Figure 2. IL-32γ TG mice show increased expression of osteogenic markers in OB precursor cells.

(a,b) Calvarial OB precursor cells were induced to differentiate for 2 and 4 weeks, and mRNAs were isolated. The expression of OB-specific genes, including runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteocalcin (OCN), integrin β3, and collagen type 1 alpha 2 (Col1A2), was examined by qRT-PCR in differentiated OBs from WT and IL-32γ TG mice (a). Expression of RANKL was determined by qRT-PCR in WT and IL-32γ TG mice (b). (c–e) The amounts of RANKL (c) and OPG (d) secreted into the culture media from OBs of WT and IL-32γ TG mice at the indicated times were measured using ELISA kits, and the ratio of RANKL/OPG (e) was calculated. (f) Mouse bone marrow cells were co-cultured with calvarial osteoblastic precursor cells from WT and IL-32γ TG mice in the presence of 1α, 25(OH)₂D₃ (10⁻⁸ M) + PGE₂ (10⁻⁶ M) for 5 days. The cells were then fixed and stained with TRAP. Representative images are shown in the left panel. The TRAP-positive (TRAP⁺) multinucleated cells (MNCs) containing three or more nuclei were counted under the light microscope. The quantitative data are expressed as means ± SD in the right panel. *p < 0.05 versus co-cultures from wild-type. Representative data of at least three independent experiments are shown.