Abstract
Purpose: :
Corneal stroma contains proteoglycans modified with corneal keratan sulfate (KS), a unique glycosaminoglycan important for corneal transparency. Biosynthesis of KS is lost in healing wounds and in vitro, but the mechanism of KS downregulation is not fully understood. Keratocytes contain cadherin 11 (Cad11) a cell surface protein which forms calcium-dependent cell-cell junctions between mesenchymal cells. This study sought to determine the role of Cad11 junctions in regulating keratocyte phenotype as demonstrated by KS synthesis.
Methods: :
Primary bovine keratocytes were cultured in serum-free conditions on fibronectin or recombinant Cad11. Cad11 and ß-catenin were detected using immunoblotting and immunohistochemistry. Cad11 junctions were disrupted in calcium-free culture, in low density culture, or after treatment with Cad11 siRNA. Sulfated KS biosynthesis was determined using fluorophore assisted carbohydrate electrophoresis (FACE), 35S-sulfate labeling, immunoblotting with anti-KS monoclonal and qRT-PCR for mRNA of KS biosynthetic enzymes.
Results: :
Primary bovine keratocytes in serum-free culture contained Cad11, ß-catenin, and filamentous actin localized to regions of cell-cell contact. Calcium-free or low cell density culture reduced these junctions and reduced KS synthesis. Keratocytes plated on recombinant Cad11 formed ß-catenin- and actin-containing adhesions to the substrate and had increased KS synthesis. Knockdown of Cad11 with siRNA reduced KS synthesis as well as expression of mRNA for KS biosynthetic enzymes. Treatment with serum resulted in loss of KS biosynthesis over a 24 hr period simultaneous with translocation of ß-catenin to the nucleus and cytoskeletal rearrangement. GSK-3ß inhibitor LiCl also caused translocation of ß-catenin and downregulation of KS biosynthesis.
Conclusions: :
Keratocytes form functional adherens junctions in vitro involving Cad11. Disruption of those junctions using a variety of effectors resulted in downregulation of KS synthesis and nuclear translocation of the adaptor protein ß-catenin. These results suggest that canonical Wnt signaling pathway may be involved in down-regulation of KS synthesis during keratocyte activation.
Keywords: cell adhesions/cell junctions • proteoglycans/glycosaminoglycans • gene/expression