to induce inflammatory bone resorption in Dap12 -/-mice. Mechanistically, RBP-J inhibited induction of NFATc1, BLIMP1, and c-FOS by suppressing PLCγ2 expression and activity and downstream calcium-CaMKK-PYK2 signaling via the TGF-β pathway during osteoclastogenesis. Thus, RBP-J deficiency allows RANK or TNFR signaling to induce osteoclast differentiation independently of ITAM-mediated costimulation. These results indicate that RBP-J imposes a requirement for ITAM-mediated costimulation and limits the crosstalk between ITAM and RANK signaling, thereby restraining both pathways in osteoclastogenesis.
Results
RBP-J deficiency reverses the osteopetrotic bone phenotype of Dap12-
Dap12-/-or TKO chimeric mice relieved the osteopetrotic phenotype caused by lack of DAP12 or both DAP12 and FcRγ, respectively. Rbpj
ΔM/ΔM
Dap12-/-mice exhibited trabecular bone volume, number, thickness, and spacing similar to those of control mice (Figure 1, A Figure 1C). Notably, serum TRAP levels in Rbpj
Dap12-/-mice were comparable to those in control mice ( Figure 1C and
Fcrg-/-cells leads to attenuation of PLCγ2 activation followed by impaired NFATc1 induction and osteoclast differentiation even in the presence of RANKL (18, 19). Thus, ITAM-mediated costimulatory signals and PLCγ2 activity, which determine downstream calcium signaling, are required for osteoclast differentiation. RANK signaling induces a transient increase in PLCγ2 activity, but does not regulate its expression and is not required for basal PLCγ2 activity in osteoclast precursors. Instead, basal PLCγ2 activity, which has been observed in myeloid cells including osteoclast precursors (17, 21-24), appears to be maintained by tonic ITAM signaling mediated by engagement of ITAM-associated receptors by constitutively expressed ligands (13); consistently, addition of ligands/agonists for ITAMassociated receptors is not required for osteoclast differentiation in vitro. Importantly this basal level of costimulation and PLCγ2 activation together with RANKL is sufficient for NFATc1 induction and osteoclast differentiation (16,18,19). The ligands for FcRγ-and DAP12-associated receptors that mediate tonic signaling are suggested to be expressed by osteoblasts/stromal cells and osteoclast precursors, respectively (18, 24). Thus, cell-cell interaction could provide basal/"tonic" ITAM-mediated PLCγ2/ calcium costimulatory signaling during osteoclastogenesis. There has been great interest in delineating the mechanisms by which ITAM signals crossregulate heterologous receptors, such as TLRs and cytokine receptors (13), but less is known about mechanisms/factors in the cells that fine tune ITAM-mediated PLCγ2/calcium signaling.Recombinant recognition sequence binding protein at the J κ site (RBP-J, also called RBP-J κ , CSL, or CBF1) functions as a central transcription factor that receives input from several signaling pathways, such as the canonical NOTCH pathway, the WNT/ β-catenin pathway, the NF-κB pathway, and the TLR-and TNFsignaling pathways (25-31), to mediate diverse cellula...
