Annette Kohl

Establishment of oral mucosa phenotype in vitro in correlation to epithelial anchorage.

Abstract Cell-matrix interactions and the ordered deposition of basement membrane (BM) components are of major importance for the maintenance of tissue homeostasis in complex epithelia. This aspect was studied in vitro in a coculture system designed as an oral mucosa model. As crucial epithelial features the kinetics of proliferation, expression of site-specific keratins as well as integrin patterns in correlation to synthesis of BM components were assessed by immunohistochemistry and in situ hybridization. Comparison with non-cornified gingiva as tissue of origin revealed different stages of epithelial development, eventually leading to complete reconstruction within a time frame of 1–3 weeks. First, the initial activated stage up to 1 week was characterized by (a) high keratinocyte proliferation, (b) extended expression of the basal cell-specific keratin K5 and (c) a patchy pattern of the differentiation-specific keratins K4 and K13. Second, after 2 weeks the improvement of histoarchitecture correlated to (a) predominant K5 expression in the basal and (b) extension of K4 and K13 within the suprabasal cell compartment, (c) high expression of integrins α3β1 and α6β4 including their ligand laminin-5 and (d) accumulating deposition of basement membrane components. Third, virtually complete tissue normalization at 3 weeks was indicated by (a) restriction of K5 to the basal cell area, (b) regular suprabasal localization of K4 and K13, (c) polarization of integrins to basal and parabasal cells and (d) linear codistribution of collagen IV, “classical” laminin (-1 or -10) and laminin-5 underneath the basal cells. Thus, these organotypic cocultures represent relevant equivalents for non-keratinized oral mucosa with typical gingival differentiation features and in addition suitable models for preclinical trials such as prospective dental material testing.

Mechano-transduction in periodontal ligament cells identifies activated states of MAP-kinases p42/44 and p38-stress kinase as a mechanism for MMP-13 expression

BackgroundMechano-transduction in periodontal ligament (PDL) cells is crucial for physiological and orthodontic tooth movement-associated periodontal remodelling. On the mechanistic level, molecules involved in this mechano-transduction process in PDL cells are not yet completely elucidated.ResultsIn the present study we show by western blot (WB) analysis and/or indirect immunofluorescence (IIF) that mechanical strain modulates the amount of the matrix metalloproteinase MMP-13, and induces non-coherent modulation in the amount and activity of signal transducing molecules, such as FAK, MAP-kinases p42/44, and p38 stress kinase, suggesting their mechanistic role in mechano-transduction. Increase in the amount of FAK occurs concomitant with increased levels of the focal contact integrin subunits β3 and β1, as indicated by WB or optionally by IIF. By employing specific inhibitors, we further identified p42/44 and p38 in their activated, i.e. phosphorylated state responsible for the expression of MMP-13. This finding may point to the obedience in the expression of this MMP as extracellular matrix (ECM) remodelling executioner from the activation state of mechano-transducing molecules. mRNA analysis by pathway-specific RT-profiler arrays revealed up- and/or down-regulation of genes assigning to MAP-kinase signalling and cell cycle, ECM and integrins and growth factors. Up-regulated genes include for example focal contact integrin subunit α3, MMP-12, MAP-kinases and associated kinases, and the transcription factor c-fos, the latter as constituent of the AP1-complex addressing the MMP-13 promotor. Among others, genes down-regulated are those of COL-1 and COL-14, suggesting that strain-dependent mechano-transduction may transiently perturbate ECM homeostasis.ConclusionsStrain-dependent mechano-/signal-transduction in PDL cells involves abundance and activity of FAK, MAP-kinases p42/44, and p38 stress kinase in conjunction with the amount of MMP-13, and integrin subunits β1 and β3. Identifying the activated state of p42/44 and p38 as critical for MMP-13 expression may indicate the mechanistic contribution of mechano-transducing molecules on executioners of ECM homeostasis.

