Retinoic acid delays the terminal differentiation of keratinocytes in suspension culture.

The effect of retinoic acid (RA) on the terminal differentiation of guinea pig keratinocytes maintained in suspension culture was studied. Keratinocytes obtained from trypsinized guinea pig skin were suspended in medium containing 20% calf serum and 1.2% methyl cellulose. RA, which was added at the beginning of culture, delayed differentiation as judged by a decrease in the percent of cells that developed disulfide cross-linked keratin (sodium dodecyl sulfate insoluble cells) and cornified envelopes (sodium dodecyl sulfate and 2-mercaptoethanol insoluble cells). RA inhibited differentiation maximally at 5 microgram/ml on day 3 of 5 day culture; concentrations as low as .005 microgram/ml were also inhibitory. Because the disulfide cross-linking of keratin and the formation of cornified envelopes are thought to occur when the cell membrane becomes permeable, we determined whether RA inhibited these processes by stabilizing the cell membrane. Two agents (ionophore X537A and Triton X-100) which permeate cell membranes rapidly reversed the inhibitory effect of RA on cornified envelope formation. In addition, when cultured with RA, the percent of cells which became permeable to trypan blue was reduced, also suggesting that RA acts on the cell membrane. These studies show that RA can inhibit keratinocyte differentiation by stabilizing the cell membrane thereby delaying transition from a living epidermal cell to a dead cornified cell.