TREM2- and DAP12-dependent activation of PI3K requires DAP10 and is inhibited by SHIP1

Abstract

The activation and fusion of macrophages and of osteoclasts require the adaptor molecule DNAX-activating protein of 12 kD (DAP12), which contains immunoreceptor tyrosine-based activation motifs (ITAMs). TREM2 (triggering receptor expressed on myeloid cells-2) is the main DAP12-associated receptor in osteoclasts and, similar to DAP12 deficiency, loss of TREM2 in humans leads to Nasu-Hakola disease, which is characterized by bone cysts and dementia. Furthermore, in vitro experiments have shown that deficiency in DAP12 or TREM2 leads to impaired osteoclast development and the formation of mononuclear osteoclasts. Here, we demonstrate that the ligation of TREM2 activated phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase 1 (ERK1) and ERK2, and the guanine nucleotide exchange factor Vav3; induced the mobilization of intracellular calcium (Ca(2+)) and the reorganization of actin; and prevented apoptosis. The signaling adaptor molecule DAP10 played a key role in the TREM2- and DAP12-dependent recruitment of PI3K to the signaling complex. Src homology 2 (SH2) domain-containing inositol phosphatase-1 (SHIP1) inhibited TREM2- and DAP12-induced signaling by binding to DAP12 in an SH2 domain-dependent manner and preventing the recruitment of PI3K to DAP12. These results demonstrate a previously uncharacterized interaction of SHIP1 with DAP12 that functionally limits TREM2- and DAP12-dependent signaling and identify a mechanism through which SHIP1 regulates key ITAM-containing receptors by directly blocking the binding and activation of PI3K.

Fig. 1

SHIP1 inhibits enhanced osteoclastogenesis stimulated by the ligation of TREM2. Bone marrow–derived macrophage precursors were isolated from WT or SHIP1-deficient mice. Cells were treated with plate-bound antibody against TREM2 or control antibody together with RANKL (50 ng/ml) and M-CSF (20 ng/ml) for 5 days. The number of TRAP-positive osteoclasts that contained more than two nuclei were counted per well. (A) Osteoclast cultures were fixed and stained for TRAP. (B) Compared to cultures of WT cells, cultures of SHIP1-deficient cells formed twice as many osteoclasts (*P < 0.01). Ligation of TREM2 led to a twofold increase in the number of WT osteoclasts (**P < 0.01) and a fourfold increase in SHIP1-deficient osteoclasts (^#P < 0.002). Data are representative of two experiments.

Fig. 2

SHIP1 is recruited to DAP12 after ligation of TREM2 and it inhibits TREM2 and DAP12 signaling and Ca²⁺ flux. (A) Activation of TREM2 and DAP12 induces the association of SHIP1 with DAP12 in J774 cells stimulated with antibody against TREM2 (T2 Ab) but not in cells stimulated with control Ab (Con Ab). Samples from immunoprecipitations (IP) with antibody against pDAP12 were analyzed by Western blotting for the presence of SHIP1 and DAP12. Whole-cell lysates (WCL) were also analyzed similarly for the presence of SHIP1 and DAP12. Vanadate (Van) increased the amount of SHIP1 that coimmunoprecipitated with pDAP12. (B) BMMs stimulated with antibody against TREM2 or with control antibody were used in immunoprecipitation reactions with antibody against pDAP12 and were analyzed by Western blotting for the presence of SHIP1. (C) J774 cells were stimulated with antibodies against TREM2, SIRPβ, or MDL-1 and then used in immunoprecipitation reactions with antibody against pDAP12. Stimulation with antibodies against TREM2 or MDL-1 induced the association of SHIP1 with DAP12, whereas stimulation with antibody against SIRPβ did not. (D) BMMs derived from WT (+/+) or SHIP1-deficient (−/−) mice were treated by ligation of TREM2 and WCLs were analyzed by Western blotting for the presence of pAkt, pERK, and SHIP1. Membranes were stripped and incubated with antibodies against total ERK and Akt. Cell lysates were used in immunoprecipitation reactions with an antibody against phosphorylated tyrosine (pY) residues and analyzed by Western blotting for the presence of pVav3 and pSyk. (E) Ca²⁺ flux was induced in WT and SHIP1-deficient BMMs by antibody against TREM2 (red lines), whereas control antibody (black lines) had no effect. Ligation of TREM2 induced enhanced Ca²⁺ flux in SHIP1-deficient cells (P = 0.0123 for area under the curve). Data are representative of two studies for (B) and (C) and three studies for (A), (D), and (E).

