Publications

UNLABELLED

Deficiency of the signaling adapter protein DAP12 is associated with bony abnormalities in both mice and humans. We identify specific DAP12-associated receptors expressed by osteoclasts and examine function of DAP12 in murine osteoclasts in vivo and in vitro. These data show a new role for DAP12 signaling in regulating formation of multinucleated osteoclasts.

INTRODUCTION

Osteoclasts are bone-resorbing cells derived from hematopoietic precursors in the myeloid lineage. In other myeloid cell types, the signaling adapter protein DAP12 transmits activating signals on ligation of a DAP12-associated receptor (DAR). The aim of this study was to clarify the role of DAP12 signaling during osteoclast development.

MATERIALS AND METHODS

Osteoclasts from DAP12 -/- or control mice were analyzed in vitro for morphology, function, and for osteoclast markers. DARs were identified in osteoclast cultures through reverse transcriptase-polymerase chain reaction (RT-PCR). Bone density of DAP12 -/- and control mice were analyzed by microcomputed tomography. DAP12 -/- osteoclasts were retrovirally reconstituted with DAP12. RAW264.7 cells were transfected with FLAG-tagged DAP12 or TREM2 and stimulated by anti-FLAG antibody during in vitro osteoclastogenesis.

RESULTS

C57BL/6 DAP12-deficient mice have higher bone mass than C57BL/6 wildtype controls. We verified the presence of DAP12 in pre-osteoclasts and osteoclasts derived from C57BL/6 or the pre-osteoclast line RAW 264.7 and identified the DARs expressed. DAP12 -/- osteoclasts developed in vitro with macrophage colony-stimulating factor (M-CSF) and RANKL formed only intensely TRACP+ mononuclear cells and failed to generate multinuclear osteoclasts. These mononuclear cells are functional osteoclast-like cells because, by RT-PCR, they express other osteoclast markers and generate resorption pits on dentine slices, although quantitative assessment of bone resorption shows decreased resorption by DAP12 -/- osteoclasts compared with C57BL/6 osteoclasts. Restoration of DAP12 expression by retroviral transduction of DAP12 -/- osteoclast precursors rescued in vitro osteoclast multinucleation. Direct stimulation of DAP12 expressed in RAW264.7 during in vitro osteoclastogenesis led to a marked increase in the number of TRACP+ multinucleated osteoclast-like cells formed.

CONCLUSION

Our studies indicate that stimulation of the DAP12 adapter protein plays a significant role in formation of multinuclear osteoclasts and that DAP12 and DARs likely participate in the regulation of bony remodeling.

By homology to triggering receptor expressed by myeloid cells-2, we screened the mouse expressed sequence tag database and isolated a new single Ig domain receptor, which we have expressed and characterized. The receptor is most similar in sequence to the human CMRF-35 receptor, and thus we have named it CMRF-35-like molecule (CLM)-1. By screening the mouse genome, we determined that CLM-1 was part of a multigene family located on a small segment of mouse chromosome 11. Each contains a single Ig domain, and they are expressed mainly in cells of the myeloid lineage. CLM-1 contains multiple cytoplasmic tyrosine residues, including two that lie in consensus immunoreceptor tyrosine-based inhibitory motifs, and we demonstrate that CLM-1 can associate with Src-homology 2 containing phosphatase-1. Expression of CLM-1 mRNA is down-regulated by treatment with receptor activator of NF-kappaB ligand (RANKL), a cytokine that drives osteoclast formation. Furthermore, expression of CLM-1 in the osteoclastogenic cell line RAW (RAW.CLM-1) prevents osteoclastogenesis induced by RANKL and TGF-beta. RAW.CLM-1 cells fail to multinucleate and do not up-regulate calcitonin receptor, but they express tartrate-resistant acid phosphatase, cathepsin K, and beta(3) integrin, suggesting that osteoclastogenesis is blocked at a late-intermediate stage. Thus, we define a new family of myeloid receptors, and demonstrate that the first member of this family, CLM-1, is an inhibitory receptor, able to block osteoclastogenesis.

OBJECTIVE

To estimate the incidence of lipoatrophy and lipohypertrophy among HIV-infected and HIV-uninfected women from the Women's Interagency HIV Study.

DESIGN

Eight hundred fifteen women with semiannual data on self-report of bidirectional change in body fat, anthropometric measurements, weight, and bioelectric impedance analysis were included in a 30-month incidence analysis.

METHODS

Lipoatrophy and lipohypertrophy in both peripheral (arms, legs, and buttocks) and central (waist, chest, and upper back) sites were defined by self-report of either a decrease or an increase in a body fat region over the previous 6 months that was confirmed by a corresponding change in anthropometric measurement.

RESULTS

Weight and total body fat increased in HIV-uninfected women but remained stable in HIV-infected women over 30 months. Among HIV-infected women, the incidence of peripheral (relative hazard, 2.1; 95% confidence interval [CI], 1.4-3.3) and central (relative hazard, 1.9; 95% CI, 1.2-2.8) lipoatrophy was about double that among HIV-uninfected women, after adjustment for age and race. The incidence of peripheral lipohypertrophy appeared lower among HIV-infected women than among HIV-uninfected women (relative hazard, 0.8; 95% CI, 0.6-1.1), while the incidence of central lipohypertrophy did not differ by HIV status. Of HIV-infected women with 2 of 4 lipodystrophy outcomes, most (81%) had combined peripheral and central lipoatrophy or combined peripheral and central lipohypertrophy. Only 14% of these women had both peripheral lipoatrophy and central lipohypertrophy.

CONCLUSIONS

These prospective data suggest that lipoatrophy, affecting both peripheral and central sites, predominates in HIV-infected women. The simultaneous occurrence of peripheral lipoatrophy and central lipohypertrophy was uncommon.