Uniform expression of alcohol dehydrogenase 3 in epithelia regenerated with cultured normal, immortalised and malignant human oral keratinocytes

The human oral epithelium is a target for damage from the inhalation of formaldehyde. However, most experimental studies on this chemical have relied on laboratory animals that are obligatory nose breathers, including rats and mice. Therefore, in vitro model systems that mimic the structure of the human oral epithelium and which retain normal tissue-specific metabolic competence are desirable. Based on the established role of alcohol dehydrogenase 3 (ADH3), also known as glutathione-dependent formaldehyde dehydrogenase, as the primary enzyme catalysing the detoxification of formaldehyde, the aim of this study was to investigate the expression of ADH3 in organotypic epithelia regenerated with normal (NOK), immortalised (SVpgC2a) and malignant (SqCC/Y1) human oral keratinocytes. Organotypic epithelia, usually consisting of 5-10 cell layers, were produced at the air-liquid interface of collagen gels containing human oral fibroblasts, after culture for 10 days in a standardised serum-free medium. Immunochemical staining demonstrated uniform expression of ADH3 in these organotypic epithelia, as well as in the epithelial cells of oral tissue. The specificity of the ADH3 antiserum was ascertained from the complete neutralisation of the immunochemical reaction with purified ADH3 protein. Assessment of the staining intensities indicated that the expression levels were similar among the regenerated epithelia. Furthermore, the regenerated epithelia showed similar ADH3 expression to the epithelium in oral tissue. Therefore, a tissue-like expression pattern for ADH3 can be generated from the culture of various oral keratinocyte lines in an organotypic state. Similar expression levels among the various cell lines indicate the preservation of ADH3 during malignant transformation, and therefore that NOK, SVpgC2a and SqCC/Y1 represent functional models for in vitro studies of formaldehyde metabolism in human oral mucosa.     

Keratinocyte growth factor (KGF) promotes keratinocyte cell attachment and migration on collagen and fibronectin

Keratinocyte growth factor (KGF) induction of keratinocyte attachment and migration on provisional and basement membrane proteins was examined. KGF-treated keratinocytes showed increased attachment to collagen types I and IV and fibronectin, but, not to laminin-1, vitronectin, or tenascin. This increase was time- and dose-dependent. Increase in attachment occurred with 2 10 microg/ml of ECM proteins. This KGF-stimulated cell attachment was beta1 integrin-dependent but was not associated with stimulation of the cell surface expression nor affinity (activity) of the collagen integrin receptor (alpha2beta1) nor the fibronectin integrin receptors (alpha5beta1 or alphav). At the basal layer of KGF-treated cells significant accumulation of beta1 integrins was found at the leading edges, and actin stress fibers colocalized with beta1. KGF also induced migratory phenotype and stimulated keratinocyte migration on both fibronectin and collagen types I and IV but not on laminin-1, vitronectin nor tenascin. The results suggest that in addition to its proliferation promoting activity. KGF is able to modulate keratinocyte adhesion and migration on collagen and fibronectin. Our data suggest that KGF induced integrin avidity (clustering), a signaling event, which is not dependent on the alteration of cell surface integrin numbers.     

Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human epidermal keratinocytes and non-differentiating keratinocytes

