Low dose IR-induced IGF-1-sCLU expression: a p53-repressed, TGFß1-induced expression cascade that interferes with TGFß1 signaling to confer survival

To 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 (>2 cGy) exposure induced hCLUp-Luc activity with peak levels at 96 h, consistent with endogenous sCLU levels. As doses increased (>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 hCLUpLuc 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 >10 cGy. Tissue-specific, non-linear doseand 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 α-TGFß1 (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 TGFß1 after IR. Mechanistically, media with elevated sCLU levels suppressed signaling, blocked apoptosis and increased survival of TGFß1-exposed tumor or normal cells. Thus, sCLU is a TGF-ß1-induced pro-survival, potential bystander factor expressed in certain exposed tissues that, in turn, abrogates TGFß1 signaling and may promote wound healing and likely contributes to a pro-tumor growth microenvironment. Introduction DNA damageand 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 ultimately 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. To date, few pathways activated in vivo in response to low doses of IR have been elucidated. Recently, 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. Regulation of IGF-1 expression, and thus sCLU, was rate-limited by p21 in an ill-defined ATM-dependent modification of NF-YA, even though p53 stabilization and protein induction occurred (Goetz et al 2011). IGF-1-sCLU expression was linked to cytoprotection of irradiated cells in vitro and tissues in vivo, however, the underlying mechanisms of cytoprotection are unknown (Cardona-Gomez et al 2001). sCLU is a known low dose IR-inducible secreted protein that is regulated by IGF-1 signaling and can confer an ‘adaptive response’ (Criswell et al 2003, Criswell et al 2005). Since numerous cancers constitutively over-express sCLU as a pro-survival and pro-growth factor, strategies using antisense oligomers (Chi et al , Zoubeidi et al) or siRNA nanoparticles (Sutton et al 2006) have been developed to enhance chemoand radio-therapies against human prostate and lung cancers. sCLU can bind and sequester BAX in the endoplasmic reticulum (ER) of cells, thereby preventing drugand radiation-induced apoptosis (Araki et al 2005, Shannan et al 2006, Zhang et al 2005). Thus, low dose IR-inducible IGF-1-sCLU expression from cells in vitro or in tissues in vivo could afford significant cytoprotective bystander functions. Since we previously linked IGF-1 signaling to expression of pro-survival sCLU levels, we hypothesized that expression of the IGF-1-sCLU axis would: (i) allow development of a potent and ultrasensitive biodosimeter of live cells using the human clusterin (CLU) promoter linked to firefly luciferase (hCLUp-Luc) expression for bioluminescence imaging (BLI); BLI allows for non-invasive temporal quantitative imaging of cells and tissue in real time and greatly increases detection sensitivities in vitro and in vivo; and (ii) result in expression of a secreted protein expression axis that would have significant pro-survival bystander effects in vivo. Here, we demonstrate that cells in vitro and transgenic mice in vivo containing the hCLUpLuc reporter can be used as an extremely sensitive and potentially important reporter system to repeatedly image responses of irradiated live cells and tissues. We present compelling evidence that sCLU expression in specific radiation-sensitive tissues in vivo is controlled by low dose IR activation of TGFß1. The IGF-1-sCLU expression axis is long-lived and extremely responsive to low doses of IR, as well as to other cytotoxic agents (Goetz et al 2011), making it ideal for future use as a ‘biomarker’ for biological responses to low dose IR exposures. The exogenous reporter responses matched endogenous sCLU protein expression in doseresponse and temporal kinetics, and tissues differed in doseand time-responses to low (i.e., 2 cGy) versus higher IR doses. Importantly, induction of sCLU at low doses of IR, from 1-100 cGy, were linear for cells in vitro and tissue in vivo, however, expression of sCLU could not be extrapolated from high doses, and non-linear responses