Mice Lacking PLAP-1/Asporin Show Alteration of Periodontal Ligament Structures and Acceleration of Bone Loss in Periodontitis.

Periodontal ligament-associated protein 1 (PLAP-1), also known as Asporin, is an extracellular matrix protein expressed in the periodontal ligament and plays a crucial role in periodontal tissue homeostasis. Our previous research demonstrated that PLAP-1 may inhibit TLR2/4-mediated inflammatory responses, thereby exerting a protective function against periodontitis. However, the precise roles of PLAP-1 in the periodontal ligament (PDL) and its relationship to periodontitis have not been fully explored. In this study, we employed PLAP-1 knockout mice to investigate its roles and contributions to PDL tissue and function in a ligature-induced periodontitis model. Mandibular bone samples were collected from 10-week-old male C57BL/6 (WT) and PLAP-1 knockout (KO) mice. These samples were analyzed through micro-computed tomography (µCT) scanning, hematoxylin and eosin (HE) staining, picrosirius red staining, and fluorescence immunostaining using antibodies targeting extracellular matrix proteins. Additionally, the structure of the PDL collagen fibrils was examined using transmission electron microscopy (TEM). We also conducted tooth extraction and ligature-induced periodontitis models using both wild-type and PLAP-1 KO mice. PLAP-1 KO mice did not exhibit any changes in alveolar bone resorption up to the age of 10 weeks, but they did display an enlarged PDL space, as confirmed by µCT and histological analyses. Fluorescence immunostaining revealed increased expression of extracellular matrix proteins, including Col3, BGN, and DCN, in the PDL tissues of PLAP-1 KO mice. TEM analysis demonstrated an increase in collagen diameter within the PDL of PLAP-1 KO mice. In line with these findings, the maximum stress required for tooth extraction was significantly lower in PLAP-1 KO mice in the tooth extraction model compared to WT mice (13.89 N ± 1.34 and 16.51 N ± 1.31, respectively). In the ligature-induced periodontitis model, PLAP-1 knockout resulted in highly severe alveolar bone resorption, with a higher number of collagen fiber bundle tears and significantly more osteoclasts in the periodontium. Our results demonstrate that mice lacking PLAP-1/Asporin show alteration of periodontal ligament structures and acceleration of bone loss in periodontitis. This underscores the significant role of PLAP-1 in maintaining collagen fibrils in the PDL and suggests the potential of PLAP-1 as a therapeutic target for periodontal diseases.

Identification of disease-associate variants of aggressive periodontitis using genome-wide association studies.

Aggressive periodontitis (AgP), Stage III or IV and Grade C according to the new periodontitis classification, is characterized by the rapid destruction of periodontal tissues in the systemically healthy population and often causes premature tooth loss. The presence of familial aggregation suggests the involvement of genetic factors in the pathogenesis. However, the genes associated with the onset and progression of the disease and details of its pathogenesis have not yet been fully identified. In recent years, the genome-wide approach (GWAS), a comprehensive genome analysis method using bioinformatics, has been used to search for disease-related genes, and the results have been applied in genomic medicine for various diseases, such as cancer. In this review, we discuss GWAS in the context of AgP. First, we introduce the relationship between single-nucleotide polymorphisms (SNPs) and susceptibility to diseases and how GWAS is useful for searching disease-related SNPs. Furthermore, we summarize the recent findings of disease-related genes using GWAS on AgP inside and outside Japan and a possible mechanism of the pathogenesis of AgP based on available literature and our research findings. These findings will lead to advancements in the prevention, prognosis, and treatment of AgP.

CD40-CD40 ligand interaction between periodontal ligament cells and cementoblasts enhances periodontal tissue remodeling in response to mechanical stress.

