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Biomedical subjects

S Hanazawa

Publications and source records attributed to S Hanazawa.

At least 19 recordsLinked to original sources
PubMed2001

A possible mechanism of maxillofacial abscess formation: involvement of Porphyromonas endodontalis lipopolysaccharide via the expression of inflammatory cytokines.

In a previous study, we developed a specific monoclonal antibody against Porphyromonas endodontalis lipopolysaccharide, and demonstrated that this lipopolysaccharide was detected in bacterially infected root canal fluid. We suggest here that P. endodontalis lipopolysaccharide in the infectious materials plays a stimulatory role in maxillofacial abscess formation via the expression of inflammatory cytokines. Our epidemiological study showed that this lipopolysaccharide was detected in significant levels the infectious material of patients with periapical periodontitis and odontogenic abscesses. Interestingly, infectious material-induced expression of tumor necrosis factor-alpha, interleukin-1beta, or neutrophil chemoattractant KC genes in mouse macrophages, was significantly neutralized by monoclonal antibody against the lipopolysaccharide. In addition, we also detected a significant amount of tumor necrosis factor-alpha in the infectious material. These results suggest that P. endodontalis lipopolysaccharide plays an important role in the pathogenic mechanism of maxillofacial abscess formation via the expression of inflammatory cytokines.

Adolescent↗
PubMed2001

Porphyromonas gingivalis fimbriae inhibit caspase-3-mediated apoptosis of monocytic THP-1 cells under growth factor deprivation via extracellular signal-regulated kinase-dependent expression of p21 Cip/WAF1.

Apoptotic regulation of monocytes/macrophages appears to be closely associated with chronic inflammatory reactions. Since it was demonstrated earlier that certain bacterial cell components are involved in apoptotic regulation of these cells, in the present study, we investigated whether the bacterial fimbria, an important cell structure involved in bacterial adherence to host cells, regulates apoptosis of human monocytic THP-1 cells induced under growth factor deprivation. To investigate this point, we used fimbriae of Porphyromonas gingivalis, a pathogen causing periodontal disease, which is a chronic inflammatory disease. The fimbriae inhibited apoptosis of the cells under growth factor deprivation. This inhibitory action of the fimbriae was completely neutralized by anti-fimbrial antibody. The fimbriae stimulated activation of extracellular signal-regulated kinase (ERK) and expression of cyclin-dependent kinase inhibitor p21 Cip/WAF1 (p21) in the cells. The stimulatory effect of the fimbriae on the expression of the p21 protein was inhibited by treatment with PD98059, a specific inhibitor of ERK. The cell apoptosis was inhibited by treatment with Ac-DEVD-CHO, an inhibitor of caspase-3. The fimbriae inhibited the serum withdrawal-induced cleavage of the caspase-3 proform and poly(ADP-ribose) polymerase, one of the caspase-3 substrates. Furthermore, PD98059 and antisense p21 oligonucleotide blocked the fimbrial inhibition of apoptosis and caspase-3 activation of the cells induced by serum withdrawal. These results show that the bacterial fimbriae inhibited apoptosis of THP-1 cells induced under growth factor deprivation via ERK-dependent expression of p21. The present study suggests that bacterial fimbriae act as potent regulators of chronic inflammatory disease, e.g., periodontal disease, through blocking apoptosis of monocytes/macrophages.

Apoptosis↗
PubMed2000

Selective stimulation by ceramide of the expression of the alpha isoform of retinoic acid and retinoid X receptors in osteoblastic cells. A role of sphingosine 1-phosphate-mediated AP-1 in the ligand-dependent transcriptional activity of these receptors.