Assessment of acute cyto- and genotoxicity of corrosion eluates obtained from orthodontic materials using monolayer cultures of immortalized human gingival keratinocytes

Whilst a patient is undergoing orthodontic treatment, dental appliances based on non-precious metals or titanium remain in the oral cavity for up to several years. Throughout this period the appliance is in either direct or indirect contact with the oral mucosa.To investigate the possibility of cell damage occurring as a result of appliance corrosion, monolayer cultures of immortalized human gingival keratinocytes were assessed for acute cyto- and genotoxicity using the hexosaminidase assay and the Comet assay respectively. The materials tested included 1. a nickel-free wire, 2. a UK-1 bond, 3. nickel-free as well as nickel-containing brackets with and without color signature and 4. a titanium expansion screw. Each of the test materials was corroded in a solution consisting of equal amounts of lactic acid and sodium chloride (0.1 M) for 1, 3, 7 and 14 days. The cell cultures were then exposed to eluates exhibiting the highest ion concentrations.None of the eluates was found to exhibit acute cytotoxicity, regardless of the type of test system used. Qualitative assessment using neutral red dye for live cells and either trypan blue or propidium iodide to disclose dead cells failed to reveal any significant increase in cell damage when exposed cells were compared to control cultures. Unrestricted cell vitality was confirmed by quantifying viable cells through measurement of hexosaminidase enzyme activity. Furthermore, assessment of genotoxicity revealed no apparent DNA damage to immortalized gingival keratinocytes following exposure to the test eluates.Because the materials tested in this study were corroded using the exacting methods normally applied to precious metals or gold-containing alloys, the lack of either acute cyto- or genotoxic effects following exposure to the test eluates indicates that the materials tested exert no adverse effects on cells similar to those of the target tissue exposed to the materials in situ.ZusammenfassungDie während der kieferorthopädischen Behandlung eingesetzten Apparaturen, die aus Nichtedelmetallegierungen oder Titan bestehen, verbleiben häufig über Jahre in der Mundhöhle. Während des gesamten Zeitraums steht die Apparatur in direktem oder indirektem Kontakt mit der Oralmukosa.Um zu untersuchen, ob eine mögliche Zellschädigung durch Korrosion der Apparatur hervorgerufen wird, wurden Monolayerkulturen von immortalisierten humanen Gingivakeratinozyten mit dem Hexosaminidasetest und dem Comet-Assay (Einzelzellgelelektrophorese) auf akute Zyto- und Gentoxizität hin untersucht. Untersucht wurden 1. ein nickelfreier Draht, 2. ein UK-1-Band, 3. nickelfreie und nickelhaltige Brackets mit und ohne Farbmarkierung und 4. eine Dehnschraube aus Titan. Jedes der Untersuchungsmaterialien wurde für einen, drei, sieben und 14 Tage in einer Lösung, die zu gleichen Teilen aus Milchsäure und Natriumchlorid (0,1 M) bestand, korrodiert. Danach wurden die Zellkulturen mit den Eluaten, die die höchsten lonenkonzentrationen aufwiesen, inkubiert.Unabhängig vom Testsystem zeigte keines der Eluate eine akute Zytotoxizität. Die qualitative Untersuchung mit dem Farbstoff Neutralrot, der lebende Zellen färbt, und Trypanblau oder Propidiumjodid, die tote Zellen anfärben, ergab keinerlei Zunahme der Zellschädigung im Vergleich von exponierten zu nichtexponierten Kontrollkulturen. Die uneingeschränkte Zellvitalität wurde durch die Quantifizierung lebender Zellen mittels Messung der Aktivität des Enzyms Hexosaminidase bestätigt. Darüber hinaus ließ die Untersuchung der Gentoxizität nach Eluatexposition keine sichtbare DNA-Schädigung an immortalisierten Gingivakeratinozyten erkennen.Die in dieser Studie untersuchten Materialien wurden unter Bedingungen korrodiert, die normalerweise auf Edelmetalle oder goldhaltige Legierungen angewendet werden. Das Ausbleiben zyto- oder gentoxischer Effekte nach Eluatexposition zeigt, daß die getesteten Materialien keine schädigenden Effekte auf Zellen ausüben, die große Ähnlichkeit zu denen des Zielgewebes aufweisen, das den Materialien in situ ausgesetzt ist.

The HPV-16 E5 Protein Inhibits TRAIL- and FasL-Mediated Apoptosis in Human Keratinocyte Raft Cultures

By using raft cultures of the polyclonal HaCaT cell lines stably transfected either with E5 (HaCaT/E5) or the empty vector (HaCaT/pMSG) as reference, we investigated the effect of the human papillomavirus type 16 (HPV-16) E5 protein on apoptosis. In comparison to conventional monolayer cultures this model system allows analysis of apoptosis under more tissue-like conditions by mimicking the stratified organization of a normal surface epithelium. Apoptosis was triggered either by FasL or TRAIL. Execution of the death program was checked at early and late stages by monitoring procaspase-3 cleavage and DNA fragmentation, respectively. Rafts of E5-expressing keratinocytes were completely protected from apoptosis and showed a background of apoptotic cells as low as the untreated cultures. In contrast, the HaCaT/pMSG cultures revealed a dramatic increase in apoptotic cells upon ligand treatment throughout the epithelial compartment. We conclude that the presence of the HPV-16 E5 protein in our tissue-like model prevents FasL- or TRAIL-mediated apoptosis.

