title: Nonsecreted insulin-like growth factor binding protein-3 (IGFBP-3) can induce apoptosis in human prostate cancer cells by IGF-independent mechanisms without being concentrated in the nucleus.
abstract: Insulin-like growth factor binding protein-3 (IGFBP-3), a secreted protein, has the intrinsic ability to induce apoptosis directly without binding insulin-like growth factors. Previous studies suggested that IGFBP-3 must be secreted to exert its biological functions. IGFBP-3 contains a nuclear localization signal (NLS), and exogenous IGFBP-3 is translocated into the nucleus, suggesting that both secretion and nuclear localization may play important roles in IGFBP-3 action. To address these questions, we fused yellow fluorescent protein (YFP) to mature IGFBP-3 lacking its signal peptide so that it would remain intracellular and mutated the C-terminal NLS of IGFBP-3, (228)KGRKR(232), to MDGEA. Following transfection of PC-3 human prostate cancer cells with these constructs, Western blots indicated that YFP-IGFBP-3 lacking a signal peptide was cell-associated and not present in the extracellular media. Moreover, the fusion protein was not N-glycosylated, indicating that it had not entered the secretory pathway. Confocal imaging showed that intracellular YFP-MDGEA-IGFBP-3 was predominantly cytoplasmic. Transient transfection of nonsecreted YFP-wild-type IGFBP-3 decreased cell viability, as assessed by staining with annexin V followed by flow cytometry. Induction of cell death was caspase-dependent, indicative of apoptosis. Apoptosis also was induced by the nonsecreted NLS mutant (YFP-MDGEA-IGFBP-3) alone and when the IGF-binding site also had been mutated. These results indicate that IGFBP-3 can induce apoptosis in an IGF-independent manner without being secreted or concentrated in the nucleus.
authors.affiliation: Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Nisan
authors.initials: N
authors.lastname: Bhattacharyya
authors.forename: Klaus
authors.initials: K
authors.lastname: Pechhold
authors.forename: Hanief
authors.initials: H
authors.lastname: Shahjee
authors.forename: Giovanna
authors.initials: G
authors.lastname: Zappala
authors.forename: Cem
authors.initials: C
authors.lastname: Elbi
authors.forename: Bruce
authors.initials: B
authors.lastname: Raaka
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Wiench
authors.forename: Jiang
authors.initials: J
authors.lastname: Hong
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Rechler
mesh_terms: D017209:Apoptosis; D001665:Binding Sites; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000378:metabolism; D008297:Male; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D011485:Protein Binding; D021381:Protein Transport
chemical_list: D018972:Insulin-Like Growth Factor Binding Protein 3; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II