OBJECTIVE: We analyzed the localization and expression of Cluster of differentiation 40 ligand (CD40L) in murine periodontal tissue applied with the orthodontic force to determine the CD40L-expressing cells under mechanical stress. Furthermore, we investigated whether CD40-CD40L interaction played an important role in transducing mechanical stress between periodontal ligament (PDL) cells and cementoblasts and remodeling the periodontal tissue for its homeostasis. BACKGROUND: PDL is a complex tissue that contains heterogeneous cell populations and is constantly exposed to mechanical stress, such as occlusal force. CD40 is expressed on PDL cells and upregulated under mechanical stress. However, whether its ligand, CD40L, is upregulated in periodontal tissue in response to mechanical stress, and which functions the CD40-CD40L interaction induces by converting the force to biological functions between the cement-PDL complex, are not fully understood. METHODS: The orthodontic treatment was applied to the first molars at the left side of the upper maxillae of mice using a nickel-titanium closed-coil spring. Immunohistochemistry was performed to analyze the localization of CD40L in the periodontal tissue under the orthodontic force. Human cementoblasts (HCEM) and human PDL cells were stretched in vitro and analyzed CD40L and CD40 protein expression using flow cytometry. A GFP-expressing CD40L plasmid vector was transfected into HCEM (CD40L-HCEM). CD40L-HCEM was co-cultured with human PDL cells with higher alkaline phosphatase (ALP) activity (hPDS) or lower ALP (hPDF). After co-culturing, cell viability and proliferation were analyzed by propidium iodide (PI) staining and bromodeoxyuridine (BrdU) assay. Furthermore, the mRNA expression of cytodifferentiation- and extracellular matrix (ECM)-related genes was analyzed by real-time PCR. RESULTS: Immunohistochemistry demonstrated that CD40L was induced on the cells present at the cementum surface in periodontal tissue at the tension side under the orthodontic treatment in mice. The flow cytometry showed that the in vitro-stretching force upregulated CD40L protein expression on HCEM and CD40 protein expression on human PDL cells. Co-culturing CD40L-HCEM with hPDF enhanced cell viability and proliferation but did not alter the gene expression related to cytodifferentiation and ECM. In contrast, co-culturing CD40L-HCEM with hPDS upregulated cytodifferentiation- and ECM-related genes but did not affect cell viability and proliferation. CONCLUSION: We revealed that in response to a stretching force, CD40L expression was induced on cementoblasts. CD40L on cementoblasts may interact with CD40 on heterogeneous PDL cells at the necessary time and location, inducing cell viability, proliferation, and cytodifferentiation, maintaining periodontal tissue remodeling and homeostasis.

Lysosomal acid lipase regulates bioenergetic process during the cytodifferentiation of human periodontal ligament cells.

Lipid metabolism is one of energy metabolic pathways that produce adenosine triphosphate (ATP). In this pathway, lysosomal acid lipase (LAL) encoded by Lipase A (LIPA), plays an important role in catalyzing lipids to fatty acids (FAs), which drive oxidative phosphorylation (OXPHOS) and generate ATP. Previously, we found that a LIPA single nucleotide polymorphism rs143793106, which decreases the LAL activity, suppressed the cytodifferentiation of human periodontal ligament (HPDL) cells. However, the mechanisms underlying that suppression are still not fully clarified. Thus, we aimed to investigate the mechanisms regulating the cytodifferentiation of HPDL cells by LAL in terms of energy metabolism. We performed the osteogenic induction of HPDL cells with or without Lalistat-2, a LAL inhibitor. To visualize lipid droplet (LD) utilization, we performed confocal microscopy on HPDL cells. We also performed real-time PCR to analyze the gene expression of calcification-related and metabolism-related genes. Furthermore, we measured the ATP production rate from two major energy production pathways, OXPHOS and glycolysis, and OXPHOS-related parameters of HPDL cells during their cytodifferentiation. We found that LDs were utilized during the cytodifferentiation of HPDL cells. Alkaline phosphatase (ALPL), collagen type 1 alpha 1 chain (COL1A1), ATP synthase F1 subunit alpha (ATP5F1A), and carnitine palmitoyltransferase 1A (CPT1A) mRNA expressions were upregulated, whereas lactate dehydrogenase A (LDHA) mRNA expression was downregulated. Additionally, total ATP production rate was significantly increased. In contrast, in the presence of Lalistat-2, LD utilization was inhibited and ALPL, COL1A1, and ATP5F1A mRNA expression was downregulated. Additionally, ATP production rate and spare respiratory capacity of the OXPHOS pathway were decreased in HPDL cells during their cytodifferentiation. Collectively, the defect of LAL in HPDL cells decreased LD utilization and OXPHOS capacity, resulting in reduced energy to sustain the adequate ATP production required for the cytodifferentiation of HPDL cells. Thus, LAL is important for periodontal tissue homeostasis as a regulator of bioenergetic process of HPDL cells.

