Fig. 2.

Interaction between Wnt and BMP signaling in osteoblasts. (A) Effects of BMP2 and Wnt3a and their antagonists on BMP signaling reporter activity. C2C12 cells, which were transfected with 12SBE-Luc reporter, were treated with BMP2 at 100 ng/mL, or Wnt3a at 40 ng/mL, in the presence or absence of noggin at 500 ng/mL or DKK1 at 100 ng/mL, for 36 h. Relative luciferase activity in the cell lysates was determined with β-gal normalization. *: P<0.01, BMP2 or Wnt3a vs. vehicle; #: P<0.01 noggin vs. BMP2, or noggin, DKK1 vs. Wnt3a (n=6). (B) Effects of BMP2 and Wnt3a and their antagonists on alkaline phosphatase (ALP) activity in C2C12 cells. C2C12 cells were treated with BMP2 or Wnt3a, with or without noggin or DKK1 as described above for 48 h. ALP activity in the cell lysates was determined using a Sigma ALP kit with normalization by total cell proteins. *: P<0.05 noggin or DKK1 or BMP2 or Wnt3a vs. vehicle; #: P<0.05, noggin vs. BMP2 or Wnt3a, or DKK1 vs. Wnt3a (n=6). (C) Effects of BMP2 on ALP activity of calvarial osteoblasts. Primary calvarial cells isolated from newborn mice were treated with BMP2 in the absence or presence of noggin or DKK1 at the doses as described above, for 48 h. ALP activity was quantitated as described above. *: P<0.01 BMP2 vs. vehicle;#: P<0.01 BMP2 vs. BMP2+noggin (n=6). (D) Effects of Wnt3a on Col1a1 and Runx2 expression in calvarial cells. The calvarial osteoblasts were incubated with Wnt3a at doses of 20, 40 and 80 ng/mL for 24 h. mRNA levels of Col1a1 and Runx2 were determined by real time PCR with GAPDH normalization. *: P<0 .01, Wnt3a vs. vehicle (n=6). (E) Effects of DKK1 on ALP activity induced by a combination of Wnt- and BMP2-induced signaling in neonatal calvariae. Hemi-calvariae were incubated ex vivo for 4 days with either Wnt3a (80 ng/ml) or BMP2 (100 ng/ml) or a combination of both in the presence or absence of DKK1. Conditioned media were harvested on day 4 and relative ALP levels determined. Data represent mean±SD (n≥3 calvariae/group). *: P<0.05, Wnt3a/BMP2 vs. Wnt3a or BMP2, or vehicle alone; #: P<0.05 Wnt3a/BMP2+DKK1 vs. Wnt3a/BMP2 (n≥3). (F) Soluble Kremen enhances BMP2-induced bone formation in neonatal mouse calvariae. Hemi-calvariae were treated with sKremen, BMP2 or a combination of both for 7 days with media and recombinant proteins completely replenished on day 4. Bones were processed for histology and stained with hematoxylin and eosin. Although soluble Kremen on its own did not have any effect, there was increased cellular proliferation and accumulation of mature osteoblasts adjacent to new bone in representative calvariae treated with BMP2 in the presence of sKremen compared with BMP2 alone. Representative calvariae from n≥3 calvariae/group are presented.