Korean J Physiol Pharmacol.  2012 Feb;16(1):31-36. 10.4196/kjpp.2012.16.1.31.

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

• Son A
• Kim MS
• Jo H
• Byun HM
• Shin DM

• KMID: 2285437
• DOI: http://doi.org/10.4196/kjpp.2012.16.1.31

Abstract

The receptor activator of NF-kappaB ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-kappaB 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) delta, 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.

Keyword

Inositol 1,4,5-trisphosphate; RANKL; Osteoclastogenesis; Ca2+ signaling

MeSH Terms

Animals
Blood Proteins
Boron Compounds
Calcium-Transporting ATPases
Cell Membrane
Dentin
Estrenes
Inositol
Inositol 1,4,5-Trisphosphate
Inositol 1,4,5-Trisphosphate Receptors
Macrophages
Mice
NF-kappa B
Osteoclasts
Phosphoproteins
Proteins
Pyrrolidinones
Receptor Activator of Nuclear Factor-kappa B
Reticulum
Signal Transduction
Tumor Necrosis Factor-alpha
Type C Phospholipases
Blood Proteins
Boron Compounds
Calcium-Transporting ATPases
Estrenes
Inositol
Inositol 1,4,5-Trisphosphate
Inositol 1,4,5-Trisphosphate Receptors
NF-kappa B
Phosphoproteins
Proteins
Pyrrolidinones
Receptor Activator of Nuclear Factor-kappa B
Tumor Necrosis Factor-alpha
Type C Phospholipases

Figure

• 

  Fig. 1 Effect of receptor activator of NF-κB ligand (RANKL) on the expression of Ca2+ signaling proteins. RAW264.7 cells and mouse bone marrow derived monocyte/macrophage precursor cells (BMMs) were treated with RANKL (50 ng/ml). Levels of three types of inositol 1,4,5-trisphosphate receptors (IP3Rs), plasma membrane Ca2+ ATPase (PMCA), and sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) 2b were measured at different time points from 24 h to 6 days after RANKL treatment.

• 

  Fig. 2 Effect of PLC and IP3 inhibition on RANKL-induced [Ca2+]i oscillations in BMMs. (A) Generation of Ca2+ oscillations after 48 h with RANKL treatment. (B, C) Inhibitory effect of RANKL-induced [Ca2+]i oscillations by the PLC inhibitor, U73122 and IP3R inhibitor, 2APB. (D) Inhibition of RANKL-induced [Ca2+]i oscillations by elimination of IP3.

• 

  Fig. 3 Localization of phospholipase C (PLC) δ-PH-GFP in RAW264.7 cells after RANKL stimulation. RAW264.7 cells were treated with RANKL following their transfection with the PLC δ-PH-GFP domain. After 24 h with RANKL treatment, GFP intensity was measured using confocal laser microscopy. The graph shows the fluorescence intensity along the lines indicated in the images.

• 

  Fig. 4 Effects of IP3R inhibitor and PLC inhibitor on RANKL-induced multinucleated cell formation and pit formation in BMMs. (A) BMMs were seeded in 48-well plates and cultured for 6 days with RANKL and M-CSF. RANKL was simultaneously added to 2-aminoethoxydiphenyl borate (2APB)-, U73122-, and U73343-treated cells. Multinucleated cells (MNCs; ≥3 nuclei) in a well are presented as MNCs per well. Data from three independent experiments are represented as mean±SD (**p<0.005 compared with RANKL-induced MNC counts). (B) BMMs were seeded on OAAS plates, the bottom of which was covered with dentin slices, and cultured for 15 days with RANKL and M-CSF. RANKL was simultaneously added to 2APB- and U73122-treated cells. Data show the relative pit area compared to RANKL-treated BMMs and mean¡¾SD obtained from three independent experiments (**p<0.005 compared with RANKL-induced pit area).

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