Cilostazol Attenuates Ovariectomy-Induced Bone Loss by Inhibiting Osteoclastogenesis

<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^phox 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 attenuates OVX-induced bone loss in mice.

<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 10³ cells/well) (B) and whole bone marrow (2 x 10⁴ cells/well) (C) stimulated with RANKL/M-CSF and 1,25(OH)₂D₃, 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. ^#, <i>p</i><0.05; ^###, <i>p</i><0.001 compared with vehicle-treated OVX. Similar results were obtained in three independent experiments.</p

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

<p>(A) BMM (5 x 10⁶ 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. ^##, <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 decreases OC formation and bone resorption induced by RANKL.

<p>(A) BMM (10⁴ 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. ^#, <i>P</i> <0.05; ^##, <i>P</i> <0.01; ^###, <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 10⁵ 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. ^##, <i>P</i> <0.01; ^###, <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. ^##, <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

Tranilast attenuates OVX-induced bone loss in mice.

<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)₂D₃ (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

Trabecular microarchitecture and biochemical markers of OVX and SHAM mice treated with vehicle or Tranilast at 8 week after surgery.

<p>Data are represented as mean±SEM. Differences between each groups were analyzed by one-way ANOVA, followed by Bonferroni post tests. a, <i>p</i><0.05; a’, <i>p</i><0.01; a”, <i>p</i><0.001 compared with vehicle-treated SHAM. b, <i>p</i><0.05 compared with vehicle-treated OVX. No significantly difference between vehicle-treated SHAM vs. Tranilast-treated SHAM.</p

Tranilast decreases OC formation induced by RANKL.

<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 impairs a RANKL signaling.

<p>(A) BMM (5×10⁶ cells/plate) was stimulated with vehicle (V) (lane 2) or RANKL (lane 3) with Tranilast (30 µM, lane 4; 70 µM, lane 5) for 1 h. Hundred-fold excess of unlabeled probe (lane 1) was used as a negative control. NF-Y DNA binding activity was measured as an internal control. (B–F) BMM with or without Tranilast (T, 70 µM) or TGF-β (10 ng/ml) were incubated with M-CSF and RANKL for 48 h (B–D, F) and 72 h for counting TRAP-positive MNCs (E). Total RNA was extracted and subjected to qPCR analysis for NFAT2 (B, F) or TGF-β (D). The expression level before RANKL treatment was set to be 1. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001 compared with V. 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 the normalization of cytoplasmic and nuclear extracts, respectively. Numbers between the panels are ratios of the intensity of NFAT2 over β-actin (total and cytosolic) or lamin B1 (nucleus) (C). ***, <i>P</i><0.001 compared with V. Numbers above the histograms are ratios of the numbers of MNC formed in the presence of Tranilast to the numbers formed in its absence (E). There was a significant difference between TGF-β and TGF-β/TL (*** <i>P</i><0.001; E, * <i>P</i><0.05; F), whereas no significant difference between V and TGF-β/TL (E, F). Similar results were obtained in three independent experiments.</p

Tranilast decreases RANKL-induced ROS level.

<p>(A) Intracellular levels of ROS upon stimulation with RANKL in the presence or absence of Tranilast (30, 60 µM) for 48 h were determined using H₂DCFDA. ROS levels were quantified by flow cytometry. (B) BMM were stimulated with M-CSF and RANKL in the presence of Tranilast (30, 50, 70 µM) with or without DPI (5 nM) for 3 d for measurement of TRAP-positive MNCs. Means of Tranilast-treated 4 groups in the presence or absence of DPI are significantly different (<i>P</i><0.001). *** <i>P</i><0.001 compared with each vehicle (V)-treated cells. Numbers above the histograms are ratios of the number of MNC formed in the presence of Tranilast to the number formed in its absence. (C) BMMs were incubated with 50 µM of Tranilast in the presence of M-CSF and RANKL for the indicted periods; total RNA was extracted and subjected to qPCR analysis for HO-1 (8 h), Gpx-1 or PRX1 (24 h). Expression level before RANKL treatment was set to be 1. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001 compared to V. (D, E) BMMs were transfected with siPRX1 or scRNA. Down-regulation of PRX1 by siRNA was confirmed by RT-PCR and qPCR. The expression levels obtained from scRNA-treated cells were set to be 1. ***, <i>P</i><0.001 compared to scRNA (D). After 24 h of transfection with siRNA cells were treated with Tranilast and stimulated with RANKL for 48 h for determination of ROS level or 72 h for counting TRAP-positive MNCs. * <i>P</i><0.05, ** <i>P</i><0.01 compared with V of scRNA-transfected cells. There was a significant difference between siPRX1-treanfected V and Tranilast (* <i>P</i><0.05; ROS, ** <i>P</i><0.01; TRAP-positive MNC), whereas no significant difference between scRNA-transfected V and siPRX1-transfected Tranilast (ROS, TRAP-positive MNC). (E). (F) BMM from HO-1^+/+ and HO-1^−/− mice were incubated in the presence of M-CSF and RANKL with Tranilast (30, 50, 70 µM). After 3 d, cells were fixed and stained for TRAP. Means of Tranilast-treated 4 groups in the presence or absence of HO-1 are significantly different (<i>P</i><0.001). ** <i>P</i><0.01, ***, <i>P</i><0.001 compared with V-treated cells. Numbers above the histograms are ratios of the number of MNC formed in the presence of Tranilast to the number formed in its absence. Similar results were obtained in three independent experiments.</p

Science and society: in the sixteenth and seventeenth centuries

eIF2α phosphorylation in hepatocytes is dispensable for survival of adult mice. (a) Diagram depicting the four genotypes of mice used in these experiments. S/A and A/A represent heterozygous and homozygous eIF2α Ser51Ala (*) mutation(s) in exon 2 of one eIF2α allele and both eIF2α alleles, respectively. fTg/0 represents the floxed wild type (WT) eIF2α transgene driven by the CMV enhancer and chicken β-actin promoter (Enh-Pro). The loxP sequences (black arrowheads) allow excision of the WT eIF2α floxed transgene (fTg) and expression of EGFP, an indicator of recombination. CRE Hep /0 represents the Cre recombinase transgene driven by the promoter (Alfp) of Alb1 (encoding albumin) and the enhancer of Afp (encoding alpha-fetoprotein). (b) Efficiency of deletion of the fTg in liver tissues. Results from quantitative RT-PCR analyses of transgenic and total eIF2α mRNAs are shown. Data are means ± SEM (n = 4 ~ 5 mice per group); ### p < 0.001; Cont. vs A/A Hep . (c) Western blot analysis of eIF2α protein expression driven by the fTg in liver tissues. To quantity expression of eIF2α, blots were incubated with anti-eIF2α antibody followed by IRDye-800 goat anti-rabbit IgG (LI-COR). Membranes were scanned on an Odyssey scanner (LI-COR) (lower two panels in left panels) and quantified with the Odyssey Software package. (d) Western blot analysis of liver lysates in Cont. and A/A Hep mice at the indicated times after Tm injection. Cont. mice and A/A Liv mice were injected with vehicle or tunicamycin (Tm, 1 mg/kg). (e) Body weight measurements of fTg -deleted A/A Liv mice. At the weeks, body weight was measured in both male and female mice. Data are means ± SEM (n = 6-14 mice per group). (PDF 1987 kb