Figure 1.
Bone remolding markers and uterine weights.
(A) Uterine weight was measured at the end of the experiment when mice were sacrificed. Four weeks after operations, urine DPD (B) and serum osteocalcin (C) were measured. WT: wild-type mice; DKO: mice lacking both DAP12 and FcRγ. N = 5 in each group. *: p<0.05; **: p<0.01; ***: p<0.001; when compared to SHAM groups, respectively.
Figure 2.
OVX-induced bone remodeling in lumbar vertebra and cortical bone.
Six weeks after operations, the sixth lumbar vertebra (LVB6), and tibia were processed and analyzed by µCT, as described in Materials and Methods. Trabecular bone volume/tissue volume (A), trabecular thickness (B), and trabecular number (C) of LVB6, and cortical thickness of tibia at distal tibiofibular junction (D) are shown. WT: wild-type mice; DKO: mice lacking both DAP12 and FcRγ. N = 5 in each group. *: p<0.05; **: p<0.01; ***: p<0.001; when compared to SHAM groups, respectively.
Figure 3.
OVX-induced bone remodeling of trabeculae in distal femur.
Six weeks after operations, femurs were processed and analyzed by µCT. Representative figures of 3D reconstructed µCT results are shown in (A), with average BV/TV indicated below. Trabecular BV/TV (B); mean bone mineral density of TV (C); mean bone mineral density of trabeculae (D); trabecular number (E); trabecular spacing (F) and trabecular thickness (G) are shown. Scale bar in (A) shows 1 mm physical size. WT: wild-type mice; DKO: mice lacking both DAP12 and FcRγ. N = 5 in each group. *: p<0.05; **: p<0.01; ***: p<0.001; when compared to SHAM groups, respectively. ** in (G) indicates p<0.01 when DKO sham group were compared to sham group of all three other mice. N = 5 for each group.
Table 1.
Bone loss in the trabecular region of the distal femur.
Figure 4.
Increased osteoclast formation in vivo in DAP12-/-FcRγ-/- OVX group.
(A) TRACP staining and DAPI staining was performed on paraffin section of decalcified femur of SHAM and OVX groups of DKO mice. Brightfield and fluorescence images were taken with 40× objective. Red staining in brightfield indicates TRACP+cells. Magenta arrow indicates mononuclear TRACP+ osteoclasts; red asterisk indicates TRACP+ multinuclear osteoclasts (MNCs). (B) Mononuclear, multinuclear, and total osteoclast number per section was counted. *: p<0.05; ***: p<0.001; when compared between groups indicated by dotted line. N = 3 each group.
Figure 5.
OVX-induced osteoclast activity is not self-sustained in osteoclasts from DAP12-/- and DAP12-/-FcRγ-/- mice.
At six weeks post-OVX, non-adherent bone marrow macrophages were cultured with 10 ng/mL M-CSF and 25 ng/mL RANKL on BD BioCoat™ Osteologic™ Bone Cell Culture dishes. (A) Representative images of resorption pits where white represents pits and black in unresorbed substrate. Total resorption area (B) and pit number (C) were calculated and shown. Standard error is shown. WT: wild-type mice; DKO: mice lacking both DAP12 and FcRγ. N = 5 in each group. Experiment was repeated three times. Representative images and results were shown.
Figure 6.
RANKL-independent osteoclast differentiation induced by TGFβ and TNF-α cytokines.
BMMs from WT, DAP12-/- and DAP12-/-FcRγ-/- were separated and non-adherent preosteoclasts were cultured with RANKL (A, E, I); or pretreated by 2 ng/ml TGFβ (B, F, J) for 2 days, followed by 30 ng/ml TNF-α treatment (C, G, K) or 25 ng/ml RANKL (D, H, L) for another 2 day. TRACP staining was performed at the end of the experiment, as described. TRACP staining is indicated by dark purple. WT: wild-type mice; DKO: mice lacking both DAP12 and FcRγ. Experiment was repeated three times with representative results shown.
Figure 7.
Stimulatory effect of FSH has no effect on DAP12-deficient osteoclastogenesis.
BMMs from WT, DAP12-/-, and DAP12-/-FcRγ-/- were separated and non-adherent BMMs were cultured with 30 ng/ml FSH alone (Aa, Ad, Ag), 3 ng/ml RANKL alone (Ab, Ae, Ah), or FSH and RANKL (Ac, Af, Ai) for 4 days. TRACP staining was performed at the end of the experiment, as described. Dark purple staining indicates that cells are TRACP positive. The number of multinucleated (>3nuclei) -osteoclasts were calculated and shown in (B). DKO: mice lacking of both DAP12 and FcRγ. Experiment was repeated three times with representative results shown.