Fig. 3

SHIP1 binds directly to the phosphorylated ITAM in DAP12. (A) Biotinylated DAP12 ITAM peptides are listed. The specific phosphorylated tyrosine is noted by a lowercase p. Abbreviations for the amino acids are as follows: D, Asp; E, Glu; G, Gly; L, Leu; P, Pro; Q, Gln; R, Arg; S, Ser; V, Val; and Y, Tyr. (B) Lysates from resting RAW 264.7 cells were incubated with biotinylated peptides (10 μM) overnight at 4°C. Peptide-protein complexes were precipitated with NeutrAvidin-conjugated beads and analyzed by Western blotting with antibodies against SHIP or Syk. WCLs were analyzed for the presence of SHIP1 as a loading control. (C) Schematic representation of the expression constructs that encode GST-SH2 fusion proteins of WT SHIP1 or an SH2 domain mutant of SHIP1 that contains a glycine in the place of arginine at position 34 (R34G) (GST-mSH2). Coomassie blue staining of purified GST-fusion proteins is shown on the right. GST is ~25 kD, SH2 and mSH2 are ~37 kD. (D) The purified fusion proteins, GST, SH2, and mSH2 were incubated with biotinylated WT or phosphorylated Y⁶⁵ ITAM peptides overnight at 4°C, precipitated with NeutrAvidin beads, and analyzed by Western blotting with an antibody against GST. Purified GST-fusion proteins were incubated with antibody against GST to reveal the amount of fusion proteins added to the peptides. Data are representative of three studies.

Fig. 4

DAP10-dependent recruitment of PI3K p85 mediates TREM2- and DAP12-dependent activation of Akt and ERK1/2. (A) WT (+/+) and SHIP1-deficient (−/−) BMMs were stimulated with antibody against TREM2 or with control antibody, lysed, subjected to immunoprecipitation with an antibody against pDAP12, and analyzed by Western blotting with antibodies against PI3K p85 or pDAP12. (B) Biotinylated DAP12 pITAM peptides coprecipitated with p85 in BMM lysates. (C) WT and DAP10-deficient BMMs were stimulated with antibody against TREM2. Samples subjected to immunoprecipitation with antibody against pDAP12 were analyzed by Western blotting for the presence of SHIP1, p85, and Grb2. (D) WCLs were analyzed by Western blotting with antibodies against pAkt, ERK1/2, and DAP12. Blots were stripped and incubated with antibodies against total Akt and ERK1/2. (E) WT macrophages were lysed in digitonin buffer and then subjected to immunoprecipitation reactions with antibodies against TREM2, DAP12, or DAP10 or with control antibody. Immunoprecipitated complexes were analyzed by Western blotting for each of these proteins. TREM2 was immunoprecipitated with DAP12 and DAP10. Data are representative of two experiments.

Fig. 5

Inhibition of PI3K activity enhances the recruitment of SHIP1 to DAP12 after ligation of TREM2. (A) J774 cells were treated with DMSO or wortmannin (0.2 μM) before stimulation with antibody against TREM2 or with control antibody. Samples subjected to immunoprecipitation with antibody against pDAP12 were analyzed by Western blotting for the presence of SHIP1, Grb2, p85, and pDAP12. Lysates were subsequently subjected to immunoprecipitation with antibody against pY and analyzed by Western blotting for the presence of SHIP1, Vav3, and Syk. WCLs were analyzed by Western blotting for the presence of pAkt and total Akt. Data are representative of three experiments. (B) BMMs derived from WT or SHIP1-deficient mice were treated with wortmannin or DMSO and incubated with antibody against TREM2 or with control antibody. Lysates were subjected to immunoprecipitation with antibody against pDAP12 and analyzed by Western blotting for the presence of p85, Syk, Grb2, pDAP12, and SHIP1. WCLs were similarly analyzed for the presence of pAkt and pERK. Membranes were stripped and incubated with antibodies against total Akt or ERK. Data are representative of two experiments.

