Bone remodeling is elaborately controlled by two established processes: bone resorption by osteoclasts, and bone formation by osteoblasts. The balanced interplay between the osteocytes, osteoclasts and osteoblasts is responsible for the replacement and recycling of as much as 10% of the total adult human bone content each year (Manolagas, 2000).
Osteoclasts are derived from hematopoietic precursor cells in the bone marrow, and differentiate into mature osteoclasts when exposed to receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) (Boyle
Terminal differentiation of osteoclasts is characterized by acquisition of mature phenotypic markers which include expression of tartrate-resistant acid phosphatase (TRAP), calcitonin receptor, matrix metalloproteinase 9 (MMP9) and cathepsin K, as well as morphological conversion into large multinucleated cells and the capability to facilitate bone resorption (Fujisaki
The fruits of the
Poncirin, a flavanone glycoside, is one of the biologically active components contained in the
We therefore undertook to explore the effect of poncirin on osteoclast differentiation and evaluate the involved signaling pathways in RANKL-stimulated RAW264.7 cells. Our results demonstrate that poncirin significantly suppresses RANKL-induced osteoclast differentiation by suppressing osteoclast-specific gene expression with modulation of RANKL-mediated signal transduction. Furthermore, we demonstrate that poncirin considerably inhibits bone erosion in a mouse model.
Cell culture media and supplements were purchased from Invitrogen (Carlsbad, CA, USA). Soluble recombinant mouse RANKL (sRANKL) was purchased from Peprotech (NJ, USA). RNAzol and all PCR reagents were obtained from Takara Bio Inc (Shiga, Japan). TRAP stain kit was from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for p38 MAPK, ERK, JNK, phospho-p38MAPK (Thr180/Tyr182), phospho-ERK, phospho-JNK (Thr183/Tyr185), and anti-β-actin were procured from Cell Signaling Technology (Danvers, MA, USA). Poncirin was provided by the Korea Food and Drug Administration (KFDA, Ochang, Korea). Imprinting Control Region (ICR) mice were purchased from ORIENT Bio (Seongnam, Korea).
Mouse monocyte/macrophage RAW264.7 cells (ATCC #CRL-TBI-71) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM). Cells were supplemented with 10% fetal bovine serum (FBS), 100 µg/mL streptomycin, and 100 IU/mL penicillin. The cells were maintained in a humidified incubator of 5% CO2 and at 37°C, and fresh medium was replenished every 3 days. To differentiate RAW264.7 cells into osteoclasts, cultured cells were suspended in α-minimal essential media (α-MEM) (10% FBS) supplemented with 100 ng/mL sRANKL. The cells were plated in a 96-well plate (5×103 cells/well). Multinucleation of osteoclasts was observed from differentiation day 4.
The cells were (1×104 cells/well) cultured in DMEM/10% FBS. After 24 h, the cells were incubated with various concentrations of poncirin for 48 h. Cell viability was measured using the MTT (3 [4,5 dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, as described by Kim
At differentiation day 4, cells were fixed with 10% formalin by incubating for 10 min, following which they were stained using the Leukocyte Acid Phosphatase kit-387A according to the manufacturer’s instructions (Sigma-Aldrich). The images of TRAP-positive cells were captured under a microscope with DC controller (Olympus Optical, Tokyo, Japan).
Total RNA was isolated using the RNAzol reagent according to the manufacturer’s protocol. The concentrations of RNA were determined using ND1000 (Thermo Scientific, Wilmington, DE, USA).
First-strand cDNA was acquired from 1 μg of total RNA; qPCR was then performed using the SYBR Premix Ex Taq (Takara Bio Inc.), according to the manufacturer’s protocol. All reactions were run in triplicate, and the relative expression levels were analyzed by the 2–ΔΔCT method. β-actin is known to maintain a constant basal level during osteoclastogenesis, and was used as an internal standard. The primer sets utilized in the study are listed in Table 1.
Cells were washed with ice-cold phosphate buffered saline (PBS) containing 1 mM sodium vanadate, after which they were solubilized in lysis buffer (20 mM Tris–HCl (pH 7.5), 1 mM EGTA, 150 mM NaCl, 50 mM NaF, 1% Triton X-100, 1 mM EDTA, 1 mM β-glycerophosphate, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, and 1 μg/mL leupeptin). After a freeze–thaw cycle with vortexing, the lysate was centrifuged at 12,000×g for 15 min at 4°C. The cell lysates were resolved by SDS-PAGE and transferredonto a nitrocellulose membrane. The membrane was soaked in a blocking solution (5% non-fat dry milk/Tris-buffered saline, 0.1% Tween-20) for 1 h at room temperature, after which it was incubated with the relevant antibodies overnight at 4°C. The membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 1 h and then the bands were detected using ECL (Amersham Biosciences, Pittsburgh, PA, USA).
