The expressions of claudin‐1 and E‐cadherin in junctional epithelium

Fujita T, Hayashida K, Shiba H, Kishimoto A, Matsuda S, Takeda K, Kawaguchi H, Kurihara H. The expressions of claudin‐1 and E‐cadherin in junctional epithelium. J Periodont Res 2010; 45: 579–582. © 2010 John Wiley & Sons A/S Background and Objective: The epithelium provides an important barrier against microbial invasion. Tight junction structural proteins called claudins are known to contribute to the epithelial cell barrier. Junctional epithelium is located at a strategically important interface between gingival sulcus and is interconnected by desmosomes and gap junctions, but not by tight junctions. Although claudins are tight junction‐associated proteins, they are also expressed in the epithelium despite its lack of tight junctions in invertebrates. Therefore, claudins may play an important role in junctional epithelium without tight junctions. E‐cadherin is a key molecule in the formation of adherence junctions and desmosomes. In the present study, we aimed to investigate the expressions of claudin‐1,claudin‐3, claudin‐7 and E‐cadherin in the junctional epithelium of Fischer 344 rats. Material and Methods: Gingival tissues from Fischer 344 rats were analyzed by immunohistochemical staining for claudin‐1, claudin‐3, claudin‐7, and E‐cadherin. Results: Intense staining for claudin‐1 and E‐cadherin were observed in the junctional epithelium. In contrast to claudin‐1, claudin‐3 was mainly expressed in oral gingival epithelium and claudin‐7 could not be detected on immunohistochemical analysis of the rat gingiva. Conclusion: These data suggest that claudin‐1 and E‐cadherin exist in the junctional epithelium and may play an important role in epithelial barrier function.     

Loss of claudin-1 in lipopolysaccharide-treated periodontal epithelium

Fujita T, Firth JD, Kittaka M, Ekuni D, Kurihara H, Putnins EE. Loss of claudin‐1 in lipopolysaccharide‐treated periodontal epithelium. J Periodont Res 2012; 47: 222–227. © 2011 John Wiley & Sons A/S Background and Objective: The epithelial barrier is a critical component of innate immunity and provides protection against microbial invasion. Claudin‐1, a tight junction protein, is known to contribute to the epithelial cell barrier. An experimentally induced rat periodontal disease model was used to study the effects of lipopolysaccharide (LPS) on the expression of tight junction‐associated molecule genes in the junctional epithelium. Material and Methods: LPS was applied for 8 wk in the gingival sulcus, and junctional epithelium was collected by laser‐capture microdissection and subjected to microarray analysis. Results: Microarray analysis identified that expression of the claudin‐1 gene was decreased in the epithelium by chronic LPS challenge. Immunohistochemical analysis confirmed the expression of claudin‐1 protein in junctional epithelium and that 8 wk of chronic LPS topical application significantly reduced claudin‐1 expression. The effect of LPS on claudin‐1 protein expression was validated using a porcine junctional epithelial cell culture Transwell model. The epithelial barrier, as measured using transmembrane resistance, was significantly reduced after 3 wk of LPS challenge and this was associated with a decreased level of expression of claudin‐1 protein. Conclusion: These results confirm that the initiation of experimental periodontal disease is associated with reduction in the expression of claudin‐1 gene and protein. This decreased level of a critical tight junction protein may result in the disruption of barrier function and may play an important role in the initiation of periodontal disease.     

Establishment of gingival epithelial cell lines from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene

We established two gingival epithelial cell lines (GE1 and GE6), originating from transgenic mice harboring the temperature-sensitive simian virus 40 large T-antigen gene. GE1 and GE6 grew at a permissive temperature (33 degrees C) in a pavement arrangement and solely formed multilayers that exhibited morphological features similar to those of the stratified oral epithelium, with neither the use of stromal equivalents nor feeder layers. Both GE cells underwent apoptosis at a non-permissive temperature (39 degrees C). Characteristic keratin peptides, keratin 4 and 13, for mucosal epithelium were obviously expressed in the suprabasal cells, and keratohyalin granules and involucrin were present in the surface flat cells in the multilayered culture. Keratin 10 (one of the markers for higher keratinized gingival epithelium) was rarely found in some uppermost cells, and filaggrin (a component of keratohyalin granules) appeared sparsely in uppermost desquamating cells in the older cultures. These observations indicated that GE1 and GE6 cells exhibited the phenotype characterizing nonkeratinized sulcular epithelium, which possessed the potency undergoing keratinization in such highly stratified cultures as oral gingival epithelium. GE cells increased the expression levels of mRNA of interleukin-1beta and tumor necrosis factor alpha by the stimulation of lipopolysaccharide and extracellular substances of oral streptococci. The GE cell lines thus could serve as an excellent experimental system for further studies on the physiology of gingival epithelium and corresponding diseases, such as periodontal disease, epithelial hyperplasia, and gingival tumors.     

