Emi Shimizu

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.

Histologic observation of a human immature permanent tooth with irreversible pulpitis after revascularization/regeneration procedure.

INTRODUCTION Histological studies of immature human permanent necrotic teeth with or without apical periodontitis after revascularization have not been reported. This case report describes the histological findings of tissue formed in the canal space of an immature permanent tooth #9 with irreversible pulpitis without apical periodontitis after revascularization. METHODS An immature human permanent tooth #9 was fractured 3.5 weeks after revascularization and could not be retained. The tooth was extracted and prepared for routine histological and immunohistochemical evaluation in order to examine the nature of tissue formed in the root canal following the revascularization procedure. RESULTS At 3.5 weeks after revascularization, more than one half of the canal was filled with loose connective tissue similar to the pulp tissue. A layer of flattened odontoblast-like cells lined along the predentin. Layers of epithelial-like cells, similar to the Hertwigs epithelial root sheath, surrounded the root apex. No hard tissue was formed in the canal. CONCLUSIONS Based on the histological findings in the present case, regeneration of pulp-like tissue is possible after revascularization. In this case, both the apical papilla and the Hertwigs epithelial root sheath survived in an immature permanent tooth despite irreversible pulpitis but without apical periodontitis.

EGFR signaling suppresses osteoblast differentiation and inhibits expression of master osteoblastic transcription factors Runx2 and Osterix.

The epidermal growth factor receptor (EGFR) and its ligands regulate key processes of cell biology, such as proliferation, survival, differentiation, migration, and tumorigenesis. We previously showed that, EGFR signaling pathway is an important bone regulator and it primarily plays an anabolic role in bone metabolism. In this study, we demonstrated that EGF‐like ligands strongly inhibited osteoblast differentiation and mineralization in several lines of osteoblastic cells. Real‐time RT‐PCR and promoter reporter assays revealed that EGF‐like ligands suppressed the expression of both early and late bone marker genes at the transcriptional level in the differentiating osteoblasts via an EGFR‐dependent manner. This inhibitory effect of EGFR signaling was not dependent on its mitogenic activity. Furthermore, we demonstrated that EGFR signaling reduced the expression of two major osteoblastic transcription factors Runx2 (type II) and Osterix in osteoblast differentiating cells. EGFR‐induced decrease in Runx2 transcriptional activity was confirmed by Runx2 reporter and chromatin immunoprecipitation assays. EGFR signaling increased the protein amounts of transcription co‐repressors HDAC4 and 6 and over‐expression of HDAC4 decreased Runx2 amount in differentiating osteoblasts, implying that HDACs contribute to the down‐regulation of Runx2 by EGFR. Moreover, activation of EGFR in undifferentiated osteoprogenitors attenuated the expression of early bone markers and Osterix and decreased Runx2 protein amounts. Together with our previous data, that EGFR stimulates osteoprogenitor proliferation and that blocking EGFR activity in osteoblast lineage cells results in fewer osteoprogenitors and an osteopenic phenotype, we conclude that EGFR signaling is important for maintaining osteoprogenitor population at an undifferentiated stage. J. Cell. Biochem. 112: 1749–1760, 2011.

HDAC4 Represses Matrix Metalloproteinase-13 Transcription in Osteoblastic Cells, and Parathyroid Hormone Controls This Repression

Parathyroid hormone (PTH) is a hormone regulating bone remodeling through its actions on both bone formation and bone resorption. Previously we reported that PTH induces matrix metalloproteinase-13 (MMP-13) transcription in osteoblastic cells. Here, we show that histone deacetylase 4 (HDAC4) interacts with Runx2, binds the MMP-13 promoter, and suppresses MMP-13 gene transcription in the rat osteoblastic cell line, UMR 106-01. PTH induces the rapid cAMP-dependent protein kinase-dependent release of HDAC4 from the MMP-13 promoter and subsequent transcription of MMP-13. Knock-out of HDAC4 either by siRNA in vitro or by gene deletion in vivo leads to an increase in MMP-13 expression, and overexpression of HDAC4 decreases the PTH induction of MMP-13. All of these observations indicate that HDAC4 represses MMP-13 gene transcription in bone. Moreover, PTH stimulates HDAC4 gene expression and enzymatic activity at times corresponding to the reassociation of HDAC4 with the MMP-13 promoter and a decline in its transcription. Thus, HDAC4 is a basal repressor of MMP-13 transcription, and PTH regulates HDAC4 to control MMP-13 promoter activity. These data identify a novel and discrete mechanism of regulating HDAC4 levels and, subsequently, gene expression.

