Studies on the Cellular Uptake of Retinol Binding Protein and Retinol Francesca Tosetti,* ,1 Francesco Campelli,* and Giovanni Levi* , † *Molecular Biology Laboratory, National Cancer Institute (I.S.T.)–Advanced Biotechnology Center (ABC-CBA), Largo Rosanna Benzi 10, 16132 Genoa, Italy; and †Museum National d’Histoire Naturelle, CNRS URA 90, 7 rue Cuvier, Paris, France The uptake and release of 125 I-RBP and of holoRBP labeled with [ 3 H]retinol ( 3 H-ROH) were studied in two cell lines which synthesize and secrete RBP, the HepG2 hepatocarcinoma cell line and the Caki-1 kid- ney adenocarcinoma cell line, and in HeLa cells that do not express the endogenous RBP gene. In all three cell lines a part of endocytosed 125 I-RBP is recycled to the extracellular medium and part is degraded. Non- specific endocytosis of 125 I-RBP was estimated to be approximately 10% of total endocytosed 125 I-RBP. In HepG2 cells the 3 H-ROH from the [ 3 H]retinol–RBP complex ( 3 H-ROH–RBP) is recycled bound to RBP into serum-free chase medium. This 3 H-ROH recycling is blocked in HepG2 cells by cyclohexymide and by brefeldin A, an inhibitor of protein export from the main secretory route, and is absent in HeLa cells, which do not synthesize RBP. These data suggest that at least part of retinol taken up from exogenous ho- loRBP is delivered to newly synthesized RBP. 3 H-ROH recycled by HeLa cells is bound to serum albumin, as is a portion of that recycled by HepG2 cells. Transfer of 3 H-ROH from RBP to serum albumin does not occur in the absence of cells. We conclude that RBP is endocy- tosed through a specific pathway and that the RBP- associated retinol is transferred to newly synthesized RBP or to serum albumin. © 1999 Academic Press Key Words: HepG2; Caki-1; Hela; retinol recycling; endocytosis; serum albumin. INTRODUCTION Despite extensive in vivo studies on retinol metabo- lism, little is known on the mechanisms which underlie retinol homeostasis at the molecular level. In particu- lar, two main processes need further elucidation: the regulation of retinol mobilization from storage tissues and retinol uptake by target cells. Although most epi- thelia require vitamin A to maintain differentiation, only a few cell types carry out specific metabolic func- tions such as absorption of dietary retinol from the lumen of the small intestine and packaging into chylo- microns (enterocytes) or storage of retinol as retinyl esters (liver stellate cells and adipocytes) [1]. Retinol is mainly mobilized from liver stores as a complex with the retinol binding protein (holoRBP) bound to trans- thyretin (TTR) and in this form it is distributed to peripheral extrahepatic tissues. Studies in the rat have shown that plasma retinol levels are controlled homeo- kinetically by retinol recycling among liver and extra- hepatic tissues in response to changes in nutritional or metabolic conditions [2]. The molecular mechanisms regulating the redistribution of retinol among tissues are not well defined. Retinol release as holoRBP is a regulated process [3] and it is likely that a comparable mechanism controls retinol uptake by the cell. A non- specific receptor-independent mechanism has been proposed for retinol uptake by cells [4 – 6]; this model, however, would not explain in which way a regulated retinol transfer into the cell and between different cell types could be accomplished. Several studies have shown a cellular uptake of RBP and TTR through receptor-mediated endocytosis [7–13]. Convincing evidence supports the existence of a cell membrane RBP receptor [14 –18]; the exact iden- tity of this receptor is, however, still controversial [19]. Furthermore, it has been shown that a novel cellular structure, the caveolae, could be involved in RBP en- docytosis (potocytosis) [20]. While it has been shown that TTR endocytosis can increase the uptake of its hormonal ligand T4 [12, 13], the role of RBP endocyto- sis on retinol uptake by target cells still remains un- clear. In this study we have analyzed the uptake and re- lease of 125 I-RBP in the hepatocarcinoma HepG2 cell line, in the kidney adenocarcinoma Caki-1 cell line, and in human uterine cervical carcinoma HeLa cells. We show that in these three different human cell lines, part of the endocytosed 125 I-RBP is recycled as apopro- tein and part is degraded. These cell lines were se- lected in order to reproduce an in vitro model of the main sites of retinol metabolism: the liver, where reti- nyl esters contained in chylomicron remnants are taken up and retinol is stored and redistributed to the peripheral tissues as holoRBP (HepG2 cells), and the kidney, which is the organ deputed to RBP catabolism and to retinol resorption by the S1 segment of the proximal convoluted tubules (Caki-1 cells), and a tissue 1 To whom reprint requests should be addressed. Fax: (10)- 5737224. E-mail: Tosetti@sirio.cba.unige.it. Experimental Cell Research 250, 423– 433 (1999) Article ID excr.1999.4520, available online at http://www.idealibrary.com on 0014-4827/99 $30.00 423 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.