DNA methylation of GJA1, BMP2 and BMP4 in a human cementoblast cell line induced by lipopolysaccharide.

AIM: To examine DNA methylation of GJA1, BMP2 and BMP4 in human cementoblasts (HCEM) induced by lipopolysaccharide (LPS). METHODOLOGY: HCEM were cultured in osteoinduction medium. After 24 h, Escherichia coli LPS (1 µg/mL) was added to the medium, which was changed every 2-3 days. Untreated samples were used as controls. Messenger RNA was extracted after 4 weeks, and quantitative real-time polymerase chain reaction (qRT-PCR) for GJA1, BMP2, BMP4 and DNMT1 was performed. Genomic DNA was extracted after 4 weeks, and quantitative methylation-specific polymerase chain reaction was carried out for GJA1, BMP2 and BMP4. To detect mineralization, alizarin red and alkaline phosphatase staining were performed. The cells were also treated with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5Aza) and examined. The significance of differences amongst groups was assessed using a two-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test with P < 0.05 being significant. RESULTS: Decreased expression of mRNA was seen in GJA1, BMP2 and BMP4 after 4 weeks (P < 0.05). DNA hypermethylation was detected in GJA1, BMP2 and BMP4 (P < 0.05). Alizarin red staining and alkaline phosphatase staining revealed decreased mineralization levels in HCEM stimulated with LPS. 5Aza abolished the effects of DNA methylation in HCEM stimulated with LPS. CONCLUSIONS: These results suggest that long-term LPS stimulation induces DNA methylation of GJA1, BMP2 and BMP4 in HCEM.

Alkaline extracellular conditions promote the proliferation and mineralization of a human cementoblast cell line.

AIM: To investigate the proliferation and mineralization of a human cementoblast cell line under alkaline conditions. METHODOLOGY: A human cementoblast cell line was cultured in alkaline media with several pHs (pH 7.6, 8.0 and 8.4) without CO2 . Cell numbers, phospho-p44/42 expression, alkaline phosphatase (ALP) activity and mineralization were evaluated. The significance of differences between groups was assessed using two-way analysis of variance 15 (ANOVA) followed by Bonferroni's multiple comparison test (α = 0.01). RESULTS: Cell numbers increased in a time-dependent manner in the high pH medium groups. Western blot analysis revealed the upregulated expression of phospho-p44/42 under alkaline conditions. ALP activity was also increased at pH 8.0 and 8.4. Alizarin red staining revealed increased mineralization in the high pH medium groups. The incorporation of the transient receptor potential ankyrin subfamily member 1 (TRPA1) antagonist HC030031 markedly negated the effect on proliferation and mineralization. CONCLUSIONS: Extracellular alkaline conditions promoted the proliferation and mineralization of human cementoblasts in vitro via TRPA1.

Response of porcine epithelial rests of Malassez to stimulation by interleukin-6.

AIM: To investigate the proliferation and migration of epithelial cell rests of Malassez (ERM) after stimulation with IL-6. METHODOLOGY: Porcine-derived ERM were seeded on Dulbecco's modified Eagle's Medium, and IL-6 (100 pg mL-1 ) was incorporated into the culture medium. The WST-1 assay was performed to evaluate cell proliferation, and absorption was measured at 450 nm. A wound-healing assay and immunofluorescence assay for integrin α3 were conducted to investigate migration. The Kruskal-Wallis test and the Mann-Whitney U-test with Bonferroni correction were used to analyse data of WST-1 and wound-healing assays. RESULTS: Cell proliferation following the stimulation by IL-6 increased over time, with a significant increase being observed at 6 h (P < 0.05), but not in a concentration-dependent manner. Cell proliferation was significantly greater in IL-6-treated ERM than in nontreated ERM (P < 0.05). The results of the wound-healing assay revealed earlier closure in IL-6-treated ERM (P < 0.05). In the immunofluorescence assay, integrin α3 was detected at the edge of cell processes adjacent to the wound area. A neutralized antibody abrogated the effects of the IL-6 stimulation in cell proliferation and migration. CONCLUSION: IL-6 promoted the proliferation and migration of porcine ERM in vitro.

Actin stabilization induces apoptosis in cultured porcine epithelial cell rests of Malassez.

