Sumi Nakao

Tumor necrosis factor alpha (TNF-alpha)-induced prostaglandin E2 release is mediated by the activation of cyclooxygenase-2 (COX-2) transcription via NFkappaB in human gingival fibroblasts.

Nuclear factor kappaB (NFκB) is a transcription factor and plays a key role in the expression of several genes involved in the inflammatory process. Cyclooxygenase (COX) is the key regulatory enzyme of the prostaglandin/eicosanoid synthetic pathway. COX-2 is a highly inducible enzyme by proinflammatory cytokines, of which gene expression is regulated by NFκB. TNF-α is a pro-inflammatory cytokine. In this paper, we investigated the involvement of NFκB on TNF-α-mediated prostaglandin E2 (PGE2) release and COX-2 gene expression in human gingival fibroblasts (HGF). TNF-α- induced PGE2 release and COX-2 mRNA accumulation in a time- and concentration-dependent manner in HGF. The results of transient transfection assays using a chimeric construct of the human COX-2 promoter (nts –1432 ∼ +59) ligated to a luciferase reporter gene indicated that TNF-α stimulated the transcriptional activity ∼ 1.4-fold. Gel mobility shift assays with a radiolabelled COX-2-NFκB oligonucleotide (nts –223 to –214) revealed an increase in the binding of nuclear proteins from TNF-α-stimulated HGF. The COX-2-NFκB DNA-protein complex disappeared after treatment with pyrrolidine dithiocarbamate (PDTC; an antioxidant) or herbimycin A (a tyrosine kinase inhibitor). PDTC and herbimycin A attenuated TNF-α-stimulated PGE2 release. These results suggest that NFκB transcription factor is a key regulator of COX-2 expression in TNF-α-induced PGE2 production, which is mediated through a tyrosine kinase pathway in HGF.

Effect of calcium-channel blockers on cell proliferation, DNA synthesis and collagen synthesis of cultured gingival fibroblasts derived from human nifedipine responders and non-responders.

Human gingival fibroblasts from six patients who developed gingival hyperplasia as a result of nifedipine medication and five who did not were studied for the effects of calcium-channel blockers (nifedipine, diltiazem, verapamil and nicardipine) on cell proliferation, DNA synthesis and collagen synthesis. Phenytoin was used as a positive control. The fibroblasts from reactive patients gave trends toward better cell proliferation rates, DNA synthesis ([3H]-thymidine incorporation), and collagen synthesis ([3H]-proline incorporation) than those from non-reactive patients in the presence of 1 microM of calcium-channel blockers or phenytoin.

Activation of NFκB is necessary for IL-1β-induced cyclooxygenase-2 (COX-2) expression in human gingival fibroblasts

The immediate-early cyclooxygenase-2 (COX-2) gene encodes an inducible prostaglandin synthase enzyme which is implicated in inflammatory and proliferative diseases. COX-2 is highly induced during cell activation by various factors, including mitogens, hormones and cytokines. Since pro-inflammatory cytokine IL-1β has been shown to induce prostaglandin E2 (PGE2) release in human gingival fibroblasts (HGF), here we analyzed the effect of IL-1β on the expression of COX-2 and the activation of NFκB in HGF. Northern hybridization analysis revealed that IL-1β (200 pg/ml) increased the expression of COX-2 mRNA in HGF. The effect of IL-1β was abrogated by herbimycin A, a protein tyrosine kinase inhibitor, and enhanced by orthovanadate, a protein tyrosine phosphatase inhibitor. IL-1β-induced PGE2 release was blocked by the tyrosine kinase inhibitor and increased by the tyrosine phosphatase inhibitor. The results of transient transfection assays using chimeric constructs of the human COX-2 promoter (nt -1432 ~ +59) ligated to a luciferase reporter gene indicated that IL-1β stimulated the transcriptional activity ~ 1.5-fold. Gel mobility shift assays with a radiolabelled COX-2-NFκB oligonucleotide (nts -223 to -214) revealed an increase in the binding of nuclear proteins from IL-1β-stimulated HGF. This increase of DNA-protein complex formation induced by IL-1β was blocked by herbimycin A and another tyrosine kinase inhibitor, genistein. These results suggest that NFκB is an important transcription factor for IL-1β-induced COX-2 gene expression, and is involved in inducing COX-2 gene transcription through tyrosine phosphorylation in HGF.

