Fig. 1.

Bmp signaling is diminished in Lgr6-null mesenchymal cells: (A) Representative Alizarin Red stained wells show extracellular calcium deposition after BMP2 stimulation of hMSCs for ten days. The graph depicts LGR4, LGR5, and LGR6 expression levels after BMP2 stimulation for three days in the osteopermissive media. Each colored dot represents cells from a unique hMSC donor. (B and C) Alizarin Red S staining of MC3T3-E1 and murine calvarial osteoblasts stimulated with BMP2 in osteopermissive media for 12 and 16 days, respectively. Expression of Lgr4, Lgr5, and Lgr6 was determined after BMP2 stimulation for three days in osteopermissive media for both cell types. (D) Temporal expression of Id1, Noggin, and Lgr6 in response to Bmp2 (50 ng/ml and 100 ng/ml) following 24 h serum starvation of WT calvarial cultures was determined by RT-qPCR. (E) Control and Lgr6-null calvarial cultures derived from 5-day-old pups after one passage were serum starved for 24 h and then treated with Bmp2 (50 ng/ml), Dkk1 (50 ng/ml), or a combination of both for 24 h. The expression of Id1 and Noggin was examined using RT-qPCR. (F) Calvarial cultures were grown to confluence, and serum starved for 24 h, followed by stimulation with Bmp2 (50 ng/ml) for 5 and 20 min. Protein expression of phospho-SMAD1, phospho-AKT, and phospho-ERK was determined by western blotting. Blots were stripped and reprobed with anti-SMAD1, anti-AKT, or anti-ERK antibodies to determine the phosphorylated/total protein ratio. (G) Cultures from Lgr6-null and control mice were grown to confluence and treated with 10 % FBS or osteogenic media (OM; 50 μg/ml ascorbic acid and 8 mM β-glycerophosphate) or treated with Bmp2 (50 ng/ml), or Rspo2 (50 ng/ml), or combination of both factors (50 ng/ml each). All growth factors were added to media containing no serum. Media was changed every day for five days. Cells were stained using a commercial kit for ALP activity to visualize osteogenesis. Cells from four-five independent donors were used for the experiment using hMSCs. A representative of four independent experiments is shown for MC3T3-E1 cells. A representative experiment of three separate experiments is shown for all murine calvarial culture experiments. Data were analyzed by Student’s t-test; **p < 0.01, ***p < 0.001; ****p < 0.0001 vs untreated. White bars indicate controls and red bars represent Lgr6-null samples. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)