No increase in the STAT3 phosphorylation with increasing mOSM concentrations is seen in the mock lanes because at 5 ng/ml mOSM the cellular response is already saturated. To test the inhibitory activity of mOSM-RFP on sustained STAT3 activation Fao rat hepatoma cells were stimulated with mOSM for prolonged periods of times (Determine ?(Figure2C).2C). Conclusions mOSM-RFP consisting of D1-D4 of mOSMR and D2-D3 of mgp130 is usually a highly potent and specific inhibitor of mOSM. Since mOSM-RFP is usually encoded by a single gene it offers numerous possibilities for specific cytokine inhibition in gene delivery methods based on viral vectors, transgenic animals and finally gene therapy. Background Cytokines are central mediators of the immune system. Anti-cytokine therapies are aimed at the specific inhibition Vincristine sulfate of a cytokine that has been identified to be critically involved in the initiation, maintenance or progression of a disease. Most cytokines signal through heteromeric receptors consisting of two different receptor chains. We have developed a new class of cytokine inhibitors based on the fusion of the ligand-binding domains of cytokine receptors by a flexible linker [1]. The prototypic receptor fusion protein (RFP) directed against human interleukin-6 (hIL-6-RFP) turned out to be a highly specific and highly potent inhibitor of hIL-6 [2]. Based on this original approach further RFP have been generated by others for the inhibition of human oncostatin M [3] and most recently human interleukin-31 [4]. In a different but related approach so called cytokine traps have been generated by the fusion of soluble receptors through Fc-fragments [5]. For the validation of the RFP approach in murine animal models in vivo RFP directed against murine cytokines are required. RFPs based on human Rabbit Polyclonal to TALL-2 receptor proteins are not useful for this purpose because murine cytokines usually do not bind to the human receptors. Therefore, we concentrated on the generation of receptor fusion proteins for the inhibition of murine cytokines. We described Vincristine sulfate mLIF-RFP [6] for the inhibition of murine leukemia inhibitory factor (mLIF) and recently mIL-6-RFP [7] for the inhibition of murine IL-6 (mIL-6). Oncostatin M (OSM) is a pro-inflammatory cytokine of the IL-6 family implicated in rheumatoid arthritis [8], lung fibrosis [9] and skin disease [10]. OSM is secreted by activated T-cells [11], macrophages [12], neutrophils [13] and synovial fibroblasts from patients with rheumatoid arthritis [14]. The murine OSM receptor consists of two receptor proteins [15], the OSM-specific OSMR and gp130, Vincristine sulfate the common signalling receptor subunit of the IL-6 family of cytokines. OSM signals through the Jak/STAT pathway resulting in the activation of STAT3 and STAT5. ERK1/2 and p38 MAP kinases are also activated in response to OSM [16]. Here we describe the generation of a novel inhibitor for murine OSM, mOSM-RFP, that is based on the fusion of murine OSMR and murine gp130 fragments. mOSM-RFP will be a useful tool for the investigation of the role of OSM in murine models of human diseases. Results 1. Design and expression of murine oncostatin M receptor fusion proteins (mOSM-RFPs) We generated four different murine oncostatin M receptor fusion proteins (mOSM-RFPs) (Figure ?(Figure1A).1A). The first protein (mOSM-RFP) is built up in analogy to the recently published receptor fusion protein for the inhibition of murine LIF (mLIF-RFP) [6]. It consists of the four N-terminal domains of the murine OSM receptor Vincristine sulfate (mOSMR) and domains D2 and D3 of murine gp130 (mgp130) connected by a flexible polypeptide linker. We [17] and others [18] have shown that the N-terminal domain D1 of gp130 is dispensable for signal transduction in response to OSM. Another report suggests a functional role of D1 of gp130 in OSM-binding [19]. Moreover, we have shown that the addition of a single domain, even if not involved in ligand-binding, can strongly enhance the expression of a receptor fusion protein [7]. Therefore, we decided to construct another fusion protein that includes D1 of mgp130 (mOSM-RFP+D1, Figure ?Figure1A).1A). To assess the importance of the order of the receptor fragments we also constructed inverted receptor fusion proteins with the mgp130 fragment preceding the mOSMR fragment (i-mOSM-RFP and.