciferase activities were measured in cells without and after treatment with LIF for 4 h and values were adjusted to b-galactosidase activity. The relative inductions of reporter activities in LIF-treated wildtype HepG2 cells and HepG2 cells transfected with cDNA for Stat5a were calculated (Table 1). Although the cells responded with an increase in transcriptional activity after treatment with LIF even in the absence of exogenous Stat5a, the response was significantly higher in cells cotransfected with the expression construct for Stat5a. The increase of transcriptional activities correlated in a dose-dependent manner with the amount of exogenous Stat5a (Table 1). Simultaneously galactosidase activity increased from 118 light units in HepG2 cells transfected without Stat5a cDNA to 4063 light units in cells transfected with 15 mg cDNA for Stat5a. We currently can not explain this remarkably transcriptional activitation. Despite of this general transcriptional activity the values of relative induction were clearly increased by treatment of the cells with LIF. The LIF-induced relative induction was 3.7 in cells transfected with 5 mg Stat5a cDNA and 6.1 in cells transfected with 15 mg of the expression construct (Table 1). Therefore, HepG2 cells transfected with 15 mg Stat5a cDNA expressed a 2.7-fold higher LIF-induced response than HepG2 cells missing exogenous Stat5a (Fig. 4). This finding demonstrated that Stat5a was also activated in hepatoma cells after treatment of the cells with LIF. In summary, Stat5a can be activated to bind DNA and to confer transcriptional activity in several cells types responsive to IL-6-types cytokines.
TABLE 1
Stat5a (mg)
0 5 10 15
LIF
Luc
Gal
Relative activity (Luc/Gal)
0 / 0 / 0 / 0 /
1587 4458 1473 5517 3009 15028 5659 39683
118 144 225 229 904 894 4063 4637
13.4 30.9 6.5 24.1 3.3 16.8 1.4 8.6
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Note. HepG2 cells were cotransfected with a luciferase reporter construct containing binding sites for Stat5, a b-galactosidase construct as internal control, and increasing amounts of an expression vector for Stat5a. Relative induction was defined as cytokine-induced relative activity divided by the activity of untreated cells.
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FIG. 4. Transfection of HepG2 cells with increasing amounts of Stat5a leads to a dose-dependent increase in LIF-induced transcriptional activity. HepG2 cells were transfected and transcriptional activity was measured as described in Table 1. Relative increases were defined as LIF-induced activity of HepG2 cells cotransfected with an expression construct for Stat5a divided by activity of HepG2 cells without exogenous Stat5a. The ordinate shows the relative increases in multiples of the values obtained for HepG2 cells without exogenous Stat5a.
DISCUSSION The main conclusions drawn from the presented data were: 1) Treatment of M1 myeloid leukemic cells with LIF or IL-6 led to activation and DNA-binding of Stat factors 1, 3 and 5a. 2) LIF-treatment of embryonal stem cells stably transfected with a cDNA for Stat5a caused tyrosine phosphorylation and DNA-binding of this factor. 3) LIF-treatment of hepa toma cells transfected with a cDNA for Stat5a led to a dose-dependent increase of transcriptional activity. Various cell types respond to treatment with IL-6type cytokines with activation of transcription factors Stat1 and Stat3 (6, 10). DNA-binding of Stat5b to the IL-6/LIF-RE occured in rat liver cells after induction of an acute phase response which is mainly triggered by IL-6 (11). In accordance with this finding exogenous Stat5b confered transcriptional activation in COS-1 cells using chimeric receptors consisting of the extracellular domain of the receptor for granulocyte-colony stimulating factor (G-CSF) and the intracellular domain of gp130 (26). Contradictory to these results transfection studies in COS-7 cells with chimeric receptors containing modulations of the intracellular domain of gp130 led to the conclusion that the common receptor chain gp130 is not able to transduce the cytokine signal to DNA-binding of Stat5a (25). However, the authors observed a weak Stat5a activation after exchange of
the box 3 motif of gp130 by a tyrosine module of the ligand binding chain of the LIF-receptor. We studied activation of Stat5 in the myelomonocytic cell line M1, which expresses high levels of most of the components of the Jak/Stat pathway (27). Treatment of these cells with LIF or IL-6 caused tyrosine phosphorylation and DNA-binding not only of Stat1 and Stat3, but in addition of Stat5a (Fig. 1B and 2). Interestingly, the amount of tyrosine phosphorylated Stat5b was not increased (Fig. 2B), similar to treatment of M1 cells with G-CSF (28). The G-CSF-receptor displays homology to gp130 (29) and should activate the Jak/ Stat pathway in a similar way. Mutated forms of this receptor were analyzed in M1 cells. Even in the absence of any activated tyrosine modules in the intracellular part Stat5 was activated to bind DNA (28). Therefore activation of Stat5 may not only be triggered by tyrosine phosphorylation directly via gp130, but may depend alternatively on the presence of adaptor protein(s), which ‘‘crosstalk’’ between gp130, Jak-kinase(s) and Stat5. In line with this assumption it was recently demonstrated that a direct interaction of Jak kinases and Stat5 resulted in activation of Stat5 (30). Activation of Stat5a can therefore be reached by diverse mechanisms ((15, 30), this work). In two other cell types which contain a complete Jak/ Stat cascade but no activated Stat5a the signaling pathway under study was capable to activate exogenous Stat5a. Treatment of the cells with LIF led to tyrosine phosphorylation and DNA-binding of exogenous Stat5a causing modulation of transcriptional activity (Tab. 1). These findings suggest that cells containing the important components of the Jak/Stat signal cascade at sufficient levels are able to activate Stat5a after cytokine treatment. In summary, despite of the critical role of the Jak/ Stat signal pathway in general, alterations in the spectrum of activated Stat factors may not only be correlated with, but responsible for some of the events triggered by treatment of various cells with IL-6-type cytokines. ACKNOWLEDGMENTS The expert technical assistance of Brigitte Go¨pfert is gratefully acknowledged. This work was supported by Research Grants Deutsche Forschungsgemeinschaft (DFG) Hi 291/5-2 TP6 and Thyssen Stiftung (awarded to G.M.H.). R.P.P. and C.N. were recipients of graduate student fellowships from the Graduiertenkolleg ‘‘RNASynthese’’ supported by the DFG.
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