AIM: To test whether actin stabilization by jasplakinolide induces inhibition of cell viability and apoptosis in epithelial cell rests of Malassez (ERM). METHODOLOGY: ERM derived from porcine were spread in a 96-well dish (5 × 10(4) /well) using Dulbecco's modified Eagle's medium. The actin-specific stabilization reagent, jasplakinolide, was incorporated into the culture medium and incubated for 24 h. To evaluate cell viability, the WST-1 assay was carried out and absorption (450 nm) was measured. To detect apoptotic cells, monoclonal antibody to single-strand DNA (ssDNA) was used and absorption (405 nm) was measured. Actin stabilization and apoptosis induced by jasplakinolide were morphologically investigated by staining with Alexa Fluor 568 phalloidin and observed under a fluorescent microscope. As a negative control, DMSO was used instead of jasplakinolide. Differences between the jasplakinolide-treated group and the control group were analysed statistically using the Student's t-test. RESULTS: Cell viability decreased in a concentration-dependent manner, and cell viability in the jasplakinolide-treated ERM was lower than that in nontreated ERM (n = 16, P < 0.01). Apoptotic cells in the jasplakinolide-treated ERM were more frequently detected compared to that in nontreated ERM (n = 16, P < 0.01). Morphologically, shrinkage, irregular forms and fragmentation of nuclei suggesting apoptotic bodies were observed in jasplakinolide-treated ERM, whilst actin filaments were extended in non-treated ERM. CONCLUSION: Actin stabilization by jasplakinolide inhibited cell viability and induced apoptosis in epithelial cell rests of Malassez.

Factors associated with self-assessed masticatory ability among community-dwelling elderly Japanese.

UNLABELLED: Self-assessed masticatory ability has been shown to be significantly related to general health among elderly persons. OBJECTIVE: To identify oral factors associated with the self-assessed masticatory ability. BASIC RESEARCH DESIGN: Cross-sectional study. PARTICIPANTS: A total of 736 community-dwelling elderly persons. MAIN OUTCOME MEASURES: Data on background factors and the self-assessed masticatory ability were collected by questionnaire. An intraoral examination examined the pattern of posterior occluding pairs of natural teeth (POPs), the WHO Community Periodontal Index of Treatment Needs (CPI) and denture-related factors such as use of dentures, pain when using dentures and stability and retention of dentures. Chi-squared tests examined the relationships between the self-assessed masticatory ability and the background factors and oral conditions. Ordinal regression models were constructed with the self-assessed masticatory ability as the dependent variable and oral conditions as the principal independent variables, to adjust for the potential confounding variables. RESULTS: Self-assessed impairment of masticatory ability was associated with lost POPs (p < 0.001) and CPI (p = 0.012). In the participants with lost POPs, self-assessed impairment of masticatory ability was associated with not using dentures and pain when using dentures (p < 0.001). In the totally edentulous subjects, impairment of masticatory ability was not associated with stability and retention of dentures (p = 0.070). CONCLUSIONS: Factors affecting self-assessed masticatory ability include the pattern of POPs, periodontal status, denture use and pain when using dentures.

Some biochemical properties of thymus leukemia antigens solubilized from cell membranes by papain digestion.

Thymus leukemia (TL) alloantigenic activity was solubilized by papain proteolytic digestion from intact RADA1 tumor cells. If the cells were labeled with amino acids and fucose, the TL alloantigen could be isolated as a doubly labeled glycoprotein fragment by indirect precipitation from the papain digest. This TL glycoprotein fragment was approximately the same mol wt as the papain-digested H-2.4 alloantigen fragment as judged by chromatography on Sephadex G-150 in sodium dodecyl sulfate. The carbohydrate chain of the TL glycoprotein obtained by exhaustive pronase digestion behaved as a glycopeptide of approximately 4,500 mol wt, as compared with the glycopeptide of the H-2.4 alloantigen that had a mol wt of about 3,500. Thus, the TL alloantigen can be solubilized by papain digestion as a glycoprotein fragment similar in mol wt to the H-2 alloantigen glycoprotein fragment. The carbohydrate chain of the TL glycoprotein is larger than the H-2 carbohydrate chain.

Expression and function of laminin and integrins on adhesion/migration of primary culture cells derived from rat oral epithelium.

