This action might not be possible to undo. Are you sure you want to continue?
ISSN 0898-6568/99 $–see front matter PII S0898-6568(98)00049-7
Role of STAT5 in InterferonSignal Transduction in Ba/F3 Cells
Robert Jaster,* Edda Tschirch, Thomas Bittorf and Josef Brock
Institute of Medical Biochemistry, Medical Faculty of the University Rostock, Schillingallee 70, 18057 Rostock, Germany
ABSTRACT. In interferon- (IFN- ) signalling, the essential role of the transcription factors STAT1 and STAT2 is well established. In contrast, the involvement of other STAT proteins, including STAT5, is much less well understood. Here we show that, in IFN- -responsive Ba/F3 cells, this cytokine stimulates the DNAbinding of STAT5A and B but that IL-3 is a much more potent activator of both STAT5 isoforms. A stably expressed dominant-negative mutant of JAK2 suppressed the IL-3- but not the IFN- -dependent DNA binding of STAT5, suggesting independent mechanisms of its activation. Northern blots revealed that IL-3 strongly induced the expression of two STAT5-regulated genes, pim-1 and oncostatin-M, whereas IFN- had a weak stimulatory effect on pim-1 expression only. In summary, our results suggest that, despite the capability of IFN- to stimulate DNA binding of STAT5, this transcription factor does not play a pivotal role in IFN- signalling in Ba/ F3 cells. cell signal 11;5:331–335, 1999. © 1999 Elsevier Science Inc. KEY WORDS. Interferon- , Signal transduction, STAT5, Ba/F3 cells
INTRODUCTION Cytokines regulate survival, growth and differentiation of their target cells through binding to specific cell surface receptors and the activation of intracellular signalling cascades . Recent data from many laboratories indicate that intracellular tyrosine kinases of the Janus kinase (JAK) family and their key effectors, the signal transducer and activator of transcription (STAT) proteins, are essential in signalling by the interferons (IFNs) and other cytokines [1–4]. The specific set of genes induced by individual cytokine receptors is determined, at least in part, by the specific combination of the JAKs and STAT proteins that are activated [1–4]. In IFN- signal transduction, for example, ligand binding to the receptor induces the activation of the Janus kinases JAK1 and TYK2, which stimulate tyrosine phosphorylation and activation of the transcription factors STAT1 and 2 [1–3]. Subsequently, STAT1-STAT2 heterodimers can form and associate with p48, an IFN regulatory factor family protein. The resulting transcription factor complex termed ISGF3 then binds to DNA sequences designated the IFN-stimulated response element (ISRE) in the promoter of target genes. Alternatively, STAT1 homodimers can bind to IFN- activation site (GAS) sequences. A targeted disruption of the STAT1 gene revealed that
*Author to whom all correspondence should be addressed. E-mail: jaster@ med.uni-rostock.de Abbreviations: JAK–Janus kinase; STAT–signal transducer and activator of transcription; IFN–interferon; ISRE–interferon-stimulated response element; GAS–interferon- activation site; IL-3–interleukin-3; DN-JAK2– dominant-negative Janus kinase 2; EMSA–electrophoretic mobility shift assay; WT–wild type. Received 5 May 1998; and accepted 1 June 1998.
