KMUP-1 Suppresses RANKL-Induced Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss: Roles of MAPKs, Akt, NF-κB and Calcium/Calcineurin/NFATc1 Pathways

<div><p>Background</p><p>KMUP-1 is a xanthine derivative with inhibitory activities on the phosphodiesterase (PDE) 3,4 and 5 isoenzymes to suppress the degradation of cyclic AMP and cyclic GMP. However, the effects of KMUP-1 on osteoclast differentiation are still unclear. In this study, we investigated whether KMUP-1 inhibits osteoclastogenesis induced by RANKL in RAW 264.7 cells and bone loss induced by ovariectomy in mice, and the underlying mechanisms.</p><p>Principal Findings</p><p><i>In vitro</i>, KMUP-1 inhibited RANKL-induced TRAP activity, the formation of multinucleated osteoclasts and resorption-pit formation. It also inhibited key mediators of osteoclastogenesis including IL-1β, IL-6, TNF-α and HMGB1. In addition, KMUP-1 inhibited RANKL-induced activation of signaling molecules (Akt, MAPKs, calcium and NF-κB), mRNA expression of osteoclastogensis-associated genes (TRAP, MMP-9, Fra-1, and cathepsin K) and transcription factors (c-Fos and NFATc1). Furthermore, most inhibitory effects of KMUP-1 on RANKL-mediated signal activations were reversed by a protein kinase A inhibitor (H89) and a protein kinase G inhibitor (KT5823). <i>In vivo</i>, KMUP-1 prevented loss of bone mineral content, preserved serum alkaline phosphate and reduced serum osteocalcin in ovariectomized mice.</p><p>Conclusions</p><p>KMUP-1 inhibits RANKL-induced osteoclastogenesis <i>in vitro</i> and protects against ovariectomy-induced bone loss <i>in vivo</i>. These effects are mediated, at least in part, by cAMP and cGMP pathways. Therefore, KMUP-1 may have a role in pharmacologic therapy of osteoporosis.</p></div

Effects of KMUP-1 on RANKL-induced activations of MAPKs and Akt pathways.

<p>(<b>A</b>) Time course analysis of RANKL-induced phosphorylation of MAPKs and Akt showed that their activations were maximized at 15 min or 30 min, respectively. (<b>B</b>–<b>E</b>) RAW264.7 cells were pretreated with KMUP-1 for 24 h followed by stimulation with RANKL (10 ng/ml) for 15 min (MAPKs) or 30 min (Akt). The cell lysates were analyzed by Western blotting. Each value represents the mean ± S.E.M. of three independent experiments, with triplicate determinations in each experiment. ^##<i>P</i><0.01 compared with control; *<i>P</i><0.05, **<i>P</i><0.01 compared with RANKL alone.</p

Effects of KMUP-1 on RANKL-induced activations of c-Fos and the calcium/calcineurin/NFATc1 pathway.

<p>(<b>A</b>) RAW264.7 cells were cultured for 24 h with RANKL (10 ng/ml) and KMUP-1 (0–10 µM). Cell lysates were then analyzed by Western blotting with antibodies against c-Fos, NFATc1, calcineurin and actin. (<b>B</b>–<b>D</b>) The expressions of these proteins were quantified by densitometry. (<b>E</b>) In RAW264.7 cells, RANKL did not induce Ca²⁺ oscillation. Each color indicates an individual cell in the same field. (<b>F, G)</b> In osteoclasts, KMUP-1 inhibited Ca²⁺ oscillation evoked by RANKL. Pretreatment with KMUP-1 (10 µM), similar to calcium chelator BAPTA (10 µM), significantly reduced the amplitude of oscillation induced by RANKL. Each value represents the mean ± S.E.M. of three independent experiments, with triplicate determinations in each experiment. ^##<i>P</i><0.01 compared with control; *<i>P</i><0.05, **<i>P</i><0.01 compared with RANKL alone.</p

Effects of KMUP-1 on bone loss in ovariectomized (OVX) mice.

<p>(<b>A</b>) OVX mice were sacrificed after 30 days of KMUP-1 treatment. Images of the longitudinal and transverse sections of the proximal tibia were obtained with a µCT. (<b>B</b>, <b>C</b>) Tibial trabecular bone mineral content (BMC) and bone volume/tissue volume (BV/TV, %) were quantified from data obtained by the µCT. All values are expressed as mean ± S.E.M. ^#<i>P</i><0.05 compared with the Sham group;^*<i>P</i><0.05 compared with the OVX group.</p

Effects of KMUP-1 on RANKL-induced pit formation in mature osteocalsts.

