Non-secreted 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

doi:10.1074/jbc.M509463200

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Non-secreted 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

Nisan Bhattacharyya, Klaus Pechhold, Hanief Shahjee, Giovanna Zappala, Cem Elbi, Bruce Raaka, Malgorzata Wiench, Jiang Hong, and Matthew M. Rechler

Diabetes Branch, NIDDK/NIH, Bethesda, MD 20892-1758

Corresponding Author: mrechler{at}helix.nih.gov

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 non-secreted YFP-wild-type-IGFBP-3 decreased cell viability, as assessed by staining with annexin V-APC followed by flow cytometry. Induction of cell death was caspase-dependent, indicative of apoptosis. Apoptosis also was induced by the non-secreted 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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