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. 2013 Jan;52(1):145-56.
doi: 10.1016/j.bone.2012.09.029. Epub 2012 Sep 29.

Wnt/β-catenin signaling activates bone morphogenetic protein 2 expression in osteoblasts

Rongrong Zhang  1 , Babatunde O Oyajobi, Stephen E Harris, Di Chen, Christopher Tsao, Hong-Wen Deng, Ming Zhao
Affiliations

Wnt/β-catenin signaling activates bone morphogenetic protein 2 expression in osteoblasts

Rongrong Zhang et al. Bone. 2013 Jan.

Abstract

The BMP and Wnt/β-catenin signaling pathways cooperatively regulate osteoblast differentiation and bone formation. Although BMP signaling regulates gene expression of the Wnt pathway, much less is known about whether Wnt signaling modulates BMP expression in osteoblasts. Given the presence of putative Tcf/Lef response elements that bind β-catenin/TCF transcription complex in the BMP2 promoter, we hypothesized that the Wnt/β-catenin pathway stimulates BMP2 expression in osteogenic cells. In this study, we showed that Wnt/β-catenin signaling is active in various osteoblast or osteoblast precursor cell lines, including MC3T3-E1, 2T3, C2C12, and C3H10T1/2 cells. Furthermore, crosstalk between the BMP and Wnt pathways affected BMP signaling activity, osteoblast differentiation, and bone formation, suggesting Wnt signaling is an upstream regulator of BMP signaling. Activation of Wnt signaling by Wnt3a or overexpression of β-catenin/TCF4 both stimulated BMP2 transcription at promoter and mRNA levels. In contrast, transcription of BMP2 in osteogenic cells was decreased by either blocking the Wnt pathway with DKK1 and sFRP4, or inhibiting β-catenin/TCF4 activity with FWD1/β-TrCP, ICAT, or ΔTCF4. Using a site-directed mutagenesis approach, we confirmed that Wnt/β-catenin transactivation of BMP2 transcription is directly mediated through the Tcf/Lef response elements in the BMP2 promoter. These results, which demonstrate that the Wnt/β-catenin signaling pathway is an upstream activator of BMP2 expression in osteoblasts, provide novel insights into the nature of functional cross talk integrating the BMP and Wnt/β-catenin pathways in osteoblastic differentiation and maintenance of skeletal homeostasis.

