Article Figures & Data
Figures
- FIGURE 1.
SHIP1−/− BMMs proliferate at an accelerated rate in response to M-CSF. A and B, Equal numbers of WT (+/+) and SHIP1-deficient (−/−) BMMs (1 × 105 per 100-mm plate) were grown for the indicated time periods in the presence of 20 ng/ml M-CSF. Cells were trypsinized, stained with trypan blue, and counted for viable (A) and dead (B) cells. C, Equal numbers of WT and SHIP1−/− BMMs (1 × 104/well) were grown in medium supplemented with the indicated concentrations of M-CSF for 2 days. Cells were labeled with BrdU for the last 4 h and assayed for BrdU incorporation. D, Similar to C, except 10 ng/ml M-CSF with or without 25 ng/ml RANKL was used (*, p < 0.05; **, p < 0.005 vs +/+; #, p < 0.05, only vs M-CSF).
- FIGURE 2.
SHIP1 deficiency results in enhanced M-CSF-dependent activation of Akt, but not ERK, JNK, or p38. BMMs were grown in the absence of serum and M-CSF for 6 h and then stimulated with 20 ng/ml M-CSF for the indicated time periods (A and B) or with the indicated concentrations of M-CSF for 5 min (C) or 100 ng/ml RANKL for indicated time periods (D). Cell lysates were subjected to immunoblotting with Abs, as indicated.
- FIGURE 3.
The PI3K/Akt pathway mediates BMM proliferation. A, WT BMMs were grown in the absence of serum and M-CSF for 6 h and subsequently treated with or without LY294002 (10 μM) or wortmannin (0.2 μM) 1 h before stimulation with 20 ng/ml M-CSF for 5 min. Cell lysates were analyzed by immunoblotting with indicated Abs. B and C, Growth-arrested WT or SHIP1−/− BMMs were grown for 24 h in the presence of 20 ng/ml M-CSF and the indicated concentration of LY294002 or wortmannin. Proliferation was determined by BrdU incorporation (*, p < 0.001 vs cells not treated with inhibitors). D, WT BMMs were grown for 24 h in the presence of 20 ng/ml M-CSF with indicated concentrations of LY294002 or wortmannin. Cell death was assayed by cell death ELISA, which quantitates DNA fragmentation. E, WT BMMs were grown in the presence of 10 ng/ml M-CSF, and WT osteoclasts were generated with 10 ng/ml M-CSF and 100 ng/ml RANKL. Cells were treated with LY294002 for 8 h at the indicated concentration and assayed for DNA fragmentation by cell death ELISA (*, p < 0.001 vs cells not treated with LY294002).
- FIGURE 4.
Cell cycle progression in the absence of SHIP1 or by inhibition of PI3K correlates with alterations in levels of cyclin D1, D2, D3, and p27. A, D, and F, WT (+/+) and SHIP1-deficient (−/−) BMMs were grown in the absence of M-CSF for 16–24 h, followed by stimulation with 20 ng/ml M-CSF. Cell lysates were collected at the indicated times and subjected to immunoblotting with Abs, as indicated in the figure (A and D). Cells were also assayed for BrdU incorporation at the indicated time points (F) (p < 0.05 vs +/+ from 6 to 14 h). B, C, and E, WT BMMs were grown in the absence of M-CSF for 20 h. A total of 10 μM LY294002 or 10 μM U0126 was added for 1 h, as indicated, followed by 20 ng/ml M-CSF for 12 h. B and E, Cell lysates were subjected to immunoblotting, as indicated, or C, RNA expression was analyzed by RT-PCR for cyclin D1, D2, and D3. GAPDH serves as loading control.
- FIGURE 5.
Retroviral expression of SHIP1 in SHIP1−/− BMMs normalizes Akt activation, proliferation, and osteoclastogenesis. WT (+/+) and SHIP1-deficient (−/−) BMMs were infected with retrovirus coding for only vector or the full-length SHIP1 gene. Cells were selected for 3 days. A, Transduced cells were serum and cytokine starved for 6 h, followed by addition of the indicated concentrations of M-CSF for 5 min. Immunoblotting was performed using indicated Abs. B, Selected cells were grown for 2 days in medium supplemented with 10 ng/ml M-CSF. Cells were labeled with BrdU for 4 h and then assayed for BrdU incorporation (*, p < 0.01 vs −/− vector). C and D, The same transductants were maintained in the presence of 10 ng/ml M-CSF and 25 ng/ml RANKL for 5 days. The cells were then stained for TRAP activity to visualize osteoclasts (C), and the numbers of osteoclasts were counted (D) (*, p < 0.02 vs +/+ vector).
- FIGURE 6.
Osteoclastogenesis and bone erosion in inflammatory arthritis are enhanced by the absence of SHIP1. Lethally irradiated mice transplanted with either WT (WT > WT) or SHIP1−/− (KO > WT) marrow were injected with PBS or arthrogenic serum (A). Paw thickness was measured with time. B, Six days later, the mice were sacrificed and histological sections of paws were stained for TRAP activity (red reaction product). Arrows indicate inflammatory response. C, Histomorphometric quantitation of the percentage of bone surface covered by osteoclasts in paws of each group of mice (*, p < 0.005 vs WT > WT PBS; **, p < 0.001 vs WT > WT serum).
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