Lectin binding as a probe of proliferative and differentiative phases in primary monolayer cultures of cutaneous keratinocytes

Abstract

The surface of cells in the cutaneous epidermis of the newborn rat exhibits a discrete change in lectin-binding specificity from Griffonia simplicifolia I-B4 (GS I-B4), specific for α- d-galactosyl residues, to Ulex europeus agglutinin I (UEA), specific for α- l-fucose, as the cell leaves the basal layer and differentiates. Primary monolayer cultures of rat keratinocytes maintained in low Ca 2+ medium (0.08 m M) exhibited a characteristic unimodal pattern in the ratio of bound UEA to bound GS I-B4 (UEA/B4 ratio) over a 7-day culture period as determined by a quantitative fluorometric assay. The UEA/B4 ratio was initially low between Days 1 and 2 (0.56±0.05), steadily increased to a maximum of 0.84±0.09 between Days 2 and 4, and then gradually decreased to 0.41 ±0.07 between Days 6 and 7. Estimation of DNA synthesis showed (a) a higher [ 3H]thymidine incorporation when the UEA/B4 ratio was low and (b) a steady but lower incorporation between Days 3 and 4, coincident with the higher UEA/B4 ratio. Autoradiographic results further showed that cells stained intensely with UEA failed to incorporate [ 3H]thymidine into their nuclei. Electrophoresis of [ 3H]fucose-labeled material isolated on UEA-Sepharose 4B revealed that the changes in labeling by [ 3H]fucose, bound UEA, and the UEA/B4 ratio in the monolayer were related in part to variable expression of “96K-associated UEA-bound” radioactivity corresponding to a major class of lectin-specific cell-surface glycoproteins (GP96 fraction) identified in situ. Overall, the results suggest that (a) the increase in the UEA/B4 ratio between Days 2 and 4 reflects the progression of a proportion of the cells in the monolayer to an early spinous cell stage, the ultimate fate of which is desquamation into the medium and (b) the decrease in the UEA/B4 ratio between Days 5 and 7 reflects a consequent proliferative response to this loss of cells. This system should be useful for studying environmental influences on the homeostasis of cell proliferation and differentiation in the cutaneous epidermis.