Fig. 6

Ligation of TREM2 induces the intracellular colocalization of SHIP1, F-actin, and DAP12 in osteoclasts. Osteoclasts derived from WT or SHIP1-deficient BMMs were prepared on eight-well paradox chamber slides, as described in Materials and Methods. Osteoclasts were starved of serum for 4 hours and treated with antibody against TREM2 for 15 min. Unstimulated (0 min) and stimulated cells were fixed and incubated with antibody against DAP12 (shown in red), phalloidin to detect F-actin (shown in blue), and antibody against SHIP1 (shown in green). The overlay shows the areas of colocalization of DAP12, F-actin, and SHIP1 at the plasma membrane. Data are representative of five studies.

Fig. 7

Stimulation with RANKL and M-CSF leads to the recruitment of SHIP1 to DAP12. (A) RAW 264.7 cells were treated with control medium or with medium containing M-CSF (20 ng/ml), RANKL (50 ng/ml), or both for 3 days. Adherent cells were lysed on the plates and subjected to immunoprecipitation with antibody against DAP12. Complexes were analyzed by Western blotting for the presence of SHIP1 and DAP12. (B) J774 cells in suspension were treated with RANKL (100 ng/ml), M-CSF (100 ng/ml), or both or were treated with antibody against TREM2 for up to 20 min. Samples subjected to immunoprecipitation with antibody against pDAP12 were analyzed by Western blotting for the presence of SHIP1 and pDAP12. (C) J774 cells were stimulated in suspension with antibody against TREM2 alone, or in the presence of RANKL or M-CSF. Cells were subjected to immunoprecipitation with antibody against pDAP12 and immunoprecipitates were analyzed by Western blotting with antibodies against SHIP1 or pDAP12. (D) A different antibody against pDAP12 (2426) was used to immunoprecipitate pDAP12 from RAW 264.7 cells and the resulting immunoprecipitates were analyzed by Western blotting for pDAP12. Data are representative of three experiments.

Fig. 8

M-CSF and RANKL differentially induce the colocalization of SHIP1 and DAP12. WT and SHIP1-deficient BMMs were differentiated to osteoclasts by culturing with M-CSF and RANKL for 3 days. After being starved of serum for 4 hours, cells were treated with M-CSF (100 ng/ml) or RANKL (100 ng/ml) for 4 hours, fixed, and incubated with antibody against DAP12 (shown in red), phalloidin (shown in blue), or antibody against SHIP1 (shown in green).

Fig. 9

Proposed DAP12 signaling pathway in macrophages and osteoclasts. In response to ligation of TREM2, Src family kinases phosphorylate the ITAM of DAP12 and the YINM motif of DAP10, which form docking sites for the Syk and p85, with the subsequent recruitment of PLC-γ2 and Grb2. This signaling complex then leads to the activation of Akt, ERK1/2, and Vav3. Simultaneously, SHIP1 is recruited to the DAP12 ITAM where it may dislodge or prevent the further recruitment of SH2 domain–containing proteins, including p85 and Syk. This leads to an arrest in the activation of PI3K, ERK, Akt, and VAV3. The absence of SHIP1 enables prolonged signaling in response to the activation of DAP12, which leads to enhanced osteoclastogenesis. Low-avidity ligands of macrophages or bacteria may preferentially recruit SHIP1, whereas high-avidity ligands or receptor cross-linking may induce a strong activation of PI3K and Syk.