Gou Yamamoto

Frequent silencing of a putative tumor suppressor gene melatonin receptor 1 A (MTNR1A) in oral squamous‐cell carcinoma

Array‐based comparative genomic hybridization (array‐CGH) has good potential for the high‐throughput identification of genetic aberrations in cell genomes. In the course of a program to screen a panel of 21 oral squamous‐cell carcinoma (OSCC) cell lines for genome‐wide copy‐number aberrations by array‐CGH using our in‐house bacterial artificial chromosome arrays, we identified a frequent homozygous deletion at 4q35 loci with approximately 1 Mb in extent. Among the seven genes located within this region, the expression of the melatonin receptor 1 A (MTNR1A) messenger RNA (mRNA) was not detected or decreased in 35 out of the 39 (89%) OSCC cell lines, but was detected in immortalized normal oral epithelial cell line, and was restored in gene‐silenced OSCC cells without its homozygous loss after treatment with 5‐aza‐2′‐deoxycytidine. The hypermethylation of the CpG (cytosine and guanine separated by phosphate) island in the promoter region of MTNR1A was inversely correlated with its expression in OSCC lines without a homozygous deletion. Methylation of this CpG island was also observed in primary OSCC tissues. In an immunohistochemical analysis of 50 primary OSCC tumors, the absence of immunoreactive MTNR1A was significantly associated with tumor size and a shorter overall survival in patients with OSCC tumors, and seems to be an independent prognosticator in a multivariate analysis. Exogenous restoration of MTNR1A expression inhibited the growth of OSCC cells lacking its expression. Together with the known tumor‐suppressive function of melatonin and MTNR1A in various tumors, our results indicate MTNR1A to be the most likely target for epigenetic silencing at 4q35 and to play a pivotal role during oral carcinogenesis. (Cancer Sci 2008; 99: 1390–1400)

Association of genetic, psychological and behavioral factors with sleep bruxism in a Japanese population.

Sleep bruxism is a sleep‐related movement disorder that can be responsible for various pains and dysfunctions in the orofacial region. The aim of the current case–control association study was to investigate the association of genetic, psychological and behavioral factors with sleep bruxism in a Japanese population. Non‐related participants were recruited and divided into either a sleep bruxism group (n = 66) or control group (n = 48) by clinical diagnoses and 3‐night masseter electromyographic recordings by means of a portable miniature device. The Epworth Sleepiness Scale, Temperament and Character Inventory, NEO‐Five Factor Inventory and custom‐made questionnaires that asked about familial aggregation, alcohol intake, caffeine intake, cigarette smoking, past stressful life events, daytime tooth‐contacting habit, temporomandibular disorder, daily headache, snoring, apnea/hypopnea symptoms, leg‐restlessness symptoms and nocturnal‐myoclonus symptoms were administered. In addition, 13 polymorphisms in four genes related to serotonergic neurotransmission (SLC6A4, HTR1A, HTR2A and HTR2C) were genotyped. These factors were compared between case (sleep bruxism) and control groups in order to select potential predictors of sleep‐bruxism status. The statistical procedure selected five predictors: Epworth Sleepiness Scale, leg‐restlessness symptoms, rs6313 genotypes, rs2770304 genotypes and rs4941573 genotypes. A multivariate stepwise logistic regression analysis between the selected predictors and sleep‐bruxism status was then conducted. This analysis revealed that only the C allele carrier of HTR2A single nucleotide polymorphism rs6313 (102C>T) was associated significantly with an increased risk of sleep bruxism (odds ratio = 4.250, 95% confidence interval: 1.599–11.297, P = 0.004).This finding suggests a possible genetic contribution to the etiology of sleep bruxism.

