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• W2080473403 abstract "Fusion proteins of the extracellular parts of cytokine receptors, also known as cytokine traps, turned out to be promising cytokine inhibitors useful in anti-cytokine therapies. Here we present newly designed cytokine traps for murine and human leukemia inhibitory factor (LIF) as prototypes for inhibitors targeting cytokines that signal through a heterodimer of two signaling receptors of the glycoprotein 130 (gp130) family. LIF signals through a receptor heterodimer of LIF receptor (LIFR) and gp130 and induces the tyrosine phosphorylation of STAT3 leading to target gene expression. The analysis of various receptor fusion and deletion constructs revealed that a truncated form of the murine LIF receptor consisting of the first five extracellular domains was a potent inhibitor for human LIF. For the efficient inhibition of murine LIF, the cytokine-binding module of murine gp130 had to be fused to the first five domains of murine LIFR generating mLIF-RFP (murine LIFR fusion protein). The tyrosine phosphorylation of STAT3 and subsequent gene induction induced by human or murine LIF are completely blocked by the respective inhibitor. Furthermore, both inhibitors are specific and do not alter the bioactivities of the closely related cytokines interleukin (IL)-6 and oncostatin M. The gained knowledge on the construction of LIF inhibitors can be transferred to the design of inhibitors for related cytokines such as IL-31, IL-27, and oncostatin M for the treatment of inflammatory and malignant diseases. Fusion proteins of the extracellular parts of cytokine receptors, also known as cytokine traps, turned out to be promising cytokine inhibitors useful in anti-cytokine therapies. Here we present newly designed cytokine traps for murine and human leukemia inhibitory factor (LIF) as prototypes for inhibitors targeting cytokines that signal through a heterodimer of two signaling receptors of the glycoprotein 130 (gp130) family. LIF signals through a receptor heterodimer of LIF receptor (LIFR) and gp130 and induces the tyrosine phosphorylation of STAT3 leading to target gene expression. The analysis of various receptor fusion and deletion constructs revealed that a truncated form of the murine LIF receptor consisting of the first five extracellular domains was a potent inhibitor for human LIF. For the efficient inhibition of murine LIF, the cytokine-binding module of murine gp130 had to be fused to the first five domains of murine LIFR generating mLIF-RFP (murine LIFR fusion protein). The tyrosine phosphorylation of STAT3 and subsequent gene induction induced by human or murine LIF are completely blocked by the respective inhibitor. Furthermore, both inhibitors are specific and do not alter the bioactivities of the closely related cytokines interleukin (IL)-6 and oncostatin M. The gained knowledge on the construction of LIF inhibitors can be transferred to the design of inhibitors for related cytokines such as IL-31, IL-27, and oncostatin M for the treatment of inflammatory and malignant diseases. Proinflammatory cytokines such as tumor necrosis factor (TNF), 2The abbreviations used are:TNFtumor necrosis factorCBMcytokine-binding moduleDdomainFNIIIfibronectin type III-likeGAPDHglyceraldehyde-3-phosphate dehydrogenasegpglycoproteinhhumanILinterleukinLBPLIF-binding proteinLIFleukemia inhibitory factormmurineMEFmurine embryonic fibroblastsOSMoncostatin MRreceptorSOCSsuppressor of cytokine signalingSTATsignal transducer and activator of transcriptionPBSphosphate-buffered salineELISAenzyme-linked immunosorbent assaysnsupernatant.2The abbreviations used are:TNFtumor necrosis factorCBMcytokine-binding moduleDdomainFNIIIfibronectin type