Title: Regulatory mechanisms of secretory clusterin (sCLU) expression: a low doseinducible ‘bystander’ cytoprotective protein.

Authors: Tracy Criswell, Eva Cataldo, Shinako Araki, Konstantin Leskov, Vijay Thakur, Lakshmi Sampath, David Wilson, Lindsey Mayo, and David A. Boothman.

Institutions: Departments of Oncology, Pharmacology, and Radiation Oncology, Simmons Comprehensive Cancer Center, University of Southwestern Medical Center, Dallas, TX.

Secretory clusterin protein (sCLU) is a low dose ionizing radiation (IR)-inducible, prosurvival gene product implicated in aging, obesity, heart disease and cancer. Recently, we have elucidated that regulatory signal transduction processes that control sCLU expression after both IR versus TGF-ß1 exposures. Here, we report that induction of sCLU is delayed, peaking 72 h after low doses of IR (>2 cGy), and is dependent on the up-regulation of insulin-like growth factor-1, as well as phosphorylation-dependent activation of its receptor (IGF-1 and IGF-1R, respectively). Activated IGF-1R then stimulates the downstream Src-Mek-Erk signal transduction cascade to ultimately transactivate the early growth response-1 (Egr-1) transcription factor, required for sCLU expression. Interestingly, p53 suppresses sCLU expression and mechanisms of p53 control are being elucidated. Thus, IR exposure causes stress-induced activation of IGF-1R-Src-Mek-Erk-Egr-1 signaling that regulates the sCLU pro-survival cascade pathway, important for radiation resistance in cancer therapy.

In contrast, TGF-ß1 exposure, which can be activated by IR treatment, can over-ride p53 suppression of sCLU, allowing massive sCLU protein expression. The mechanism by which TGF-ß1 over-rides p53 suppression of sCLU expression involved induction of the human double minute-2 (Hdm-2) protein, which acts to degrade p53 expression. Thus, in animals following low doses of IR exposure, sCLU expression is high in the colon, spleen, thymus, and bone marrow, presumably via the IR-activation of TGF-ß1.

Importantly, we now demonstrate that sCLU over-expression can act to feedback
regulate TGF-ß1-induced signaling and growth suppression. Using siRNA specific to
full-length CLU mRNA, which expresses sCLU, we should that knocking down sCLU
expression can enhance TGF-ß1-induced DNA synthesis inhibition and growth
suppression. Furthermore, adding sCLU to the medium suppresses TGF-ß1-induced
growth suppression. These data suggest that sCLU can be a radioprotector, and can
work in a bystander manner protecting both irradiated expressing or non-expressing
cells, as well as non-irradiated cells. We are exploring the biological consequences oflow dose IR-inducible sCLU expression both in cell culture in vitro and in animals in vivo, wherein we have developed a CLU promoter-firefly luciferase ‘low dose IR indicator’ mouse.

This work was supported by grant # DE-FG-022179 to DAB.