Transcriptional regulation of human odontogenic ameloblast-associated protein gene by tumor necrosis factor-α.

OBJECTIVE: Odontogenic ameloblast-associated protein (ODAM) is produced by maturation stage ameloblasts and junctional epithelium (JE). The function of ODAM is thought to be involved in the attachment of teeth and JE. To elucidate transcriptional regulation of human ODAM gene in inflamed gingiva, we have analyzed the effects of TNF-α on the expression of ODAM gene in Ca9-22 and Sa3 gingival epithelial cells. MATERIALS AND METHODS: Total RNAs were extracted from Ca9-22 and Sa3 cells after stimulation by TNF-α (10 ng/ml). ODAM mRNA and protein levels were analyzed by qPCR and Western blotting. Luciferase (LUC) analyses were performed using LUC constructs inserted in various lengths of ODAM gene promoter. Gel shift and chromatin immunoprecipitation (ChIP) assays were carried out. RESULTS: TNF-α increased ODAM mRNA and protein levels at 3 to 24 h. TNF-α induced LUC activities of the ODAM gene promoter constructs, and the activities were inhibited by protein kinase A, tyrosine kinase, MEK1/2, PI3-kinase and NF-κB inhibitors. Gel shift and ChIP assays revealed that TNF-α increased CCAAT/enhancer-binding protein (C/EBP) ß and Yin Yang1 (YY1) binding to three kinds of C/EBPs and YY1 elements. CONCLUSION: These results demonstrate that TNF-α stimulates ODAM gene transcription via C/EBPs and YY1 elements in the human ODAM gene promoter.

Biphasic Functions of Sodium Fluoride (NaF) in Soft and in Hard Periodontal Tissues.

Sodium fluoride (NaF) is widely used in clinical dentistry. However, the administration of high or low concentrations of NaF has various functions in different tissues. Understanding the mechanisms of the different effects of NaF will help to optimize its use in clinical applications. Studies of NaF and epithelial cells, osteoblasts, osteoclasts, and periodontal cells have suggested the significant roles of fluoride treatment. In this review, we summarize recent studies on the biphasic functions of NaF that are related to both soft and hard periodontal tissues, multiple diseases, and clinical dentistry.

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.

Odontogenic Ameloblast-associated Protein (ODAM) Mediates Junctional Epithelium Attachment to Teeth via Integrin-ODAM-Rho Guanine Nucleotide Exchange Factor 5 (ARHGEF5)-RhoA Signaling.

Adhesion of the junctional epithelium (JE) to the tooth surface is crucial for maintaining periodontal health. Although odontogenic ameloblast-associated protein (ODAM) is expressed in the JE, its molecular functions remain unknown. We investigated ODAM function during JE development and regeneration and its functional significance in the initiation and progression of periodontitis and peri-implantitis. ODAM was expressed in the normal JE of healthy teeth but absent in the pathologic pocket epithelium of diseased periodontium. In periodontitis and peri-implantitis, ODAM was extruded from the JE following onset with JE attachment loss and detected in gingival crevicular fluid. ODAM induced RhoA activity and the expression of downstream factors, including ROCK (Rho-associated kinase), by interacting with Rho guanine nucleotide exchange factor 5 (ARHGEF5). ODAM-mediated RhoA signaling resulted in actin filament rearrangement. Reduced ODAM and RhoA expression in integrin ß3- and ß6-knockout mice revealed that cytoskeleton reorganization in the JE occurred via integrin-ODAM-ARHGEF5-RhoA signaling. Fibronectin and laminin activated RhoA signaling via the integrin-ODAM pathway. Finally, ODAM was re-expressed with RhoA in regenerating JE after gingivectomy in vivo. These results suggest that ODAM expression in the JE reflects a healthy periodontium and that JE adhesion to the tooth surface is regulated via fibronectin/laminin-integrin-ODAM-ARHGEF5-RhoA signaling. We also propose that ODAM could be used as a biomarker of periodontitis and peri-implantitis.

The genes Scgb1a1, Lpo and Gbp2 characteristically expressed in peri-implant epithelium of rats.