III-likeGAPDHglyceraldehyde-3-phosphate dehydrogenasegpglycoproteinhhumanILinterleukinLBPLIF-binding proteinLIFleukemia inhibitory factormmurineMEFmurine embryonic fibroblastsOSMoncostatin MRreceptorSOCSsuppressor of cytokine signalingSTATsignal transducer and activator of transcriptionPBSphosphate-buffered salineELISAenzyme-linked immunosorbent assaysnsupernatant. interleukin-1β (IL-1β), or interleukin-6 (IL-6) have been identified as promising therapeutic targets in the treatment of chronic inflammation. A dimeric soluble TNF receptor is currently used for the treatment of inflammatory diseases caused by elevated TNF expression (1Goldenberg M.M. Clin. Ther. 1999; 21: 75-87Abstract Full Text PDF PubMed Scopus (131) Google Scholar). Whereas TNF signals through a receptor homotrimer, most cytokines signal through receptor complexes consisting of two or more different receptor subunits. In this case, the respective cytokine can be inhibited by using fusion proteins composed of the different soluble receptors, as we and others showed for the inhibition of IL-6 (2Ancey C. Küster A. Haan S. Herrmann A. Heinrich P.C. Müller-Newen G. J. Biol. Chem. 2003; 278: 16968-16972Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 3Metz S. Wiesinger M. Vogt M. Lauks H. Schmalzing G. Heinrich P.C. Müller-Newen G. J. Biol. Chem. 2007; 282: 1238-1248Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 4Economides A.N. Carpenter L.R. Rudge J.S. Wong V. Koehler-Stec E.M. Hartnett C. Pyles E.A. Xu X. Daly T.J. Young M.R. Fandl J.P. Lee F. Carver S. McNay J. Bailey K. Ramakanth S. Hutabarat R. Huang T.T. Radziejewski C. Yancopoulos G.D. Stahl N. Nat. Med. 2003; 9: 47-52Crossref PubMed Scopus (336) Google Scholar). tumor necrosis factor cytokine-binding module domain fibronectin type III-like glyceraldehyde-3-phosphate dehydrogenase glycoprotein human interleukin LIF-binding protein leukemia inhibitory factor murine murine embryonic fibroblasts oncostatin M receptor suppressor of cytokine signaling signal transducer and activator of transcription phosphate-buffered saline enzyme-linked immunosorbent assay supernatant. tumor necrosis factor cytokine-binding module domain fibronectin type III-like glyceraldehyde-3-phosphate dehydrogenase glycoprotein human interleukin LIF-binding protein leukemia inhibitory factor murine murine embryonic fibroblasts oncostatin M receptor suppressor of cytokine signaling signal transducer and activator of transcription phosphate-buffered saline enzyme-linked immunosorbent assay supernatant. All cytokines signaling through the common receptor subunit gp130 belong to the family of IL-6-type cytokines (5Heinrich P.C. Behrmann I. Müller-Newen G. Schaper F. Graeve L. Biochem. J. 1998; 334: 297-314Crossref PubMed Scopus (1743) Google Scholar), which includes IL-6, IL-11, IL-27, LIF, OSM, ciliary neurotrophic factor, cardiotrophin-1, cardiotophin-like cytokine, and neuropoietin. IL-6-type cytokines contain distinct receptor-binding sites that were discovered by mutagenesis studies on IL-6, ciliary neurotrophic factor, and LIF (6Grötzinger J. Kurapkat G. Wollmer A. Kalai M. Rose-John S. Proteins. 1997; 27: 96-109Crossref PubMed Scopus (96) Google Scholar, 7Simpson R.J. Hammacher A. Smith D.K. Matthews J.M. Ward L.D. Protein Sci. 1997; 6: 929-955Crossref PubMed Scopus (300) Google Scholar, 8van Dam M. Müllberg J. Schooltink H. Stoyan T. Brakenhoff J.P. Graeve L. Heinrich P.C. Rose-John S. J. Biol. Chem. 1993; 268: 15285-15290Abstract Full Text PDF PubMed Google Scholar). The IL-6-type cytokines can be subdivided into those containing three (I, II, and III) or two (II and III) receptor-binding sites. Site I determines the specificity of α-receptor binding. The α-receptor is not capable