Lipase-a single-nucleotide polymorphism rs143793106 is associated with increased risk of aggressive periodontitis by negative influence on the cytodifferentiation of human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Aggressive periodontitis (AgP) is characterized by general health and rapid destruction of periodontal tissue. The familial aggregation of this disease highlights the involvement of genetic factors in its pathogeny. We conducted a genome-wide association study (GWAS) to identify AgP-related genes in a Japanese population, and the lipid metabolism-related gene, lipase-a, lysosomal acid type (LIPA), was suggested as an AgP candidate gene. However, there is no report about the expression and function(s) of LIPA in periodontal tissue. Hence, we studied the involvement of how LIPA and its single-nucleotide polymorphism (SNP) rs143793106 in AgP by functional analyses of LIPA and its SNP in human periodontal ligament (HPDL) cells. MATERIALS AND METHODS: GWAS was performed using the genome database of Japanese AgP patients, and the GWAS result was confirmed using Sanger sequencing. We examined the mRNA expression level of LIPA and the protein expression level of the encoded protein lysosomal acid lipase (LAL) in periodontium-composing cells using conventional and real-time polymerase chain reaction (PCR) and western blotting, respectively. Lentiviral vectors expressing LIPA wild-type (LIPA WT) and LIPA SNP rs143793106 (LIPA mut) were transfected into HPDL cells. Western blotting was performed to confirm the transfection. LAL activity of transfected HPDL cells was determined using the lysosomal acid lipase activity assay. Transfected HPDL cells were cultured in mineralization medium. During the cytodifferentiation of transfected HPDL cells, mRNA expression of calcification-related genes, alkaline phosphatase (ALPase) activity and calcified nodule formation were assessed using real-time PCR, ALPase assay, and alizarin red staining, respectively. RESULTS: The GWAS study identified 11 AgP-related candidate genes, including LIPA SNP rs143793106. The minor allele frequency of LIPA SNP rs143793106 in AgP patients was higher than that in healthy subjects. LIPA mRNA and LAL protein were expressed in HPDL cells; furthermore, they upregulated the cytodifferentiation of HPDL cells. LAL activity was lower in LIPA SNP-transfected HPDL cells during cytodifferentiation than that in LIPA WT-transfected HPDL cells. In addition, ALPase activity, calcified nodule formation, and calcification-related gene expression levels were lower during cytodifferentiation in LIPA SNP-transfected HPDL cells than those in LIPA WT-transfected HPDL cells. CONCLUSION: LIPA, identified as an AgP-related gene in a Japanese population, is expressed in HPDL cells and is involved in regulating cytodifferentiation of HPDL cells. LIPA SNP rs143793106 suppressed cytodifferentiation of HPDL cells by decreasing LAL activity, thereby contributing to the development of AgP.

Evaluation of periodontitis-related tooth loss according to the new 2018 classification of periodontitis.

The new 2018 classification of periodontal diseases is reported to be related to tooth loss due to periodontal disease (TLPD) during supportive periodontal therapy (SPT). However, few reports have evaluated this relationship for Asians or have analyzed the association of the new classification and TLPD by distinguishing between active periodontal therapy (APT) and SPT. In this study, we retrospectively applied the new classification to 607 Japanese periodontitis patients and examined the relationship between the new classification and annual TLPD rates per patient during the respective periods. TLPD rates were higher in patients in stage IV and/or grade C during both APT and SPT. TLPD during SPT was not associated with the presence or absence of TLPD during APT. Multivariate analysis revealed that stage IV and grade C as independent variables were significantly associated with the number of instances of TLPD not only during the total treatment period, but also during APT or SPT. Our results suggest that the new classification has a significantly strong association with TLPD during both APT and SPT, and that patients diagnosed with stage IV and/or grade C periodontitis had a higher risk of TLPD during both periods.