Modulation of epithelial tight junctions by TGF-beta 3 in cultured oral epithelial cells

Background: Previous studies have indicated that transforming growth factor beta 3 (TGF‐β3) was strongly expressed both in the gingival epithelium and the poorly structured pocket epithelium. Methods: A comprehensive analysis of the profile of tight junction proteins was carried out by quantitative real‐time RT‐PCR, Western blot and paracellular permeability assays. Results: Active TGF‐β3 protein added to monolayers of cultured oral epithelial cells initially reduced the permeability to dextran (10 kDa), followed by an increase in permeability. Three hours after the addition of TGF‐β3, expression of genes encoding tight junction components was selectively up‐ or down‐regulated. In addition, up‐ or down‐regulation of expression of several tight junction associated proteins was observed, although the protein changes did not parallel changes in gene expression. To confirm that TGF‐β3 plays a role in epithelial barrier function, a selective Src family kinase inhibitor saracatinib (AZD0530) was added to cells treated with active TGF‐β3. Tight junction proteins claudins‐2, ‐20 and ZO‐2 were significantly decreased, but claudin‐4 and ‐18 were significantly increased. Conclusions: These results suggest that TGF‐β3 is involved in the modulation of epithelial barrier function by regulating assembly of tight junctions.     

Histomorphological and biochemical differentiation capacity in organotypic co-cultures of primary gingival cells

To establish a three-dimensional in vitro test system mimicking the physiological situation of the oral cavity, organotypic co-cultures consisting of primary gingival cells on a collagen matrix with fibroblasts were generated. The histomorphological development after 7 and 14 d revealed close similarity with the non-keratinized gingiva epithelium. Furthermore, as epithelial specific markers synthesis and localization of keratins as well as the deposition of basement membrane components were assessed on frozen sections by immunofluorescence and keratin expression by in situ hybridization. Primary keratinocytes in conventional culture stained positive for keratin K14 and the mucosal differentiation-specific keratins K4 and K13, while primary fibroblasts, isolated from the same tissue source, and also some keratinocytes, were positive for vimentin. In organotypic co-cultures the keratinocytes formed a multilayered epithelium within 14 d containing basal cells and flattened cells in the uppermost layers. Comparable to native non-keratinized gingiva keratin 14 gene expression was clearly detectable in the basal cell compartment but showed extending immunolocalization. In addition, particularly at the early stage (7d), basally located keratinocytes were also vimentin positive. According to morphological differentiation K4 and K13 were detectable in suprabasal position at the RNA and protein level. The major basement membrane constituents collagen type IV and laminin increased with time revealing first an interrupted and later a fully extended staining underneath the basal cells. Maintenance of basal cell function was further demonstrated by cell proliferation (BrdU incorporation) which was initially high (7d) but declined towards the later stages (14–21 d). The results demonstrate i) that this co-culture system leads to a stratified surface epithelium with morphological and biochemical characteristics of the non-keratinized gingiva epithelium and ii) that a state of physiological tissue balance was reached, thus rendering a suitable model for tissue compatibility studies.     

Cyclic mechanical pressure‐loading alters epithelial homeostasis in a three‐dimensional in vitro oral mucosa model: clinical implications for denture‐wearers