OBJECTIVES: The peri-implant epithelium (PIE) plays an important role in the prevention against initial stage of inflammation. To minimize the risk of peri-implantitis, it is necessary to understand the biological characteristics of the PIE. The aim of this study was to investigate the characteristic gene expression profile of PIE as compared to junctional epithelium (JE) using laser microdissection and microarray analysis. METHODS: Left upper first molars of 4-week-old rat were extracted, and titanium alloy implants were placed. Four weeks after surgery, samples were harvested by laser microdissection, and total RNA samples were isolated. Comprehensive analyses of genes expressed in the JE and PIE were performed using microarray analysis. Confirmation of the differential expression of selected genes was performed by quantitative real-time polymerase chain reaction and immunohistochemistry. RESULTS: The microarray analysis showed that 712 genes were more than twofold change upregulated in the PIE compared with the JE. Genes Scgb1a1 were significantly upregulated more than 19.1-fold, Lpo more than 19.0-fold, and Gbp2 more than 8.9-fold, in the PIE (P < 0.01). Immunohistochemical localization of SCGB1A1, LPO, and GBP2 was observed in PIE. CONCLUSION: The present results suggested that genes Scgb1a1, Lpo, and Gbp2 are characteristically expressed in the PIE.

Ultrastructural immunolocalization of laminin 332 (laminin 5) at dento-gingival interface in Macaca fuscata monkey.

Although laminin 332 (laminin 5), an extracellular matrix molecule involved in cell adhesion and migration, has been localized at the interface between the tooth enamel and junctional epithelium, its ultrastructural localization remains to be fully clarified. The purpose of the present study was to investigate the ultrastructural distribution of laminin 332 at the dento-gingival interface in Japanese monkey (Macaca fuscata) using pre- and post-embedding immunoelectron microscopy. Pre-embedding immunoelectron microscopy revealed a broad band of internal basal lamina together with supplementary lamina densa, and both showed immunolabeling for laminin 332. Immunoreaction products for laminin 332 were observed in the rough-surfaced endoplasmic reticulum of the junctional epithelial cells close to the tooth enamel. Post-embedding immunoelectron microscopy revealed an increase in the number of immunogold particles toward the coronal portion, resulting in a large accumulation of particles on the basal lamina, preferentially on the lamina densa. Concomitantly the dental cuticle at the dento-gingival interface was sporadically, but specifically, immunogold-labeled with anti-laminin 332 antibody. These data suggest that junctional epithelium actively produces laminin 332, and that the products accumulate at the dento-gingival interface during cell migration coronally towards the gingival sulcus.

Perlecan-enriched intercellular space of junctional epithelium provides primary infrastructure for leukocyte migration through squamous epithelial cells.

The aim of this study was to demonstrate the presence of intraepithelial stroma represented by extracellular matrix (ECM) deposits in the junctional epithelium to clarify its function as a scaffold for leukocyte migration through epithelial cells. Twenty-three biopsy specimens from the gingiva including the junctional epithelium were examined to determine comparative protein and gene level expression profiles for keratin and ECM molecules between the junctional epithelium and the gingival epithelium using immunohistochemistry and in situ hybridization. Intraepithelial leukocyte types and frequencies were also determined and compared between the junctional and gingival epithelia. In the junctional epithelium, which was positive for keratin 19, perlecan was strongly deposited in intercellular space of the whole epithelial layer, while it was faintly positive around the parabasal layer of the gingival epithelium. Perlecan mRNA signals were enhanced to a greater degree in both epithelial and inflammatory cells within the junctional epithelium. In the junctional epithelium, greater numbers of neutrophils and macrophages were found as compared with the gingival epithelium. Our results showed that perlecan is the primary ECM molecule comprising intraepithelial stroma of the junctional epithelium, in which leukocytes may migrate on ECM scaffolds in intercellular space toward the surface of the gingival sulci or pockets.

Developmental relationship between junctional epithelium and epithelial rests of Malassez.

Keratin 17 (K17) is thought to be a candidate target gene for regulation by Lymphoid Enhancer Factor-1 (Lef-1). K17 is a marker that distinguishes junctional epithelium (JE) from epithelial rests of Malassez (ERM). However, the relationship of Lef-1 to K17 is not clear in this context. Moreover, the expression of other keratins such as K5, K6, K7 and K16 is not reported. Therefore, the aim of our study was to assay the expression of K5, K6, K7, K14, K16, K17 and Lef-1 in postnatal developing teeth, and clarify the corresponding immunophenotypes of the JE and ERM. Upper jaws of Wistar rats aged from postnatal (PN) day 3.5 to PN21 were used and processed for immunohistochemistry. K5 and K14 were intensely expressed in inner enamel epithelium (IEE), reduced enamel epithelium (REE), ERM and JE. There was no staining for K16 in the tissue, except for strong staining in the oral epithelium. Specifically, at PN3.5 and PN7, K17 was initially strongly expressed and then negative in the IEE. At PN16 and PN21, both REE and ERM were strongly stained for K17, whereas K17 was negative in the JE. In addition, K6, K7 and Lef-1 were not detected in any tissue investigated. REE and ERM have an identical keratin expression pattern before eruption, while JE differs from ERM in the expression of K17 after eruption. The expression of K17 does not coincide with that of Lef-1. These data indicate that JE has a unique phenotype different from ERM, which is of odontogenic origin.

