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ABSTRACT: The dental follicle is an ectomesenchymal tissue that surrounds developing tooth germ and that contains osteoblastic-lineage-committed stem/progenitor cells. We examined the osteogenic potential of human dental follicle cells (hDFC) by microarray analysis. We first compared the characteristics of hDFC with those of human bone marrow mesenchymal stem cells (hMSC). Like hMSC, hDFC expressed stem cell markers such as STRO-1 and Notch-1 and differentiated not only into the osteoblastic lineage, but also into the adipogenic lineage. We analyzed the gene expression profiles of hDFC and hMSC that were not differentiated toward the osteogenic lineage. The expression of cell markers and growth factor receptors by hDFC and hMSC was similar, whereas the expression pattern of homeobox genes differed between hDFC and hMSC. Next, we investigated gene expression in hDFC during osteogenic differentiation. Gene expression profiles were analyzed in hDFC cultured in osteogenic induction medium (OIM) or in growth medium (GM) for 3 and 10 days. Many genes whose expression was regulated under these conditions were functionally categorized as "transcription" genes. Osteogenic markers were up-regulated in hDFC during osteogenic differentiation, whereas neurogenic markers were down-regulated. The genes whose expression was regulated in hDFC during osteogenic differentiation were further analyzed by ingenuity pathway analysis and real-time polymerase chain reaction. Bone morphogenetic protein and transforming growth factor-β signaling pathways were activated in hDFC cultured in OIM for 3 days. This study indicates that the dental follicle contains stem cells and/or osteoblastic progenitor cells and is a potential cellular resource for bone regeneration therapy.
Cell and Tissue Research 08/2012; 350(2):317-31. · 3.11 Impact Factor
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ABSTRACT: Synovitis, which is characterized by the infiltration of inflammatory cells, often accompanies progression of temporomandibular joint disorder (TMD) symptoms. Because IL-1β is elevated in synovial fluids obtained from TMDs, we hypothesized that IL-1β-responsive genes in synoviocytes may help identify the putative genes associated with synovitis. Using microarray analysis, we found that monocyte chemoattractant protein-1 (MCP-1) mRNA levels were elevated in IL-1β-stimulated synoviocytes. MCP-1 is a member of the chemokine superfamily. The production of MCP-1 was increased in synoviocytes treated with IL-1β. When IL-1β was injected into the cavities of rat TMJs, inflammatory cells and MCP-1-positive cells were detected in the synovial tissues. Furthermore, MCP-1 levels were higher in synovial fluids from individuals with pain compared with those without pain. Inhibitors of MAP-kinases and NF-κB reduced IL-1β-induced MCP-1 production. These results suggest that MCP-1 stimulated by IL-1β is one of the factors associated with the inflammatory progression of TMDs.
Journal of dental research 10/2010; 89(10):1117-22. · 3.46 Impact Factor
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ABSTRACT: In this study, we analyzed the gene expression profile of fibroblast-like synoviocyte (FLS) cultures from the temporomandibular joint (TMJ) to identify candidate genes associated with intracapsular pathologic conditions of TMJ. Cyclooxygenase (COX)-2 was one of the genes in FLS upregulated following stimulation by interleukin (IL)-1beta, a cytokine thought to play a key role in several pathological conditions. This study investigated the expression of COX-1 and COX-2 in cultured human FLS and rat TMJ synovium following stimulation with IL-1beta.
RNA was isolated from human FLS after IL-1beta treatment. COX-1 and -2 expression was examined using a GeneChip and real-time polymerase chain reaction. Prostaglandin E(2) (PGE(2)) levels in conditioned media from FLS were measured using enzyme-linked immunosorbent assay. Synovial tissues from TMJs of IL-1beta-injected rats were examined for COX-1 and COX-2 expression by immunohistochemical staining.
Following treatment of FLS with IL-1beta, expression of the COX-2 gene increased up to 8 h and peaked at 4 h, whereas COX-1 expression did not change. Stimulation with IL-1beta increased the level of PGE(2) in conditioned media of cultured FLS in a time-dependent manner up to 48 h. Immunohistochemistry showed a strong positive staining for COX-2 in the lining and sub-lining synovial tissues of the TMJ of IL-1beta-injected rats. In contrast, staining for COX-1 was the same in synovial tissues with and without IL-1beta injection.
These data suggest that COX-2 expression stimulated by IL-1beta stimulates the production of PGE(2) in FLS and plays important roles in the progression of inflammation in TMJ.
Journal of Oral Pathology and Medicine 01/2009; 38(7):584-90. · 1.63 Impact Factor
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ABSTRACT: Synovial fibroblasts of temporomandibular joint (TMJ) are poorly characterized, although they have important roles in progression of temporomandibular disorders (TMD). In this study, we investigated responses of synovial fibroblasts to interleukin (IL)-1beta.