Denture‐wearing affects the quality and quantity of epithelial cells in the underlying healthy oral mucosa. The physiologic mechanisms, however, are poorly understood. This study aimed to compare histologic changes and cellular responses of an epithelial cell layer to cyclic mechanical pressure‐loading mimicking denture‐wearing using an organotypic culture system to develop a three‐dimensional in vitro oral mucosa model (3DOMM). Primary human oral keratinocytes and fibroblasts were serially grown in a monolayer culture, and cell viability was measured under continuous cyclic mechanical pressure (50 kPa) for 7 days (cycles of 60 min on, 20 s off to degas and inject air). Upon initiation of an air–liquid interface culture for epithelial stratification, the cyclic pressure, set to the mode above mentioned, was applied to the 3DOMMs for 7 days. Paraffin‐embedded 3DOMMs were examined histologically and immunohistochemically. In the monolayer culture, the pressure did not affect the viability of oral keratinocytes or fibroblasts. Few histologic changes were observed in the epithelial layer of the control and pressure‐loaded 3DOMMs. Immunohistochemical examination, however, revealed a significant decrease in Ki‐67 labelling and an increase in filaggrin and involucrin expression in the suprabasal layer of the pressure‐loaded 3DOMMs. Pressure‐loading attenuated integrin β1 expression and increased matrix metalloproteinase‐9 activity. Incomplete deposition of laminin and type IV collagen beneath the basal cells was observed only in the pressure‐loaded 3DOMM. Cyclic pressure‐loading appeared to disrupt multiple functions of the basal cells in the 3DOMM, resulting in a predisposition towards terminal differentiation. Thus, denture‐wearing could compromise oral epithelial homeostasis.     

Nicotine modulates gelatinase B (MMP‐9) and epilysin (MMP‐28) expression in reconstituted human oral epithelium

J Oral Pathol Med (2011) 40 : 33–36 Oral epithelial keratinocytes express nicotinic cholinergic receptors which activation modulates keratinocytes differentiation and migration through different metabolic pathways. Matrix metalloproteinases (MMPs) are Zn‐dependent enzyme involved in cell migration. Among them, gelatinase B (MMP‐9) and epilysin (MMP‐28) are two MMPs expressed by human keratinocytes during both wound healing and proliferation. Their expression has been investigated in a reconstituted human oral epithelium (HOE) exposed to nicotine (Nic, 1–50 μM) for 72 h both in the absence and presence of the nicotinic antagonist mecamylamine (Mec), H7, a PKC inhibitor and PD98059, a MAPK inhibitor (PD). At the end of treatment, MMP‐28 expression has been analyzed in epithelium sections using an anti‐MMP‐28 antibody, whereas MMP‐9 presence and activity has been measured in cell‐conditioned medium analyzed by gelatine zymography. The expression of MMP‐9 was reduced by Nic in a dose‐dependent fashion and this effect was antagonized by Mec, H7 and PD. On the other hand, Nic increased the expression of MMP‐28, and this effect was blocked both by H7 and PD, whereas Mec even enforced it. Nic effects on MMP‐9 and MMP‐28 expression by oral keratinocytes were not previously reported and these data suggest MMPs expression mediated by PKC and MAPK as a possible target for Nic toxicity in oral epithelium.     

Lysophosphatidic acid regulates adhesion molecules and enhances migration of human oral keratinocytes

Oral keratinocytes are connected via cell‐to‐cell adhesions to protect underlying tissues from physical and bacterial damage. Lysophosphatidic acids (LPAs) are a family of phospholipid mediators that have the ability to regulate gene expression, cytoskeletal rearrangement, and cytokine/chemokine secretion, which mediate proliferation, migration, and differentiation. Several forms of LPA are found in saliva and gingival crevicular fluid, but it is unknown how they affect human oral keratinocytes (HOK). The aim of the present study was therefore to examine how different LPA forms affect the expression of adhesion molecules and the migration and proliferation of HOK. Keratinocytes were isolated from gingival biopsies obtained from healthy donors and challenged with different forms of LPA. Quantitative real‐time RT‐PCR, immunocytochemistry, and flow cytometry were used to analyze the expression of adhesion molecules. Migration and proliferation assays were performed. Lysophosphatidic acids strongly promoted expression of E‐cadherin and occludin mRNAs and translocation of E‐cadherin protein from the cytoplasm to the membrane. Occludin and claudin‐1 proteins were up‐regulated by LPA. Migration of HOK in culture was increased, but proliferation was reduced, by the addition of LPA. This indicates that LPA can have a role in the regulation of the oral epithelial barrier by increasing the expression of adhesion molecules of HOK, by promotion of migration and by inhibition of proliferation.     

