Altered glycosylation and cell surface expression of β1 integrin receptors during keratinocyte activation

Abstract

We studied the mechanism by which cell adhesiveness becomes activated when keratinocytes are removed from skin and placed into cell culture. Our results suggest that activation involves altered β1 integrin subunit glycosylation accompanied by an increase in cell surface 01 integrin receptors. Activated keratinocytes contained two forms of the β1 integrin subunit, ∼93 kDa and ∼113 kDa. As shown by pulse-chase experiments, the smaller represented the cytoplasmic precursor of the larger, and only the 113 kDa mature form was detected in integrin receptors expressed at the cell surface. Preactivated keratinocytes contained 01 integrin subunits ranging from ∼97 to 110 kDa. These β1 subunits had been processed through the Golgi, based on resistance to endoglycosidase-H treatment, and were not converted to 113 kDa subunits during subsequent cell culture. Experiments with endoglycosidase-F showed that differences in the apparent sizes of β1 integrin subunits observed in pre-activated and activated keratinocytes could be attributed to differences in subunit glycosylation. Smaller β1 subunits found in pre-activated keratinocytes, like the precursor 01 subunits of activated cells, appeared to be less efficient in reaching the cell surface. Overall, a ∼10-fold increase in the level of cell surface integrin receptors occurred concomitant with the increased proportion of 113 kDa 01 subunits found in activated cells. Endoglycosidase-F experiments also indicated that there were changes in keratinocyte a subunits associated with 01. In related experiments, keratinocytes cultured in low Ca2+, serum-free MCDB medium for 4 days proliferated but their adhesiveness did not become activated. Therefore, keratinocyte proliferation and activation of adhesion are regulated separately. Finally, substantial activation of keratinocytes was observed when serum was added to cells cultured in MCDB with serum, indicating a role for serum factors in the activation process.