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.

OBJECTIVE

To describe the experiences of postmenopausal women who try to stop hormone therapy and to identify characteristics associated with inability to stop.

METHODS

We conducted telephone interviews with 377 randomly selected female members of the Kaiser Foundation Health Plan, aged 50-69 years, who regularly used hormone therapy for at least 1 year before July 1, 2002 and had attempted to stop between July 2002 and March 2003.

RESULTS

Of the 377 women, 280 (74%) successfully stopped and 97 (26%) resumed taking hormone therapy. The major predictor of resuming hormone therapy use was the development of troublesome withdrawal symptoms (odds ratio 8.8; 95% confidence interval 4.9, 16.0). Report of hysterectomy, hormone therapy prescribed by a nongynecologist, and perception of high risk of hip or spine fracture were independently associated with a higher likelihood of unsuccessful stopping. Women with a hysterectomy who had used hormone therapy for 10 or more years and who started hormone therapy mainly for reasons other than health promotion were more likely (P <.001) to be unsuccessful in quitting (44%) compared with those with one or two (25%) or none (9%) of these three characteristics. Most successful stoppers (71%) stopped hormone therapy abruptly, but 29% tapered off hormone therapy; there was no difference in the incidence of troublesome withdrawal symptoms or successful quitting between these two groups.

CONCLUSION

Approximately one quarter of women who try to stop report that they are unable to discontinue postmenopausal hormone therapy, primarily because they develop troublesome withdrawal symptoms. Effective approaches to reducing hormone therapy withdrawal symptoms should be a priority for future research.

OBJECTIVE

To examine whether women who were using postmenopausal hormone therapy (HT) before publication of Women's Health Initiative findings about risks associated with HT had been informed about the findings once published; and to estimate how knowledge of these findings was associated with their decision to discontinue HT.

METHODS

We performed a telephone survey of 670 female members of a large health maintenance organization, aged 50-69 yr, who had regularly used HT from July 1, 2001, through June 30, 2002.

RESULTS

Most women (93%) reported hearing about the new findings; however, only 57% considered the quality of this information to be good, regardless of its source: mass media (21%), the health plan (32%), or a health care practitioner (34%). Women's knowledge of Women's Health Initiative findings was generally poor; 64% did not know what the findings were, 7% were unsure of their knowledge, 6% had incorrect knowledge, and 23% had correct knowledge of Women's Health Initiative findings. On a simple, five-question, true-or-false quiz about HT risks, 30% of respondents answered four to five questions correctly. Although not well informed, 56% reported attempting to discontinue HT in the 6 to 8 months after July 2002. Our multivariable model included five statistically significant predictors of attempting to stop HT: having been sent a letter about Women's Health Initiative findings (odds ratio [OR] 2.7; 95% confidence interval [CI] 1.8, 3.9), reporting good-quality information from media (OR 2.1; 95% CI 1.3, 3.3), having started HT for health promotion (OR 2.0; 95% CI 1.2, 3.3), using a lower-than-standard dosage of estrogen (OR 1.9; 95% CI 1.1, 3.1), and correctly answering four or more items on the HT quiz (OR 1.9; 95% CI 1.2, 2.8).

CONCLUSION

During the 6-8 months after publication of Women's Health Initiative trial findings, most regular postmenopausal HT users tried to stop using HT, despite not being well informed about the Women's Health Initiative findings.