PROPIONIC ACID STIMULATES SUPEROXIDE GENERATION IN HUMAN NEUTROPHILS

Short‐chain carboxylic acids are the metabolic by‐products of pathogenic anaerobic bacteria and are found at sites of infection in millimolar quantities. We previously reported that propionic acid, one of the short‐chain carboxylic acids, induces an increase in intracellular Ca2+([Ca2+]i) in human neutrophils. Here we investigate the effect of propionic acid on superoxide generation in human neutrophils. Propionic acid (10mm) induced inositol 1,4,5‐trisphosphate (IP3) formation and a rapidly transient increase in [Ca2+]i, but not superoxide generation, whereas 1μm formylmethionyl‐leucyl‐phenylalanine (fMLP), a widely used neutrophil‐stimulating bacterial peptide, stimulated not only IP3formation and Ca2+mobilization but also superoxide generation. The IP3level induced by propionic acid was slightly lower than that induced by fMLP. The transient increase in [Ca2+]iinduced by propionic acid immediately returned to the basal level, whereas a sustained increase in [Ca2+]i, which was higher than the basal level, following a transient increase in [Ca2+]iwas induced by fMLP. The peak level induced by propionic acid was lower than that with fMLP. In the absence of extracellular Ca2+, thapsigargin, a potent inhibitor of endoplasmic reticulum Ca2+‐ATPase, induced an increase in [Ca2+]ieven after propionic acid stimulation, but not after fMLP. The Ca2+ionophore A23187 and thapsigargin induced superoxide generation by themselves. Propionic acid enhanced the superoxide generating effect of A23187 and thapsigargin. These results suggest that Ca2+mobilization induced by propionic acid is much weaker than that with fMLP, and propionic acid is able to generate superoxide in the presence of a Ca2+ionophore and a Ca2+influx activator.

Nicotine stimulates the expression of cyclooxygenase-2 mRNA via NFκB activation in human gingival fibroblasts

Nicotine, a major component in tobacco smoke, stimulates the synthesis of prostaglandin E(2). We investigated the involvement of the transcription nuclear factor kappa B (NFkappaB) in the nicotine-induced expression of cyclooxygenase-2 (COX-2), a key enzyme for prostaglandin synthesis, in human gingival fibroblasts. Nicotine-stimulated release of prostaglandin E(2) and expression of COX-2 mRNA in a time- and dose-dependent manner. The nicotine-stimulated release of prostaglandin E(2) and expression of COX-2 mRNA and protein were inhibited by an NFkappaB inhibitor, pyrrolidine dithiocarbamate (PDTC), by approximately 50%. Nicotine stimulation of IkappaBalpha, an inhibitor of NFkappaB degradation, was also characterized by Western blotting. Mecamylamine, a specific antagonist of the nicotinic acetylcholine receptor, failed to inhibit the effect of nicotine on prostaglandin E(2) release. When human gingival fibroblasts were incubated with [3H]-nicotine, uptake of nicotine was observed. These results suggest that nicotine is taken up by human gingival fibroblasts and that it then stimulates COX-2 expression via the activation of NFkappaB and the subsequent release of prostaglandin E(2).

Platelet‐derived growth factor‐induced arachidonic acid release for enhancement of prostaglandin E2 synthesis in human gingival fibroblasts pretreated with interleukin‐1β

Platelet‐derived growth factor (PDGF) is a biological mediator for connective tissue cells and plays a critical role in a wide variety of physiological and pathological processes. We here investigated the effect of PDGF on arachidonic acid release and prostaglandin E2 (PGE2) synthesis in human gingival fibroblasts (HGF). PDGF induced arachidonic acid release in a time‐ and dose‐dependent manner, and simultaneously induced a transient increase in intracellular Ca2+ concentration ([Ca2+]i), but less provoked PGE2 release and cyclooxygenase‐2 (COX‐2) mRNA expression. When [Ca2+]i was increased by Ca2+‐mobilizaing reagents, arachidonic acid release was increased. The PDGF‐induced arachidonic acid release and increase in [Ca2+]i were prevented by a tyrosine kinase inhibitor. On the other hand, in the HGF pre‐stimulated with interleukin‐1β (IL‐1β), PDGF clearly increased PGE2 release. The PDGF‐induced PGE2 release was inhibited by a tyrosine kinase inhibitor. In the HGF pretreated with IL‐1β, arachidonic acid strongly enhanced PGE2 release and COX‐2 mRNA expression. These results suggest that PDGF stimulates arachidonic acid release by the increase in [Ca2+]i via tyrosine kinase activation, and which contributes to PGE2 production via COX‐2 expression in HGF primed with IL‐1β.

Plasmin is involved in inflammation via protease-activated receptor-1 activation in human dental pulp.

