Hisao Yagishita

Contribution of sialic acid-binding adhesin to pathogenesis of experimental endocarditis caused by Streptococcus gordonii DL1.

ABSTRACT An insertional mutation in hsa, the gene encoding the sialic acid-binding adhesin of Streptococcus gordonii DL1, resulted in a significant reduction of the infection rate of the organism and an inflammatory reaction in the rat aortic valve with experimental endocarditis, suggesting that the adhesin contributes to the infectivity of the organism for heart valves.

Molecular mechanism for connective tissue destruction by dipeptidyl aminopeptidase IV produced by the periodontal pathogen Porphyromonas gingivalis

ABSTRACT Porphyromonas gingivalis is a pathogen associated with adult periodontitis. It produces dipeptidyl aminopeptidase IV (DPPIV), which may act as a virulence factor by contributing to the degradation of connective tissue. We investigated the molecular mechanism by which DPPIV contributes to the destruction of connective tissue. DPPIV itself did not show gelatinase or collagenase activity toward human type I collagen, but it promoted the activity of the host-derived matrix metalloproteinase 2 (MMP-2) (gelatinase) and MMP-1 (collagenase). DPPIV bound to fibronectin and mediated the adhesion of P. gingivalis to fibronectin. Mutant DPPIV with catalytic Ser mutagenized to Ala (DPPSA) did not accelerate the degradation of collagen and gelatin by MMPs but retained fibronectin-binding activity. The adhesion of human gingival fibroblasts and NIH 3T3 cells to fibronectin was inhibited by DPPIV. Strain 4351ADPPSA exhibited an intermediate level of virulence in mice, between that of the strain expressing wild-type DPPIV (4351ADPP) and that of the strain harboring only the plasmid vector (4351AVEC). It is suggested that both activity promoting the degradation of collagen and gelatin and binding to fibronectin are required for full virulence. These results reveal novel biological functions of DPPIV and suggest a pathological role in the progression of periodontitis.

Enamel Mineralization and an Initial Crystalline Phase

In this communication, we summarized our recent experimental approaches to an unsettled issue, i.e., the nature and role of an acidic precursor in enamel mineralization. The objectives we specially focused our attention on are: the composition, structure and high resolution images of enamel crystals at various developmental stages, thermodynamic and kinetic consideration of octacalcium phosphate (OCP) vs hydroxyapatite (HA) precipitation in physiological media simulating the enamel fluid, reversible changes in the composition and structure of OCP, effects of fluoride at low concentrations and enamel proteins on OCP hydrolysis, and adsorption of enamel proteins onto OCP and fluoridated hydrolysates at neutral pH and room temperature. On the basis of all experimental evidence, we propose that enamel crystal growth comprises two events: the two-dimensional growth of an OCP-like precursor in a narrow outermost zone adjacent to the ameloblasts and the subsequent overgrowth of apatite units on the template under discrete fluid environment in the underlying region distant from the cell layer. The experimental data also support the concept that the whole process of enamel mineralization is modulated substantially through interaction between enamel proteins and crystals including the acidic precursor.

Histopathological Studies on Virulence of Dipeptidyl Aminopeptidase IV (DPPIV) of Porphyromonas gingivalis in a Mouse Abscess Model: Use of a DPPIV-Deficient Mutant

ABSTRACT To elucidate the role of dipeptidyl aminopeptidase IV (DPPIV) in the virulence of Porphyromonasgingivalis, mice were infected with either a wild-type strain or a DPPIV-deficient mutant using an abscess model. Histopathological analysis of the resulting lesions indicated that DPPIV participates in virulence through the destruction of connective tissue and the less effective mobilization of inflammatory cells.

Three-Dimensional Reconstruction of Oral Tongue Squamous Cell Carcinoma at Invasion Front

We conducted three-dimensional (3D) reconstruction of oral tongue squamous cell carcinoma (OTSCC) using serial histological sections to visualize the architecture of invasive tumors. Fourteen OTSCC cases were collected from archival paraffin-embedded specimens. Based on a pathodiagnostic survey of whole cancer lesions, a core tissue specimen (3 mm in diameter) was dissected out from the deep invasion front using a paraffin tissue microarray. Serial sections (4 μm thick) were double immunostained with pan-cytokeratin and Ki67 antibodies and digitized images were acquired using virtual microscopy. For 3D reconstruction, image registration and RGB color segmentation were automated using ImageJ software to avoid operator-dependent subjective errors. Based on the 3D tumor architecture, we classified the mode of invasion into four types: pushing and bulky architecture; trabecular architecture; diffuse spreading; and special forms. Direct visualization and quantitative assessment of the parenchymal-stromal border provide a new dimension in our understanding of OTSCC architecture. These 3D morphometric analyses also ascertained that cell invasion (individually and collectively) occurs at the deep invasive front of the OTSCC. These results demonstrate the advantages of histology-based 3D reconstruction for evaluating tumor architecture and its potential for a wide range of applications.

Proteomic identification of keratin alterations with enhanced proliferation of oral carcinoma cells by loss of mucosa-associated lymphoid tissue 1 expression

Progression of oral carcinomas associates with aberrant activation and inactivation of molecules that work in established or unknown pathways. Although mucosa‑associated lymphoid tissue 1 (MALT1) expressed in normal oral epithelium is inactivated in the aggressive subset of carcinomas with worse prognosis, phenotypic changes of carcinoma cells upon the loss of expression is unknown. We performed a proteomic analysis to identify MALT1‑regulated proteins in oral carcinoma cells. Four different keratins were included in the ten most abundantly changed proteins. K8/18 were upregulated in MALT1 stably‑expressing carcinoma cells and K5/14 in MALT1‑marginal control cells. K8/18 upregulation and K5/14 downregulation were MALT1 dose‑dependent and observed in a series of oral carcinoma cells. MALT1 suppressed cell proliferation (0.52-fold, P<0.01) and its dominant-negative form stimulated it (1.33-fold, P<0.01). The decreased proliferation associated with reduction of cyclin D1, which was recovered by the short interfering RNA against MALT1. Taken together, loss of MALT1 expression alters keratin expression and enhances proliferation of carcinoma cells, and may progress oral carcinomas into the advanced state.