of transferring the signal into the cell but is crucial for increasing the binding affinity of the cytokine to its signaling receptors. Site II seems to be the universal gp130-binding site of all IL-6-type cytokines. Depending on the cytokine, site III is used for the recruitment of LIFR, OSMR, or a second gp130 molecule (5Heinrich P.C. Behrmann I. Müller-Newen G. Schaper F. Graeve L. Biochem. J. 1998; 334: 297-314Crossref PubMed Scopus (1743) Google Scholar). The IL-6 inhibitor IL-6-RFP (2Ancey C. Küster A. Haan S. Herrmann A. Heinrich P.C. Müller-Newen G. J. Biol. Chem. 2003; 278: 16968-16972Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 3Metz S. Wiesinger M. Vogt M. Lauks H. Schmalzing G. Heinrich P.C. Müller-Newen G. J. Biol. Chem. 2007; 282: 1238-1248Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) was designed to block a cytokine containing all three receptor-binding sites. However, there are also IL-6-type cytokines, which do not need to recruit an α-receptor analogous to human IL-6Rα, and thus do not seem to have a functional site I. One example for a cytokine belonging to this group is the leukemia inhibitory factor (LIF). In this study we present an approach to construct inhibitory receptor fusion proteins for human and murine LIF as prototypes of inhibitors targeting cytokines whose receptors only bind to the site II and III of the cytokine without occupying site I. We designate these inhibitors “site II/III inhibitors.” LIF signals through a heterodimer of LIFR and gp130. Janus tyrosine kinases that are constitutively associated with the cytoplasmic parts of gp130 (9Giese B. Au-Yeung C.K. Herrmann A. Diefenbach S. Haan C. Kuster A. Wortmann S.B. Roderburg C. Heinrich P.C. Behrmann I. Müller-Newen G. J. Biol. Chem. 2003; 278: 39205-39213Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and LIFR (10Hermanns H.M. Radtke S. Haan C. Schmitz-Van de Leur H. Tavernier J. Heinrich P.C. Behrmann I. J. Immunol. 1999; 163: 6651-6658PubMed Google Scholar) are activated upon ligand binding and phosphorylate the receptors and the recruited transcription factor STAT3. Activated STAT3 dimerizes and translocates into the nucleus, where it induces LIF target genes (11Heinrich P.C. Behrmann I. Haan S. Hermanns H.M. Müller-Newen G. Schaper F. Biochem. J. 2003; 374: 1-20Crossref PubMed Scopus (2502) Google Scholar). We wanted to integrate only those receptor domains of gp130 and LIFR into the inhibitory receptor fusion proteins that are necessary for LIF binding. For gp130, which includes six extracellular domains (D1–D6), it has been clearly shown that domains D2 and D3 forming the cytokine-binding module (CBM) are necessary and sufficient for LIF binding (12Boulanger M.J. Bankovich A.J. Kortemme T. Baker D. Garcia K.C. Mol. Cell. 2003; 12: 577-589Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). In contrast, there are contradictory statements in the literature with regard to the domains of LIFR involved in LIF binding. The LIFR is a protein of 190 kDa, which includes eight extracellular domains (D1–D8); these are an N-terminal CBM (D1 and D2), an Ig-like domain (D3), a C-terminal CBM (D4 and D5), and three fibronectin-type III-like (FNIII) domains (D6–D8) (13Gearing D.P. Thut C.J. VandeBos T. Gimpel S.D. Delaney P.B. King J. Price V. Cosman D. Beckmann M.P. EMBO J. 1991; 10: 2839-2848Crossref PubMed Scopus (518) Google Scholar) (see left scheme in Fig. 1A). The LIFR belongs to the family of tall cytokine receptors. Within this family the three membrane-proximal FNIII domains are dispensable for ligand binding, as it was shown by mutagenesis studies of the granulocyte colony-stimulating factor receptor (14Fukunaga R. Ishizaka-Ikeda E. Pan C.X. Seto Y. Nagata S. EMBO J. 1991; 10: 2855-2865Crossref PubMed Scopus (271) Google Scholar) and gp130 (15Horsten U. Schmitz-Van de Leur H. Müllberg J. Heinrich P.C. Rose-John S. FEBS Lett. 1995; 360: 43-46Crossref PubMed Scopus (44) Google Scholar). In the literature, different parts of the LIFR are proposed as the minimal requirement for LIF binding. Apart from a few studies in which the binding between hLIF and mLIFR was investigated (16Layton M.J. Cross B.A. Metcalf D. Ward L.D. Simpson R.J. Nicola N.