BACKGROUND AND OBJECTIVE: It remains controversial whether or not the junctional epithelium cells that are directly attached to teeth migrate on the enamel surface, as those cells are able to adhere firmly to the enamel. The aim of this study was to investigate the expression patterns of laminin gamma(2), integrin beta(4) and integrin alpha(3), and to examine their potential function in cell migration. MATERIAL AND METHODS: Oral epithelium cells obtained from Sprague-Dawley rats were established in primary culture. We employed a wound-healing assay to characterize the direction of cell extension at the start of cell migration, and observed different localizations of laminin and integrins using immunofluorescence. For functional analyses of integrins, we employed a phosphatidylinositol-3-kinase (PI3K) activator to promote integrin beta(4) function and used P1B5 to inhibit integrin alpha(3) function, and we analyzed the percentage of re-epithelialization as the migration function. RESULTS: Marked accumulation of laminin gamma(2) was detected in the peripheral cytoplasm of cells adjacent to the wound area, as shown by the results of the migration assay. Integrin beta(4) was detected in the distal cell processes of actively migrating cells, while integrin alpha(3) was found in cell membranes of cells adjacent to the wound area. In the functional analyses, the percentage of re-epithelialization was significantly lower in the PI3K-activator group and in the P1B5-treated group (2.5% and 7.2%, respectively) than in the control group (39.0%) (p < 0.01). CONCLUSION: The results suggest that laminin gamma(2) is secreted as a foothold for cell migration, that integrin beta(4) participates in cell adhesion and that integrin alpha(3) is involved in cell migration in the primary culture cells.

Proliferation, migration and apoptosis of periodontal ligament cells after tooth replantation.

OBJECTIVE: The aim of this study was to investigate the proliferation, migration and death of periodontal ligament (PDL) cells after tooth replantation. MATERIALS AND METHODS: Maxillary first molars were extracted from 4-week-old male (n = 28) Sprague-Dawley rats and immediately replanted, after which, proliferation, migration and death of PDL cells were investigated. RESULTS: At 3 days after tooth replantation, many proliferative cell nuclear antigen (PCNA)-positive PDL cells were observed on the alveolar bone side, but fewer on the root side. However, while a gradual decrease was observed in number of PCNA-positive PDL cells on the alveolar bone side until 7 days, an increase was seen on the root side. At 3 weeks, cells labeled with PKH26 (fluorescent dye into plasma membrane) were located in the middle of the PDL space. However, these PKH26-labeled cells did not spread to the surface of the cementum or the alveolar bone. TUNEL-positive cells were observed on both the bone and root sides at 3 days. Number of apoptotic cells increased until 7 days on the bone sides, but decreased on root sides. CONCLUSION: These results suggest that both cell proliferation and apoptosis occur in different patterns and at different times to maintain regular spacing of the PDL after tooth replantation.

Midkine (MK), a heparin-binding growth/differentiation factor, is regulated by retinoic acid and epithelial-mesenchymal interactions in the developing mouse tooth, and affects cell proliferation and morphogenesis.

Midkine (MK) is the first cloned gene in a new family of heparin-binding growth/differentiation factors involved in the regulation of growth and differentiation. We have analyzed the expression of MK mRNA and protein during tooth development in mouse embryos and studied the regulation of MK expression and the biological effects of MK protein in organ cultures. MK expression was restricted and preferential in the tooth area as compared to the rest of the developing maxillary and mandibular processes suggesting specific functions for MK during tooth morphogenesis. MK mRNA and protein were expressed during all stages of tooth formation (initiation, morphogenesis, and cell differentiation), and shifts of expression were observed between the epithelial and mesenchymal tissue components. However, the expression of mRNA and protein showed marked differences at some stages suggesting paracrine functions for MK. Tissue recombination experiments showed that MK gene and protein expression are regulated by epithelial-mesenchymal interactions, and, moreover, that dental tissue induces the ectopic expression of MK protein in non-dental tissue. The expression of MK gene and protein in the mandibular arch mesenchyme from the tooth region were stimulated by local application of retinoic acid in beads. Cell proliferation was inhibited in dental mesenchyme around the beads releasing MK, but this effect was modulated by simultaneous application of FGF-2. Morphogenesis and cell differentiation were inhibited in tooth germs cultured in the presence of neutralizing antibodies for MK, whereas the development of other organs (e.g., salivary gland, kidney) was unaffected. These results suggest important roles for MK in the molecular cascade that regulates tooth development.

