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Smad7 is inactivated through a direct physical interaction with the LIM protein Hic-5/ARA55

A Correction to this article was published on 24 October 2022

This article has been updated

Abstract

We recently reported that hydrogen peroxide-inducible clone-5 (Hic-5, also named androgen receptor-associated protein 55) can bind to the transforming growth factor-β (TGF-β)-signaling regulator Smad3, thereby inhibiting certain Smad3-dependent TGF-β responses. We now show that Hic-5 can also control TGF-β responses through an alternative mechanism involving Smad7, a key negative regulator of TGF-β signaling. Hic-5 binds directly to Smad7. This interaction requires the LIM3 domain of Hic-5, and enhances TGF-β signaling through causing loss of Smad7 protein but not mRNA. Enforced expression of Hic-5 reverses the ability of Smad7 to suppress TGF-β-induced phosphorylation of Smads 2 and 3 and activation of the plasminogen activator inhibitor-1 promoter (in NRP-154 and PC3 prostate carcinoma and WPMY-1 prostate myofibroblast cell lines). Lentiviral-mediated small-hairpin RNA silencing of endogenous Hic-5 reduced TGF-β responses in PC3 and WPMY-1 cells. Further work suggests that the level of Smad7 is modulated by its physical interaction with Hic-5 and targeted to a degradation pathway not likely to be proteasomal. Our findings support that Hic-5 functions as a cell-type-specific activator of TGF-β signaling through its ability to physically interact with and neutralize Smad7.

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Abbreviations

AR:

androgen receptor

ARA55:

androgen receptor-associated protein 55

ARE-lux:

Smad2-responsive reporter containing activin response element

co-IP:

co-immunoprecipitation

DMEM/F-12:

Dulbecco's modified Eagle's medium/Ham's F-12

ECM:

extracellular matrix

EMT:

epithelial-mesenchymal transition

FAST-1:

forkhead activin signal transducer-1

FBS:

fetal bovine serum

GST:

glutathione S-transferase

HEK293:

human embryonic kidney cell line 293

Hic-5:

hydrogen peroxide-inducible clone-5

PAI-1:

plasminogen activator inhibitor-1

shRNA:

small-hairpin RNA

TβRI:

TGF-β type I receptor

TβRII:

TGF-β type II receptor

TGF-β:

transforming growth factor-β

3TP-lux:

TGF-β-responsive reporter containing PAI-1 promoter region

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Acknowledgements

We thank Dr Joan Massagué for 3TP-lux plasmid, Dr Bert Vogelstein for SBE4BV-luciferase plasmid, Dr Kohei Miyazono for full-length mouse Flag-Smad7-pcDNA3 plasmid, Dr Bing-Cheng Wang for the pcDNA3-Myc plasmid, Dr Ryosuke Takahashi and Dr Yu-Chung Yang for HA-ubiquitin plasmid, Dr Malcolm Whitman for ARE-lux and Myc-FAST-1. This work was supported by NCI grants 1R01CA102074 and 1R01CA092102.

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Correspondence to D Danielpour.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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Wang, H., Song, K., Krebs, T. et al. Smad7 is inactivated through a direct physical interaction with the LIM protein Hic-5/ARA55. Oncogene 27, 6791–6805 (2008). https://doi.org/10.1038/onc.2008.291

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