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8616-8620Crossref PubMed Scopus (130) Google Scholar, 17Owczarek C.M. Zhang Y. Layton M.J. Metcalf D. Roberts B. Nicola N.A. J. Biol. Chem. 1997; 272: 23976-23985Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), most studies focused on the binding of hLIF to hLIFR. Whereas two groups (17Owczarek C.M. Zhang Y. Layton M.J. Metcalf D. Roberts B. Nicola N.A. J. Biol. Chem. 1997; 272: 23976-23985Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 18Voisin M.B. Bitard J. Daburon S. Moreau J.F. Taupin J.L. J. Biol. Chem. 2002; 277: 13682-13692Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar) described that the domains D1–D5 of LIFR are needed to bind LIF, another group assumes that the N-terminal CBM and the Ig-like domain of LIFR are sufficient for a functional LIF receptor complex (19Aasland D. Oppmann B. Grötzinger J. Rose-John S. Kallen K.J. J. Mol. Biol. 2002; 315: 637-646Crossref PubMed Scopus (27) Google Scholar). In contrast, He et al. (20He W. Gong K. Zhu G. Smith D.K. Ip N.Y. FEBS Lett. 2002; 514: 214-218Crossref PubMed Scopus (11) Google Scholar, 21He W. Gong K. Smith D.K. Ip N.Y. FEBS Lett. 2005; 579: 4317-4323Crossref PubMed Scopus (8) Google Scholar) state that LIF only binds to the Ig-like domain and the C-terminal CBM of LIFR. The N-terminal CBM does not bind LIF at all. The N-terminal CBM is required for ciliary neurotrophic factor binding and signaling (20He W. Gong K. Zhu G. Smith D.K. Ip N.Y. FEBS Lett. 2002; 514: 214-218Crossref PubMed Scopus (11) Google Scholar, 21He W. Gong K. Smith D.K. Ip N.Y. FEBS Lett. 2005; 579: 4317-4323Crossref PubMed Scopus (8) Google Scholar). In another study (12Boulanger M.J. Bankovich A.J. Kortemme T. Baker D. Garcia K.C. Mol. Cell. 2003; 12: 577-589Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), only the Ig-like domain of LIFR is proposed to mediate LIF binding to the LIFR. In view of these hypotheses we fused the proposed domains of murine LIFR to the CBM (D2 and D3) of human or murine gp130 to generate putative human or murine LIF inhibitors, respectively. It had already been demonstrated that the natural inhibitor of murine LIF, namely the LIF-binding protein (LBP), a truncated, extracellular form of the mLIFR, binds mLIF with relatively low affinity (about 600–2000 pm) (16Layton M.J. Cross B.A. Metcalf D. Ward L.D. Simpson R.J. Nicola N.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8616-8620Crossref PubMed Scopus (130) Google Scholar, 17Owczarek C.M. Zhang Y. Layton M.J. Metcalf D. Roberts B. Nicola N.A. J. Biol. Chem. 1997; 272: 23976-23985Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) similar to the membrane-bound mLIFR. It was our aim to increase the binding affinity of LBP to mLIF by fusing distinct domains of the mLIFR to the CBM of murine gp130 and thereby generating a high affinity inhibitor for murine LIF. For the construction of potent human LIF inhibitors, we made use of the unusual species cross-reactivity of mLIFR toward human LIF. Although hLIF binds to the hLIFR with a relatively low affinity (about 600–2000 pm), it binds to the mLIFR with a much higher affinity (about 10–20 pm). This phenomenon is primarily mediated by the Ig-like domain (17Owczarek C.M. Zhang