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(Investigative Ophthalmology and Visual Science. 2003;44:3317-3325.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-1146
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Production of Fibronectin and Tenascin Isoforms and Their Role in the Adhesion of Human Immortalized Corneal Epithelial Cells

Sissi Filenius,1,2 Timo Tervo,2 and Ismo Virtanen1

1From the Institute of Biomedicine and Anatomy, University of Helsinki, Helsinki, Finland; and the 2Department of Ophthalmology, Helsinki University Central Hospital, Helsinki, Finland.

PURPOSE. To study the production and deposition of fibronectin (Fn) isoforms and tenascin-C (Tn-C) by immortalized human corneal epithelial (HCE) cells and their integrin-dependent adhesion characteristics.

METHODS. Indirect immunofluorescence with isoform-specific monoclonal antibodies (mAbs) was used to study extracellular matrix (ECM) protein composition and their integrin receptors in HCE cells. The synthesis of proteins was studied by Western blot analysis and adhesion by quantitative adhesion assay.

RESULTS. HCE cells deposited fibrillar matrix containing extradomain EDA-Fn and sparser deposits of Onc-Fn, whereas Tn-C was deposited diffusely. EDA-Fn was present both in ECM and in culture medium, whereas Tn-C was present only in ECM. Fn-binding integrin (Int) {alpha}5 subunit was present in subconfluent cells in focal adhesions (FAs) and matrix adhesions, whereas Int {alpha}vß5 was present in FAs in sparse cultures and as ringlike structures in denser cultures. Int {alpha}vß6 was colocalized with Int {alpha}5 in FAs, only in cells adhering to growth substratum coated with Fn or Fn/Tn-C. Ints {alpha}5ß1 and {alpha}vß6 mediated adhesion to Fn and Int {alpha}vß5 mediated adhesion to Vn, and both were inhibited by RGD peptide. The cells failed to adhere to Tn-C but adhered to Fn/Tn-C and were then more efficiently inhibited by the function-blocking integrin mAbs and RGD peptide.

CONCLUSIONS. The results suggest corneal epithelial cells as the possible source for Fn isoforms and Tn-C in wound healing and pathologic conditions. The presence of Tn-C only in ECM suggest a vectorial deposition and adhesion experiments also indicate a role for Tn-C in Fn functioning.






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