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Figures

Fig. 1
Fig. 1
Wnt/β-catenin signaling in osteoblasts. (A and B) Effects of Wnt3a on β-catenin expression. 2T3, C2C12, C3H10T1/2, and MC3T3-E1 cells were treated with Wnt3a at 40 ng/mL for 24 h. β-catenin in cell lysates was detected by Western blot using an anti-β-catenin antibody. β-actin was used as control (A). The relative β-catenin levels were quantitated using Quantity One gel imaging system with normalization by β-actin (B). *: P<0.05 Wnt3a vs. vehicle (n=4). (C and D) Effects of β-catenin/TCF4 on Wnt reporter activity. C2C12 cells, which were transfected with TOPFLASH (C) or9×6-OC-Luc reporter (D), were transfected with expression vectors for wild-type and mutant β-catenin and TCF4at a dose of 0.2 µg DNA/well in 24-well plates for 36 h. Relative luciferase activity in the cell lysates was measured and normalized by β-galactosidase (β-gal) activity. #: P<0.05 β-catenin or TCF4 vs. vector; *: P<0 .01 β-catenin+TCF4 vs. β-catenin (n=6). (E) Effects of DKK1 on β-catenin activation of Wnt signaling activity. C2C12 cells carrying TOPFLASH reporter were incubated with DKK1 at 100 ng/mL for 36 h. Relative luciferase activity was measured with normalization by Renilla. *: P<0 .01 β-catenin vs. vector; #: P<0.01 β-catenin vs. β-catenin+DKK1 (n=6). (F) Effects of Wnt3a on Wnt signaling activity. C2C12 cells carrying TOPFLASH reporter were incubated with Wnt3a in a dose range of 10–80 ng/mL for 36 h. Relative luciferase activity was measured as described in (E). *: P<0 .01, Wnt3a vs. vehicle (n=6).
Fig. 2
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.
Fig. 3
Fig. 3
Effects of Wnt ligand and antagonists on BMP2 expression. (A and B) Effects of Wnt3a on BMP2 mRNA expression in C2C12 cells. (A) BMP2 PCR. C2C12 cells were treated with Wnt3a at a dose range from 20 to 80 ng/mL for 24 h. BMP2 mRNA levels in the cell lysates were determined by RT-PCR using mouse BMP2 primers, with GAPDH normalization. (B) Quantitative real time PCR of BMP2. C2C12 and 2T3 cells were treated with Wnt3a as described above. Relative BMP2 mRNA concentrations in the cell lysates were quantitated by real time PCR using TaqMan mouse BMP2 probe, and normalized by GAPDH. *: P<0.05 Wnt3a vs. vehicle (n=4). (C) Effects of Wnt3a on BMP2 mRNA expression in calvarial cells. Primary calvarial osteoblasts were incubated with Wnt3a at doses as described above. mRNA levels of BMP2 were determined as described above. *: P<0 .01 Wnt3a vs. vehicle (n=6). (D) Effects of Wnt3a on BMP2 promoter activity. C2C12 cells with BMP2 promoter reporter −2712/+165-Luc were treated with Wnt3a at 40 ng/mL for 36 h. Relative luciferase activity in the cell lysates was measured and normalized by Renilla activity. *: P<0.01, Wnt3a vs. vehicle (n=6). (E) Effects of Wnt antagonists on BMP2 promoter activity. C2C12 cells were transfected with BMP2 promoter reporter −2712/+165-Luc, and treated with DKK1 at 100 ng/mL or sFRP4 at 200 ng/mL for 36 h. Relative luciferase activity in the cell lysates was measured and normalized by β-gal activity. *: P<0.01 DKK1 or sFRP4 vs. vehicle (n=6).
Fig. 4
Fig. 4
Effects of overexpression of β-catenin on BMP2 expression. (A and B) Effects of β-catenin/TCF4 on BMP2 mRNA expression. (A) BMP2 PCR. C2C12 and 2T3 cells were transfected with expression vectors for β-catenin and TCF4 at a dose of 0.5 µg DNA/well in 6-well plates for 24 h. BMP2 mRNA levels in the cell lysates were determined by RT-PCR using mouse BMP2 primers, with GAPDH normalization. (B) Quantitative real time PCR of BMP2. C2C12 and 2T3 cells were transfected with β-catenin/TCF4 as described above. BMP2 mRNA concentrations in the cell lysates were quantitated by real time PCR using TaqMan mouse BMP2 probe, and normalized to GAPDH. *: P<0.05 β-catenin/TCF4 vs. vector (n=4). (C and D) Effects of β-catenin/TCF4 on BMP2 promoter activity. C2C12 cells, which were transfected with −2712/+165-Luc reporter, were co-transfected with expression vectors for wild-type or mutant β-catenin at a dose of 0.2 µg DNA/well in 24-well plates (C) or co-transfected with β-catenin/TCF4 or β-catenin(S33Y)/TCF4 at a dose range of 0.05–0.2 µg DNA/well in 24-well plates (D) for 36 h. Relative luciferase activity in the cell lysates was measured and normalized by β-gal activity. *: P<0.05 β-catenin or its mutants vs. vector (n=6). (E) β-Catenin protein level correlates with amount of plasmid vector transfected. C2C12 cells in 6-well plates were transfected with β-catenin expression vector at varying doses (0.5, 2.0 and 10 µg DNA/well). After 36 h, cells were harvested and β-catenin protein levels in the respective cell lysates were determined by Western blot using anti-β-catenin antibody. GAPDH was used as an internal control.
Fig. 5
Fig. 5
Effects of down-regulation of β-catenin activity on BMP2 expression. (A) Effects of BMP or Wnt antagonists on β-catenin/TCF4 activation of BMP2 promoter activity. C2C12 cells were co-transfected with −2712/+165-Luc reporter and expression vectors for β-catenin and TCF4. Cells were treated with noggin at 500 ng/mL or DKK1 at 100 ng/mL for 36 h. Relative luciferase activity in the cell lysates was measured and normalized by β-gal activity. *: P<0.01 β-catenin/TCF4 vs. vector; #: P<0.05 noggin or DKK1 vs. vehicle (n=6). (B and C) Effects of FWD1/β-TrCP on Wnt signaling activity or BMP2 promoter activity. C2C12 cells were co-transfected with TOPFLASH (B) or −2712/+165-Luc reporter (C) and expression vectors for β-catenin and TCF4, and expression vectors for wild-type β-TrCP (FWD1) or mutant β-TrCP (FWD1ΔF) for 36 h. Relative luciferase activity in the cell lysates was measured and normalized by β-gal activity. In (B): *: P<0.01 FWD1ΔF vs. vector, or FWD1ΔF+β-catenin/TCF4 vs. vector+β-catenin/TCF4; #: P<0.05 FWD1+β-catenin/TCF4 vs. vector+β-catenin/TCF4. In (C): *: P<0.05 FWD1 vs. vector, or FWD1+β-catenin/TCF4 vs. vector+β-catenin/TCF4 (n=6).
Fig. 6
Fig. 6
Transactivation of BMP2 promoter by Wnt/β-catenin signaling through Tcf/Lef response elements. (A and B) Effects of inhibitor of interaction of β-catenin and TCF4 on BMP2 promoter activity. C2C12 cells were co-transfected with −2712/+165-Luc reporter and expression vectors for β-catenin and TCF4, and expression vectors for ICAT (A) or ΔTCF4 (B) for 36 h. Relative luciferase activity in the cell lysates was measured and normalized by β-gal activity. *: P<0.05 ICAT or ΔTCF4 vs. vector; #: P<0.05 ICAT+β-catenin/TCF4 or ΔTCF4+β-catenin/ TCF4 vs. vector+β-catenin/TCF4 (n=6). (C) Mutagenesis of TREs in the BMP2 promoter. The core nucleotides (bold dashes) of putative TREs at −2269/−2263 and −1824/−1804 (bold) in the mouse BMP2 promoter reporter −2712/+165-Luc were deleted using synthesized mutagenesis DNA oligonucleotides. (D) Effects of mutated TREs on β-catenin/TCF4 activation of BMP2 promoter activity. C2C12 cells were co-transfected with−2712/+165-Luc reporter containing wild-type TREs or mutated TREs (Δ-2269/−2263, Δ-1824/−1804) with expression vectors for β-catenin and TCF4 for 36 h. Relative luciferase activity in the cell lysates was measured and normalized by β-gal activity.*:P<.01 β-catenin/TCF4 vs. vector (n=6).
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