We have compared the adhesive properties and integrin expression profiles of cultured human epidermal keratinocytes and a strain of nondifferentiating keratinocytes (ndk). Both cell types adhered to fibronectin, laminin, and collagen types I and IV, but ndk adhered more rapidly and at lower coating concentrations of the proteins. Antibody blocking experiments showed that adhesion of both cell types to fibronectin was mediated by the alpha 5 beta 1 integrin and to laminin by alpha 3 beta 1 in synergy with alpha 2 beta 1. Keratinocytes adhered to collagen with alpha 2 beta 1, but an antibody to alpha 2 did not inhibit adhesion of ndk to collagen. Both cell types adhered to vitronectin by alpha v-containing integrins. Immunoprecipitation of surface-iodinated and metabolically labeled cells showed that in addition to alpha 2 beta 1, alpha 3 beta 1, and alpha 5 beta 1, both keratinocytes and ndk expressed alpha 6 beta 4 and alpha v beta 5. ndk expressed all these integrins at higher levels than normal keratinocytes. ndk, but not normal keratinocytes, expressed alpha v beta 1 and alpha v beta 3; they also expressed alpha 1 beta 1, an integrin that was not consistently detected on normal keratinocytes. Immunofluorescence experiments showed that in stratified cultures of normal keratinocytes integrin expression was confined to cells in the basal layer; terminally differentiating cells were unstained. In contrast, all cells in the ndk population were integrin positive. Our observations showed that the adhesive properties of ndk differ from normal keratinocytes and reflect differences in the type of integrins expressed, the level of expression and the distribution of integrins on the cell surface. ndk thus have a number of characteristics that distinguish them from normal basal keratinocytes.     

Analysis of heterogeneity of human keratinocyte populations by their adhesion to substrate and the keratin 19 to actin ratio

Keratin 19 is reported to be a marker for skin stem cells and that assumes an independent subpopulation of these cells in keratinocyte population. In addition, keratinocytes have natural affinity to extracellular matrix proteins. The aim of this work was to reveal subpopulations ofkeratinocytes cultivated on the substrates of collagen I type, laminin-2/4 and fibronectin, and distinguished by keratin 19/actin ratio (G/R). The area of cell projection on a substrate, perimeter, the spreading coefficient and G/R were measured. Both on fibronectin and laminin-2/4, keratinocyte populations were morphologically homogeneous with large cells comprising 12 and 20 percents of the population, respectively. On fibronectin, correlation between morphological parameters of the cells and the keratin 19/actin ratio was not found. The cells growth on collagen behaved as a heterogeneous population, with the large cells compressing more than 50 percents of the population. An average, the size of the cells growing on collagen was twice as large as than of the cells cultivated on fibronectin. On the other hand, correlation between the cell size and G/R was revealed in the cells growth both on collagen and laminin-2/4. The cells with lower G/R value display a larger size and extent of spreading. We assume that this correlation may be determined by alphal and alpha2, integrins characteristic of these cells. Our results cast doubts on whether stem cells are present in the culture of human skin keratinocytes.     

Ganglioside GM3 inhibits matrix metalloproteinase-9 activation and disrupts its association with integrin

Gangliosides GM3 and GT1b both inhibit epithelial cell adhesion and migration on fibronectin. GT1b binds to integrin alpha5beta1 and blocks the integrin-fibronectin interaction; GM3 does not interact with integrin, and its effect is poorly understood. We evaluated the effects of endogenous modulation of GM3 expression on epithelial cell motility on several matrices and the mechanism of these effects. Endogenous accumulation of GM3 decreased cell migration on fibronectin, types I, IV, and VII collagen matrices; depletion of GM3 dramatically increased cell migration, regardless of matrix. GM3 overexpression and depletion in vitro correlated inversely with the expression and activity of matrix metalloproteinase-9; consistently, the cell migration stimulated by GM3 depletion is reversed by inhibition of matrix metalloproteinase-9 activity. Accumulation and depletion of GM3 in epithelial cells grown on fibronectin also correlated inversely with epidermal growth factor receptor and mitogen activated protein kinase phosphorylation and with Jun expression. Ganglioside depletion facilitated the co-immunoprecipitation of matrix metal-loproteinase-9 and integrin alpha5beta1, while endogenous accumulation of GM3, but not GT1b, blocked the co-immunoprecipitation. These data suggest modulation of epidermal growth factor receptor signaling and dissociation of integrin/matrix metalloproteinase-9 as mechanisms for the GM3-induced effects on matrix metalloproteinase-9 function.     

Modulation of growth factor responsiveness of murine mammary carcinoma cells by cell matrix interactions: correlation of cell proliferation and spreading.

