Abstract 5091: Increased susceptibility of Nrf2 deficient mice to radiation-induced pulmonary fibrosis
Authors: Travis Elizabeth L. , Rachakonda Girish , Zhou Xinhui , Sekhar Konjeti R. , Korhonen Katrina E. , Freeman Michael L. 
Published: June 2012

Abstract
The development of late normal tissue damage secondary to radiation therapy can be a significant problem for cancer survivors. Pulmonary toxicity resulting from radiation-induced fibrosis represents an insidious injury that can manifest itself 6 to 24 months after irradiation and continue to progress over a period of years. While dosimetric parameters that affect risk for radiation-induced pulmonary fibrosis are well described, less is known about genetic susceptibility. The transcription factor Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) regulates the expression of Phase II/antioxidant genes such as Sod2 and is a useful surrogate for estimating the intracellular ROS burden. We found that administration of 16 Gy to the thorax of C57BL/6J mice resulted in elevation of pulmonary Nrf2 levels 10 to 16 weeks after irradiation, indicative of elevated oxidative stress in injured tissue. Therefore, we compared the susceptibility of Nrf2−/− C57BL/6J mice to Nrf2+/− mice and to wild type (WT) littermates following thoracic irradiation (16 Gy). Significant levels of cleaved Caspase 3, a marker for apoptosis, were observed in both genotypes 24hrs after x-irradiation. Compared to WT littermates, Nrf2−/− mice responded to this injury by rapidly expressing plasminogen activator inhibitor-1 (PAI-1), a well established etiological agent for development of pulmonary fibrosis. One hundred percent of irradiated male Nrf2−/−mice developed fibrotic lesions that reduced survival time to a mean of 174 days, compared to irradiated WT mice that lived an average of 212 days before succumbing to fibrosis (p = 0.0001). Loss of a single Nrf2 allele resulted in a similar phenotype in irradiated male Nrf2+/− mice (p = 0.0001 compared to WT). Comparison of irradiated female to male mice yielded results consistent with that obtained using male mice, as well as the expected gender susceptibility. We investigated the role of Nrf2 in regulating TGF-β1/Smad3-dependent PAI-1 expression in IMR-90 lung fibroblasts. Nrf2 and pSmad3 reside in an immunoprecipitable nuclear complex. ChIP analysis of the PAI-1 promoter demonstrated that Nrf2 resides along with Smad3 at CAGA cis elements. Use of CAGA-Luciferase reporter vectors demonstrated that Nrf2 but not dominate negative Nrf2, inhibited TGF-β/R-Smad-dependent luciferase activity. In summary, these experiments indicate that Nrf2 suppresses TGF-β/R-Smad-mediated PAI-1 expression. Loss of Nrf2 would allow exuberant PAI-1 expression, as well as elevated ROS, that would contribute to the development of pulmonary fibrosis. The knowledge that there are functional SNPs in the promoter of NRF2 that impair expression and activity and that NRF2 expression diminishes with aging may help to explain the interpatient variation of irradiation-mediated late effects.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5091. doi:10.1158/1538-7445.AM2011-5091