Periodontal tissue regeneration by transplantation of autologous adipose tissue-derived multi-lineage progenitor cells.

Periodontitis is a chronic inflammatory disease that destroys tooth-supporting periodontal tissue. Current periodontal regenerative therapies have unsatisfactory efficacy; therefore, periodontal tissue engineering might be established by developing new cell-based therapies. In this study, we evaluated the safety and efficacy of adipose tissue-derived multi-lineage progenitor cells (ADMPC) autologous transplantation for periodontal tissue regeneration in humans. We conducted an open-label, single-arm exploratory phase I clinical study in which 12 periodontitis patients were transplanted with autologous ADMPCs isolated from subcutaneous adipose tissue. Each patient underwent flap surgery during which autologous ADMPCs were transplanted into the bone defect with a fibrin carrier material. Up to 36 weeks after transplantation, we performed a variety of clinical examinations including periodontal tissue inspection and standardized dental radiographic analysis. A 36-week follow-up demonstrated no severe transplantation-related adverse events in any cases. ADMPC transplantation reduced the probing pocket depth, improved the clinical attachment level, and induced neogenesis of alveolar bone. Therapeutic efficiency was observed in 2- or 3-walled vertical bone defects as well as more severe periodontal bone defects. These results suggest that autologous ADMPC transplantation might be an applicable therapy for severe periodontitis by inducing periodontal regeneration.

Mouse Model of Loeys-Dietz Syndrome Shows Elevated Susceptibility to Periodontitis via Alterations in Transforming Growth Factor-Beta Signaling.

Loeys-Dietz syndrome (LDS) is a syndromic connective tissue disorder caused by a heterozygous missense mutation in genes that encode transforming growth factor (TGF)-ß receptor (TGFBR) 1 and 2. We encountered a patient with LDS, who had severe periodontal tissue destruction indicative of aggressive periodontitis. The patient had a missense mutation in the glycine and serine-rich domain of TGFBR1 exon 3. This G-to-T mutation at base 563 converted glycine to valine. We established an LDS model knock-in mouse that recapitulated the LDS phenotype. Homozygosity of the mutation caused embryonic lethality and heterozygous knock-in mice showed distorted and ruptured elastic fibers in the aorta at 24 weeks of age and died earlier than wildtype (WT) mice. We stimulated mouse embryonic fibroblasts (MEFs) from the knock-in mouse with TGF-ß and examined their responses. The knock-in MEFs showed downregulated Serpine 1 mRNA expression and phosphorylation of Smad2 to TGF-ß compared with WT MEFs. To clarify the influence of TGF-ß signaling abnormalities on the pathogenesis or progression of periodontitis, we performed pathomolecular analysis of the knock-in mouse. There were no structural differences in periodontal tissues between WT and LDS model mice at 6 or 24 weeks of age. Micro-computed tomography revealed no significant difference in alveolar bone resorption between WT and knock-in mice at 6 or 24 weeks of age. However, TGF-ß-related gene expression was increased significantly in periodontal tissues of the knock-in mouse compared with WT mice. Next, we assessed a mouse periodontitis model in which periodontal bone loss was induced by oral inoculation with the bacterial strain Porphyromonas gingivalis W83. After inoculation, we collected alveolar bone and carried out morphometric analysis. P. gingivalis-induced alveolar bone loss was significantly greater in LDS model mice than in WT mice. Peritoneal macrophages isolated from Tgfbr1 G188V/+ mice showed upregulation of inflammatory cytokine mRNA expression induced by P. gingivalis lipopolysaccharide compared with WT macrophages. In this study, we established an LDS mouse model and demonstrated that LDS model mice had elevated susceptibility to P. gingivalis-induced periodontitis, probably through TGF-ß signal dysfunction. This suggests that TGF-ß signaling abnormalities accelerate the pathogenesis or progression of periodontitis.

