Transfer of retinol-binding protein from HepG2 human hepatoma cells to cocultured rat stellate cells.
Rat liver stellate cells were cocultured with HepG2 human hepatoma cells, which are known to synthesize and secrete retinol-binding protein (RBP). Transfer of human RBP from HepG2 cells to stellate cells was studied by cryoimmunoelectron microscopy. In stellate cells, human RBP was found on the cell surface and within endosomes. The transfer of human RBP from HepG2 cells to stellate cells was blocked by addition of RBP antibodies to the culture medium. Very little uptake of RBP was observed when fibroblasts were cocultured with HepG2 cells. In a series of experiments, RBP was bound to its putative cell surface receptor at 4 degrees C, and the stellate cells were washed and then incubated at 37 degrees C in order to allow them to internalize a pulse of RBP. About 50% of the RBP was internalized after 6 min of incubation. The RBP-positive vesicles were initially (after 1-2 min) located close to the cell surface and later were found deeper in the cytoplasm. During the first 10 min, RBP was mainly observed in close association with membranes. After 2 hr, however, most RBP was localized in intracellular vesicles at a distance from the vesicular membranes, suggesting that RBP had been released from its receptor. Saturable binding of RBP to liver cells was demonstrated when cells were incubated with 125I-RBP at 4 degrees C and cell-associated radioactivity was determined. The calculated dissociation constant for the specific binding was 12.7 +/- 3.2 nM. A binding assay was also developed for determination of solubilized RBP receptor. Solubilized proteins from the nonparenchymal liver cells bound about 30 times more 125I-labeled RBP than did parenchymal cells (based on mass of cell protein). These data suggest that RBP mediates the paracrine transfer of retinol from hepatocytes to perisinusoidal stellate cells in liver and that stellate cells bind and internalize RBP by receptor-mediated endocytosis.
Institute for Nutrition Research, School of Medicine, University of Oslo, Norway.
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Senoo
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Smeland
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Malaba
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Bjerknes
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Stang
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Roos
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Berg
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Norum
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Blomhoff
D000818:Animals; D000906:Antibodies; D001692:Biological Transport; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002450:Cell Communication; D002478:Cells, Cultured; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D008113:Liver Neoplasms / Q000378:metabolism*; D008297:Male; D016253:Microscopy, Immunoelectron; D051381:Rats; D017208:Rats, Wistar; D012177:Retinol-Binding Proteins / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000906:Antibodies; D012177:Retinol-Binding Proteins