We examined gene expression profiles of synovial fibroblasts in response to IL-1beta, using Affymetrix GeneChip. Regulated upon activation normal T-cell expressed and secreted (RANTES) gene expression was confirmed by polymerase chain reaction (PCR) and real-time PCR. RANTES protein levels were measured by enzyme-linked immunosorbent assay (ELISA).
The RANTES was preferentially up-regulated in synovial fibroblasts by IL-1beta. The increase in RANTES gene expression in response to IL-1beta was confirmed by PCR and real-time PCR. Protein level of RANTES in synovial fibroblasts was also increased by IL-1beta.
The RANTES, a cc-type chemokine, has chemotactic effects on lymphocytes and monocytes. Increased gene expression and protein production of RANTES in synovial fibroblasts, in response to IL-1beta, may play an important role in recruitment of inflammatory cells into synovium and progression of synovitis in TMD.
Journal of Oral Pathology and Medicine 12/2004; 33(10):629-33. · 1.63 Impact Factor
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ABSTRACT: To measure the activities of plasminogen activator (PA), plasmin and kallikrein, multiple synovial fluid samples were taken from 32 patients with internal derangement (ID) and osteoarthrosis (OA), and nine asymptomatic volunteers. The enzyme activity in synovial fluid from the temporomandibular joint (TMJ) was quantitated by a fluorogenic substrate assay using an enzyme substrate. In fluid samples from the patient group, PA was detected in 24 (31.5%), plasmin in 20 (26.3%) and kallikrein in 53 (96.4%), while none of these enzymes were found in the synovial fluid samples from the control group. There were positive correlations found among PA, plasmin and kallikrein. These results clearly demonstrated increased levels of PA, plasmin and kallikrein activities in the synovial fluid of patients with ID and OA, and suggest that these enzymes may be involved in the pathogenesis of synovitis, as well as the resorption of cartilage and bone in TMJ.
International Journal of Oral and Maxillofacial Surgery 09/2001; 30(4):323-8. · 1.51 Impact Factor
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ABSTRACT: The binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR) in various cell types has been proposed as an important feature of many cellular processes requiring extracellular proteolysis, cell adhesion, motility, and invasion. uPAR attaches to the cell surface with a glycosylphophatidylinositol (GPI) anchor, and serves to localize and accelerate the proteolysis cascade. In this study, we examined both uPA and uPAR levels in human gingival fibroblasts treated with an inflammatory cytokine, interleukin-1beta (IL-1beta). PA activity in the cell lysate was increased by treatment with IL-1beta. Further, PA activity released by phosphatidylinositol-specific phospholipase C, which detaches the GPI anchor, was also increased by IL-1beta. The activity was inhibited by amiloride, a specific inhibitor of uPA. In addition, IL-1beta increased the protein and mRNA levels of both uPA and uPAR in gingival fibroblasts. These findings suggest that the enhancement of uPA and uPAR levels by IL-1beta may play an important role in the progression of periodontal diseases through pericellular proteolysis, and subsequent cellular behavior.
International Union of Biochemistry and Molecular Biology Life 07/2001; 51(6):381-5. · 3.51 Impact Factor
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ABSTRACT: Urokinase-type plasminogen activator receptor (uPAR) presenting on the cell surface with glycosylphosphatidylinositol (GPI) anchor is a key component in the plasminogen activator (PA)-plasmin system and plays a critical role in extracellular matrix degradation. It is believed that uPAR serves to localize and accelerate this generation system. In this study, we examined the levels of both uPA and uPAR in human gingival fibroblasts treated with Porphyromonas gingivalis lipopolysaccharide (LPS).
Human gingival fibroblasts from the seventh to tenth doubling passages were plated at 5x10(4) cells per well in 24-well plates. The confluent-stage cells were cultured for 24 hours in alpha-MEM medium containing 2% fetal calf serum, after which they were incubated with P. gingivalis LPS. PA activity was measured using plasminogen and plasmin substrate S2251.
PA activity in the cell lysate was increased by LPS and reached maximum at 1 microg/ml LPS after being incubated for 8 hours. PA activity released by phosphatidylinositol-specific phospholipase C, which detaches the GPI anchor, was also increased by LPS. The activity was inhibited by amiloride, which is a specific inhibitor for uPA. LPS increased the protein and mRNA levels of both uPA and uPAR in gingival fibroblasts.
These findings suggest that increase of the uPAR level by LPS may play an important role in the progression of periodontal diseases through pericellular proteolysis.