Plasmin is a proteolytic enzyme produced from plasminogen by plasminogen activators. We investigated the function of plasmin in human dental pulp fibroblast-like cells. Plasmin induced an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in a concentration-dependent manner. Expression of mRNA for protease-activated receptor-1 (PAR-1) was detected, and the PAR-1 activating peptide SFLLRN induced an increase in [Ca(2+)](i) in the cells. The plasmin-induced increase in [Ca(2+)](i) was inhibited in the presence of the PAR-1 antagonist SCH79797. Plasmin stimulated the expression of interleukin-8 (IL-8) mRNA and prostaglandin E(2) release, which are involved in inflammation. These effects of plasmin on expression of IL-8 mRNA and prostaglandin E(2) release were inhibited in the presence of the PAR-1 antagonist SCH79797. These results suggest that plasmin activates PAR-1 and is involved in inflammation in human dental pulp.

Bradykinin regulates the histamine-induced Ca2+ mobilization via protein kinase C activation in human gingival fibroblasts.

We previously demonstrated that histamine and bradykinin evoke an increase in intracellular Ca2+ ([Ca2+]i) in human gingival fibroblasts by using a fluorescent Ca2+ indicator Fura-2. In this paper, we further demonstrate the regulation of the histamine-induced Ca2+ mobilization by bradykinin. In fibroblasts stimulated with bradykinin (1 microM), subsequent stimulation with histamine (100 microM) failed to mobilize Ca2+, whereas bradykinin induced an increase in [Ca2+]i in the cells pre-stimulated with histamine. The attenuation of the histamine response was dependent on the concentration of bradykinin for the first stimulation. Histamine also failed to induce the formation of inositol 1,4,5-trisphosphate in fibroblasts pretreated with bradykinin. In fibroblasts pretreated with bradykinin (1 microM) for 3 min and then washed with fresh medium, the effect of histamine on [Ca2+]i quickly returned to the control level. The activation of protein kinase C by phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (PMA) elicited a marked decrease in histamine-induced Ca2+ mobilization. When the protein kinase C activity was inhibited with H7, a protein kinase C inhibitor, or was down-regulated by pretreatment with PMA for 20 h, the inhibitory effect of PMA on the histamine response was relieved. In the fibroblasts pretreated with H7 or PMA for 20 h, histamine evoked Ca2+ mobilization even after bradykinin stimulation. These results suggest that the histamine response is regulated by bradykinin receptor activation via the activation of protein kinase C in human gingival fibroblasts.

Effect of organic germanium compound (Ge-132) on experimental osteoporosis in rats

1. The therapeutic effect of organic germanium compound, 2-carboxyethylgermaniumsesquioxide (Ge-132), for experimental osteoporosis was studied using ovariectomized rats maintained on a low calcium containing diet. 2. Serum calcitonin (sCT) level was decreased and serum parathyroid hormone (sPTH) level was increased by ovariectomy and the decrement and increment rates, respectively, were reduced by administration of Ge-132. Thus, the sCT/sPTH ratio was greater in the groups given Ge-132, indicating that the resorption was somehow inhibited by Ge-132. 3. The transverse strength of femur bone was significantly enhanced by Ge-132. 4. A trend was found in the group given Ge-132 for a larger femur cortical bone index. 5. The relative femur bone wet weight was greater in the group given Ge-132. 6. These results indicate that Ge-132 prevents decreased bone strength, and affects the femur cortical bone index, and bone mineral mass caused by osteoporosis.

Bradykinin potentiates prostaglandin E2 release in the human gingival fibroblasts pretreated with interleukin-1β via Ca2+ mobilization

Interleukin-1beta, a proinflammatory cytokine, causes a slow increase in prostaglandin E(2) release. On the other hand, bradykinin, a chemical mediator for inflammation, induces a rapid prostaglandin E(2) release. Simultaneous stimulation with interleukin-1beta (200 pg/ml) and bradykinin (1 microM) evoked a moderately synergistic increase in prostaglandin E(2) release in human gingival fibroblasts. However, in the human gingival fibroblasts pretreated with interleukin-1beta, bradykinin drastically enhanced prostaglandin E(2) release. NS-398, a specific inhibitor of cyclooxygenase-2, inhibited not only interleukin-1beta-induced prostaglandin E(2) release but also bradykinin-induced prostaglandin E(2) release in the human gingival fibroblasts pretreated with interleukin-1beta. Transcriptional and translational inhibitors such as actinomycin D, cycloheximide, and dexamethasone also suppressed the interleukin-1beta-induced prostaglandin E(2) release and the bradykinin-induced prostaglandin E(2) release in interleukin-1beta-pretreated human gingival fibroblasts. In the fibroblasts pretreated with interleukin-1beta, Ca(2+)-mobilizing reagents such as ionomycin and thapsigargin mimicked the potentiating effect of bradykinin on prostaglandin E(2) release. These results suggest that interleukin-1beta- and bradykinin-induced prostaglandin E(2) release is dependent on cyclooxygenase-2 and the potentiated effect of bradykinin in the human gingival fibroblasts primed with interleukin-1beta is caused by Ca(2+) mobilization.