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Lack of evidence for nuclear IGFBP5 in mammary epithelial cells

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Background

IGFBP5 plays a role in mediating the effects of IGFs, which are important in mammary gland development [1] and carcinogenesis [2]. IGF-independent effects of IGFBP5 have also been described and it has been postulated that these are at least partially mediated via IGFBP5 localized in the nucleus [3, 4].

Methods

The cellular localization of IGFBP5 was analyzed by confocal microscopy after either applying exogenous fluorescent-labeled recombinant protein or applying immunostaining of cells ectopically expressing IGFBP5. HC11, MCF10A mammary epithelial and T47D mammary carcinoma cell lines were used in this study.

Results

Nuclear localization of IGFBP5 was observed under two conditions: fluorescent-labeled IGFBP5 added to cells with selectively permeabilized plasma but not nuclear membrane; and cells transfected with IGFBP5 expression vectors lacking the coding region for the signal peptide. By contrast, non-permeabilized cells could be stimulated to take up IGFBP5 only into intracellular vesicles outside the nucleus and this was enhanced by adding a conjugate of polylysine and transferrin, indicating an endocytotic uptake route. In addition, cells transfected with IGFBP5 containing the signal peptide secreted IGFBP5 into the medium but did not show any detectable nuclear staining.

Conclusions

Nuclear localization of IGFBP5 in mammary epithelial cells required the crossing of the plasma membrane, which does not appear to occur under normal cell culture conditions. Exit of IGFBP5 from endosomal vesicles into the cytosol followed by nuclear uptake was never observed. Our results indicate a minor role or no role of nuclear IGFBP5 in mediating its IGF-independent effect in the mammary epithelium and in breast cancer.

References

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    Allan GJ, Beattie J, Flint DJ: The role of IGFBP-5 in mammary gland development and involution. Domest Anim Endocrinol. 2004, 27: 257-266. 10.1016/j.domaniend.2004.06.009.

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    Pollak MN, Schernhammer ES, Hankinson SE: Insulin-like growth factors and neoplasia. Nat Rev Cancer. 2004, 4: 505-518. 10.1038/nrc1387.

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    Schedlich LJ, Le Page SL, Firth SM, Briggs LJ, Jans DA, Baxter RC: Nuclear import of insulin-like growth factor-binding protein-3 and -5 is mediated by the importin beta subunit. J Biol Chem. 2000, 275: 23462-23470. 10.1074/jbc.M002208200.

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    Xu Q, Li S, Zhao Y, Maures TJ, Yin P, Duan C: Evidence that IGF binding protein-5 functions as a ligand-independent transcriptional regulator in vascular smooth muscle cells. Circ Res. 2004, 94: e46-e54. 10.1161/01.RES.0000124761.62846.DF.

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Acknowledgement

Supported by the Austrian Science Fund FWF, SFB021 'Cell proliferation and cell death in tumors'.

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Berlato, C., Jurgeit, A., Geley, S. et al. Lack of evidence for nuclear IGFBP5 in mammary epithelial cells. Breast Cancer Res 7, P4.12 (2005) doi:10.1186/bcr1142

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Keywords

  • Signal Peptide
  • Transferrin
  • Nuclear Localization
  • Mammary Epithelial Cell
  • Mammary Epithelial