Shigemasa Hanazawa

Adiponectin inhibits Toll‐like receptor family‐induced signaling

Recent studies have shown that adiponectin, an adipocyte‐derived cytokine, acts as a potent inhibitor of inflammatory responses. It has been also demonstrated that bacterial and viral signalings in host cells are triggered via Toll‐like receptor (TLR) molecules. Therefore, in the present study, we investigated whether globular adiponectin (gAd) would be able to inhibit TLR‐mediated nuclear factor‐κB (NF‐κB) signaling in mouse macrophages (RAW264). gAd predominantly bound to the AdipoR1 receptor and suppressed TLR‐mediated NF‐κB signaling. gAd‐mediated inhibition of TLR‐mediated IκB phosphorylation and NF‐κB activation was eliminated by the pretreatment of cycloheximide. Also their inhibitions of gAd were blocked by preincubation of the cells with an antibody against AdipoR1, but not with an antibody against AdipoR2. Taken together, these findings indicate that adiponectin negatively regulates macrophage‐like cell response to TLR ligands via an unknown endogenous product(s).

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.

Adiponectin inhibits induction of TNF-α/RANKL-stimulated NFATc1 via the AMPK signaling

We investigated here whether adiponectin can exhibit an inhibitory effect on tumor necrosis factor‐alpha (TNF‐α)‐ and receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis by using RAW264 cell D clone with a high efficiency to form osteoclasts. Globular adiponectin (gAd) strongly inhibited TNF‐α/RANKL‐induced differentiation of osteoclasts by interfering with TNF receptor‐associated factor 6 production and calcium signaling; consequently, the induction of nuclear factor of activated T cells c1 (NFATc1) was strongly inhibited. Moreover, we observed that inhibition of AMP‐activated protein kinase abrogated gAd inhibition for TNF‐α/RANKL‐induced NFATc1 expression. Our data suggest that adiponectin acts as a potent regulator of bone resorption observed in diseases associated with cytokine activation.

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.

Transforming growth factor-beta inhibits lipopolysaccharide-stimulated expression of inflammatory cytokines in mouse macrophages through downregulation of activation protein 1 and CD14 receptor expression.

ABSTRACT The septic shock that occurs in gram-negative infections is caused by a cascade of inflammatory cytokines. Several studies showed that transforming growth factor-β1 (TGF-β1) inhibits this septic shock through suppression of expression of the lipopolysaccharide (LPS)-induced inflammatory cytokines. In this study, we investigated whether TGF-β1 inhibition of LPS-induced expression of inflammatory cytokines in the septic shock results from downregulation of LPS-stimulated expression of CD14, an LPS receptor. TGF-β1 markedly inhibited LPS stimulation of CD14 mRNA and protein levels in mouse macrophages. LPS-stimulated expression of CD14 was dramatically inhibited by addition of antisense, but not sense, c-fosand c-jun oligonucleotides. Since TGF-β1 pretreatment inhibited LPS-stimulated expression of c-fos and c-jun genes and also the binding of nuclear proteins to the consensus sequence of the binding site for activation protein 1 (AP-1), a heterodimer of c-Fos and c-Jun, in the cells, TGF-β1 inhibition of CD14 expression may be a consequence of downregulation of AP-1. LPS-stimulated expression of interleukin-1β and tumor necrosis factor alpha genes in the cells was inhibited by addition of CD14 antisense oligonucleotide. Also, TGF-β1 inhibited the LPS-stimulated production of both inflammatory cytokines by the macrophages. In addition, TGF-β1 inhibited expression of the two cytokines in several organs of mice receiving LPS. Thus, our results suggest that TGF-β1 inhibition of LPS-stimulated inflammatory responses resulted from downregulation of CD14 and also may be a possible mechanism of TGF-β1 inhibition of LPS-induced septic shock.

Preventive effect of bis-eugenol, a eugenol ortho dimer, on lipopolysaccharide-stimulated nuclear factor kappa B activation and inflammatory cytokine expression in macrophages

Eugenol exhibits antioxidant and anti-inflammatory activities, but at higher concentrations acts as an oxidant and potent allergen. It was earlier shown that bis-eugenol synthesized by the oxidation of eugenol was less cytotoxic and more highly antioxidative than eugenol. But its anti-inflammatory mechanism remains yet unclear. Since nuclear factor-kappa B (NF-kappa B) is a key transcriptional factor in the expression of inflammatory cytokines, we examined whether eugenol and bis-eugenol are inhibitors of NF-kappa B activation. We observed that bis-eugenol, but not eugenol, clearly inhibited the degradation of inhibitory kappa B-alpha in RAW264.7 murine macrophages stimulated with lipopolysaccharide and, consequently, the transcriptional activity of the stimulated NF-kappa B in the cells. In addition, bis-eugenol actually inhibited LPS-stimulated expression of inflammatory cytokines at both gene and protein levels. These findings suggest that bis-eugenol acts as a potent inhibitor of NF-kappa B.

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.