Expression of Fibronectin and Integrins in Cultured Periodontal Ligament Epithelial Cells

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Abstract
The process of attachment of epithelial cells obtained from the porcine periodontal ligament (cell rests of Malassez) to different extracellular matrix proteins and their expression of fibronectin and integrin receptors were studied by means of immunocytochemistry, in situ hybridization, and time-lapse cinemicrography techniques. The cell lines of periodontal ligament epithelial cells (PLE cells) attached to and spread rapidly on fibronectin, vitronectin, and type I collagen. One of the cell lines also attached to laminin, while the other cell line showed poor attachment to both laminin and Matrigel®, a basement membrane material. By use of the in situ hybridization technique, some PLE cells were found to express the fibronectin gene strongly. Immunocytochemical staining localized fibronectin in extracellular fibrils and intracellular granules. Fibronectin was also found in the tracks left behind by the cells migrating on the substratum. Arg-gly-asp-ser peptide inhibited the attachment of the PLE cells to fibronectin, laminin, type I collagen, and vitronectin by 47%, 43%, 83%, and 94%, respectively, suggesting that the cell-matrix interactions were partly mediated by receptors related to the integrin family. Antibodies against the β1-integrin subunit stained the cell bodies and the plasma membrane projections of spreading cells. After 24 h or longer in culture, β1-integrins were localized to the regions of cell-cell contact. Cinemicrography of the arg-gly-asp-ser-peptide-treated cells demonstrated that the spreading and migration of isolated cells were prevented by the peptide. The peptide did not appear to dissociate the cell-cell contacts or interfere with migration of spread-cell colonies. By immunoprecipitation with anti-β1 antibodies of the integrins from extracts of the radiolabeled cells, two bands characteristic of a- and B-subunits of β 1-integrins were resolved by SDS-gel electrophoresis. The results suggest that PLE cells synthesize and utilize fibronectin-containing material for their attachment, spreading, and migration. The cell-substratum and cell-cell contacts are at least partly mediated through integrin-type cell-surface receptors. During the different stages of cell growth and spreading, the integrins appeared to become reorganized, which facilitated cell-cell contact and allowed for migration of the cell colonies.

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