Effects of SHH on MAP kinase activity and osteoclast differentiation.

<p>(<b>A</b>) Detection of p-ERK, ERK, p-p38, and p38 by immunoblot analysis after RANKL (50 ng/ml) in murine macrophage RAW264.7 cells with or without SHH (500 ng/ml). (<b>B</b>) Immunoblot analysis of NFATc1 in RAW264.7 cells 0–48 h after RANKL (30 ng/ml) or SHH (500 ng/ml) treatment. (<b>C</b>) The mature osteoclast-differentiated cells were treated with SHH (500 ng/ml) or RANKL (30 ng/ml). Protein expression of TRAP and Cathepsin K was analyzed by immunoblot analysis. Similar results were obtained in three different experiments.</p

Hedgehog stimulated osteoclast formation of RAW264.7 cells, CD11b⁺ cells in bone marrow, and bone marrow macrophages.

<p>(<b>A</b>) RAW264.7 cells were inoculated onto a 96-well plate at different cell densities and then treated with increasing concentrations of RANKL +/- SHH for 6 days. Osteoclast formation was evaluated by TRAP staining. (<b>B and C</b>) CD11b⁺ cells isolated from the bone marrow of 5-week-old mice were cultured in the presence of 30 ng/ml M-CSF and 50 ng/ml RANKL with or without 500 ng/ml SHH in 96-well plate for 6 days. Cultures were also treated with or without SHH and hedgehog inhibitor cyclopamine for 6 days (C). Bone marrow macrophages were cultured with indicated amount of SHH in the presence of 30 ng/ml M-CSF and 50 ng/ml RANKL in 48-well plate for 7 days. TRAP-positive multinucleated cells (nuclear number > 3) were counted as osteoclasts. The data from a typical experiment are presented. Data are shown as the mean ± SD. Statistically significant differences (*P < 0.05, **P < 0.01) between the indicated groups are marked by asterisks.</p

Immunohistochemical staining for Sonic hedgehog (SHH), Patched, CD68 and Gli-2 in normal epithelium and osteolytic mandibular squamous cell carcinoma.

<p>(A) SHH, Patched, CD68 and Gli-2 expression in the normal epithelium at the edges of area were the mandible was resected. Each right photo is a magnification of the rectangle delimited area of normal epithelium in the corresponding left photo. Scale bar, 50 μm. (B) SHH, Patched, CD68 and Gli-2 expression in a resected mandible. Scale bar, 50 μm. Arrow: osteoclast. Arrowhead: osteoclasts progenitor cell.</p