A retinoic acid responsive gene MK found in the teratocarcinoma system is expressed in spatially and temporally controlled manner during mouse embryogenesis.

A newly identified gene MK is transiently expressed in early stages of retinoic acid-induced differentiation of embryonal carcinoma cells (Kadomatsu, K., M. Tomomura, and T. Muramatsu, 1988. Biochem. Biophys. Res. Commun. 151:1312-1318). MK gene has been predicted to code a polypeptide that is rich in basic amino acids and cysteine and is not related to any other peptides so far reported. In the present study, we investigated MK expression during mouse embryogenesis by in situ hybridization. The MK transcript was detected all over the embryo proper of the 7-d embryo, while it was not detectable in the 5-d embryo. The ubiquitous expression continued in the 9-d embryo proper. On the 11th-13th d of gestation, the sites where MK gene was intensely expressed became progressively restricted; these sites were the brain ectoderm around the lens and brain ventricles, the anterior lobe of the pituitary gland, the upper and lower jaw, the caudal sclerotomic half of vertebral column, the limbs, the stomach, and the epithelial tissues of the lung, the pancreas, the small intestine, and the metanephros. These areas include the region where secondary embryonic induction is prominent. In the 15-d embryo, only the kidney expressed MK significantly. These data suggest that MK gene plays a fundamental role in the differentiation of a wide variety of cells; MK gene may also play some specific roles in generation of epithelial tissues, and remodeling of mesoderm.

Effect of the dental adhesive, 4-META/MMA-TBB resin, on adhesion and keratinization of regenerating oral epithelium.

BACKGROUND AND OBJECTIVE: The 4-META/MMA-TBB [4-(2-methacryloxyethyl)trimellitic anhydride/methyl methacrylate-tributylborane] resin is widely used as a dental adhesive. It has also been applied in the dressing of gingival wound surfaces following periodontal surgery. However, its effect on the regeneration and/or cell attachment of the oral epithelium remains to be clarified. To evaluate the effect of the resin applied as a wound dressing, we investigated expression of laminin 5, integrin beta(4) and cytokeratin 14 in regenerating oral epithelium treated with this resin following gingivectomy from the viewpoint of cell attachment and differentiation. MATERIAL AND METHODS: The resin was applied to the entire wound surface in rats after gingival surgery, and regenerating epithelium was examined immediately and at 1, 3, 5, 7 and 14 days later. The resin was removed 2 weeks after application in some animals and tissue further examined at 1, 3, 5 and 7 days later. RESULTS: Regenerating epithelium under the resin was not keratinized, but became keratinized immediately after removal of the resin. Laminin 5 and integrin beta(4) were immunolocalized in the basal lamina, the internal basal lamina, in marginal cells of the regenerating epithelium and at the resin-regenerating epithelium interface. Cytokeratin 14 localized in the regenerating epithelium underneath the resin, as well as in healthy and regenerated junctional epithelial cells. CONCLUSION: These results suggest that this resin covers the wound surface and that the regenerating epithelium biologically adheres to the resin during the initial process of its regeneration.

Immunolocalization of laminin and integrin in regenerating junctional epithelium of mice after gingivectomy.

BACKGROUND AND OBJECTIVE: The expression patterns of adhesive proteins and extracellular matrix proteins in regenerating gingival epithelium after gingivectomy are unknown. The aim of this study was to examine the expression of laminin 1, laminin gamma(2) (a specific component of laminin 5), integrin beta(4) and integrin alpha(3) in the regenerating gingival epithelium in order to understand the mechanism of wound healing during reconstitution of the sulcular environment. MATERIAL AND METHODS: The palatal gingivae of the maxillary molars of Institute of Cancer Research mice were excised, and the regenerating tissues were examined 1, 3, 5, 7 and 14 days later. Fresh, non-fixed and non-decalcified frozen sections were prepared and stained using immunofluorescence. RESULTS: At 1 day post-surgery, intense expression of laminin gamma(2), integrin beta(4) and integrin alpha(3) was distinct in the frontal margin of the regenerating oral epithelium. Laminin gamma(2) was diffusely detected on the root surface and in connective tissues beneath the regenerating oral epithelium at 3 and 5 days. At 7 days, laminin gamma(2) was intermittently recognizable in the internal basal lamina (IBL) close to tooth-facing cells, while laminin gamma(2), integrin beta(4) and integrin alpha(3) were observed in the IBL and in the external basal lamina (EBL) of the regenerating junctional epithelium at 14 days. CONCLUSION: These results suggest that secretion of laminin 5 in the connective tissue may induce epithelial cell migration, and that binding of laminin 5 to integrin alpha(6)beta(4) and integrin alpha(3)beta(1) in the IBL may provoke cell adhesion and migration of cells facing the tooth on the enamel surface of the regenerating junctional epithelium.

