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• W2014356768 abstract "The receptor for interleukin-5 (IL-5R) is composed of a unique α chain (IL-5Rα) expressed on eosinophils and basophils, associated with a βc subunit, which is shared by the receptors for IL-3 and granulocyte macrophage-colony stimulating factor. One of the molecular events activated via the IL-5R is the JAK/STAT signaling pathway. Recent reports have shown that IL-5 induces tyrosine phosphorylation of JAK2 followed by the subsequent cell type-specific activation of either STAT1α or STAT5. To identify additional STAT proteins activated by IL-5, we co-transfected the IL-5R with STAT cDNAs in COS cells. We found that IL-5 induces binding of STAT3 to the intercellular adhesion molecule-1 pIRE, and activates STAT3-dependent transcription. Moreover, endogenous STAT3 was tyrosine phosphorylated and activated in human IL-5-stimulated BaF3 cells ectopically expressing the human IL-5R (BaF3/IL5R). These data imply that multiple STAT proteins are involved in gene regulation by IL-5 in a cell type-specific manner. We further demonstrate using C-terminal truncations of the α and βc subunits of the IL-5R that the membrane-proximal regions of both subunits are required for STAT activation. Interestingly, a βc receptor mutant lacking intracellular tyrosine residues is able to mediate STAT3 activation, suggesting that tyrosine phosphorylation of the βc receptor is not essential for STAT3 activation. The receptor for interleukin-5 (IL-5R) is composed of a unique α chain (IL-5Rα) expressed on eosinophils and basophils, associated with a βc subunit, which is shared by the receptors for IL-3 and granulocyte macrophage-colony stimulating factor. One of the molecular events activated via the IL-5R is the JAK/STAT signaling pathway. Recent reports have shown that IL-5 induces tyrosine phosphorylation of JAK2 followed by the subsequent cell type-specific activation of either STAT1α or STAT5. To identify additional STAT proteins activated by IL-5, we co-transfected the IL-5R with STAT cDNAs in COS cells. We found that IL-5 induces binding of STAT3 to the intercellular adhesion molecule-1 pIRE, and activates STAT3-dependent transcription. Moreover, endogenous STAT3 was tyrosine phosphorylated and activated in human IL-5-stimulated BaF3 cells ectopically expressing the human IL-5R (BaF3/IL5R). These data imply that multiple STAT proteins are involved in gene regulation by IL-5 in a cell type-specific manner. We further demonstrate using C-terminal truncations of the α and βc subunits of the IL-5R that the membrane-proximal regions of both subunits are required for STAT activation. Interestingly, a βc receptor mutant lacking intracellular tyrosine residues is able to mediate STAT3 activation, suggesting that tyrosine phosphorylation of the βc receptor is not essential for STAT3 activation. INTRODUCTIONHematopoiesis is tightly regulated by a complex network of stromal interactions and by soluble polypeptide factors named cytokines. IL-3, 1The abbreviations used are: ILinterleukinGM-CSFgranulocyte macrophage-colony stimulating factorIL-5Rreceptor for IL-5JAKjanus kinaseSTATsignal transducer and activator of transcriptionSH domainSrc homology domainIFNinterferonhILhuman interleukinGRRγ responsive regionICAM-1intercellular adhesion molecule-1pIREpalindromic IL-6/IFN-γ response elementSIESis-inducible element. IL-5, and GM-CSF are cytokines that have diverse effects on the proliferation, differentiation, and activation of blood cells and their precursors (1Arai K.I. Lee F. Miyajima A. Miyatake S. Arai N. Yokota T. Annu. Rev. Biochem. 1990; 59: 783-836Crossref PubMed Scopus (1168) Google Scholar, 2Lopez A.F. Elliott M.J. Woodcock J. Vadas M.A. Immunol. Today. 