TLT-1s, Alternative Transcripts of Triggering Receptor Expressed on Myeloid Cell-like Transcript-1 (TLT-1), Inhibits the Triggering Receptor Expressed on Myeloid Cell-2 (TREM-2)-mediated Signaling Pathway during Osteoclastogenesis*

Background: The triggering receptor expressed on myeloid cell (TREM)-mediated signaling is essential for osteoclastogenesis. Results: The alternative transcripts of triggering receptor expressed on myeloid cell-like transcript-1 (TLT-1s) inhibits osteoclast formation by counteracting the TREM-2 signaling pathway. Conclusion: TLT-1s is a negative regulator of osteoclastogenesis, by constitutively associating with SHP-1 and SHIP-1 phosphatases, abrogating the TREM-2 signaling upon RANKL stimulation. Significance: We discovered that an alternative transcript of TLT-1, namely TLT-1s, negatively regulates osteoclastogenesis. Triggering receptor expressed on myeloid cells (TREM)-like transcript-1 (TLT-1) is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-baring TREM family protein. In this study, we identified an alternative transcript form of TLT-1, namely TLT-1s, which has very short extracellular immunoglobulin domain consisting of only 202 amino acids. TLT-1s was mainly expressed in macrophages and osteoclast precursor cells. Upon receptor activator of nuclear factor-κB ligand stimulation, TLT-1s mRNA and protein levels were gradually decreased in BMMs. We also showed the TLT-1s is localized to the cytoplasmic membrane in osteoclast precursor cells. TLT-1s silencing strongly enhanced the formation and resorption activity of osteoclast. In addition, forced expression of TLT-1s showed reduced formation of osteoclast. Because ITIM-baring proteins inhibit immunoreceptor tyrosine-based activation motif (ITAM)-mediated receptor signaling, we tested whether TLT-1s physically interacted with TREM-2, the ITAM-associated co-stimulatory receptor essential for osteoclast differentiation. We showed that TLT-1s is associated with TREM-2 in osteoclast precursor cells. TLT-1s is also associated with tyrosine Src homology 2 domain-containing phosphatase-1 and SH2 domain-containing inositol phosphatase-1 and recruited them to the TREM2-ITAM signaling complex. In addition, knockdown of TLT-1s markedly elevated the intracellular calcium concentration and oscillation in osteoclast precursor cells. In addition, calcium-mediated induction of nuclear factor of activated T cells was also increased by TLT-1s silencing. Furthermore, TREM-2-mediated Akt activation and proliferation of osteoclast precursor cells were also enhanced in TLT-1s silenced cells. In this paper, we found the noble ITIM-baring inhibitory membrane protein; TLT-1s, which regulates ITAM-mediated signaling on osteoclastogenesis.

Triggering receptor expressed on myeloid cells (TREM) 2 is a member of the activating immunoreceptor expressed on monocytes, macrophages, microglia, and neutrophils (1)(2)(3). To date, 3 activating TREM genes have been identified, clustered on human chromosome 6 and mouse chromosome 17 (4). TREM is characterized by a single V-set immunoglobulin domain, short cytoplasmic domain, and transmembrane domain capable of interaction with an immunoreceptor tyrosine-based activation motif (ITAM)-baring protein, DNAX activating protein of 12 (DAP12) (5). In addition to 3 activating TREM receptors, the TREM gene cluster includes an inhibitory receptor, TREM-like transcript-1 (TLT-1). Unlike other TREMs, TLT-1 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain capable of recruiting protein-tyrosine phosphatases (6). TLT-1 expression has been reported in ␣-granules of platelets and megakaryocytes, regulating platelet activation and inflammation (7). TLT-1 null mice exhibit deficiency in platelet aggregation and are susceptible to lipopolysaccharide-induced septic shock (8). Recently, two splice variants of TLT-1 with short cytoplasmic domains were reported (9), suggesting that isoforms of TLT-1 may have different cellular functions.
