Abstract
Osteoclasts are multinucleated giant cells responsible for bone resorption. Various mediators involved in osteoclast differentiation have been investigated as possible therapeutic targets for osteoporosis and rheumatoid arthritis (RA). Although transforming growth factor beta1 (TGFβ1) has been described as one such multifunctional cytokine essential for bone remodeling, its effect on osteoclastogenesis remains controversial. Therefore, we sought to examine the effect of TGFβ1 on osteoclast generation induced by receptor activator of nuclear factor (NF)-κB ligand (RANKL) in humans. Peripheral blood monocytes, isolated using magnetic bead sorting, were cultured with macrophage-colony stimulating factor (M-CSF) or RANKL with or without TGFβ1. Tartrate-resistant acid phosphatase (TRAP) staining, as well as bone resorption assays, revealed that TGFβ1 suppressed RANKL-mediated human osteoclast development. Real-time reverse transcription PCR and Western blotting revealed that TGFβ1 reduced the gene and protein expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), the master regulator of osteoclast differentiation, respectively. Luciferase assays indicated that TGFβ1 inhibited the NF-κB p65-stimulated promoter activity of NFATc1. Immunofluorescence analysis demonstrated that TGFβ1 abrogated RANKL-induced nuclear translocation of p65. Thus, TGFβ1 regulates human RANKL-induced osteoclastogenesis via downregulation of NFATc1 by blocking nuclear translocation of NF-κB, suggesting that TGFβ1 may be a potential therapeutic target for RA.
Highlights
Osteoclasts are multinucleated giant cells with the unique ability of bone resorption [1,2,3,4,5]
The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) in the peripheral blood monocytes (PBMs) cultured with both macrophage-colony stimulating factor (M-CSF) and RANKL was considerably higher than that in the PBMs cultured with M-CSF alone (Figure 1a,b)
The number of TRAP-positive MNCs in PBMs cultured with M-CSF, RANKL, and TGFβ1 was considerably lower than that in PBMs cultured with M-CSF and RANKL
Summary
Osteoclasts are multinucleated giant cells with the unique ability of bone resorption [1,2,3,4,5]. Previous studies suggest that osteoclasts differentiate from bone marrow monocyte/macrophage lineage cells in the presence of two indispensable cytokines: macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-κB ligand (RANKL) [11,12]. In vitro osteoclast generation following M-CSF and RANKL stimulation of CD14-positive cells (monocytes) isolated from peripheral blood mononuclear cells (PBMCs) has been reported [13]. Transcription factors and related genes are markedly upregulated in the early stages (monocytes and osteoclast precursor). Cathepsin K is a cysteine protease specific for osteoclasts and plays an important role in degrading the bone matrix proteins during bone resorption [15]
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