Histologic and Histobacteriologic Observations of Failed Revascularization/Revitalization Therapy: A Case Report

INTRODUCTION Mechanical debridement plays an important role in eliminating intracanal bacteria, such as biofilm on the canal walls and bacteria in the dentinal tubules. Mechanical debridement is not recommended for root canal disinfection in revascularization/revitalization therapy. Here we report a failed revascularization/revitalization case, which could be due to inadequate root canal disinfection without mechanical removal of biofilm and bacteria in dentinal tubules. METHODS A 6-year-old boy had a traumatic injury to tooth #9, which was avulsed and replanted within 40 minutes. The tooth subsequently developed a local swelling in the periapical area. The patient was referred to the Postgraduate Endodontic Clinic for revascularization/revitalization therapy on tooth #9. The treated tooth remained asymptomatic for 16 months and then developed pain and local periapical swelling. The oral surgeon extracted the revascularized/revitalized tooth. On request, the extracted tooth was processed for histologic and histobacteriologic examination. RESULTS The tissue in the canal was completely destroyed. Most bacteria were observed in the apical portion and not in the coronal portion of the canal and formed biofilm on the canal walls and penetrated into the dentinal tubules. CONCLUSIONS On the basis of histobacteriologic observations, the failure of revascularized/revitalized tooth could be due to inadequate root canal disinfection without mechanical debridement. It may be important to perform mechanical debridement as part of the revascularization/revitalization therapy to disrupt the biofilm on the canal walls and remove bacteria in the dentinal tubules because revascularization/revitalization therapy is able to increase thickening of the canal walls.

Activation of bone sialoprotein gene transcription by flavonoids is mediated through an inverted CCAAT box in ROS 17/2.8 cells

Bone sialoprotein (BSP) is a major noncollagenous protein of the mineralized bone extracellular matrix that has been implicated in the nucleation of hydroxyapatite. Recent studies have shown that BSP is also expressed by osteotropic cancers suggesting BSP might play a role in the pathogenesis of bone metastases. The present study investigates regulation of BSP transcription in rat osteosarcoma ROS 17/2.8 cells by flavonoids: genistein (an inhibitor of protein tyrosine kinases), daidzein (an inactive compound of genistein), flavone, and flavanone. Genistein, daidzein, and flavone (50 μM) increased steady state levels of BSP mRNA about 1.7‐fold at 12 h. From transient transfection assays using various sized BSP promoter‐luciferase constructs, genistein increased luciferase activities within 12 h. Constructs including the promoter sequence nucleotides (nts) −116 to −43 (pLUC3) were found to enhance transcriptional activity ∼2.6‐fold in ROS 17/2.8 cells treated with genistein (50 μM). Daidzein, flavone, and flavanone (50 μM) also increased luciferase activities. In contrast, the tyrosine kinase inhibitors, herbimycin A and lavendustin A, which do not have a flavonoid structure, did not stimulate BSP transcription. Transcriptional stimulation by genistein was almost completely abrogated in a construct that included 2 bp mutations in the inverted CCAAT box. A monoclonal antibody against NF‐YA, a CCAAT box‐binding transcription factor, inhibited formation of DNA‐NF‐Y protein complex in gel shift assays formed by nuclear extracts of ROS 17/2.8 cells. These data suggest that the inverted CCAAT box is required for flavonoid‐induced BSP expression and that the stimulatory action is dependent on the flavone structure and does not involve an inhibitory action on protein tyrosine kinase. J. Cell. Biochem. 86: 35–44, 2002.

