ITAM-mediated signaling has been shown to be critical for osteoclast differentiation and function under normal physiological conditions. ITAM signaling requires multiple components including ITAM-containing adapter proteins, adapter-associated downstream signal mediators, receptors, and their putative ligands. Variation in the local expression any of these ITAM signaling components may contribute to the changes in bone remodeling observed in unique bone microenvironments. The deficiency of ITAM-containing adapter proteins leads to a disconnection of intracellular signaling from the osteoclast microenvironment sensed by the receptors mediating these ITAM signals. Here we show that acute estrogen-deficiency induced osteoporosis is differentially affected by deficiency of ITAM adapter proteins. ITAM-adapter deficiency (DAP12-/-FcRγ-/-) leads to a complete blockade OVX induced bone loss in vertebrae and cortical bones. However, quite unexpectedly, ITAM signaling is bypassed in the estrogen-deficient state with losses of significant amounts of trabecular bone in the femur and tibia. More interesting, the mechanism bypassing ITAM signaling was not readily explained by our current understanding of requirements for osteoclastogenesis.
Severe osteopetrosis in the DAP12-/-FcRγ-/-
mice suggests that ITAM-adapter signaling plays an important role in normal bone remodeling. Unlike other severely osteopetrotic mice, such as those deficient in MCSF-1 
, PTHrP 
, c-fos 
, or RANKL 
mice have tooth eruption, suggesting that there are mechanisms that allow normal osteoclastogenesis in the absence of ITAM-adapter signals. Thus, even under normal physiological conditions, distinct localized regulation of bone remodeling in DAP12-/-FcRγ-/-
mice already exists. Signals that can bypass the need for ITAM-adapter signaling to induce the necessary osteoclast differentiation activity for tooth eruption are not known. Severe osteopetrosis in the long bones in DAP12-/-FcRγ-/-
mice suggests that during normal physiological conditions osteoclastogenesis is largely controlled by ITAM-adapter signals transduced through a ligand-receptor-adapter axis, presumably involving interactions with the microenvironment. The exception of tooth eruption and bone mobilization during estrogen-deficiency suggests that there is an alternate signaling pathway that can be elicited in specific sites during certain stresses to induce bone remodeling.
Here we showed that during bone remodeling following acute estrogen-deficiency, the defect of osteoclastogenesis and function could be rescued in ITAM-adapter deficient mice in trabecular long bone. Our data suggest that during a physiological state of estrogen-deficiency, such as lactation, DAP12-/-FcRγ-/-
mice would be able to mobilize calcium to nutritionally support bone growth of the next generation. This emergency signaling pathway, which is quiescent during normal physiological conditions, activates osteoclasts and mobilizes mineral without utilizing ITAM-adapter signaling. We applied different models and theories to expand our understanding about how ITAM-adapter signaling is bypassed during estrogen-deficiency in ITAM-adapter deficient mice. Estrogen-deficiency can rescue the osteoclast defect of ITAM-adapter deficient mice stimulating osteoclast multinucleation and promoting bone resorption in vivo
. However, unlike wild-type mice, this enhanced osteoclastogenesis was not intrinsic to BMMs or preosteoclasts in ITAM-adapter deficient mice, as shown in the in vitro
cultures. Secondly, estrogen-deficiency induces changes in cytokine and hormone levels, including increased levels of TNFα and FSH. Both TNFα and FSH have been shown to stimulate or even substitute for RANKL during osteoclastogenesis through different mechanisms. TNFα, in combination with TGFβ, can induce osteoclast differentiation and multinucleation in the absence of RANKL in normal osteoclasts derived in vitro 
. Increasing levels of FSH can cooperate with RANKL to have a synergistic effect on osteoclast differentiation during estrogen-deficiency in vitro 
. However, as shown in this study, these two new models could not explain the increase in multinucleated osteoclasts seen in the DAP12-/-FcRγ-/-
Although we investigated alternative methods of osteoclast activation, one important mechanism that we did not address is the role of T lymphocytes in acute estrogen-deficiency induced bone loss. However, Pierre Jurdic and colleagues (Lyons, France) have recently investigated the role of mature lymphocytes and ITAM-adapter signaling in bone remodeling induced by OVX (A. Anginot, et. al., PLoS One, in press). They have completed similar studies examining the role of DAP12 signaling and mature T and B cells in the acute estrogen deficient state in constitutively inactive DAP12 (KΔ75), RAG1 (RAG1-/-), or double deficient (RAG1-/-; KΔ75) mice. Their results demonstrate increased bone loss in the double deficient (RAG1-/-; KΔ75) mice suggesting that DAP12 ITAM signals negatively regulate bone remodeling in RAG1-/- mice consistent with our observations. Furthermore, RAG1-/- and RAG1-/-;KΔ75 mice lose significant bone after OVX indicating that mature T and B cells are not needed for estrogen-deficiency induced bone loss and do not provide the missing signal for osteoclastogenesis. Combined with our own studies, these results strongly suggest a novel, still to be discovered, osteoclast activation mechanism in vivo.