Lipopolysaccharide induces a stromal-epithelial signalling axis in a rat model of chronic periodontitis.

AIM: Lipopolysaccharide is a bacterial virulence factor implicated in chronic periodontitis, which may penetrate the junctional epithelial barrier and basement membrane to insult underlying stroma. We sought to identify lipopolysaccharide-induced global gene expression changes responsible for signalling between stroma and epithelium during disease onset. MATERIALS AND METHODS: Using a rat lipopolysaccharide periodontitis model, junctional epithelium and underlying stromal tissue were separately collected from healthy and diseased animals by laser-capture microdissection and subject to gene expression microarray analysis. Key gene products identified were validated in gingival epithelial and fibroblast cell cultures. RESULTS: Global gene expression patterns distinguishing health versus disease were found in and between both tissue types. In stroma, the most significantly altered gene ontology function group (Z ≥ 4.00) was cytokines, containing most significantly (±2-fold; p < 0.05) upregulated genes amphiregulin, IL1-ß and Fas ligand, all positive, diffusible modulators of the epithelial growth factor receptor pathway. In epithelium, the most significant changes were in downregulated FOS-related antigen-1 gene, somatostatin receptor-2 gene and mucin-4 gene, all negative modulators of the epithelial growth factor receptor pathway. CONCLUSION: These results establish a periodontitis model for studying gene product interactions and suggests that the onset of junctional epithelial disease hyperproliferation involves a concerted stromal-epithelial signalling axis.

Lipopolysaccharide induces rapid loss of follicular dendritic cell-secreted protein in the junctional epithelium.

UNLABELLED: Oshiro A, Iseki S, Miyauchi M, Terashima T, Kawaguchi Y, Ikeda Y, Shinomura T. Lipopolysaccharide induces rapid loss of follicular dendritic cell-secreted protein in the junctional epithelium. J Periodont Res 2012; 47: 689-694. © 2012 John Wiley & Sons A/S Background and Objective: We have previously reported that mRNA encoding follicular dendritic cell-secreted protein (FDC-SP) is expressed specifically in the junctional epithelium at the gingival crevice. Other tissues, such as tonsil, prostate gland and trachea, also express high levels of FDC-SP. These tissues participate in a range of functions closely related to innate immunity. Therefore, it is hypothesized that FDC-SP plays a crucial role in close association with the host defense system within the gingival crevice. Accordingly, the main aim of this study was to investigate the expression and localization of FDC-SP in and around the junctional epithelium and to observe the dynamic changes of FDC-SP in experimental inflammation. MATERIAL AND METHODS: We examined, immunohistochemically, the expression of FDC-SP in the junctional epithelium using a specific antibody raised in rabbit after immunization with a synthetic peptide derived from the hydrophilic region of FDC-SP. Experimental inflammation was induced in the upper molars of Wistar rats by applying bacterial lipopolysaccharide (LPS; 5 mg/mL in sterile saline) for 1 h. RESULTS: We confirmed that FDC-SP is present in the junctional epithelium in a pattern that is consistent with the expression of FDC-SP mRNA. Of special interest is that no FDC-SP was detectable in the junctional epithelium 3 h after transient topical treatment with LPS. CONCLUSION: The presence of FDC-SP in the junctional epithelium and its loss after LPS treatment strongly support our hypothesis of FDC-SP playing a crucial role in close association with the host defense system within the gingival crevice.

Distribution of carcinoembryonic antigen-related cellular adhesion molecules in human gingiva.

