All-trans-retinoic acid regulates retinol and 3,4-didehydroretinol metabolism in cultured human epidermal keratinocytes

The potential for all-trans-retinoic acid to regulate the metabolism of ³H-retinol and ³H-3,4-didehydroretinol was examined in cultured human epidermal keratinocytes. Confluent cultures were treated daily with medium containing 5% fetal bovine serum or the same medium supplemented with nanomolar concentrations of all-trans-retinoic acid for up to 3 d. During the last 24 h of treatment, cells were incubated with ³H-retinol or ³H-3,4-didehydroretinol for 24 h (isotopic steady state) to label the endogenous retinoids. After the labeling period, one group of cells was harvested and another group was allowed to incubate for an additional 24 h in the absence of medium retinol for the determination of endogenous ³H-retinoid utilization. The ³H-retinoids present in cells were extracted and quantitated by reverse-phased high-pressure liquid chromatography. Keratinocytes treated with retinoic acid and labeled with ³H-retinol exhibited time- and concentration-dependent (i) increases in retinyl ester mass, (ii) increases in the rate of retinyl ester synthesis, (iii) decreases in retinyl ester utilization, and (iv) decreases in the cellular concentrations of retinoic and 3,4-didehydroretinoic acids. There was no effect of exogenous retinoic acid on its own metabolism. Cells labeled with ³H-3,4-didehydroretinol exhibited exclusive labeling of vitamin A₂-related retinoids suggesting that the A₁ to A₂ conversion is not reversible. Treatment of cells with low nanomolar concentrations of retinoic acid decreased the utilization of 3,4-didehydroretinyl esters, decreased the production of 3,4-didehydroretinoic acid but had no effect on the synthesis of 3,4-didehydroretinol or its esters. The results demonstrate that keratinocytes respond to extracellular retinoic acid by decreasing endogenous production of active retinoids, sequestering extracellular substrate retinol as retinyl ester, and decreasing ester utilization.