Recent studies have demonstrated that sphingosine 1-phosphate (SPP) plays a functional role as a signaling molecule in gene expression in several kinds of cells. The present study demonstrates selective expression by ceramide of retinoic acid receptor-alpha (RAR-alpha) and retinoic X receptor-alpha (RXR-alpha) in osteoblastic MC3T3-E1 cells and a functional role of SPP-mediated AP-1 in the signaling mechanism of ligand-dependent transcriptional activity of heterodimers of these receptors in the cells. C(2)- and C(6)-ceramides selectively stimulated the expression of RAR-alpha and RXR-alpha genes, but not that of beta and gamma isoform genes of RAR and RXR, in the cells. The C(2)-ceramide-induced stimulation was clearly inhibited by dl-threo-dihydrosphingosine, an inhibitor of sphingosine kinase. SPP also selectively stimulated the expression of both receptors and increased the specific binding of the nuclear proteins to direct repeat 5 (DR-5), a consensus sequence of RAR-RXR. In addition, SPP markedly stimulated transient chloramphenicol acetyltransferase (CAT) activity of retinoic acid-dependent transcriptional activity in the cells transfected with a DR-5-CAT reporter gene. The SPP stimulation was activation protein-1 (AP-1)-dependent, because the SPP stimulatory action toward these nuclear gene expressions and the transient CAT activity were inhibited by antisense c-fos and c-jun oligonucleotides. We observed that SPP actually stimulated AP-1 transcriptional activity in the cells. This study suggests an important role of SPP-mediated AP-1 in the selective expression of RAR-alpha and RXR-alpha in osteoblastic cells via the sphingosine pathway.

Animals↗
PubMed2000

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.

Animals↗
PubMed2000

Prostaglandin E2 stimulates AP-1-mediated CD14 expression in mouse macrophages via cyclic AMP-dependent protein kinase A.

PGs play a functional role in the early stage of Gram-negative bacterial infections, because this prostanoid is produced rapidly by epithelial cells after a bacterial infection. CD14, one of the LPS receptors, is a key molecule in triggering the response to bacterial LPS in association with a Toll-like molecule. Therefore, in this study, we investigated the effect of PG on CD14 expression in mouse macrophages. PGE1, PGE2, and PGA1 among the PGs tested strongly stimulated the expression of the CD14 gene in the cells. The stimulatory action also was observed by Western blot analysis. cAMP-elevating agents stimulated expression of CD14 gene as well. Protein kinase A inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), but not protein kinase C inhibitor 3-(1-[3-(dimethylamino)propyl]-1H-indol-3-yl)-4-(1H-indol-3-yl)-1H-py rrole-2,5-dione (GF109203X), abolished the stimulated expression of CD14. A run-on assay showed that PGE2 stimulated the CD14 gene expression at the transcriptional level via protein kinase A. PGE2 also stimulated activation of AP-1, a heterodimer of c-Jun and c-Fos, because the prostanoid increased specific binding of nuclear proteins to the AP-1 consensus sequence and stimulated AP-1-promoted luciferase activity. PGE2-stimulated expression of CD14 was inhibited by antisense c-fos and c-jun oligonucleotides, but not by their sense oligonucleotides. Finally, PGE2 pretreatment synergistically stimulated LPS-induced expression of IL-1beta and IL-6 genes in mouse macrophages. Therefore, the present study demonstrates that PGE2 has the ability to stimulate AP-1-mediated expression of CD14 in mouse macrophages via cAMP-dependent protein kinase A.

Animals↗
PubMed2000

Role of transcriptional factor activation protein-1 in endogenous expression of the interleukin-1 beta gene involved in Porphyromonas gingivalis fimbria-stimulated bone resorption in the mouse calvarial system.

We previously suggested that endogenous interleukin-1 beta (IL-1 beta) produced by Porphyromonas gingivalis fimbriae stimulation in calvarial bone cell cultures plays a role in bone resorption as a major cytokine. Therefore, in the present study, we initiated experiments to clarify the stimulatory mechanism of IL-1 beta gene expression in fimbria-stimulated bone resorption. Fimbria-stimulated bone resorption was dramatically inhibited by curcumin, a potent inhibitor of activation protein-1 (AP-1). In fact, the fimbriae induced markedly both the expression of c-fos and c-jun genes and their protein production in the calvarial cells. In addition, a mixture of antisense oligonucleotides against c-fos and c-jun significantly inhibited not only the fimbria-induced expression of the IL-1 beta gene but also the fimbria-induced bone resorption. Therefore, the present study demonstrates that transcriptional factor AP-1 plays a functional role in P. gingivalis fimbria-stimulated bone resorption via endogenous IL-1 beta in the mouse calvarial system.

