TNF-α in combination with TGF-β induces preosteoclasts at a low concentration of M-CSF.

<p>(A) Induction of TRAP⁺ mononuclear cells in the presence of various concentrations of TNF-α or TGF-β in the presence of M-CSF (3 ng/ml). Each value represents the mean ± SEM of three cultures. (B) Representative photomicrograph of TRAP⁺ mononuclear cells induced by M-CSF (3 ng/ml), TNF-α (20 ng/ml), and TGF-β (1 ng/ml) (top); or TRAP⁺ MNCs induced from preosteoclasts by RANKL (30 ng/ml) as illustrated in (C) (middle); or TRAP⁺ mononuclear cells induced by RANKL (3 ng/ml) and M-CSF (10 ng/ml) (bottom). Bar = 50 µm. (C) Illustration of sequence of culture conditions. (D) RT-PCR analysis of mRNA expression of osteoclast-specific genes and genes encoding cell surface proteins in cultures of BMM, preosteoclasts (pre-OC), and MNCs. (E) Relative expression levels of <i>ctsk, calcr, tnfrs11a</i> (RANK), <i>nfatc,</i> and <i>c-fos</i> were normalized with β<i>-actin</i>. (F) Real-time RT-PCR analysis of expression levels of <i>ctsk, tm7sf4</i> (DC-STAMP), and <i>csf-1r</i> (c-fms). Expression levels were normalized with <i>gapdh</i>. NABMCs were cultured for 3 days in the presence of various concentrations of TNF-α, TGF-β, M-CSF, and RANKL. To induce TRAP⁺ MNCs, RANKL was added to preosteoclasts induced by treatment with TNF-α, TGF-β, and M-CSF, and cultures were continued for 2 days. At the end of culture, TRAP-staining was performed and TRAP⁺ mononuclear cells were counted. Total RNA from cells was prepared and analyzed by semi-quantitative RT-PCR or real-time RT-PCR using specific primers as in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047930#s2" target="_blank">Materials and Methods</a>.</i> Each value represents the mean ± SEM (n = 3). Statistical significance was determined by Student’s <i>t</i> test; *P<0.05, **P<0.01 compared with BMM. Experiments were performed three times using three different rats, and similar results were obtained.