Journal of Periodontology 05/1999; 70(4):402-8. · 2.60 Impact Factor
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ABSTRACT: A B-cell line producing a human monoclonal antibody (HuMAb) against a recombinant 40-kDa outer membrane protein (OMP) of Porphyromonas gingivalis was constructed by in vivo immunization of a severe combined immunodeficiency C.B.-17/Icr mouse, which had been injected with human peripheral blood lymphocytes, with recombinant 40-kDa OMP and subsequent Epstein-Barr virus immortalization of B cells isolated from the spleen of the mouse. This HuMAb inhibited coaggregation between P. gingivalis vesicles and Actinomyces naeslundii cells.
Infection and Immunity 10/1997; 65(9):3966-9. · 4.16 Impact Factor
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ABSTRACT: The effect of extracellular oxygen radicals on cultured gingival fibroblast cells (Gin cells) was investigated in the plasminogen activator (PA)/plasmin system. The activation of the PA/plasmin system in Gin cells exposed to a sublethal oxygen radical [hypoxanthine (HX) 0.1 mg ml-1/xanthine oxidase (XOD) 5 munit ml-1] system was examined. Following a 1 h exposure, washed cells were cultured for up to 24 h in fresh medium containing 2% fetal calf serum. The exogenous addition of superoxide dismutase, an oxygen radical scavenger, abolished the PA/plasmin activity enhanced by the HX/XOD system. The PA produced by Gin cells was found to be urokinase-type PA (uPA), as preincubation of Gin cell-conditioned medium with anti-uPa serum completely inhibited PA activity. These findings suggest that extracellular oxidant targetting to Gin cells may be involved in the progression of inflammation and the invasion of periodontium through stimulation of the PA/plasmin system.
Archives of Oral Biology 05/1997; 42(4):263-70. · 1.60 Impact Factor
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ABSTRACT: The production of interleukin-6 (IL-6) in human gingival fibroblasts (Gin cells) is increased by lipopolysaccharide (LPS) from Campylobacter rectus (C. rectus), which is associated with adult periodontitis; however, the age-related changes in the susceptibility of Gin cells to C. rectus LPS remain unclear. We examined the influence of in vitro senescence on C. rectus LPS-stimulated IL-6 production in Gin cells. LPS was prepared from C. rectus ATCC 33238 using hot phenol-water. The Gin cells were established from healthy gingival tissue removed from three patients, aged 10-12 years. The cells were cultured until confluence then stimulated with LPS (0.01, 0.1, 1.0 and 10.0 micrograms/ml). Levels of IL-6 released in the medium were measured after incubation for 3, 6, 9, 12, and 24 h. In both young (5-6 population doublings) and senescent (17-20 population doublings) cells, LPS stimulated IL-6 production in a dose- and time-dependent manner. In response to 0.01-10.0 micrograms/ml of LPS, IL-6 production in the senescent cells was higher than that in the young cells. Using cells from each of the three donors, we found that this phenomenon of higher LPS-stimulated IL-6 production in senescent cells was reproducible. The greater capacity of the senescent cells to synthesize IL-6 in response to LPS was a higher production of mRNA for IL-6. This increase of IL-6 production induced by C. rectus LPS in senescent Gin cells could help to explain the increased susceptibility to periodontal diseases shown by aged individuals.
Mechanisms of Ageing and Development 06/1996; 87(1):47-59. · 3.44 Impact Factor
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ABSTRACT: The plasminogen activator (PA)-plasmin system is implicated in the degradation of the extracellular matrix in inflammation through activation of metalloproteases and prekallikrein. We examined the activation of the PA-plasmin system in human gingival fibroblast cells (Gin-1 cells) following treatment with lipopolysaccharide (LPS) from Campylobacter rectus, which is frequently detected at sites of periodontal disease. The C. rectus LPS stimulated the plasmin activity in the conditioned medium of Gin-1 cells in a time- and dose-dependent manner, and C. rectus LPS also stimulated the PA activity in the conditioned medium. The PA produced by Gin-1 cells was determined to be urokinase PA (uPA), as preincubation of Gin-1 conditioned medium with anti-uPA antiserum completely inhibited the PA activity while that with anti-tPA antiserum had no inhibitory effect. The concentration of PA inhibitor-1 (PAI-1) in the conditioned medium was decreased by the addition of C. rectus LPS. Therefore, the enhancement of plasmin activity in the conditioned medium was dependent on increased uPA activity via the decrease of the PAI-1 level of Gin-1 cells treated with C. rectus LPS. Furthermore, the conditioned medium of Gin-1 cells treated with C. rectus LPS showed significantly increased kallikrein activity, indicating the conversion of prekallikrein to kallikrein, which converts kininogen into kinin. These findings suggest that C. rectus LPS is a potent stimulator of inflammation of gingival tissue which acts through stimulation of the PA-plasmin system.