Y. Layton M.J. Metcalf D. Roberts B. Nicola N.A. J. Biol. Chem. 1997; 272: 23976-23985Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Therefore, we integrated domains of the mLIFR not only into the possible murine but also into the possible human LIF inhibitors. In this study we describe the development of specific inhibitors for human and murine LIF based on the ligand-binding domains of the corresponding soluble receptors. Cloning of LIF Inhibitors—All LIF inhibitors were constructed in the same way; the desired domains of mLIFR were located behind the N-terminal signal sequence and fused through a peptide linker to D2 and D3 of human or murine gp130. C-terminally a FLAG tag was added. Human LIF inhibitors contained the linker stalk-49 (2Ancey C. Küster A. Haan S. Herrmann A. Heinrich P.C. Müller-Newen G. J. Biol. Chem. 2003; 278: 16968-16972Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) and murine LIF inhibitors the linker AGS-41 (2Ancey C. Küster A. Haan S. Herrmann A. Heinrich P.C. Müller-Newen G. J. Biol. Chem. 2003; 278: 16968-16972Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) consisting of 49 and 41 amino acids, respectively. The domains of the murine LIFR were flanked by XbaI and XmaI restriction sites, the linkers by XmaI and NheI, the respective gp130(D2–D3) fragment by NheI and ApaI, and the FLAG tag by ApaI and BamHI restriction sites. All LIF-RFP constructs were cloned into the eukaryotic expression vector pSVL that contains an SV40 promoter (GE Health-care). For the amplification of human gp130(D2–D3), the plasmid pSVL-hgp130 was used as a template, which contains the complete cDNA of human gp130. For generating constructs containing domains of murine LIFR or murine gp130, whole mRNA isolated from mouse liver was reversely transcribed into cDNA using random primers. The cDNA served as a template for amplifying the desired sequences of mLIFR or mgp130. For cloning of the construct mLIFR(D2–D4)-mgp130(D2–D3), the plasmid pSVL-mLIFR(D2–D4)-hgp130(D2–D3) was cut with XmaI and NdeI, and the released DNA insert was replaced by a fragment derived from pSVL-mLIF-RFP cut with the same enzymes. In the same fashion, mLIFR(D3–D5)-mgp130(D2–D3) was cloned using pSVL-mLIFR(D3–D5)-hgp130(D2–D3) and the restriction enzymes XmaI and SalI. For the stable expression of mLIFR(D1–D5) and mLIF-RFP in Hek293 Flp-In T-Rex expression cell lines (see below), the constructs were subcloned into the expression vector pcDNA5/FRT/TO that contains a doxycycline-inducible cytomegalovirus promoter (Invitrogen). Constructs and primers used for cloning are as follows: pSVL-mLIFR(D1–D3)-hgp130(D2–D3), primers 1–4; pSVL-mLIFR(D3)-hgp130(D2–D3), primers 5–8; pSVL-mLIFR(D1–D5)-hgp130(D2–D3), primers 9 and 10; pSVL-mLIFR(D2–D4)-hgp130(D2–D3), primers 5, 6, 11, and 12; pSVL-mLIFR(D3–D5)-hgp130(D2–D3), primers 5, 6, 7, and 12; pSVL-mLIFR(D1–D5), primers 9 and 15; pSVL-mLIFR(D1–D3)-mgp130(D2–D3), primers 15 and 16; pSVL-mLIFR(D3)-mgp130(D2–D3), primers 15 and 16; pSVL-mLIF-RFP = pSVL-mLIFR(D1–D5)-mgp130(D2–D3), primers 14 and 15; pcDNA5/FRT/TO-mLIF-RFP, primers 17 and 18. The primer sequences are as follows: 1) mLIFRXbaIs 5′-TCTAG AATGG CAGCT TACTC ATGGT G-3′; 2) mLIFRXmaIas 5′-CCCGG GCAGC TTCTG AGGAA CATCG-3′; 3) hgp130D2–D3NheIs 5′-GCTAG CTCAG GCTTG CCTCC AGAAA AACC-3′; 4) hgp130D2–D3ApaIas 5′-GGGCC CTGGT CTATC TTCAT AGGTG ATCCC AC-3′; 5) pSVL1240–1259s 5′-ACAAT GGTGA GACAA GTAGC-3′; 6) SIG(mLIFR)SacIas 5′-GCTAA GGAGC TCACC GTTTG CATGC ATCGT CAG-3′; 7) mLIFRD3SacIs 5′-GCTAA GGAGC TCGAG ACTAA TGTTT TTCCT CAAGA C-3′; 8) hgp130D2as 5′-AGGTT TTTCT GGAGG CAAGC CTGA-3′; 9) mLIFRD1–D5XbaIs 5′-GCTAA GTCTA GAATG GCAGC TTACT CATGG TGG-3′; 10) mLIFRD1–D5XmaIas 5′-CTTAG CCCCG GGGTC TGGTC CCTTT GAAGG AG-3′; 