Animals↗
PubMed2000

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

The septic shock that occurs in gram-negative infections is caused by a cascade of inflammatory cytokines. Several studies showed that transforming growth factor-beta1 (TGF-beta1) inhibits this septic shock through suppression of expression of the lipopolysaccharide (LPS)-induced inflammatory cytokines. In this study, we investigated whether TGF-beta1 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-beta1 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-fos and c-jun oligonucleotides. Since TGF-beta1 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-beta1 inhibition of CD14 expression may be a consequence of downregulation of AP-1. LPS-stimulated expression of interleukin-1beta and tumor necrosis factor alpha genes in the cells was inhibited by addition of CD14 antisense oligonucleotide. Also, TGF-beta1 inhibited the LPS-stimulated production of both inflammatory cytokines by the macrophages. In addition, TGF-beta1 inhibited expression of the two cytokines in several organs of mice receiving LPS. Thus, our results suggest that TGF-beta1 inhibition of LPS-stimulated inflammatory responses resulted from downregulation of CD14 and also may be a possible mechanism of TGF-beta1 inhibition of LPS-induced septic shock.

Animals↗
PubMed1999

TGF-beta inhibits lipopolysaccharide-stimulated activity of c-Jun N-terminal kinase in mouse macrophages.

Transforming growth factor-beta (TGF-beta) is a potent anti-inflammatory cytokine. Although this cytokine inhibits lipopolysaccharide (LPS)-mediated septic shock, the molecular mechanism of TGF-beta is not well known. Since recent studies showed that c-Jun N-terminal kinase (JNK), one of the mitogen-activated protein kinases, plays an important role in LPS signalling, we focused here on the inhibitory action of TGF-beta1 on LPS-stimulated JNK activity in mouse macrophages. TGF-beta1 inhibited LPS-stimulation of phosphorylated JNK1 and JNK2 and consequently of JNK activity in the cells. This JNK activity resulted in a decreased level of phosphorylated c-Jun protein. Using Western blotting, we also observed TGF-beta1 inhibition of newly synthesized c-Jun protein in LPS-stimulated cells. These results demonstrate that TGF-beta1 inhibits LPS-stimulated JNK activity in mouse macrophages. Also, our present study suggests a possible inhibitory mechanism of TGF-beta in signalling of LPS-induced inflammatory responses.

Animals↗
PubMed1999

CpG motifs in Porphyromonas gingivalis DNA stimulate interleukin-6 expression in human gingival fibroblasts.

We suggest here that Porphyromonas gingivalis DNA may function as a virulence factor in periodontal disease through expression of inflammatory cytokine. The bacterial DNA markedly stimulated in a dose-dependent manner interleukin-6 (IL-6) production by human gingival fibroblasts. The stimulatory action was eliminated by treatment with DNase but not RNase. The stimulatory effect was not observed in the fibroblasts treated with eucaryotic DNAs. The bacterial DNA also stimulated in dose- and treatment time-dependent manners the expression of the IL-6 gene in the cells. In addition, the stimulatory effect was eliminated when the DNA was methylated with CpG motif methylase. Interestingly, a 30-base synthetic oligonucleotide containing the palindromic motif GACGTC could stimulate expression of the IL-6 gene and production of its protein in the cells. Furthermore, the synthetic oligonucleotide-induced expression of this cytokine gene was blocked by pyrrolidine dithiocarbamate and N-acetyl-L-cystine, potent inhibitors of transcriptional factor NF-kappaB. Gel mobility shift assay showed increased binding of NF-kappaB to its consensus sequence in the synthetic oligonucleotide-treated cells. Also, using specific antibody against p50 and p65, which compose NF-kappaB, we showed the consensus sequence-binding proteins to be NF-kappaB. These results are the first to demonstrate that the internal CpG motifs in P. gingivalis DNA stimulate IL-6 expression in human gingival fibroblasts via stimulation of NF-kappaB.

