Figure 5. PTH increases the access of BMP2 to receptors by exposure of cell surface BMPRII.

(A) PTH did not induce endocytosis of BMPRII. YFP-BMPRII was expressed in HEK293 cells and visualized at the cell membrane in green (left column). PTH^TMR (red) internalized in a time-dependent manner (middle column). No co-localization (yellow) of BMPRII-YFP expressed at membrane in green with PTH^TMR in red was observed after PTH treatment (right column). Scale bar = 10 μm.

(B,C) PTH did not decrease the level of cell surface BMPRII in GFP-labeled Sca-1⁺CD45⁻CD11b⁻ MSCs harvested from the bone marrow cavity transplantation shown by representative one-parameter histograms for FASCs anaylsis (B) with quantification of BMPRII positive GFP-labeled Sca-1⁺CD45⁻CD11b⁻ MSCs presented as percentage of GFP positive cells. Data are presented as mean ± SEM. n = 6.

(D) LRP6 siRNA knockdown increased cell surface exposure of BMPRII in C2C12 cells. Flag-BMPRII expression was examined using biotin-streptavidin blotting system.

(E,F) PTH stimulated cell surface binding of ¹²⁵I-BMP2 in a dose (E) and time (F) dependent manner in C2C12 cells. Cells were incubated with varying concentrations of PTH at 37 °C for 30 minutes (E) or with 100 nM PTH at 37 °C for varying time intervals (F). After PTH exposure, all cells were then incubated with ¹²⁵I-BMP2 at 4 °C for 4 hours. Data are presented as mean ± SEM. n = 3.

(G) PTH increased the binding of BMP2 ligand to its receptor on GFP-labeled Sca-1⁺CD45⁻CD11b⁻ MSCs in FACS analysis. Cells were incubated with 250 ng/ml Alexa 647-BMP2 with or without 100nM PTH at 37 °C for 30 minutes, then incubated at 4 °C for 4 hours. One representative FACS analysis of three was shown.