Fig. 5.

Impairment of sclerostin and Wise suppression of OB differentiation in Lrp4-deficient BMSCs. (A) Schematic of Lrp4 structure and its potential ligands. (B and C) Sclerostin and Wise, but not DKK1, reduced ALP activity in an Lrp4-dependent manner. The control, sclerostin, DKK1, and agrin condition medium (CM) were mixed with the OB differentiation medium (DM) at a ratio of 1:1 and incubated with control and Lrp4 mutant BMSCs to induce OB differentiation for 1 wk. The Wise recombinant protein (100 ng/mL) was tested. OB-like cells were subjected to ALP-staining analysis. Representative images are shown in B. (Scale bars, 80 μm.) Quantitative analyses (mean ± SD from three different cultures) are presented in C. Data were normalized by WT controls. *P < 0.05, significant difference. (D) Sclerostin reduced osteocalcin expression in an Lrp4-dependent manner. BMSCs were treated with control CM or sclerostin CM for 24 h. mRNAs were subjected to real-time PCR analyses. The values were normalized to β-actin and WT controls. Means ± SD values from three different experiments are presented. *P < 0.05, significant difference. (E–G) Reduction of the sclerostin/Wise inhibitory effects by Lrp4–ECD. As in B, the indicated CM were mixed with the DM (1:1), and then incubated with Lrp4 mutant and control BMSCs to induce OB differentiation for 1 wk. Representative images (E) and quantitative analyses (mean ± SD from three different cultures) (F and G) are shown. Data were normalized by WT controls. *P < 0.05, significant difference. (Scale bars, 80 μm.) (H) Illustration of a working model for Lrp4-binding protein’s effect on OB differentiation. Note that sclerostin and Wise inhibit OB in an Lrp4-dependent manner; DKK1 inhibition of OB is Lrp4-independent; and agrin has little to no effect on this event.