The complexity of the different roles of ITAM-adapter signaling in different local microenvironments is consistent with the nature of the associated receptors. TREM2 and OSCAR are immunoglobulin superfamily innate immune receptors expressed on osteoclasts. TREM2, associated with DAP12, is considered a putative pattern-recognition receptor (PRR) because it recognizes a wide variety of anionic ligands, including dextran sulfate and bacterial products such as lipoteichoic acid and peptidoglycan 
. PRRs are genetically coded and able to recognize conserved structural features in a large range of pathogens. Being able to sense these environmental changes, PRRs could respond upon ligand binding as a first line of defense. Presently, specific ligands in the bone for these two receptors are still unidentified.
Recent findings convincingly show that ITAM-containing adapter-receptor protein complexes can also send inhibitory signals, although the mechanism is still unclear 
. Despite the general concept that the ITAM in the intracellular domain of DAP12 will transduce an activation signal upon ligand binding of the associated receptor, it has been shown by multiple groups that DAP12-deficient macrophages have an enhanced response to TLR stimulation 
. It has also been shown that TREM2, but not other DAP12-associated receptors, is responsible for transducing the inhibitory signal  
. It has been suggested that the type of downstream signals induced (activating or inhibitory) is dependent on the affinity and avidity of the extracellular ligands. High affinity or avidity ligands have been proposed to initiate activation, whereas low affinity or avidity ligands may cause inhibition 
We have observed evidence of a dual function of ITAM-adapter signaling in osteoclasts. The absence of downstream activation signal in ITAM-adapter deficient mice accounts for the osteopetrosis and defective osteoclastogenesis in ITAM-adapter deficient mice during normal physiological conditions. In our study, DAP12-/-FcRγ-/- mice lose about 40% trabecular long bone following OVX, which is similar to wild type, DAP12-/-, and FcRγ-/- mice. However DAP12-/-FcRγ-/- and DAP12-/- mice have much higher baseline bone mass. If the absolute bone loss is taken into consideration, DAP12-/-FcRγ-/- mice and DAP12-/- quantitatively lose more bone compared to wild-type mice. One possible explanation is that a low affinity ligand in the bone marrow compartment is engaged by TREM2/DAP12 that initiates an inhibitory signal in osteoclasts through DAP12 to fine-tune the resorption magnitude. In the absence of both ITAM-adapter proteins, the “inhibitory” signals cannot be used to regulate bone resorption, which leads to an extremely high absolute trabecular bone loss in DAP12-/-FcRγ-/- and DAP12-/- OVX mice. Thus, ITAM signaling adapters may have multiple functions in the regulation of osteoclasts. It will be necessary to identify these putative ligands in the bone to gain further insight into signals regulating these responses to local microenvironmental changes at distinct location and different stimuli in bone remodeling.