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Effects of Inositol 1,4,5-triphosphate on Osteoclast Differentiation in RANKL-induced Osteoclastogenesis

The receptor activator of NF-κB ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-κB and other signal transduction pathways essential for osteoclastogenesis, such as Ca2+ signaling. However, the intracellular levels of inositol 1,4,5-trisphosphate (IP3) and IP3-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of IP3 and evaluated IP3-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of Ca2+ signaling proteins such as IP3 receptors (IP3Rs), plasma membrane Ca2+ ATPase, and sarco/endoplasmic reticulum Ca2+ ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of IP3 was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) δ, a probe specifically detecting intracellular IP3 levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)] and of IP3Rs with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of IP3Rs) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular IP3 levels and the IP3-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis.


INTRODUCTION
Bone is a connective tissue that constructs the skeleton and is composed of the intracellular matrix proteoglycans, osteoblasts, osteoclasts, and osteocytes. It is a dynamic tissue that is constantly renewed by bone remodeling. Bone remodeling is an active process and is regulated by the activity of bone-forming osteoblasts and bone-resorbing osteoclasts [1,2]. In fact, an imbalance in bone remodeling can cause pathological conditions such as rheumatoid arthritis, osteoporosis, and osteopetrosis.
Cells grow and differentiate through cell-to-cell interactions, and extracellular signals lead to specific cell reactions through signal transduction. Cell-to-cell signaling of mesenchymal cells, including osteoblasts, induces the differentiation of osteoclast precursor cells into mature osteoclasts [3,4]. Receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) is expressed on the surfaces of osteoclasts, and an essential factor for osteoclast differentiation. RANKL, a member of the tumor necrosis factor (TNF) family, is expressed in the bone, bone marrow, and lymphoid tissue during osteoclastogenesis. RANKL binds to RANK and transmits a differentiation signal [5]. It is known that RANKL stimulation initially activates tumor necrosis factor receptor-associated factor 6 (TRAF6), and then sequentially activates NF-κB, c-fos, JNK, ERK, and PI3K in osteoclast precursor cells [6,7]. Recent studies of osteoclastogenesis have focused on TRAF6, JNK, and NF-κB signaling pathways triggered by the binding of RANKL to RANK on osteoclast precursors. During osteoclastogenesis, auto-amplification of NFATc1 induces osteoclast-specific genes including AP-1, tartrate-resistant acid phosphatase (TRAP), calcitonin receptor, and cathepsin K [6]. Recently, Ca 2＋ signaling has been recognized as an essential pathway in the differentiation of osteoclasts. In particular, a pre-vious study has shown that treatment with a Ca 2＋ chelator inhibits osteoclastogenesis in osteoclast precursor cells [8], suggesting that Ca 2＋ signaling is important in osteoclastogenesis.
Hormone and neurotransmitter-induced increases in intracellular Ca 2＋ concentration ([Ca 2＋ ]i) regulate gene expression, growth, differentiation, muscle contraction, memory, and learning [9]. Particularly, in osteoclast differentiation, Ca 2＋ was reported to play an important role by sequentially activating calcineurin and NFATc1 [5,8,10] ]i occurs through the activation of Ca 2＋ channels or Ca 2＋ release from the ER into the cytoplasm, and is then followed by activation of the sarco/endoplasmic reticulum Ca 2＋ ATPase (SERCA) and the plasma membrane Ca 2＋ ATPase (PMCA) that remove Ca 2＋ from the cytosol [11,12]. In most cells, extracellular signals such as hormones and neurotransmitters stimulate phospholipase C (PLC) and then hydrolyze a plasma membrane lipid phosphatidyl-inositol bisphosphate (PIP2) to generate inositol 1,4,5-trisphosphate (IP3) in the cytosol. Increased levels of IP3 promote Ca 2＋ release into the cytosol by changing Ca 2＋ permeability of the IP3Rs in the ER, which is the intracellular Ca 2＋ store. Ca 2＋ release from the ER can change the conductance of Ca 2＋ release-activated Ca 2＋ channels. It induces Ca 2＋ influx from an environment of high concentration of extracellular Ca 2＋ [12]. An induced increase in [Ca 2＋ ]i is removed by the Ca 2＋ pump. Nevertheless, there are no reports on the intracellular levels of IP3 in osteoclast differentiation. In the present study, we assessed the levels of IP3 and the associated IP3-induced osteoclast differentiation and activity during RANKL-mediated osteoclastogenesis in osteoclast precursor cells.