11) mLIFRD2–D4SacIs 5′-GCTAA GGAGC TCCCA GAGAC TCCCG AGATC CTG-3′; 12) mLIFRD2–D4XmaIas 5′-AGCCT TCCCG GGCGA AGTCG GATCA TGAGG AGC-3′; 13) mLIFRD1–D5ApaIas 5′-CTTAG CGGGC CCGTC TGGTC CCTTT GAAGG AG-3′; 14) mgp130D2–D3NheIs 5′-GCTAA GGCTA GCTCA GGCTT TCCTC CAGAT AAACC-3′; 15 mgp130D2–D3ApaIas 5′-CTTAG CGGGC CCTGG TCTGT CTTCG TATGT GG-3′; 16) AGS-41XmaIs 5′-TTCAA AGGGA CCAGA CCCCG GGGGA AGTG-3′; 17) mLIFRFPs 5′-GCTAA GCTCG AGTCT AGAAT GGCAG CTTAC TCATG G-3′; and 18) mLIFRFPas 5′-CTTAG CGATA TCGGA TCCTC ACTTG TCATC GTCGT C-3′. Cytokines, Cytokine Receptors—Human and murine LIF was purchased from Chemicon International (Temecula, CA) and murine OSM from R & D Systems (Minneapolis, MN). Recombinant human IL-6 was expressed in Escherichia coli, refolded, and purified as described (22Arcone R. Pucci P. Zappacosta F. Fontaine V. Malorni A. Marino G. Ciliberto G. Eur. J. Biochem. 1991; 198: 541-547Crossref PubMed Scopus (129) Google Scholar). The specific activity of IL-6 was measured in the B9 cell proliferation assay (23Aarden L.A. De Groot E.R. Schaap O.L. Lansdorp P.M. Eur. J. Immunol. 1987; 17: 1411-1416Crossref PubMed Scopus (1013) Google Scholar). sIL-6Rα was expressed in insect cells and purified as described previously (24Weiergräber O. Hemmann U. Küster A. Müller-Newen G. Schneider J. Rose-John S. Kurschat P. Brakenhoff J.P. Hart M.H. Stabel S. Heinrich P.C. Eur. J. Biochem. (Tokyo). 1995; 234: 661-669Crossref PubMed Scopus (85) Google Scholar). Cell Culture of MEF and COS-7 Cells and Transfection of COS-7 Cells—COS-7 simian monkey kidney cells (kindly provided by I. M. Kerr, Cancer Research UK, London) and murine embryonic fibroblasts (MEF) (kindly provided by B. Neel, Boston) were grown in Dulbecco's modified Eagle's medium with GlutaMAX™-I, 4.5 g/liter glucose and pyruvate (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (Cytogen, Princeton, NJ), 100 μg/ml streptomycin, and 100 units/ml penicillin (Cambrex BioScience, Verviers, Belgium). Cells were grown at 37 °C in a water-saturated atmosphere in 5% CO2. Plasmids were transiently transfected into COS-7 cells using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's instructions. Cell Culture and Transfection of HepG2 Cells—HepG2 human hepatoma cells (purchased from ATCC, Manassas, VA) were grown in Dulbecco's modified Eagle's medium F-12 1:1 mix with GlutaMAX™-I (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum, 100 μg/ml streptomycin, and 100 units/ml penicillin. Cells were grown at 37 °C in a water-saturated atmosphere in 5% CO2. Plasmids were transiently transfected into HepG2 cells using FuGENE6 (Roche Applied Science) according to the manufacturer's instructions. Preparation of Cell Lysates, SDS-PAGE, Western Blotting, and Immunodetection—COS-7 cells were transiently transfected with expression plasmids (pSVL) coding for the respective LIF inhibitor and grown for 48 h to allow protein production. Subsequently cells were lysed with radioimmune precipitation assay lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40, 1 mm NaF, 15% glycerol, 20 mm β-glycerophosphate, 1 mm Na3VO4, 0.25 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, and 1 μg/ml leupeptin). The lysates were analyzed with SDS-PAGE, Western blotting, and immunodetection using a 1:1,000 diluted antibody directed against the FLAG tag (Sigma). Coimmunoprecipitation Studies—COS-7 cells were transiently transfected with expression plasmids (pSVL) coding for the respective LIF inhibitor and grown for 48 h to allow protein production. Afterward cell supernatants were harvested and cleaned by centrifugation and sterile filtration. For immunoprecipitation the supernatants were incubated overnight at 4 °C with a FLAG-specific antibody (Sigma), which