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.
Highlights
Receptor homotrimer, most cytokines signal through receptor complexes consisting of two or more different receptor subunits
Design and Expression of Different Fusion Proteins of LIF receptor (LIFR) and gp130 as Potential leukemia inhibitory factor (LIF) Inhibitors—The antibody directed against phosphotyrosine [705]-STAT3 (Cell potential human and murine LIF inhibitors were designed to Signaling, Danvers, MA) or STAT3 (H190, Santa Cruz Biotech- contain the minimal parts of LIFR and gp130 required for high nology, Santa Cruz, CA)
LIF binding are controversially discussed, we constructed six Reporter Gene Assay in HepG2 Cells—HepG2 cells were possible inhibitors for human LIF containing the proposed seeded onto 6-well plates (9.6 cm2/well) and transiently domains of mLIFR needed for LIF binding (Fig. 1A)
Summary
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. 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). 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. 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. The generation of stable HEK293 Flp-In T-Rex expression cell lines for LIF inhibitors was performed according to the manufacturer’s instructions. 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. Primers for RT-PCR are as follows: mouse SOCS3, 5Ј-GGGTG GCAAA GAAAA GGAG-3Ј and 5Ј-GTTGA GCGTC AAGAC CCAGT-3Ј; mouse GAPDH, 5Ј-ACCAC AGTCC ATGCC ATCAC-3Ј and 5Ј-TCCAC CACCC TGTTG CTGTA-3Ј [25]
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