Figure 5
Dkk1 attenuates Wnt3a-dependent inhibition of RANKL in C2C12 cells treated with 1,25(OH)2D3or left untreated. A, Mitigation of Wnt3a-dependent suppression of RANKL message by Dkk1. Cells were pre-incubated for 3 h in Dkk1-CM or M-CM supplemented with 100 ng/ml Noggin. Plain M-CM was used for the two samples at the top (not indicated). Cells were grown for 3 days in Wnt3a-CM or L-CM without Wnt3a (control). Total RNA was extracted and mRNA levels of RANKL were determined using real-time RT-PCR. Data are shown as means ± SD based on 5 independent experiments. Number signs and asterisks designate a significant drop (p < 0.005) compared to the control and Wnt3a-treated cells, respectively. B, Dkk1 partially restores the Wnt3a-mediated drop of RANKL mRNA in cells treated with 1,25(OH)2D3. Cells were pre-incubated as desribed in panel A and grown for 3 days in L-CM (no Wnt3a) or Wnt3a-CM supplemented with 10 nM 1,25(OH)2D3. Data are shown as means ± SD based on 5 independent experiments. Number signs, asterisks and paragraph symbols designate significant changes (p < 0.005) compared to untreated cells, 1,25(OH)2D3-treated cells and Wnt3a/1,25(OH)2D3-treated cells, respectively. C, Wnt3a does not affect BMP2-dependent RANKL expression. Cells were pre-incubated as described in panel A and grown for 3 days in Dkk1-CM (2nd bar from bottom), M-CM supplemented with 100 ng/ml Noggin (bottom bar), or M-CM (3 top bars). Cells were grown for 3 days in L-CM (top bar), L-CM containing 100 ng/ml BMP2 (2nd bar from top), or Wnt3a-CM supplemented with 100 ng/ml BMP2 (three bars at bottom). Data are shown as means ± SD from 3 independent experiments. Number signs and asterisks designate a significant elevation compared to untreated cells and a reduction compared to BMP2-treated cells (p < 0.005), respectively. Note that the bar labeled Control (green) in this panel is identical to corresponding bars in panels A and B.