Density-dependent variations in the lipid content and metabolism of cultured human keratinocytes

Cultured human foreskin keratinocytes have been utilized extensively to study modulations in protein content during epidermal differentiation. In this study we examined their usefulness as a model system for differentiation-linked changes in lipid content and metabolism. First-to-third passage keratinocytes were grown in 10% fetal calf serum on a mitomycin-treated 3T3 feeder layer and harvested at intervals before, during, and after reaching confluence for determination of lipid, protein, and DNA content. Lipid synthesis, determined as acetate incorporation into lipid, was most active in pre-confluent cultures and at all times closely paralleled the growth activity of the cultures. Post-confluent cultures were characterized by an increase in total lipid content and by increased triglyceride content and synthesis. Pulse-chase studies demonstrated that labeling of the triglyceride pool was labile and suggested that even in post-confluent cultures, triglycerides provide a fatty acid reservoir for phospholipid biosynthesis. A novel band, which co-migrated with monoalkyldiacylglycerol in two solvents systems was present in confluent and post-confluent cultures, but absent in pre-confluent cultures. Sphingolipids constituted less than 10% of total lipid at all stages of growth, and cholesterol sulfate was present only in small quantities. These studies illustrate the relationship of lipid synthesis to growth and demonstrate that human foreskin keratinocytes, cultured under standard conditions, reproduce incompletely the lipid composition of epidermis in vivo.