Periodontal inflamed surface area is associated with hs-CRP in septuagenarian Japanese adults in cross-sectional findings from the SONIC study.

Periodontal disease is a chronic inflammatory condition that affects various peripheral organs. The periodontal inflamed surface area (PISA) quantifies periodontitis severity and the spread of inflammatory wounds. This study aimed to investigate the association between PISA and high-sensitivity C-reactive protein (hs-CRP), a systemic inflammation marker. This study included 250 community-dwelling septuagenarians (69-71 years). We collected information on their medical (e.g., diabetes and dyslipidemia) and dental examinations (e.g., measurement of the probing pocket depth). Generalized linear model analysis was used to explore the association between PISA and hs-CRP levels. There was a significant difference in hs-CRP levels between groups with PISA ≥ 500 and < 500 (p = 0.017). Moreover, the generalized linear model analysis revealed a significant association between PISA and hs-CRP levels (risk ratio = 1.77; p = 0.033) even after adjusting other factors. Further, we found a correlation between PISA and hs-CRP (Spearman's rank correlation coefficient, rs = 0.181; p = 0.023). Our findings suggest that PISA is an effective index for estimating the effect of periodontitis on the whole body, enabling medical-dental cooperation.

Zbp1-positive cells are osteogenic progenitors in periodontal ligament.

Periodontal ligament (PDL) possesses a stem/progenitor population to maintain the homeostasis of periodontal tissue. However, transcription factors that regulate this population have not yet been identified. Thus, we aimed to identify a molecule related to the osteogenic differentiation of PDL progenitors using a single cell-based strategy in this study. We first devised a new protocol to isolate PDL cells from the surface of adult murine molars and established 35 new single cell-derived clones from the PDL explant. Among these clones, six clones with high (high clones, n = 3) and low (low clones, n = 3) osteogenic potential were selected. Despite a clear difference in the osteogenic potential of these clones, no significant differences in their cell morphology, progenitor cell marker expression, alkaline phosphatase activity, proliferation rate, and differentiation-related gene and protein expression were observed. RNA-seq analysis of these clones revealed that Z-DNA binding protein-1 (Zbp1) was significantly expressed in the high osteogenic clones, indicating that Zbp1 could be a possible marker and regulator of the osteogenic differentiation of PDL progenitor cells. Zbp1-positive cells were distributed sparsely throughout the PDL. In vitro Zbp1 expression in the PDL clones remained at a high level during osteogenic differentiation. The CRISPR/Cas9 mediated Zbp1 knockout in the high clones resulted in a delay in cell differentiation. On the other hand, Zbp1 overexpression in the low clones promoted cell differentiation. These findings suggested that Zbp1 marked the PDL progenitors with high osteogenic potential and promoted their osteogenic differentiation. Clarifying the mechanism of differentiation of PDL cells by Zbp1 and other factors in future studies will facilitate a better understanding of periodontal tissue homeostasis and repair, possibly leading to the development of novel therapeutic measures.

