Shigeru Amano

Stimulatory effect of curcumin on osteoclast apoptosis

Curcumin is a potent inhibitor of the transcriptional factors activator protein-1 and nuclear factor-kappaB. Since transcriptional factors may play a functional role in the survival of osteoclasts, it was of interest to us to examine the effect of curcumin on osteoclast apoptosis. We observed that curcumin is a potent stimulator of this process in rabbit osteoclasts, as evidenced by morphological changes in nuclei and DNA fragmentation as criteria of apoptosis. The curcumin stimulation of the osteoclast apoptosis was dose-and treatment time-dependent. In addition, curcumin dramatically inhibited osteoclastic bone resorption, supporting our data that curcumin is a potent stimulator of osteoclast apoptosis.

Biological characterization of interleukin-1-like cytokine produced by cultured bone cells from newborn mouse calvaria

SummaryWe have investigated the role of interleukin-1 (IL-1) and IL-1-like factor in the regulatory mechanisms of a bone remodeling system. To determine whether the bone cell itself produces IL-1-like cytokine, we examined bone cells cultured from newborn mouse calvaria. Bone cells migrating from fragments of newborn mouse calvaria were used in this study. We also used bone cells obtained by consecutive digestion of the calvaria with enzymes. These bone cells were cultured in fetal calf serum-containing alpha-MEM. IL-1-like cytokine activity was measured by incorporation of [3H]thymidine into C3H/HeJ thymocytes stimulated with PHA. When treated with lipopolysaccharide (LPS) fromEscherichia coli 0111 B4, the cultured bone cells produced a significant amount of IL-1-like cytokine. The maximum concentration of IL-1-like cytokine was observed in culture supernatants of the bone cells cultured for 24 horus with the LPS in serum-free medium. The IL-1-like cytokine closely resembles IL-1 in some of its biological characteristics: (1) stimulation of mitogen-induced thymocyte proliferation, (2) stimulation of fibroblast proliferation, (3) pyrogenicity, and (4) molecular weight. These results show that cultured bone cells from newborn mouse calvariae produce an IL-1-like cytokine that closely resembles IL-1.

Cellular Events Involved in Butyric Acid-Induced T Cell Apoptosis

We have previously demonstrated that butyric acid induces cytotoxicity and apoptosis of murine thymocytes, splenic T cells, and human Jurkat T cells. Therefore, to determine the apoptotic signaling pathway induced by butyric acid, we investigated the contribution of reactive oxygen species (ROS), mitochondria, ceramide, and mitogen-activated protein kinases in butyric acid-induced human Jurkat cell apoptosis. After exposure of cells to butyric acid, a pronounced accumulation of ROS was seen. Pretreatment of cells with the antioxidant N-acetyl-cysteine or 3-aminobenzamide attenuated butyric acid-induced apoptosis through a reduction of ROS generation. Cytochrome c, apoptosis-inducing factor, and second mitochondria-derived activator of caspases protein release from mitochondria into the cytosol were detected shortly after butyric acid treatment. Exposure of cells to butyric acid resulted in an increase in cellular ceramide in a time-dependent fashion. In addition, butyric acid-induced apoptosis was inhibited by DL-threo-dihidrosphingosine, a potent inhibitor of sphingosine kinase. Using anti-extracellular signal-regulated kinase (ERK), anti-c-Jun N-terminal kinase (JNK), and anti-p38 phosphospecific Abs, we showed a decrease in ERK, but not in JNK and p38 phosphorylation after treatment of cells with butyric acid. Pretreatment of cells with the JNK inhibitor SP600125 attenuated the effect of butyric acid on apoptosis, whereas no effect was seen with the p38 inhibitor SB202190 or the ERK inhibitor PD98059. Taken together, our results indicate that butyric acid-induced T cell apoptosis is mediated by ceramide production, ROS synthesis in mitochondria, and JNK activation in the mitogen-activated protein kinase cascade. Finally, these results were further substantiated by the expression profile of butyric acid-treated Jurkat cells obtained by means of cDNA array.

Spontaneous production of interleukin-1-like cytokine from a mouse osteoblastic cell line(MC3T3-E1)

We have investigated the role of interleukin-1 in the regulatory mechanisms of a bone remodeling system. Osteoblastic cell line (MC3T3-E1) established from newborn mouse calvaria spontaneously produced interleukin-1-like cytokine. Although the interleukin-1-like activity was not observed in culture supernatants of the cells during their exponential phase of growth, a most remarkable interleukin-1-like activity was detected in the supernatants of cells cultured in serum-free alpha-MEM on day 5 after the cells had formed a confluent monolayer. Gel filtration analysis indicated that the interleukin-1-like cytokine exhibits some heterogeneity in size.