The onset risk of carcinoma in patients continuing tacrolimus topical treatment for oral lichen planus: a case report

Oral lichen planus is a chronic inflammatory mucocutaneous disease. Topical use of steroids and other immuno-modulating therapies have been tried for this intractable condition. Nowadays, tacrolimus ointment is used more commonly as a choice for treatment. However, a number of discussions have taken place after tacrolimus was reported to be carcinogenic. This report describes a patient who applied tacrolimus ointment to the lower lip after being diagnosed with oral lichen planus in 2008, and whose lesion developed squamous cell carcinoma in 2010. Since the relationship between tacrolimus and cancer development has been reported in only a few cases, including this case report, the clinician must be careful selecting tacrolimus as a second-line treatment for oral lichen planus.

Minimal essential region for krüppel-like factor 5 expression and the regulation by specificity protein 3-GC box binding

Krüppel-like factor 5 (KLF5) transcriptionally controls the proliferation-differentiation balance of epithelium and is overexpressed in carcinomas. Although genomic region modifying KLF5 expression is widespread in different types of cells, the region that commonly regulates basal expression of the genes across cell-types is uncertain. In this study we determined the minimal essential region for the expression and its regulatory transcription factors using oral carcinoma cells. A reporter assay defined a 186bp region downstream of the transcription start site and a cluster of six GC boxes (GC1-GC6) as the minimal essential region. Mutation in the GC1 or GC6 regions but not other GC boxes significantly decreased the reporter expression. The decrease by the GC1 mutation was reproduced in the 2kbp full-length promoter, but not by the GC6 mutation. Additionally, specificity proteins (Sp) that can be expressed in epithelial cells and bind GC box, Sp3 co-localized with KLF5 in oral epithelium and carcinomas and chromatin immunoprecipitation analyses showed Sp3 as the prime GC1-binding protein. Inhibition of Sp-GC box binding by mithramycin A and knockdown of Sp3 by the short interfering RNA decreased expression of the reporter gene and endogenous KLF5. These data demonstrate that a 186bp region is the minimal essential region and that Sp3-GC1 binding is essential to the basal expression of KLF5.

MALT1 Inhibition of Oral Carcinoma Cell Invasion and ERK/MAPK Activation

The expression of mucosa-associated lymphoid tissue 1 (MALT1) that activates nuclear factor (NF)–κB in lymphocyte lineages is rapidly inactivated in oral carcinoma cells at the invasive front and the patients with worst prognosis. However, its mechanism to accelerate carcinoma progression remains unknown, and this study was carried out to examine the role in invasion. HSC2 oral carcinoma cells stably expressing wild-type MALT1 (wtMALT1) reduced the invasion of basement membrane matrices and collagen gels, and the dominant-negative form (∆MALT1)–expressing cells aggressively invaded into collagen gels. MALT1 decelerated proliferation and migration of cells and downregulated expression of matrix metalloproteinase 2 and 9, which were confirmed by short interfering RNA transfections. Reporter assays and immunoblot analysis showed that MALT1 does not affect the NF-κB pathway but inhibits ERK/MAPK activation. This was confirmed by endogenous MALT1 expression in oral carcinoma cell lines. Orthotopic implantation of ∆MALT1-expressing HSC2 cells in mice grew rapid expansive and invasive tongue tumors in contrast to an absence of tumor formation by wtMALT1-expressing cells. These results demonstrate that MALT1 suppresses oral carcinoma invasion by inhibiting proliferation, migration, and extracellular matrix degradation and that the ERK/MAPK pathway is a target of MALT1 and further suggests a role as a suppressor of carcinoma progression.

Histomorphometric Analysis of Development and Morphogenesis of Mouse Temporomandibular Joints

Abstract The temporomandibular joint (TMJ) is a multicomponent organ including the condyle, mandibular fossa, articular disc and clefts, and surrounding tissues. In the present study, we reported the chronology of TMJ development and associated tissue-marker expression in C57BL/6 mice. Wild-type mice of embryonic day 12.5 (E12.5) through postnatal day 7 were examined by histochemistry (HE, metachromasia by toluidine blue), enzyme-histochemistry for alkaline and acid phosphatases (ALP and TRAP) and immunohistochemistry for BrdU and type II collagen. The results confirmed that: 1) the condylar blastema was first designated as a troop of ALP-positive cells at E13.5, and some of those cells also displayed BrdU incorporation, 2) after differentiation of the blastema, condylar morphogenesis progressed from the anterior to the posterior direction, while the mandibular fossa of discrete origin developed inversely, and 3) development of the articular disk and superior/inferior clefts appeared to link intimately with the articular function. Histomorphometric analysis, in conjunction with BrdU administration, further revealed that condylar growth during development can be divided into three stages: the initial slow growth due to cell proliferation by E13.5, the second rapid growth between E14.5 and E17.5 due to production of extracellular matrices instead of acceleration of cell proliferation, and the last functional adaptation after birth that progresses at a steady growth rate coupled with systemic body growth. The current observation also supported the spatial linkage of the condylar blastema with a group of the mesenchymal cell population that is involved in mandibular bone development. Further analysis in conjunction with three-dimensional reconstruction of ALP-positive cell condensation is required to clarify the origin and spatio-temporal development of condylar blastema in detail.