Integrin and phosphotyrosine expression in normal and migrating newt keratinocytes.
BACKGROUND
Cells interact with the extracellular matrix through a family of cell surface receptors known as integrins. Ligand specificity of a given integrin is determined in part by the type of alpha and the type of beta subunit comprising it. Accumulating evidence suggests that integrin-ligand 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 beta 1 and beta 4 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 beta 1 integrins in migrating and nonmigrating keratinocytes.


RESULTS
In normal epidermis, beta 1 and beta 4 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, beta 1, beta 4, 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 beta 1 subunits from normal and migrating keratinocytes showed no increase in amount of beta 1, nor did the apparent size of beta 1 change in migrating compared to normal cells.


CONCLUSIONS
The heavy accumulation of beta 1 and beta 4 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 beta 1 in migrating keratinocytes is larger and therefore more mature than beta 1 in normal keratinocytes. By comparison, beta 1 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.
Donaldson
D J
DJ
Department of Anatomy and Neurobiology, University of Tennessee, Memphis 38163.
Mahan
J T
JT
Yang
H
H
Yamada
K M
KM
D000818:Animals; D002465:Cell Movement; D003094:Collagen / Q000502:physiology; D005353:Fibronectins / Q000502:physiology; D016023:Integrins / Q000032:analysis* / Q000502:physiology; D015603:Keratinocytes / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D008297:Male; D008856:Microscopy, Fluorescence; D009673:Notophthalmus viridescens; D019000:Phosphotyrosine; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000502:physiology; D014945:Wound Healing / Q000502:physiology
D005353:Fibronectins; D016023:Integrins; D019000:Phosphotyrosine; D014443:Tyrosine; D003094:Collagen