Insulin-like growth factor binding protein (IGFBP)-3 levels in conditioned media of Hs578T human breast cancer cells are post-transcriptionally regulated.
Hs578T human breast cancer cells secrete insulin-like growth factor binding protein (IGFBP)-3 (41-kDa and 39-kDa) and IGFBP-4 (24-kDa) as major BP species. In addition, cell surface-associated IGFBP-3 is demonstrable by use of cell monolayer affinity cross-linking or by employing immunoperoxidase staining of the cell surface with specific polyclonal anti-human IGFBP-3 antibodies (alpha IGFBP-3gl and alpha IGFBP-3ngl). In this study, we have demonstrated that regulation of Hs578T IGFBP-3 by IGF peptides is specific, non-receptor mediated, and post-translational by showing: 1) dose-dependent increase of IGFBP-3 in conditioned media(CM) following addition of IGF-I and IGF-II (maximum 8-13-fold increase at 100 ng/ml concentration), but not by insulin up to 1 microgram/ml; 2) confirmation of IGF-induced increases in CM concentrations of IGFBP-3 by means of Western ligand blot, affinity cross-linking, and IGFBP-3-specific radioimmunoassay; 3) increase of IGFBP-3 in CM by addition of IGF analogs which retain full affinity for IGFBPs ([Leu27]IGF-II and [Tyr55,Gln56]IGF-I), but not by IGF analogs which have significantly decreased affinity for IGFBPs ([Gln3,Ala4,Tyr15,Leu16]IGF-I, [Gln6,Ala7,Tyr18,Leu19,Leu27]IGF-II and IGF-I/insulin hybrid); 4) no change in IGFBP-3 mRNA following addition of IGFs; 5) existence of metal ion-dependent IGFBP-3 specific protease in CM and protection of IGFBP-3 from protease by formation of [IGF:IGFBP-3] complexes; and 6) release of cell surface-associated IGFBP-3 into CM by addition of IGF peptides. These studies demonstrate that IGF peptides regulate CM concentrations of IGFBP-3 through non-receptor mediated events, including dissociation of cell surface-associated IGFBP-3 and protection of IGFBP-3 from protease activity.
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Department of Pediatrics, Stanford University School of Medicine, CA 94305.
Müller
H L
HL
Pham
H
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Lamson
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Rosenfeld
R G
RG
D015152:Blotting, Northern; D015153:Blotting, Western; D001943:Breast Neoplasms; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D017077:Culture Media, Conditioned; D003720:Densitometry; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000502:physiology; D018969:Insulin-Like Growth Factor Binding Proteins; D014176:Protein Biosynthesis; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D013002:Somatomedins / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D007328:Insulin; D018969:Insulin-Like Growth Factor Binding Proteins; D017527:Receptor, IGF Type 2; D013002:Somatomedins; D010450:Endopeptidases; C080473:insulin-like growth factor binding protein-3 protease