Osteoclasts are bone-resorbing cells differentiated from monocyte/macrophage lineage cells in the presence of receptor activator of nuclear factor-B ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) (9,10). RANKL activates signaling pathways involving the induction of the nuclear factor of activated T cells (NFATc1) upstream of osteoclastogenic genes such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K. In addition to RANKL, co-stimulatory receptors such as TREM-2 and osteoclast-associated receptor provide calcium signals required for the optimal NFATc1 activation (11). The ITAM bearing proteins DAP12 and FcR␥ recruit SYK kinase that activates calcium signaling through phospholipase C␥ (PLC␥) (11)(12)(13). On the other hand, phosphatases such as tyrosine Src homology 2 (SH2) domain-containing phosphatase-1 (SHP-1) and SH2 domain-containing inositol phosphatase-1 (SHIP-1) binds to ITIMs to negate the ITAMmediated signaling (14,15). During osteoclast differentiation, LILRB and PIR-B down-regulates the osteoclastogenesis by recruiting SHP-1 via their ITIMs (16). In the present report, we discovered an alternative transcript form of TLT-1, namely TLT-1s, which has a very short extracellular Ig domain. Here we show that TLT-1s is a negative regulator of osteoclastogenesis, by constitutively associating with SHP-1 and SHIP-1 phosphatases, abrogating the TREM-2 signaling upon RANKL stimulation.

EXPERIMENTAL PROCEDURES
Reagents and Antibodies-Anti-TLT-1 antibody was from Novous Biologicals (Littleton, CO). Anti-NFATc1, TREM-2, and osteoclast-associated receptor antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other antibodies were purchased from Cell Signaling Technology (Beverly, MA). Human soluble RANKL and M-CSF were from PeproTech (Rocky Hill, NJ). Lipofectamine 2000 was purchased from Invitrogen. Anti-actin antibody and all other chemicals were purchased from Sigma.
Bone Marrow-derived Macrophage Culture and Osteoclast Differentiation-Bone marrow-derived macrophages (BMMs) were generated as described previously (15,16). In brief, isolated bone marrow cells from mouse femurs were cultured overnight in ␣-modified essential medium containing 10% FBS on culture dishes. Nonadherent cells were further cultured for 3 days in the presence of 5 ng/ml of M-CSF to generate BMMs. Osteoclasts were obtained by culturing cells in ␣-MEM containing 10% FBS, 30 ng/ml of M-CSF, and 200 ng/ml of RANKL for 3 days.
Preparation of Platelet-To harvest platelets, mice were anesthetized with isoflurane and 500 l of blood was collected into a tube containing 3.8% sodium citrate (1/9, v/v) by cardiac puncture. Platelet-rich plasma was obtained by centrifugation at 200 ϫ g for 7 min. The plasma and buffy coat were transferred to a fresh tube. Platelet was isolated by centrifugation at 850 ϫ g for 7 min.
In Vitro Resorption Pit Formation Assay-BMMs were cultured on dentin slices for 5 days in the presence of RANKL and M-CSF. After removing the cells by sonication, dentin slices were stained with hematoxylin and observed under a light microscope. The bone resorption area was measured with image analysis software (Image Pro-Plus, Media Cybernetics).
Gene Knock-down by Small Interfering RNA Oligonucleotides-The 22-nucleotide small interfering RNA (siRNA) duplexes for TLT-1s and negative control were purchased from Invitrogen. The TLT-1s target sequence was 5Ј-ACATGTGGAATGTC-CGAGGGTAGT-3Ј. BMMS were transfected with siRNA oligonucleotides using Lipofectamine 2000 following the manufacturer's instructions.
Retroviral Transduction-Mouse TLT-1s was cloned into pMX-IRES vector. Retroviral particles were packaged by transfecting Plat-E cells with DNA plasmids using Lipofectamine 2000 according to the manufacturer's instructions. After a 48-h culture in DMEM supplemented with 10% FBS, viral supernatants were collected and filtered through a 0.45-m syringe filter. BMMs were infected with viral supernatants in the presence of Polybrene (10 g/ml) and M-CSF for 12 h.
5Ј-Rapid Amplification of cDNA Ends (5Ј-RACE)-Total RNA was isolated from mouse bone marrow-derived macrophages using the PARIS TM Kit (Ambion, TX). The 5Ј-RACE was performed using the System for Rapid Amplification of cDNA Ends, version 2 (Invitrogen).
Reverse Transcriptase-Polymerase Chain Reaction Analysis-Total RNA prepared using TRIzol (Invitrogen) were reverse transcribed using SuperScript II reverse transcriptase (Invitrogen). One l of cDNA synthesized from 1 g of total RNA was amplified with the specific primers.
