PARENT SESSION

Signaling Mediated by Reactive Oxygen Species

(SY018) TGF beta suppression of Nrf2-mediated transcription elevates ROS.

Bakin, Andrei¹, Sekhar, Konjeti², Arteaga, Carlos³, Freeman, Michael ^*,2, ¹ Cancer Genetics, Buffalo, NY, USA² Radiation Oncology, Nashville, TN, USA³ Medical Oncology, Nashville, TN, USA

ABSTRACT- TGF-1 appears to be central to the pathogenic progress of oxidant-induced pneumonitis and chronic progressive fibrosis. Reactive oxygen species (ROS) formed during Radiation Therapy are postulated to activate latent TGF-1. Following irradiation, there is a rapid rise in TGF- concentrations. TGF-1 signaling, in turn, increases intracellular ROS levels which can continue to activate latent TGF-, as well as mediate TGF-1 signaling, eg., induction of plasminogen activator inhibitor-1. This investigation examined TGF-1 regulation of Phase II gene expression, the cellular detoxification system used to remove ROS. Phase II gene expression is regulated by the transcription factor Nrf2 which is sequestered in the cytosol by association with the Cul3 adaptor protein Keap1. Keap1 directs Nrf2 ubiquitination and degradation. As intracellular ROS levels increase, critical cysteine residues in Keap1 become oxidized, Nrf2 dissociates from Keap1, Nrf2 becomes stabilized, heterodimerizes with transcription factors such as Maf or Jun, binds to antioxidant response elements (ARE) located in the proximal promoters of Phase II genes, and induces gene expression with subsequent detoxification of ROS. The importance of this pathway is illustrated in gene disruption experiments: loss of Nrf2 signaling sensitizes animals to oxidant-mediated fibrosis. Using microarray profiling, semi-quantitative RT PCR, immunoblotting, and flow cytometry, we found that in the presence of TGF-1, Phase II gene expression was suppressed, allowing intracellular ROS to increase. Ectopic expression of constitutive active or dominant negative proteins demonstrated that suppression required Smad3, which in turn, increased expression of ATF3. Immuno-precipitation experiments, GST/Nrf2 fusion pull-downs, and DNA precipitation experiments demonstrated that increasing ATF3 expression resulted in the formation of an ATF3/Nrf2 heterodimer that bound AREs and suppressed Phase II gene transcription. These observations potentially provide critical insight into the mechanism underlying TGF--mediated processes.

Key words: tgf beta, ros, Nrf2