Low dose IR-induced IGF-1-sCLU expression: a p53-repressed expression cascade that interferes with TGF&amp;x00DF;1 signaling to confer survival

David Willson,Eva Goetz,Yonglong Zou,Dmitry Klokov,David Boothman,Xiuquan Luo,Konstantin Leskov,Shinako Araki

doi:10.1038/npre.2011.6451.1

Abstract

AbstractTo better understand tissue responses after low IR doses, we generated a reporter system using human clusterin promoter fused to firefly luciferase (hCLUp-Luc). Secretory clusterin (sCLU), an extra-cellular molecular chaperone, induced by low doses of cytotoxic agents, clears cell debris promoting survival. Low dose IR (&gt;2 cGy) exposure induced hCLUp-Luc activity with peak levels at 96 h, consistent with endogenous sCLU levels. As doses increased (&gt;1 Gy), sCLU induction amplitudes increased and time to peak response decreased. sCLU expression was stimulated by IGF-1, but suppressed by p53. Responses in transgenic hCLUp-Luc reporter mice after low IR doses showed that specific tissues (i.e., colon, spleen, mammary, thymus, bone marrow) of female mice induced hCLUp-Luc activity more than male mice after whole body &gt;10 cGy. Tissue-specific, non-linear dose- and time-responses of hCLUp-Luc and endogenous sCLU levels were noted. Colon maintained homeostatic balance after 10 cGy. Bone marrow responded with delayed, but prolonged and elevated expression. Intraperitoneal administration of the &x03B1;-TGF&x00DF;1 (1D11) antibody, but not a control antibody (13C4), immediately following IR exposure abrogated CLU induction responses. Induction in vivo also correlated with Smad signaling via activated TGF&x00DF;1 after IR. Mechanistically, media with elevated sCLU levels suppressed signaling, blocked apoptosis and increased survival of TGF&x00DF;1-exposed tumor or normal cells. Thus, sCLU is a TGF-&x00DF;1-induced pro-survival, potential bystander factor expressed in certain exposed tissues that, in turn, abrogates TGF&x00DF;1 signaling and may promote wound healing and likely contributes to a pro-tumor growth microenvironment.

Highlights

DNA damage- and senescence-induced secreted proteinshave been identified as an important response to low doses of ionizing radiation (IR) (Arufe et al 2011, Freund et al 2011, Rodier et al 2011)
We showed that IGF-1 was suppressed by p53/NF-YA complexes in its basal state at one unique NF-Y regulatory site in the IGF-1 promoter, whose binding was lost after low dose IR exposures in an ATM-dependent manner in cells deficient in p53 function, resulting in secretory clusterin expression
Since secretory clusterin (sCLU) can be induced by activated TGFß1 (Jin and Howe 1999), and low dose IR exposure can stimulate TGFß1 (Nguyen et al 2011), we examined whether sCLU responses in vivo by hCLUp-Luc reporter and endogenous sCLU induction were prevented by intraperitoneal administration of a αTGFß1 (1D11, 10 mg/Kg) versus control IgG antibody (13C4, 10 mg/kg) (Figure 3C); the 1D11 antibody neutralizes all three forms of TGFß (Dasch et al 1989)

Summary

Introduction

DNA damage- and senescence-induced secreted proteins (i.e., an ‘induced secretome’)have been identified as an important response to low doses of ionizing radiation (IR) (Arufe et al 2011, Freund et al 2011, Rodier et al 2011). Identifying low dose IR-responsive expression pathways that result in secreted proteins that could have biological impacts on exposed, as well as non-exposed, cells would strongly suggest that not all cells have to be irradiated for entire tissues to respond in vivo. We delineated a unique pathway of regulation of insulin-like growth factor 1 (IGF1) expression in response to low doses of IR in vitro (Criswell et al 2003, Criswell et al 2005, Goetz et al 2011). We showed that IGF-1 was suppressed by p53/NF-YA complexes in its basal state at one unique NF-Y regulatory site in the IGF-1 promoter, whose binding was lost after low dose IR exposures in an ATM-dependent manner in cells deficient in p53 function, resulting in secretory clusterin (sCLU) expression. IGF-1-sCLU expression was linked to cytoprotection of irradiated cells in vitro and tissues in vivo, the underlying mechanisms of cytoprotection are unknown (Cardona-Gomez et al 2001)

Methods

Results

Conclusion