Abstract
TREM2‐DAP12 stimulation of macrophages and pre‐osteoclasts (OC) leads to enhanced in vitro osteoclastogenesis. To further define the regulation of TREM2‐DAP12 signals, we studied proteins that associated with DAP12 following TREM2 stimulation. We found that TREM2 crosslinking of RAW264.7 cells leads to a novel association of Src homology region 2(SH2)‐containing inositol phosphatase (SHIP1) with DAP12 at 1 and 5 min, which was not seen after stimulation with isotype control Ab. Recombinant GST‐SHIP1‐SH2 protein, but not mutated GST‐SH2(R34G) protein, could bind phosphorylated DAP12 indicating that the SHIP1‐DAP12 interaction is mediated via the SH2 domain of SHIP1. Biotinylated‐DAP12 ITAM phosphopeptide, but not un‐phosphorylated or Y65/76F mutated peptides, interacted with SHIP1. SHIP1 co‐immunoprecipitated with DAP12 in OC generated from C57BL6 bone marrow macrophages (BMM) treated with RANKL and M‐CSF. To determine the functional significance of this interaction, we evaluated TREM2 stimulation of SHIP1 deficient OC precursors during osteoclastogenesis. Crosslinking with TREM2 monoclonal Ab but not control Ab lead to a 4 fold increase in OC generated from SHIP1−/− BMM compared to wild type BMM. Together these data reveal a novel association of SHIP1 with DAP12 that is dependent on the DAP12 ITAM and the SH2 domain of SHIP1 and this association inhibits DAP12 signaling during osteoclastogenesis.
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