Loss of claudin-1 in lipopolysaccharide-treated periodontal epithelium.

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.

Matrilysin (matrix metalloproteinase-7) expression in human junctional epithelium.

Matrilysin is a matrix metalloproteinase expressed in exocrine and mucosal epithelium in many human tissues. Immunohistochemical staining showed that matrilysin is expressed in suprabasal cells of junctional epithelium facing the teeth and in epithelial cell rests of Malassez. No matrilysin expression was seen in the periodontal pocket tissue. In a tissue culture model mimicking junctional epithelium, matrilysin expression was also observed in suprabasal epithelial cells. Of 13 anaerobic oral bacterial species tested, F. nucleatum, F. necrophorum, P. endodontalis, and P. denticola stimulated matrilysin expression in porcine periodontal ligament epithelial cells from 2.5- to 5.7-fold, compared with untreated cells. The enzyme was localized in intracytoplasmic vesicles that also reacted with antibodies against lysosomal membrane protein h-lamp-1. The results indicate that matrilysin may play an important role in the normal physiology of junctional epithelium.

Regulation of epithelial cell growth factor receptor protein and gene expression using a rat periodontitis model.

BACKGROUND AND OBJECTIVE: Regulation of epithelial cell behavior associated with periodontitis is not well elucidated but many responses will ultimately be regulated by growth factor receptors. Using a rat experimental periodontitis model, protein and gene expression of select growth factor receptors in junctional and pocket epithelium were examined. MATERIAL AND METHODS: Periodontal disease was induced by daily topical application of lipopolysaccharide using an established protocol. Animals were killed at time 0 (control), and at 2 and 8 wk. Frozen tissue samples were collected from the right palatal gingival soft tissue, and the left periodontal tissues were decalcified and embedded in paraffin. Laser microdissection and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to quantify keratinocyte growth factor receptor (KGFR), hepatocyte growth factor receptor (HGFR), epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor 1 (FGFR1) gene expression, and in situ RT-PCR localized these increases to specific epithelial cells. Receptor protein expression was examined immunohistochemically. In cell culture, induction of HGFR and KGFR protein expression by serum, lipopolysaccharide and pro-inflammatory cytokines were examined using flow cytometry. RESULTS: Eight-week tissue samples exhibited histological changes consistent with periodontitis. KGFR and HGFR gene and protein expression were significantly induced at the 8 wk time point. KGFR expression was significantly up-regulated in basal and parabasal pocket epithelial cells, but HGFR was up-regulated throughout the pocket epithelium. In cell culture serum, lipopolysaccharide and pro-inflammatory cytokines, interleukin-1beta and tumour necrosis factor-alpha significantly induced KGFR protein receptor expression, but HGFR expression was only induced by serum. CONCLUSION: KGFR and HGFR are highly up-regulated in this model of periodontal disease and may play a significant role in regulating the proliferation and migration of pocket epithelium.

Reciprocal interactions between human ovarian surface epithelial cells and adjacent extracellular matrix.

The human ovarian surface epithelium (OSE), or ovarian mesothelium, is functionally complex as seen by its capacity to proliferate, migrate, and contribute to ovulation and ovulatory repair in response to cyclic hormonal and environmental changes. We wished to determine whether this phenotypic versatility is reflected in cell-extracellular matrix interactions in primary and low-passage culture. Comparisons of cultures maintained on different substrata revealed that these cells form cohesive monolayers on plastic, while fibrin clots enhance cell dispersion, and thus may provide a migratory cue. The cells invaded Matrigel as multicellular aggregates, while collagen gels mediated a morphologic epithelial-mesenchymal conversion. On plastic, the cells produced extracellular matrix components characteristic of epithelial basement membrane (laminin and collagen type IV), as well as stroma (collagen types I and III). In addition, ovarian surface epithelial cells secreted serine proteases and matrix metalloproteinases. The levels of chymotrypsin- and elastase-like proteases were dictated by the substratum: low levels were secreted by cells grown on plastic, intermediate levels on collagen gels and fibrin clots, and most protease was produced on Matrigel. The rate of cell proliferation varied with the substrata and was inversely related to protease secretion. Integrin expression was greatest on plastic and least on collagen gels where integrins were downregulated with time. alpha 6/beta 4 was absent from all cells while varying levels of alpha 2, alpha 3, alpha 5, beta 1, and vitronectin receptor were detected depending on the culture substratum employed. In low-passage cultures of human ovarian surface epithelial cells, then, cell shape, growth, protease production, and integrin expression are modulated by the extracellular matrix. The cells, in turn, alter extracellular matrix by synthesis, lysis, and physical remodeling, and express both stromal and epithelial characteristics. The broad repertoire of these functions may be related to their mesodermal origin, and may reflect the expression of a dual, epithelio-mesenchymal phenotype by relatively immature, uncommitted cells. The results demonstrate the great complexity and versatility of these interactions which render OSE cells capable of participating in numerous physiological and pathological processes.

