Oncostatin M (OSM) is a prominent member of the IL-6 cytokine family. It signals through a heterodimeric OSM receptor/gp130 (OSMR) complex, specific for thiscytokine and driving its many biological effects. Especially interesting are its potential therapeutic applications, as OSM has been beneficial in several murine cardiovascular disease models. Unfortunately, the translatability of these results to the human situation is not assured due to the different receptor activation abilities of human and mouse OSM (hOSM/mOSM). While mOSM is specific for the OSMR, hOSM signals through the leukemia inhibitory factor receptor/gp130 (LIFR) in addition to the OSMR, which could result in additional effects besides those reflected in preclinical mouse models. Taken together, there is a clear need for more information on these ligand-receptor interactions, the exclusive features of OSM governing OSMR activation and the divergence between hOSM and mOSM.This thesis contributes new insights into these topics by means of molecular biology tools: in the first place, a mammalian expression system was established to obtain recombinant hOSM, mOSM and human leukemia inhibitory factor (hLIF) as well as an array of mutant variants of these cytokines. Cytokine stimulation experiments were then carried out in cell lines with specific readouts for each receptor complex, such as A375 cells for hOSMR, JAR cells for hLIFR, NIH3T3 cells for mOSMR or MH-S cells for mLIFR. Both shorter- and longer-term readouts were used, monitoring the phosphorylation of a key downstream signaling component (STAT3) 10 minutes after stimulation and measuring expression levels of well-known target genes (e.g. TIMP1) after 24 hours.In a first approach, different binding site regions in hOSM or hLIF were exchanged to create hOSM/hLIF chimeras. Cell-stimulation experiments using these chimeric variants pointed to the N-terminal AB loop and N-terminal helix D regions of hOSM as responsible for hOSMR signaling, enabling hOSMR activation by a hLIF-based chimera with these two hOSM regions. Subsequently, site-directed mutagenesis studies individually targeting all the residues within these regions identified the amino acids Y34, Q38, G39, L45 and P153 as critical for hOSMR activation. Similar methods were applied to study the receptor specificity of mOSM, which was mapped to its N-terminal AB loop by means of chimeric mouse/human OSM cytokines. In this case, point mutagenesis experiments revealed residues N37, T40 and D42 of the murine cytokine to be responsible for mOSMR specificity. This specificity was lost upon replacing these amino acids by their hOSM counterparts, resulting in a mOSM variant able to activate mLIFR as well as mOSMR. Finally, phylogenetic analyses indicated that the acquisition of specificity likely constitutes the final stage in OSM evolution, as it is associated with a larger evolutionary distance from the ancestral form of the protein.In sum, new key features of OSM enabling human and murine OSMR signaling have been identified and characterized in detail by using a variety of recombinant mutant cytokines. These results illustrate a novel path for cytokine evolution within the IL-6 family, in which an initial gene duplication event in the LIF/OSM ancestral gene was followed first by the acquisition of a new function (OSMR activation) and finally by the loss of the ancestral function (LIFR activation) to attain specificity. In addition, some of the mOSM mutants generated are expected to improve the translatability of future research on the therapeutic applications of OSM. Given that these mutant variants possess a human-like receptor signaling profile, their use in murine preclinical disease models will more closely reflect the potential effect of hOSM in the human situation, with LIFR being activated along with the OSMR.
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