Fig 2: OSM gene expression is induced in hypoxia through a HIF-1α dependent manner. a Thioglycollate-elicited peritoneal macrophages (TEPMs) were isolated from hemtopoietic/endothelial-specific HIF-1α knockout mice (HIF-1α KO) or cre negative littermates as a control (cont). The TEPMs were exposed to hypoxia (1% O2), and the relative expression level of Osm mRNA was analyzed. The one-way ANOVA and Dunnett’s multiple comparisons test were used for the statistical analysis (F (2, 6) = 90.20). †p < 0.05 vs 0 h. The two-way ANOVA and Sidak’s multiple comparison test was performed for the statistical analysis (F (2, 12) = 94.66). *p < 0.05 vs cont. b HIF-1α binding to the promoter of Osm gene was studied by chromatin immunoprecipitation coupled to detection by quantitative PCR. Primer set 1 and 2 were designed to detect the promoter legion (−500 and −170 bp) of Pgk1 gene. Primer set 3 and 4 were designed to detect the hypoxia response element (HRE, −4 kb) of Osm gene. Data show the mean and the standard deviation (effort bar) of technical triplicates from a representative experiment. Quantitative PCR analysis were repeated at least three independent experiments. Two-tailed t-test with Welch’s correction was used for the statistical analysis (Primer 1 (t = 1.527, df = 3.863), Primer 2 (t = 93.69, df = 2.011), Primer 3 (t = 197.7, df = 3.202), Primer 4 (t = 75.41, df = 2.505)). n.s. not statistically significant. *p < 0.05

Fig 3: Oncostatin M from hypoxic MΦ inhibits fibroblast activation. a Supernatants were collected from thioglycollate-elicited peritoneal macrophages (TEPMs) under normoxic or hypoxic condition. After pretreatment with the supernatants, C3H/10T1/2 cells were stimulated with TGF-β1 (2.5 ng per ml, 12 h) and the relative expression of αSMA mRNA was calculated (right). The one-way ANOVA and Dunnett’s multiple comparisons test were used for the statistical analysis (F (2, 6) = 6.019). b We performed transcriptome analysis in isolated primary TEPMs from hematopoietic/endothelial-specific HIF-1α conditional knockout mice (HIF-1αflox/flox; Tie2-cre+/− mice; HIF-1α KO) or cre negative littermate control. Flow chart shows the strategy to identify hypoxia-inducible secretory factors (left). Table illustrates the identified 11 hypoxia-inducible secretory factors (right). c The effect of each hypoxic inducible secretory factor in fibroblasts activation was examined (20 ng per ml for CTGF, CXCL2, CXCL3, LIF and 10 ng per ml for OSM). Thirty minutes after pretreatment with hypoxia-inducible secretory factors, C3H/10T1/2 cells were stimulated with TGF-β1 (2.5 ng per ml, 12 h) and the relative expression of αSMA mRNA was calculated. The one-way ANOVA and Dunnett’s multiple comparisons test were used for the statistical analysis (F (5, 12) = 68.07). d The effect of oncostatin M (OSM) (10 ng per ml) in primary cardiac fibroblasts activation was examined. Thirty minutes after pretreatment with OSM (10 ng per ml, 12 h), C3H/10T1/2 cells were stimulated with TGF-β1 (2.5 ng per ml, 12 h) and the relative expression of αSMA mRNA was calculated. Two-tailed t-test with Welch’s correction was used for the statistical analysis (t = 10.89, df = 2.208). Data show the mean and the standard deviation (error bar) of technical triplicates from a representative experiment. e Immunohistological staining were performed using cardiac tissues from TAC operated mice (day 3). OSM (left), CD11b (right). Scale bar = 50 μm. *p < 0.05

Fig 4: OSM suppresses SMAD signaling. a After pretreatment with the OSM (10 ng per ml) or IL6 (20 ng per ml), C3H/10T1/2 cells were stimulated with TGF-β1 (2.5 ng per ml, 12 h) and the relative expression level of αSMA mRNA was calculated. The one-way ANOVA and Dunnett’s multiple comparisons test were used for the statistical analysis (F (2, 6) = 97.35). b Immunoblot analysis of phospho-ERK1/2, total ERK1/2, phospho-SMAD2 linker (Ser245/250/255) and total SMAD2 protein. Thirty minutes after U0126 (20 nM) pretreatment, C3H/10T1/2 cells were stimulated with OSM (10 ng per ml) or IL6 (20 ng per ml) for 30 min. and collected for the analysis. U0126: a selective MEK1/2 inhibitor. c Immunoblot analysis of phospho-SMAD2 C-terminal (Ser465/467) and Lamin A/C. Thirty minutes after OSM (10 ng per ml) pretreatment, C3H/10T1/2 cells were stimulated with TGF-β1 (2.5 ng per ml, 12 h) and collected at indicated time points. d After pretreatment with U0126 (20 μM, 60 min) and OSM (10 ng per ml, 30 min), C3H/10T1/2 cells were stimulated with TGF-β1 (2.5 ng per ml, 12 h) and the relative expression level of αSMA mRNA was calculated. Two-tailed t-test with Welch’s correction was used for the statistical analysis (t = 3.212, df = 3.172). Data show the mean and the standard deviation (error bar) of technical triplicates from a representative experiment (a, d). *p < 0.05

Fig 5: OSM exposure and OSMR loss differentially affect the expression of genes associated with lipolysis. (a) Fully differentiated 3T3-L1 adipocytes were exposed to 0.5 nM OSM in lipolysis incubation medium for 4 h. Cells were harvested for RNA synthesis and quantitative RT-PCR was performed for the genes indicated. (b) Primary adipocytes isolated from Osmrfl/fl and OsmrFKO mice were fully differentiated and then harvested for RNA synthesis. Quantitative RT-PCR was performed for the genes indicated. This experiment was conducted in duplicate on a single batch of adipocytes. Significant differences are denoted as follows: * p < 0.1, ** p < 0.01, *** p < 0.001, **** p < 0.0001 between groups.