Bacterial phospholipase C upregulates matrix metalloproteinase expression by cultured epithelial cells.

Phospholipase C (PLC) is a putative virulence factor of several pathogenic bacteria. We studied if exogenous PLC would perturb epithelial behavior in infected tissues. Gelatin and casein zymography of cell culture medium indicated that the broad-spectrum PLC of Bacillus cereus induced matrix metalloproteinase (MMP) production in epithelial cells of human skin (NHEK), human gingiva (HGE), and porcine periodontal ligament (PLE). In all three cell types, the strongest increase (ninefold) at 0.1 U/ml was seen in the MMP-9 (92-kDa gelatinase) activity, and the effect was dose dependent in the range of 0.1 to 1.0 U/ml. A relatively weaker increase (twofold) in MMP-2 (72-kDa gelatinase) was also observed in each cell type. PLC induction of MMP-3 (48-kDa stromelysin) was also seen in NHEK and HGE on gelatin and more sensitively for PLE by casein zymography (fivefold). Total gelatinolytic activity as measured by degradation of 14C-labeled denatured type I collagen increased by about 18-fold (NHEK), 12-fold (HGE), and 14-fold (PLE). Northern analysis showed a clear increase in the MMP-9, and a minor increase in MMP-3 mRNA levels but no significant increase in MMP-2 mRNA levels. Further studies with PLE revealed that MMP-9 induction by PLC progressively increased with the length of cell culture time in the absence of serum. PLC induction of MMPs was polar, with MMP-9 and MMP-3 secreted primarily in the apical direction and MMP-2 secreted mainly in the basal direction. The PLC effect was blocked by neomycin, an inhibitor of the phosphoinositol signal pathway. No significant effects were observed in MMP expression with the calcium ionophore A23187 or phospholipase A2. Morphologically, PLC treatment resulted in reduced contacts between the cultured cells and loss of the cell surface microvilli. These results suggest that PLC secreted by bacterial pathogens may disrupt epithelium of infected tissue and increase the subepithelial tissue destruction through induction of MMPs.

Effects of titanium on transcriptional and post-transcriptional regulation of fibronectin in human fibroblasts.

The effects of commercially pure titanium (Ti) on the regulation of fibronectin gene expression and synthesis were investigated in early-passage human gingival fibroblasts. The fibroblasts were cultured on 50 nm Ti-coated silicon wafers treated with radio-frequency glow discharge prior to use and on Falcon tissue culture plastic (TCP) dishes as a control. Northern hybridization analysis revealed that fibroblasts cultured on Ti reduced the fibronectin mRNA level by 58% at 16 h, but increased it by 2.6-fold at 90 h, although the cell numbers and house-keeping gene GAPD mRNA levels on these two surfaces were essentially the same. The amount of total RNA was slightly less on the Ti surface. While the total [35S]methionine incorporation was essentially unaltered, the amount of [35S]methionine-labeled fibronectin was significantly increased in cells cultured on a Ti surface in early cultures but decreased in the late cultures. The apparent discrepancy between the increased fibronectin mRNA levels and decreased translation could be explained by a 30% reduction in fibronectin mRNA half life in cells cultured on Ti. The distribution of fibronectin between the medium and the cell layer also was altered on Ti surfaces, with a approximately 100-fold increase of fibronectin assembled in extracellular matrix at 16 h, but a 36% reduction at 90 h. In contrast, the amount of fibronectin recovered in the medium was essentially unchanged. The total amount of protein assembled into the extracellular matrix by cells on Ti increased 2.1-fold at 16 h but decreased by 19% in 90-h cultures. These significant changes in fibronectin gene activity and gene product distribution by cells cultured on Ti surfaces demonstrate that the surface chemistry of biomaterials can selectively regulate the cellular behavior at the molecular level and, conversely, that molecular biological techniques provide sensitive indicators of the molecular biocompatibility of implant materials.

