Comparison of the uptake and metabolism of retinol delivered to primary mouse keratinocytes either free or bound to rat serum retinol-binding protein.
Serum retinol-binding protein (RBP) is believed to be responsible for the transport of retinol from its storage site in the liver to vitamin A requiring target cells such as keratinocytes. We have used primary mouse keratinocytes as a model system to compare the uptake and metabolism of [3H] retinol delivered to them either free in solution or bound to RBP. RBP was purified from rat serum, loaded with [3H]retinol, and the [3H]retinol-RBP complex purified by affinity chromatography on human transthyretin-Sepharose. Keratinocytes incubated with either free [3H]retinol or [3H]retinol-RBP complex accumulated [3H]retinol in a time and temperature dependent manner. However, cells incubated with free [3H]retinol acquired 15- to 20-fold more ligand than if the retinol was delivered via RBP. The uptake of free [3H]retinol or [3H]retinol from RBP was not inhibited by excess unlabeled free retinol. The uptake of [3H]retinol from RBP was inhibited by high concentrations of holo-RBP, with half maximal inhibition occurring at 3 microM holo-RBP. However, no specific binding of 125I-labeled RBP to monolayers of keratinocytes or membranes prepared from them was found indicating the absence of a high affinity RBP receptor on keratinocytes. Surprisingly, 50% of the [3H]retinol delivered to the keratinocytes during a 30-min uptake period was released from them within 30-min irrespective of whether or not it was initially delivered to them as free [3H]retinol or bound to RBP. The remaining 50% was lost at a much slower rate, but only 20% remained 24-h after delivery. Studies on retinol metabolism demonstrated that 7%-12% of the total cell-associated [3H]retinol delivered during a 90-min uptake period was esterified (mostly as retinyl palmitate) whether or not it was given free in solution or bound to RBP. Additionally, [3H]retinol taken up by the keratinocytes during the initial 90-min incubation was not chased into a stable retinyl ester pool in a subsequent 9.5-h incubation, but instead, retinyl ester was lost from the cells with kinetics similar to those of total cell-associated radioactivity. These results suggest that a function of RBP is to protect cells from a rapid accumulation of the vitamin which occurs when it is delivered free in solution. However, the cellular fate and metabolism of retinol appears to be the same whether the vitamin is delivered free in solution or bound to RBP.
Creek
K E
KE
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20892.
Silverman-Jones
C S
CS
De Luca
L M
LM
D000818:Animals; D000078404:Epidermal Cells; D004817:Epidermis / Q000378:metabolism*; D007633:Keratins; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats / Q000097:blood; D012177:Retinol-Binding Proteins / Q000097:blood* / Q000378:metabolism; D012996:Solutions; D014801:Vitamin A / Q000097:blood / Q000378:metabolism / Q000493:pharmacokinetics*
D012177:Retinol-Binding Proteins; D012996:Solutions; D014801:Vitamin A; D007633:Keratins