Carcinoembryonic antigen-related cellular adhesion molecules (CEACAMs) are glycoproteins produced in epithelial, endothelial, lymphoid, and myeloid cells. Carcinoembryonic antigen-related cellular adhesion molecules mediate cell-cell contact and host-pathogen interactions. The aims of this study were to map the distribution and examine the regulation of CEACAMs in human gingival sites. Quantitative real-time PCR performed on human gingival biopsies from periodontitis sites revealed mRNA coding for CEACAM1, -5, -6, and -7. Immunohistochemistry showed that CEACAMs were not found in oral gingival epithelium, except for CEACAM5 in periodontitis. Carcinoembryonic antigen-related cellular adhesion molecules 1, 5, and 6 were present in the oral sulcular epithelium of periodontitis but not in that of healthy gingiva. In junctional epithelium, all three molecules were present in healthy gingiva, but in periodontitis only CEACAM1 and -6 were detected. Staining for CEACAM1 and -6 was also seen in the inflammatory cell infiltrate in periodontitis. No staining for CEACAM7 was found. Proinflammatory mediators, including lipopolysaccharide (LPS), tumour necrosis factor-α (TNF-α)/interleukin-1ß (IL-1ß), and interferon-γ (IFN-γ), increased the expression of CEACAM1 and CEACAM6 mRNAs in cultured human oral keratinocytes. CEACAM1 and CEACAM6 mRNAs were also strongly up-regulated upon stimulation with lysophosphatidic acid. In conclusion, the distribution of different CEACAMs was related to specific sites in the gingiva. This might reflect different functional roles in this tissue.

An SCPPPQ1/LAM332 protein complex enhances the adhesion and migration of oral epithelial cells: Implications for dentogingival regeneration.

Common periodontal disease treatment procedures often fail to restore the structural integrity of the junctional epithelium (JE), the epithelial attachment of the gum to the tooth, leaving the tooth-gum interface prone to bacterial colonization. To address this issue, we introduced a novel bio-inspired protein complex comprised of a proline-rich enamel protein, SCPPPQ1, and laminin 332 (LAM332) to enhance the JE attachment. Using quartz crystal microbalance with dissipation monitoring (QCM-D), we showed that SCPPPQ1 and LAM332 interacted and assembled into a protein complex with high-affinity adsorption of 5.9e-8 [M] for hydroxyapatite (HA), the main component of the mineralized tooth surfaces. We then designed a unique shear device to study the adhesion strength of the oral epithelial cells to HA. The SCPPPQ1/LAM332 complex resulted in a twofold enhancement in adhesion strength of the cells to HA compared to LAM332 (from 31 dyn/cm2 to 63 dyn/cm2). In addition, using a modified wound-healing assay, we showed that gingival epithelial cells demonstrated a significantly high migration rate of 2.7 ± 0.24 µm/min over SCPPPQ1/LAM332-coated surfaces. Our collective data show that this protein complex has the potential to be further developed in designing a bioadhesive to enhance the JE attachment and protect the underlying connective tissue from bacterial invasion. However, its efficacy for wound healing requires further testing in vivo. STATEMENT OF SIGNIFICANCE: This work is the first functional study towards understanding the combined role of the enamel protein SCPPPQ1 and laminin 332 (LAM332) in the epithelial attachment of the gum, the junctional epithelium (JE), to the tooth hydroxyapatite surfaces. Such studies are essential for developing therapeutic approaches to restore the integrity of the JE in the destructive form of gum infection. We have developed a model system that provided the first evidence of the strong interaction between SCPPPQ1 and LAM332 on hydroxyapatite surfaces that favored protein adsorption and subsequently oral epithelial cell attachment and migration. Our collective data strongly suggested using the SCPPPQ1/LAM332 complex to accelerate the reestablishment of the JE after surgical gum removal to facilitate gum regeneration.

Expression and localization of amelotin, laminin γ2 and odontogenesis-associated phosphoprotein (ODAPH) on the basal lamina and junctional epithelium.

At maturation stage of enamel development, a specialized basal lamina (sBL) was built between ameloblasts and enamel. After the teeth eruption, the ameloblasts transform into the inner cell layer of junctional epithelium. The inner cell layer forms the internal basal lamina of junctional epithelium. However, the composition of the sBL and internal basal lamina was not clarified. The objective of our study was to make a description of the localization of amelotin (AMTN), laminin γ2 (LAMC2) and Odontogenesis-associated phosphoprotein (ODAPH) on the sBL and internal basal lamina. In immunohistochemical study, AMTN, LAMC2 and ODAPH were detected on the sBL at maturation stage. AMTN was also detected in ameloblasts at maturation stage. The expression of AMTN decreased from early-to-late maturation stage. In contrast, the expression of LAMC2 and ODAPH was stable. Immunofluorescence double-staining showed the localization of AMTN was close to enamel surface. However, the localization of ODAPH was close to ameloblasts. LAMC2 and ODAPH were observed on internal basal lamina of junctional epithelium. In contrast, no expression of AMTN was detected on internal basal lamina of junctional epithelium. Our results suggested that ODAPH might participate in enamel maturation and periodontal health, which might provide a better understanding of enamel defects and periodontal disease in clinic.