Western Blotting-Cells were washed with ice-cold PBS, scraped with a rubber policeman, and lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.5 mM PMSF, 1 g/ml of aprotinin, 1 g/ml of leupeptin, and 1 g/ml of pepstatin). After protein quantification with a protein assay kit (Bio-Rad), whole cell extracts were separated on polyacrylamide gels and transferred onto nitrocellulose membranes. After blocking for 1 h with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20, membranes were incubated overnight at 4°C with primary antibodies in TBST containing 2% skim milk. Membranes were washed, incubated with secondary antibodies conjugated with horseradish peroxidase, and developed using an enhanced chemiluminescence system.
TRAP Staining-Cells were fixed in 3.7% formaldehyde solution and permeabilized with 0.1% Triton X-100. After washing with PBS, cells were stained using the Leukocyte Acid Phosphatase Assay Kit (Sigma) following the manufacturer's instruction. TRAP-positive multinuclear osteoclasts containing three or more nuclei were counted under a light microscope and photographed.
Measurement of Intracellular Calcium Concentration and Oscillations-For measurement of total intracellular calcium concentration, cells were loaded with Fluo-4 NW dye (Molecular Probes, Eugene, OR) at 37°C for 30 min followed by an additional incubation for 30 min at room temperature. The calcium-dependent fluorescence was measured in a CytoFluor plate reader (Applied Biosystems, Foster City, CA) with a 485/ 535 nm excitation/emission filter pair. Calcium oscillations were measured as described previously (17).
BrdU Incorporation Assay-Bromodeoxyuridine (BrdU) incorporation into cells was measured using the BrdU Cell Proliferation Assay (Calbiochem, San Diego, CA) following the manufacturer's instruction.
Cell Viability Assay-Cell viability was measured using the CCK-8 assay (Dogindo Laboratories) according to the manufacturer's protocol. Cells were incubated with the CCK-8 reagent for 1 h and optical density was determined at 450 nm.
Statistics-To determine the significance of results, Student's t test was used. Differences with p Ͻ 0.05 were regarded as significant.

Cloning and Characterization of the Alternative Transcript of
Mouse TLT-1-To discover new molecules that regulate osteoclast differentiation, we monitored gene expression profiles during osteoclast differentiation of human peripheral blood mononuclear cells (18). Microarray analysis revealed that the TLT-1 mRNA level negatively correlated with osteoclastogenesis. Interestingly, RT-PCR amplification in mouse BMMs failed to gain the full-length TLT-1 mRNA, resulting in only the C-terminal 400 base pairs of TLT-1. Both in BMMs and osteoclasts, the N-terminal region of TLT-1 could not be amplified using primer sets designed with the known TLT-1 sequences. So, we hypothesized that transcript variants of TLT-1 with alternative N-terminal sequences existed in osteoclast precursors. To test this, a 5Ј-RACE was carried out in BMMs. Indeed, a cDNA clone, designated as TLT-1s, containing a 5Ј-untranslated region (UTR) distinct from that of TLT-1 was identified. Comparison of the TLT-1s 5Ј-UTR sequence with the mouse TLT-1 gene (GenBank TM accession number AY078502) revealed that the TLT-1s 5Ј-UTR was derived from intron 2 of the TLT-1 gene (Fig. 1A). Exons 1 and 2 were totally missing in TLT-1s, starting with the intervening 16-bp sequence of intron 2 immediately before exon 3 of TLT-1. As a result, full-length TLT-1 consists of 322 amino acids, TLT-1s was predicted to have only 202 amino acids mostly lacking the N-terminal extracellular Ig domain of TLT-1. To gain further insights into the nature of TLT-1s, the tissue expression pattern was investigated by RT-PCR using specific primer sets. Strong TLT-1 mRNA expression was detected in bone, spleen, and to a lesser extent in heart (Fig. 1B). Notably, only TLT-1s was expressed in BMMs, although both TLT-1 and TLT-1s were detected in bone. Comparison of mRNA expression in whole bone marrow cells (WBMs), BMMs (Mac), and platelets (Plat) revealed that TLT-1s was expressed exclusively only in BMMs (Fig. 1C). On the other hand, TLT-1 expressed in WBMs seemed to disappear during the M-CSF-derived differentiation into macrophages. Western blotting using an antibody detecting a common C-terminal region confirmed that the 37-kDa TLT-1 was detected in WBMs and platelets, whereas 27-kDa TLT-1s was exclusively detected in BMMs (Fig. 1D).
