Epidermal keratinocyte growth: changes in protein composition and synthesis of keratins in differentiating cultures

The epidermal skin layer undergoes extensive changes in all areas of metabolism as the lowermost basal cell differentiates into a dead structure that forms the upper, protective stratum corneum. Epidermal keratinocyte differentiation was studied in vitro using primary epidermal keratinocyte cultures that grow from a basal cell monolayer to a differentiating and proliferating, multilayered keratinocyte structure. Specific groups of proteins that form complex structures as keratinocyte differentiation occurs were studied. The quantities and synthesis of the proteins forming the tonofilamentous bundles, keratohyaline granules, and cornified cell envelopes in these cultures were determined. The selective solubilities of these proteins in a series of buffers were exploited to separate the proteins into 6 fractions. Protein assays, [3H]-amino acid pulse labeling, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography were used to quantitate and characterize the proteins extracted from basal monolayers and from stratifying and fully differentiated keratinocyte cultures. The results showed that multilayered, more differentiated cultures accumulated the greatest amount of keratin, keratohyaline granule, and cell envelope proteins, although there was no apparent increase in the synthesis of these proteins in the more differentiated cultures. These cultures showed extensive disulfide cross-linking of the keratins. Covalent keratin bonding occurred at least 6 hr after the synthesis and rapid noncovalent bonding of the polypeptides into keratins; thus various stages of keratin formation were identified. The differentiation of the epidermis appeared to be a complex, orderly, and regulated process that can be studied in vitro using this epidermal cell culture system.