1992; 13: 495-500Abstract Full Text PDF PubMed Scopus (127) Google Scholar, 3Ogawa M. Blood. 1993; 81: 2844-2853Crossref PubMed Google Scholar). Whereas IL-3 and GM-CSF also have effects on other hematopoietic lineages(3Ogawa M. Blood. 1993; 81: 2844-2853Crossref PubMed Google Scholar, 4Clutterbuck E.J. Hirst E.M. Sanderson C.J. Blood. 1989; 73: 1504-1512Crossref PubMed Google Scholar), the effect of IL-5 in humans is restricted to eosinophils and basophils. IL-5 is essential for eosinophil differentiation (5Campbell H.D. Tucker W.Q.J. Hort Y. Martinson M.E. Mayo G. Clutterbuck E.J. Sanderson C.J. Young I.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6629-6633Crossref PubMed Scopus (266) Google Scholar, 6Sanderson C.J. Warren D.J. Strath M. J. Exp. Med. 1985; 162: 60-74Crossref PubMed Scopus (225) Google Scholar) and plays an important role in the function of mature eosinophils(7Silberstein D.S. Owen W.F. Gasson J.C. DiPersio J.F. Golde D.W. Bina J.C. Soberman R. Austen K.F. David J.R. J. Immunol. 1986; 137: 3290-3294PubMed Google Scholar, 8Lopez A.F. Sanderson C.J. Gamble J.R. Campbell H.D. Young I.G. Vadas M.A. J. Exp. Med. 1988; 167: 219-224Crossref PubMed Scopus (801) Google Scholar, 9Fujisawa T. Abu Ghazaleh R. Kita H. Sanderson C.J. Gleich G.J. J. Immunol. 1990; 144: 642-646PubMed Google Scholar, 10van der Bruggen T. Kok P.T.M. Raaijmakers J.A.M. Verhoeven A.J. Kessels R.G.C. Lammers J.W.J. Koenderman L. J. Leukocyte Biol. 1993; 53: 347-353Crossref PubMed Scopus (49) Google Scholar, 11van der Bruggen T. Kok P.T.M. Raaijmakers J.A.M. Lammers J.W.J. Koenderman L. J. Immunol. 1994; 153: 2729-2735PubMed Google Scholar). The specific effects of IL-5 on eosinophils and basophils are due to restricted expression of the low affinity IL-5Rα receptor on these cell types(12Chihara J. Plumas J. Gruart V. Tavernier J. Prin L. Capron A. Capron M. J. Exp. Med. 1990; 172: 1347-1351Crossref PubMed Scopus (93) Google Scholar, 13Ingley E. Young I.G. Blood. 1991; 78: 339-344Crossref PubMed Google Scholar). The high affinity receptor for IL-5 (IL-5R) is composed of a unique α subunit associated with a βc subunit that is shared with the receptors for IL-3 and GM-CSF(14Tavernier J. Devos R. Cornelis S. Tuypens T. Van der Heyden J. Fiers W. Plaetinck G. Cell. 1991; 66: 1175-1184Abstract Full Text PDF PubMed Scopus (493) Google Scholar). The βc subunit is essential for signal transduction(15Kitamura T. Miyajima A. Blood. 1992; 80: 84-90Crossref PubMed Google Scholar, 16Miyajima A. Mui A.L. Ogorochi T. Sakamaki K. Blood. 1993; 82: 1960-1974Crossref PubMed Google Scholar), but also, the α chain transduces intracellular growth signals(17Takaki S. Kanazawa H. Shiiba M. Takatsu K. Mol. Cell. Biol. 1994; 14: 7404-7413Crossref PubMed Google Scholar). Therefore, the common use of the βc subunit by IL-3, IL-5, and GM-CSF explains the partial observed functional redundancy of these cytokines(16Miyajima A. Mui A.L. Ogorochi T. Sakamaki K. Blood. 1993; 82: 1960-1974Crossref PubMed Google Scholar, 18Miyajima A. Hara T. Kitamura T. Trends Biochem. Sci. 1992; 17: 378-382Abstract Full Text PDF PubMed Scopus (104) Google Scholar). However, postreceptor signal transduction pathways are not well defined and are likely to be composed of both mitogenic and differentiation signals. It is known that the βc subunit, like other cytokine receptors, does not contain intrinsic tyrosine kinase activity(19Sato N. Sakamaki K. Terada N. Arai K. Miyajima A. EMBO J. 1993; 12: 4181-4189Crossref PubMed Scopus (329) Google Scholar, 20Mui A.L. Miyajima A. Proc. Soc. Exp. Biol. Med. 1994; 206: 284-288Crossref PubMed Scopus (10) Google Scholar). However, one of the earliest events to occur after IL-3, IL-5, and GM-CSF stimulation is induction of protein tyrosine phosphorylation(19Sato N. Sakamaki K. Terada N. Arai K. Miyajima A. EMBO J. 1993; 12: 4181-4189Crossref PubMed Scopus (329) Google Scholar, 21Sakamaki K. Miyajima I. Kitamura T. Miyajima A. EMBO J. 1992; 11: 3541-3549Crossref PubMed Scopus (295) Google Scholar). This tyrosine phosphorylation is caused by the activation of several cytoplasmic protein tyrosine kinases such as Lyn (22Torigoe T. O'Connor R. Santoli D. Reed J.C. Blood. 