Effects of oxidative stress-induced increases in Zn2+ concentrations in human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Previous studies have reported that oxidative stress increases intracellular Zn2+ concentrations and induces cytotoxicity. However, no studies have investigated whether oxidative stress induces such changes in periodontal tissue cells. In the present study, we investigated the effect of oxidative stress on intracellular Zn2+ concentration in periodontium constituent cells and its potential relationship with periodontal disease. METHODS: We analyzed changes in intracellular Zn2+ concentrations in human gingival epithelial (epi4) cells treated with hydrogen peroxide (H2 O2 ). The fluorescent probes FluoZin-3 AM and CellTracker Green CMFDA were used to detect intracellular Zn2+ and thiol groups, respectively. Western blot analyses, luciferase reporter assays, and real-time polymerase chain reaction (PCR) analyses were performed to examine the effect of intracellular Zn2+ on epi4 cells. RESULTS: H2 O2 treatment increased intracellular concentrations of Zn2+ in epi4 cells by facilitating the movement of Zn2+ from cellular nonprotein thiols to the cytoplasm and promoting cell membrane permeability to Zn2+ . Furthermore, H2 O2 -induced increases in intracellular Zn2+ activated the p38 cAMP response element-binding protein/mitogen-activated protein kinase (p38 CREB/MAPK) cascade, upregulated nuclear factor kappa B (NF-κB) DNA binding, and increased the expression of inflammatory cytokines and matrix metallopeptidase-9 (MMP-9). CONCLUSION: Increases in intracellular Zn2+ induced by oxidative stress activate signaling pathways involved in inflammation, potentially contributing to the progression of periodontal disease.

Association of periodontal disease with atherosclerosis in 70-year-old Japanese older adults.

Periodontal disease and arteriosclerotic disease are greatly affected by aging. In this study, the association of conventional risk factors and periodontal disease with atherosclerosis was longitudinally examined in Japanese older adults. Subjects in this study were 490 community-dwelling septuagenarians (69-71 years) randomly recruited from the Basic Resident Registry of urban or rural areas in Japan. At the baseline examination, all subjects underwent socioeconomic and medical interviews; medical examinations, including examinations for carotid atherosclerosis, hypertension, diabetes mellitus, and dyslipidemia; and conventional dental examinations, including a tooth count and measurement of probing pocket depth (PPD). After 3 years, 182 septuagenarians who had no atherosclerosis at the baseline examination were registered and received the same examination as at the baseline. In the re-examination conducted 3 years after the baseline survey, 131 (72.0%) of the 182 participants who had no atherosclerosis at the baseline examination were diagnosed with carotid atherosclerosis. Adjusting and analyzing the mutual relationships of the conventional risk factors for atherosclerosis by multiple logistic regression analysis for the 171 septuagenarians with a full set of data, the proportion of teeth with PPD ≥ 4 mm was independently related to the prevalence of atherosclerosis (odds ratio: 1.029, P < 0.022). This longitudinal study of Japanese older adults suggests that periodontal disease is associated with the onset/progression of atherosclerosis. Maintaining a healthy periodontal condition may be an important factor in preventing the development and progression of atherosclerosis.

Fibroblast growth factor-2 inhibits CD40-mediated periodontal inflammation.

Fibroblast growth factor-2 (FGF-2) stimulates periodontal regeneration by a broad spectrum of effects on periodontal ligament (PDL) cells, such as proliferation, migration, and production of extracellular matrix. A critical factor in the success of periodontal regeneration is the rapid resolution of inflammatory responses in the tissue. We explored an anti-inflammatory effect of FGF-2 during periodontal regeneration and healing. We found that FGF-2 on mouse periodontal ligament cells (MPDL22) markedly downregulated CD40 expression, a key player of inflammation. In addition, FGF-2 inhibited CD40 signaling by the non-canonical nuclear factor-kappa B2 (NFκB2) pathway, resulting in decreased production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which have the potential to recruit immune cells to inflamed sites. Furthermore, in vivo treatment of FGF-2 enhanced healing of skin wounds by counteracting the CD40-mediated inflammation. These results reveal that FGF-2 has an important function as a negative regulator of inflammation during periodontal regeneration and healing.

Identification of genetic risk factors of aggressive periodontitis using genomewide association studies in association with those of chronic periodontitis.

To identify the genetic risk factors for aggressive periodontitis (AgP), it is important to understand the progression and pathogenesis of AgP. The purpose of this review was to summarize the genetic risk factors for AgP identified through a case-control genomewide association study (GWAS) and replication study. The initial studies to identify novel AgP risk factors were potentially biased because they relied on previous studies. To overcome this kind of issue, an unbiased GWAS strategy was introduced to identify genetic risk factors for various diseases. Currently, three genes glycosyltransferase 6 domain containing 1 (GLT6D1), defensin α1 and α3 (DEFA1A3), and sialic acid-binding Ig-like lectin 5 (SIGLEC5) that reach the threshold for genomewide significance have been identified as genetic risk factors for AgP through a case-control GWAS.