was previously immobilized to protein A-Sepharose (Amersham Biosciences) through a mouse antibody (Dako, Hamburg, Germany). The next day, the Sepharose with precipitated LIF inhibitors was centrifuged and incubated overnight at 4 °C in PBS containing 50 nm human LIF or murine LIF to allow coimmunoprecipitation. Proteins were eluted from the Sepharose with 2× Laemmli buffer and analyzed with SDS-PAGE, Western blotting, and immunodetection using a 1:1,000 diluted antibody directed against human LIF (Biodesign, Saco, ME) or murine LIF (Sigma). After stripping the membrane in stripping buffer (2% SDS, 62.5 mm Tris-HCl, pH 6.7, 76 μl of β-mercaptoethanol per 10 ml) for 25 min at 70 °C, a second immunodetection was performed using a FLAG antibody. Generation of Stable HEK293 Flp-In T-Rex Expression Cell Lines for the Production of LIF-RFPs—For production of the LIF inhibitors, the Flp-In T-Rex protein expression system (Invitrogen) was used. The generation of stable HEK293 Flp-In T-Rex expression cell lines for LIF inhibitors was performed according to the manufacturer's instructions. In brief, HEK293 Flp-In T-Rex host cells were cotransfected with 3.6 μg of the plasmid pOG44 (Invitrogen) coding for the Flp-In recombinase and 0.4 μg of the plasmid pcDNA5/FRT/TO (Invitrogen) containing the coding sequence for the desired LIF inhibitor. Cotransfection was carried out by using FuGENE 6 according to the manufacturer's instructions. 48 h after transfection stably transfected cell were selected with 50–200 μg/ml hygromycin B (Perbio/HyClone, Logan, UT). Single clones were expanded and treated with 10 ng/ml doxycycline (Sigma) for 24 h without serum and hygromycin B to induce protein production. The supernatants were cleaned by centrifugation and sterile filtration, and the production of the respective LIF-RFP was checked by SDS-PAGE, Western blotting, and immunodetection using a FLAG antibody. Concentration of LIF-RFPs—After doxycycline treatment, supernatants of the respective HEK293 Flp-In T-Rex expression cells were harvested, cleaned by centrifugation and sterile filtration, and concentrated about 20-fold using Vivaspin concentrators (Vivascience AG, Hannover, Germany). The same was done with supernatants from control HEK293 Flp-In T-Rex cells, which were stably transfected with an empty vector. ELISA-based Binding Assay—ELISA plates with a polystyrene surface (Nunc) were coated with mLIF (50 ng/well) overnight. Blocking of free binding sites was carried out using PBS supplemented with 3% bovine serum albumin and 10% fetal calf serum for 30 min. After three washing steps with 250 μl of PBS containing 0.005% Tween 20, the wells were incubated with varying concentrations of the indicated FLAG-tagged LIF inhibitor or control supernatant and a FLAG antibody (20 ng/well, Sigma) for 3.5 h. The plates were washed three times with 250 μl of PBS/Tween and incubated with a horseradish peroxidase-conjugated secondary antibody (20 ng/well) for 30 min. After three washing steps the horseradish peroxidase-catalyzed color reaction was initiated using a 0.1 m sodium acetate solution, pH 5.5, containing 100 μg/ml tetramethylbenzidine and 0.003% H2O2. The reaction was stopped by the addition of 2 m H2SO4. The absorption was measured with an ELISA reader. All incubation steps were carried out at room temperature. STAT3 Tyrosine Phosphorylation in MEF Cells—For investigation of STAT3 tyrosine phosphorylation, MEF cells were grown on 6-well plates (9.6 cm2/well) and stimulated for 20 min with hLIF, mLIF, hIL-6/shIL-6Rα, or mOSM or the combination of cytokine and LIF-RFP in the absence of serum. Subsequently, the cells were lysed with radioimmune precipitation assay lysis buffer (see above). The lysates were analyzed