1992; 80: 617-624Crossref PubMed Google Scholar, 23Pazdrak K. Schreiber D. Forsythe P. Justement L. Alam R. J. Exp. Med. 1995; 181: 1827-1834Crossref PubMed Scopus (150) Google Scholar) and c-Fps/Fes(24Hanazono Y. Chiba S. Sasaki K. Mano H. Miyajima A. Arai K. Yazaki Y. Hirai H. EMBO J. 1993; 12: 1641-1646Crossref PubMed Scopus (144) Google Scholar). Recent studies have demonstrated that the family of Janus kinases (JAKs), containing four members (JAK1, JAK2, JAK3, and TYK2), is associated with different cytokine receptors and is phosphorylated after ligand binding(25Ihle J.N. Kerr I.M. Trends Genet. 1995; 11: 69-74Abstract Full Text PDF PubMed Scopus (818) Google Scholar). We and others have shown that only JAK2 is activated in response to IL-3, IL-5, and GM-CSF (26Silvennoinen O. Witthuhn B.A. Quelle F.W. Cleveland J.L. Yi T. Ihle J.N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8429-8433Crossref PubMed Scopus (437) Google Scholar, 27van der Bruggen T. Caldenhoven E. Kanters D. Coffer P. Raaijmakers J.A.M. Lammers J.W.J. Koenderman L. Blood. 1995; 85: 1442-1448Crossref PubMed Google Scholar, 28Sato S. Katagiri T. Takaki S. Kikuchi Y. Hitoshi Y. Yonehara S. Tsukada S. Kitamura D. Watanabe T. Witte O. Takatsu K. J. Exp. Med. 1994; 180: 2101-2111Crossref PubMed Scopus (163) Google Scholar) and specifically associates with the membrane-proximal region of the β chain(29Quelle F.W. Sato N. Witthuhn B.A. Inhorn R.C. Eder M. Miyajima A. Griffin J.D. Ihle J.N. Mol. Cell. Biol. 1994; 14: 4335-4341Crossref PubMed Google Scholar). The JAK protein-tyrosine kinases activate members of a novel family of transcription factors named signal transducers and activators of transcription (STATs)(25Ihle J.N. Kerr I.M. Trends Genet. 1995; 11: 69-74Abstract Full Text PDF PubMed Scopus (818) Google Scholar).STAT proteins were first described as intermediates in the interferon-α/β (IFN-α/β) signaling pathway(30Fu X.Y. Kessler D.S. Veals S.A. Levy D.E. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8555-8559Crossref PubMed Scopus (335) Google Scholar, 31Fu X.Y. Schindler C. Improta T. Aebersold R. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7840-7843Crossref PubMed Scopus (445) Google Scholar). At present, eight different STATs (STAT1α, STAT1β, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6) have been described and are involved in specific cytokine regulation.(31Fu X.Y. Schindler C. Improta T. Aebersold R. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7840-7843Crossref PubMed Scopus (445) Google Scholar, 32Zhong Z. Wen Z. Darnell J.E.J. Science. 1994; 264: 95-98Crossref PubMed Scopus (1691) Google Scholar, 33Yamamoto K. Quelle F.W. Thierfelder W.E. Kreider B.L. Gilbert D.J. Jenkins N.A. Copeland N.G. Silvennoinen O. Ihle J.N. Mol. Cell. Biol. 1994; 14: 4342-4349Crossref PubMed Scopus (204) Google Scholar, 34Jacobsen N.G. Szabo S.J. Weber-Nordt R.M. Zhong Z. Schreiber R.D. Darnell J.E.J. Murphy K.M. J. Exp. Med. 1995; 181: 1755-1762Crossref PubMed Scopus (578) Google Scholar, 35Gouilleux F. Wakao H. Mundt M. Groner B. EMBO J. 1994; 13: 4361-4369Crossref PubMed Scopus (524) Google Scholar, 36Mui A.L. Wakao H. O'Farrell A.M. Harada N. Miyajima A. EMBO J. 1995; 14: 1166-1175Crossref PubMed Scopus (537) Google Scholar, 37Gouilleux F. Pallard C. Dusanter-Fourt I. Wakoa H. Haldosen L.