STAT1 plays an essential and specific role in the induction of IFN-dependent biological responses . The attractive model of a JAK/STAT pathway-mediated specificity of cytokine action, however, has become more and more complex with the discovery that many STATs are activated in response to a much broader range of cytokines and growth factors than originally expected. For example, IFN- has also been shown to activate STAT3 and STAT5 in some cell types [6–9]. The biological consequences of STAT5 activation by interferons are only partly understood. Some data suggest that STAT5 might be part of the IFN-induced differentiation response of myeloid cells . The specific role of the two STAT5 isoforms A and B is currently unclear. Most cytokine receptors that are known to activate STAT5, including, for example, the receptors for erythropoietin, granulocyte-macrophage colony-stimulating factor and interleukin-3 (IL-3) [10, 11], stimulate both growth and differentiation of haematopoietic progenitor cells [12, 13]. In contrast, interferons are inhibitors of cell proliferation . How IFN- activates STAT5 has not been addressed so far. According to the current model of cytokine receptor signalling, STAT5 activation is mediated by JAK2, but the Janus kinases activated by IFN- are JAK1 and Tyk2 [1, 2]. Although there is evidence for a principal interchangeability of different JAKs in STAT activation , which Janus kinase is the physiological mediator of the IFN- -dependent STAT5 activation still remains to be shown. Furthermore, the STAT5-binding site(s) of the IFN- receptor have not been identified so far. In this study, we analysed the role of STAT5 in IFNreceptor signal transduction in Ba/F3 cells. The prolifera-
however. Minneapolis. 0.. 10 mM Tris-HCl (pH 7.332 R.. Jaster et al. Eggenstein. MATERIAL AND METHODS Cell Culture Ba/F3 is a murine IL-3-dependent pro-B cell line . Ba/F3 cells were depleted of IL-3 for 8 h.1% NP40. 1 mM dithiothreitol. Preparation of Nuclear Extracts and EMSAs Nuclear extracts from Ba/F3 were prepared essentially as described previously . Note that the intensities of band-shift signals in (A) and (B) are not comparable. Stable expression of DN-JAK2 was confirmed by immunoblot analysis. Electrophoretic mobility shift assays (EMSAs) with the use of a double-stranded. 1 mM Pefabloc FIGURE 2. For induction studies.. Recombinant murine IL-3 (R&D Systems. . 0.1 mM EDTA.. Eggenstein. Effects of DN-JAK2 on the IL-3. is capable of stimulating the DNAbinding of both STAT5A and STAT5B and that this effect is not mediated by JAK2. 32P-labelled oligonucleotide derived from the bovine -casein promoter (5 -AGA TTT CTA GGA ATT CAA ATC-3 ) as a STAT5-binding probe have been described before . Briefly.-dependent formation of -casein oligonucleotide probe-binding complexes. were treated with (A) IL-3 (100 pM) or (B) IFN(1000 U/mL) for various times. The indicated BA/F3 subclones. like IL-3. depleted of cytokine for 8 h. 50 mM potassium chloride. Generation and characteristics of Ba/F3 subclones stably expressing a dominant-negative form of JAK2 (DN-JAK2) truncated at amino acid 829 have been described before . Cells were maintained in RPMI 1640 (Life Technologies. Nuclear extracts from 2 105 cells per sample were subjected to gel mobility shift analysis by using a labelled -casein oligonucleotide probe. FIGURE 1. 1 mg/mL BSA. Ba/F3 cells were IL-3-deprived for 8 h and stimulated with IL-3 (100 pM) or IFN. Binding reaction mixtures (20 L) contained nuclear extracts from 2 105 cells. at least in BA/F3 cells. Germany) were used to stimulate the cells where indicated. followed by G418 selection of resistant cells. is questionable. also suggest that the physiological relevance of STAT5 activation by IFN. and -casein oligonucleotide probe-binding activities in nuclear extracts from 2 105 cells per sample were analysed by EMSA. These cells were cultured in complete medium supplemented with G418 at 1 mg/mL. Germany) medium supplemented with 10% foetal calf serum and 10% conditioned medium from WEHI-3B cells as a source of murine IL-3. 5% glycerol.and IFN. MN. wild-type cells were co-transfected with DN-JAK2 in pBOS along with pSVneo by electroporation.5). Our data. owing to different times of X-ray exposure. tion of this murine pro-B cell line depends on the presence of IL-3 and is inhibited by IFN. USA) and IFN.(1000 U/mL) for the indicated periods of time. Induction of -casein oligonucleotide probe-binding activities by IL-3 and IFN. We provide evidence that IFN.(Life Technologies.
