The overall goal of the application is to identify the molecular pathways responsible for elevated reactive oxygen specie (ROS) levels that follow in response to TGF-beta signaling in order to aid the development of therapeutic strategies needed to treat this significant clinical problem. ROS contribute to the progression of degenerative pulmonary injury in patients with thoracic malignancies who undergo radiation therapy. Phase II detoxification proteins are responsible for removal of ROS. The transcription factor Nrf2 is a master regulator of Phase II gene expression. Loss of Nrf2 signaling ablates Phase II gene expression, elevates ROS, and enhances progression of pulmonary fibrosis. We show that TGF-beta signaling suppresses Nrf2- regulated Phase II gene expression and elevates ROS. We hypothesize that suppression of Nrf2-regulated transcription is ATF3 mediated, a consequence of TGF-beta signaling through SmadS.
Aim 1 will identify the mechanism by which ATF3 suppresses Nrf2-regulated gene expression. The approach will identify the domains required for ATF3 Nrf2 interaction in vitro and in vivo and determine if such an interaction alters Nrf2 function in terms of either Nrf2 DNA binding activity or transcriptional activation. Binding of deletion constructs to GST fusion molecules, co-immunoprecipitation, two hybrid systems, DNA precipitation and ChIP assays will be used.
Aim 2 will determine if loss of ATF3 abrogates TGF-beta-mediated suppression of Nrf2-regulated Phase II gene expression and lowers ROS levels. Two approaches will be used to down regulate ATF3: Dominant negative ATF3 molecules and siRNA directed against ATF3.
Aim 3 will test the hypothesis that TGF-beta-mediated signaling suppresses Nrf2-regulated gene expression in vivo and that Nrf2 impacts radiation-induced pulmonary fibrosis. The well characterized model of radiation-induced fibrosis will be used to determine if loss of Nrf2 signaling enhances pulmonary injury. Nrf2 wild type and null animals will be used.
|Travis, Elizabeth L; Rachakonda, Girish; Zhou, Xinhui et al. (2011) NRF2 deficiency reduces life span of mice administered thoracic irradiation. Free Radic Biol Med 51:1175-83|
|Mukhopadhyay, Sutapa; Sekhar, Konjeti R; Hale, Ashley B et al. (2011) Loss of NRF2 impairs gastric nitrergic stimulation and function. Free Radic Biol Med 51:619-25|
|Zhao, Zhenyang; Chen, Yan; Wang, Jian et al. (2011) Age-related retinopathy in NRF2-deficient mice. PLoS One 6:e19456|
|Dworski, Ryszard; Han, Wei; Blackwell, Timothy S et al. (2011) Vitamin E prevents NRF2 suppression by allergens in asthmatic alveolar macrophages in vivo. Free Radic Biol Med 51:516-21|
|Rachakonda, G; Sekhar, K R; Jowhar, D et al. (2010) Increased cell migration and plasticity in Nrf2-deficient cancer cell lines. Oncogene 29:3703-14|
|Thotala, Dinesh K; Geng, Ling; Dickey, Amy K et al. (2010) A new class of molecular targeted radioprotectors: GSK-3beta inhibitors. Int J Radiat Oncol Biol Phys 76:557-65|
|Segal, Brahm H; Han, Wei; Bushey, Jennifer J et al. (2010) NADPH oxidase limits innate immune responses in the lungs in mice. PLoS One 5:e9631|
|Thotala, Dinesh K; Hallahan, Dennis E; Yazlovitskaya, Eugenia M (2008) Inhibition of glycogen synthase kinase 3 beta attenuates neurocognitive dysfunction resulting from cranial irradiation. Cancer Res 68:5859-68|
|Tu, Tianxiang; Thotala, Dinesh; Geng, Ling et al. (2008) Bone marrow X kinase-mediated signal transduction in irradiated vascular endothelium. Cancer Res 68:2861-9|
|Brown, Stephan L; Sekhar, Konjeti R; Rachakonda, Girish et al. (2008) Activating transcription factor 3 is a novel repressor of the nuclear factor erythroid-derived 2-related factor 2 (Nrf2)-regulated stress pathway. Cancer Res 68:364-8|