Cilostazol impairs activation of two key transcription factors for osteoclastogenesis, NF-κB and NFAT2.


<p>(A) BMM (5 x 10<sup>6</sup> cells/plate) were stimulated with vehicle (V) (lane 1) or RANKL (lane 2) along with cilostazol (10 μM, lane 3; 30 μM, lane 4) for 1 h. Hundred-fold excess of unlabeled probe (lane 5) was used as a negative control. NF-Y DNA binding activity was measured as an internal control. (B-D) BMM with cilostazol (30 μM) in the presence or absence of BAY 11–7082 (1 μM) were incubated with M-CSF and RANKL for 72 h to count TRAP-positive MNCs (B) and for 48 h to determine ROS level (C) and extract RNA (D). Numbers above the histograms are ratios of the number of MNC (B) or ROS-positive cells (C) in the presence of cilostazol to in its absence. Total RNA was extracted and subjected to qPCR analysis for NFAT2. The expression level before RANKL treatment was set at 1 (D). **, <i>P</i><0.01, ***, <i>P</i><0.001 compared with V. <sup>##</sup>, <i>p</i><0.01 compared with V in the presence of BAY 11–7082. (E) Whole cell extracts, cytoplasmic fractions, and nuclear fractions were harvested from cultured cells and subjected to Western blot analysis with specific Abs as indicated. Abs for β-actin and lamin B1 were used for normalization of cytoplasmic and nuclear extracts, respectively. Numbers between the panels are ratios of the intensity of NFAT2 to β-actin (total and cytosolic) or lamin B1 (nucleus). (F) BMMs were cultured with M-CSF and RANKL for 42 h and then treated with cilostazol (30 μM) or sp-cAMP (10 μM) for 6 h. Whole cell lysates were immunoprecipitated with anti-NFAT2 and subjected to Western blot analysis with a phosphorylated PKA substrate-specific Ab. Similar results were obtained in three independent experiments.</p

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