Figure 6. RBP-J suppresses PLCγ2/calcium-CaMKK/Pyk2 signaling in osteoclast differentiation.

(A) Immunoblot analysis of phospho-PLCγ2, NFATc1, Blimp1, and c-Fos expression at basal levels or stimulated with RANKL for 24 hours in the BMMs. The cells were pretreated for 30 minutes with PLCγ2 inhibitor U-73122 (10 μM) or its vehicle ethanol; inhibitor or vehicle was completely washed away before addition of RANKL. (B) Quantitation of TRAP-positive MNCs (≥ 10 nuclei per cell) that were pretreated for 30 minutes with U-73122 or ethanol and then cultured with RANKL for 6 days. (C and G) Immunoblot analysis of NFATc1, Blimp1, and c-Fos expression at basal levels or after being stimulated with RANKL for 24 hours with CaMKK inhibitor STO-609 (5 μg/ml) (C) or PYK2 inhibitor AG 17 (3 μM) (G) or vehicle DMSO in the BMMs. (D and H) Quantitation of TRAP-positive MNCs (≥ 10 nuclei per cell) that were stimulated with RANKL with STO-609 (D), AG 17 (H), or DMSO for 6 days. (E and F) Immunoblot analysis of phospho-PYK2 and total PYK2 expression in the indicated conditions (E) or at basal levels (F). The lanes separated by a black line were run on the same gel but were noncontiguous. Quantification of the immunoblot bands by densitometry were shown in the right panels of E and F. β-Tubulin (A) or p38α (C and E–G) was measured as a loading control. (I) A model showing that RBP-J suppresses ITAM-mediated basal PLCγ2 expression/activity and downstream calcium-CaMKK-PYK2 signaling in induction of c-FOS, BLIMP1, and NFATc1 in a TGF-β dependent pathway.