15 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

    Physiological measurements of sham and OVX of WT and MCP-1-KO mice 12 weeks after operation.

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    <p>Data are expressed as mean ± SEM. ND, non-detectible. Differences between groups were analyzed by two-way ANOVA, followed by Bonferroni post-tests (Increased body weight, subcutaneous fat, visceral fat, serum H<sub>2</sub>O<sub>2</sub>, and blood insulin; <i>P</i><0.001, serum M-CSF; <i>P</i><0.01, blood glucose; <i>P</i><0.05, effect of surgery. Increased body weight, subcutaneous fat, and visceral fat; <i>P</i><0.001, serum M-CSF and blood insulin; <i>P</i><0.01, serum ROS; <i>P</i><0.05, effect of MCP-1). WT OVX vs. MCP-1-KO OVX;</p>*<p><i>P</i><0.05,</p>**<p><i>P</i><0.01,</p>***<p><i>P</i><0.001.</p

    MCP-1-deficiency decreased OVX-induced immune cell infiltration in AT.

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    <p>SVCs from visceral fat were extracted from WT (open bar) and MCP-1-KO mice (oblique-lined bar) 12 weeks after sham or OVX surgery. SVCs were labeled with conjugated Abs to CD11bF4/80 (A), CD11cF4/80 (B), CD4 (C), and CD8 (D) and quantified by flow cytometry. Data are expressed as mean ± SEM. Differences between groups were analyzed by two-way ANOVA, followed by Bonferroni post-tests (CD11bF4/80, CD11cF4/80, CD4; <i>P</i><0.01, CD8; <i>P</i><0.05, effect of surgery. CD11cF4/80; <i>P</i><0.05, effect of MCP-1). *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001 compared with WT OVX mice. Similar results were obtained in three independent experiments.</p

    MCP-1-deficiency decreased CD11c-expressing cells via impairing the production of ROS and decreased activation of PLCγ2, Akt, and ERK upon M-CSF stimulation in BMM.

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    <p>BMMs from WT (open bar) and MCP-1-KO mice (oblique-lined bar) were incubated in the presence of M-CSF (30 ng/ml) with U73122 (10 µM), Akt inhibitor IV (0.3 µM), PD098059 (5 µM), DPI (50 nM), NAC (3 mM), or H<sub>2</sub>O<sub>2</sub> (300 µM) for 4 d (A). *, <i>P</i><0.05; ***, <i>P</i><0.001 compared with vehicle-treated WT cells. There was no significant difference between WT and MCP-1-KO cells except upon vehicle treatment. Treatment with U73122, Akt inhibitor IV, PD98059, DPI, NAC, or H<sub>2</sub>O<sub>2</sub> abolished the decrease in CD11cF4/80 observed in MCP-1-KO cells. BMMs were serum-starved for 8 h and stimulated with M-CSF for 1, 2, or 3 d (B). Phosphorylation of PLCγ2 was determined by Western blotting. Total protein level served as the loading control. Relative ratios of phosphorylated forms to total forms were plotted. **, <i>P</i><0.01; ***, <i>P</i><0.001 compared with WT cells. Intracellular levels of ROS upon stimulation in the presence of M-CSF (M) or/and MCP-1 with control IgG (3 µg/ml) or anti-MCP-1 Ab (3 µg/ml) for 2 d were determined in WT cells and MCP-1-KO cells using H2DCFDA (C, D). ROS levels were quantified by flow cytometry. *, <i>P</i><0.05; **, <i>P</i><0.01 compared with M-stimulated WT cells. No significant difference between WT and MCP-1-KO cells stimulated with M+MCP-1 (C). *, <i>P</i><0.05; ***, <i>P</i><0.001 compared with IgG-treated WT cells. No significant difference between IgG- and anti-MCP-1 Ab-treated MCP-1-KO cells (D). BMMs were transfected with sip47<sup>phox</sup> or scRNA. Downregulation of p47<sup>phox</sup> by siRNA was confirmed by RT-PCR and qPCR (E). The expression level obtained from scRNA-treated cells was set to be 1. After 24 h of transfection with siRNA, cells were stimulated with M-CSF for 2 d (mRNA) or 4 d (FACS) in order to determine CD11c (F) and for 2 d to measure ROS (G). *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001 compared with scRNA-transfected WT cells. No significant difference was found in MCP-1-KO cells (F, G). Similar results were obtained in three independent experiments.</p

    The absence of MCP-1 reduced fat mass and improved metabolic perturbation induced by OVX.

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    <p>WT mice (open bar) and MCP-1-KO (oblique-lined bar) mice were subjected to OVX or sham surgery, and then held for 12 weeks. Whole body weight change up to 12 weeks after surgery (A) and average daily food intake (B) were measured. *, <i>P</i><0.05; WT OVX vs. MCP-1-KO OVX mice. Adipocyte volume was calculated from photograph of hematoxylin-eosin staining of visceral fat, assuming that an adipocyte is a sphere (magnification, ×200). Scale bar, 100 µm (C). Glucose clearance (D) and insulin sensitivity (E) were determined 12 weeks after sham or OVX, following an intraperitoneal injection of glucose (1 mg/kg) and insulin (0.75 munits/kg), respectively. AUC was measured for each group for D and E. *, <i>P</i><0.05, ***, <i>P</i><0.001; WT OVX vs. MCP-1-KO OVX. Data are expressed as mean ± SEM. Differences between groups were analyzed by two-way ANOVA, followed by Bonferroni post-tests (C, D) (adipocyte volume; <i>P</i><0.001, AUC for glucose tolerance; <i>P</i><0.01, effect of surgery. adipocyte volume; <i>P</i><0.001, AUC for glucose tolerance; <i>P</i><0.05, effect of MCP-1). *, <i>P</i><0.05; ***, <i>P</i><0.001 compared with WT OVX mice. Similar results were obtained in three independent experiments.</p

    Tranilast decreases OC formation induced by RANKL.

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    <p>(A) BMM were incubated with Tranilast (30, 50, 70 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. Means of 4 groups are significantly different (<i>P</i><0.001). ***, <i>P</i><0.001 compared with V (vehicle)-treated cells. (B) Representative photos of A. Scale bar, 200 µm. (C) BMMs were incubated with Tranilast (70 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 (CTR), and c-Fos. *, <i>P</i><0.05; **, <i>P</i><0.01 with V. (D) RANKL-induced mature OC (∼1000 cells) was incubated with or without Tranilast (70 µM) on dentine slices for 24 h, and stained for pit formation. Representative photos of the resorption pits in V- and Tranilast-treated slices are shown. Scale bar, 50 µm. There was no significant difference between them in the areas of the resorption pits as determined with the ImageJ 1.37v program. Similar results were obtained in three independent experiments.</p

    Tranilast attenuates OVX-induced bone loss in mice.

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    <p>Bone densities of the femora were measured from vehicle-treated (sham, n = 6; OVX, n = 6), Tranilast (200 mg/kg/d)-treated (sham, n = 6; OVX, n = 6) mice 8 weeks after surgery. The representative µCT images of distal femora (1.0 mm from the growth plate of the distal femur) (A). Number of OCs in the cultures of enriched BMM (B) and whole bone marrow (C) stimulated with RANKL/M-CSF and 1,25(OH)<sub>2</sub>D<sub>3</sub> (C), respectively. a, <i>p</i><0.05; a’, <i>p</i><0.01 compared with vehicle-treated SHAM. b, <i>p</i><0.05 compared with vehicle-treated OVX. No significant difference between vehicle-treated SHAM and Tranilast-treated OVX (B, C).</p
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