Laminin alpha5 chain adhesion and signaling in conjunctival epithelial cells.
OBJECTIVE
To identify peptides of the LG4 module of the laminin alpha5 chain that mediate human conjunctival epithelial cell adhesion to the laminin-10 isoform.


METHODS
A peptide corresponding to a major heparin- and cell-binding domain of the LG4 module of the laminin alpha5 chain was analyzed. The attachment of conjunctival epithelial cells to the peptide compared with laminin-10 was determined by colorimetric adhesion assay. The role of glycosaminoglycans in mediating adhesion to the peptide was determined by altering their function at the cell surface and by blocking adhesion with exogenous glycosaminoglycans. The role of syndecan-4 in cell adhesion to the peptide was examined by adhesion assay. The role of the peptide in cell signaling was examined by phosphotyrosine Western blot analysis.


RESULTS
The peptide facilitated the adhesion of conjunctival epithelium, although not as efficiently as laminin-10. Heparinase had no effect on adhesion to the peptide. In contrast, adhesion to the peptide decreased in glycosaminoglycan-deficient cells, heparatinase-treated cells, cells blocked with exogenous heparan sulfate proteoglycan, and cells treated with antibodies to the ectodomain of syndecan-4. Cell adhesion to the peptide for 90 or 120 minutes resulted in a significant increase in focal adhesion kinase (FAK) tyrosine phosphorylation, compared with the nonadherent control.


CONCLUSIONS
Human conjunctival epithelial cells use a heparin- and cell-binding peptide of the LG4 module of laminin alpha5 chain in adhesion to laminin-10. Syndecan-4 is one mechanism by which the peptide facilitates adhesion. In addition to adhesion, the peptide may function in cell-signaling events.
Lin
Lin
L
Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Kurpakus-Wheater
Michelle
M
D015153:Blotting, Western; D002448:Cell Adhesion / Q000502:physiology; D018929:Cell Culture Techniques; D003228:Conjunctiva / Q000166:cytology*; D004847:Epithelial Cells / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006025:Glycosaminoglycans / Q000502:physiology; D006801:Humans; D007797:Laminin / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D053671:Syndecan-4; D014443:Tyrosine / Q000378:metabolism
D006025:Glycosaminoglycans; D007797:Laminin; D008562:Membrane Glycoproteins; D009842:Oligopeptides; D011509:Proteoglycans; C508304:SDC4 protein, human; D053671:Syndecan-4; C119013:laminin 10; C097043:laminin alpha5; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human