DKK-1 inhibition of Wnt3a induced OPG mRNA and protein in osteoblast cells. C2C12 (A) and Saos-2 (B) cells were stimulated with or without Wnt3a for 8 hours after prior treatment with recombinant DKK-1 for 1 hour at indicated concentrations and then lysed. A total of 0.5 mg of protein from cell lysates was subjected to the GST-E-cadherin assay. After SDS-PAGE, uncomplexed β-catenin was detected with anti–β-catenin antibody. The cells were cultured at 105/well in 6-well plate for 24 hours, and 100 ng/mL of Dkk1 was added for 1 hour followed by addition of 100 ng/mL of rWnt3a for 48 or 72 hours. Total RNA was isolated from treated C2C12 (C) and Saos-2 (D) cells after 48 hours and OPG mRNA was quantified. The supernatant of C2C12 (E) and Saos-2 (F) cells treated, as above for 72 hours, was harvested and subjected to ELISA for measurement of OPG. The results are shown as means plus or minus SD (n = 3). Results are representative of 3 independent experiments (**P < .01, ***P < .001, vs control).
