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

被引:61
作者
Bhattacharyya, Nisan
Pechhold, Klaus
Shahjee, Hanief
Zappala, Giovanna
Elbi, Cem
Raaka, Bruce
Wiench, Malgorzata
Hong, Jiang
Rechler, Matthew M. [1 ]
机构
[1] NIDDK, Diabet Branch, NIH, Bethesda, MD 20892 USA
[2] NIDDK, Islet Autoimmun Branch, NIH, Bethesda, MD 20892 USA
[3] NIDDK, Clin Endocrinol Branch, NIH, Bethesda, MD 20892 USA
[4] NCI, Lab Receptor Biol & Gene Express, NIH, Bethesda, MD 20892 USA
关键词
D O I
10.1074/jbc.M509463200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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, 228KGRKR232, 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.
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页码:24588 / 24601
页数:14
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