Effect of chemical modification of keratinocytes and collagen in keratinocyte-collagen interactions.

Abstract

Neonatal and adult keratinocytes isolated from thin sections of split-thickness skin by trypsin-release show a preferential and strong attachment to collagen when compared to plastic, fibronectin-coated plastic, glass, or agar gels. We have investigated the reactive groups of keratinocytes and collagen required for this interaction and have determined the kinetics of attachment. At 37 degrees C both neonatal and adult keratinocytes show a rapid and irreversible attachment to collagen, reaching a plateau phase at 30-60 minutes. The cells cannot be replaced from the gel by extensive washing or by conditions normally expected to break ionic bonds. Chilling to 0 degree C before plating completely inhibits attachment, and heating at 37 degrees C reverses the inhibition. One cycle of freezing and thawing of cells inhibits the interaction. Removal of sialic residues from keratinocytes before plating with neuraminidase, or oxidation of sugars with periodate, does not inhibit attachment or growth, indicating that cell carbohydrates are not required for interaction with collagen. Neither denaturation of collagen with 8 M urea nor oxidation of sugar side chains on the gel with periodic acid affects attachment or growth. However, reaction of free-SH groups with iodoacetic acid or -NH2 groups with dinitrofluorobenzene of the gel completely inhibits growth. Blocking the guanidyl residues of collagen arginine with cyclohexanedione markedly alters all aspects of attachment, growth, and morphology, producing new and completely unique growth patterns. These studies indicate that specific chemical groups on collagen affect keratinocyte-matrix interactions and that the availability of specific residues in collagen directly influences growth and maturation. Most vertebrate cells remain closely associated with extracellular collagenous substances throughout their lifespan. The collagen may be present in both collagen fibers and in reticular fibers as well as in basement membranes. The way cells interact with and are anchored to these various substrata influences a number of important cellular functions including growth and maturation and the synthesis of extracellular matrix components. Skin epithelial cells display a particularly striking and strong dependence on collagen for growth. When plated on a collagen gel, the plating efficiency and growth is increased several-fold compared to other substrates such as glass, plastic, or agar. More recently, the initial observations on the selective attachment of keratinocytes to collagen gels have been extended by Murray et al., who demonstrated that guinea pig keratinocytes show increased plating efficiencies on Type IV collagen gels. In these studies, we have examined the mechanisms for keratinocyte-collagen interaction, and described the kinetics of attachment, the reactive sites on the cell and collagen, and the effects of chemical modification of collagen on the expression of the keratinocytes phenotype.

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