Shunsuke Furuyama

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.

Transforming growth factor‐β1 regulation of bone sialoprotein gene transcription: Identification of a TGF‐β activation element in the rat BSP gene promoter

Transforming growth factor‐β (TGF‐β) increases steady‐state mRNA levels of several extracellular matrix proteins in mineralized connective tissues. Bone sialoprotein (BSP) is a major constituent of the bone matrix, thought to initiate and regulate the formation of mineral crystals. To determine the molecular pathways of TGF‐β1 regulation of bone proteins, we have analyzed the effects of the TGF‐β1 on the expression of the BSP in the rat osteosarcoma cell line (ROS 17/2.8). TGF‐β1 at 1 ng/ml, increased BSP mRNA levels in ROS 17/2.8 cells ∼8‐fold; the stimulation was first evident at 3 hr, reached maximal levels at 12 hr and slowly declined thereafter. Since the stability of the BSP mRNA was not significantly affected by TGF‐β1, and nuclear “run‐on” transcription analyses revealed only a ∼2‐fold increase in the transcription of the BSP gene, most of the increase in BSP mRNA appeared to involve a nuclear post‐transcriptional mechanism. Moreover, the effects of TGF‐β1 were indirect, since the increase in BSP mRNA was abrogated by cycloheximide (28 μg/ml). To identify the site of transcriptional regulation by TGF‐β1, transient transfection analyses were performed using BSP gene promoter constructs linked to a luciferase reporter gene. Constructs that included nt −801 to −426 of the promoter sequence were found to enhance transcriptional activity ∼1.8‐fold in cells treated with TGF‐β1. Within this sequence, ∼500 nt upstream of the transcription start site, a putative TGF‐β activation element (TAE) was identified that contained the 5′‐portion of the nuclearfactor‐1 (NF‐1) canonical sequence (TTGGC) overlapping a consensus sequence for activator protein‐2 (AP‐2). The functionality of the TAE was shown by an increased binding of a nuclear protein from TGF‐β1 stimulated cells in gel mobility shift assays and from the attenuation of TGF‐β1‐induced luciferase activity when cells were co‐transfected with a double‐stranded TAE oligonucleotide. Competition gel mobility shift analyses revealed that the nuclear protein that binds to the TAE has similar properties to, but is distinct from, NF‐1 nuclear protein. These studies have therefore identified a TGF‐β activation element (TAE) in the rat BSP gene promoter that mediates the stimulatory effects of TGF‐β1 on BSP gene transcription. J. Cell. Biochem. 65:501–512.

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.

Association of FcγRII with Low-Density Detergent-Resistant Membranes Is Important for Cross-Linking-Dependent Initiation of the Tyrosine Phosphorylation Pathway and Superoxide Generation

IgG immune complexes trigger humoral immune responses by cross-linking of FcRs for IgG (FcγRs). In the present study, we investigated role of lipid rafts, glycolipid- and cholesterol-rich membrane microdomains, in the FcγR-mediated responses. In retinoic acid-differentiated HL-60 cells, cross-linking of FcγRs resulted in a marked increase in the tyrosine phosphorylation of FcγRIIa, p58lyn, and p120c-cbl, which was inhibited by a specific inhibitor of Src family protein tyrosine kinases. After cross-linking, FcγRs and tyrosine-phosphorylated proteins including p120c-cbl were found in the low-density detergent-resistant membrane (DRM) fractions isolated by sucrose-density gradient ultracentrifugation. The association of FcγRs as well as p120c-cbl with DRMs did not depend on the tyrosine phosphorylation. When endogenous cholesterol was reduced with methyl-β-cyclodextrin, the cross-linking did not induce the association of FcγRs as well as p120c-cbl with DRMs. In addition, although the physical association between FcγRIIa and p58lyn was not impaired, the cross-linking did not induce the tyrosine phosphorylation. In human neutrophils, superoxide generation induced by opsonized zymosan or chemoattractant fMLP was not affected or increased, respectively, after the methyl-β-cyclodextrin treatment, but the superoxide generation induced by the insoluble immune complex via FcγRII was markedly reduced. Accordingly, we conclude that the cross-linking-dependent association of FcγRII to lipid rafts is important for the activation of FcγRII-associated Src family protein tyrosine kinases to initiate the tyrosine phosphorylation cascade leading to superoxide generation.