Cdc42 is required for chondrogenesis and interdigital programmed cell death during limb development

Cdc42, a member of the Rho subfamily of small GTPases, is known to be a regulator of multiple cellular functions, including cytoskeletal organization, cell migration, proliferation, and apoptosis. However, its tissue-specific roles, especially in mammalian limb development, remain unclear. To investigate the physiological function of Cdc42 during limb development, we generated limb bud mesenchyme-specific inactivated Cdc42 (Cdc42(fl/fl); Prx1-Cre) mice. Cdc42(fl/fl); Prx1-Cre mice demonstrated short limbs and body, abnormal calcification of the cranium, cleft palate, disruption of the xiphoid process, and syndactyly. Severe defects were also found in long bone growth plate cartilage, characterized by loss of columnar organization of chondrocytes, and thickening and massive accumulation of hypertrophic chondrocytes, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed that expressions of Col10 and Mmp13 were reduced in non-resorbed hypertrophic cartilage, indicating that deletion of Cdc42 inhibited their terminal differentiation. Syndactyly in Cdc42(fl/fl); Prx1-Cre mice was caused by fusion of metacarpals and a failure of interdigital programmed cell death (ID-PCD). Whole mount in situ hybridization analysis of limb buds showed that the expression patterns of Sox9 were ectopic, while those of Bmp2, Msx1, and Msx2, known to promote apoptosis in the interdigital mesenchyme, were down-regulated. These results demonstrate that Cdc42 is essential for chondrogenesis and ID-PCD during limb development.

Comprehensive analysis of gene expression in the junctional epithelium by laser microdissection and microarray analysis

BACKGROUND AND OBJECTIVE The junctional epithelium attaches to the tooth enamel at the dentogingival junction. The attachment mechanisms of the junctional epithelium have been studied histologically, but the molecular functions of the junctional epithelium have not been elucidated. The aim of this study was to perform a comprehensive analysis of gene expression in the junctional epithelium and to search for specific genetic markers of the junctional epithelium. MATERIAL AND METHODS A comprehensive analysis of genes expressed in the mouse junctional epithelium and oral gingival epithelium was performed using laser microdissection and microarray analysis. To extract high-quality RNA from these tissues, we made frozen sections using a modified film method. Confirmation of the differential expression of selected genes was performed by quantitative real-time PCR and immunohistochemistry. RESULTS The modified method produced RNA of sufficient quality for microarray analysis. The result of microarray analysis showed that 841 genes were up-regulated in the junctional epithelium compared with the oral gingival epithelium, and five were increased more than 50-fold in the junctional epithelium. These five genes were secretory leukocyte protease inhibitor (Slpi), keratin 17 (Krt17), annexin A1 (Anxa1), myosin light peptide 6 (Myl6) and endoplasmic reticulum protein 29 (Erp29). In particular, Slpi expression in the junctional epithelium was approximately 100-fold higher than in the oral gingival epithelium by real-time PCR. Additionally, immunohistochemistry indicated that the Slpi protein is highly expressed in the junctional epithelium. CONCLUSION We developed a method for generating fresh-frozen tissue sections suitable for extraction of good-quality RNA. We determined that Slpi is characteristically expressed in the junctional epithelium. Our results provide a substantial advance in the analysis of gene expression in the junctional epithelium.

A New Method for Alveolar Bone Repair Using Extracted Teeth for the Graft Material

BACKGROUND In the clinical field of jawbone formation, the use of autogenous bone as the graft material is the gold standard. However, there are some problems with this technique, such as risk of infection on the donor side, the limited amount of available bone mass, and marked resorption of the grafted bone. We investigated the potential for using teeth as a bone graft material for jawbone formation because the dental pulp contains stem cells, including undifferentiated neural crest-derived cells. METHODS Alveolar bone defects were created in Wistar rats, and the defects were filled with either tooth or iliac bone graft material, or left as controls. The potential for using teeth as a bone graft material for jawbone formation was measured using real-time polymerase chain reaction, microcomputed tomography, and histologic analysis. RESULTS Polymerase chain reaction revealed that the expressions of P75, P0, nestin, and musashi-1 were significantly higher in teeth than in mandibular bone and iliac bone grafts. Hematoxylin and eosin staining and microcomputed tomography showed that at 8 weeks, tooth graft material produced a similar amount of new bone compared to iliac bone graft material. Osteopontin was expressed in both the tooth and iliac bone graft material at 6 and 8 weeks after surgery. Dentin sialoprotein was expressed in the tooth graft material in the new bone at 6 weeks only. CONCLUSION These results indicate that teeth may be an alternative material to autogenous bone for treating alveolar bone defects by grafting.