Acetylcysteine↗
PubMed1999

Intracellular interleukin-1 alpha production in human gingival fibroblasts is differentially regulated by various cytokines.

Interleukin-1 (IL-1) may play a critical role in immune and inflammatory responses in inflamed gingiva, and it is synthesized by a wide variety of host cells. In this study, we examined the regulatory effects of various cytokines on bioactive membrane IL-1 and intracellular IL-1 alpha production in cultured human gingival fibroblasts (HGF). Recombinant human (rh) IL-1 beta stimulated membrane IL-1 activity, which was mainly attributed to IL-1 alpha. rhIL-1 beta and rh tumor necrosis factor (TNF)-alpha stimulated HGF to produce intracellular IL-1 alpha, whereas rh interleukin-6 (IL-6), rh interleukin-4 (IL-4), and rh interferon (IFN)-gamma did not do so. Intracellular IL-1 alpha production induced by rhIL-1 beta or rhTNF-alpha may be partially related to protein kinase C (PKC) activation, because rhIL-1 beta or rhTNF-alpha-induced intracellular IL-1 alpha production was stimulated by pre-treatment with 12-o-tetradecanoylphorbol 13-acetate (TPA), a PKC activator, but was suppressed by the pre-treatment with 1-(5-isoquinoline-sulfonyl) -2-methylpiperazine dihydrochloride (H-7), which is a PKC inhibitor. rhIL-4 inhibited rhIL-1 beta- or rhTNF-alpha-induced intracellular IL-1 alpha production, but rhIL-6 had no effect on this production. Pre-treatment with rh IFN-gamma remarkably enhanced intracellular IL-1 alpha production induced by subsequent treatment with rhIL-1 beta or rhTNF-alpha. Simultaneous treatment with rhIFN-gamma and rhIL-1 beta inhibited rhIL-1 beta-induced intracellular IL-1 alpha production, but co-treatment with rhIFN-gamma and rhTNF-alpha enhanced rhTNF-alpha-induced intracellular IL-1 alpha production. These results suggest that in inflamed gingiva, pro-inflammatory cytokines such as IL-1 beta and TNF-alpha may induce bioactive intracellular IL-1 alpha production in human gingival fibroblasts and that this production can be differentially modulated by T-cell-derived cytokines such as IFN-gamma or IL4.

Adult↗
PubMed1998

1alpha,25-dehydroxyvitamin D3 synergism toward transforming growth factor-beta1-induced AP-1 transcriptional activity in mouse osteoblastic cells via its nuclear receptor.

The present study demonstrates 1alpha,25-dehydroxyvitamin D3 (1alpha-25-(OH)2D3) synergism toward transforming growth factor (TGF)-beta1-induced activation protein-1 (AP-1) activity in mouse osteoblastic MC3T3-E1 cells via the nuclear receptor of the vitamin. 1alpha-25-(OH)2D3 synergistically stimulated TGF-beta1-induced expression of the c-jun gene in the cells but not that of the c-fos gene. We actually showed by a gel mobility shift assay 1alpha-25-(OH)2D3 synergism of TGF-beta1-induced AP-1 binding to the 12-(O-tetradecanoylphorbol-13-acetate response element (TRE). 1alpha-25-(OH)2D3 markedly stimulated the transient activity of TGF-beta1-induced AP-1 in the cells transfected with a TRE-chloramphenicol acetyltransferase (CAT) reporter gene. Also, a synergistic increase in TGF-beta1-induced CAT activity was observed in the cells cotransfected with an expression vector encoding vitamin D3 receptor (VDR) and the reporter gene. However, the synergistic CAT activity was inhibited by pretreatment with VDR antisense oligonucleotides. In addition, in a Northern blot assay, we observed 1alpha-25-(OH)2D3 synergism of TGF-beta1-induced expression of the c-jun gene in the cells transfected with the VDR expression vector and also found that the synergistic action was clearly blocked by VDR antisense oligonucleotide pretreatment. The present study strongly suggests a novel positive regulation by 1alpha-25-(OH)2D3 of TGF-beta1-induced AP-1 activity in osteoblasts via "genomic action."

