Abstract
The major signaling pathway in human cells is related to the antioxidant defense system. The main component of this system is a transcription factor, Nuclear Factor Erythroid 2-Related Factor 2 (NRF2). It regulates this system in different cellular situations under stimulation by oxidative stress or antioxidants. Thus, detecting the stimulation of NRF2 via a screening strategy may enable us to discover stimulating agents of NRF2-related signaling pathway. With this in mind, we designed a whole cell bioreporter containing the NRF2 response elements that are inserted in a luciferase vector, immediately upstream of a luciferase gene whose promoter has been removed. This bioreporter is activated by stimulators such as 3H-1,2-dithiole-3-thione (D3T), butyl hydroxyanisole (BHA) and ascorbic acid reacting as antioxidant agents. It was observed that the regulatory region of the NRF2 gene, which is identified by NRF2 protein, is located inside its coding region. This designed bioreporter can detect the presence of antioxidant agents. It also exhibits a significant linear correlation over different doses of these agents ranging from 0.8 to 80 μM for ascorbic acid, 0.1 to 100 μM for D3T, and 0.1 to 100 μM for BHA. This detection system is proven to be more sensitive than Real-time PCR, suggesting it to be a highly sensitive system among the available methods.
Highlights
The major signaling pathway in human cells is related to the antioxidant defense system
Ascorbic acid at concentrations of about 70 and 80 μM can significantly increase the expression of the Nuclear Factor Erythroid 2-Related Factor 2 (NRF2), but at lower concentrations, no change in the expression of the NRF2 in treated cells compared to non-treated ones was observed (Fig. 2)
In the steady state of cells, KEAP1 proteins inhibit the NRF2 nuclear translocation, but they lead to its ubiquitination and degradation, whereas the increase in cytosolic free NRF2 may result in its nuclear translocation and NRF2/DNA complex formation
Summary
The major signaling pathway in human cells is related to the antioxidant defense system. The activation of the transcription involves NRF2 recognizing its own promoter and establishing an effective interaction with it and the newly formed and accumulated NRF2 in the nucleus binds to promoters of other specific genes Such genes encode detoxifying enzymes/proteins including Glutathione-S-Transferases(GSTs), Superoxide Dismutase(SOD), Catalase, NAD(P) H: Quinoneoxidoreductase-1(NQO1) as well as stress response proteins such as heme oxygenase-1 (hmox1) and -2 (hmox2), metallothioneins and heat shock proteins. Antioxidants perform as the accelerator of this protective system through two major mechanisms: first, they have specific functional groups that are capable of disrupting the NRF2-KEAP1 complex leading to the release of the latter part form the former This happens via changing the conformation of the KEAP1 and disrupting the ubiquitination of the NRF2 which result in successful transcription of the antioxidant defense gene[14]. That means that they can neutralize the free radicals such as reactive oxygen and nitrogen species via reducing them to stable compounds and break the molecular chain oxidation reactions, both in cells and extra-cellular environments[15]
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