Long Isoforms of NRF1 Contribute to Arsenic-Induced Antioxidant Response in Human Keratinocytes

Abstract

Background

Human exposure to inorganic arsenic (iAs), a potent oxidative stressor, causes various dermal disorders, including hyperkeratosis and skin cancer. Nuclear factor–erythroid 2–related factor 1 (NRF1, also called NFE2L1) plays a critical role in regulating the expression of many antioxidant response element (ARE)-dependent genes.

Objectives

We investigated the role of NRF1 in arsenic-induced antioxidant response and cytotoxicity in human keratinocytes.

Results

In cultured human keratinocyte HaCaT cells, inorganic arsenite (iAs ³⁺) enhanced the protein accumulation of long isoforms (120–140 kDa) of NRF1 in a dose- and time-dependent fashion. These isoforms accumulated mainly in the nuclei of HaCaT cells. Selective deficiency of NRF1 by lentiviral short-hairpin RNAs in HaCaT cells [ NRF1-knockdown (KD)] led to decreased expression of γ-glutamate cysteine ligase catalytic subunit (GCLC) and regulatory subunit (GCLM) and a reduced level of intracellular glutathione. In response to acute iAs ³⁺ exposure, induction of some ARE-dependent genes, including NAD(P)H:quinone oxidoreductase 1 ( NQO1), GCLC, and GCLM, was significantly attenuated in NRF1-KD cells. However, the iAs ³-induced expression of heme oxygenase 1 ( HMOX-1) was unaltered by silencing NRF1, suggesting that HMOX-1 is not regulated by NRF1. In addition, the lack of NRF1 in HaCaT cells did not disturb iAs ³⁺-induced NRF2 accumulation but noticeably decreased Kelch-like ECH-associated protein 1 (KEAP1) levels under basal and iAs ³⁺-exposed conditions, suggesting a potential interaction between NRF1 and KEAP1. Consistent with the critical role of NRF1 in the transcriptional regulation of some ARE-bearing genes, knockdown of NRF1 significantly increased iAs ³⁺-induced cytotoxicity and apoptosis.

Conclusions

Here, we demonstrate for the first time that long isoforms of NRF1 contribute to arsenic-induced antioxidant response in human keratinocytes and protect the cells from acute arsenic cytotoxicity.