Yohei Nakayama

Comparative temporospatial expression profiling of murine amelotin protein during amelogenesis.

Tooth enamel is formed in a typical biomineralization process under the guidance of specific organic components. Amelotin (AMTN) is a recently identified, secreted protein that is transcribed predominantly during the maturation stage of enamel formation, but its protein expression profile throughout amelogenesis has not been described in detail. The main objective of this study was to define the spatiotemporal expression profile of AMTN during tooth development in comparison with other known enamel proteins. A peptide antibody against AMTN was raised in rabbits, affinity purified and used for immunohistochemical analyses on sagittal and transverse paraffin sections of decalcified mouse hemimandibles. The localization of AMTN was compared to that of known enamel proteins amelogenin, ameloblastin, enamelin, odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4. Three-dimensional images of AMTN localization in molars at selected ages were reconstructed from serial stained sections, and transmission electron microscopy was used for ultrastructural localization of AMTN. AMTN was detected in ameloblasts of molars in a transient fashion, declining at the time of tooth eruption. Prominent expression in maturation stage ameloblasts of the continuously erupting incisor persisted into adulthood. In contrast, amelogenin, ameloblastin and enamelin were predominantly found during the early secretory stage, while odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4 expression in maturation stage ameloblasts paralleled that of AMTN. Secreted AMTN was detected at the interface between ameloblasts and the mineralized enamel. Recombinant AMTN protein did not mediate cell attachment in vitro. These results suggest a primary role for AMTN in the late stages of enamel mineralization.

Enamel Hypomineralization and Structural Defects in Amelotin-deficient Mice

Amelotin (AMTN) is a relatively recently discovered enamel protein that is predominantly expressed by ameloblasts during the maturation stage of amelogenesis and is present at lower levels in the junctional epithelium of erupted teeth. Previous studies have suggested a function of this protein in enamel mineralization and cell attachment. Genetic mouse models have been instrumental in defining the role of many enamel-related proteins, but a genetic mouse model lacking the Amtn gene has not been reported. Here, we describe the generation of amelotin-deficient mice and the analysis of their enamel phenotype in comparison with that of wild-type animals. Ablation of AMTN expression resulted in mechanically inferior enamel of mandibular incisors that showed chipping and fractures at the incisal edge. Enamel mineralization was delayed, resulting in hypomineralized inner enamel and structural defects in the outer enamel. Erupted enamel close to the gingival margin showed increased surface roughness. The expression levels of the enamel matrix proteins AMEL, AMBN, ENAM, and ODAM and the enamel proteases MMP-20 and KLK-4 were not significantly altered, although the expression of KLK-4 was delayed. The morphology of ameloblasts showing prominent Tomes’ processes during the secretory stage was not altered, and there was no indication of disruption of cell structures or activities, but a residual layer, presumably consisting of organic material, remained at the enamel surface close to the gingival margin. The integrity of the dentogingival attachment at the junctional epithelium appeared unaffected by AMTN deficiency. These observations indicate that AMTN plays a subtle yet critical role in enamel biomineralization, particularly during the establishment of the outer and surface enamel layers. This role appears to be largely independent of other enamel proteins.

Salivary pathogen and serum antibody to assess the progression of chronic periodontitis: a 24-mo prospective multicenter cohort study