Each value was represented as the mean ± standard deviation (SD). Significant differences were determined using Student’s
We evaluated the effect of poncirin (Fig. 1, the structure of poncirin) on osteoclast differentiation in murine monocyte/macrophage cell line RAW264.7 cells. Cells were incubated with sRANKL (100 ng/mL) for 4 days to induce differentiation. Differentiation was assessed by counting the number of multinucleated TRAP-positive cells which is prominent morphological features of mature osteoclasts. As shown in Fig. 2A, osteoclastic differentiation was inhibited by exposure to poncirin in a concentration-dependent manner. Poncirin effectively reduced the number of TRAP-positive multinucleated cells at concentrations as low as 0.2 μg/mL and exerted up to 75.19 ± 3.56% inhibition at 5 µg/mL (Fig. 2B).
To assess if the inhibitory effect of poncirin on osteoclastogenesis resulted from its cytotoxicity, RAW264.7 cells were treated with varying concentrations of poncirin for 48 h, following which the cell viability was assessed by the MTT assay. Poncirin did not affect the rate of cell growth even as high as 50 μg/mL, indicating that the poncirin-mediated suppression of osteoclastogenesis is not due to cytotoxic effects (Fig. 2C).
Osteoclast differentiation is positively regulated by osteogenic genes, such as
c-Fos and NFATc1 are the key transcription factors involved in osteoclast differentiation. Induction of c-Fos, a critical component of the activator protein (AP)-1, is required for the robust expression of NFATc1. Therefore, we further undertook to evaluate if poncirin regulates osteoclast differentiation by modulating the activities of c-Fos and NFATc1. Incubation for 4 days with sRANKL elevated the gene expression of both
RANKL activates NFATc1 via a variety of key signal transducers, including p38, ERK, JNK, and NF-κB. To elucidate the role of poncirin in signal transduction of osteoclast differentiation, we examined the effects of poncirin on the RANKL-induced early activation of MAPKs. RAW264.7 cells were pretreated with poncirin for 1 h and stimulated with sRANKL for 15 min, after which we determined the phosphorylation levels of p38, JNK, ERK, and NF-κB by immunoblot analysis. We observed that sRANKL markedly induces the activation of all three MAPKs. However, pretreatment with poncirin inhibited the sRANKL-induced acute JNK activation without significantly affecting the expressions of ERK or p38 (Fig. 5A). Furthermore, poncirin also suppresses the sRANKL-induced NF-κB activation (Fig. 5B).
Based on the
Bone homeostasis requires a finely-tuned balance between bone resorption and bone formation, in which osteoblasts stimulate bone formation while osteoclasts promote bone resorption. An imbalance in bone homeostasis can occur due to a loss in osteoblast functions, or the abnormal activation of osteoclasts which enhances abnormal bone resorption. The imbalance due to osteoclast activation is closely related to most bone-related metabolic diseases such as osteoporosis, rheumatoid arthritis, periodontitis, multiple myeloma, and metastatic cancers (Boyle
We observed that poncirin treatment suppressed the NFATc1 gene expression (Fig. 4) which is considered to be mediated by the decreased activities of NF-κB and JNK (Fig. 5). The inhibitory effect of poncirin on NF-κB phosphorylation at 5 μg/mL, which effectively inhibited the differentiation of osteoclast, was unclear but was observed more clearly at higher concentrations of 50 μg/mL. This suggests that poncirin may regulate NF-κB at any interval between 5 and 50 μg/mL. Although the effect is unclear at low concentrations, it seems clear that poncirin controls phosphorylation of NF-κB. It is noteworthy that poncirin specifically suppresses the JNK activity without modulating the activities of p38 and ERK. NF-κB and AP-1 are immediately activated following RANKL binding, and mediate the signal from RANKL to NFATc1 during osteoclast differentiation resulting in transactivation of the NFATc1 promoter (Asagiri
We also found that down-regulation of c-Fos, a component of AP-1, might be a key event in poncirin-mediated inhibition in osteoclast differentiation of RAW264.7 cells. RANKL activates AP-1 partly through the induction of one of the critical components, c-Fos. c-Fos is known to play critical roles in bone-related diseases as is evident in c-Fos-deficient cells in which the RANKL-induced NFATc1 activation is completely abrogated (Grigoriadis
Osteoclasts are responsible for bone destruction in inflammatory bone diseases. LPS, a bacteria-derived cell wall product, has long been recognized as a key factor in the development of bone loss (Smith
In conclusion, this study demonstrates that poncirin is a potent inhibitor of RANKL-induced osteoclast differentiation. We also provide the molecular mechanisms of this inhibition, involving transcription factors such as NF-κB, NFATc1 and c-Fos. Inhibitory effects of poncirin are found to be associated with JNK inactivation, but not p-38 MAPK and ERK. Further, the
The authors wish to acknowledge the financial support of Gachon University Research Fund (GCU 2012-R003).