Expression of TLT-1s during RANKL-mediated Osteoclastogenesis-Because TLT-1s highly expressed in macrophages, the expression of TLT-1s during RANKL-induced osteoclastogenesis was investigated. TLT-1s mRNA levels gradually decreased during the 24-h treatment of RANKL ( Fig. 2A). Fulllength TLT-1 was not detected in BMMs before and after RANKL stimulation. TLT-1s expression was also down-regulated by RANKL in RAW 264.7 mouse macrophage cell lines (data not shown). Western blotting corroborated the decrease in TLT-1s expression during RANKL-dependent osteoclastogenesis (Fig. 2B). It was previously shown that TLT-1 moved to cytoplasmic membranes upon stimulation in platelets (9). The examination of subcellular localization of TLT-1s by confocal microscopy in Fig. 2C revealed that TLT-1s was dispersed in the cytoplasm in BMMs and localized in the plasma membrane region in pre-osteoclasts. TLT-1s was hardly detectable in the mature osteoclast.
Suppression of RANKL-mediated Osteoclastogenesis and Bone Resorption by TLT-1s-To determine the role of TLT-1s during osteoclast differentiation, TLT-1s knockdown was performed using siRNA oligonucleotides specific for TLT-1s that efficiently reduced the TLT-1s mRNA levels in BMMs (Fig. 3A). The knockdown of TLT-1s significantly increased the number of multinuclear osteoclasts upon RANKL stimulation of BMMs A, schematic representation of TLT-1 and TLT-1s genes. The TLT-1 gene consists of 6 exons and 5 introns. Boxes represent individual exons. The primers used to amplify TLT-1 (P1 and P3) and TLT-1s (P2 and P3) were indicated by arrows. B, the expression of TLT-1 and TLT-1s in mouse tissues was assessed by RT-PCR. C, mouse BMMs were differentiated from WBM by incubation with 10 ng/ml of M-CSF for 3 days. The expression of TLT-1 and TLT-1s was detected by RT-PCR in WBMs, BMMs (Mac), and platelet (Plat). D, the protein levels of TLT-1 and TLT-1s were measured by Western blotting using an antibody against the C terminus of TLT-1. (Fig. 3B, left panel). Similarly, the silencing of TLT-1s in BMMs significantly enhanced osteoclastogenesis in BMM-osteoblast co-culture experiments (Fig. 3B, right panel). In accordance with enhanced osteoclast differentiation, TLT-1 knockdown markedly elevated NFATc1 expression in RANKL-stimulated BMMs (Fig. 3C). In addition, the bone resorption activity of osteoclasts cultured on dentin slices was also significantly increased by TLT-1s knockdown (Fig. 3D). To further clarify the role of TLT-1s on osteoclastogenesis, TLT-1s was ectopically expressed in BMMs by retrovirus-mediated gene transfer (Fig. 4A). As expected, the overexpression of TLT-1s significantly reduced the number of osteoclasts compared with control (Fig. 4B). We also showed the inhibitory effect of TLT-1 on osteoclastogenesis in the RAW 265.7 macrophage cell line (supplemental Fig. S1). These inhibitory effects of TLT-1s on osteoclast differentiation were eliminated by the addition of TLT-1s-siRNA oligonucleotides (Fig. 4, C and D), suggesting the specific role of TLT-1s in osteoclastogenesis.