Journal of Periodontal Research 04/1995; 30(2):132-40. · 1.69 Impact Factor
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ABSTRACT: Interleukin-6 (IL-6), which is a multifunctional cytokine, has important roles in acute and chronic inflammation and may also be implicated in bone resorption. We examined the IL-6 production in periodontal ligament (PDL) cells which were treated with lipopolysaccharide (LPS) from several oral inflammatory pathogens. The LPS from Porphyromonas endodontalis, which was isolated from infected root canals and radicular cyst fluids, was more potent than the LPS from any other periodontal organisms examined. P. endodontalis LPS stimulated IL-6 release from PDL cells in a time- and dose-dependent manner. Northern blot hybridization analysis revealed that the IL-6 mRNA level in PDL cells was increased by P. endodontalis LPS. These results suggest that stimulation of the IL-6 release of PDL cells by P. endodontalis LPS may have a role in the progression of inflammation and alveolar bone resorption in periodontal and periapical diseases.
Biochemical Medicine and Metabolic Biology 01/1995; 53(2):130-6.
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ABSTRACT: 1. Plasmin activity in the conditioned medium of Gin-1 cells, a human gingival fibroblast cell line, was stimulated by Porphyromonas endodontalis, a putative pathogen of oral submucous abscesses, in a time- and dose-dependent manner. 2. P. endodontalis stimulated the activity of plasminogen activator in both the conditioned medium and the cell lysate. The plasminogen activator in Gin-1 cells was approx. 50 kDa by zymography. 3. The conditioned medium of Gin-1 cells exposed to P. endodontalis stimulated the conversion of human serum prekallikrein to kallikrein. 4. These results suggested that P. endodontalis stimulates the plasminogen activator-plasmin system in Gin-1 cells, and that activated plasmin plays a role in the progress of periodontal tissue inflammation.
International Journal of Biochemistry 10/1993; 25(9):1227-31.
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ABSTRACT: Interleukin-1(IL-1), a cytokine present in the gingiva and crevicular fluid of patients with periodontitis and in the periodontal ligament (PDL) of experimentally moved teeth, has multiple biological activities, including the ability to elicit bone resorption. Interleukin-6, also found in the gingiva of patients with periodontitis, may induce osteoclastic bone resorption through an effect on osteoclastogenesis. Here IL-6 production and its gene expression in response to recombinant IL-1 beta were examined in primary cultures of PDL cells. IL-1 beta stimulated IL-6 production by these cells in a dose- and time-dependent manner; this increase in IL-6 production was much higher than that in human gingival fibroblasts. In situ hybridization, using a synthetic oligonucleotide DNA probe of the IL-6 gene, revealed that most PDL cells expressed IL-6 mRNA in response to IL-1 beta treatment. The finding that IL-6 is produced by PDL cells and is regulated by IL-1 beta has revealed a potentially important mechanism for controlling alveolar bone resorption.
Archives of Oral Biology 10/1992; 37(9):743-8. · 1.60 Impact Factor
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ABSTRACT: We found that platelet-activating factor (PAF) stimulated the production of prostaglandin (PG) E2 in MC3T3-E1 cells in a time- and dose-dependent manner. 1.0 microM PAF gave a maximal stimulation of PGE2 production by MC3T3-E1 cells after a 4 hr PAF-treatment. Furthermore, the PAF-induced PGE2 production was abolished by the pre-treatment of the cells with a PAF receptor antagonist, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho(N,N,N-trimethyl)hexanolamine, which occupied the same receptor site as PAF. These results suggest that PAF stimulates the PGE2 synthesis through a PAF receptor mediated pathway. Possibly PAF modulates bone metabolism by stimulating PGE2 synthesis.
Life Sciences 02/1991; 49(15):1103-9. · 2.53 Impact Factor
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ABSTRACT: 1. The amount of apolipoprotein B (apo B) was measured using slit-immunoblotting in 20 specimens of radicular cyst fluids. Apo B was detected in all the cyst fluids with varying amounts. 2. Relationship between the amounts of apo B and free cholesterol or activity of heat-stable cholesterol-binding protein (HCBP) were examined. The amount of apo B was correlated well with the activity of HCBP (n = 20, r = 0.72, P less than 0.01) and with the amount of free cholesterol (n = 20, r = 0.45, P less than 0.05). 3. Anti-human apo B antibody inhibited cholesterol-binding activity in radicular cyst fluid. 4. When human-serum was chromatographed on a HPLC ion-exchange column, both cholesterol-binding activity and apo B had exactly the same retention time. 5. These results suggest that HCBP originates from beta-lipoprotein, and beta-lipoprotein may have an important role in cholesterol accumulation on radicular cysts.
International Journal of Biochemistry 02/1990; 22(2):165-9.