Role of the junctional epithelium in periodontal innate defense and homeostasis

BACKGROUND AND OBJECTIVE   The junctional epithelium provides the front-line defense against periodontal bacterial infection. The migration of neutrophils into the junctional epithelium might represent a protective reaction against bacterial infections. However, neutrophils penetrate into the junctional epithelium even under sterile conditions. In this study, we analyzed and compared the number of neutrophils and the cytokine expression related to neutrophil migration in the junctional epithelium in conventional and germ-free mice. MATERIAL AND METHODS   Germ-free and conventional ICR mice were used at 12 wk of age. Frozen sections were used for the detection of Gr-1, macrophage inflammatory protein-2 (MIP-2/CXCL2) and proliferating cell nuclear antigen-positive cells in the two groups of mice. Laser capture microdissection and RT-PCR analysis were used to evaluate the expression of keratinocyte-derived chemokine (KC/CXCL1), MIP-2, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) mRNAs in the two groups of mice. RESULTS   Morphometric examination indicated an increase in the area of the junctional epithelium upon bacterial infection. Immunohistochemical studies also detected an increased number of neutrophils in the junctional epithelium upon bacterial infection. Higher up-regulation of KC and MIP-2 were detected in the junctional epithelium of conventional mice than in germ-free mice, whereas the expression of Il-1β and Tnfα mRNAs was not affected. CONCLUSION   Junctional epithelium cells constitutively expressed several types of chemokines and cytokines and the expression of chemokines was augmented by bacterial infection. Therefore, the constitutive expression of cytokines in junctional epithelium might be related to the morphological and functional homeostasis of the junctional epithelium in addition to the defense against the bacterial infection.

Expression of matrix metalloproteinases MMP-2, MMP-9 and their tissue inhibitors TIMP-1 and TIMP-2 in the epithelium and stroma of salivary gland pleomorphic adenomas

Aims:  The balance between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) is involved in the morphogenesis of normal salivary gland as well as in the mechanisms of tumour invasion and metastasis. The role of MMPs and TIMPs in pleomorphic adenoma has not been elucidated sufficiently. Our aim was to analyse the mRNA and protein expression of MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 in the epithelium and stroma of pleomorphic adenoma and to evaluate their roles.

Transplantation of side population cells restores the function of damaged exocrine glands through clusterin

Stem cell‐based therapy has been proposed as a promising strategy for regenerating tissues lost through incurable diseases. Side population (SP) cells have been identified as putative stem cells in various organs. To examine therapeutic potential of SP cells in hypofunction of exocrine glands, SP cells isolated from mouse exocrine glands, namely, lacrimal and salivary glands, were transplanted into mice with irradiation‐induced hypofunction of the respective glands. The secretions from both glands in the recipient mice were restored within 2 months of transplantation, although the transplanted cells were only sparsely distributed and produced no outgrowths. Consistent with this, most SP cells were shown to be CD31‐positive endothelial‐like cells. In addition, we clarified that endothelial cell‐derived clusterin, a secretory protein, was an essential factor for SP cell‐mediated recovery of the hypofunctioning glands because SP cells isolated from salivary glands of clusterin‐deficient mice had no therapeutic potential, whereas lentiviral transduction of clusterin restored the hypofunction. In vitro and in vivo studies showed that clusterin had an ability to directly inhibit oxidative stress and oxidative stress‐induced cell damage. Thus, endothelial cell‐derived clusterin possibly inhibit oxidative stress‐induced hypofunction of these glands. Stem Cells2012;30:1925–1937

