T. Kajikawa

Effects of L-ascorbic acid 2-phosphate magnesium salt on the properties of human gingival fibroblasts.

BACKGROUND AND OBJECTIVE L-Ascorbic acid 2-phosphate magnesium salt (APM) is an L-ascorbic acid (AsA) derivative developed to improve AsA stability and display effective biochemical characteristics. This study aimed to investigate the effects of APM on the functions and properties of human gingival fibroblasts with respect to the prevention of periodontal disease in comparison with those of AsA. MATERIAL AND METHODS Human gingival fibroblasts were incubated in the presence or absence of APM or L-ascorbic acid sodium salt (AsANa). Intracellular AsA was analysed by HPLC. Collagen synthesis was measured by ELISA and real-time RT-PCR. Intracellular reactive oxygen species (ROS) induced by hydrogen peroxide (H(2)O(2)) were quantified using a fluorescence reagent, and cell damage was estimated with calcein acetoxymethyl ester. Furthermore, intracellular ROS induced by tumor necrosis factor-α (TNF-α) were quantified, and expression of TNF-α-induced interleukin-8 expression, which increases due to inflammatory reactions, was measured by ELISA and real-time RT-PCR. RESULTS APM remarkably and continuously enhanced intracellular AsA and promoted type 1 collagen synthesis and mRNA expression. Furthermore, APM decreased cell damage through the suppression of H(2)O(2)-induced intracellular ROS and inhibited interleukin-8 production through the suppression of TNF-α-induced intracellular ROS. These effects of APM were superior to those of AsANa. CONCLUSION These results suggest that APM is more effective than AsANa in terms of intake, collagen synthesis, decreasing cell damage and inhibiting interleukin-8 expression in human gingival fibroblasts. This suggests that local application of APM can help to prevent periodontal disease.

Suppressive effects of nicotine on the cytodifferentiation of murine periodontal ligament cells.

OBJECTIVES Tobacco smoking has been suggested to be one of the important risk factors of developing periodontal disease. Although epidemiological studies have shown the detrimental effects of smoking on periodontal disease, the effects of smoke compounds on gingival tissue are not well understood. The aim of this study was to evaluate the effects of nicotine, which is the major component of the thousands of chemicals that constitute cigarette smoke, for cytodifferentiation of murine periodontal ligament (MPDL) cell. MATERIALS AND METHODS Expression of nAChR subunits on MPDL cells was examined using RT-PCR. The effects of nicotine on gene expression of extracellular matrices and osteoblastic transcription factors were evaluated by quantitative RT-PCR. Mineralized nodule formation of nicotine-treated MPDL cells was characterized by alizarin red staining. RESULTS Murine periodontal ligament cells expressed several subunits of nAChR, which have functional calcium signals in response to nicotine. Gene expression of extracellular matrices and osteoblastic transcription factors were reduced in nicotine-treated MPDL cells. In addition, mineralized nodule formation was inhibited in MPDL cells in the presence of nicotine. CONCLUSION Our findings indicate that nicotine may negatively regulate the cytodifferentiation and mineralization of MPDL cells.

Characterization of a novel periodontal ligament-specific periostin isoform.

Periostin is a mesenchymal cell marker predominantly expressed in collagen-rich fibrous connective tissues, including heart valves, tendons, perichondrium, periosteum, and periodontal ligament (PDL). Knockdown of periostin expression in mice results in early-onset periodontitis and failure of cardiac healing after acute myocardial infarction, suggesting that periostin is essential for connective tissue homeostasis and regeneration. However, its role(s) in periodontal tissues has not yet been fully defined. In this study, we describe a novel human isoform of periostin (PDL-POSTN). Isoform-specific analysis by reverse-transcription polymerase chain-reaction (RT-PCR) revealed that PDL-POSTN was predominantly expressed in the PDL, with much lower expression in other tissues and organs. A PDL cell line transfected with PDL-POSTN showed enhanced alkaline phosphatase (ALPase) activity and calcified nodule formation, compared with cells transfected with the full-length periostin isoform. A neutralizing antibody against integrin-αv inhibited both ALPase activity and calcified nodule formation in cells transfected with PDL-POSTN. Furthermore, co-immunoprecipitation assays revealed that PDL-POSTN bound to integrin αvβ3 more strongly than the common isoform of periostin, resulting in strong activation of the integrin αvβ3-focal adhesion kinase (FAK) signaling pathway. These results suggest that PDL-POSTN positively regulates cytodifferentiation and mineralization in PDL cells through integrin αvβ3.