-A. Norstedt G. Levy D. Groner B. EMBO J. 1995; 14: 2005-2013Crossref PubMed Scopus (332) Google Scholar, 38Hou J. Schindler U. Henzel W.J. Ho T.C. Brasseur M. McKnight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (725) Google Scholar, 39Quelle F.W. Shimoda K. Thierfelder W. Fischer C. Kim A. Ruben S.M. Cleveland J.L. Pierce J.H. Keegan A.D. Nelms K. Paul W.E. Ihle J.N. Mol. Cell. Biol. 1995; 15: 3336-3343Crossref PubMed Scopus (301) Google Scholar). This family of STAT proteins comprises a new class of transcription factors that contain Src homology 2 (SH2), SH3-like domains, and a carboxyl-terminal tyrosine phosphorylation site(25Ihle J.N. Kerr I.M. Trends Genet. 1995; 11: 69-74Abstract Full Text PDF PubMed Scopus (818) Google Scholar). STAT proteins normally exist as inactive monomers in the cytoplasm, but after phosphorylation on tyrosine, they form homo- or heterodimers, which translocate to the nucleus and bind to specific DNA motifs(40Fu X.Y. Zhang J.J. Cell. 1993; 74: 1135-1145Abstract Full Text PDF PubMed Scopus (272) Google Scholar, 41Larner A.C. David M. Feldman G.M. Igarashi K. Hackett R.H. Webb D.S. Sweitzer S.M. Petricoin E.F. Finbloom D.S. Science. 1993; 261: 1730-1733Crossref PubMed Scopus (314) Google Scholar, 42Wegenka U.M. Buschmann J. Lutticken C. Heinrich P.C. Horn F. Mol. Cell. Biol. 1993; 13: 276-288Crossref PubMed Scopus (485) Google Scholar, 43Caldenhoven E. Coffer P. Yuan J. van de Stolpe A. Horn F. Kruijer W. van der Saag P.T. J. Biol. Chem. 1994; 269: 21146-21154Abstract Full Text PDF PubMed Google Scholar, 44Coffer P. Lutticken C. van Puijenbroek A. Klop-de Jonge M. Horn F. Kruijer W. Oncogene. 1995; 10: 985-994PubMed Google Scholar). We recently found that STAT3 is also phosphorylated on serine residues(45Lutticken C. Coffer P. Yuan J. Schwartz C. Caldenhoven E. Schindler C. Kruijer W. Heinrich P.C. Friedemann H. FEBS Lett. 1995; 360: 137-143Crossref PubMed Scopus (93) Google Scholar). This serine phosphorylation seems to be necessary for the regulation of the transactivation potential of STAT3.We have previously shown that IL-5 activates STAT1α together with an unidentified DNA-binding protein in human eosinophils(27van der Bruggen T. Caldenhoven E. Kanters D. Coffer P. Raaijmakers J.A.M. Lammers J.W.J. Koenderman L. Blood. 1995; 85: 1442-1448Crossref PubMed Google Scholar). Therefore, we were interested whether other known STAT proteins can be activated by IL-5. For this purpose, we reconstituted both subunits of the IL-5 receptor together with different STAT cDNAs in COS cells. We demonstrated that besides STAT1α, STAT3 was also activated by IL-5 in these transfected COS cells. In addition, endogenous STAT3 was tyrosine phosphorylated and activated by hIL-5 in BaF3 cells stably expressing the hIL-5R (BaF3/IL5R). Finally we demonstrate, using carboxyl-terminal truncations of both the α and β subunits, that the membrane-proximal region of both subunits are necessary for STAT3 activation.MATERIALS AND METHODSCell Culture, Reagents, and AntibodiesMonkey COS-1 cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% heat inactivated fetal calf serum. A mouse IL-3-dependent pro-B cell line, BaF3, and BaF3 cells expressing the wild type human IL-5 receptor α and β subunits (BaF3/IL5R) were maintained in RPMI 1640 supplemented with 10% Hyclone serum and the appropriate cytokine. Parental BaF3 cells were grown in medium with 10-10M mouse IL-3 (Genzyme, Cambridge, MA), BaF3/IL5R cells were grown in medium with 10-10M hIL-5 (a kind gift of Dr. D. Fattah, Glaxo Group Research, Greenford, United