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Introduction
Reactive oxygen species (ROS) [e.g., superoxide anions, hydroxyl radicals, and hydrogen peroxide (H2O2)] can harm cellular proteins, lipids and DNA (1,2). Long-term effects of oxidative damage to the skin include premature aging, cancer and inflammation (3–5).
Heme oxygenase-1 (HO-1, one of three heme oxygenase isoforms) is an extensively studied phase II enzyme that is essential for the rate-limiting step of heme catabolism. Heme catabolism leads to the formation of carbon monoxide, ferrous iron (Fe2+) and biliverdin, which is converted into bilirubin by biliverdin reductase (6). The end products of heme catabolism exert antioxidant effects by neutralizing intracellular ROS (7,8). Thus, HO-1 demonstrates potent cytoprotective properties against oxidative damage (9–11), and the induction of its expression increases cellular resistance to oxidative stress.
HO-1 expression is primarily regulated at the transcriptional level and is induced by the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) (12,13). Under basal conditions, Nrf2 is constitutively docked to Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm, culminating in the ubiquitination and proteosomal degradation of Nrf2 (14). However, Keap1 is oxidized under conditions of electrophilic stress, which blocks Nrf2 degradation and allows Nrf2 phosphorylation, nuclear translocation, and association with the antioxidant response element (ARE) within the HO-1 gene promoter region. The net result of ARE binding by Nrf2 is the activation of multiple antioxidant genes, including HO-1 (15).
Similar to Keap1, extracellular signal-regulated kinase (ERK) and protein kinase B (PKB, Akt) are involved in the transduction of various signals from the cell surface to the nucleus. Both of their signaling pathways are associated with the modulation of ARE-driven gene expression via Nrf2 activation (16,17).
The polyphenolic compound 7,8-dihydroxyflavone (DHF) is a member of the flavonoid family with antioxidant properties (18–21).
In a previous study, we reported that DHF protected cells against oxidative stress-induced genotoxicity by scavenging intracellular ROS and enhancing Akt activity (22).
In the present study, we investigated the cytoprotective mechanisms of DHF against oxidative stress-induced cell damage in human keratinocytes with respect to its stimulatory effects on HO-1 expression and activity. We also investigated the molecular mechanisms involved in DHF-mediated cytoprotection and HO-1 induction, with a particular focus on ERK- and Akt-Nrf2 signaling cascades.
Materials and methods
Materials
[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] bromide (MTT) and...