Chymotrypsin-like enzyme secretion is stimulated in cultured epithelial cells during proliferation and in response to Actinobacillus actinomycetemcomitans.

A chymotrypsin-like enzyme was partially purified from culture medium of epithelial cells of human skin, human gingiva and porcine periodontal ligament by aprotinin-affinity chromatography. The enzyme levels from all three cell types were low in quiescent cultures but increased markedly when the cells were allowed to proliferate. The biphasic elution profile of the enzyme from the affinity column closely matched that of alpha-chymotrypsin and the protein comigrated with it on polyacrylamide gels at 27,000 ML. Synthetic substrate tests of purified fractions showed strong chymotrypsin-like but no trypsin-like or elastase-like activity. Inhibition of protease activity and pH optimum in the range of 7.5-8.0 were consistent with chymotrypsin-like enzymes. Secreted activity was found to be significantly increased by phorbol myristate acetate treatment in a time-course that differed from that of elastase-like activity. Keratinocyte growth factor and epidermal growth factor but not transforming growth factor-beta increased the chymotrypsin-like activity in a concentration-dependent manner. The enzyme secretion by epithelial cells was strongly elevated by exposure to 5 of 6 Actinobacillus actinomycetemcomitans strains isolated from plaque samples of juvenile periodontitis patients. These results indicate that chymotrypsin-like enzymes are secreted by proliferative phenotypes of normal epithelial cells. This enzyme may, therefore, play a role in epithelial physiology and in cell response to certain pathogenic bacteria.

Effects of titanium substratum and grooved surface topography on metalloproteinase-2 expression in human fibroblasts.

The chemical and topographic effects of commercially pure titanium on cell morphology and the regulation of matrix metalloproteinase-2 (MMP-2) gene expression, synthesis, and activity were investigated in early passage human gingival fibroblasts. Scanning electron microscopy showed that on smooth titanium (Ti), fibroblasts remained well spread and randomly oriented throughout the culture period. In contrast, cells on V-shaped grooved titanium (VTi) were oriented along the grooves by 16 h and proliferated in this organization throughout the culture period. The effects of substratum surface chemistry on MMP-2 expression were found to be distinct from those of topography. Northern hybridization analysis of fibroblasts cultured on Ti revealed an MMP-2 mRNA time-course expression pattern parallel to that observed on the tissue culture plastic (TCP) dishes, but at significantly lower levels at each time-point. The Ti mRNA levels were decreased by 34% at 16 h, 55% at 40 h, and 45% at 90 h relative to TCP. In contrast, MMP-2 mRNA expression on VTi showed both an altered time-course expression pattern and altered levels compared to Ti and TCP. Relative to TCP, VTi MMP-2 mRNA levels were approximately 80% less at 16 h and approximately 50% less at 40 h, but not significantly different at 90 h. Relative to Ti, VTi MMP-2 mRNA levels were approximately 75% less at 16 h, but approximately 40% greater at 40 h and approximately 70% greater at 90 h. These differences may be explained in part by the observed changes in MMP-2 mRNA half-life which decreased by approximately 40% on Ti but increased by over fourfold on VTi relative to TCP. The smooth Ti also showed an approximate twofold increase of MMP-2 secretion in the late cultures over TCP controls. These results indicate that substratum surface chemistry and topography-induced changes in cell shape can alter MMP-2 expression in normal fibroblasts. The molecular approach to investigating the major molecules involved in tissue degradation may provide sensitive indicators of tissue remodeling at the tissue-biomaterial interface.

Substratum surface topography alters cell shape and regulates fibronectin mRNA level, mRNA stability, secretion and assembly in human fibroblasts.