Preparation of BM M s
Four-week-old male mice (Koatec, Pyeongtaek, Korea) were sacrificed, and the femur and tibia were separated.
Bone marrow cells derived from the femur and tibia were collected and cultured in α-MEM containing 10% FBS and 10 ng/ml M-CSF. The next day, non-adherent cells in the media were collected and seeded on adequate number of plates with 50 ng/ml M-CSF. After 2 days, non-adherent cells were washed out with fresh media, and the adherent cells were used as BMMs.

Expression of IP3 Sponge and PLC δ-PH-GFP expression in RAW264.7 cells
RAW264.7 cells were transiently transfected with a green fluorescent protein (GFP)-tagged high affinity (R441Q) or low affinity (K508A) IP3-sequestering sponge [13], or were transfected with PLC-δ1-PH-GFP construct provided by Dr. Shmuel Muallem (NIH/NIDCR, USA) using Lipofectamine 2000 reagent (invitrogen), according to the manufacturer's instructions. Cells were incubated for 48 h at 37 o C, in a 5% CO2 atmosphere with saturated humidity to allow expression of each protein was confirmed by GFP fluorescence.

cells
After RANKL treatment, GFP-positive cells were identified, and the GFP density in PLC δ-PH-GFP cDNA-transfected RAW264.7 cells was measured using a confocal laser scanning microscope (Leica, Buffalo, NY, USA) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

TRAP stain assay
BMMs were seeded in 48-well plates at a concentration of 1×10 4 cells per well and pretreated with indicated compounds. Cells were then stimulated with 50 ng/ml sRANKL. After 6 days, a TRAP stain assay was performed to confirm the cell differentiation rate. TRAP-positive cells were stained using a Leukocyte Acid Phosphate Assay Kit (Sigma-Aldrich) by following the manufacturer's procedure. TRAP-positive multinucleated cells (containing ≥3 nuclei) were then counted.

M easurement of bone-resorption rate (pit assay)
BMMs were collected from 4-week-old mice, as described previously [14]. Collected BMMs were seeded on 48-well bone-slice covered plates, coated with an osteoclast activity assay substrate (OAAS   TM   ; OCT, Cheonan, Korea). BMMs in bone-sliced plates were maintained with 50 ng/ml M-CSF and sRANKL for 15 days. After 15 days, cells were washed out with sodium hypochlorite solution for 1 h at room temperature. The bone slices were imaged, and the pits in the bone slices were identified based on pixel area calculations. The data were expressed relative to the pit areas in RANKL-stimulated BMMs by using the MetaMorph software (Molecular Devices).

Statistical analyses
Data from at least three independent experiments were expressed as mean±SD. Statistically significant differences between groups were determined using the Student t-test.

RANKL-induced expression of Ca 2＋ signaling proteins
To assess the effect of RANKL stimulation on the expression levels of Ca 2＋ signaling proteins such as IP3R, SERCA, and PMCA, RAW264.7 cells and BMMs were treated with RANKL for 24 h, 48 h, 72 h, and 6 days. There were no differences in the expression levels of these Ca 2＋ signaling proteins after RANKL treatment (Fig. 1, n=3).

RANKL-mediated changes in intracellular IP3 levels
Because there were no differences in expression levels of Ca 2＋ signaling proteins after RANKL treatment (Fig. 1), we hypothesized that RANKL-mediated activation of PLC induced an increase in intracellular IP3 levels, and then sequentially generated Ca 2＋ oscillations. To test our hypothesis, we transfected RAW264.7 cells with the PLC δ-PH-GFP domain. The localization of PLC δ-PH-GFP domain was confirmed using confocal laser microscopy after 24 h of RANKL treatment. In the steady state, the PLC δ PHdomain was located in the cell membrane in the absence of RANKL treatment; however, after 24 h of RANKL treatment, the PH-domain bound to IP3 and then moved into the cytoplasm (Fig. 3, n=3).

