TREM2, a DAP12-Associated Receptor, Regulates Osteoclast Differentiation and Function

Abstract

Deficiency of the signaling adapter protein DAP12 or its associated receptor TREM2 is associated with abnormal OC development in humans. Here we examine the role of TREM2 in mouse OC development and function, including migration and resorption in vitro. These results provide new evidence that TREM2 regulates OC function independent of its effects on multinucleated OC differentiation.Introduction: TREM2 (triggering receptor expressed in myeloid cells-2) associates with the signaling adapter DAP12 in osteoclasts (OCs). Genetic mutation or deletion of either the TYROBP (DAP12) or TREM2 gene is associated with the human disorder of brain and bone, Nasu-Hakola disease. We and others recently showed the critical requirement for immunoreceptor tyrosine-based activation motif (ITAM) signals through DAP12 and the Fc Receptor ␥ chain (FcR␥) during OC development. Here, we further define the role of TREM2 in OC differentiation and describe a role for TREM2 in OC migration and bone resorption. Materials and Methods:We generated monoclonal anti-mouse TREM2 antibodies (mAb), analyzed preosteoclasts and mature OCs for TREM2 surface expression, and determined the effect of antibody ligation on in vitro OC differentiation, resorption, and migration. TREM2 RNA interference (RNAi) was used to disrupt expression of TREM2 in pre-osteoclasts. Results: Using flow cytometry, our studies reveal that TREM2 is weakly expressed on C57BL/6 bone marrow macrophages (BMMs) and is upregulated during culture with RANKL and macrophage-colony stimulating factor (M-CSF). The expression of TREM2 is unaltered in DAP12-deficient OCs. Using C57BL/6 BMMs or RAW264.7 precursors, anti-TREM2 mAb treatment with RANKL and M-CSF enhances the formation of multinuclear TRACP + OCs compared with control mAb treatment. In contrast, these agents have no effect on DAP12-deficient precursors. Monoclonal Ab blockade of TREM2 on OCs generated from C57BL/6 BMMs results in decreased resorption of artificial calcium-phosphate substrate and dentine. Reduction of TREM2 expression in RAW264.7 cells by RNAi results in loss of OC formation in response to RANKL and M-CSF. Anti-TREM2 cross-linking enhances migration of C57BL/6 OCs and RAW246.7 OCs in response to M-CSF. Conclusions: Our studies indicate that the TREM2 receptor regulates OC multinucleation as well as resorption and migration of mature OCs. Thus, TREM2-DAP12 signals regulate both OC formation and function.

Cutting Edge: Inhibition of TLR and FcR Responses in Macrophages by Triggering Receptor Expressed on Myeloid Cells (TREM)-2 and DAP12

“…The amount of TREM-2 was only slightly lower in DAP12-deficient than in wild-type macrophages (Fig. 2D) (5). When the TREM-1ϩDAP12 or TREM-2ϩDAP12 chimeras were transduced into a mouse T cell line expressing an NFAT-lacZ reporter, both were efficiently expressed and cross-linking of the chimeras with mAbs resulted in NFAT activation, confirming that both chimeras were functional (data not shown).…”

“…As association with DAP12 would predict, ligation of TREM-2 using mAbs induced NO production by a macrophage cell line (8) and promoted partial maturation of human immature monocyte-derived dendritic cells (9). TREM-2 signaling through DAP12 is required for efficient osteoclast development and function (10 -14), and TREM-2 mAb-mediated cross-linking enhances the formation of osteoclasts in vitro (5).…”

Innate Immune Responses to TREM-1 Activation: Overlap, Divergence, and Positive and Negative Cross-Talk with Bacterial Lipopolysaccharide

“…TREM-1 activation, therefore, through the production of M-CSF and SPP1 could potentially contribute to osteoclast maintenance and function (64). Interestingly, DAP12 knockout mice have severe osteopetrosis (65)(66)(67) and although currently this is attributed to defective TREM-2 function in osteoclasts (68,69), our results raise the possibility that defective TREM-1 function may also contribute to this phenotype. Other genes preferentially induced by TREM-1 activation were the metallothionein family members MT1K, MT1E, and MT1F, which are cysteine-rich heavy metal-binding proteins implicated in binding, thereby reducing intracellular levels of available zinc (70).…”

Mechanism and Function of Monoclonal Antibodies Targeting Siglec-15 for Therapeutic Inhibition of Osteoclastic Bone Resorption

“…Notably, DAP12 expression was not dramatically altered during RAW264.7 osteoclast differentiation. Another DAP12-associated immunoglobulin-like receptor, TREM-2, is expressed in osteoclasts and is important for their differentiation and activity (23). We were able to detect a TREM-2⅐DAP12 complex that was present at similar levels in control and differentiated RAW264.7 cells (data not shown), indicating that the abundant Siglec-15⅐DAP12 complexes present in osteoclasts do not reduce TREM-2⅐DAP12 binding.…”