Fibronectin receptors of human keratinocytes and their expression during cell culture

Abstract

Keratinocyte attachment to fibronectin (FN) substrata was inhibited by the peptide Gly-Arg-Gly-Asp-Ser-Pro-Cys, but not by the variant peptide Gly-Arg-Gly-Glu-Ser-Pro. The RGDS-containing peptide did not inhibit keratinocyte adhesion to collagen. Keratinocyte adhesion to FN substrata also was inhibited by polyclonal anti-FN receptor antibodies originally prepared against the 140-kD FN receptors of Chinese hamster ovary (CHO) cells. Anti-CHO FN receptor antibodies did not, however, inhibit keratinocyte adhesion to collagen substrata. A monoclonal antibody designated VM-1 that was prepared against human basal keratinocytes inhibited keratinocyte adhesion to collagen but not to FN. Based on these results, we conclude that keratinocytes have distinct FN and collagen receptors. Experiments were performed to compare the expression of FN receptors on keratinocytes freshly isolated from skin and keratinocytes harvested from cell cultures. Cells harvested from keratinocyte cultures were able to neutralize the inhibitory activity of anti-CHO FN receptor antibodies and were able to attach and spread on anti-CHO FN receptor-coated substrata. Cells freshly harvested from skin, however, did not neutralize the antibodies, nor did they attach and spread on antibody-coated substrata. To learn more about the biochemical nature of the keratinocyte FN receptors, we performed immunoaffinity chromatography and immunoprecipitation experiments using the anti-CHO FN receptor antibodies. Extracts from metabolically radiolabeled, 10-d cultured keratinocytes contained FN receptors that had a 135-kD component under reducing conditions and 115- and 155-kD components under nonreducing conditions. Similar components were observed in extracts from surface- radiolabeled cells indicating that the FN receptors were expressed on keratinocyte cell surfaces. On the other hand, extracts from metabolically radiolabeled, 1-d cultured keratinocytes lacked intact FN receptors but contained a component that migrated at 48 kD under reducing conditions and 50 kD under nonreducing conditions. Because this fragment was not detected in surface-radiolabeled keratinocytes that were freshly isolated from skin, it seems likely that the fragment was located inside the cells rather than on the cell surface. A 50-kD FN receptor fragment also was observed in extracts from 10-d cultured keratinocytes if leupeptin and pepstatin were omitted from the extraction buffer. The results suggested that human keratinocytes cultured for 10 d express the 140-kD class of FN receptors, but that these receptors are not expressed on the surfaces of keratinocytes freshly isolated from skin.(ABSTRACT TRUNCATED AT 400 WORDS)

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