Kingdom) and 1 mg/ml G418 (Life Technologies, Inc.). Anti-phosphotyrosine monoclonal antibody (4G10) was obtained from UBI (Lake Placid, NY). The monoclonal antibody directed against STAT1α/β was purchased from Transduction Laboratories (Lexington, Kentucky). The STAT3 rabbit polyclonal antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, California).Synthetic Oligonucleotides and Plasmid ConstructionOligonucleotides with the following sequences were used in this study (only the top strands are shown): the human ICAM-1 pIRE, 5′-AGCTTAGGTTTCCGGGAAAGCAC-3′; a mutant pIRE, 5′-AGGCGCGAGGTTAGCGGTCAAGCAGCACCGC-3′; the c-fos SIEm67, 5′-GTGCATTTCCCGTAAATCTTGTCTACAATTC-3′; and the FcγRI GRR, 5′-AGCTTGAGATGTATTTCCCAGAAAAGA-3′. The 2xpIREtkluc reporter construct has been described by us previously(43Caldenhoven E. Coffer P. Yuan J. van de Stolpe A. Horn F. Kruijer W. van der Saag P.T. J. Biol. Chem. 1994; 269: 21146-21154Abstract Full Text PDF PubMed Google Scholar); also the pSV-lacZ expression vector has been described previously(46Shen S. van der Saag P.T. Kruijer W. Mech. Dev. 1993; 40: 177-189Crossref PubMed Scopus (23) Google Scholar). The reporter plasmids 4xIREtkCAT and 4xGRRtkCAT were constructed by cloning four oligonucleotides containing either the pIRE or GRR in the HindIII site upstream of the thymidine kinase minimal promoter in pBLCAT2. pSGhSTAT1α was constructed by inserting the cDNA for human STAT1α from pMNC91 (47Shuai K. Stark G.R. Kerr I.M. Darnell J.E.J. Science. 1993; 261: 1744-1746Crossref PubMed Scopus (682) Google Scholar) into the NotI/BamHI sites of pSG513(48Green S. Issemann I. Sheer E. Nucleic Acids Res. 1988; 16: 369Crossref PubMed Scopus (544) Google Scholar). pSGhSTAT3 was constructed by inserting the human STAT3 cDNA isolated from a human eosinophil cDNA library into PSG513. pSGmSTAT4 was constructed by inserting the cDNA for mouse STAT4 from Rc/CMV-STAT4 (32Zhong Z. Wen Z. Darnell J.E.J. Science. 1994; 264: 95-98Crossref PubMed Scopus (1691) Google Scholar) into the HindIII/SmaI sites of pSG513. pSGshSTAT5 was constructed by inserting the cDNA for sheep STAT5 (MGF) from pXM-STAT5 (49Wakao H. Gouilleux F. Groner B. EMBO J. 1994; 13: 2182-2191Crossref PubMed Scopus (712) Google Scholar) into the HindIII/NotI sites of pSG513. pSGhSTAT6 was constructed by inserting the cDNA for human STAT6 (IL-4STAT) from TPU231 (38Hou J. Schindler U. Henzel W.J. Ho T.C. Brasseur M. McKnight S.L. Science. 1994; 265: 1701-1706Crossref PubMed Scopus (725) Google Scholar) into the EcoRI/XhoI sites of pSG513. pSGhIL5Rα was constructed by inserting the cDNA for the human IL-5α receptor from pBKhIL5Rα (50Zanders E.D. Eur. Cytokine Netw. 1994; 5: 35-42PubMed Google Scholar) into the NotI/KpnI sites of pSG513. pSGhIL5Rβ was constructed by inserting the cDNA for the human βc subunit from pSV532 (14Tavernier J. Devos R. Cornelis S. Tuypens T. Van der Heyden J. Fiers W. Plaetinck G. Cell. 1991; 66: 1175-1184Abstract Full Text PDF PubMed Scopus (493) Google Scholar) into the EcoRI sites of pSG513. For construction of the c-terminal deletions of the IL-5 receptor α and β chains, stop codons where introduced at the indicated amino acids using PCR.Generation of Stable TransfectantsPlasmid DNAs of the human IL-5Rα and βc containing the neomycin resistance gene were transfected into the BaF3 cells by using electroporation as described previously(21Sakamaki K. Miyajima I. Kitamura T. Miyajima A. EMBO J. 1992; 11: 3541-3549Crossref PubMed Scopus (295) Google Scholar), and transfectants were selected using G418 (1 mg/ml). Expression of the α and βc subunits was verified by PCR analysis and by growth in the presence of hIL-5.Transient TransfectionFor transfection experiments, COS cells were split 1:3 in 6-well plates (Costar), and 2 h later the cells were transfected with 10 μg of supercoiled plasmid DNA by the calcium phosphate coprecipitation technique(51Graham F.L. van der Eb A.J. Mol. Cell. Biol. 1973; 2: 607-616Google Scholar). Following 16-20 h of exposure to the calcium-phosphate precipitate, medium was refreshed, and cells were incubated for 16 h with IL-5. Transfected cells were subsequently harvested for luciferase assay (52Brasier A.R. Tate J.E. Habener J.F. BioTechniques. 