We have examined the role of growth factors and extracellular matrix in the proliferation and cell adhesion of a murine mammary carcinoma, SP1, and a stable highly metastatic variant, SP1-3M. On fibronectin, both cell types proliferated strongly in response to basic fibroblast growth factor (bFGF) and platelet-derived growth factor BB (PDGF-BB) after culture for 24 h and 72 h. In contrast, on collagen type I, SP1 cells proliferated only weakly to PDGF-BB at either time, and SP1-3M cells showed a response to PDGF-BB only at 72 h. The proliferative response to bFGF was also consistently lower when the cells were cultured on collagen than on fibronectin. No significant proliferative responses were detected to epithelial growth factor (EGF), transforming growth factor-beta (TGF-beta), or estrogen on any substratum. The lack of responsiveness to PDGF-BB of cells cultured on collagen type I was not due to differences in numbers or affinity of PDGF receptors. We therefore examined the adhesion and spreading properties of SP1 and SP1-3M cells. Without exogenous growth factors, both cell lines adhered to fibronectin and laminin. SP1-3M cells did not bind to collagen type I, whereas SP1 cells did. Attachment to all three substrata was inhibited by anti-beta 1 integrin IgG, suggesting that the primary adhesion to these substrata is mediated by beta 1 integrins. SP1 and SP1-3M cells showed similar integrin patterns following immunoprecipitation by anti-beta 1 integrin IgG. bFGF stimulated increased adhesion and spreading of both SP1 and SP1-3M cells to collagen type I within 24 h, whereas PDGF-BB was less capable of this effect. Our results suggest that the proliferative response of SP1 and SP1-3M cells to PDGF-BB and bFGF is dependent on the extracellular matrix environment, and imply that modification of extracellular matrix and/or surface integrin receptors may regulate responsiveness to these growth factors in the SP1 tumor model.     

Alpha v beta 6 integrin down-regulates the MMP-13 expression in oral squamous cell carcinoma cells

The integrin alphavbeta6, a receptor for fibronectin, vitronectin, tenascin and TGF-beta latency-associated peptide (LAP), is not detectable on normal oral epithelium but is neo-expressed in oral squamous cell carcinomas (OSCC) and epithelial dysplasia. Previously it has been shown that alphavbeta6 integrin can up-regulate MMP-3 and -9 expression in OSCC cells. Using beta6-transfected and control OSCC cells we demonstrate that alphavbeta6 integrin down-regulates MMP-13 expression at both mRNA and protein level. Although expressing less MMP-13, beta6-transfected cells were found to have similar collagenolytic activity as control cells and invade at similar levels through type I collagen. Growth of the tumour cells in organotypic culture and confocal microscopy confirmed low levels of MMP-13 in cells with high alphavbeta6 expression. Furthermore, human squamous cell carcinomas of the tongue with high expression of alphavbeta6 showed lower MMP-13 levels than carcinomas with low levels of alphavbeta6. Our results suggest that alphavbeta6 down-regulates MMP-13 expression in OSCC cells and that MMP-13 is not essential for the degradation of type I collagen by OSCC cells.     

Ultrastructural and immunocytochemical detection of keratins and extracellular matrix proteins in lizard skin cultured in vitro

The present study shows the localization of epidermal and dermal proteins produced in lizard skin cultivated in vitro. Cells from the skin have been cultured for up to one month to detect the expression of keratins, actin, vimentin and extracellular matrix proteins (fibronectin, chondroitin sulphate proteoglycan, elastin and collagen I). Keratinocytes and dermal cells weakly immunoreact for Pan-Cytokeratin but not with the K17-antibody at the beginning of the cell culture when numerous keratin bundles are present in keratinocyte cytoplasm. The dense keratin network disappears after 7-12 days in culture, and K17 becomes detectable in both keratinocytes and mesenchymal cells isolated from the dermis. While most epidermal cells are lost after 2 weeks of in vitro cultivation dermal cells proliferate and form a pellicle of variable thickness made of 3-8 cell layers. The fibroblasts of this dermal equivalent produces an extracellular matrix containing chondroitin sulphate proteoglycan, collagen I, elastic fibers and fibronectin, explaining the attachment of the pellicle to the substratum. The study indicates that after improving keratinocyte survival a skin equivalent for lizard epidermis would be feasible as a useful tool to analyze the influence of the dermis on the process of epidermal differentiation and the control of the shedding cycle in squamates.     