Periostin, discovered by nano-flow liquid chromatography and mass spectrometry, is a novel marker of diabetic retinopathy

Diabetes can lead to serious microvascular complications including proliferative diabetic retinopathy (PDR), the leading cause of blindness in adults. Recent studies using gene array technology have attempted to apply a hypothesis-generating approach to elucidate the pathogenesis of PDR, but these studies rely on mRNA differences, which may or may not be related to significant biological processes. To better understand the basic mechanisms of PDR and to identify potential new biomarkers, we performed shotgun liquid chromatography (LC)/tandem mass spectrometry (MS/MS) analysis on pooled protein extracts from neovascular membranes obtained from PDR specimens and compared the results with those from non-vascular epiretinal membrane (ERM) specimens. We detected 226 distinct proteins in neovascular membranes and 154 in ERM. Among these proteins, 102 were specific to neovascular membranes and 30 were specific to ERM. We identified a candidate marker, periostin, as well as several known PDR markers such as pigment epithelium-derived factor (PEDF). We then performed RT-PCR using these markers. The expression of periostin was significantly up-regulated in proliferative membrane specimens. Periostin induces cell attachment and spreading and plays a role in cell adhesion. Proteomic analysis by LC/MS/MS, which permits accurate quantitative comparison, was useful in identifying new candidates such as periostin potentially involved in the pathogenesis of PDR.

Tumor necrosis factor‐α enhances RANKL expression in gingival epithelial cells via protein kinase A signaling

OBJECTIVE AND BACKGROUND Periodontitis is an inflammatory disorder of the supporting tissue of teeth, which is composed of gingival soft tissue, cementum covering the tooth root, alveolar bone and periodontal ligament. The receptor activator of nuclear factor kappa B ligand (RANKL) is known to be an essential factor for osteoclastogenesis. Recent clinical studies indicate that levels of RANKL in the gingival crevicular fluid are increased while levels of its decoy receptor, osteoprotegerin (OPG), are decreased in patients with periodontitis. Although the gingival sulcus is composed of gingival tissue, RANKL and OPG expression in gingival epithelial cells is not fully understood. The aim of this study is to investigate the expression of RANKL and OPG in gingival tissue and which factors regulate RANKL expression in gingival epithelial cells. MATERIAL AND METHODS Reverse transcriptase polymerase chain reaction analysis, western blotting and immunohistochemistry were performed to confirm RANKL and OPG expression in gingival epithelial cells (GECs) and in gingival tissue. Immunostaining was also examined to confirm tumor necrosis factor (TNF)-α and TNF receptor type 1 (TNFR1) expression in gingival tissue. Ca9-22 cells, a human gingival epithelial cell line and human primary GECs were treated with TNF-α. Ca9-22 cells were treated by antibodies against TNF receptors, an inhibitor and an activator of protein kinase A (PKA) signaling and inhibitors of p38, Erk and NF-κB signaling to examine TNF-α-RANKL signaling pathways. RESULTS RANKL mRNA and protein were expressed in GECs. Immunohistochemistry also showed RANKL expression in gingival tissue. On the other hand, the reverse transcriptase polymerase chain reaction and immunohistochemistry assay showed that GECs did not express OPG. In addition, TNF-α and TNFR1 proteins were expressed in junctional epithelium. TNF-α increased RANKL expression in GECs. TNF-α-induced RANKL expression was inhibited by an antibody against TNFR1 and an inhibitor of PKA signaling. Surprisingly, forskolin, a PKA activator, increased TNF-α-induced RANKL expression. CONCLUSION RANKL, TNF and TNFR1 were coexpressed in junctional epithelium of gingival tissue. TNF-α induced RANKL expression via TNFR1 and PKA signaling in GECs of junctional epithelium.