1989; 7: 1116-1122PubMed Google Scholar) and lacZ determination (53Kress C. Vogels R. de Graaf W. Bonnerot C. Meijlink F. Nicolas J.F. Deschamps J. Development. 1990; 109: 775-786PubMed Google Scholar).Gel Retardation AssayNuclear extracts were prepared from unstimulated and stimulated COS and BaF3 cells following a previously described procedure(54Fried M. Crothers D.M. Nucleic Acids Res. 1981; 9: 6505-6525Crossref PubMed Scopus (1683) Google Scholar). Oligonucleotides were labeled by filling in the cohesive ends with [α-32P]dCTP using Klenow fragment of DNA polymerase I. Gel retardation assays were carried out according to published procedures with slight modifications (30Fu X.Y. Kessler D.S. Veals S.A. Levy D.E. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8555-8559Crossref PubMed Scopus (335) Google Scholar). Briefly, nuclear extracts (10 μg) were incubated in a final volume of 20 μl, containing 10 mM HEPES, pH 7.8, 50 mM KCL, 1 mM EDTA, 5 mM MgCl2, 10% (v/v) glycerol, 5 mM dithiothreitol, 2 μg of poly(dI-dC) (Pharmacia Biotech Inc.), 20 μg of bovine serum albumin, and 1.0 ng of 32P-labeled ICAM1-pIRE oligonucleotide for 20 min at room temperature. In competition experiments, extracts were incubated for 5 min with the indicated molar excess of unlabeled oligonucleotide prior to the addition of labeled oligonucleotide. Identification of the different DNA binding proteins was performed by preincubating nuclear extracts for 30 min on ice with antisera before addition of the probe.Immunoprecipitation and Western BlottingUnstimulated and IL-5 stimulated BaF3 cells were incubated with lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 5 mM EDTA, Na3VO4, 10 μg/ml aprotinine, 1 mM phenylmethylsulfonylfluoride, 1 mM leupeptin) for 15 min on ice. The lysate was centrifuged to remove nuclei and cellular debris. The cell lysates were incubated with the anti-STAT3 polyclonal antibody for 1 h at 4°C. Immune complexes were then precipitated with protein A-Sepharose for 1 h at 4°C, washed 3 times with lysis buffer, and boiled in 1.0 × Laemmli's sample buffer. The proteins were electrophoresed on a SDS-polyacrylamide gel and transferred to nitrocellulose membrane. After blocking in TBST (150 mM NaCl, 10 mM Tris, pH 8.0, 0.3% Tween 20) with 5% bovine serum albumin, the membrane was either incubated with the anti-phosphotyrosine (4G10) monoclonal antibody or with the polyclonal anti-STAT3 antibody. Between the incubation with 4G10 and STAT3 antibodies, the membrane was stripped with 1% SDS, 30 mM Tris, pH 8.0, 50 mM β-mercaptoethanol for 2 × 15 min at 55°C. After washing 3 times with TBST, the membrane was incubated for 1 h with peroxidase-conjugated rabbit anti-mouse or swine anti-rabbit antibodies, respectively. In both cases, the membrane was washed 5 times with TBST, and immunoprecipitated proteins were visualized with enhanced chemiluminescence (ECL, Amersham Corp.).RESULTSReconstitution of the IL-5 Receptor in COS Cells Results in STAT3 Activation by IL-5We have previously shown that IL-5 treatment of human eosinophils activates the binding of multiple protein complexes to the pIRE from the ICAM-1 promoter(27van der Bruggen T. Caldenhoven E. Kanters D. Coffer P. Raaijmakers J.A.M. Lammers J.W.J. Koenderman L. Blood. 