<p>(<b>A</b>) Mature osteoclasts were treated with RANKL (10 ng/ml) and KMUP-1 <b>(</b>1–10<b> </b>µM) for 48 h. Pit formation on the disc was observed by optical microscopy. (<b>B</b>) Pit areas were quantified using Image Pro Plus analyzer Version 4.6 (Media Cybernetics Inc., MD). Each value represents the mean ± S.E.M. of three independent experiments, with triplicate determinations in each experiment. *<i>P</i><0.05, **<i>P</i><0.01 compared with RANKL alone.</p

Effects of KMUP-1 on changes in histomorphometric and biochemical markers of bone turnover in OVX mice.

<p>Histomorphometric data and serum biochemical markers were compared in sham-operated, OVX mice, and OVX+KMUP-1 mice. (<b>A</b>) Tb.N, trabecular number, (<b>B</b>) Tb.Th, trabecular thickness, (<b>C</b>) Tb.Sp, trabecular separation, (<b>D</b>) ALP, serum alkaline phosphatase and (<b>E</b>) osteocalcin. All values are expressed as mean ± S.E.M. ^#<i>P</i><0.05 compared with the Sham group;^*<i>P</i><0.05, ^**<i>P</i><0.01 compared with the OVX group.</p

Effects of KMUP-1 on mRNA expression of osteoclastogenesis-related genes.

<p>Total RNA was extracted from RAW264.7 cells cultured for 24 h in the presence of RANKL (10 ng/ml) and KMUP-1 (0–10 µM), or with pretreatment of PKA inhibitor H89 (10 µM) and PKG inhibitor KT5823 (3 µM) for 30 min. The mRNA expression of the indicated genes was analyzed by real time RT-PCR. Each value represents the mean ± S.E.M. of three independent experiments, with triplicate determinations in each experiment. ^##<i>P</i><0.01 compared with control; *<i>P</i><0.05 compared with RANKL alone; ⁺<i>P</i><0.05 compared with KMUP-1 10 µM plus RANKL.</p

Effects of KMUP-1 on RANKL-induced production of inflammatory cytokines.

<p>(<b>A</b>) TNF- α, (<b>B</b>) IL-1 β, (<b>C</b>) IL-6, and (<b>D</b>) IL-10 were determined 24 hr after co-incubation with RANKL (10 ng/ml) and KMUP-1 (0–10 µM). Each value represents the mean ± S.E.M. of three independent experiments, with triplicate determinations in each experiment. ^##<i>p</i><0.01 compared with control; *<i>P</i><0.05, **<i>P</i><0.01 compared with that treated with RANKL alone.</p

Effects of KMUP-1 on RANKL-induced osteoclastogenesis.

<p>RAW264.7 cells were cultured with RANKL (10 ng/ml) for 5 days to induce osteoclast differentiation, with treatment of KMUP-1 (0–10 µM) to assess its anti-osteoclastogenic effects. In addition, cells treated with KMUP-1 (10 µM) had pretreatment of PKA inhibitor H89 (10 µM) and PKG inhibitor KT5823 (3 µM) to determine underlying mechanisms. (<b>A</b>) After 5 days of culture, TRAP positive cells and multinucleated osteoclasts were counted under the microscope. (<b>B</b>) The number of TRAP-positive multinucleated cells was counted. (<b>C</b>) TRAP activity was measured at 405 nm. Each value represents the mean ± S.E.M. of three independent experiments, with triplicate determinations in each experiment. ^##<i>P</i><0.01 compared with control; *<i>P</i><0.05, **<i>P</i><0.01 compared with RANKL alone; ⁺<i>P</i><0.05 compared with KMUP-1 10 µM plus RANKL.</p

Effects of KMUP-1 on RANKL-induced NF-κB activation.

<p>Cells were pretreated with KMUP-1 (0–10 µM) for 24 h before the stimulation by RANKL (10 ng/ml) for 30 min. (<b>A</b>) Using Western blotting, the nuclear fractions were analyzed for protein content of p65, a subunit of NF-κB protein, and (<b>B</b>) the cytosolic fractions were analyzed for protein content of IκB-α and phosphorylated IκB-α. (<b>C, D</b>) Confocal microscopy demonstrated that KMUP-1 inhibited RANKL-induced nuclear translocation of p65 as shown by the location of anti-p65 stain within the nucleus stained with DAPI. Scale bar: 5 µm. Each value represents the mean ± S.E.M. of three independent experiments, with triplicate determinations in each experiment. ^##<i>P</i><0.01 compared with control; *<i>P</i><0.05, **<i>P</i><0.01 compared with RANKL alone.</p