Osteoblasts induce Ca ²⁺ oscillation-independent NFATc1 activation during osteoclastogenesis

Abstract

Intercellular cross-talk between osteoblasts and osteoclasts is important for controlling bone remolding and maintenance. However, the precise molecular mechanism by which osteoblasts regulate osteoclastogenesis is still largely unknown. Here, we show that osteoblasts can induce Ca 2+ oscillation-independent osteoclastogenesis. We found that bone marrow-derived monocyte/macrophage precursor cells (BMMs) lacking inositol 1,4,5-trisphosphate receptor type2 (IP 3 R2) did not exhibit Ca 2+ oscillation or differentiation into multinuclear osteoclasts in response to recombinant receptor activator of NF-κB ligand/macrophage colony-stimulating factor stimulation. IP 3 R2 knockout BMMs, however, underwent osteoclastogenesis when they were cocultured with osteoblasts or in vivo in the absence of Ca 2+ oscillation. Furthermore, we found that Ca 2+ oscillation-independent osteoclastogenesis was insensitive to FK506, a calcineurin inhibitor. Taken together, we conclude that both Ca 2+ oscillation/calcineurin-dependent and -independent signaling pathways contribute to NFATc1 activation, leading to efficient osteoclastogenesis in vivo .

Molecular Understanding of Osteoclast Differentiation and Physiology

“…In addition, NFATc1 seems to be activated by osteoblasts through a Ca 2+ oscillation-independent signal pathway during osteoclastogenesis and this clearly differs from that seen during RANKL/M-CSF-induced osteoclastogenesis [57]. Recently, it has been found that NFATc1 functions in the osteoclast fusion process via up-regulation of the dendritic cell-specific transmembrane protein (DC-STAMP) and the d2 isoform of vacuolar ATPase V0 domain (Atp6v0d2) through co-activation with MEF2 and MITF, which would be followed [58,59].…”

Caffeic acid 3,4-dihydroxy-phenethyl ester suppresses receptor activator of NF-κB ligand–induced osteoclastogenesis and prevents ovariectomy-induced bone loss through inhibition of mitogen-activated protein kinase/activator protein 1 and Ca2+–nuclear factor of activated T-cells cytoplasmic 1 signaling pathways

“…RANKL-induced intracellular Ca 2þ oscillation requires the PI3K/ AKT signaling pathway (11,19,40) and RANKL activates PI3K/AKT through the TRAF6 and c-Src complex in osteoclastogenesis. (15,16) In our study, we verified that CADPE inhibited the RANKL-induced interaction between TRAF6 and c-Src, suppressed the phosphorylation of AKT, and abrogated the oscillation of Ca 2þ , suggesting that CADPE modulated the TRAF/c-Src/PI3K/Ca 2þ signaling pathway in osteoclastogenesis.…”

“…After washing with PBS, cells were incubated with 5 mM Fura-2/AM at 378C, and then imaged at 340 and 380 nM excitation wavelengths to detect intracellular free calcium (with an Olympus IX71 and LAMBDA DG-4) and recorded by InVivo software; analysis was by Image-Pro Analyzer 6.2 software (Media Cybernetics, Bethesda, MD, USA). (19) Statistical analysis All experimental data are presented as the mean AE SD, with values from more than three experiments. Statistical significance was determined by the Student t test.…”

“…Because release of intracellular Ca 2þ requires activation of the PI3K/AKT signaling pathway, (17)(18)(19) and RANKL activates PI3K/AKT through the TRAF6 and c-Src complex. (15,16) we then assessed the effect of CADPE on the RANKL-induced association of TRAF6 with c-Src by endogenous coimmunoprecipitation assay and the effect of CADPE on the RANKL-induced phosphorylation of AKT by Western blot analysis.…”

“…(13,14) In particular, RANK-TRAF6 complex can lead to the activation of phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT) signaling pathway by recruiting Src family kinases. (2,11,15,16) Activation of PI3K finally releases intracellular Ca 2þ , (17)(18)(19) which can activate the central transcriptional factor in osteoclastogenesis, nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). (20) NFATc1 then translocates to the nucleus and activates the expression of multiple osteoclastogenesis-related genes, such as tartrate-resistant acid phosphatase (TRAP), latent transforming growth factor beta binding protein3 (LTBP3), chloride channel (ClC7), MMP9, calcitonin receptor (CTR), cathepsin K, and c-Src.…”