Effect of Curcumin on Systemic T Helper 17 Cell Response; Gingival Expressions of Interleukin‐17 and Retinoic Acid Receptor‐Related Orphan Receptor γt; and Alveolar Bone Loss in Experimental Periodontitis

Background: Curcumin has anti‐inflammatory and antioxidant effects and is reported to have many biologic activities. The current study examines effect of curcumin on: 1) systemic T helper 17 (Th17) cell response; 2) gingival expressions of interleukin (IL)‐17 and retinoic acid receptor‐related orphan receptor (ROR) γt; and 3) alveolar bone loss (ABL) in experimental periodontitis. Methods: Thirty‐eight male albino Wistar rats were divided into four groups: 1) group 1 = periodontitis; 2) group 2 = periodontitis with curcumin treatment; 3) group 3 = periodontally healthy with curcumin treatment; and 4) group 4 = periodontally healthy. Curcumin was administered via oral gavage (30 mg/kg/d) for 15 days. After sacrifice via exsanguination, the following serum levels were determined using enzyme‐linked immunosorbent assay: 1) IL‐1β; 2) IL‐6; 3) IL‐17A; 4) IL‐23; and 5) transforming growth factor‐ β. Morphometric evaluation of ABL was conducted and expression levels of IL‐17 and RORγt in gingival tissues were evaluated immunohistochemically. Results: Group 2 had significantly lower ABL than group 1 (P <0.0125). Highest expression levels of IL‐17 and RORγt were observed in group 1 and were significantly higher than those in all other groups (P <0.0125). The only serum biochemical parameter significantly different among groups was level of IL‐23 (P <0.05). Serum IL‐23 levels were higher in groups 1 and 2 than groups 3 and 4 (P <0.0125); however, they were not significantly different for groups 1 and 2 (P >0.0125). Conclusion: Curcumin seems to be a promising host modulatory agent in periodontal disease pathogenesis regarding IL‐17/IL‐23 axis, with a decreasing effect on ABL and gingival expressions of IL‐17 and RORγt.     

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‐1β (IL‐1β), 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.     

Expression of keratins 8, 18, and 19 in epithelia of atrophic oral lichen planus

Keratins form intermediate filaments of the cytoskeleton in keratinocytes and have roles in cell structure, signaling, intracellular transport, and cell death. Oral lichen planus (OLP) is an oral inflammatory disease with derangements in basal keratinocytes and disruption of the basal membrane. Here, we focused on epithelial expression of keratins 8, 18, and 19 because these proteins are known to modulate cell death. Biopsies were taken from buccal oral mucosa of persons with normal oral mucosa (n = 10) or atrophic OLP (n = 10). Cultured normal oral keratinocytes (n = 4) showed expression of mRNA and protein for keratins 8, 18, and 19. Immunohistochemistry showed consistent staining for keratins 8 and 18 in basal keratinocytes of normal oral mucosa. In OLP, staining for keratin (K)8 was mostly negative and staining for K18 was weak. Keratin 19 was expressed irregularly in most biopsies of normal oral mucosa and not at all in OLP. Several mononuclear leukocytes in the cellular infiltrate showed membrane staining for K8 and K18. Positive staining for K16 confirmed partial collapse of the basal cell layer in OLP. The basal cell niche in OLP therefore appeared to be partly populated with keratinocytes demonstrating a higher degree of differentiation (K8⁻ K18⁻ K19⁻ K16⁺); consequently, such areas may be more susceptible to the action of cell death factors released from the cell infiltrate as a result of lacking the protective, normal keratin present in the basal epithelial cell layer of normal oral mucosa.     