BACKGROUND AND OBJECTIVE A diagnosis of periodontitis progression is presently limited to clinical parameters such as attachment loss and radiographic imaging. The aim of this multicenter study was to monitor disease progression in patients with chronic periodontitis during a 24-mo follow-up program and to evaluate the amount of bacteria in saliva and corresponding IgG titers in serum for determining the diagnostic usefulness of each in indicating disease progression and stability. MATERIAL AND METHODS A total of 163 patients with chronic periodontitis who received trimonthly follow-up care were observed for 24 mo. The clinical parameters and salivary content of Porphyromonas gingivalis, Prevotella intermedia and Aggregatibacter actinomycetemcomitans were assessed using the modified Invader PLUS assay, and the corresponding serum IgG titers were measured using ELISA. The changes through 24 mo were analyzed using cut-off values calculated for each factor. One-way ANOVA or Fishers exact test was used to perform between-group comparison for the data collected. Diagnostic values were calculated using Fishers exact test. RESULTS Of the 124 individuals who completed the 24-mo monitoring phase, 62 exhibited periodontitis progression, whereas 62 demonstrated stable disease. Seven patients withdrew because of acute periodontal abscess. The ratio of P. gingivalis to total bacteria and the combination of P. gingivalis counts and IgG titers against P. gingivalis were significantly related to the progression of periodontitis. The combination of P. gingivalis ratio and P. gingivalis IgG titers was significantly associated with the progression of periodontitis (p = 0.001, sensitivity = 0.339, specificity = 0.790). CONCLUSIONS It is suggested that the combination of P. gingivalis ratio in saliva and serum IgG titers against P. gingivalis may be associated with the progression of periodontitis.

Nicotine suppresses bone sialoprotein gene expression

BACKGROUND AND OBJECTIVE Tobacco smoking is a risk factor for periodontitis and osteoporosis. Nicotine is a major component of tobacco, and has been reported to inhibit proliferation and differentiation of osteoblasts. Bone sialoprotein (BSP) is a mineralized tissue-specific protein expressed by differentiated osteoblasts that appears to function in the initial mineralization of bone. The purpose of this study was to determine the effects of nicotine on bone metabolism. MATERIAL AND METHODS We used rat osteobast-like UMR106 and ROS 17/2.8 cells and rat stromal bone marrow RBMC-D8 cells. To determine the molecular basis of the transcriptional regulation of the BSP gene by nicotine, we conducted Northern hybridization, transient transfection analyses with chimeric constructs of the BSP gene promoter linked to a luciferase reporter gene and gel mobility shift assays. RESULTS Nicotine (250 microg/mL) decreased the BSP mRNA levels at 12 and 24 h in UMR106 and ROS 17/2.8 cells. From transient transfection assays using various sized BSP promoter-luciferase constructs, nicotine decreased the luciferase activities of the construct, including the promoter sequence nucleotides -116 to +60, in UMR106 and RBMC-D8 cells. Nicotine decreased the nuclear protein binding to the cAMP response element (CRE), fibroblast growth factor 2 response element (FRE) and homeodomain protein-binding site (HOX) at 12 and 24 h. CONCLUSION This study indicates that nicotine suppresses BSP transcription mediated through CRE, FRE and HOX elements in the proximal promoter of the rat BSP gene.

Prevalence and risk factors for peri-implant diseases in Japanese adult dental patients

We investigated the prevalences and risk factors for peri-implant diseases in Japanese adult dental patients attending a follow-up visit at dental hospitals or clinics as part of their maintenance program. This cross-sectional multicenter study enrolled patients with dental implants who attended regular check-ups as part of a periodontal maintenance program during the period from October 2012 through September 2013. Patients with implants with at least 3 years of loading time were included in the study. The condition of peri-implant tissue was examined and classified into the following categories: healthy, peri-implant mucositis, and peri-implantitis. Patients were also evaluated for implant risk factors. A total of 267 patients (110 men, 157 women; mean age: 62.5 ± 10.7 years) were analyzed. The prevalence of patient-based peri-implant mucositis was 33.3% (n = 89), and the prevalence of peri-implantitis was 9.7% (n = 26). Poor oral hygiene and a history of periodontitis were strong risk factors for peri-implant disease. The present prevalences were lower than those previously reported. The quality of periodontal therapy before and after implant installation and patient compliance and motivation, as indicated by plaque control level, appear to be important in maintaining peri-implant tissue health.

Protamine stimulates bone sialoprotein gene expression.

