Insulin-like growth factor-binding protein-3 (IGFBP-3) concentration in rat Sertoli cell-conditioned medium is regulated by a pathway involving association of IGFBP-3 with cell surface proteoglycans.
Rat Sertoli cells in culture produce insulin-like growth factor-I (IGF-I), and IGF-binding protein-3 (IGFBP-3) is the predominant IGFBP. Previous studies suggested that the concentration of IGFBP-3 in Sertoli cell-conditioned medium is regulated by a clearance pathway involving association of IGFBP-3 with the Sertoli cell surface. The purpose of these studies was to characterize further the nature of the interaction of IGFBP-3 with the cell surface and to examine how this interaction might be regulating IGFBP-3 concentrations in conditioned medium. Recombinant nonglycosylated human IGFBP-3 was added to cultured Sertoli cells derived from 15-day-old rats, and the change in concentration over time in the medium was measured by [125I]IGF-I ligand blot analysis and Western blot analysis using an antiserum specific for human IGFBP-3. Chloroquine, an inhibitor of lysosomal enzymes involved in internalization of proteins and reduction of temperature, inhibited IGFBP-3 reduction in Sertoli cell medium. Soluble heparin, at a half-maximal concentration of approximately 5 microgram/ml, inhibited the decrease in IGFBP-3 in the medium. In addition, soluble heparin decreased the amount of IGFBP-3 detectable on the cell surface, as determined by performing ligand blots on plasma membrane preparations. Finally, pretreatment of Sertoli cells with sodium chlorate, an inhibitor of cell surface proteoglycan sulfation, also retarded the decrease in recombinant IGFBP-3. Taken together, these data suggest that an important mechanism influencing the concentration of IGFBP-3 3 in Sertoli cell-conditioned medium is a pathway involving IGFBP-3 interaction with the cell surface proteoglycans.
Smith
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EP
Division of Endocrinology, Children's Hospital Medical Center, Cincinnati, Ohio 45229.
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Chernausek
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Klein
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D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism* / Q000493:pharmacokinetics; D002462:Cell Membrane / Q000378:metabolism; D002704:Chlorates / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000378:metabolism; D004705:Endocytosis; D006493:Heparin / Q000494:pharmacology; D018969:Insulin-Like Growth Factor Binding Proteins; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D009994:Osmolar Concentration; D011509:Proteoglycans / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins; D012708:Sertoli Cells / Q000378:metabolism*
D002352:Carrier Proteins; D002704:Chlorates; D017077:Culture Media, Conditioned; D018969:Insulin-Like Growth Factor Binding Proteins; D011509:Proteoglycans; D011994:Recombinant Proteins; D006493:Heparin; C032706:sodium chlorate