4 research outputs found

    Cilostazol Attenuates Ovariectomy-Induced Bone Loss by Inhibiting Osteoclastogenesis

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    <div><p>Background</p><p>Cilostazol has been reported to alleviate the metabolic syndrome induced by increased intracellular adenosine 3’,5’-cyclic monophosphate (cAMP) levels, which is also associated with osteoclast (OC) differentiation. We hypothesized that bone loss might be attenuated via an action on OC by cilostazol.</p><p>Methodology and Principal Findings</p><p>To test this idea, we investigated the effect of cilostazol on ovariectomy (OVX)-induced bone loss in mice and on OC differentiation in vitro, using μCT and tartrate-resistant acid phosphatase staining, respectively. Cilostazol prevented from OVX-induced bone loss and decreased oxidative stress in vivo. It also decreased the number and activity of OC in vitro. The effect of cilostazol on reactive oxygen species (ROS) occurred via protein kinase A (PKA) and cAMP-regulated guanine nucleotide exchange factor 1, two major effectors of cAMP. Knockdown of NADPH oxidase using siRNA of p47<sup>phox</sup> attenuated the inhibitory effect of cilostazol on OC formation, suggesting that decreased OC formation by cilostazol was partly due to impaired ROS generation. Cilostazol enhanced phosphorylation of nuclear factor of activated T cells, cytoplasmic 1 (NFAT2) at PKA phosphorylation sites, preventing its nuclear translocation to result in reduced receptor activator of nuclear factor-κB ligand-induced NFAT2 expression and decreased binding of nuclear factor-κB-DNA, finally leading to reduced levels of two transcription factors required for OC differentiation.</p><p>Conclusions/Significance</p><p>Our data highlight the therapeutic potential of cilostazol for attenuating bone loss and oxidative stress caused by loss of ovarian function.</p></div

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

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    <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

    Cilostazol attenuates OVX-induced bone loss in mice.

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    <p>Bone densities of femora were measured on vehicle (V)-treated (SHAM, n = 5; OVX, n = 7), cilostazol (0.5 mg/kg/d)-treated (SHAM, n = 6; OVX, n = 7) mice 8 weeks after surgery. Representative μCT images of distal femora (1.0 mm from the growth plate of the distal femur) (A). Numbers of OCs in cultures of enriched BMM (5 x 10<sup>3</sup> cells/well) (B) and whole bone marrow (2 x 10<sup>4</sup> cells/well) (C) stimulated with RANKL/M-CSF and 1,25(OH)<sub>2</sub>D<sub>3</sub>, respectively were counted by an experienced observer who was blinded to each treatment for quantification of TRAP-positive MNC/ each well using an eye piece graticule at a magnification of Χ100. Results were expressed as means ± SEM of 3–6 cultures per variable. ***, <i>p</i><0.001 compared with vehicle-treated SHAM. <sup>#</sup>, <i>p</i><0.05; <sup>###</sup>, <i>p</i><0.001 compared with vehicle-treated OVX. Similar results were obtained in three independent experiments.</p

    Cilostazol decreases OC formation and bone resorption induced by RANKL.

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    <p>(A) BMM (10<sup>4</sup> cells/well) from sham and OVX mice were incubated with cilostazol (0, 15, 25 μM) in the presence of M-CSF (20 ng/ml) and RANKL (40 ng/ml). After 3 d, cells were fixed and stained for TRAP. Numbers of OCs were counted by an experienced observer who was blinded to cilostazol dose for quantification of TRAP-positive MNC/each well using an eye piece graticule at a magnification of Χ100. Results were expressed as means ± SEM of 3–6 cultures per variable. Frequency distribution of OCs according to number of nuclei. (B) Representative photos of A. Scale bar, 200 μm. Means of the 3 groups are significantly different (<i>P</i> <0.001). **, <i>P</i> <0.01; ***, <i>P</i> <0.001 compared with vehicle (V)-treated cells in sham and OVX. <sup>#</sup>, <i>P</i> <0.05; <sup>##</sup>, <i>P</i> <0.01; <sup>###</sup>, <i>P</i> <0.001 sham vs. OVX. Numbers above the histogram are ratios of the number of TRAP-positive MNC in the presence of cilostazol to in its absence for each group. (C) BMMs (5 x 10<sup>5</sup> cells/well) from sham and OVX mice were incubated with cilostazol (25 μM) in the presence of M-CSF and RANKL for 48 h; total RNA was extracted and subjected to qPCR analysis for TRAP, calcitonin receptor, cathepsin K, DC-STAMP, and ATP6v0d2. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i> <0.001 compared with V in sham and OVX. <sup>##</sup>, <i>P</i> <0.01; <sup>###</sup>, <i>P</i> <0.001 sham vs. OVX. No significant difference was observed between sham vs. OVX in the presence of cilostazol. (D) RANKL-induced mature OC (~1000 cells) from sham and OVX mice were incubated with or without cilostazol (25 μM) on dentine slices for 24 h, and the slices were stained for pit formation. Representative photos of the resorption pits in V- and cilostazol-treated slices are shown. Scale bar, 50 μm. **, <i>P</i><0.01; ***, <i>P</i><0.001 compared with V in sham and OVX. <sup>##</sup>, <i>P</i> <0.01 sham vs, OVX. Numbers above the histogram are ratios of pit area of in the presence of cilostazol to in its absence for each group. The areas of the resorption pits per dentine slice were quantified blind using the ImageJ 1.37v program. Similar results were obtained in three independent experiments.</p
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