RESULTS To investigate whether IFN. Figure 3A shows that IL-3 stimulated the DNA binding of both STAT5A and STAT5B because two different supershift bands (indicated by arrows) were observed if the binding reaction was incubated with an antibody detecting both STAT5 isoforms . To study more directly the role of this Janus kinase in the mediation of the IFN. Previous experiments have shown that. the IFN. The indicated cDNA probes were labelled with [ -32P]dCTP (Amersham. The presence of STAT5 in the -casein promoter probebinding complexes was examined by supershift analysis with the use of specific antibodies (Fig. does not induce the tyrosine phosphorylation of JAK2 . Eggenstein. 1 g of poly(dI-dC) and 16 fmol of end-labelled oligonucleotide. we performed EMSAs by using nuclear extracts from IFN. The IL-3 effect. In the presence of DN-JAK2. This phenomenon may be. at least in part. however. The concentrations of cytokine applied in the experiments corresponded to a maximum stimulation (IL-3) or inhibition (IFN. Germany) and hybridised as described previously .25 TBE buffer. For supershift analysis. Moreover. On long-run gels (see also Fig. which lacks the kinase domain of the enzyme . 0. unlike IL-3.Signalling 333 (Boehringer Mannheim. The maximum stimulation of DNA-binding activity was observed after 20–60 min.was compared with the effect of IL-3.effect is even enhanced in Ba/F3 cells stably transfected with the DN-JAK2 cDNA. blots were rehybridised with a glyceraldehyde-3phosphate dehydrogenase cDNA probe. Figure 2B indicates that DNJAK2 does not suppress the IFN.stimulates the binding of protein complexes to a consensus DNA-binding sequence of STAT5. Germany) according to the manufacturer’s instructions. Supershift analysis of IL-3.-stimulated Ba/F3 cells and an oligonucleotide derived from the bovine -casein promoter. and supershift analysis was performed by incubating the EMSA binding reaction mixtures with the indicated STAT antibodies as described. The effect of IFN. Northern Blot Analysis Total RNA from Ba/F3 cells was isolated by using TRIzolreagent (Life Technologies. Germany). Amersham.. a known activator of the JAK2/STAT5 pathway [1. in Ba/F3 cells.. UK) by using a Prime Kit (Boehringer Mannheim. the DNA–protein complexes separate into at least two distinguishable components. RNA samples (20 g) were electrophoresed on denaturating formaldehyde gels. blotted onto nylon membranes (Hybond-N.and IL-3dependent induction of -casein probe-binding activity. Nuclear extracts were prepared. IFNalso induced protein complex binding to the -casein promoter probe.5 g of the indicated STAT-antibody (Santa Cruz Biotechnology. CA. IFN. In contrast. we compared the effects of these cytokines in wild-type (WT) Ba/F3 cells with the effects in Ba/F3 cells stably expressing a dominant-negative deletion mutant of JAK2. Ihle (Memphis. Binding was for 30 min on ice. The cDNA probes for murine oncostatin-M and pim-1 were kindly provided by J. 11] and essential growth factor for Ba/F3 cells . unpublished data). Figure 2A shows that the IL-3-dependent induction of protein binding to the -casein oligonucleotide is much weaker in the DN-JAK2-expressing clone than in WT-Ba/F3 cells. Both the kinetics of stimulation of DNA-binding activity and the position of the band shifts on the gel were the same as those after IL-3 stimulation. Figure 1 indicates that IL-3 strongly induced the rapid and transient formation of protein complexes binding to the -casein promoter probe.Role of STAT5 in Interferon. To test for loading equivalence. Jaster. pressed . 3).) of cell growth (data not shown). activation of WT-JAK2 by IL-3 is strongly sup- FIGURE 3.and IFN. due to the fact that this mutant cell line expresses higher protein levels of both STAT5A and STAT5B than WT-Ba/F3 cells do (R. USA) was added afterwards.-induced complex binding. TN. USA). UK) and immobilised by ultraviolet cross-linking. Complexes were analysed by electrophoretic separation on a 6% polyacrylamide gel in 0.-induced -casein oligonucleotide probe-binding activities (A) Parental Ba/F3 cells or (B) DN-JAK2 expressing Ba/F3 were starved for 8 h and stimulated with cytokine for 60 min. and the incubation continued for an additional 20 min. was much more pronounced. 1).
