Abstract
Purpose: :
During wound healing keratocytes become activated and migrate into the wounded area resulting in corneal haze or permanent scarring. Previously we showed hyaluronan (HA) to be induced during keratocyte activation by transforming growth factor beta (TGFB). HA mediates expression of scar-associated extracellular matrix components and cell motility. In the current study we examined roles for CD44, the major cell surface receptor of HA, in the migration of stromal fibroblasts.
Methods: :
Isoforms of CD44 were detected using RT-PCR and immunoblotting with isoform-specific antibodies. Migration was induced by scratch-wounding of confluent low-passage human corneal fibroblasts in the presence of inhibitors, 4-methylumbelliferone (4MU), 4-methylesculetin (4ME), function-blocking antibodies to CD44, or a peptide containing 10-amino acids of the CD44V6 splice form. Both rate and directionality of migration was quantified by individual cell-tracking using real time imaging of DiO stained live cells.
Results: :
CD44 was rapidly upregulated in keratocytes by TGFB. All 10 alternately-spiced regions of CD44, V1-V10, were identified in bovine keratocyte mRNA. Inhibitors 4ME and 4MU markedly reduced the CD44 upregulation. These inhibitors or the presence of any of three CD44 function-blocking antibodies (IM7, BRIJ35, and V6-specific CD44 antibody) significantly reduced the rate of human corneal fibroblast migration. A peptide containing sequence from the CD44V6-region resulted in a loss of directional migration.
Conclusions: :
CD44 function is important for keratocyte migration as it is in other cell types. We show for the first time that 4MU and 4ME prevent CD44 synthesis, and that CD44V6 spice form is involved in mediating migration. HA is not present in normal corneal matrix, but may play an important role in cell signaling during healing, including mediation of both migration and matrix synthesis. The simultaneous upregulation of both HA and its receptor CD44 during differentiation of keratocytes to myofibroblasts seems likely to be essential for migration of the cells into the wound. Targeting this migration using inhibitors such as 4MU may be useful approach to prevent haze and scarring resulting from corneal wound healing.
Keywords: proteoglycans/glycosaminoglycans • extracellular matrix • cornea: stroma and keratocytes