Production of fibronectin and tenascin isoforms and their role in the adhesion of human immortalized corneal epithelial cells.
OBJECTIVE
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(v)beta(5) was present in FAs in sparse cultures and as ringlike structures in denser cultures. Int alpha(v)beta(6) was colocalized with Int alpha(5) in FAs, only in cells adhering to growth substratum coated with Fn or Fn/Tn-C. Ints alpha(5)beta(1) and alpha(v)beta(6) mediated adhesion to Fn and Int alpha(v)beta(5) 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.
Filenius
Sissi
S
Institute of Biomedicine and Anatomy, University of Helsinki, Helsinki, Finland. sissi.filenius@helsinki.fi
Tervo
Timo
T
Virtanen
Ismo
I
D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002448:Cell Adhesion / Q000502:physiology*; D002461:Cell Line, Transformed; D004847:Epithelial Cells; D019573:Epithelium, Corneal / Q000166:cytology* / Q000502:physiology*; D005353:Fibronectins / Q000096:biosynthesis*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D020033:Protein Isoforms; D019063:Tenascin / Q000096:biosynthesis*; D014945:Wound Healing
D000911:Antibodies, Monoclonal; D005353:Fibronectins; D020033:Protein Isoforms; D019063:Tenascin