Shigeo Kitano

NF-κB inhibitors stimulate apoptosis of rabbit mature osteoclasts and inhibit bone resorption by these cells

Interesting, recent studies have suggested a possibility that transcriptional factor NF‐κB may play a functional role in the survival of mouse osteoclasts. However, it has not been known whether NF‐κB is involved in apoptosis of and bone resorption by mature osteoclasts. Thus, using NF‐κB inhibitors, we examined the functional role of NF‐κB in the induction of apoptosis in rabbit mature osteoclasts. PDTC, a potent inhibitor of NF‐κB, stimulated markedly apoptosis of the osteoclasts and inhibited bone resorption by these cells. These effects also was observed when three other inhibitors of NF‐κB were used. And a gel mobility shift assay showed that PDTC also inhibited NF‐κB binding to its consensus sequence in the cells. These results suggest a regulatory role for NF‐κB in apoptosis in and bone resorption by rabbit mature osteoclasts.

Transcriptional regulation by transforming growth factor beta of the expression of retinoic acid and retinoid X receptor genes in osteoblastic cells is mediated through AP-1.

We now report that transforming growth factor β1 (TGF-β1), a potent regulatory cytokine of bone remodeling, is a powerful stimulator for gene expression of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) in osteoblastic MC3T3-E1 cells. TGF-β1 transcriptionally stimulated the expression of RARα, RARγ, and RXRα genes, but did not do so for RARβ, RXRβ, and RXRγ genes. We also observed that AP-1, a transcriptional factor, plays an important role in the signal pathway for expression of RARα, RARγ, and RXRα genes stimulated by TGF-β1 because stimulation of the expression of these genes in the cytokine-treated cells was markedly inhibited by a mixture of antisense c-fos and c-jun. A gel mobility shift assay demonstrated that TGF-β1 is able to increase, in a dose-dependent manner, the binding of nuclear proteins to direct repeat 5, a consensus sequence with high affinity for RAR-RXR heterodimers. The mobility shift assay, using specific antibody for each receptor, showed that direct repeat 5-binding proteins may be RAR and RXR isoforms. The stimulated binding to direct repeat 5 was inhibited strongly by H-7, an inhibitor of serine/threonine kinase, and by curcumin, an inhibitor of AP-1. The present study suggests a novel pathway for TGF-β1 action in osteoblastic cells via stimulation of RAR-RXR transcriptional activity in a ligand-dependent fashion.

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.

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.

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.

Sphingomyelinase and ceramide inhibit formation of F-actin ring in and bone resorption by rabbit mature osteoclasts.

Recent studies have demonstrated that ceramide plays an important role as a second messenger in many kinds of cells. However, it is not known whether apoptosis of and bone resorption by mature osteoclasts are mediated via sphingomyelinase (SMase) and ceramide. Thus, we examined the possible involvement of SMase and ceramide in the induction of apoptosis in and bone resorption by rabbit mature osteoclasts. SMase and C2‐ceramide inhibited strongly F‐actin ring formation of and bone resorption by the osteoclasts. However, the osteoclast apoptosis was not induced by C2‐ceramide. The ceramide inhibition of the bone resorption was suppressed by dl‐threo‐dihydrosphingosine, an inhibitor of sphingosine kinase. In addition, we observed that sphingosine‐1‐phosphate is able to inhibit bone resorption by the osteoclasts. These results suggest an important role of the sphingomyelin pathway in bone resorption by rabbit mature osteoclasts.

Transforming growth factor-β-induced gene expression of monocyte chemoattractant JE in mouse osteoblastic cells, MC3T3-E1

A recent study demonstrated that PDGF-inducible JE is an inflammatory cytokine that directs chemotactic activity of monocytes. Accumulation of monocyte/macrophage lineage cells at site of bone tissue sites is very important for formation of multinucleate osteoclasts, which mediate bone resorption. Since transforming growth factor-beta (TGF-beta) is a potent regulator in bone remodeling, we examined whether TGF-beta induced JE gene expression in mouse osteoblastic cells, MC3T3-E1. TGF-beta induced a maximum JE mRNA expression at 3 hr after initiation of the cytokine treatment. This maximal expression was observed in when TGF-beta was used at a concentration of 1 ng/ml. The chemotactic activity for human monocytes was detected in conditioned medium of TGF-beta-treated cells, and the chemotactic activity was neutralized by anti-JE serum treatment.