The regulation of cell shape, fibronectin mRNA level, secretion and assembly by substratum surface topography was investigated in early passage human gingival fibroblasts cultured on titanium-coated smooth or V-shaped grooved substrata produced by micromachining. Cells on grooved surfaces were significantly elongated and orientated along the grooves of the substratum, while cell height, measured using confocal scanning laser microscopy, was approximately 1.5-fold greater than that of cells on smooth surfaces. Northern hybridization analysis revealed that on a per cell basis the grooved surface increased the amounts of fibronectin mRNA/cell approximately 3.5-fold at 16 hours, approximately 1.9-fold at 40 hours and approximately 2.2-fold at 90 hours, while the mRNA levels of the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPD) were constant. The amounts of secreted fibronectin on the grooved surface were increased approximately 2-fold for all time points. The stability of fibronectin mRNA was also altered by substratum surface topography. The half-life of fibronectin mRNA on smooth surfaces was estimated to be approximately 5 hours, but on the grooved surfaces the half-life of fibronectin mRNA showed a two-phase response: a rapid 60% reduction in the first half-life (t1/2 approximately 2 hours) and a 2.4-fold increase in the second half-life (t1/2 approximately 12 hours) relative to that observed on the smooth surface. The GAPD mRNA half-lives were essentially unaffected by the surface topography of the substrata. The grooved surface was also found to alter the amount of fibronectin assembled into the extracellular matrix, producing a approximately 2-fold increase in the cultures at all time points. It thus appears that substratum surface topography alters cell shape and modulates fibronectin at the transcriptional and post-transcriptional levels, as well as the amount of fibronectin assembled into extracellular matrix. Micromachining, which has the ability to precisely control surface topography over a wide range of dimensions and shapes, appears to be a useful technique in investigating the relationship between cell shape and function.

Multilayer culture of periodontal ligament epithelial cells: a model for junctional epithelium.

The unique features of junctional epithelium involve lack of keratinization, limited differentiation and a relatively permeable structure. In order to study the relationship between differentiation and permeability of stratified epithelium a model system was developed. Porcine periodontal ligament epithelial cells were cultured on the polycarbonate nucleopore membrane of the Transwell two-compartment culture system. Within 5 days of culture the cells formed a confluent multilayered structure. Subsequently, maturation of the structure and differentiation of surface cells took place. Transmission electron microscopy showed that the cells were arranged into basal and suprabasal layers with sparse desmosomal attachments and wide intercellular spaces resembling the organization of junctional epithelium. The basal cells attached to a subepithelial basal lamina through numerous hemidesmosomes. The cytokeratin profile of the cultured epithelium (K5, 6, 14, 16, 19) resembled that of the cells of junctional epithelium attached to the tooth surface. The older cultures expressed differentiation markers, K4, K13 and involucrin, thereby resembling sulcular epithelium. The epithelial permeability, measured by diffusion of phenol red, radioactive dextran or methionine tracers, and as transepithelial electrical resistance, decreased with the increased cell number and maturation of the cultures. The new model provides an organotypic culture system which allows to control differentiation of a multilayered periodontal epithelium. It thus may serve as a valuable new tool for studies on the permeability and behaviour of periodontal epithelium under the influence of exogenous and endogenous factors.

Collagenase-3 (matrix metalloproteinase-13) expression is induced in oral mucosal epithelium during chronic inflammation.

Increased proliferation of mucosal epithelium during inflammation is associated with degradation of subepithelial connective tissue matrix and local invasion of the epithelial cells. Here we have studied, whether collagenase-3 (MMP-13), a collagenolytic matrix metalloproteinase with an exceptionally wide substrate specificity, is expressed in the epithelium of chronically inflamed mucosa. Examination of human gingival tissue sections from subjects with chronic adult periodontitis with in situ hybridization revealed marked expression of MMP-13 in basal cells of some epithelial rete ridges expanding into connective tissue. Immunohistochemical staining demonstrated that these cells also expressed strongly laminin-5, suggesting that they are actively migrating cells. A strong signal for MMP-13 mRNA was occasionally also noted in the suprabasal epithelial cells facing the gingival pocket, whereas no collagenase-1 (MMP-1) mRNA was detected in any areas of the epithelium. MMP-13 expression was also detected in fibroblast-like cells associated with collagen fibers of the inflamed subepithelial connective tissue. In organ culture of human oral mucosa, MMP-13 mRNA expression was observed in epithelial cells growing into connective tissue of the specimens. Regulation of MMP-13 expression was examined in cultured normal nonkeratinizing epithelial cells isolated from porcine periodontal ligament. In these cells, MMP-13 expression at the mRNA and protein level was potently enhanced (up to sixfold) by tumor necrosis factor-alpha, transforming growth factor-beta(1), and transforming growth factor-alpha and by keratinocyte growth factor in the presence of heparin. In addition, plating periodontal ligament epithelial cells on type I collagen stimulated MMP-13 expression (sevenfold) as compared with cells grown on tissue culture plastic. The results of this study show, that expression of MMP-13 is specifically induced in undifferentiated epithelial cells during chronic inflammation due to exposure to cytokines and collagen. Thus, it is likely that MMP-13 expression is instrumental in the subepithelial collagenolysis and local invasion of the activated mucosal epithelium into the connective tissue.