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Intermittent administration of PTH stimulates bone formation, but the precise mechanisms responsible for PTH responses in osteoblasts are only incompletely understood. Here we show that binding of PTH to its receptor PTH1R induced association of LRP6, a coreceptor of Wnt, with PTH1R. The formation of the ternary complex containing PTH, PTH1R, and LRP6 promoted rapid phosphorylation of LRP6, which resulted in the recruitment of axin to LRP6, and stabilization of beta-catenin. Activation of PKA is essential for PTH-induced beta-catenin stabilization, but not for Wnt signaling. In vivo studies confirmed that PTH treatment led to phosphorylation of LRP6 and an increase in amount of beta-catenin in osteoblasts with a concurrent increase in bone formation in rat. Thus, LRP6 coreceptor is a key element of the PTH signaling that regulates osteoblast activity.
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Figure 2.
Formation of complexes of LRP6 with PTH–PTH1R. (A) LRP6-specific siRNA reduced the amount of LRP6 protein in HEK 293 cells as determined by Western blotting. siRNA directed against GFP was used as an siRNA control. (B) LRP6-specific siRNA reduced PTH-induced β-catenin stabilization in HEK293 cells as determined by Western blotting analysis. (C) LRP6-specific siRNA reduced PTH-stimulated TCF/LEF activity in UMR-106 cells as determined using a luciferase assay. (*) P < 0.01 (in comparison with control), n = 3; (n.s.) not significant (in comparison with control), n = 3. (D,E) Real-time PCR analysis of Osteocalcin (D) and RANKL (E) mRNA expression. C2C12 cells expressing siGFP (control) or siLRP6 together with PTH1R were treated with or without PTH (1–34) in osteogenic induction medium (100 nM ascorbic acid, 10 mM glycerophosphate, and 100 ng/mL BMP2) and harvested at day 3 for RNA extraction. (F) Co-IP of endogenous LRP6 with PTH1R in UMR-106 cells. Cells were serum deprived and treated with 10−8 M PTH (1–84). The LRP6-associated PTH1R was determined separately by Western blotting of the anti-LRP6 immunoprecipitates. (WCL) Whole-cell lysates. (G) PTH enhances binding of PTH1R to LRP6, but not LRP5. HEK 293 cells were transfected with VSVG-tagged LRP6 or HA-LRP5 together with PTH1R and treated with 10−8 M PTH (1–84). The PTH1R-associated LRP5 or LRP6 was determined by Western blotting analysis of the anti-PTH1R immunoprecipitates. (WCL) Whole cell lysates. (H) Ternary complex of LRP6, PTH, and PTH1R. HEK 293 cells were transfected with VSVG-tagged LRP6 and HA-PTH1R and treated with 10−8 M PTH (1–84). The LRP6-associated PTH ligand was determined by Western blotting analysis of the anti-VSVG immunoprecipitates. (WCL) Whole cell lysates. (I–K) PTH brings PTH1R and LRP6 into close proximity as demonstrated by FRET. (I) A photobleaching-based FRET (pbFRET) system was generated by transiently expressing two constructs in HEK293 cells in which CFP and YFP were fused at the C terminus of PTH1R and LRP6, respectively. The interactions of YFP-fused LRP6 with CFP-fused BMPRII or CFP-fused PTH1R with YFP-fused mLRP4T100 were also examined as controls. (J) Representative confocal imaging of the association of CFP-PTH1R with YFP-LRP6 at 5 min after PTH treatment in HEK293 cells by pbFRET. The total photobleached area (ROI_1) is marked with a green square. Quantification of fluorescent intensities of each chosen point within (ROI_2∼ROI_6) or outside of the marked bleached area (ROI_7∼ROI_9) by averaging fluorescence before and after the bleach was conducted. (K) Comparison of the FRET efficiencies (FRET Eff%) before and after photobleaching in the absence or presence of PTH. (*) P < 0.001, compare with unbleached, n = 6; (n.s.) not significant compare with unbleached. (L,M) Ventral injection of RNA for PTH (2 pg) plus PTH1R (50 pg) promotes LRP6 (200 pg)-induced axis duplication. (n) Numbers of embryos scored.
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