Inhibitory Effects of PLAP-1/asporin on Periodontal Ligament Cells

PLAP-1/asporin is an extracellular matrix protein that is predominantly expressed in the human periodontal ligament (PDL) and has an aspartic acid (D) repeat polymorphism in its N-terminal region. In this study, we hypothesized that the D repeat polymorphism of PLAP-1/asporin may affect the physiological functions of periodontal ligaments. We established periodontal ligament cell lines transfected with the D13- or D14-PLAP-1 gene. Alkaline phosphatase staining and alizarin red staining revealed that the cytodifferentiation of the D14-PLAP-1-expressing PDL cells was more repressed compared with that of the D13-PLAP-1-expressing cells. Furthermore, the D14-PLAP-1-expressing cells inhibited BMP-2-induced cytodifferentiation more strongly than did the D13-PLAP-1-expressing cells. Western blotting analysis and luciferase assay revealed that D14-PLAP-1 suppressed BMP-2 signal transduction more efficiently than did D13-PLAP-1, and co-immunoprecipitation demonstrated the stronger affinity of the D14-PLAP-1 protein to BMP-2 compared with the D13-PLAP-1 protein. Analysis of these data suggests that the D repeat polymorphism of PLAP-1/asporin has a significant influence on the functions of PDL cells.

PLAP-1/Asporin Positively Regulates FGF-2 Activity

PLAP-1 is an extracellular matrix protein that is predominantly expressed in the periodontal ligament within periodontal tissue. It was previously revealed that PLAP-1 negatively regulates bone morphogenetic protein 2 and transforming growth factor β activity through direct interactions. However, the interaction between PLAP-1 and other growth factors has not been defined. Here, we revealed that PLAP-1 positively regulates the activity of fibroblast growth factor 2 (FGF-2), a critical growth factor in tissue homeostasis and repair. In this study, we isolated mouse embryonic fibroblasts (MEFs) from Plap-1-/- mice generated in our laboratory. Interestingly, Plap-1-/- MEFs exhibited enhanced responses to bone morphogenetic protein 2 but defective responses to FGF-2, and Plap-1 transfection into Plap-1-/- MEFs rescued these defective responses. In addition, binding assays revealed that PLAP-1 promotes FGF-2–FGF receptor 1 (FGFR1) complex formation by direct binding to FGF-2. Immunocytochemistry analyses revealed colocalization of PLAP-1 and FGF-2 in wild-type MEFs and reduced colocalization of FGF-2 and FGFR1 in Plap-1-/- MEFs compared with wild-type MEFs. Taken together, PLAP-1 positively regulates FGF-2 activity through a direct interaction. Extracellular matrix–growth factor interactions have considerable effects; thus, this approach may be useful in several regenerative medicine applications.

Role of ferritin in the cytodifferentiation of periodontal ligament cells.

This study investigated the expression and functions of ferritin, which is involved in osteoblastogenesis, in the periodontal ligament (PDL). The PDL is one of the most important tissues for maintaining the homeostasis of teeth and tooth-supporting tissues. Real-time PCR analyses of the human PDL revealed abundant expression of ferritin light polypeptide (FTL) and ferritin heavy polypeptide (FTH), which encode the highly-conserved iron storage protein, ferritin. Immunohistochemical staining demonstrated predominant expression of FTL and FTH in mouse PDL tissues in vivo. In in vitro-maintained mouse PDL cells, FTL and FTH expressions were upregulated at both the mRNA and protein levels during the course of cytodifferentiation and mineralization. Interestingly, stimulation of PDL cells with exogenous apoferritin (iron-free ferritin) increased calcified nodule formation and alkaline phosphatase activity as well as the mRNA expressions of mineralization-related genes during the course of cytodifferentiation. On the other hand, RNA interference of FTH inhibited the mineralized nodule formation of PDL cells. This is the first report to demonstrate that ferritin is predominantly expressed in PDL tissues and positively regulates the cytodifferentiation and mineralization of PDL cells.