Interleukin-18 is regulated by parathyroid hormone and is required for its bone anabolic actions

Interleukin-18 (IL-18) can regulate osteoblast and osteoclast function. We have identified, using cDNA microarray technology, that IL-18 expression is increased in UMR 106-01 rat osteoblastic cells in response to parathyroid hormone (PTH) treatment. Confirmation of these data using real-time reverse transcription-PCR showed that steady-state levels of IL-18 mRNA increased by 2 h (3-fold), peaked by 4 h (10-fold), and had diminished after 12 h (4.4-fold) and that this regulation was via the protein kinase A signaling pathway and did not involve activation of the PKC signal cascade. PTH regulation of IL-18 was confirmed at the protein level, and analysis of differentiating primary rat calvarial osteoblasts verified that both IL-18 mRNA and protein are regulated by PTH in primary rat osteoblasts. Promoter reporter assays revealed that PTH regulated the upstream IL-18 promoter and induced the exon 1 containing 1.1-kb IL-18 mRNA transcript in primary osteoblast cells. The in vivo physiological role of IL-18 in the anabolic actions of PTH on bone was then assessed using IL-18 knock-out mice. Female IL-18 null mice and wild-type littermate controls were injected with vehicle or 8 μg/100 g of human 1–38 PTH for 4 weeks. In IL-18 knock-out animals the anabolic effect of PTH (determined by bone mineral density changes in the proximal tibia) was abolished in trabecular bone but not in the cortical component. These data characterize the PTH regulation of IL-18 expression in osteoblastic cells and suggest that this cytokine is involved in the anabolic actions of PTH.

Identification and characterization of Runx2 phosphorylation sites involved in matrix metalloproteinase‐13 promoter activation

Matrix metalloproteinase‐13 (MMP‐13) plays a critical role in parathyroid hormone (PTH)‐induced bone resorption. PTH acts via protein kinase A (PKA) to phosphorylate and stimulate the transactivation of Runx2 for MMP‐13 promoter activation. We show here that PTH stimulated Runx2 phosphorylation in rat osteoblastic cells. Runx2 was phosphorylated on serine 28 and threonine 340 after 8‐bromo cyclic adenosine mono phosphate (8‐Br‐cAMP) treatment. We further demonstrate that in the presence of 8‐Br‐cAMP, the wild‐type Runx2 construct stimulated MMP‐13 promoter activity, while the Runx2 construct having mutations at three phosphorylation sites (S28, S347 and T340) was unable to stimulate MMP‐13 promoter activity. Thus, we have identified the Runx2 phosphorylation sites necessary for PKA stimulated MMP‐13 promoter activation and this event may be critical for bone remodeling.

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.

Alendronate Affects Osteoblast Functions by Crosstalk through EphrinB1-EphB

Bisphosphonates are therapeutic agents in the treatment of post-menopausal osteoporosis. Although they have been associated with delayed healing in injured tissues, inappropriate femoral fractures, and osteonecrosis of the jaw (ONJ), the pathophysiological mechanisms involved are not clear. Our hypothesis is that alendronate, a member of the N-containing bisphosphonates, indirectly inhibits osteoblast function through the coupling of osteoclasts to osteoblasts by ephrinB-EphB interaction. We found that alendronate increased gene and protein expression of ephrinB1 and EphB1, as well as B3, in femurs of adult mice injected with alendronate (10 µg/100 g/wk) for 8 weeks. Alendronate suppressed the expression of bone sialoprotein (BSP) and osteonectin in both femurs and bone marrow osteoblastic cells of mice. After elimination of pre-osteoclasts from bone marrow cells, alendronate did not affect osteoblast differentiation, indicating the need for pre-osteoclasts for alendronate’s effects. Alendronate stimulated EphB1 and EphB3 protein expression in osteoblasts, whereas it enhanced ephrinB1 protein in pre-osteoclasts. In addition, a reverse signal by ephrinB1 inhibited osteoblast differentiation and suppressed BSP gene expression. Thus, alendronate, through its direct effects on the pre-osteoclast, appears to regulate expression of ephrinB1, which regulates and acts through the EphB1, B3 receptors on the osteoblast to suppress osteoblast differentiation.