Epithelium and fibroblast-like phenotypes derived from HPV16 E6/E7-immortalized human gingival keratinocytes following chronic ethanol treatment.

Epithelial-mesenchymal transition (EMT) may be critical for neoplastic progression and its eventual tumorigenicity of epithelia. In this context, we investigated whether EMT and EMT-associated features occurred after chronic ethanol treatment of human gingival keratinocytes immortalized with the E6/E7 oncogenes of human papillomavirus (HPV) type 16. Following a nine-week treatment of cells with 30 mM ethanol in keratinocyte growth medium, they were cultured in normal DMEM with 10% serum. These cell populations were able to proliferate in this medium gradually exhibiting elongated morphology indicating that these cells underwent EMT. Control cells without ethanol treatment did not survive subcultures in DMEM. Upon long-term subcultures of ethanol-treated cells, two phenotypes were obtained exhibiting epithelium-like and spindle-shape fibroblast-like morphology (respectively, termed as EPI and FIB cells), the latter indicating EMT. In comparison to EPI cells, the phenotypic transition to FIB cells was concomitant with a decrease in the expression of keratins, desmoplakins and a complete loss of K14. Moreover, FIB cell transition strongly correlates with an increase in the expression of vimentin and simple epithelial keratin K18. These alterations in FIB cells were associated with the ability of these cells to exhibit anchorage-independent growth, while EPI cells exhibited anchorage-dependent growth. Concerning the transformation stage, FIB cells represent a progressively more advanced transformed phenotype which may reflect an early step during HPV- and ethanol-dependent multi-step carcinogenesis.

Modulation of the epidermal growth factor receptor by the human papillomavirus type 16 E5 protein in raft cultures of human keratinocytes.

It has been shown that the E5 protein of the human papillomavirus type 16 modulates epidermal growth factor receptor downregulation in monolayer cultures of human keratinocytes and mouse fibroblasts. We have now analysed the effect of this protein on the expression, the distribution and the activation of EGF receptors in raft cultures derived from an E5-transfected human keratinocyte cell line. The epithelia generated in these cultures were stratified and exhibited suprabasal expression of cytokeratins 1 and 10, which are known markers of early epidermal differentiation. In situ hybridization with an antisense riboprobe to the human papilloma virus type 16 E5 protein revealed a homogeneous gene expression within the entire epithelium of E5-transfected but not empty vector-transfected control cultures. Treatment of serum-starved rafts with EGF for 48 hours led to a strong decrease of suprabasal EGF receptors in control cultures, but not in rafts of E5-expressing cells. Under these conditions, no activated receptors were observed in control cultures, but activated receptors were still present in E5-raft cultures. Our results indicate that human papilloma virus type 16 E5-mediated modulation of EGF receptor expression occurs in a time- and structure-dependent manner in epithelial equivalents of human keratinocytes.

Connexin 43 expression is downregulated in raft cultures of human keratinocytes expressing the human papillomavirus type 16 E5 protein.

Abstract. A decrease in gap junction-mediated cell-to-cell communication has previously been observed in monolayer cultures of human keratinocytes (HaCaT cells) expressing the human papillomavirus type 16 E5 (HPV16 E5) gene and attributed to the reduced phosphorylation of connexin 43, the most abundant connexin in HaCaT cells. In line with this observation, we have now analyzed the effect of HPV16 E5 on connexin 43 expression in raft cultures produced by transfected HaCaT cells. These keratinocytes transcribe HPV16 E5 under the control of a dexamethasone-inducible promoter. Our results show that treatment with dexamethasone leads to an almost complete disappearance of connexin 43 in rafts expressing the E5 gene but not in control rafts. In our study we discuss the possible effects of this downregulation on cell-cell communication and cellular malignant transformation.

Elevated gene expression of MMP-1, MMP-10, and TIMP-1 reveal changes of molecules involved in turn-over of extracellular matrix in cyclosporine-induced gingival overgrowth.