Presence of a Complex Containing Vesicle-associated Membrane Protein 2 in Rat Parotid Acinar Cells and Its Disassembly upon Activation of cAMP-dependent Protein Kinase

Amylase release from parotid acinar cells is mainly induced by the accumulation of intracellular cAMP, presumably through the phosphorylation of substrates by cAMP-dependent protein kinase (PKA). However, the molecular mechanisms of this process are not clear. In a previous study (Fujita-Yoshigaki, J., Dohke, Y., Hara-Yokoyama, M., Kamata, Y., Kozaki, S., Furuyama, S., and Sugiya, H. (1996) J. Biol. Chem. 271, 13130–13134), we reported that vesicle-associated membrane protein 2 (VAMP2) is localized at the secretory granule membrane and is involved in cAMP-induced amylase secretion. To study the formation of the solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex containing VAMP2 in parotid acinar cells, we prepared rabbit polyclonal antibody against the peptide corresponding to Arg47-Asp64 of VAMP2 (anti-SER4256). The recognition site of anti-SER4256 overlaps the domain involved in binding target membrane SNAREs (t-SNARES). Then we examined the condition of VAMP2 by immunoprecipitation with anti-SER4256. VAMP2 was not included in the immunoprecipitate from solubilized granule membrane fraction under the control conditions, but incubation with cytosolic fraction and cAMP caused immunoprecipitation of VAMP2. The effect of cytosolic fraction and cAMP was reduced by addition of PKA inhibitor H89. Addition of both the catalytic subunit of PKA and the cytosolic fraction allowed immunoprecipitation of VAMP2, whereas the PKA catalytic subunit alone did not. These results suggest that (1) the t-SNARE binding region of VAMP2 is masked by some protein Xand activation of PKA caused the dissociation of X from VAMP2; and (2) the effect of PKA is not direct phosphorylation ofX, but works through phosphorylation of some other cytosolic protein.

Lipid Peroxidation as a Possible Cause of Benzoyl Peroxide Toxicity in Rabbit Dental Pulp—A Microsomal Lipid Peroxidation in vitro

The toxicity of composite resin on rabbit dental pulp was investigated biochemically. A microsomal fraction of rabbit dental pulp was incubated with each of the components of composite resins, and the formation of peroxide was determined by the thiobarbituric acid reaction. Benzoyl peroxide (BPO), the most widely used catalyst, was the most effective on peroxidation, but monomers were not. Cations such as Cu2+ or Fe2+ were required for acceleration of this reaction. Authentic polyunsaturated fatty acids and phospholipids were extensively converted into their peroxides by BPO, but amino acids and carbohydrates were not. Among the active oxygens, hydroxyl radicals were thought to be responsible for BPO-dependent peroxidation. The results presented in this paper indicate that the lipid portion of the cells may be attacked by hydroxyl radicals produced by BPO and copper or iron. Therefore, BPO is considered to be the major factor responsible for the toxicity of composite resins.

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.

Involvement of aquaporin-5 water channel in osmoregulation in parotid secretory granules.

Aquaporins (AQPs) are a family of channel proteins that allow water or very small solutes to pass, functioning in tissues where the rapid and regulated transport of fluid is necessary, such as the kidney, lung, and salivary glands. Aquaporin-5 (AQP5) has been demonstrated to localize on the luminal surface of the acinar cells of the salivary glands. In this paper, we investigated the expression and function of AQP5 in the secretory granules of the rat parotid gland. AQP5 was detected in the secretory granule membranes by immunoblot analysis. The immunoelectron microscopy experiments confirmed that AQP5 was to be found in the secretory granule membrane. Anti-AQP5 antibody evoked lysis of the secretory granules but anti-aquaporin-1 antibody did not and AQP1 was not detected in the secretory granule membranes by immunoblot analysis. When chloride ions were removed from the solution prepared for suspending secretory granules, the granule lysis induced by anti-AQP5 antibody was inhibited. Furthermore, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, an anion channel blocker, blocked the anti-AQP5 antibody-induced secretory granule lysis. These results suggest that AQP5 is, expressed in the parotid gland secretory granule membrane and is involved in osmoregulation in the secretory granules.

