Figure 5.

OSM and, to a lesser extent, LIF stimulate bone resorption and RANKL production in calvarial bones, calvarial osteoblasts and stromal cells. (A) Mouse calvarial bones were cultured in the absence or the presence of LIF or OSM (both 0.1–100 ng/mL) and bone resorption was assessed by ⁴⁵Ca release after a 5-day culture period. (B) RT-qPCR was performed using mRNA extracted from calvarial bones treated with either LIF or OSM (both at 100 ng/mL) for 24 h to assess the expression of Tnfsf11. (C) Protein expression of RANKL after 48 h was also analyzed in calvarial bone treated with LIF or OSM (both at 100 ng/L). (D) Mouse calvarial osteoblasts were incubated in the absence (Co) or the presence of LIF (100 ng/mL) or OSM (100 ng/mL) for 48 h and expression of Tnfsf11 was analyzed by semi-quantitative RT-PCR. (E) The expression of Tnfsf11 mRNA in calvarial osteoblasts stimulated by LIF and OSM a different concentrations (0.1-100 ng/mL) was performed using quantitative RT-PCR. (F) The mRNA expression of Osmr and Lifr in osteoblasts was compared at three different time points. (G) The receptor components Il6st, Lifr and Osmr are expressed in ST-2 stromal cells as assessed by RT-PCR. (H) The mRNA expression of Tnfsf11 in ST-2 cells cultured without (Co) or with LIF or OSM (both at 100 ng/mL) for 48 h was analyzed. Values represent means for six bones (calvarial bones) or four wells (cell culture experiments) and SEM is shown as vertical bars. ^*, ^**, and ^***, indicate significant difference compared to untreated (Co) cells, ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001, respectively. Statistical significance was determined by ANOVA using Levene's homogeneity test followed by Dunnett's T3 post-hoc tests vs. Co. In (F), Tukey post-hoc test was used to compare all groups and no statistical difference was observed.