Tumor Necrosis Factor-α Induces Differentiation of and Bone Resorption by Osteoclasts

Yoshiaki Azuma,Keisuke Kaji,Rei Katogi,Sunao Takeshita,Akira Kudo

doi:10.1074/jbc.275.7.4858

Abstract

Osteoclast progenitors differentiate into mature osteoclasts in the presence of receptor activator of NF-kappaB (RANK) ligand on stromal or osteoblastic cells and monocyte macrophage colony-stimulating factor (M-CSF). The soluble RANK ligand induces the same differentiation in vitro without stromal cells. Tumor necrosis factor-alpha (TNF-alpha), a potent cytokine involved in the regulation of osteoclast activity, promotes bone resorption via a primary effect on osteoblasts; however, it remains unclear whether TNF-alpha can also directly induce the differentiation of osteoclast progenitors into mature osteoclasts. This study revealed that TNF-alpha directly induced the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), which produced resorption pits on bone in vitro in the presence of M-CSF. The bone resorption activity of TNF-alpha-induced MNCs was lower than that of soluble RANK ligand-induced MNCs; however, interleukin-1beta stimulated this activity of TNF-alpha-induced MNCs without an increase in the number of MNCs. In this case, interleukin-1beta did not induce TRAP-positive MNC formation. The osteoclast progenitors expressed TNF receptors, p55 and p75; and the induction of TRAP-positive MNCs by TNF-alpha was inhibited completely by an anti-p55 antibody and partially by an anti-p75 antibody. Our findings presented here are the first to indicate that TNF-alpha is a crucial differentiation factor for osteoclasts. Our results suggest that TNF-alpha and M-CSF play an important role in local osteolysis in chronic inflammatory diseases.

Highlights

Osteoclasts, which differentiate from hematopoietic stem cells [1,2,3], have a crucial role in physiological bone remodeling [4], but they function in the local bone destruction that occurs in association with chronic inflammatory diseases [5]
TNF-␣ Induces Osteoclast Differentiation of MDBM Cells and Bone Marrow Cells—To search for the dominant cytokines involved in local bone destruction, we examined the effects of proinflammatory cytokines such as IL-1 and TNF-␣ on osteoclast differentiation using the newly established osteoclast progenitors (MDBM cells) described under “Experimental Procedures” as well as bone marrow cells
The results demonstrate that TNF-␣ can induce the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), though not to the extent found with soluble RANKL (sRANKL)

Summary

EXPERIMENTAL PROCEDURES

Animals and Chemicals—Male ddY mice, 6 –9 weeks of age, were used MDBM cells (1 ϫ 104 cells/ well) in ␣-MEM containing 10% fetal calf serum were added into each well and incubated for different periods at 37 °C in a humidified atmo-. The MDBM cells were incubated for different periods of time at 37 °C in a humidified atmosphere of 5% CO2 in air, after which the discs were examined by phase-contrast microscopy. These discs were washed in a solution of 6% NaClO and 5.2% NaCl for removal of the cells to observe the resorption pits more clearly. The stained cells were incubated further with fluorescein isothiocyanate-conjugated streptavidin or goat anti-rat IgG and analyzed by flow cytometry (FACS-Calibur instruments and software, Becton Deckinson Inc., San Jose, CA)

RESULTS

No treatmenta

DISCUSSION