Comprehensive Landscape of Nrf2 and p53 Pathway Activation Dynamics by Oxidative Stress and DNA Damage

A quantitative
dynamics pathway map of the Nrf2-mediated oxidative stress response
and p53-related DNA damage response pathways as well as the cross-talk
between these pathways has not systematically been defined. To allow
the dynamic single cell evaluation of these pathways, we have used
BAC-GFP recombineering to tag for each pathway’s three key
components: for the oxidative stress response, Keap1-GFP, Nrf2-GFP,
and Srxn1-GFP; for the DNA damage response, 53bp1-GFP, p53-GFP, and
p21-GFP. The dynamic activation of these individual components was
assessed using quantitative high throughput confocal microscopy after
treatment with a broad concentration range of diethyl maleate (DEM;
to induce oxidative stress) and etoposide (to induce DNA damage).
DEM caused a rapid activation of Nrf2, which returned to baseline
levels at low concentrations but remained sustained at high concentrations.
Srxn1-GFP induction and Keap1-GFP translocation to autophagosomes
followed later, with upper boundaries reached at high concentrations,
close to the onset of cell death. Etoposide caused rapid accumulation
of 53bp1-GFP in DNA damage foci, which was later followed by the concentration
dependent nuclear accumulation of p53-GFP and subsequent induction
of p21-GFP. While etoposide caused activation of Srxn1-GFP, a modest
activation of DNA damage reporters was observed for DEM at high concentrations.
Interestingly, Nrf2 knockdown caused an inhibition of the DNA damage
response at high concentrations of etoposide, while Keap1 knockdown
caused an enhancement of the DNA damage response already at low concentrations
of etoposide. Knockdown of p53 did not affect the oxidative stress
response. Altogether, the current stress response landscapes provide
insight in the time course responses of and cross-talk between oxidative
stress and DNA-damage and defines the tipping points where cell injury
may switch from adaptation to injury.