Identification of integrins in cultured corneal fibroblasts and in isolated keratocytes

Purpose. The integrins are a family of transmembrane glycoproteins that function in attachment of cells to one another and to the extracellular matrix. When cell-cell and cell-matrix interactions are altered, the population of integrins may change. In particular, removing cells from their normal environment may be used as a model of wounding. The current study reports the identification of the integrins expressed at the cell surface of noncultured keratocytes and of cultured corneal fibroblasts, which are derived from keratocytes grown in primary culture. Methods. For integrin identification, the surface proteins of keratocytes and cultured corneal fibroblasts were labeled with biotin, and the integrins were immunoprecipitated using anti-integrin antibodies. Attachment assays determined (1) the extracellular matrix preference of the cultured corneal fibroblasts and (2) the effects of function-perturbing antibodies against the fibronectin receptor (α₅β₁) or against other β₁-containing integrins. Results. The integrins of noncultured keratocytes were present as heterodimeric α,β surface proteins that were immunoprecipitated by anti- β₁, anti-α(v), anti-α₆, anti-α₃, anti-α₁, and anti-β₃. Furthermore, when the keratocytes were placed in culture, the integrin pattern changed. The classic fibronectin receptor, α₅β₁, is then expressed along with additional integrins that bind to fibronectin. Using attachment assays, we determined that the cultured corneal fibroblasts prefer fibronectin to collagen, vitronectin, or laminin as extracellular matrix substrate. In addition, function-perturbing antibodies against the fibronectin receptor (α₅β₁) or against β₁ inhibit attachment of cultured corneal fibroblasts to fibronectin. Conclusions. Receptors for fibronectin and other extracellular matrix molecules are expressed at the cell surface in cultured corneal fibroblasts, and are in position to play a significant functional role as seen in attachment to extracellular matrix.