Recruitment of SHIP and SHP-1 to TREM-2 by TLT-1s-Costimulatory receptor signaling is critical for RANKL-mediated calcium oscillations and NFATc1 induction during osteoclastogenesis. Because TLT-1s contain ITIM, we asked whether TLT-1s inhibited osteoclast differentiation by affecting FIGURE 2. The TLT-1s expression was reduced by RANKL. A, BMMs were stimulated with 200 ng/ml of RANKL for the indicated times. The mRNA levels of TLT-1 and TLT-1s were determined by RT-PCR. B, BMMs were stimulated with 200 ng/ml of RANKL for the indicated days. The protein level of TLT-1s was determined by Western blotting using an antibody against the C terminus of TLT-1. NFATc1 expression was used as a marker for osteoclast differentiation. C, BMMs were cultured with 200 ng/ml of RANKL for 1 (pOC, preosteoclasts) or 3 days (OS, osteoclasts). Cells were immunostained using an antibody against the C terminus of TLT-1 followed by Cy3-conjugated secondary antibody. Nuclei and actin filaments were stained with DAPI and FITCconjugated phalloidin.  co-stimulatory receptor signaling in osteoclast precursors. Although TLT-1s did not associate with osteoclast-associated receptor (data not shown), FLAG-tagged TLT-1s co-immunoprecipitated with TREM-2 (Fig. 5A) independently of RANKL stimulation in BMMs. Notably, SHIP-1 and SHP-1 phosphatases co-immunoprecipitated with TLT-1s in association with TREM-2 (Fig. 5B) indicating that TLT-1s inhibited TREM-2mediated signaling by recruiting SHP-1 and SHIP-1 to TREM-2 in osteoclast precursor cells.

Modulation of Calcium Oscillations by TLT-1s-Because
TREM-2 activation has been shown to facilitate PLC␥2-mediated calcium oscillations, the effect of TLT-1s knockdown on PLC␥2 activation and calcium oscillations was investigated. After TLT-1s knockdown, pre-osteoclasts were serum-starved and re-stimulated with RANKL. As shown in Fig. 6A, TLT-1s knockdown significantly elevated the RANKL-mediated PLC␥2 phosphorylation. In addition, the total intracellular calcium concentration was also elevated in TLT-1s-silenced preosteoclasts (Fig. 6B). Furthermore, the frequency and amplitude of RANKL-dependent calcium oscillations were also dramatically increased by TLT-1s knockdown (Fig. 6C). These results suggest that TLT-1s is a transmembrane adaptor molecule that regulates RANKL-mediated calcium oscillations.
Modulation of Osteoclast Precursor Proliferation by TLT-1s-TREM-2 signaling has been shown to enhance the proliferation of osteoclast precursors via a phosphatidylinositol 3-kinasemediated activation of Akt, the proliferation of osteoclast precursors was tested after TLT-1s knock-down. Fig. 7A showed that RANKL-induced Akt phosphorylation was significantly increased in TLT-1s-silenced BMMs. However, the phosphorylation of p38, ERK, and JNK was marginally altered by TLT-1s knockdown. The BrdU incorporation was significantly higher in BMMs treated with TLT-1s-siRNA following both M-CSF and M-CSF plus RANKL stimulation, compared with control cells treated with scrambled siRNA (Fig. 7B). The BrdU incor- A, BMMs were infected with viruses harboring empty vector (pMX) or FLAGtagged TLT-1s (pMX-FLAG-TLT-1s) and further cultured with RANKL for 2 days. After serum starvation for 3 h, cells were stimulated with 200 ng/ml of RANKL for 30 min. The FLAG-tagged TLT-1s was immunoprecipitated using an anti-FLAG antibody. Immunoprecipitated (IP) proteins or whole cell lysates were subjected to Western blotting. B, control or TLT-1s-overexpressing BMMs were cultured with RANKL for 2 days. After serum starvation for 3 h, cells were stimulated with 200 ng/ml of RANKL for the indicated times. Whole cell lysates were subjected to immunoprecipitation using an anti-FLAG antibody followed by Western blotting. FIGURE 6. TLT-1s modulates calcium oscillations. BMMs were transfected with scrambled control siRNA or TLT-1s-siRNA. A, siRNA-transfected BMMs were incubated with RANKL for 2 days and the whole cell lysates were subjected to Western blotting. B, TLT-1s-silenced BMMs were seeded on 96-well plates, incubated with RANKL for 2 days, and loaded with Fluo-4 NW. The calcium-dependent fluorescence was measured using a plate reader. C, BMMs were plated on coverslips after TLT-1s knockdown. After incubation with RANKL for 2 day, cells were loaded with Fura-2/AM and the calcium oscillations were monitored using a confocal microscope. *, p Ͻ 0.05 versus control.

