Nuclear factor erythroid 2-related factor-2 (Nrf2) is a redox-sensitive transcription factor that activates several antioxidant and cytoprotective genes in response to oxidative stress. The role of Nrf2 activators and the intracellular regulation of Nrf2 have been studied extensively. In comparison, little is known about the self-regulation of Nrf2 due to experimental techniques commonly used to synchronize cellular signaling. Here we report that endogenous Nrf2 was downregulated in the nucleus of HeLa and MDA-MB-231 cells serum starved for 24hrs. Nrf2 expression was rescued by the addition of unconditioned media irrespective of its serum content. No concomitant change was observed in the expression of the primary inhibitor of Nrf2, Kelch-like ECH-associated protein-1 (Keap1). Nrf2 was upregulated by tert-butyl hydroquinone, although there was limited increase in Nrf2 in conditioned media-treated cells as compared to unconditioned media-treated cells. Decreasing the fraction of conditioned media in culture resulted in a dose-dependent increase in Nrf2 protein level. Taken together, our data suggests the existence of a complex self-regulatory mechanism for endogenous Nrf2 signaling.
Keywords: Keap1; Nrf2; cell synchronization; conditioned media; serum-free media; starvation.