TNF-α suppresses bone sialoprotein (BSP) expression in ROS17/2.8 cells

Tumor necrosis factor‐alpha (TNF‐α) is a major mediator of inflammatory responses in many diseases that inhibits bone formation and stimulates bone resorption. To determine molecular mechanisms involved in the suppression of bone formation we have analyzed the effects of TNF‐α on BSP gene expression. Bone sialoprotein (BSP) is a mineralized tissue‐specific protein that appears to function in the initial mineralization of bone. Previous studies have demonstrated that BSP mRNA expression is essentially restricted to fully‐differentiated cells of mineralized connective tissues and that the expression of BSP is developmentally regulated. Treatment of rat osteosarcoma ROS 17/2.8 cells with TNF‐α (10 ng/ml) for 24 h caused a marked reduction in BSP mRNA levels. The addition of antioxidant N‐acetylcysteine (NAC; 20 mM) 30 min prior to stimulation with TNF‐α attenuated the inhibition of BSP mRNA levels. Transient transfection analyses, using chimeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene, revealed that TNF‐α (10 ng/ml) suppressed expression in all constructs, including a short construct (pLUC3; nts −116 to +60), transfected into ROS17/2.8 cells. Further deletion analysis of the BSP promoter showed that a region within nts −84 to −60 was targeted by TNF‐α, the effects which were inhibited by NAC and the tyrosine kinase inhibitor, herbimycin A (HA). Introduction of 2bp mutations in the inverted CCAAT box (ATTGG; nts −50 and −46), a putative cAMP response element (CRE; nts −75 to −68), and a FGF response element (FRE; nts −92 to −85) showed that the TNF‐α effects were mediated by the CRE. These results were supported by gel mobility shift assays, using a radiolabeled double‐stranded CRE oligonucleotide, which revealed decreased binding of a nuclear protein from TNF‐α‐stimulated ROS 17/2.8 cells. Further, the inhibitory effect of TNF‐α on CRE DNA–protein complex was completely abolished by NAC or HA treatment. These studies, therefore, show that TNF‐α suppresses BSP gene transcription through a tyrosine kinase‐dependent pathway that generates reactive oxygen species and that the TNF‐α effects are mediated by a CRE element in the proximal BSP gene promoter. J. Cell. Biochem. 87: 313–323, 2002.

Histamine H1 receptor-induced Ca2+ mobilization and prostaglandin E2 release in human gingival fibroblasts: Possible role of receptor-operated Ca2+ influx☆

Stimulation of human gingival fibroblasts with histamine elicited an increase in the intracellular concentration of free calcium ([Ca2+]i) and the formation of inositol 1,4,5-trisphosphate (InsP3) in a concentration- and time-dependent manner. The histamine-induced increase in [Ca2+]i was attenuated completely by chlorpheniramine, an H1 antagonist, but not by cimetidine, an H2 antagonist. The histamine-induced Ca2+ response consisted of an initial transient peak response and a subsequent sustained increase. The transient phase can be largely attributed to Ca2+ release from intracellular InsP3-sensitive stores since the increased [Ca2+]i effect of histamine completely disappeared after depletion of intracellular Ca2+ stores with thapsigargin in the absence of extracellular Ca2+. The sustained phase was due to Ca2+ influx which was attenuated in the absence of extracellular Ca2+. The Ca2+ influx required the continuous binding of histamine to the receptor, since chlorpheniramine attenuated the increase in [Ca2+]i observed when extracellular Ca2+ was re-applied to the cells after stimulation with histamine in the absence of extracellular Ca2+. Pretreatment with the Ca2+ channel blocker SK&F96365 inhibited the Ca2+ influx component, suggesting that histamine stimulates Ca2+ influx through an H1 receptor-operated Ca2+ channel. Histamine also evoked a concentration- and time-dependent release of prostaglandin E2 (PGE2). The histamine-evoked PGE2 release was reduced markedly by exclusion of extracellular Ca2+ or pretreatment with SK&F96365 or an H1 antagonist. These results indicate that histamine stimulates both the intracellular Ca2+ release from InsP3-sensitive stores and the H1 receptor-operated Ca2+ influx from extracellular sites. The increased [Ca2+]i due to the Ca2+ influx causes PGE2 release in human gingival fibroblasts.