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Hic-5 controls BMP4 responses in prostate cancer cells through interacting with Smads 1, 5 and 8

Abstract

Hydrogen peroxide-inducible clone-5 (Hic-5, or androgen receptor-associated protein 55) is a transforming growth factor-β-inducible LIM protein whose deregulation is implicated in the progression of prostate cancer. Here, we report that Hic-5 binds to Smads 1, 5 and 8, and represses bone morphogenetic protein (BMP) signaling responses. Myc-Hic-5 but not Myc-paxillin was specifically immunoprecipitated with anti-FLAG IgG1 from lysates of HEK293 co-transfected with either Myc-Hic-5 or Myc-paxillin and FLAG-tagged Smads 1, 5 or 8. We showed that such interactions require the LIM3 domain of Hic-5 and the MH2 domain of those Smads. Anti-Hic-5 antibody specifically pulled down endogenous Smad1 in both the PC3 human prostate cell line and primary cultures of rat prostate fibroblasts, supporting that Hic-5 binds to Smad1 at the endogenous level. Bacterially expressed glutathione S-transferase (GST)–Smads 1, 5 or 8, but not GST alone, pulled down in vitro transcribed and translated Hic-5, implicating that Hic-5 binds directly to Smads 1, 5 and 8. Significantly, using Hic-5 small hairpin RNA silencing and overexpression systems, we show that Hic-5 (at both the endogenous and exogenous levels) represses the ability of BMP4 to induce expression of the inhibitor of differentiation-1 (Id1; a downstream target gene of BMP), activate the Id1 gene promoter and induce apoptosis in human and rat prostate epithelial cells. Moreover, silencing of Hic-5 in PC3 cells as well as in the WPMY-1 human prostate stroma cell line greatly enhances the levels of endogenous phospho-Smad1/5/8. Finally, we provide fluorescent microscopic imaging to support that Smad1 and Hic-5 mutually interact also at the level of their nuclear export mechanisms. Collectively, these results provide the first evidence for a physical and mutual functional interaction between Hic-5 and the BMP signaling pathway.

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Acknowledgements

This work was supported by NIH Grants R01CA102074 and R01CA134878 (D Danielpour), a pre-doctoral fellowship (R Wahdan-Alaswad) from Case Comprehensive Cancer Center's Research Oncology Training Grant 5T32CA059366-15 and National Research Service Award Individual Fellowship Application 1F31CA142311, and the Case Comprehensive Cancer Center P30CA43703 (for the Cytometry Core facility).

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Correspondence to D Danielpour.

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Shola, D., Wang, H., Wahdan-Alaswad, R. et al. Hic-5 controls BMP4 responses in prostate cancer cells through interacting with Smads 1, 5 and 8. Oncogene 31, 2480–2490 (2012). https://doi.org/10.1038/onc.2011.422

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  • DOI: https://doi.org/10.1038/onc.2011.422

Keywords

  • Hic-5
  • BMP
  • Smad1
  • Smad
  • apoptosis
  • prostate

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