Effect of PLC and IP3R on RANKL-mediated osteoclast differentiation and osteoclast activity
Based on our finding that RANKL induced an increase in intracellular IP3 levels, we further investigated the role of PLC and IP3R activation on RANKL-mediated osteoclast differentiation and osteoclast activity using a TRAP staining assay and pit assay. The blockage 10 μM U73122, and 75 μM 2APB inhibited the generation of RANKL-induced multinucleated cells to 60% and 84.3% of the controls, respectively (Fig. 4A, n=3). In addition, we measured osteoclast activity in RAW264.7 cells and BMMs using dentin slices following U73122 and 2APB treatment. Pit areas in RANKL-induced osteoclastogenesis were decreased to 48.4% and 93.6% of the controls, respectively (Fig. 4B, n=3).

DISCUSSION
Osteoclastogenesis requires costimulatory receptor signaling mediated through adaptor proteins that contain immunoreceptor tyrosine-based activation motifs (ITAMs), such as the Fc receptor γ (FcR γ) and the 12-kDa DNAXactivating protein [5]. The active signal transmitted through adaptor proteins then induces Ca 2＋ signaling, which sequentially activates NFATc1 and induces osteoclast differentiation [5,10]. In addition, the signal via TRAF6 activates transcription factors such as c-fos and NF-κB [4] and the Ca 2＋ signal and activation of NFATc1 are transduction signals occurring after the middle stage of osteoclast differentiation in most cells. It is likely that activation of IP3Rs in the ER may induce Ca 2＋ oscillations. Because there was no way to directly measure the intracellular concentration of IP3, we transfected cells with a PLC δ-PH domain [16]. The transfected PLC δ-PH domain binds to PIP2 with high specificity and affinity during the resting state. However, it is translocated to the cytoplasm and binds to IP3 when IP3 accumulates via an external signal because the PLC δ-PH domain has a stronger affinity for IP3 than for PIP2 [16]. We demonstrated that RANKL induces an increase in IP3 levels at 24 h after RANKL treatment, after which   IP3 binds to the PH-domain and translocates from the membrane to the cytosol. On the other hand, during osteoclastogenesis, PLCγ activation is important to generate Ca 2＋ signaling, and this has been confirmed by using the PLC inhibitor U73122 [15]. In our study, treatment of cells with U73122 inhibited the generation of RANKL-induced Ca 2＋ oscillations, multinucleated cells, and reduced the bone-resorption rate. In addition, we observed that treatment with 2APB, an IP3R inhibitor, inhibited the generation of RANKLinduced Ca 2＋ oscillations, multinucleated cells, and reduced bone-resorption rate. This result is consistent with a previous finding that, Ca 2＋ oscillations were not generated and in vitro osteoclastogenesis was impaired in IP3R2/3-deficient mice [17]. Therefore, IP3-mediated Ca 2＋ signaling is essential for osteoclastogenesis.
Previous studies showed that Ca 2＋ influx channels located on the plasma membrane play an important role in osteoclastogenesis [18,19]. Transient receptor potential cation channel subfamily V member 4 (TRPV4)-deficient mice led to an increase in bone mass; Ca 2＋ oscillations were normal in these cells at the early stage of osteoclastogenesis; however, they gradually disappeared at the later stages. These results suggest that Ca 2＋ influx via TRPV4 is necessary for Ca 2＋ signaling [19]. TRPV5-deficient mice also showed an increase in bone mass, although increased number and size of osteoclasts in vivo, suggesting the importance of Ca 2＋ influx for resorption activity in mature osteoclasts [18]. Therefore, it remains to be determined how Ca 2＋ oscillations and Ca 2＋ channel-mediated Ca 2＋ influx relate to osteoclastogenesis.
In summary, these results suggest that RANKL induces increases in intracellular IP3 and IP3 plays an important role in osteoclstogenesis. Therefore, manipulation of this signaling pathway may provide a target for treatment of bone-related disease.