were stimulated with IL-3 (100 pM) or IFN(1000 U/mL) for the indicated periods of time. The expression of the pim-1 mRNA was stimulated by both cytokines with similar kinetics. but the IFN. whereas IFN. 3A and B).and .on oncostatin-M and pim-1 mRNA expression. and Northern blot analysis was performed as described by using 32P-labelled probes specific for oncostatin-M. The capability of IFN. in Ba/F3 cells. 25]. A and B. To study the role of STAT5 in IFN.activates the DNA binding of both STAT5A and STAT5B. We previously showed that. Jaster et al. IFNs induce the transcription of specific target genes through the activation of intracellular signalling cascades including the JAK/STAT pathway. suggesting that this complex contains only the B isoform of STAT5. the IFN. we took advantage of the enhanced IFN. Our data indicate that IFN. RNA samples from IL-3-stimulated Ba/F3 cells were included in the investigations. stimulates the binding of both STAT5A and STAT5B to the -casein promoter probe. Therefore. Figure 4 shows that IL-3 strongly induced the rapid and transient expression of oncostatin-M. They can be subdivided into two distinct types: type I IFNs (comprising IFN. The results obtained from DN-JAK2-expressing Ba/F3 cells were confirmed by the additional analysis of nuclear extracts from WT-Ba/F3 cells (data not shown). cultured for 8 h in the absence of IL-3. IFN. DISCUSSION IFNs are a family of cytokines with pleiotropic effects on target cells. including induction of an anti-viral state.action is poorly understood.effect in DN-JAK2 expressing Ba/F3 cells (Fig. Ba/F3 cells. inhibition of cell growth and modulation of the immune response . like IL-3. the specific involvement of the Janus kinases JAK1 and TYK2 and the STAT proteins 1 and 2 is well established [1.) and type II IFN or IFN. Again using antibodies detecting either both STAT5A and STAT5B (lane 2) or STAT5A (lane 3) only.signal transduction.on the activation of both isoforms of STAT5. Northern blot anal- ysis of the effects of IL-3 and IFN. 2]. After receptor binding. Northern blots were performed to study the IFN.-induced probebinding complexes (Fig. pim-1 and oncostatin-M [20–22]. 2B).stimulation of the cells (R. we observed the same band-shift pattern as that described for the IL-3activated probe-binding complexes (compare lanes 2 and 3 in Fig. but only one was observed if a STAT5A-specific antibody was applied (lane 3). For comparison..to induce DNA binding of STAT5 raised the question of possible effects on the expression of genes known to be regulated by STAT5. In contrast. the contribution of other STATs to the mediation of IFN. in murine Ba/F3 cells. but. Jaster. No supershift was detected with anti-STAT1 or 3 antibodies (Fig. IFN. lanes 4 and 5).signal transduction. indicating that the IFN. indicating an activation of the JAK1/ Tyk2 and STAT1/2 signalling cascade . which facilitated the supershift analysis. In conclusion. Furthermore. which recognise structurally related but not identical cell surface receptors [24.effect is weak. In IFN. Total RNA was isolated. IFN.-dependent expression of two members of this gene family. only a small fraction of cellular STAT5 becomes tyrosine phosphorylated subsequent to IFN.induced the expression of a luciferase reporter with an ISRE enhancer element. the fastermigrating complex was supershifted exclusively by the antibody directed against both STAT5A and STAT5B but not by the STAT5A-specific one. we analysed the effects of IFN.334 R..had no effect. .is a potent suppressor of the IL-3-dependent growth of these cells . pim-1 and glyceraldehyde-3-phosphate dehydrogenase. To study the composition of the IFN.-induced protein complex binding to the -casein promoter probe does not contain these STAT proteins. 3B). Furthermore. . unpublished data). In contrast.. (lane 2).effect was very weak compared with the response to IL-3 stimulation. compared with the IL-3 response. 3B. FIGURE 4.
