BMP-2 acts at multiple steps of β-catenin signaling to modulate β-catenin levels. Our current findings suggest that BMP-2 may regulate β-catenin protein levels through stimulating Lrp5 expression and inhibiting the expression of E3 ubiquitin ligase β-TrCP in osteoblasts.

β-Catenin signaling is active in bone cells. A–C: Expression of β-catenin protein was detected in osteoblasts on trabecular (red arrow) and cortical (green arrow) bones in tibiae of 3-day-old (P3) C57BL/6J mice by immunostaining using the anti-β-catenin monoclonal antibody (A, 4×; B, 20×; C, 40×). D: The mRNA expression of Tcf and Lef was detected in primary osteoblasts isolated from 3-day-old C57BL/6J mice by RT-PCR. Tcf3 and Tcf4 were highly expressed and Tcf1 was only weakly expressed in osteoblasts. E: The protein expression of TCF3 and TCF4 in osteoblasts was detected by Western blot. TCF3 protein was highly expressed in 2T3 and MC3T3 cells and in primary osteoblasts and was weakly expressed in C2C12 cells. TCF4 is highly expressed in MC3T3 cells but weakly expressed in primary osteoblasts.

BMP-2 enhances β-catenin protein levels in osteoblasts. A: 2T3 osteoblast precursor cells were treated with BMP-2 (100 ng/ml) for 3, 12, and 24 h. BMP-2 significantly increased the β-catenin protein level (non-phosphorylated active form) after 24 h treatment. B: 2T3 cells were treated with BMP-2 at 50 and 100 ng/ml concentrations for 24 h. Wnt3a (100 ng/ml) was used as a positive control. BMP-2 at both concentrations significantly increased β-catenin protein level (non-phosphorylated active form) after 24 h treatment. C: 2T3 cells were treated with BMP-2 for different periods of time (0–24 h) and nuclear protein was extracted. BMP-2 enhanced nuclear β-catenin protein level (active form) at 24 h time-point. D: MC3T3-E1 cells were treated with BMP-2 (50 and 100 ng/ml) and Wnt3a (100 ng/ml) for 24 h. Similar to 2T3 cells, BMP-2 significantly increased β-catenin protein levels in MC3T3-E1 cells. E: 2T3 cells were treated with different concentrations of BMP-2 (0–200 ng/ml) for 24 h and total RNA was extracted. BMP-2 had no significant effect on β-catenin mRNA expression.

BMP-2 stimulates Lrp5 expression and inhibits β-TrCP expression. A,B: 2T3 cells were treated with BMP-2 (100 ng/ml) for 2, 24, and 48 h. BMP-2 stimulated Lrp5 expression at 24 and 48 h cultures, detected by Northern blotting. C: 2T3 cells were treated with BMP-2 (100 ng/ml) with or without addition of the MAP kinase inhibitor SB 220025 (1 μM) or transfection of Smad6 for 24 h. Lrp5 mRNA expression was measured by real-time PCR assay. Treatment with BMP-2 significantly increased Lrp5 mRNA expression which was inhibited by addition of MAP kinase inhibitor. Transfection of Smad6 had no effect on BMP-2-induced Lrp5 expression. D: 2T3 cells were treated with BMP-2 (100 ng/ml) for 24 h with or without transfection of Lrp5 siRNA. β-catenin expression (active form) was measured by Western blotting. BMP-2 significantly increased β-catenin expression and transfection of Lrp5 siRNA significantly inhibited BMP-2-induced up regulation of β-catenin protein levels. E: 2T3 cells were treated with DKK1 (300 ng/ml) with or without BMP-2 (100 ng/ml). The BMP-2-induced β-catenin expression (active form) was significantly inhibited by addition of DKK1. F–H: 2T3 cells were treated with BMP-2 (100 ng/ml) for 24 h and mRNA expression of canonical Wnt ligands Wnt1, Wnt3a, and Wnt4 was examined by real-time PCR. BMP-2 significantly up regulated the expression of Wnt1, Wnt3a, and Wnt4 in 2T3 cells.

BMP-2 inhibits β-TrCP expression. A: 2T3 cells were plated into six-well culture plates and transfected with 0.5 and 1 μg of β-TrCP expression plasmid. Western blot analysis showed that β-TrCP induced β-catenin degradation in 2T3 cells. B: 2T3 cells were treated with different concentrations of BMP-2 (0–100 ng/ml) for 24 h. Western blotting result demonstrated that BMP-2 inhibited β-TrCP expression in a dose-dependent manner in 2T3 cells. C: 2T3 cells were treated with BMP-2 (100 ng/ml) and transfected with β-TrCP or empty vector. β-catenin protein levels were examined by western blotting 24 h after BMP-2 treatment. BMP-2 significantly up regulated β-catenin protein levels which was completely reversed by the transfection of β-TrCP. D: 2T3 cells were transfected with β-TrCP or empty vector and HA-Ub and treated with BMP-2 (100 ng/ml) for 24 h. Immunoprecipitation (IP) was performed using the anti-β-catenin antibody followed by Western blotting using the anti-HA antibody. Treatment with BMP-2 significantly inhibited β-catenin ubiquitination. Transfection of β-TrCP completely reversed the inhibitory effect of BMP-2 on β-catenin ubiquitination. E: 2T3 cells were treated with BMP-2 (100 ng/ml) for 24 h with or without addition of MAP kinase inhibitor SB 220025 or transfection of Smad6. β-TrCP and β-catenin protein levels were examined by Western blotting. BMP-2 down regulated β-TrCP protein levels and up regulated β-catenin protein levels. Addition of MAP kinase inhibitor significantly reversed BMP-2-regulated β-TrCP and β-catenin protein levels. In contrast, transfection of Smad6 had minor effect on BMP-2 regulated β-TrCP and β-catenin protein levels.

Deletion of the β-catenin gene in osteoblasts impairs osteoblast proliferation and differentiation. Primary osteoblasts were isolated from 3-day-old β-catenin^fx/fx mice and infected with same amounts of Ad-Cre or Ad-GFP (control). A: Genomic structure of floxed and deleted β-catenin gene. B: Over 80% infection efficiency was achieved when Ad-GFP was infected into primary mouse osteoblasts. C: The genotyping showed that Cre-mediated recombination (631-bp PCR product) was only observed in the cells infected with Ad-Cre (lane 1, far left). The PCR product of floxed β-catenin gene is shown in lane 2 and 3 (324-bp, middle panel). D: Western blotting result showed that β-catenin protein level was significantly reduced in osteoblasts infected with Ad-Cre. E,F: Osteoblast proliferation detected by BrdU labeling was decreased 40% in β-catenin-deleted osteoblasts. G: The expression of cell cycle protein cyclin D1 was reduced in β-catenin-deleted osteoblasts. H: Treatment with Wnt3a (100 ng/ml) increased cyclin D1 expression in WT primary mouse osteoblasts. I,J: ALP activity and the expression of type I collagen (Col-1) were determined. The ALP activity (I) and the expression of Col-1 (J) were significantly increased by the treatment with BMP-2 (100 ng/ml) which was inhibited by deletion of the β-catenin gene in osteoblasts. K: The formation of mineralized bone matrix was further analyzed. The BMP-2 (100 ng/ml)-induced mineralized bone matrix formation was significantly inhibited in β-catenin-deleted osteoblasts in long-term culture, demonstrated by von Kossa staining. ^*P <0.05, compared to the Ad-GFP control group (n = 3), unpaired Student t-test.