Integrin and phosphotyrosine expression in normal and migrating newt keratinocytes

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Abstract
Background: Cells interact with the extracellular matrix through a family of cell surface receptors known as integrias. Ligand specificity of a given integrin is determined in part by the type of α and the type of β subunit comprising it. Accumulating evidence suggests that integrinligand binding in some systems influences cell behavior through tyrosine phosphorylation of intracellular proteins. Methods: In this study, we utilized immunohistochemistry to examine the expression of β1 and β4 integrin subunits as well as tyrosine phosphorylation in normal keratinocytes and in keratinocytes migrating to form a wound epithelium. An adhesion assay was used to determine if freshly isolated keratinocytes could interact with fibronectin and collagen. Polyacrylamide gel electrophoresis followed by immunoblotting was employed to compare β1 integrins in migrating and nonmigrating keratinocytes. Results: In normal epidermis, β1 and β4 localized primarily to basal cells, where both subunits were generally distributed over all parts of the cell periphery. Except for a modest presence in suprabasal cells and a minimal presence adjacent to the epidermal basement membrane, phosphotyrosine (ptyr) had a similar distribution. In migrating keratinocytes, β1, β4, and ptyr localized most heavily at the interface between the forming wound epithelium and the wound bed. Adhesion assays using keratinocytes from normal epidermis revealed a population of cells that could specifically adhere and spread on fibronectin and type I collagen. Immunoblots of β1 subunits from normal and migrating keratinocytes showed no increase in amount of β1, nor did the apparent size of β1 change in migrating compared to normal cells. Conclusions: The heavy accumulation of β1 and β4 at the wound bed interface in migrating cells suggests that these subunits may be involved in attachments of migrating cells to extracellular matrix proteins in the wound. The accumulation of ptyr in the same region further suggests that integrin‐ligand interaction in keratinocytes modulates cell behavior through phosphorylated proteins. The fact that freshly isolated newt keratinocytes could adhere and spread on fibronectin or collagen shows that these cells are constitutively activated. This view is supported by the absence of any evidence that the β1 in migrating keratinocytes is larger and therefore more mature than β1 in normal keratinocytes. By comparison, β1 integrins on human keratinocytes are not constitutively activated (Takashima and Grinnell, 1985; Toda et al., 1987; Guo et al., 1990, 1991), a difference that may explain why epidermal wound healing is faster in newts than in humans.

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