Protamine is a small, arginine-rich, nuclear protein that replaces histone late in the haploid phase of spermatogenesis and is believed to be essential for sperm head condensation and DNA stabilization. Protamine has many biological activities and has roles in hematopoiesis, immune responses, the nervous system and bone metabolism. Bone sialoprotein (BSP) is a mineralized connective tissue-specific protein expressed in differentiated osteoblasts that appears to function in the initial mineralization of bone. Protamine (71.35 ng/ml) increased BSP mRNA levels by 6h in osteoblast-like ROS 17/2.8 cells. In a transient transfection assay, protamine (71.35 ng/ml) increased luciferase activity of the construct (-116 to +60) in ROS 17/2.8 cells and rat bone marrow stromal cells. Luciferase activities induced by protamine were blocked by protein kinase A, tyrosine kinase and ERK1/2 inhibitors. Introduction of 2 bp mutations to the luciferase constructs showed that the effects of protamine were mediated by a cAMP response element (CRE), a fibroblast growth factor 2 response element (FRE) and a homeodomain protein-binding site (HOX). Gel shift analyses showed that protamine (71.35 ng/ml) increased the nuclear protein binding to CRE, FRE and HOX. CREB, phospho-CREB, c-Fos, c-Jun, JunD and Fra2 antibodies disrupted the formation of CRE-protein complexes. Dlx5, Msx2, Runx2 and Smad1 antibodies disrupted FRE- and HOX-protein complex formations. These studies demonstrate that protamine induces BSP transcription by targeting CRE, FRE and HOX sites in the proximal promoter of the rat BSP gene. Moreover, phospho-CREB, c-Fos, c-Jun, JunD, Fra2, Dlx5, Msx2, Runx2 and Smadl transcription factors appear to be key regulators of protamine effects on BSP transcription.

Evaluation of a novel immunochromatographic device for rapid and accurate clinical detection of Porphyromonas gingivalis in subgingival plaque

UNLABELLED An important goal for the improved diagnosis and management of infectious and inflammatory diseases, such as periodontitis, is the development of rapid and accurate technologies for the decentralized detection of bacterial pathogens. The aim of this prospective multicenter study was to evaluate the clinical use of a novel immunochromatographic device with monoclonal antibodies for the rapid point-of-care detection and semi-quantification of Porphyromonas gingivalis in subgingival plaque. Sixty-three patients with chronic periodontitis and 28 periodontally healthy volunteers were subjected to clinical and microbiological examinations. Subgingival plaque samples were analyzed for the presence of P. gingivalis using a novel immunochromatography based device DK13-PG-001, designed to detect the 40k-outer membrane protein of P. gingivalis, and compared with a PCR-Invader method. In the periodontitis group, a significant strong positive correlation in detection results was found between the test device score and the PCR-Invader method (Spearman rank correlation, r=0.737, p<0.0001). The sensitivity, specificity, and positive and negative predictive values of the test device were 96.2%, 91.8%, 90.4% and 96.7%, respectively. The detection threshold of the test device was determined to be approximately 10(4) (per two paper points). There were significant differences in the bacterial counts by the PCR-Invader method among groups with different ranges of device scores. With a cut-off value of ≥0.25 in device score, none of periodontally healthy volunteers were tested positive for the subgingival presence of P. gingivalis, whereas 76% (n=48) of periodontitis subjects were tested positive. There was a significant positive correlation between device scores for P. gingivalis and periodontal parameters including probing pocket depth and clinical attachment level (r=0.317 and 0.281, respectively, p<0.01). The results suggested that the DK13-PG-001 device kit can be effectively used for rapid, chair-side detection and semi-quantification of P. gingivalis in subgingival plaque. TRIAL REGISTRATION UMIN Clinical Trials Registry (UMIN-CTR) UMIN000011943.

Effects of porcine 25 kDa amelogenin and its proteolytic derivatives on bone sialoprotein expression

