Evidence for a specific cell membrane retinol-binding protein transport mechanism in a human keratinocyte line.
The epidermis is highly sensitive to retinoids, and vitamin A (retinol) is a critical factor in the regulation of skin cell differentiation and proliferation. Despite extensive knowledge of retinoid-mediated gene transcription effects on epidermal cells and evidence for retinoid-mediated suppression of carcinogenesis in skin, basic transport events, especially cellular uptake, of this vitamin remain poorly understood and controversial. Herein, evidence is presented for receptor-mediated uptake of retinol-binding protein, RBP, the specific circulatory vitamin A carrier, in the A431 human epidermal cell line. Cellular RBP uptake was significantly inhibited by anti-RBP IgG. Addition of transthyretin (TTR), a circulatory protein that can interact with RBP, to the internalization assay also significantly reduced RBP uptake to 49.4+/-4.6% (+/- SEM) of control values (p<0.01). RBP uptake was impaired by sucrose, a known inhibitor of early endocytosis, but not significantly affected by a disruptor of later trafficking events, chlorpromazine. Binding analysis indicated saturable RBP binding to the cell surface and a total of about 94,000 binding sites/cell. Based on dissociation constants, two RBP binding sites were detected with a 50-fold affinity difference: 0.7 and 35.0 nM, with 12,000 and 82,000 receptors/cell, respectively. These results indicate that high affinity RBP receptors capable of internalizing RBP independently of TTR exist in these malignant keratinocytes, and that TTR influences binding of RBP to its putative receptor(s). Overall, the data establish membrane transport parameters for RBP, and provide a basis for examining modulation of vitamin A endocytosis that may accompany changes in proliferation or differentiation state of epidermal cells.
Huang
Janet
J
Endocrine and Metabolic Research Laboratory, Faculty of Applied Sciences and Institute for Health Research (IHRE), Simon Fraser University, Burnaby, BC, Canada.
Vieira
Amandio
A
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001667:Binding, Competitive; D001692:Biological Transport / Q000187:drug effects; D001710:Biotin / Q000737:chemistry; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002746:Chlorpromazine / Q000494:pharmacology; D006801:Humans; D007074:Immunoglobulin G / Q000494:pharmacology; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D008954:Models, Biological; D011228:Prealbumin / Q000494:pharmacology; D011485:Protein Binding; D012177:Retinol-Binding Proteins / Q000276:immunology / Q000378:metabolism / Q000493:pharmacokinetics*; D013395:Sucrose / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D008565:Membrane Proteins; D011228:Prealbumin; D012177:Retinol-Binding Proteins; D013395:Sucrose; D001710:Biotin; D002746:Chlorpromazine