Co-stimulatory function in primary germinal center responses: CD40 and B7 are required on distinct antigen-presenting cells.

T cell-dependent germinal center (GC) responses require coordinated interactions of T cells with two antigen-presenting cell (APC) populations, B cells and dendritic cells (DCs), in the presence of B7- and CD40-dependent co-stimulatory pathways. Contrary to the prevailing paradigm, we found unique cellular requirements for B7 and CD40 expression in primary GC responses to vaccine immunization with protein antigen and adjuvant: B7 was required on DCs but was not required on B cells, whereas CD40 was required on B cells but not on DCs in the generation of antigen-specific follicular helper T cells, antigen-specific GC B cells, and high-affinity class-switched antibody production. There was, in fact, no requirement for coexpression of B7 and CD40 on the same cell in these responses. Our findings support a substantially revised model for co-stimulatory function in the primary GC response, with crucial and distinct contributions of B7- and CD40-dependent pathways expressed by different APC populations and with important implications for understanding how to optimize vaccine responses or limit autoimmunity.

T cell-B cell thymic cross-talk: maintenance and function of thymic B cells requires cognate CD40-CD40 ligand interaction.

Thymic development requires bidirectional interaction or cross-talk between developing T cells and thymic stromal cells, a relationship that has been best characterized for the interaction between thymocytes and thymic epithelial cells. We have characterized in this article the requirement for similar cross-talk in the maintenance and function of thymic B cells, another population that plays a role in selection of developing thymic T cells. We found that maintenance of thymic B cells is strongly dependent on the presence of mature single-positive thymocytes and on the interactions of these T cells with specific Ag ligand. Maintenance of thymic B cell number is strongly dependent on B cell-autonomous expression of CD40, but not MHC class II, indicating that direct engagement of CD40 on thymic B cells is necessary to support their maintenance and proliferation. Thymic B cells can mediate negative selection of superantigen-specific, self-reactive, single-positive thymocytes, and we show that CD40 expression on B cells is critical for this negative selection. Cross-talk with thymic T cells is thus required to support the thymic B cell population through a pathway that requires cell-autonomous expression of CD40, and that reciprocally functions in negative selection of autoreactive T cells.

Role of mechanical stress-induced glutamate signaling-associated molecules in cytodifferentiation of periodontal ligament cells.

In this study, we analyzed the effects of tensile mechanical stress on the gene expression profile of in vitro-maintained human periodontal ligament (PDL) cells. A DNA chip analysis identified 17 up-regulated genes in human PDL cells under the mechanical stress, including HOMER1 (homer homolog 1) and GRIN3A (glutamate receptor ionotropic N-methyl-d-aspartate 3A), which are related to glutamate signaling. RT-PCR and real-time PCR analyses revealed that human PDL cells constitutively expressed glutamate signaling-associated genes and that mechanical stress increased the expression of these mRNAs, leading to release of glutamate from human PDL cells and intracellular glutamate signal transduction. Interestingly, exogenous glutamate increased the mRNAs of cytodifferentiation and mineralization-related genes as well as the ALP (alkaline phosphatase) activities during the cytodifferentiation of the PDL cells. On the other hand, the glutamate signaling inhibitors riluzole and (+)-MK801 maleate suppressed the alkaline phosphatase activities and mineralized nodule formation during the cytodifferentiation and mineralization. Riluzole inhibited the mechanical stress-induced glutamate signaling-associated gene expressions in human PDL cells. Moreover, in situ hybridization analyses showed up-regulation of glutamate signaling-associated gene expressions at tension sites in the PDL under orthodontic tooth movement in a mouse model. The present data demonstrate that the glutamate signaling induced by mechanical stress positively regulates the cytodifferentiation and mineralization of PDL cells.