Animals↗
PubMed1998

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-threodihydrosphingosine, 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.

Actins↗
PubMed1998

Internalization of Mycobacterium bovis Bacillus Calmette-Guérin into osteoblast-like MC3T3-E1 cells and bone resorptive responses of the cells against the infection.

Mycobacterium bovis BCG (BCG) is a live vaccine used worldwide against tuberculosis. However, it has unfavourable side effects such as osteitis or osteomyelitis, and these sometimes lead to vertebral caries in some patients as a result of bone resorption. Osteoblasts might play a role in the bone resorption caused by BCG infection, because they are central cells in bone metabolism. Cultured osteoblast-like cell lines (MC3T3-E1) derived from C57BL mice susceptible to BCG infection cells were infected with BCG at several doses. Interestingly, internalization of BCG-enveloped phagosome-like membrane in osteoblast-like cells were observed by transmission electron microscopy (TEM). Owing to infection, the proliferation and alkaline phosphatase activity of the osteoblast-like cells were reduced in a dose-dependent manner. On the other hand, interleukin (IL)-6 production was considerably enhanced by infection. These results suggest that BCG infects osteoblasts, suppressing their proliferation and differentiation and inducing bone resorption, which may be related to osteitis/osteomyelitis and bone caries caused by BCG infection.

Animals↗
PubMed1998

Fibronectin in saliva inhibits Porphyromonas gingivalis fimbria-induced expression of inflammatory cytokine gene in mouse macrophages.

The aim of this study was to examine whether fibronectin in saliva plays a regulatory role in Porphyromonas gingivalis fimbria-mediated pathogenesis in adult periodontal disease. Our previous study demonstrated that fibronectin is one of the binding proteins of P. gingivalis fimbrillin. In the present study, we observed that fibronectin in saliva binds specifically to the fimbrillin. The fibronectin content in saliva of adult periodontal patients was significantly lower than that of healthy subject. In addition, the inhibitory action of salivary fibronectin from adult periodontal patients toward P. gingivalis fimbria-induced expression of the neutrophil chemoattractant KC gene in mouse macrophages was clearly weak compared with that of the fibronectin from healthy subjects. These results suggest that fibronectin in saliva plays an important role as a regulator in the pathogenesis of P. gingivalis fimbriae in adult periodontal disease.

Adult↗
PubMed1998

Porphyromonas gingivalis fimbriae use beta2 integrin (CD11/CD18) on mouse peritoneal macrophages as a cellular receptor, and the CD18 beta chain plays a functional role in fimbrial signaling.

In this study, we demonstrate that Porphyromonas gingivalis fimbriae use molecules of beta2 integrin (CD11/CD18) on mouse peritoneal macrophages as cellular receptors and also show that the beta chain (CD18) may play a functional role in signalling for the fimbria-induced expression of interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) genes in the cells. Using a binding assay with 125I-labeled fimbriae, we observed that fimbrial binding to the macrophages was inhibited by treatment with CD11a, CD11b, CD11c, or CD18 antibody but not by that with CD29 antibody. Western blot assays showed that the fimbriae bound to molecules of beta2 integrin (CD11/CD18) on the macrophages. Furthermore, Northern blot analyses showed that the fimbria-induced expression of IL-1beta and TNF-alpha genes in the cells was inhibited strongly by CD18 antibody treatment and slightly by CD11a, CD11b, or CD11c antibody treatment. Interestingly, intracellular adhesion molecule 1 (ICAM-1), a ligand of CD11/CD18, inhibited fimbrial binding to the cells in a dose-dependent manner. In addition, ICAM-1 clearly inhibited the fimbria-induced expression of IL-1beta and TNF-alpha genes in the cells. However, such inhibitory action was not observed with laminin treatment. These results suggest the importance of beta2 integrin (CD11/CD18) as a cellular receptor of P. gingivalis fimbriae in the initiation stage of the pathogenic mechanism of the organism in periodontal disease.

Animals↗
PubMed1997

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

Animals↗