Disruption of periodontal integrity induces expression of apin by epithelial cell rests of Malassez.

BACKGROUND AND OBJECTIVE: It has been suggested that epithelial cell rests of Malassez (ERM) may express enamel matrix proteins and play an important role in periodontal regeneration. Two novel proteins, apin (APIN) and amelotin (AMTN), produced by maturation-stage ameloblasts and junctional epithelium, have recently been identified. The objective of this study was to evaluate whether the ERM express APIN and AMTN under normal conditions and after periodontal challenge. MATERIAL AND METHODS: Gingivectomy and orthodontic tooth movement were carried out on the left side of the maxillae of rats. The control group included the untreated contralateral side of these animals and the maxillae of normal, untreated rats. Animals were sacrificed by intracardiac perfusion on days 3 and 5 after the experimental procedures and maxillary molars were decalcified and processed for paraffin embedding. Immunohistochemistry was used to evaluate the expression of various ameloblast products, including APIN, AMTN, ameloblastin (AMBN) and amelogenin (AMEL). RESULTS: At 3 and 5 days after periodontal challenge, ERM were more evident in the periodontal ligament along the root surface and in the root furcations. Immunodetection of APIN, but not of the other three proteins, was observed in the ERM following the disruption of periodontal integrity. No immunolabeling for APIN, AMTN, AMBN and AMEL was detected in the ERM under normal conditions. CONCLUSION: The expression of APIN at an early time-point following disruption of periodontal integrity suggests that this protein may be part of the cascade of events leading to the activation of ERM during periodontal healing and regeneration.

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.

Enteroids Generated from Patients with Severe Inflammation in Crohn's Disease Maintain Alterations of Junctional Proteins.

BACKGROUND: The mechanisms underlying loss of intestinal epithelial barrier [IEB] function in Crohn's disease [CD] are poorly understood. We tested whether human enteroids generated from isolated intestinal crypts of CD patients serve as an appropriate in vitro model to analyse changes of IEB proteins observed in patients' specimens. METHODS: Gut samples from CD patients and healthy individuals who underwent surgery were collected. Enteroids were generated from intestinal crypts and analyses of junctional proteins in comparison to full wall samples were performed. RESULTS: Histopathology confirmed the presence of CD and the extent of inflammation in intestinal full wall sections. As revealed by immunostaining and Western blot analysis, profound changes in expression patterns of tight junction, adherens junction and desmosomal proteins were observed in full wall specimens when CD was present. Unexpectedly, when enteroids were generated from specimens of CD patients with severe inflammation, alterations of most tight junction proteins and the majority of changes in desmosomal proteins but not E-cadherin were maintained under culture conditions. Importantly, these changes were maintained without any additional stimulation of cytokines. Interestingly, qRT-PCR demonstrated that mRNA levels of junctional proteins were not different when enteroids from CD patients were compared to enteroids from healthy controls. CONCLUSIONS: These data indicate that enteroids generated from patients with severe inflammation in CD maintain some characteristics of intestinal barrier protein changes on a post-transcriptional level. The enteroid in vitro model represents an appropriate tool to gain further cellular and molecular insights into the pathogenesis of barrier dysfunction in CD.

TRPV2 expression in rat oral mucosa.

The oral mucosa is a highly specialised, stratified epithelium that confers protection from infection and physical, chemical and thermal stimuli. The non-keratinised junctional epithelium surrounds each tooth like a collar and is easily attacked by foreign substances from the oral sulcus. We found that TRPV2, a temperature-gated channel, is highly expressed in junctional epithelial cells, but not in oral sulcular epithelial cells or oral epithelial cells. Dual or triple immunolabelling with immunocompetent cell markers also revealed TRPV2 expression in Langerhans cells and in dendritic cells and macrophages. Electron microscopy disclosed TRPV2 immunoreactivity in the unmyelinated and thinly myelinated axons within the connective tissue underlying the epithelium. TRPV2 labelling was also observed in venule endothelial cells. The electron-dense immunoreaction in junctional epithelial cells, macrophages and neural axons occurred on the plasma membrane, on invaginations of the plasma membrane and in vesicular structures. Because TRPV2 has been shown to respond to temperature, hypotonicity and mechanical stimuli, gingival cells expressing TRPV2 may act as sensor cells, detecting changes in the physical and chemical environment, and may play a role in subsequent defence mechanisms.