Effects of decorin and biglycan on human airway smooth muscle cell adhesion

Growth on a decorin matrix results in decreased human airway smooth muscle cell (HASMC) number, by decreasing proliferation and increasing apoptosis. We questioned whether these effects were related to abnormal extracellular matrix (ECM)-cell adhesion. HASMCs were seeded on decorin, biglycan, collagen type I or plastic. Actin organization and focal adhesion formation were assessed by staining for filamentous (F) and globular (G) actin, and vinculin, respectively. Gene expression for focal adhesion proteins, ECM molecules and HASMC receptors was measured. Protein levels for fibronectin, α(2), α(5), α(v) and β(3) integrin subunits and, focal adhesion kinase (FAK) were assessed. F-actin filaments were prominent in cells seeded on collagen I and plastic, less apparent in cells cultured on biglycan and faint in cells on decorin. Vinculin clustering was decreased in cells seeded on decorin and biglycan, as was vinculin gene expression. Compared to cells on plastic, cells on decorin had an increase in fibronectin gene expression. Seeding on decorin caused an increase in α(2) integrin subunit and platelet-derived growth factor receptor A gene expression. There was also an increase in α(2) and α(v) integrin subunit protein. Finally, FAK protein levels in cells seeded on decorin or biglycan were decreased compared to cells seeded on plastic or collagen I. Cells grown on proteoglycan matrices demonstrate evidence of abnormalities during many of the key processes involved in normal cell adhesion. Upregulation of cell surface receptor proteins, such as α(2) integrin subunit, may represent a compensatory mechanism to overcome poor adhesion induced by growth on these matrices.     

Collagen type I together with fibronectin provide a better support for endothelialization

Endothelialization of vascular implants is limited by the inability of cells to retain adhesion when exposed to flow. Extracellular matrix proteins, including fibronectin and collagen, enhance cell adherence on materials. This study investigated the behaviour of Human Umbilical Vein Endothelial Cells (HUVEC) on extracellular matrix coated polystyrene. Collagen and fibronectin were coated as single and double layers to analyse differences in cell proliferation, morphology, and cell-protein interactions. Significantly higher endothelial cell proliferation and migration rates were observed on the collagen and collagen+fibronectin coating compared to the uncoated or fibronectin-coated sample. Immmunofluorescent microscopy showed evidence of extracellular matrix remodelling in the double, collagen+fibronectin coating. These results strongly suggest that a double coating of collagen+fibronectin provides a better support structure for endothelial cell growth and contributes to improve the ability of vascular implants to become and remain endothelialized.     

Retinoic acid effects on an SV-40 large T antigen immortalized adult rat bone cell line

Clonal cell lines were established from adult rat tibia cells immortalized with SV-40 large T antigen. One clone (TRAB-11), in which retinoic acid (RA) induced alkaline phosphatase (AP) activity, was selected for further study. The TRAB-11 cells express high levels of type I collagen mRNA, type IV collagen, fibronectin, practically no type III collagen, little osteopontin, and no osteocalcin. RA stimulates proliferation of TRAB-11 cells (starting at 10 pM) and survival (starting at 100 pM). TRAB-11 cells synthesize fibroblast growth factor-2 (FGF-2), which has potent autocrine mitogenic effects on these cells and acts synergistically with RA. TRAB-11 cells attach better to type IV collagen than to fibronectin or laminin. Cell attachment to type IV collagen is increased by RA and decreased (65%) by an antibody directed against alpha1beta1 integrin. RA up-regulates steady-state levels of alpha1, mRNA without affecting beta1 mRNA expression. In conclusion, we report the establishment of a clonal cell line from the outgrowth of adult rat tibiae which is highly sensitive to RA in its growth and survival in culture, apparently as a result of integrin-mediated cell interaction with extracellular matrix proteins.     