Epithelial barrier and oral bacterial infection

The oral epithelial barrier separates the host from the environment and provides the first line of defense against pathogens, exogenous substances and mechanical stress. It consists of underlying connective tissue and a stratified keratinized epithelium with a basement membrane, whose cells undergo terminal differentiation resulting in the formation of a mechanically resistant surface. Gingival keratinocytes are connected by various transmembrane proteins, such as tight junctions, adherens junctions and gap junctions, each of which has a specialized structure and specific functions. Periodontal pathogens are able to induce inflammatory responses that lead to attachment loss and periodontal destruction. A number of studies have demonstrated that the characteristics of pathogenic oral bacteria influence the expression and structural integrity of different cell–cell junctions. Tissue destruction can be mediated by host cells following stimulation with cytokines and bacterial products. Keratinocytes, the main cell type in gingival epithelial tissues, express a variety of proinflammatory cytokines and chemokines, including interleukin‐1alpha, interleukin‐1beta, interleukin‐6, interleukin‐8 and tumor necrosis factor‐alpha. Furthermore, the inflammatory mediators that may be secreted by oral keratinocytes are vascular endothelial growth factor, prostaglandin E₂, interleukin‐1 receptor antagonist and chemokine (C‐C motif) ligand 2. The protein family of matrix metalloproteinases is able to degrade all types of extracellular matrix protein, and can process a number of bioactive molecules. Matrix metalloproteinase activities under inflammatory conditions are mostly deregulated and often increased, and those mainly relevant in periodontal disease are matrix metalloproteinases 1, 2, 3, 8, 9, 13 and 24. Viral infection may also influence the epithelial barrier. Studies show that the expression of HIV proteins in the mucosal epithelium is correlated with the disruption of epithelial tight junctions, suggesting a possible enhancement of human papilloma virus infection by HIV‐associated disruption of tight junctions. Altered expression of matrix metalloproteinases was demonstrated in keratinocytes transformed with human papilloma virus‐16 or papilloma virus‐18,. To summarize, the oral epithelium is able to react to a variety of exogenous, possibly noxious influences.     

Soft‐Tissue Alterations Following Exposure to Tooth‐Whitening Agents

Background: Tooth‐whitening agents are widely used, either as self‐application products or under the supervision of a dentist. These products may be associated with transient gross morphologic changes in oral soft tissues. However, their potential effects on human keratinocytes and fibroblasts in a stratified squamous epithelium have yet to be elucidated. Methods: In this study, three‐dimensional human tissue equivalents are exposed to varying concentrations of tooth‐whitening agents for increasing time periods. Tissue alterations are investigated in terms of morphology, proliferation, apoptosis, and protein expression. Results: All whitening agents tested altered tissue morphology, induced proliferation of basal keratinocytes, and caused apoptosis of cells in all epithelial strata. In addition, whitening agents induced alterations in the expression of cytokines that are linked to inflammation. Conclusions: These results suggest that whitening agents may induce similar changes in vivo and that these products should be used for limited periods of time or under the supervision of a dental professional.     

维甲酸对口腔上皮细胞内游离钙的调节

目的:探讨维甲酸(RA)对细胞内钙水平的调节作用。方法:将培养的第二代口腔上皮细胞以2×104/cm2接种于激光共聚焦测试培养皿,细胞贴壁后,分别在Ca2 浓度为0.09mmol/L和1.2mmol/L的角朊细胞培养基(K-SFM)中继续培养12h。先分别加入钙离子指示剂Fluo-3/AM负载后,再加入10-7M的维甲酸在不同时段激光共聚焦显微镜下观察细胞内钙离子分布变化并进行定量分析。结果:经含维甲酸处理的高钙环境生长的细胞内钙含量明显减少,而维甲酸对正常K-SFM中生长的细胞内钙含量无明显影响。结论:维甲酸调节上皮细胞分化的机制可能是通过减少细胞的游离钙水平而发挥作用。     

Transepithelial electrical resistance and tight junctions of human gingival keratinocvtes

Human gingival keratinocytes (HGKs) were studied by means of freeze-fracture technique, conventional electron microscopy and the transepithelial electrical resistance for the investigation of intercellular contacts. For the purpose of comparison, MDCK cells and HaCat cells were also included. An unexpected finding was the presence of tight junctions in the HGKs. In vivo the tight junctions, which were of low complexity and P-face-associated, were co-distributed with desmosomes; in one case, the strands ran directly through desmosomal plaques. Where tight junctions and desmosomes occurred together, no gap junctions were seen. In contrast, where no tight junctions were present, gap junctions and desmosomes were co-localized. However, the unfavourable fracture planes through the tissue did not allow a clearcut allocation of gap junction/tight junction occurrence to certain strata. In vitro, HGKs also expressed tight junctions which formed networks of low complexity and high P-face association. Whereas desmosomes were highly expressed, gap junctions were not observed in cultured keratinocytes. Transepithelial electrical resistances (TEER) of cultured HGKs were higher than the values in low resistance-MDCK cells and HaCat cells but considerably lower than the values in high resistance MDCK cells, supporting the fundamental correlation between tight junction morphology and TEER. The results with this cell culture model of the human gingiva provide some valuable information about in vitro differentation and concommittent changes in cellular contacts of human gingival keratinocytes.