1995; 85: 1442-1448Crossref PubMed Google Scholar). We also demonstrated that one of these complexes contained STAT1α (p91) by using STAT1α specific antibodies. However, the identity of the other DNA-binding proteins activated by IL-5 remained unknown. One approach to define whether IL-5 can also activate other known members of the STAT family is to reconstitute STAT activation by IL-5 in COS cells. We therefore transiently transfected these cells with expression vectors encoding the human IL-5Rα and IL-5Rβ subunits in combination with expression vectors encoding the different STAT members. These were co-transfected together with a reporter construct containing 2 pIRE sequences coupled to the thymidine kinase (tk) promoter and the luciferase (luc) gene (2xpIREtkluc). We previously identified this pIRE sequence from the ICAM-1 promoter to bind IL-6- and IFN-γ-induced DNA-binding proteins(43Caldenhoven E. Coffer P. Yuan J. van de Stolpe A. Horn F. Kruijer W. van der Saag P.T. J. Biol. Chem. 1994; 269: 21146-21154Abstract Full Text PDF PubMed Google Scholar). We analyzed STAT activation by IL-5 through the measurement of luciferase activity. As can be seen in Fig. 1, COS cells transfected with the pSG5 expression vector without STAT cDNAs showed an IL-5-dependent 2-fold increase in luciferase activity, which is probably due to endogenous STATs in COS cells. Overexpression of hSTAT1α resulted in an IL-5-dependent 2-fold increase of luciferase activity compared with cells transfected with pSG5. Surprisingly, IL-5 also activated hSTAT3, which resulted in a 14-fold increase of luciferase activity. As a control for ligand specificity, we transfected COS cells with the cDNA for STAT3 and treated the cells with IFN-γ. We observed that STAT3 was not activated by IFN-γ, whereas IFN-γ was able to activate STAT1α. 2E. Caldenhoven and R. de Groot, unpublished observation. Furthermore, we observed no effect of IL-5 on luciferase activity after cotransfection of the cDNAs encoding for STAT4, STAT5, and STAT6 (Fig. 1). These results show that IL-5 can activate the transactivation potential of both STAT1α and STAT3 in COS cells.Since DNA-binding is a prerequisite for transactivation, we tested nuclear extracts from untreated and IL-5-stimulated COS cells transfected with STAT3. Indeed, when COS cells were stimulated with IL-5 for 30 min, a single DNA binding complex is induced using the ICAM-1 pIRE as a probe (Fig. 2). This is a specific binding complex because it could be competed with an excess of unlabeled pIRE, whereas a nonspecific competitor (SP1) was unable to compete for binding. To confirm that this DNA-binding protein was STAT3, we preincubated nuclear extracts with antibodies against STAT3. As shown in Fig. 2, this antibody against STAT3 binds to the DNA-protein complex and is therefore not able to migrate into the gel. In contrast, a control preincubation with STAT1α antibodies had no effect on this DNA-protein complex. These results indicate that IL-5 activates STAT3 binding to the ICAM-1 pIRE.Figure 2hIL-5 induces a DNA binding complex containing STAT3. The hIL-5 receptor and STAT3 were expressed in COS cells. These cells were either untreated or treated for 30 min with IL-5, after which nuclear extracts were prepared. Nuclear extract were assayed for binding to the 32P-labeled ICAM-1 pIRE in bandshift experiments. For competition experiments, extracts were preincubated for 5 min with a 50-fold molar excess of unlabeled oligonucleotide as indicated. For identification of the STAT proteins, the extracts were incubated with either anti-STAT1α, anti-STAT3 antibodies, or nonimmune serum for 30 min before the addition of the 32P-labeled ICAM-1 pIRE. IL-5 clearly induces binding of STAT3 to the ICAM-1 pIRE.