Demethylbelamcandaquinone B from Marantodes pumilum var. alata (Blume) Kuntze inhibits osteoclast differentiation in RAW264.7 cells

Osteoclasts are multinucleated cells that resorb bones, and are derived from hematopoietic cells of the monocyte/macrophage lineage. The receptor activator of NF-κB ligand (RANKL, also called ODF/TRANCE/OPGL) stimulates both osteoclast differentiation from osteoclast progenitors and activation of mature osteoclasts. To identify genes responsible for osteoclast differentiation, we used a molecular indexing technique. Here, we report a clone of one of these genes whose transcription is induced by soluble RANKL (sRANKL) in both the RAW264.7 cells of the mouse macrophage cell line and the mouse primary bone marrow cells. The predicted protein was found to be a mouse homologue of Jun dimerization protein 2 (JDP2), a member of the AP-1 family of transcription factors, containing a basic region-leucine zipper motif. Transient transfection experiments revealed that overexpression of JDP2 leads to activation of both tartrate-resistant acid phosphatase (TRAP) and cathepsin K gene promoters in RAW264.7 cells. Infection of mouse primary bone marrow cells with retroviruses expressing JDP2-facilitated sRANKL-mediated formation of TRAP-positive multinuclear osteoclasts. Importantly, antisense oligonucleotide to JDP2 strongly suppressed sRANKL-induced osteoclast formation of RAW264.7 cells. Our findings suggest that JDP2 may play an important role in the RANK-mediated signal transduction system, especially in osteoclast differentiation.

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Osteoclastic Metabolism of 25(OH)-Vitamin D3: A Potential Mechanism for Optimization of Bone Resorption

Endocrinology ◽

10.1210/en.2010-0334 ◽

2010 ◽

Vol 151 (10) ◽

pp. 4613-4625 ◽

Cited By ~ 79

Author(s):

Masakazu Kogawa ◽

David M. Findlay ◽

Paul H. Anderson ◽

Renee Ormsby ◽

Cristina Vincent ◽

...

Keyword(s):

T Cells ◽

Bone Resorption ◽

Mononuclear Cells ◽

Osteoclast Differentiation ◽

Raw264.7 Cells ◽

Marker Genes ◽

Null Mouse ◽

Nuclear Factor ◽

Activated T Cells ◽

Mouse Splenocytes

The extrarenal synthesis of 1α,25 dihydroxyvitamin D3 (1,25D) has been demonstrated in a number of cell types including osteoblasts and cells of the monocyte/macrophage lineage. The skeleton appears responsive to serum levels of the 1,25D precursor, 25 hydroxyvitamin D3 (25D), in terms of bone mineralization parameters. The effect of metabolism of 25D into active 1,25D by osteoclast lineage cells is unknown. We found that CYP27B1 mRNA expression increased with exposure of human peripheral blood mononuclear cells (PBMCs) to macrophage colony-stimulating factor in the presence or absence of receptor activator of nuclear factor-κB ligand. Consistent with this, human osteoclast cultures incubated with 25D produced measurable quantities of 1,25D. Osteoclast formation from either mouse RAW264.7 cells or human PBMCs in the presence of physiological concentrations of 25D resulted in significant up-regulation of the key osteoclast transcription factor, nuclear factor of activated T cells-c1 in PBMCs and a number of key osteoclast marker genes in both models. The expression of the osteoblast coupling factor, ephrin-b2, was also increased in the presence of 25D. Levels of CYP27B1 and nuclear factor of activated T cells-1 mRNA correlated during osteoclastogenesis and also in a cohort of human bone samples. CYP27B1 short-hairpin RNA knockdown in RAW264.7 cells decreased their osteoclastogenic potential. 25D dose dependently reduced the resorptive capacity of PBMC-derived osteoclasts without compromising cell viability. 25D also reduced resorption by RAW264.7- and giant cell tumor-derived osteoclasts. Conversely, osteoclasts formed from vitamin D receptor-null mouse splenocytes had increased resorptive activity compared with wild-type cells. We conclude that 25D metabolism is an important intrinsic mechanism for optimizing osteoclast differentiation, ameliorating osteoclast activity, and potentially promoting the coupling of bone resorption to formation.

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