Human purified interleukin-1 inhibits DNA synthesis and cell growth of osteoblastic cell line (MC3T3-E1), but enhances alkaline phosphatase activity in the cells.

We examined the effects of human purified interleukin‐1 (IL‐1) on DNA synthesis, cell growth, and alkaline phosphatase activity in the osteoblastic cell line MC3T3‐E 1 under both preconfluent and confluent culture conditions. Addition of IL‐1 to the cells markedly inhibited their DNA synthesis and growth over the range 1–10 . Such significant inhibitory effects were observed in cells cultivated in 1 or 5% fetal calf serum (FCS)‐containing alpha modification Eagles medium (alpha‐MEM), but not in alpha‐MEM containing 10% FCS. In contrast, alkaline phosphatase activity was enhanced significantly by IL‐1 in the cell line cultivated in 1% FCS‐containing alpha‐MEM. These results demonstrate that human purified IL‐1 is effective in inducing the differentiation of osteoblastic cell MC3T3‐E1.

Effects of recombinant human interleukin 1a and interleukin 1β on cell growth and alkaline phosphatase of the mouse osteoblastic cell line MC3T3-E1

Abstract Recombinant human interleukin 1 (rhIL-1)α and rhIL-1β were examined for their effects on DNA synthesis, cell growth and alkaline phosphatase activity of the mouse osteoblastic cell line MC3T3-E1. The relative activity of rhIL-1α and rhIL-1β was compared in terms of the units which induced half-maximal [3H]thymidine uptake into mouse thymocyte cultures exposed to IL-1. Both rhIL-1α and rhIL-1β significantly inhibited DNA synthesis and division of the cells in a concentration- and cultivation time-dependent fashion. In contrast, rhIL-lα and rhIL-1β markedly increased alkaline phosphatase activity, which is a marker of osteoblastic differentiation. This activity in cells treated with rhIL-1α and rhIL-1β increased about 2.0- and 1.7-fold, respectively, compared with that of control cultures. Inhibition of the DNA synthesis and stimulation of alkaline phosphatase activity by both types of rhIL-1 were completely neutralized by treatment with their respective polyclonal antisera. Also, inhibition of DNA synthesis was unaffected by the addition of cyclooxygenase and lipoxygenase inhibitors, and stimulation of alkaline phosphatase activity was unaffected by the addition of indomethacin. These results indicate that both rhIL-1α and rhIL-1β have qualitatively similar biological effects on osteoblastic cells. They also suggest that IL-1 is an important modulator of the growth and differentiation of osteoblasts.

Functional role of endogenous CD14 in lipopolysaccharide-stimulated bone resorption.

Lipopolysaccharide (LPS) is a bacterial cell component that plays multifunctional roles in inflammatory reactions, and one of these roles is that of a powerful stimulator of bone resorption. However, the mechanism by which LPS stimulates bone resorption is not yet understood. In the present study, we show, by using mouse embryonic calvarial cells, that endogenous CD14 and interleukin‐1β (IL‐1β) play an important role in the LPS‐mediated bone resorption and that interferon‐γ (IFN‐γ)functions as a strong inhibitor of this resorption by suppressing LPS‐stimulated expression of CD14 and IL‐1β genes in the calvarial cells. We observed that LPS‐stimulated differentiation of osteoclastic cells and bone and resorption were markedly neutralized by anti‐mouse CD14 antibody were clearly inhibited by anti‐sense CD14 oligonucleotide treatment. In addition, because LPS stimulated CD14 gene expression in the calvarial cells, these observations demonstrate the precise role of endogenous CD14 in LPS‐stimulated differentiation of osteoclastic cells and boneresorption. However, the stimulation of the differentiation of osteoclastic cells and bone resorption was also inhibited by anti‐mouse IL‐1β antibody. Interestingly, anti‐sense CD14 oligonucleotide inhibited LPS‐stimulated expression of the IL‐1β gene in the calvarial cells. These observations suggest a functional role of endogenous CD14 in LPS‐stimulated expression of the IL‐1β gene in the cells. Because IFN‐γ is a potent inhibitor of bone resorption stimulated by IL‐1, in additional experiments, we examined whether IFN‐γ is able to inhibit LPS‐stimulated differentiation of osteoclastic cells and bone resorption. We found that IFN‐γ inhibited these stimulations by suppressing CD14 and IL‐1β genes in the calvarial cells. The present study thus clearly demonstrates a functional role of endogenous CD14 in LPS‐stimulated bone resorption. J. Cell. Physiol. 173:301–309, 1997.