BACKGROUND AND OBJECTIVE Amelogenins are hydrophobic proteins that are the major component of developing enamel. Enamel matrix derivative has been used for periodontal regeneration. Bone sialoprotein is an early phenotypic marker of osteoblast differentiation. In this study, we examined the ability of porcine amelogenins to regulate bone sialoprotein transcription. MATERIAL AND METHODS To determine the molecular basis of the transcriptional regulation of the bone sialoprotein gene by amelogenins, we conducted northern hybridization, transient transfection analyses and gel mobility shift assays using the osteoblast-like ROS 17/2.8 cells. RESULTS Amelogenins (100 ng/mL) up-regulated bone sialoprotein mRNA at 3 h, with maximal mRNA expression occurring at 12 h (25 and 20 kDa) and 6 h (13 and 6 kDa). Amelogenins (100 ng/mL, 12 h) increased luciferase activities in pLUC3 (nucleotides -116 to +60), and 6 kDa amelogenin up-regulated pLUC4 (nucleotides -425 to +60) activity. The tyrosine kinase inhibitor inhibited amelogenin-induced luciferase activities, whereas the protein kinase A inhibitor abolished 25 kDa amelogenin-induced bone sialoprotein transcription. The effects of amelogenins were abrogated by 2-bp mutations in the fibroblast growth factor 2 response element (FRE). Gel-shift assays with radiolabeled FRE, homeodomain-protein binding site (HOX) and transforming growth factor-beta1 activation element (TAE) double-strand oligonucleotides revealed increased binding of nuclear proteins from amelogenin-stimulated ROS 17/2.8 cells at 3 h (25 and 13 kDa) and 6 h (20 and 6 kDa). CONCLUSION These results demonstrate that porcine 25 kDa amelogenin and its proteolytic derivatives stimulate bone sialoprotein transcription by targeting FRE, HOX and TAE in the bone sialoprotein gene promoter, and that full-length amelogenin and amelogenin cleavage products are able to regulate bone sialoprotein transcription via different signaling pathways.

Transcriptional regulation of amelotin gene by proinflammatory cytokines in gingival fibroblasts

Abstract Amelotin (AMTN) is a secreted protein expressed during the late stages of enamel formation and in the junctional epithelium. Among many differentially expressed genes, we found significantly increased AMTN mRNA level in inflamed gingiva by DNA microarray. The inductions of AMTN mRNA expressions in inflamed gingiva and human gingival fibroblasts (HGF) were confirmed by real-time polymerase chain reaction. To determine the molecular basis of the expression of AMTN and its regulation by proinflammatory cytokines, we have isolated and characterized the promoter region of mouse AMTN gene. Transient transfection assays were performed using luciferase constructs including mouse AMTN gene promoter. Interleukin-1β, Interleukin-6 and tumor necrosis factor-α induced AMTN mRNA levels in HGF. These cytokines increased the luciferase activities of the AMTN promoter constructs in HGF. The results suggest that proinflammatory cytokines induce AMTN gene transcription and a role for AMTN in gingival inflammation.

Localization and expression pattern of amelotin, odontogenic ameloblast-associated protein and follicular dendritic cell-secreted protein in the junctional epithelium of inflamed gingiva

The purpose of this study is to elucidate the localization of amelotin (AMTN), odontogenic ameloblast-associated protein (ODAM) and follicular dendritic cell-secreted protein (FDC-SP) at the junctional epithelium (JE) in Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans infected mice and inflamed and non-inflamed human gingiva. We performed immunostaining to determine the localization and expression pattern of AMTN, ODAM and FDC-SP. AMTN, ODAM and FDC-SP in A. actinomycetemcomitans infected mice did not change dramatically compared with non-infected mice. AMTN and FDC-SP expressions were observed stronger in P. gingivalis infected mice at early stage. However, at the following stage, the coronal part of the AMTN expression disappeared from the JE, and FDC-SP expression decreased due to severe inflammation by P. gingivalis. ODAM expressed internal and external basal lamina, and the expression increased not only at early stage but also at the following stage in the inflammatory JE induced by P. gingivalis. In the human gingival tissues, AMTN was detected at the surface of the sulcular epithelium and JE in the non-inflamed and inflamed gingiva, and the localization did not change the process of inflammation. ODAM and FDC-SP were more widely detected at the sulcular epithelium and JE in the non-inflamed gingiva. In the inflamed gingiva, localization of ODAM and FDC-SP was spread into the gingival epithelium, compared to AMTN. These studies demonstrated that the expression pattern of AMTN, ODAM and FDC-SP at the JE were changed during inflammation process and these three proteins might play an important role in the resistance to inflammation.