Fig. 3

IL-17A modulates Wnt signaling and expression of Wnt signaling antagonists in calvarial osteoblasts. a Wnt activity, as determined by β-galactosidase absorbance levels, throughout differentiation of TOPGAL reporter calvarial osteoblasts in the presence or absence of IL-17A (50 ng/ml) or TNF (7.5 ng/ml). Data represent the mean ± SD (triplicate wells) for each condition (**p < 0.01, ****p < 0.0001 for TNF compared to same-day control; ⁺⁺ p < 0.01, ⁺⁺⁺⁺ p < 0.0001 for IL-17A compared to same-day control). b Representative secreted frizzled related protein (sFRP)1, sFRP3 and d Dickkopf (DKK)1 mRNA expression assessed by qRT-PCR in calvarial osteoblasts cultured in the presence or absence of IL-17A (5 or 50 ng/ml) over the course of differentiation. mRNA expression relative to undifferentiated osteoblasts (day 0) is shown (n = 3 independent experiments) (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 for IL-17A (5 ng/ml); ⁺ p < 0.05, ⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.001, ⁺⁺⁺⁺ p < 0.0001 for IL-17A (50 ng/ml)). c qRT-PCR showing representative expression of alkaline phosphatase and osteocalcin mRNA in differentiating calvarial osteoblasts cultured in the presence of IL-17A (50 ng/ml) plus anti-sFRP1 antibody (sFRP1 Ab; 1 μg/ml) or rabbit IgG control (IgG; 1 μg/ml) relative to undifferentiated cells (day 0) (n = 2 independent experiments) (⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.001, ⁺⁺⁺⁺ p < 0.0001 for 50 ng/ml IL-17A plus IgG control compared to unstimulated cells; *p < 0.05, ***p < 0.001 for 50 ng/ml IL-17A plus sFRP1 Ab compared to 50 ng/ml IL-17A plus IgG control). e Representative DKK1 mRNA expression assessed by qRT-PCR in calvarial osteoblasts (days 7, 14, and 21 of differentiation) treated with IL-17A (50 ng/ml) for 4, 7, or 12 hours. DKK1 expression relative to untreated osteoblasts (0 hours) is shown (n = 3 independent experiments) (*p < 0.05, **p < 0.01)