Cell-matrix entanglement and mechanical anchorage of fibroblasts in three-dimensional collagen matrices

Fibroblast-3D collagen matrix culture provides a physiologically relevant model to study cell–matrix interactions. In tissues, fibroblasts are phagocytic cells, and in culture, they have been shown to ingest both fibronectin and collagen-coated latex particles. Compared with cells on collagen-coated coverslips, phagocytosis of fibronectin-coated beads by fibroblasts in collagen matrices was found to be reduced. This decrease could not be explained by integrin reorganization, tight binding of fibronectin beads to the collagen matrix, or differences in overall bead binding to the cells. Rather, entanglement of cellular dendritic extensions with collagen fibrils seemed to interfere with the ability of the extensions to interact with the beads. Moreover, once these extensions became entangled in the matrix, cells developed an integrin-independent component of adhesion. We suggest that cell–matrix entanglement represents a novel mechanism of cell anchorage that uniquely depends on the three-dimensional character of the matrix.     

Potential synergies between matrix proteins and soluble factors on resorption and proteinase activities of rabbit bone cells

Human growth hormone (GH) has recently been found to stimulate osteoclastic resorption, cysteine-proteinase and metalloproteinase activities (MMP-2 and MMP-9) in vitro via insulin-like growth factor-I (IGF-I) produced by stromal cells. The present study investigated the effects of two extracellular matrix components (vitronectin and type-I collagen) on hGH- and hIGF-1-stimulated osteoclastic resorption and proteinase activities in a rabbit bone cell model. After 4 days of rabbit bone cell culture on dentin slices with vitronectin coating, hGH and hIGF-1 stimulated bone resorption and hIGF-1 upmodulated cysteine-proteinase activities. MMP-2 expression (but not resorption, cathepsin or MMP-9 activities) was upmodulated by hGH and hIGF-1 on dentin slices coated with type I collagen as compared to those without coating. Then, vitronectin was synergistic with hIGF-1 in the regulation of cysteine-proteinase production whereas collagen showed synergy with hGH and hIGF-1 in the regulation of MMP-2 production. Anti-alphavbeta3 totally abolished the effects of hGH and hIGF-1 on metalloproteinase release, but had no influence on cathepsin release. The results suggest that cysteine-proteinase modulation is not mediated by alphavbeta3 integrin (strongly expressed on osteoclastic surface) whereas the resorption process and metalloproteinase modulation are clearly mediated by this integrin. Our finding about the collagen coating also suggests that hGH- and hIGF-1-stimulated MMP-2 activity are mediated, along with alphavbeta3 integrin, by another adhesion molecule.     

Extracellular matrix conditions T cells for adhesion to tissue interstitium

The activation and differentiation of peripheral blood T cells (PBT) are known to correlate with increased surface expression and adhesive capacity of beta(1) integrins, which mediate adhesion to the extracellular matrix (ECM). However, little is known about the regulation of integrin expression, affinity, and avidity on tissue T cells after they are embedded in the interstitial ECM. In this study we show that tissue T cells, freshly isolated from their residence in the interstitial ECM of the intestinal lamina propria, express a distinct subset of functionally active integrins that contribute to enhanced adhesion to purified collagen, fibronectin, and cell-derived ECM when compared with freshly isolated, short term activated, and long term cultured PBT. Furthermore, integrin usage is distinct between circulating and tissue-derived T cells, in that lamina propria T cells prefer to bind to collagen, while PBT lymphoblasts choose fibronectin when presented with a complex, three-dimensional, cell-derived matrix. To identify the extrinsic factors that regulate the conversion from a nonadhesive PBT to highly adhesive tissue T cell, we demonstrate that activation of PBT in the presence of fibronectin or collagen rapidly generates a surface integrin expression profile, an integrin usage pattern, and adhesive capacity mirroring that of a tissue T cell. These results indicate that the tissue ECM microenvironment instructs newly arrived T cells for further interactions with the underlying matrix and thereby imprints them with a signature tissue adhesive phenotype.     

