Journal: The Journal of Biological Chemistry

Article Title: Engineered interleukin-6-derived cytokines recruit artificial receptor complexes and disclose CNTF signaling via the OSMR

doi: 10.1016/j.jbc.2024.107251

Figure Lengend Snippet: GIL-6 induces JAK/STAT signaling and cellular proliferation via non-natural cytokine receptor complexes. A , proliferation of Ba/F3-gp130, Ba/F3-IL-6R:gp130, Ba/F3-gp130:OSMR, Ba/F3-gp130:LIFR, Ba/F3-IL-6R:gp130:OSMR, Ba/F3-IL-6R:gp130:OSMR, Ba/F3-IL-6R:gp130:LIFR cells without cytokine (−), with 100 ng/ml HIL-6, 10 ng/ml IL-6, 10 ng/ml LIF, 10 ng/ml OSM, 100 ng/ml GIL-6, 100 ng/ml GIO-6 and 100 ng/ml IC7. Data represents mean±S.D. of three independent experiments. For statistics the treated groups were compared with the untreated group by two-way ANOVA including Dunnet’s test for correction in multiple comparison. B , STAT3 activation in Ba/F3, Ba/F3-gp130, Ba/F3-IL-6R:gp130, Ba/F3-gp130:OSMR, Ba/F3-gp130:LIFR, Ba/F3-IL-6R:gp130:OSMR, Ba/F3-IL-6R:gp130:LIFR cells without cytokine (−) and after stimulation with 100 ng/ml HIL-6, 10 ng/ml IL-6, 10 ng/ml LIF, 10 ng/ml OSM, 100 ng/ml GIL-6, 100 ng/ml GIO-6 and 100 ng/ml IC7 for 20 min. C , STAT1, STAT3, STAT5, ERK, and Akt activation in Ba/F3-IL-6R:gp130:LIFR cells with the same conditions as for the STAT3 activation. D , STAT3 activation in heart, liver, and spleen after injection of 20 μg GIL-6 or GIO-6. Mice were sacrificed 30 min after intraperitoneal cytokine injection. Equal amounts of proteins (50 μg/lane) were analyzed via specific antibodies detecting phospho-STAT3 and STAT3. Western blot data shows one representative experiment out of three. E , schematic illustration of IC7. Site 3 of IL-6 ( red ) comprising site 3-1, 3-2, and 3-3 was exchanged by site III of CNTF ( grey ) resulting in IC7. Consequently, IC7 forms tetrameric IC7:IL-6R:gp130:LIFR or IC7:IL-6R:gp130:OSMR complexes.

Article Snippet: Recombinant human OSM (catalog no.295-OM), LIF (catalog no. 7734-LF), and CNTF (catalog no. 257-NT) were purchased from R&D Systems (Minneapolis).

Techniques: Comparison, Activation Assay, Injection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Engineered interleukin-6-derived cytokines recruit artificial receptor complexes and disclose CNTF signaling via the OSMR

doi: 10.1016/j.jbc.2024.107251

Figure Lengend Snippet: CNTF signals via the alternative CNTFR:gp130:OSMR complex but not via IL-6R:gp130:OSMR. A , proliferation of Ba/F3-CNTFR:gp130:LIFR or Ba/F3-CNTFR:gp130:OSMR cells with increasing concentrations of CNTF (0.0002–100 ng/ml). One representative experiment out of four is shown. B , proliferation of Ba/F3-gp130:LIFR or Ba/F3-gp130:OSMR cells in the presence of fixed concentrations of sIL-6R (100 ng/ml) and increasing concentrations of CNTF (0.05–100 ng/ml). One representative experiment out of three is shown. C and D , CNTF dose-dependent STAT3 activation of Ba/F3-CNTFR:gp130:LIFR ( C ), Ba/F3-CNTFR:gp130:OSMR ( D ) cells without cytokine (−) or in the presence of 10 ng/ml LIF or OSM, or with 0.1, 1, 10 or 100 ng/ml CNTF for 20 min. Equal amounts of proteins (50 μg/lane) were analyzed via specific antibodies detecting phospho-STAT3 and STAT3. Western blot data shows one representative experiment out of three. E , proliferation of Ba/F3-gp130, Ba/F3-CNTFR:gp130:LIFR, Ba/F3-CNTFR:gp130:OSMR, Ba/F3-IL-6R:gp130:LIFR or Ba/F3-IL-6R:gp130:OSMR cells without cytokine (−) or in the presence of CNTF (0.5, 5 or 50 ng/ml), LIF (10 ng/ml), OSM (10 ng/ml) or IL-6 (10 ng/ml). Data represents mean ± S.D. of three independent experiments. For statistics, the treated groups were compared with the untreated group by two-way ANOVA including Dunnet’s test for correction in multiple comparisons. F , STAT3 activation Ba/F3-CNTFR:gp130:OSMR cells without cytokine (−), 10 ng/ml LIF, 10 ng/ml CNTF or 10 ng/ml OSM. Equal amounts of proteins (50 μg/lane) were analyzed via specific antibodies detecting phospho-STAT3 and STAT3. Western blot data shows one representative experiment out of three. G and H , CNTF dose-dependent STAT3 activation of Ba/F3-IL-6R:gp130:LIFR ( G ) or Ba/F3-IL-6R:gp130:OSMR ( H ) cells without cytokine (−) or in the presence of 10 ng/ml LIF or OSM, or with 0.1, 1, 10 or 100 ng/ml CNTF for 20 min. Equal amounts of proteins (50 μg/lane) were analyzed via specific antibodies detecting phospho-STAT3 and STAT3. Western blot data shows one representative experiment out of three. I , proliferation of Ba/F3-gp130:LIFR or Ba/F3-gp130:OSMR cells in the presence of increasing concentrations of Hyper-CNTF-IL-6R-Fc (0.02–8000 ng/ml). One representative experiment out of three is shown. J , schematic illustration of CNTF in tetrameric complexes consisting of CNTF:CNTFR:gp130:LIFR, CNTF:CNTFR:gp130:OSMR or CNTF:IL-6R:gp130:LIFR but not CNTF:IL-6R:gp130:OSMR.

Article Snippet: Recombinant human OSM (catalog no.295-OM), LIF (catalog no. 7734-LF), and CNTF (catalog no. 257-NT) were purchased from R&D Systems (Minneapolis).

Techniques: Activation Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Engineered interleukin-6-derived cytokines recruit artificial receptor complexes and disclose CNTF signaling via the OSMR

doi: 10.1016/j.jbc.2024.107251

Figure Lengend Snippet: CNTF recruits gp130 prior to OSMR. A , Western blotting of co-immunoprecipitation by using Protein A beads to precipitate recombinant Hyper-CNTF-Fc (2 μg), soluble OSMR (1 μg) in the presence or absence of biotinylated gp130 (1 μg). B , binding scheme of Hyper-CNTF. First Hyper-CNTF binds via site 2 to D2/D3 of gp130. The complex of Hyper-CNTF and gp130 binds hypothetically via site 3 to D2/D3 of OSMR. Hyper-CNTF is not able to bind the OSMR before recruiting gp130.

Article Snippet: Recombinant human OSM (catalog no.295-OM), LIF (catalog no. 7734-LF), and CNTF (catalog no. 257-NT) were purchased from R&D Systems (Minneapolis).

Techniques: Western Blot, Immunoprecipitation, Recombinant, Binding Assay