Nrf2 activates the PI3K/Akt pathway.

<p>(A) NHEMs were transduced with the indicated adenoviruses (10 MOI) and then assessed by Western blotting. Overexpression of Nrf2 induced phosphorylation of Akt, mTOR and S6 ribosomal protein. (B) Cells were transduced with the indicated adenoviruses and then assessed by Western blotting. Nrf2-induced phosphorylation was inhibited by overexpression of Keap1.</p

Nrf2 Negatively Regulates Melanogenesis by Modulating PI3K/Akt Signaling

<div><p>Nrf2 plays a role in protection of cells against oxidative stress and xenobiotic damage by regulating cytoprotective genes. In this study, we investigated the effect of Nrf2 on melanogenesis in normal human melanocytes (NHMCs). When NHMCs were transduced with a recombinant adenovirus expressing Nrf2, melanin synthesis was significantly decreased. Consistent with this result, overexpression of Nrf2 decreased the expression of tyrosinase and tyrosinase-related protein 1. The inhibitory effect of Nrf2 was reversed by overexpression of Keap1, an intracellular regulator of Nrf2. Interestingly, Nrf2 overexpression resulted in marked activation of PI3K/Akt signaling. Conversely, inhibition of PI3K activity by treatment with wortmannin reversed the depigmentary effects of Nrf2. Taken together, these results strongly suggest that Nrf2 negatively regulates melanogenesis by modulating the PI3K/Akt signaling pathway.</p></div

Inhibition of Nrf2-induced depigmentation by Keap1.

<p>(A) NHEMs were transduced with adenoviruses (10 MOI). Cell extracts were fractionated and Nrf2 protein was assessed by Western blotting. Lamin B was used for determination of the nuclear fraction. Whole-cell lysates were used for the detection of Keap1 and Actin. (B) NHEMs were transduced with adenoviruses (10 MOI). For detection of cell pigmentation, cells were spun down. Lower panel shows melanin content measured by spectrometer. Data are the means ± SD of triplicate measurements (*P<0.01 vs. control). (C) TYR activity was determined and expressed as a percentage of the control. Data are the means ± SD of triplicate measurements (*P<0.01 vs. control). (D) Expression of TYR and TRP1 was assessed by Western blotting. Actin was used as the loading control. (E) The TYR promoter-luciferase reporter adenovirus was co-transduced with the indicated adenoviruses. TYR promoter activity was expressed as a percentage of the control ± SD (*P<0.01 vs. control).</p

Inhibition of PI3K/AKT signaling prevents Nrf2-induced depigmentation.

<p>(A) NHEMs were transduced with the indicated adenoviruses (10 MOI) for 6 h. Cells were replenished with fresh medium containing vehicle (DMSO) or the PI3K inhibitor wortmannin (Wort, 100 nM). Cells were cultured for a 2 days, then cells were refed with fresh medium containing wortmannin again. One day after, cellular extracts were prepared and then assessed by Western blotting. Phosphorylation of Akt, mTOR and S6 ribosomal protein by Nrf2 was inhibited by PI3K inhibitor treatment. (B) Tyrosinase (TYR) activity was determined and expressed as a percentage of the control. Data are the means ± SD of triplicate measurements (*P<0.01 vs. control). Nrf2-repressed tyrosinase activity was reversed by PI3K inhibitor treatment. (C) Repression of TYR and TRP1 by Nrf2 was reversed by PI3K inhibitor treatment. (D) The TYR promoter-luciferase reporter adenovirus was co-transduced with the indicated adenoviruses. TYR promoter activity was expressed as a percentage of the control ± SD (*P<0.01 vs. control). Nrf2-repressed tyrosinase promoter activity was reversed by PI3K inhibitor treatment.</p