The Role of ROS in Nrf2 Activation Using qPCR

Phytochemicals found in plants, such as broccoli, kale, ginger, and garlic, incite the activation of the Nrf2 transcription factor. Nrf2 binds to a gene sequence called the antioxidant response element (ARE), which is located upstream of genes that encode cytoprotective proteins. These cytoprotective proteins guard cells against oxidative stress and electrophilic species that lead to certain chronic diseases. Thus, studying Nrf2 activation is highly promising for the prevention of these diseases. A majority of the phytochemicals that target and activate Nrf2 and ARE are oxidizable diphenols that are oxidized into their electrophilic forms, generating reacting oxygen species (ROS). Electrophiles have been clearly demonstrated to activate Nrf2 and the ARE. What is not as well understood is how and to what extent ROS activate Nrf2 and the ARE. The goal of this research was to elucidate the role that reactive oxygen species (ROS) play in the transcription of several ARE-regulated genes through the use of quantitative polymerase chain reactions (qPCR). Quantitative PCR is a useful tool in the comparison of mRNA accumulation in the cell under different treatment conditions. We tested the hypothesis that there is a synergistic activation of ARE-regulated genes upon the combined treatment with an electrophile and ROS. The strain of cells used is human keratinocyte HaCaT cells. The experiments included four treatment conditions: sulforaphane, hydrogen peroxide, sulforaphane+hydrogen peroxide, and a vehicle control. Sulforaphane should generate minimal ROS on its own. Hydrogen peroxide was chosen as the ROS in these experiments. The results of our qPCR experiments have shown that, not only does H₂O₂ enhance the activation of HO-1 by an electrophile, it has a synergistic effect on the transcription of this highly regulated cytoprotective protein. In addition, an ARE reporter has shown the same synergistic effect, as well as heme oxygenase 1 protein, an ARE-regulated antioxidant gene, by western blot. The goal of our project is to determine whether this observed synergy is also seen for other ARE-regulated genes. The ARE-regulated mRNA transcripts tested thus far in addition to HO-1 are AKR1C1 and AKR1C2, which encode for reductases, and GCLC and GCLM, involved in glutathione synthesis.