Osteocalcin fragment in bone matrix enhances osteoclast maturation at a late stage of osteoclast differentiation

Although 14-day-old mouse embryonic calvarial cells cultured in plastic culture dishes in the presence of 1α,25-dihydroxyvitamin D3[1α,25-(OH)2D3] for 7 days could barely resorb bone slices, the same calvarial cells cultured with an ethylenediaminetetraacetic acid (EDTA) extract from bovine bone powder under the same conditions stimulated pit formation on bone slices in a dose-dependent manner. Therefore, the present study was conducted to purify and characterize this osteoclast maturation-inducing factor(s) from the bone matrix. The protein having osteoclast maturation-inducing activity in the EDTA extract was purified by gel filtration over Superdex 75 preparation grade and chromatography on hydroxyapatite, Mono Q, and C8 reversed-phase HPLC by monitoring the ability of the eluted fractions to elicit pit formation on bone slices. The molecular weight of the purified protein estimated by high-resolution polyacrylamide gel electrophoresis was 5.7 kDa and 6.8 kDa in the respective absence and presence of 2-mercaptoethanol. The sequence of the 30-amino-acid purified protein corresponded to the 7th to 36th residues of bovine osteocalcin. The osteocalcin fragment, missing the initial 6 residues at the N-terminal region, exhibited higher osteoclast maturation-inducing ability than bovine intact osteocalcin on a per weight basis. The osteocalcin fragment had no effect on the expression of receptor activator of nuclear factor (NF)-κB ligand (RANKL) and osteoprotegerin (OPG) genes in calvarial cells, nor did it enhance the bone-resorbing activity of mature osteoclasts. When the osteocalcin fragment was added to late-stage (days 4–7) or to early-stage (days 0–3) cultures of calvarial cells pretreated with 1α,25-(OH)2D3, its stimulatory effect was observed in the late-stage cultures rather than in the early-stage ones. In addition, the osteocalcin fragment directly enhanced the formation of osteoclasts with bone-resorbing ability from Mac-1+ c-Fms+ cells in the presence of macrophage colony-stimulating factor (MCSF) and RANKL. These results suggest that the osteocalcin fragment in bone matrix is involved in osteoclast maturation, especially at a late stage of osteoclast differentiation.

Stimulatory effect on bone resorption of interleukin-1-like cytokine produced by an osteoblast-rich population of mouse calvarial cells

SummaryWe reported previously that an osteoblast-rich population of mouse calvarial cells treated with lipopolysaccharide produced on interleukin-1(IL-1)-like cytokine that closely resembles IL-1. In the present study, we examined whether or not the IL-1-like cytokine stimulates bone resorption. As a result we found that the cytokine stimulate bone resorption significantly, and the stimulatory effect was dependent on cytokine dose and length of culture time. The stimulation of bone resorption and IL-1-like cytokine activity appear to be in parallel. Since the stimulatory effect of the cytokine was not abolished in the presence of indomethacin, the cytokine may stimulate bone resorption in a prostaglandin metabolite-independent manner. These results suggest the possibility that the IL-1-like cytokine may be involved as an autoregulatory factor in the bone-remodeling system.

ERK5 activation is essential for osteoclast differentiation

The MEK/ERK pathways are critical for controlling cell proliferation and differentiation. In this study, we show that the MEK5/ERK5 pathway participates in osteoclast differentiation. ERK5 was activated by M-CSF, which is one of the essential factors in osteoclast differentiation. Inhibition of MEK5 by BIX02189 or inhibition of ERK5 by XMD 8-92 blocked osteoclast differentiation. MEK5 knockdown inhibited osteoclast differentiation. RAW264.7D clone cells, which are monocytic cells, differentiate into osteoclasts after stimulation with sRANKL. ERK5 was activated without any stimulation in these cells. Inhibition of the MEK5/ERK5 pathway by the inhibitors also blocked the differentiation of RAW264.7D cells into osteoclasts. Moreover, expression of the transcription factor c-Fos, which is indispensable for osteoclast differentiation, was inhibited by treatment with MEK5 or ERK5 inhibitors. Therefore, activation of ERK5 is required for the induction of c-Fos. These events were confirmed in experiments using M-CSF-dependent bone marrow macrophages. Taken together, the present results show that activation of the MEK5/ERK5 pathway with M-CSF is required for osteoclast differentiation, which may induce differentiation through the induction of c-Fos.