Nobuo Okahashi

Specific Inhibitors of Vacuolar H+‐ATPase Trigger Apoptotic Cell Death of Osteoclasts

Osteoclasts are multinucleated bone‐resorbing cells that play a critical role in bone remodeling. Specific inhibitors of vacuolar H+‐ATPase (V‐ATPase), concanamycin A and bafilomycin A1, abolish bone resorption by osteoclasts. In this study, we examined whether these V‐ATPase inhibitors trigger apoptotic cell death in osteoclasts, using murine osteoclast‐like multinucleated cells (OCLs) formed in vitro. Acridine orange staining revealed that the treatment of OCLs with concanamycin A resulted in chromatin condensation and alterations in nuclear morphology within a few hours. The TdT‐mediated dUTP‐nick‐end labeling (TUNEL) reaction confirmed the apoptotic features of OCLs treated with concanamycin A. The accelerated apoptotic cell death induced by concanamycin A occurred in OCLs treated with interleukin‐1α or macrophage colony‐stimulating factor as well, which are known to elongate the survival time of osteoclasts. In contrast, these inhibitors did not induce cell death of osteoblastic cells isolated from mouse calvaria. These results suggest that functional impairment of V‐ATPase triggers apoptotic cell death in osteoclasts.

Caspases (Interleukin-1β-Converting Enzyme Family Proteases) Are Involved in the Regulation of the Survival of Osteoclasts

Osteoclast-like multinucleated cells (OCLs) were prepared on collagen gels in a coculture system of mouse bone marrow cells and osteoblasts, and purified by collagenase and a subsequent pronase treatment. More than 80% of the purified OCLs were found to undergo apoptotic cell death by 48 h during the culture in a culture medium containing 10% fetal bovine serum (FBS). Withdrawal of FBS from the culture medium accelerated the cell death, which induced more than 80% of OCLs to undergo apoptotic cell death by as early as 18 h. Two peptide inhibitors of caspases (interleukin-1beta-converting enzyme family proteases), benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone (Z-DEVD-FMK), extended the survival time of OCLs in the presence and absence of 10% FBS, but the effect was rather limited in the absence of FBS. Because interleukin-1alpha (IL-1alpha) and the macrophage colony stimulating factor (M-CSF) are known to promote the survival of osteoclasts, we examined the effect of the peptide inhibitors and these cytokines. Combinations of the peptide inhibitors and IL-1alpha, or the peptide inhibitors and M-CSF, were more effective than the inhibitors alone. When endogenous caspase activities of OCLs were analyzed using fluorescence peptide substrates, the activities, in particular, caspase-3 (CPP32)-like activity, were markedly increased in OCLs by the withdrawal of FBS from the culture medium. IL-1alpha and M-CSF suppressed the activation of the caspases. In addition, western blot analysis revealed that the expression of Bcl-2, which inhibits the activation of caspases, was very weak or even negligible in OCLs. Taken together, these results suggest that the caspases are involved in the regulation of survival and apoptotic cell death of osteoclasts.

Possible involvement of protein kinases and Smad2 signaling pathways on osteoclast differentiation enhanced by activin A

Bone tissues reportedly contain considerable amounts of activin A and follistatin, an activin A‐binding protein. In the present study, we found that follistatin strongly inhibited osteoclast formation in cocultures of mouse bone marrow cells and primary osteoblasts induced by 1α,25 dihydroxyvitamin D3, prostaglandin E2, and interleukin‐1α. Antibody aganist activin A also inhibited the osteoclast formation. Furthermore, activin A synergistically stimulated osteoclast differentiation mediated by receptor activator NF‐κB ligand (RANKL). RT‐PCR analysis revealed that osteoblasts produced not only activin A but also follistatin. Western blot analysis of a panel of phosphorylated proteins revealed that activin A stimulated the phosphorylation of p44/42 mitogen activated protein (MAP) kinase (ERK1/2) and p38 MAP kinase in macrophage colony‐stimulating factor‐dependent bone marrow macrophages (M‐BMMΦs). In addition, phosphorylation of Smad2 was observed in M‐BMMΦs stimulated with activin A. These findings indicate that the phosphorylation of p44/42 MAP kinase, p38 MAP kinase, and Smad2 is involved in activin A‐enhanced osteoclast differentiation induced by RANKL. Taken together, these results suggest that both activin A and follistatin produced by osteoblasts may play an important role in osteoclast differentiation through MAP kinases and Smad2 signaling pathways.

Inhibition of experimental bone resorption and osteoclast formation and survival by 2-aminoethanesulphonic acid

It is known that bone resorption is mediated by osteoclasts, and lipopolysaccharide (LPS) and inflammatory mediators such as interleukin-1 (IL-1) and prostaglandin E2 (PGE2) induce osteoclast differentiation from haemopoietic cells, 2-aminoethanesulphonic acid, which is known as taurine, is an important nutrient and is added to most synthetic human infant milk formulas. In this study, it was found that 2-aminoethanesulphonic acid inhibits the stimulation of bone resorption mediated by LPS of the periodontopathic microorganism Actinobacillus actinomycetemcomitans Y4 in organ cultures of newborn mouse calvaria. The effect of 2-aminoethanesulphonic acid on the development and survival of osteoclast-like multinucleated cells produced in a mouse bone-marrow culture system was also examined. 2-aminoethanesulphonic acid (100 microg/ml) suppressed the formation of these osteoclast-like cells in the presence of LPS of A. actinomycetemcomitans Y4, IL-1alpha or PGE2 in mouse marrow cultures. On the other hand, 2-aminoethanesulphonic acid did not inhibit 1alpha, 25-dihydroxyvitamin D3-mediated osteoclast differentiation. Although IL-1alpha elongated the survival of the osteoclast-like cells, 2-aminoethanesulphonic acid blocked the supportive effect of IL-1alpha on osteoclast survival. 2-aminoethanesulphonic acid showed no effect on the growth of mouse osteoblasts. Finally, it was found that 2-aminoethanesulphonic acid inhibited alveolar bone resorption in experimental periodontitis in hamsters. These results suggest that 2-aminoethanesulphonic acid is an effective agent in preventing inflammatory bone resorption in periodontal diseases.

Intranasal immunization of mice with recombinant protein antigen of serotype cStreptococcus mutans and cholera toxin B subunit

The cholera toxin subunit and the recombinant cell-surface antigen (molecular mass of 190,000 Da) were administered intranasally to BALB/c mice. After 30 days, the mice were immunized intranasally with the recombinant protein antigen alone. High serum IgG and salivary IgA responses to the protein antigen were induced by the intranasal immunization.