Protective roles of NRF2 signaling pathway in cobalt chloride-induced hypoxic cytotoxicity in human HaCaT keratinocytes

Abstract Hypoxia is a key pathological process involved in many cutaneous diseases. Nuclear factor E2-related factor 2 (NRF2) is a central regulator of antioxidant response element (ARE)-dependent transcription and plays a pivotal role in the cellular adaptive response to oxidative stress. Kelch-like ECH-associated protein 1 (KEAP1) is a cullin-3-adapter protein that represses the activity of NRF2 by mediating its ubiquitination and degradation. In the present study, we examined the role of NRF2 signaling pathway in the cytotoxicity induced by cobalt chloride(CoCl 2 ), a hypoxia-mimicking agent, in human keratinocyte HaCaT cells with stable knockdown of NRF2 (NRF2- KD ) and KEAP1 (KEAP1 -KD ) . Acute CoCl 2 exposure markedly increased the levels of intracellular reactive oxygen species (ROS), and resulted in hypoxic damage and cytotoxicity of HaCaT cells. Stable knockdown of NRF2 dramatically reduced the expression of many antioxidant enzymes and sensitized the cells to acute CoCl 2 -induced oxidative stress and cytotoxicity. In contrast, KEAP1- KD cells observably enhanced the activity of NRF2 and ARE-regulated genes and led to a significant resistance to CoCl 2 -induced cellular damage. In addition, pretreatment of HaCaT cells with tert-butylhydroquinone, a well-known NRF2 activator, protected HaCaT cells from CoCl 2 -induced cellular injury in a NRF2-dependent fashion. Likewise, physical hypoxia-induced cytotoxicity could be significantly ameliorated through NRF2 signaling pathway in HaCaT cells. Together, our results suggest that NRF2 signaling pathway is involved in antioxidant response triggered by CoCl 2 -induced oxidative stress and could protect human keratinocytes against acute CoCl 2 -induced hypoxic cytotoxicity.