Naoyuki Takahashi

Activation of NF-κB Is Involved in the Survival of Osteoclasts Promoted by Interleukin-1

We previously reported that interleukin-1 (IL-1) promoted the survival of murine osteoclast-like cells (OCLs) formedin vitro and activated a transcription factor, NF-κB, of OCLs. The present study examined whether the activation of NF-κB is directly involved in the survival of OCLs promoted by IL-1. The expression of IL-1 type I receptor mRNA in OCLs was detected by the polymerase chain reaction amplification of reverse-transcribed mRNA. An electrophoretic mobility shift assay showed that IL-1 transiently activated NF-κB in the nuclei of the OCLs, and the maximal activation occurred at 30 min. The degradation of IκBα coincided with the activation of NF-κB in the OCLs. The immunocytochemical study revealed that p65, a subunit of NF-κB, was translocated from the cytoplasm into almost all of the nuclei of the OCLs within 30 min after IL-1 stimulation. The purified OCLs spontaneously died via apoptosis, and IL-1 promoted the survival of OCLs by preventing their apoptosis. The pretreatment of purified OCLs with proteasome inhibitors suppressed the IL-1-induced activation of NF-κB and prevented the survival of OCLs supported by IL-1. When OCLs were pretreated with antisense oligodeoxynucleotides to p65 and p50 of NF-κB, the expression of respective mRNAs by OCLs was suppressed, and the IL-1-induced survival of OCLs was concomitantly inhibited. These results indicate that IL-1 promotes the survival of osteoclasts through the activation of NF-κB.

Cells of BoneOsteoclast Generation

Publisher Summary This chapter focuses on the generation of osteoclasts in bone. Osteoclasts are multinucleated giant cells that resorb bone and develop from hemopoietic cells of the monocyte-macrophage lineage. Development of osteoclasts proceeds within a local microenvironment of bone and this process can be reproduced ex vivo in a coculture of mouse calvarial osteoblasts and hemopoietic cell. Multinucleated cells formed in the coculture exhibit major characteristics of osteoclasts, including tartrate-resistant acid phosphatase (TRAP) activity, expression of calcitonin receptors, c-Src (p60c-src), vitronectin receptors (αvβ3), and the ability to form resorption pits on bone and dentine slices. The discovery of the RANKL–RANK interaction has opened a wide new area in bone biology focused on the investigation of the molecular mechanism of osteoclast development and function. Membrane- or matrix-associated forms of both M-CSF and RANKL expressed by osteoblasts/stromal cells appear to be essential for osteoclast formation. Both RANKL(–/–) mice and RANK(–/–) mice show similar features of osteopetrosis with a complete absence of osteoclasts in bone. Gain-of-function mutations of RANK have been found in patients suffering from familial expansile osteolysis and familial Pagets disease of bone. These findings confirm that the RANKL–RANK interaction is indispensable for osteoclastogenesis not only in mice but also in humans. TRAF2-mediated signals are important for inducing osteoclast differentiation, and TRAF6-mediated signals are indispensable for osteoclast activation. Under physiological conditions, osteoclast formation requires cell-to-cell contact with osteoblasts/stromal cells, which express RANKL as a membrane-bound factor in response to several bone-resorbing factors.Publisher Summary nThis chapter focuses on the generation of osteoclasts in bone. Osteoclasts are multinucleated giant cells that resorb bone and develop from hemopoietic cells of the monocyte-macrophage lineage. Development of osteoclasts proceeds within a local microenvironment of bone and this process can be reproduced ex vivo in a coculture of mouse calvarial osteoblasts and hemopoietic cell. Multinucleated cells formed in the coculture exhibit major characteristics of osteoclasts, including tartrate-resistant acid phosphatase (TRAP) activity, expression of calcitonin receptors, c-Src (p60c-src), vitronectin receptors (αvβ3), and the ability to form resorption pits on bone and dentine slices. The discovery of the RANKL–RANK interaction has opened a wide new area in bone biology focused on the investigation of the molecular mechanism of osteoclast development and function. Membrane- or matrix-associated forms of both M-CSF and RANKL expressed by osteoblasts/stromal cells appear to be essential for osteoclast formation. Both RANKL(–/–) mice and RANK(–/–) mice show similar features of osteopetrosis with a complete absence of osteoclasts in bone. Gain-of-function mutations of RANK have been found in patients suffering from familial expansile osteolysis and familial Pagets disease of bone. These findings confirm that the RANKL–RANK interaction is indispensable for osteoclastogenesis not only in mice but also in humans. TRAF2-mediated signals are important for inducing osteoclast differentiation, and TRAF6-mediated signals are indispensable for osteoclast activation. Under physiological conditions, osteoclast formation requires cell-to-cell contact with osteoblasts/stromal cells, which express RANKL as a membrane-bound factor in response to several bone-resorbing factors.