DISCUSSION
ITIM-containing receptors were identified by their ability to inhibit signaling by ITAM-bearing receptors. Such ITIM-bearing receptors have been known to regulate osteoclastogenesis via the recruitment of phosphatases to co-stimulatory receptors (16,19). In the present report, we identified and characterized the novel ITIM-bearing alternative transcript TLT-1 des-ignated as TLT-1s by 5Ј-RACE analysis in BMMs. TLT-1s has a distinct N-terminal sequence from intron 2 of the TLT-1 gene. TLT-1 and TLT-1s showed the different tissue distribution patterns. TLT-1 is expressed exclusively in platelets and megakaryocytes. Interestingly, the TLT-1s was not expressed in platelets. TLT-1s was expressed in macrophages and osteoclast only. Although further investigation is required, the possible use of an alternative promoter might have resulted in the tissue-specific expression of TLT-1s. Because primary calvarial osteoblasts expressed neither TLT-1 nor TLT-1s (data not shown), TLT-1s could be a specific therapeutic target for bone erosive disease such as osteoporosis induced by unregulated osteoclastogenesis.
TLT-1, mainly expressed in platelets and megakaryocytes, is the only inhibitory receptor of the TREM cluster. In contrast to the full-length TLT-1, TLT-1s has a very short extracellular Ig domain while containing an intact transmembrane domain and FIGURE 7. TLT-1s inhibits the proliferation of osteoclast precursors. A, after TLT-1s-siRNA transfection, BMMs were serum starved for 3 h and stimulated with 200 ng/ml of RANKL for the indicated times. The phosphorylation of MAPKs was determined by Western blotting. Control cells were transfected with scrambled siRNA. B, after TLT-1s-siRNA transfection, BMMs were cultured with 30 ng/ml of M-CSF for 3 days. Cell proliferation was measured using CCK assay. C, after TLT-1s-siRNA transfection, BMMs were incubated with BrdU for 2 h. BrdU incorporation was determined by the ELISA method. D, upon ligation of RANK by RANKL, co-stimulatory receptors such as TREM-2 associate with ITAM-containing adaptor molecule including DAP12 to trigger intracellular calcium signaling that is critical for NFATc1 induction as well as proliferation signals through Akt activation. TLT-1s recruits SHP-1 and SHIP-1 phosphatases to TREM-2, thus negatively regulating osteoclast differentiation and proliferation. *, p Ͻ 0.05 versus control. cytoplasmic ITIM domain. In this study, we found that TLT-1s inhibited TREM signaling in osteoclast precursor cells. Because the ITAM-mediated calcium signaling is crucial for osteoclastogenesis, mice deficient in both ITAM-signaling adaptors DAP12 and FcR␥ are severely osteopetrotic because of the impaired formation of osteoclast (20). Both DAP12 and FcR␥ associate with the surface receptor TREM-2 in the osteoclast, which is responsible for SYK kinase and PLC␥ activation upstream of the calcium oscillations. On the other hand, ITIMbearing adaptor molecules are known to inhibit ITAM signaling via a recruitment of phosphatases such as SHP-1, SHP-2, and SHIP proteins. SHP-1 and SHIP-1 knock-out mice are reported to exhibit severe osteoporosis because of a dramatically increased number of osteoclasts (21,22). Because TLT-1s lacks the extracellular Ig domain compared with full-length TLT-1, we hypothesized that TLT-1s acts as a genuine adaptor molecule in a similar fashion with DAP12 and FcR␥ that are also deficient in the extracellular domain. Immunoprecipitation experiments revealed that TLT-1s is associated with TREM-2, SHP-1, and SHIP-1. Indeed, the knockdown of TLT-1s induced strong calcium oscillations and subsequent NFATc1 induction. Interestingly, RANKL stimulation did not affect the binding affinity of TLT-1 and TREM-2, suggesting that TLT-1s regulates the basal TREM-2 signaling. TLT-1s seems to be regulated at the transcription level by RANKL. Both TLT-1s mRNA and protein expressions significantly decreased upon RANKL stimulation during the late stages of osteoclastogenesis, allowing TREM-2 signaling to play a key role in osteoclast differentiation.
In summary, we identified a novel ITIM-bearing membrane protein, TLT-1s. TLT-1s down-regulated the TREM-2-mediated ITAM signaling that is crucial for calcium oscillations by recruiting SHP-1 and SHIP-1 phosphatases to inhibit osteoclastogenesis. To our knowledge, this is the first report providing evidence for the regulation of osteoclastogenesis by TLT-1 family proteins. The TLT-1s-mediated negative regulatory mechanism of TREM-2 may be important to prevent excessive ITAM signaling, ultimately maintaining bone homeostasis.