Involvement of laminin and integrins in adhesion and migration of junctional epithelium cells.

BACKGROUND AND OBJECTIVE: The junctional epithelium attaches to the enamel surface with hemidesmosomes (of which laminin-5 and integrin-alpha(6)beta(4) are the main components) in the internal basal lamina. Laminin-5 is also involved in cell motility with integrin-alpha(3)beta(1), although their functions have not yet been clarified.The purpose of this study was to determine the functions of those adhesive components between the tooth and the junctional epithelium during cell migration.Because an idea has been proposed that directly attached to tooth cells (DAT cells) may not contribute to cell migration, 5-bromo-2-deoxyuridine staining was performed to confirm cell migration. MATERIAL AND METHODS: We investigated laminin-gamma(2) (contained only in laminin-5), integrin-beta(4) (involved in cell-extracellular matrix contact) and integrin-alpha(3) (inducing cell migration) in the junctional epithelium, oral gingival epithelium and gingival sulcus epithelium of 6-wk-old ICR mice using laser microdissection, quantitative real-time reverse transcription-polymerase chain reaction, immunofluorescence and 5-bromo-2-deoxyuridine staining. RESULTS: Laminin and integrins were clearly immuno-localized in the basal lamina of all epithelium. Quantitative analysis of laminin and integrin mRNAs by laser microdissection showed that they were more highly expressed in DAT cells than in basal cells in the oral gingival epithelium. In particular, a 12-fold higher expression of laminin-5 was observed in the junctional epithelium compared with the oral gingival epithelium. 5-Bromo-2-deoxyuridine staining showed rapid coronal migration of DAT cells. CONCLUSION: These results suggest that the abundant expression of laminin-5 and integrin-alpha(6)beta(4) is involved in the attachment of DAT cells to teeth by hemidesmosomes. Abundant expression of laminin-5 and integrin-alpha(3)beta(1) might assist in DAT cell migration, confirmed by 5-bromo-2-deoxyuridine staining during the turnover of junctional epithelium.

Claudin rather than occludin is essential for differentiation in rat incisor odontoblasts.

Many morphological and developmental studies have demonstrated the characteristics of tight junctions (TJs) between odontoblasts. However, detailed localization of TJ-associated proteins in odontoblasts and their functions has not yet been clarified. To elucidate the relationship between the establishment of TJ structures and the differentiation of odontoblasts during early dentinogenesis, we studied the expression and localization of constituent proteins of TJs (claudin-1, occludin, ZO-1 and ZO-2) between odontoblasts in rat lower incisors using Western blotting, immunofluorescence and immunoelectron microscopy. When the expression of claudin-1 increases at the distal portion of mature odontoblasts, the TJs form complex networks of strands, and odontoblasts differentiated by developing distal membrane domains and by secreting specific molecules for mineralization. We conclude that the TJs of odontoblasts may play an important role in the differentiation of odontoblasts in rat lower incisors during early dentinogenesis.

Complete antithrombin deficiency in mice results in embryonic lethality.

