Fig. 1. STAT3-deficient mice exhibit osteoporosis. (A) Toluidine blue staining. Note the trabecular bone volume in STAT3-deficient mouse (KO) is markedly decreased compared with the control mice (WT). Left panel: magnification: 25×; right panel: magnification: 125×. (B) Histomorphometric analysis of STAT3-deficient mice (n = 8, data shown as means ± SE *p < 0.05, **p < 0.01). BV/TV, bone volume; TbTh, trabecular thickness; TbN, trabecular number; Oth, osteoid thickness; Noc/BPm, osteoclast bone surface density; and Nob/BPm, osteoblast surface density. Note a significant reduction of trabecular bone volume, accompanied by enhanced osteoclast bone surface density. However, there was no significant change in osteoblast surface density.

Fig. 2. Enhanced osteoclastogenesis in STAT3-deficient mice. (A) Splenocytes from either wild-type (WT) or STAT3-deficient mouse (KO) were cultured in the presence of 20 ng/ml CSF-1 and 20 ng/ml RANKL. Cells were stained for TRAP activity at 5 days of culture (left panel). TRAP-positive cells containing more than three nuclei were counted at indicated days (right panel). (B) Bone marrow or splenic macrophages from STAT3-deficient mice and littermate controls were cultured as described under Materials and methods. Cells were lysed and processed for Western blotting using anti-STAT3 antibody (upper panel). The membrane was then stripped and reblotted with anti-β-actin antibody as a control for protein loading (lower panel).

Fig. 3. STAT3-deficient mice exhibit enhanced expression of Mac1 and RANK. Flow cytometric analysis of cells from STAT3-deficient mice (KO) and the normal littermate (WT). Bone marrow cells were stained with Mac1-PE, rabbit anti-RANK, and FITC-anti-rabbit Ab (upper panel); splenocytes were cultured in the presence of 20 ng/ml CSF-1. At day 0 (middle panel) and day 5 (lower panel) of culture, cells were stained for Mac1 and RANK expression.

Fig. 4. Identification of osteoclastogenic population in STAT3-deficient mice. (A) Splenocytes of the mutant mice were sorted by PE-Mac1 antibody, and the Mac1 positive (left panel) and Mac1 negative (middle panel) cells were cultured by 20 ng/ml CSF-1 and 20 ng/ml RANKL. Cells were then fixed and processed for TRAP staining; Splenocytes of littermate controls were sorted by PE-Mac1 antibody and the Mac1⁻ cells were processed as described above. (B) Splenocytes of the mutant mice and littermate controls were stained with APC-c-kit antibody and analyzed by flow cytometric analysis. Splenocytes of the mutant mice were sorted by PE-Mac1 and APC-c-kit antibodies (right panel), and the percentage of c-kit⁺ cells is shown in the left panel; R2, Mac1⁺ cells; R3, c-kit⁺ cells. (C) Splenocytes of the mutant mice were sorted by PE-Mac1 and APC-c-kit antibodies as shown in (B). The Mac1⁻ cells with positive c-kit staining (left panel) or negative c-kit staining (right panel) were cultured with CSF-1 and RANKL, then fixed and processed for TRAP staining.

Fig. 5. Both protein expression and mRNA level of c-fos are increased in osteoclast precursors of STAT3-deficient mice. (A) Real time RT-PCR analysis in Mac-1 positive bone marrow cells. Genes examined were as indicated. The data shown are representatives of three different experiments. (B) Bone marrow macrophages from STAT3-deficient mice and littermate controls were cultured as described under Materials and methods. Cells were lysed and processed for Western blotting using anti-fos antibody (upper panel). The membranes were then reblotted with anti-β-actin antibody to determine the amount of protein (lower panel).