Regulated expression of nullo is required for the formation of distinct apical and basal adherens junctions in the Drosophila blastoderm.

During cellularization, the Drosophila embryo undergoes a large-scale cytokinetic event that packages thousands of syncytial nuclei into individual cells, resulting in the de novo formation of an epithelial monolayer in the cortex of the embryo. The formation of adherens junctions is one of the many aspects of epithelial polarity that is established during cellularization: at the onset of cellularization, the Drosophila beta-catenin homologue Armadillo (Arm) accumulates at the leading edge of the cleavage furrow, and later to the apicolateral region where the zonula adherens precursors are formed. In this paper, we show that the basal accumulation of Arm colocalizes with DE-cadherin and Dalpha-catenin, and corresponds to a region of tight membrane association, which we refer to as the basal junction. Although the two junctions are similar in components and function, they differ in their response to the novel cellularization protein Nullo. Nullo is present in the basal junction and is required for its formation at the onset of cellularization. In contrast, Nullo is degraded before apical junction formation, and prolonged expression of Nullo blocks the apical clustering of junctional components, leading to morphological defects in the developing embryo. These observations reveal differences in the formation of the apical and basal junctions, and offer insight into the role of Nullo in basal junction formation.

Expression of claudin-4 and -7 in porcine gingival junctional epithelium.

Junctional epithelium, a nonkeratinized stratified epithelium, extends apically in apposition to the surface of the enamel to form a seal between the epithelium and the tooth. Desmosomes and gap junctions adhere to the junctional epithelium through cell-cell contact, but no evidence of tight junctions has been found. Recently, tight junction hallmark proteins and tight junction-related structures have been identified in stratified squamous epithelium. The present study examined whether tight junction proteins were expressed in the junctional epithelium. We used immunohistochemical techniques to observe expression of claudin-1, -4, -5, -7, and occludin in porcine gingival junctional epithelium. Claudin-4 exhibited immunoreactivity in the intercellular spaces of all layers of the oral epithelium and the junctional epithelium. Stronger expression was observed in junctional epithelial cells adjacent to the inner and outer basal laminae than in the inner cell layers. Immunohistochemical positivity for claudin-7 was clearly observed in the junctional epithelium, but only a faint positivity was observed in the basal layer of the oral epithelium. No immunohistochemical positivity for claudin-1, -5, or occludin was observed in the junctional epithelium. RT-PCR assay confirmed expression of porcine claudin-4 and -7 mRNAs in the junctional epithelium. These findings indicate that claudin-4 and -7 may play a role in the junctional epithelium even in the absence of tight junctions.

A Simulated Microgravity Environment Causes a Sustained Defect in Epithelial Barrier Function.

Intestinal epithelial cell (IEC) junctions constitute a robust barrier to invasion by viruses, bacteria and exposure to ingested agents. Previous studies showed that microgravity compromises the human immune system and increases enteropathogen virulence. However, the effects of microgravity on epithelial barrier function are poorly understood. The aims of this study were to identify if simulated microgravity alters intestinal epithelial barrier function (permeability), and susceptibility to barrier-disrupting agents. IECs (HT-29.cl19a) were cultured on microcarrier beads in simulated microgravity using a rotating wall vessel (RWV) for 18 days prior to seeding on semipermeable supports to measure ion flux (transepithelial electrical resistance (TER)) and FITC-dextran (FD4) permeability over 14 days. RWV cells showed delayed apical junction localization of the tight junction proteins, occludin and ZO-1. The alcohol metabolite, acetaldehyde, significantly decreased TER and reduced junctional ZO-1 localization, while increasing FD4 permeability in RWV cells compared with static, motion and flask control cells. In conclusion, simulated microgravity induced an underlying and sustained susceptibility to epithelial barrier disruption upon removal from the microgravity environment. This has implications for gastrointestinal homeostasis of astronauts in space, as well as their capability to withstand the effects of agents that compromise intestinal epithelial barrier function following return to Earth.