In humans, pathogenesis in cyclosporine A (CsA)-induced gingival overgrowth (GO) includes the accumulation of extracellular matrix (ECM) constituents, viz., collagen type-1 and type-3 and proteoglycans, in subgingival connective tissue. However, whether this increase is associated with alterations of molecules pivotal for the turn-over of collagens and proteoglycans remains unclear. The present study explores the status of matrix metalloproteinase MMP-1 and MMP-10, which are important for fibrillar collagen and proteoglycan turn-over, and their tissue inhibitor TIMP-1, on their gene expression and protein levels in frozen sections derived from GO and matched normal tissue. In situ hybridization (ISH) revealed elevated levels of MMP-1 gene expression in the connective tissue of GO compared with normal tissue. This elevation also applied to MMP-10 and TIMP-1, the latter exhibiting the strongest gene transcription in the deep connective tissue. These differences detected by ISH were corroborated by quantitative reverse transcription/polymerase chain reaction; relative gene expression analysis indicated a 1.9-fold increase for MMP-1, a 2.3-fold increase for MMP-10, and a 4.8-fold increase for TIMP-1. Detection of the protein by indirect immunofluorescence showed that normal gingival tissue was devoid of all three proteins, although they were detectable in GO tissue, with emphasis on TIMP-1. Analysis of our data indicates elevated levels of MMP-1 and-10, and particularly TIMP-1. With respect to TIMP-1, this elevation may in turn lead to alterations in ECM turn-over by abrogating MMP-1 and MMP-10, thereby contributing to ECM accumulation associated with GO.

Topographic Changes of Focal Adhesion Components and Modulation of p125FAK Activation in Stretched Human Periodontal Ligament Fibroblasts

Mechanical stress has been shown in vitro to modulate integrin-β1-mediated activation of p125FAK/FAK. To test the hypothesis whether this also applies to periodontal ligament fibroblasts (PDLs), we subjected human PDLs to mechanical stretch and analyzed stress-induced changes of p125FAK activation by quantitative immunoprecipitation of p125FAK and changes in the topography of molecules localizing in focal adhesions by indirect immunofluorescence. Generally, all components of focal contacts under study-including detection of phosphotyrosine, i.e., integrin-β1, p125FAK, and paxillin-revealed a relative co-localization during stretch application. Under stretch, we observed a re-distribution of all components from the cell periphery to the cytoplasm following the main axes. Tyrosine phosphorylation of p125 FAK was monitored up to 72 hours under stretch. While the amount of p125FAK remained essentially constant, the activation of p125 FAK was clearly modulated. Tyrosine phosphorylation of p125FAK increased from 15 minutes up to 1 hour and declined after stretching periods of 24, 48, and 72 hours. The analysis of our data indicated a stretch-induced re-distribution of focal adhesion components and a modulation of p125 FAK activation, suggesting alterations in focal adhesions and their associated signal cascade.

Compression-dependent Up-regulation of Ephrin-A2 in PDL Fibroblasts Attenuates Osteogenesis

Members of the ephrin/Eph family have recently been shown to be involved in the regulation of bone homeostasis in a murine model. The activation of the EphB4 receptor on osteoblasts by its ligand ephrin-B2 led to stimulation of osteoblastogenesis and therefore to bone formation. The activation of ephrin-A2-EphA2 signaling on osteoblasts inhibited the activation of osteoblast-specific gene expression, leading to bone resorption. Fibroblasts within the periodontal ligament periodontal ligament may be one of the first responders to orthodontic forces. Periodontal ligament fibroblasts (PDLF) are mechanoresponsive. Members of the ephrin/Eph family might link mechanical forces received by PDLF with the regulation of osteoblastogenesis on osteoblasts of the alveolar bone. To study whether ephrin-A2 is modulated upon compression, we subjected human primary PDLF to static compressive forces (30.3 g/cm2). Static compressive forces significantly induced the expression of ephrin-A2, while the expression of ephrin-B2 was significantly down-regulated. Moreover, osteoblasts of the alveolar bone stimulated with ephrin-A2 in vitro significantly suppressed their osteoblastogenic gene expression (RUNX2, ALPL) and decreased signs of osteoblastic differentiation, as demonstrated by a significantly reduced ALP activity. Together, these findings establish a role for this ligand/receptor system linking mechanical forces with the regulation of osteogenesis during orthodontic tooth movement.