Figure 2

Effect of WFA on osteoblast signaling. (a) Proteasomal effect of WFA in calvarial osteoblast cells. 20S proteasome activities were measured 1, 3, 6 h after treatment with WFA. Values represent mean±S.E. of three (n=3) independent experiments. ***P<0.001, *P<0.001 compared with control. (b) Lower panel shows that 6 h of treatment with WFA induces ubiquitination of proteins through western blot. Protein ladders were detected by anti-ubiquitin antibody. (c) qPCR data of the osteogenic genes after WFA treatment. WFA targets BMP2 signaling by inducing mRNA levels of BMP2 responsive genes in osteoblast cells after 24 h of treatment. Data represent mean±S.E. of three independent experiments (n=3). *P<0.05, **P<0.01 ***P<0.001 when compared with control. (d) Western blot analysis of various osteogenic genes BMP2, Smad1 and its phosphorylation state in the presence of WFA. Expression of Smurf1, Smurf2 a negative regulator of osteogenesis is also shown. All blots were normalized with β-actin. (e) Densitometric analyses of western blot (d) showing fold change. Fold increase was calculated relative to control vehicle-treated cells. The values are the mean±S.E. of three experiments. ***P<0.001 versus vehicle-treated group. (f) Interaction of Smurf2 with Smad1 and RunX2 in the presence of WFA. Immuno-precipitation (IP) assays were performed using anti-Smurf antibody followed by western blot using anti-Smad1 and anti-RunX2 antibodies. (g) WFA prevents RunX2 ubiquitination and proteosomal degradation. Primary osteoblast cells were treated with 5 ng/ml TNFα for 24 h in the presence or absence of WFA, and endogenous RunX2 was immunoprecipated by anti- RunX2 antibody. Ubiquitinated RunX2 (Ub-RunX2) protein ladders were detected by anti-ubiquitin antibody (upper panel). After stripping the antibody, total un-ubiquitinated RunX2 protein levels were determined by anti- RunX2 antibody (lower panel). MW, molecular mass. (h) Western blot of RunX2 and Smurf2 in the presence of various treatments. All blots were normalized with housekeeping gene β-actin. (i) Densitometric analyses of western blot (h) showing fold change. Fold increase was calculated relative to control vehicle-treated cells. The values are the mean±S.E. of three experiments. *P<0.05, **P<0.01 ***P<0.001 versus vehicle -treated group. (j) Relative expression of Smurf2 SiRNA. Osteoblast cells were treated with Smurf2 siRNAs and mRNA levels were measured by real-time RT-PCR. The values are the mean±S.E. of three experiments. **P<0.01versus the vehicle-treated group. (k) Smurf2 knockdown reduced degradation of RunX2. Expression of RunX2 and Smurf2 was determined by western blot analysis after the osteoblast cells were treated with Smurf2 siRNA in the presence and absence of WFA and Bzb. (l) Densitometric analyses of western blot (k) showing fold change. Fold increase was calculated relative to control vehicle-treated cells. The values are the mean±S.E. of three experiments. *P<0.05, **P<0.01 ***P<0.001 versus vehicle (vehicle-treated) group. (m) Smurf2 siRNA blocks TNF-induced RunX2 degradation. Osteoblast cells were treated with Smurf2 siRNA and then treated with (5 ng/ml) TNF for 24 h. Smurf2 and RunX2 expression was determined by western blot analysis. (n) TNF-induced ubiquitination of RunX2 is reversed by Smurf2 knockdown. Endogenous Smurf2 was knocked down in primary osteoblast cells then treated with 5 ng/ml TNFα for 24 h and endogenous RunX2 was immunoprecipated by anti-RunX2 antibody. Ubiquitinated RunX2 (Ub-RunX2) protein ladders were detected by anti-ubiquitin antibody (upper panel). After stripping the antibody, total un-ubiquitinated RunX2 protein levels were determined by anti-RunX2 antibody (lower panel). MW, molecular mass. (o) Smurf2 knockdown prevents RunX2 ubiquitination and proteosomal degradation in presence of WFA. Primary osteoblast cells were treated with WFA for 24 h, and endogenous RunX2 immunoprecipated by anti- RunX2 antibody. Ubiquitinated RunX2 (Ub-RunX2) protein ladders were detected by anti-ubiquitin antibody. After stripping the antibody, total un-ubiquitinated RunX2 protein levels were determined by anti-RunX2 antibody (lower panel). (p) Smurf2 knockdown increased ALP activity in the presence of WFA and TNFα. WFA treatment of MCOs for 48 h in osteoblast differentiation medium significantly increased ALP production compared with control that was abrogated in the presence of TNFα. Treatment with Smurf2 siRNA in the presence of TNFα reversed the effect. Values represent mean±S.E. of three independent experiments (n=3). *P<0.05, ***P<0.001 compared with control vehicle group. (q) Smurf2 knockdown increased expression of osteogenic genes. mRNA expression of BMP2 and RunX2 was assessed by real-time PCR. The values are the mean±S.E. of three experiments. *P<0.05, ***P<0.001 versus control (vehicle) group