with SDS-PAGE, Western blotting, and immunodetection using an antibody directed against phosphotyrosine (705)-STAT3 (Cell Signaling, Danvers, MA) or STAT3 (H190, Santa Cruz Biotechnology, Santa Cruz, CA). Both antibodies were used in a 1:1,000 dilution. Reporter Gene Assay in HepG2 Cells—HepG2 cells were seeded onto 6-well plates (9.6 cm2/well) and transiently cotransfected with pGL3-α2M-Luc (construct with luciferase gene regulated by the STAT3-responsive α2-macroglobulin promoter) and pCRTM3-lacZ (β-galactosidase construct with a constitutively active promoter; Amersham Biosciences). Cells were stimulated with either 5 ng/ml hLIF and an appropriate volume of control supernatant (see above) or the combination of hLIF and mLIFR(D1–D5) for 16 h at molar ratios indicated. Preparation of cellular lysates and luciferase measurements were carried out according to the instructions of the manufacturer (Promega, Madison, WI). The luciferase activity values were normalized to the transfection efficiency that was determined as β-galactosidase activity. The experiments were carried out in triplicate, and the mean values and standard deviations were calculated. SOCS3 mRNA Levels in MEF Cells—MEF cells were seeded onto 6-well plates and stimulated with either 5 ng/ml mLIF and an appropriate volume of control supernatant or the combination of mLIF and mLIF-RFP for 30 min at the molar ratios indicated. Subsequently, mRNA was isolated using the RNeasy mini kit (Qiagen GmbH, Hilden, Germany). The SOCS3 and GAPDH mRNAs were amplified with the One-step RT-PCR kit (Qiagen) utilizing sequence-specific primers and analyzed with gel electrophoresis. Primers for RT-PCR are as follows: mouse SOCS3, 5′-GGGTG GCAAA GAAAA GGAG-3′ and 5′-GTTGA GCGTC AAGAC CCAGT-3′; mouse GAPDH, 5′-ACCAC AGTCC ATGCC ATCAC-3′ and 5′-TCCAC CACCC TGTTG CTGTA-3′ (25Ogata H. Kobayashi T. Chinen T. Takaki H. Sanada T. Minoda Y. Koga K. Takaesu G. Maehara Y. Iida M. Yoshimura A. Gastroenterology. 2006; 131: 179-193Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Design and Expression of Different Fusion Proteins of LIFR and gp130 as Potential LIF Inhibitors—The potential human and murine LIF inhibitors were designed to contain the minimal parts of LIFR and gp130 required for high affinity LIF binding. Because the domains of LIFR necessary for LIF binding are controversially discussed, we constructed six possible inhibitors for human LIF containing the proposed domains of mLIFR needed for LIF binding (Fig. 1A). The five receptor fusion constructs for the inhibition of hLIF contain the CBM (D2–D3) of human gp130, which was shown to be sufficient for LIF binding (12Boulanger M.J. Bankovich A.J. Kortemme T. Baker D. Garcia K.C. Mol. Cell. 2003; 12: 577-589Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). To investigate the role of hgp130 in a human LIF inhibitor containing mLIFR, we constructed the LIF inhibitor mLIFR(D1–D5) consisting of D1–D5 of mLIFR, which was lacking the CBM (D2–D3) of human gp130 and the linker. Accordingly, five potential murine LIF inhibitors were constructed by fusing the respective part of the mLIFR to the CBM of murine gp130. The two receptor fragments in the fusion proteins were connected by flexible linkers. For technical reasons, a FLAG tag was added to the C termini of all constructs. The potential human (Fig. 1B) and murine (Fig. 1C) LIF inhibitors were expressed in COS-7 cells, the lysates of which were analyzed by SDS-PAGE, Western blotting, and immunodete" @default.
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• W2080473403 title "Novel Inhibitors for Murine and Human Leukemia Inhibitory Factor Based on Fused Soluble Receptors" @default.
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