PLAP-1/Asporin Regulates TLR2- and TLR4-induced Inflammatory Responses

Periodontal ligament–associated protein 1 (PLAP-1)/asporin is an extracellular matrix protein preferentially expressed in periodontal ligaments. PLAP-1/asporin inhibits the cytodifferentiation and mineralization of periodontal ligament cells and has important roles in the maintenance of periodontal tissue homeostasis. However, the involvement of PLAP-1/asporin in inflammatory responses during periodontitis is poorly understood. This study hypothesized that PLAP-1/asporin might affect the pathogenesis of periodontitis by regulating periodontopathic bacteria-induced inflammatory responses. Proinflammatory cytokine expression induced by Toll-like receptor 2 (TLR2) and TLR4 was significantly downregulated when PLAP-1/asporin was overexpressed in periodontal ligament cells. Similarly, recombinant PLAP-1/asporin inhibited TLR2- and TLR4-induced proinflammatory cytokine expression in macrophages. We also confirmed that NF-κB activity induced by TLR2 and TLR4 signaling was suppressed by the addition of recombinant PLAP-1/asporin. Furthermore, IκB kinase α degradation induced by TLR4 was reduced by PLAP-1/asporin. Immunoprecipitation assays demonstrated the binding abilities of PLAP-1/asporin to both TLR2 and TLR4. Taken together, PLAP-1/asporin negatively regulates TLR2- and TLR4-induced inflammatory responses through direct molecular interactions. These findings indicate that PLAP-1/asporin has a defensive role in periodontitis lesions by suppressing pathophysiologic TLR signaling and that the modulating effects of PLAP-1/asporin might be useful for periodontal treatments.

Iron plays a key role in the cytodifferentiation of human periodontal ligament cells

BACKGROUND AND OBJECTIVE The periodontal ligament (PDL) is vital to maintaining the homeostasis of the tooth and periodontal tissue. The influence of iron levels on the cytodifferentiation of PDL cells has not been studied, despite evidence that iron overload or deficiency can have adverse effects on alveolar bone density. The purpose of this study was to examine the effects of altered iron levels on cytodifferentiation in human PDL cells. MATERIAL AND METHODS Human PDL cells were incubated with culture media supplemented with 10-50 μm ammonium ferric citrate or 5 μm deferoxamine (an iron chelator) during differentiation. Intracellular iron status was assessed by measuring changes in the expression of ferritin RNA and protein. PDL cell differentiation and function were evaluated by measuring osteoblast differentiation gene markers and the capacity of cultures to form mineralized nodules. RESULTS Iron accumulation resulted in upregulation of light and heavy chain ferritin proteins. Concurrently, osteoblast differentiation gene markers and mineralized nodule formation were suppressed. Iron deficiency resulted in downregulation of light and heavy chain ferritin proteins, suppression of alkaline phosphatase activity and formation of mineralized nodules during PDL cell differentiation. CONCLUSION We conclude that iron is critical for normal cell differentiation of human PDL cells.

Transcriptome Reveals Cathepsin K in Periodontal Ligament Differentiation

Periodontal ligaments (PDLs) play an important role in remodeling the alveolar bond and cementum. Characterization of the periodontal tissue transcriptome remains incomplete, and an improved understanding of PDL features could aid in developing new regenerative therapies. Here, we aimed to generate and analyze a large human PDL transcriptome. We obtained PDLs from orthodontic treatment patients, isolated the RNA, and used a vector-capping method to make a complementary DNA library from >20,000 clones. Our results revealed that 58% of the sequences were full length. Furthermore, our analysis showed that genes expressed at the highest frequencies included those for collagen type I, collagen type III, and proteases. We also found 5 genes whose expressions have not been previously reported in human PDL. To access which of the highly expressed genes might be important for PDL cell differentiation, we used real-time polymerase chain reaction to measure their expression in differentiating cells. Among the genes tested, the cysteine protease cathepsin K had the highest upregulation, so we measured its relative expression in several tissues, as well as in osteoclasts, which are known to express high levels of cathepsin K. Our results revealed that PDL cells express cathepsin K at similar levels as osteoclasts, which are both expressed at higher levels than those of the other tissues tested. We also measured cathepsin K protein expression and enzyme activity during cell differentiation and found that both increased during this process. Immunocytochemistry experiments revealed that cathepsin K localizes to the interior of lysosomes. Last, we examined the effect of inhibiting cathepsin K during cell differentiation and found that cathepsin K inhibition stimulated calcified nodule formation and increased the levels of collagen type I and osteocalcin gene expression. Based on these results, cathepsin K seems to regulate collagen fiber accumulation during human PDL cell differentiation into hard tissue-forming cells.