Expression of integrin beta 6 enhances invasive behavior in oral squamous cell carcinoma.

Oral squamous cell carcinoma (SCC) is characterized by invasive growth and the propensity for distant metastasis. The expression of specific adhesion receptors promotes defined interactions with the specific components found within the extracellular matrix (ECM). We previously showed that the alpha v beta 6 fibronectin receptor is highly expressed in oral SCC. Here we forced expression of the beta 6 subunit into poorly invasive SCC9 cells to establish the SCC9 beta 6 cell line and compared these two cell lines in several independent assays. Whereas adhesion to fibronectin was unaffected by the expression of beta 6, migration on fibronectin and invasion through a reconstituted basement membrane (RBM) were both increased. Function-blocking antibodies to alpha v beta 6 (10D5) reduced both migration on fibronectin and invasion through an RBM, whereas anti-alpha 5 antibodies were effective only in suppressing migration on fibronectin, not invasion. Expression of beta 6 also promoted tumor growth and invasion in vivo and modulated fibronectin matrix deposition. When grown as a co-culture with SCC9 cells, peritumor fibroblasts (PTF) organized a dense fibronectin matrix. However, fibronectin matrix assembly was decreased in co-cultures of SCC9 beta 6 cells and PTF and this decrease was reversed by the addition of function-blocking anti-alpha v beta 6 antibodies. The expression of beta 6 also resulted in increased levels of matrix metalloproteinase 3. Addition of the general MMP inhibitor GM6001 to SCC9 beta 6/PTF co-cultures dramatically increased fibronectin matrix assembly in a similar fashion as incubation with anti-alpha v beta 6 antibodies. These results demonstrate that expression of beta 6 (1) increases oral SCC cell motility and growth in vitro and in vivo; (2) negatively affects fibronectin matrix assembly; and (3) stimulates the expression and activation of MMP3. We suggest that the integrin alpha v beta 6 is a key component of oral SCC invasion and metastasis through modulation of MMP-3 activity.     

TGFbeta1 -mediated epithelial to mesenchymal transition is accompanied by invasion in the SiHa cell line

It has recently been suggested by several investigators that the epithelial-mesenchymal transition-inducing capacity of TGFbetas contributes to invasive transition of tumors at later stages of carcinogenesis. In the present study, we examined the possibility of TGFbeta1-stimulated epithelial-mesenchymal transition in SiHa cell line, detailed molecular events in the process, and its possible contribution to the invasive transition of tumors. TGFbeta1-induced epithelial-mesenchymal transition of SiHa cells was based on morphological and biochemical criteria; actin stress fiber formation, focal translocalization of integrin alphav, talin, and vinculin, fibronectin-based matrix assembly at the cell periphery, and translocalization and down-regulation of E-cadherin. TGFbeta1 also stimulated surface expression of integrin alphavbeta3 and FAK activation. Focal translocalization of integrin alphav preceded actin reorganization and fibronectin matrix assembly, and functional blocking of the integrin suppressed actin stress fiber formation. Furthermore, induction of actin reorganization and fibronectin matrix assembly by TGFbeta1 were shown to be mutually independent events. These changes were irreversible because 5 minutes pulse exposure to TGFbeta1 was sufficient to stimulate progress of actin reorganization and fibronectin matrix assembly. In further studies with raft culture, TGFbeta1 was found to stimulate invasion of SiHa cells into a type I collagen gel matrix. In conclusion, TGFbeta1 stimulated epithelial-mesenchymal transition of SiHa cells, indicating a positive role in the invasive transition of tumors.