Figure 4
Dkk1 and -2 inhibit Wnt3a-induced OPG expression in C2C12 cells. A, Inhibition of Wnt3a-induced OPG mRNA by Dkk1 and -2. Cells were pre-cultured in Dkk1- or Dkk2-CM for 3 h as indicated. M-CM (no Dkk) was used for the two samples at the top. Wnt3a-CM or L-CM (no Wnt 3a; control) was added and cells were grown for 3 days. Total RNA was extracted and OPG mRNA was determined using real-time RT-PCR. Message levels were normalized using β-actin mRNA as reference. Results are presented as fold induction relative to the control. Data are shown as means ± SD based on 5 independent experiments. Number signs and asterisks indicate significant changes (p < 0.005) compared to cells not treated with Wnt3a (top bar) and treated with Wnt3a (2nd bar from top), respectively. B, Dkk1 and -2 dependent inhibition of Wnt3a-induced OPG protein secreted into the extracellular milieu. Cells were treated as described above, supernatant was harvested, and OPG was determined using ELISA. Results are presented as in panel A. C, Wnt3a-dependent OPG induction is inhibited by BMP2. Cells were pre-incubated for 3 h with Dkk1-CM or M-CM supplemented with 100 ng/ml Noggin where indicated. Cells were grown for 3 days in L-CM (top panel) or Wnt3a-CM (bottom panel) that contained 100 ng/ml BMP2 where indicated. OPG mRNA was measured and results were calculated as described in panel A. Number signs, asterisks and paragraph symbols indicate significant changes (p < 0.005) compared to untreated cells (control), BMP2-treated cells and BMP2/Wnt3a-treated cells, respectively. Note that the bars labeled Control (black) and Wnt3a (green) in this panel are identical to corresponding bars in panels A and B.