Greenlund A. but not IFN. 6937–6944. Rev. 615–633. Borden E... Shi W. Haldosen L. and Steinmetz M. 1055–1063. O’Farrell A. E. 10. Kamper B.. (1997) ¨ Cell. (1989) Biochim. 4. Wohrl S. 14. Durbin J. Biol. 17. 3. L. Pfeffer L.. and Groner B.-dependent STAT5 activation remains to be elucidated. 3364–3372. Farrar M. Rodig S. Cell. Sakao.. B. Further studies in other IFN. Russell S. The exact molecular mechanism of the IFN. Langer J.-C. acts as a suppressor of cellular growth. Biochem. 15. Pellegrini S. D. Pless M.. and D’Andrea A. Raz R.-F. Zhu Y. but recent studies suggest that STAT5 is required for a maximal cell proliferation in response to haematopoietic growth factors [22. Sheehan K. Bhattacharya S. 20.. Miyajima A. (1996) EMBO J.. Mui A. (1992) N. A. 26. 85–89. Barahmand-Pour F.. Palacios R. 14.. 5. Jenkins N. Bach E. Immunol.. however. Hara T. Patel S. Biophys. Darnell J. 17. 591–594..-H. 56. D. 2. Copeland N.... Pallard C. Kaplan D. Cell. This work was supported by a grant from the Deutsche Forschungsgemeinschaft.. Meraz M.. Mui A. (1997) Eur. Zhuang H. (1995) Nature 377.to stimulate STAT5-dependent enhancer transactivation . 16..-stimulated DNA-binding of STAT5. D. Murti A. as recently suggested .. Domen J. (1994) J. Campbell D. Norstedt G.. L. Zhu Y. and Krystal G. White J. Harada N. and Schreiber R. and Pfeffer L. 24391–24395.. E. He T.. Wakao H.. 24. V. and Morgensen K. and DN-JAK2 does not suppress the IFN... only IL-3. 27]. Interferon Cytokine Res. (1995) Proc. (1996) Cell 84. activated a luciferase reporter construct with GAS elements as STAT5-binding sites. Zoon K. Acad. Wakao H. H.. M.. 269. 5557–5568. and Miyajima A. Whetton A. and Berns A. Gilbert D.. Kinjyo I. Migone T. Yoshimura A.. E. E.. Pfeffer S. 15. Murti A. Biol. J. Krantz S. The IFN. E. C. K.. K. Mathey-Prevot B.. and Yang C.. 571–611. and Dexter T. and Leonard W. Chem. Biol. 27. Biol. (1995) EMBO J.. (1986) Cell 46... Pestka S. Sci. (1997) Science 277. 2005–2013. 9. IFN.. Cell. Croze E.. raises the question of the physiological relevance of the weak but significant activation of this transcription factor by the cytokine. Gouilleux F.. Friedmann M. 21. and Brock J. M. Constantinescu S. Riley J. Stoiber D. Lutfalla G.. Jaster R.. 269. C. 1418–1420. (1995) EMBO J. Ludtke B. Mathey-Prevot B..-induced STAT5 activation seems to be mediated by a signalling cascade different from the JAK2-dependent one used by many other cytokine receptors [1. and Samuel C. Robanus-Maandag E.. H.. Ihle J. and ¨ Decker T. E. 8057–8061. G. 9. Biochem. Cutler R. Ichihara M. 431–442...-responsive cell lines will be necessary to investigate whether there is a cell-typespecific role of STAT5 and its isoforms A and B in IFNsignalling. Aguet M. Kitamura T. A. 248. Moriyama M. 3–26. M.. 2425–2433. R.. (1985) Cell 41. and Levy D. Bittorf T. Selten G. The failure of IFN. (1996) Cell 84.. 12. A. possibly related to the induction of cell differentiation. USA 93. C. 111–132. Uze G. 1630–1635. Humphries R. Natl. Basu L. Dighe A. Signal.. Acta 989. F. 22. (1992) Annu. References 1.. 603–611. 7. Engl. Meinke A...Role of STAT5 in Interferon. 19. 11. Rev. 1491–1493.... H. Jr. E. N. 13. L. Blatt L. Clark R. 326. (1987) Annu. Ihle J. 1166–1175. (1996) Mol. (1996) EMBO J. 8. 419–434. 25. (1991) Blood 77. Van Beveren C. T. J. A. Carver-Moore K. M. . C. and Schreiber R.. Niu Z... Jaster R.. 15. D... 14. (1995) J... N.. 271. (1996) Mol. J. (1994) J. M.. 2]: IFNdoes not activate JAK2. A. DuBois R. 26.. N. Chem. Wakao H.. Chem. 727–777. 2077–2082. 21411–21414. Inhorn R... Shi W. Biol. 14. and Miyajima A. Damen J. D. The role of STAT5 in the regulation of cell growth is not completely understood.. Mullersman J. 18. Verbeek J. Mullersman J. and Dusanter-Fourt I. and Miyajima A.. (1997) Mol. 11. (1997) Science 276. Cuypers H. M. J. and D’Andrea A.. Biol. along with its lacking or poor effect on the expression of STAT5-dependent genes observed in this study.. S.. 727–734.. 16. Pless M. (1996) J. 15. and Wojchowski D. Med. (1995) EMBO J. Dusanter-Fourt I. N. Krosl J. Yang C. A. E. Levy D.Signalling 335 6.. 331–334. 4808–4817. 16.. Boelens W. 23. Kinoshita T.
This action might not be possible to undo. Are you sure you want to continue?