Antithrombin is a plasma protease inhibitor that inhibits thrombin and contributes to the maintenance of blood fluidity. Using targeted gene disruption, we investigated the role of antithrombin in embryogenesis. Mating mice heterozygous for antithrombin gene (ATIII) disruption, ATIII(+/-), yielded the expected Mendelian distribution of genotypes until 14.5 gestational days (gd). However, approximately 70% of the ATIII(-/-) embryos at 15.5 gd and 100% at 16.5 gd had died and showed extensive subcutaneous hemorrhage. Histological examination of those embryos revealed extensive fibrin(ogen) deposition in the myocardium and liver, but not in the brain or lung. Furthermore, no apparent fibrin(ogen) deposition was detected in the extensive hemorrhagic region, suggesting that fibrinogen might be decreased due to consumptive coagulopathy and/or liver dysfunction. These findings suggest that antithrombin is essential for embryonic survival and that it plays an important role in regulation of blood coagulation in the myocardium and liver.

Neointima formation in a restenosis model is suppressed in midkine-deficient mice.

Neointima formation is a common feature of atherosclerosis and restenosis after balloon angioplasty. To find a new target to suppress neointima formation, we investigated the possible role of midkine (MK), a heparin-binding growth factor with neurotrophic and chemotactic activities, in neointima formation. MK expression increased during neointima formation caused by intraluminal balloon injury of the rat carotid artery. Neointima formation in a restenosis model was strongly suppressed in MK-deficient mice. Continuous administration of MK protein to MK-deficient mice restored neointima formation. Leukocyte recruitment to the vascular walls after injury was markedly decreased in MK-deficient mice. Soluble MK as well as that bound to the substratum induced migration of macrophages in vitro. These results indicate that MK plays a critical role in neointima formation at least in part owing to its ability to mediate leukocyte recruitment.

GER1, a GDSL motif-encoding gene from rice is a novel early light- and jasmonate-induced gene.

The reaction of the rice mutant HEBIBA differs from that of wild-type rice in that the mutant responds inversely to red light and is defective in the light-triggered biosynthesis of jasmonic acid (JA). Using the wild type and the HEBIBA mutant of rice in a differential display screen, we attempted to identify genes that act in or near the convergence point of light and JA signalling. We isolated specifically regulated DNA fragments from approximately 10 000 displayed bands, and identified a new early light- and JA-induced gene. This gene encodes an enzyme containing a GDSL motif, showing 38 % identity at the amino acid level to lipase Arab-1 in Arabidopsis thaliana. The GDSL CONTAINING ENZYME RICE 1 gene (GER1) is rapidly induced by both red (R) and far-red (FR) light and by JA. The results are discussed with respect to a possible role for GER1 as a negative regulator of coleoptile elongation in the context of recent findings on the impact of JA on light signalling.

Responses of rat pulp cells to heat stress in vitro.

Although heat stress can cause irritation in the dentin/pulp complex, little is known about the thermotolerance of pulp cells and their response to heat stress. We investigated cultured rat pulp cell responses to heat stress. Cells were subjected to a temperature of 42 degrees C for 30 minutes, and HSPs, alkaline phosphatase activity, and gap-junctional communication were determined at various time points. Although only low levels of HSP70 expression were detected before heat treatment, heat shock markedly induced HSP70 expression, with it gradually increasing at 1 hour after being heated. HSP25, however, showed no dramatic change. Gap junction protein connexin43 rapidly degraded after heat treatment, recovering to normal levels within the following 6 hours. Alkaline phosphatase activity decreased immediately after heat stress, recovering after 1 hour. These results indicate that dental pulp possesses protective factors, including HSPs, and that it can recover viability of intercellular communication and alkaline phosphatase activity after heat stress.

Correlation between reduction of syndecan-1 expression and clinico-pathological parameters in squamous cell carcinoma of tongue.

We examined expression of syndecan-1 in squamous cell carcinoma (SCC) of tongue using immunohistochemistry. Forty-three cases of SCC arising in lateral border of tongue were investigated. From the immunohistochemical staining pattern, the cases were divided into two groups based on expression of syndecan-1 at the supra-peripheral cells of the tumor nest: Group A, completely or mainly positive; Group B, sporadically positive or negative. Most poorly differentiated SCC cases were classified into Group B (81.8%). The number of Group B cases in T1-2 was different from that in T3-4. The number of cases where syndecan-1 expression was reduced was much greater in T3-4, and represented the majority of Group B (86.7%). More than 80% of Grade 4D cases were in Group B (83.3%) based on the Yamamoto-Kohama criteria. These results indicate that reduction of syndecan-1 correlates to histological grade, tumor size and mode of invasion in tongue SCC.