were clearly indicated at doses >1 Gy. Finally, sCLU induction is part of a TGFß1-induced negative feedback regulatory bystander loop that is induced by TGFß1, but in turn, can suppress TGFß1 signaling and promote survival, creating a microenvironment that may ultimately promote tumor growth. Materials and Methods Chemicals and plasmids. AG1024 (IGF-1R tyrosine kinase inhibitor) was obtained from EMD Chemicals (Gibbstown, NJ). IGF-1 was obtained from R&D Systems (Minneapolis, MN). TGFß1 and ultrapure luciferin were obtained from the Sigma/Aldrich Chemical Co. (St. Louis, MO), and used in luciferase assay reagent (LAR) assays (Promega, Madison, WI). The human 1403 bp CLU promoter fused to luciferase (hCLUp-Luc) was previously described (Criswell et al 2003, Criswell et al 2005, Klokov et al 2004). Flag-tagged CMV-p53 cDNA was created by subcloning p53 cDNA into the pcDNA3.1-N-term-Flag construct. Constitutive-active PTEN (PTEN CA) and kinase-deficient ATK1 (AKT KD) were obtained from Dr. Lindsey Mayo (Indiana University). Antibodies and immunoblotting. Antibodies specific to mouse (M18) and human (B5) sCLU, phosphorylated Smad3 (p-Smad3) or total Smad3 (t-Smad3), γ-H2AX, p53 and α-tubulin were purchased from Santa Cruz. Antibodies for GAPDH and α-tubulin, were used for loading. Immunoblotting was performed as described (Criswell et al 2005) and relative protein levels were quantified from x-ray films using NIH image J software as described (Criswell et al 2005). α-TGF-β1 murine monoclonal antibody (1D11) that neutralizes the three TGF-β isoforms (Dasch et al 1989), and an isotype-matched IgG1 monoclonal antibody (13C4, raised against Shigella toxin) were provided by Genzyme Corporation (Framingham, MA). Cell lines, treatments and survival assays. Human MCF-7 breast cancer cells and a stably transfected 1403 MCF-7 clone (MCF-7 cells containing stably integrated 1403 bp CLU promoter fused to firefly luciferase) were cultured in DMEM (BioWhittacker; Walkersville, MA) containing 10% fetal bovine serum (HyClone; Utah, USA). Human HCT116 cells were grown as described (Li et al 2008, Wagner et al 2008) in 10% FBS-DMEM. Human mammary epithelial cells (HMECs), life-extended using hTERT and CD4 over-expression, were kindly obtained from Dr. David Euhus (UT Southwestern). All cells and stable derivatives were maintained at 37 C at 5% CO2-95% air. For TGFß1 or IGF-1 treatments, cells were serumstarved (0.5% FBS) overnight, and exposed to IGF-1 or TGFß1 at the indicated doses in normal serum-DMEM. Cell irradiations were performed using a JL Shepherd Cs Mark I-68 irradiator (3.87 Gy/min) with appropriate shielding. All cells were free from mycoplasma infection. Transfections and luciferase assays. Cells were transiently transfected with hIGF-1p-Luc or hCLUp-Luc reporter constructs and RSV-β-gal as a transfection control using Fugene 6 (Roche) (Goetz et al 2011, Trougakos et al 2009a). Treatments, where indicated, were performed 24 h after transfection. Luciferase activities were analyzed using LAR (Promega, Madison, WI). β-Galactosidase activity was determined using Galacto-Star reagent (Life Technologies, Carlsbad, CA). All experiments were normalized for protein amounts using Bradford assays (Bio-Rad). For stable hCLUp-Luc MCF-7 cells, cells were co-transfected with the hCLUp-Luc construct together with 2-fold excess pcDNA-3 that contained a G418resistance gene. Cells were then selected with G418 and resistant clones isolated as described (Criswell et al 2005). Isolates were then treated with IR, other cytotoxic agents (Criswell et al 2005, Goetz et al 2011) or TGFß1 and analyzed for hCLUp-Luc reporter activities, as well as endogenous sCLU expression. A clone with identical hCLUp-Luc activities monitored by luciferase activities (Criswell et al 2005, Goetz et al 2011) and endogenous sCLU protein expression by Western blotting, was selected and examined for low dose IR induction. HCT116 and HMEC cells were transiently knocked down for sCLU expression using an siRNA-sCLU directed to the coding sequence for the CLU mRNA leader peptide sequence (Goetz et al 2011). siRNA-nonsense/scrambled (shRNA-Scr) was used as a control. Knockdown of sCLU levels were confirmed by Western blotting using α-tubulin as a