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We further characterized the DNA-binding specificity of the IL-5-induced STAT3 complex. These experiments were performed with nuclear extracts from COS cells transfected with the IL-5R and STAT3 and treated with IL-5 for 30 min. The ICAM-1 pIRE was used as a radioactive probe, and as competitors we used the IFN-γ activating site elements from the c-fos (SIEm67) (40Fu X.Y. Zhang J.J. Cell. 1993; 74: 1135-1145Abstract Full Text PDF PubMed Scopus (272) Google Scholar) and the FcγRI promoter (GRR)(41Larner A.C. David M. Feldman G.M. Igarashi K. Hackett R.H. Webb D.S. Sweitzer S.M. Petricoin E.F. Finbloom D.S. Science. 1993; 261: 1730-1733Crossref PubMed Scopus (314) Google Scholar). Fig. 3A shows that competition with a 50-fold molar excess of either the pIRE or SIEm67 oligonucleotides completely inhibited binding of STAT3 to the ICAM-1 pIRE, whereas a mutant ICAM-1 pIRE was unable to compete for binding. Competition with the GRR sequence resulted in a decrease of STAT3 binding to the pIRE, suggesting that the pIRE is a better binding site for STAT3 than the GRR. We also tested the transcriptional activity of STAT3 mediated on the GRR sequence. We therefore inserted four copies of the GRR sequence in front of a tkCAT reporter plasmid. This 4xGRRtkCAT reporter construct was transfected in COS cells together with cDNAs encoding the IL-5Rα and βc subunits and STAT3. As shown in Fig. 3B, overexpression of the IL-5R and STAT3 in the presence of IL-5 resulted in a 40-fold induction of luciferase activity mediated via the GRR sequence. The increase in luciferase activity is comparable with the IL-5-induced transactivation of a 4xpIREtkCAT reporter construct. Taken together, these results show that IL-5 is capable of activating STAT3 in COS cells.Figure 3hIL-5 induced STAT3 activity is not restricted to the ICAM-1 pIRE. A, COS cells expressing the hIL-5 receptor and STAT3 were stimulated with IL-5 for 30 min. Nuclear extracts were prepared and incubated with a 32P-labeled ICAM-1 pIRE. For competition experiments, extracts were pre-incubated for 5 min with a 50-fold molar excess of unlabeled oligonucleotide as indicated. IL-5-induced STAT3 can bind to other STAT binding sites. B, COS cells were transient transfected with the IL-5Rα, IL-5Rβ, and STAT3 cDNAs together with either a 4xIREtkCAT or a 4xGRRtkCAT reporter construct. 1 day after transfection, cells were stimulated with IL-5 and harvested after 16 h. Chloramphenicol acetyltransferase assays were performed with β-galactosidase-normalized samples, and -fold induction was calculated relative to untreated cells. Values represent the averages of three different experiments ± S.E. IL-5 induces activation of both IRE- and GRR-containing promoters by STAT3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)IL-5 Induces Tyrosine Phosphorylation and DNA Binding of STAT3 in BaF3/IL5R CellsSince the results described above were obtained using transiently transfected cells overexpressing STAT3, we next addressed the question of whether endogenous STAT3 could be activated by hIL-5. For this purpose, we used the IL-3-dependent murine pro B-cell line BaF3, which we stably transfected with the human IL-5Rα and βc subunits (BaF3/IL5R). Since tyrosine phosphorylation of STAT proteins is rapidly induced after cytokine stimulation, we immunoprecipita" @default.
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• W2014356768 title "Activation of the STAT3/Acute Phase Response Factor Transcription Factor by Interleukin-5" @default.
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