Mechanistic studies of the Nrf2/Keap1 signaling pathway
Oxidative stress, an imbalance between production and removal of reactive oxygen species, can damage biological macromolecules including DNA, proteins and lipids (1). Oxidative damage to biological macromolecules can have profound effects on cellular functions and has been implicated in cancer, inflammation, neurodegenerative diseases, cardiovascular diseases and aging. Eukaryotic cells have evolved anti-oxidant defense mechanisms to neutralize reactive oxygen species (ROS) and maintain cellular redox homeostasis. One of the most important cellular defense mechanisms against ROS and electrophilic intermediates is mediated through the ARE (antioxidant responsive element, or electrophile responsive elements) sequence in the promoter regions of phase II and antioxidant genes. The ARE-dependent cellular defense system is controlled by the transcription factor Nrf2. Recent advances in the mechanistic studies of this pathway have provided the following models for Nrf2 regulation: Keap1, a key player in the activation of this pathway, has been identified to function as a molecular switch to turn on and off the Nrf2-mediated antioxidant response. (i) Under basal condition, Keap1 is in the off position and functions as an E3 ubiquitin ligase, constantly targeting Nrf2 for ubiquitination and degradation. As a consequence, the constitutive levels of Nrf2 are very low. (ii) The switch is turned on when oxidative stress or chemopreventive compounds inhibit the activity of the Keap1-Cul3-Rbx1 E3 ubiquitin ligase, resulting in increased levels of Nrf2 and activation of its downstream target genes. (iii) The switch is turned off again upon recovery of cellular redox homeostasis; Keap1 travels into the nucleus to remove Nrf2 from the ARE. The Nrf2-Keap1 complex is then transported out of the nucleus by the NES in Keap1. In the cytosol, the Nrf2-Keap1 complex associates with the Cul3-Rbx1 core ubiquitin machinery, resulting in degradation of Nrf2.

The protective role of Nrf2 in arsenic-induced toxicity and carcinogenicity

Another direction of our research is to understand the molecular mechanisms of toxicity/carcinogenicity of environmental pollutants and the endogenous cellular defense systems to cope with pollutants. Drinking water contaminated with arsenic is a worldwide public health issue. Arsenic has been classified as a human carcinogen that induces tumors in the skin, lung, and bladder. Arsenic damages biological systems through multiple mechanisms, one of them being reactive oxygen species. The ARE-Nrf2-Keap1 signaling pathway, activated by compounds possessing anti-cancer properties, has been clearly demonstrated to have profound effects on tumorigenesis. More significantly, Nrf2 knockout mice display increased sensitivity to chemical toxicants and carcinogens and are refractory to the protective actions of chemopreventive compounds. Therefore, we hypothesize that activation of the ARE-Nrf2-Keap1 pathway acts as an endogenous protective system against arsenic-induced toxicity and carcinogenicity. The following Specific Aims are intended to further elucidate the mechanism of Nrf2-activation in protection from arsenic-induced toxicity/tumorigenicity. We will (1) determine the protective role of the ARE-Nrf2-Keap1 pathway in arsenic-induced toxicity and cell transformation using a model cell line UROtsa, (2) define the molecular mechanisms of activation of the ARE-Nrf2-Keap1 pathway by arsenic, sulforaphane, and tBHQ, and (3) define the protective role of the ARE-Nrf2-Keap1 pathway in arsenic-induced toxicity and tumorigenicity using Nrf2 knockout mouse as a model.