Distinctive Roles Of Nuclear Factor-E2 Related Factors In Arsenic-induced Antioxidant Response And Cytotoxicity In Human Keratinocytes

IntroductionArsenic, a potent oxidative stressor, is a natural element that ubiquitously presents in the environment in both organic and inorganic forms. Human exposure to the generally more toxic inorganic arsenic is through oral consumption of water containing elevated levels of arsenics in environmental settings and medical arsenic use. Arsenic is also a common toxicant in forensic practice. To investigate the arsenic-induced injuries will also provide evidence to judge its poision in forensic medicine though the slight sympotoms in acute poisioning and the low concentration in chronic piosioning exist in forensic practise. Chronic human exposure to inorganic arsenic causes various human diseases, which includes internal cancers, peripheral vascular disease, ischemic heart diseases, diabetes mellitus, hypertension and skin lesions. Arsenic-induced dermal disorders includes benign changes, such as dys-pigmentation and hyperkeratosis, and malignant diseases, including Bowen's disease and skin cancer. Although arsenic is a confirmed human skin toxicant, the underlying mechanism is still unclear. It has been shown that arsenic induces cellular oxidative stress and oxidative DNA injury which suggests that oxidative stress occurs in response to arsenic exposure and may be one important factor in dermal arsenic toxicity. Nuclear factor erythriod-derived factor 2-related factors (NRFs), belong to the cap'n' collar (CNC) subfamily of basic-region leucine zipper (bZIP) tranSCRiption factors, which include NRF1 (NFE2L1/LCRF1/TCF11), NRF2 (NFE2L2) and NRF3 (NFE2F3), NRFs have been shown to play critical roles in mediating cellular adaptive response to oxidative stress. The aim of the study is to investigate the role of NRFs in arsenic-induced antioxidant response and cytotoxicity in human keratinocytes and to illustrate the mechanism of arsenic-induced skin lesions.Materials and MethodsIn cultured human keratinocyte HaCaT cells, selective deficiency of NRFs and KEAP1 was made by lentiviral sh-RNAs. The gene and protein expression of NRFs, KEAP1 and parts of ARE target genes were detected by qReal time RT-PCR and Western blotting respectively. The cytotoxicity of arsenite in selective knockdown cell was measured by MTT and flow cytometry. ARE activity induced by arsenite was determined by ARE luciferase activity assay.ResultsThe epidemiologic typical skin injuries due to arsenic exposure provide evidence to identify arsenic poisioning in forensic practice. Our data showed that selective lack of NRF2 significantly increased acute cytotoxicity caused by arsenite. In contrast, knockdown of KEAP1, a negative regulator of NRF2 transcriptional activity, led to a dramatic resistance to arsenite toxicity, Deficiency of NRF1 mildly decreased the sensitivity to arsenite, whereas silencing of NRF3 showed no difference between Scramble and NRF3-knockdown cells. To investigate the mechanisms involved, expression of this transcription factor and a battery of oxidative stress-responsive genes, including NAD(P)H:quinone oxidoreductase 1 (NQO1),γ-glutamate cysteine ligase catalytic and regulatory subunits (GCLC and GCLM), heme oxygenase 1 (HMOX-1) and sulfiredoxin (SRX), was determined in response to arsenite exposure. In cultured HaCaT cells, unlike other well-known NRF2 activators, such as tert-butyl hydroxyquinone and sulforaphane, arsenite also augmented protein accumulation of NRF1 in a dose-and time-dependent fashion. The NRF1 protein was located and glycosylated in endoplasmic reticulum. The process of glycosylation affected the ARE activity caused by arsenite. NRF1 induced by arsenite was accumulated in nucleus of HaCaT cells and the slower migration and faster shift bands of NRF1 protein caused by ER stressor were also translocated into nucleus. Additionally, arsenite-induced expression of NQO1, SRX, GCLC and GCLM is regulated by NRF1 whereas induction of HMOX-1 is independent of NRF1. Arsenite exposure enhanced the expression of NRF2, even low dose of arsenite. Moreover, the induction of oxidative stress response gene which included NQO1, GCLC, GCLM, HMOX-1 and SRX, was highly dependent on NRF2, Consistent with the function of KEAP1 in regulating NRF2 activity, knockdown of KEAP1 significantly enhanced basal expression and activity of NRF2. However, deficiency of this molecule did not facilitate the induction of NRF2-target genes by arsenite which confirmed that KEAP1 was a main regulator of NRF2. Furthermore, the expression of HMOX-1 was decreased in KEAP1-KD cells which suggested a complex mechanism of HMOX-1 regulation. Though arsenite time-and dose-dependently reduced the expression of NRF3. Lack of NRF3 had no obvious effect on the toxic sensitivity caused by arsenite and the expression of ARE-regulated genes. In these studies, the crosstalks between NRFs and KEAP1 were also investigated. NRF1 and NRF3 compensate the lack of NRF2, while silence of NRF1 in HaCaT cells did not disturb arsenite induced NRF2 accumulation. Lack of NRF1 decreased KEAP1 protein levels under basal and arsenite challenged conditions which suggest the possibility of binding between NRF1 and KEAP1. Silencing of KEAP1 induces the accumulation of NRF2 while decreases the level of NRF1, suggesting the competitive binding with ARE site. Lack of NRF3 had no effect on NRF1, NRF2 and KEAP1. Only arsenite enhanced the protein accumulation of NRF1 in the chemical compounds we used in the experiments.Conclusions1. Arsenite exposure enhances the protein accumulation of NRF1 and NRF2 in a time-and dose-dependently fashion.2. NRF1-and NRF2-dependent antioxidant response play protective roles in arsenite induced cytotoxicity.3. Both NRF1 and NRF2 regulate the expression of GCLC, GCLM, SRX and NQO1, while HMOX-1 is highly dependent on NRF2.4. Lack of KEAP1 significantly enhancs basal expression of NRF2 and its dependent genes. However, deficiency of this molecule does not facilitate the induction of NRF2-target genes by arsenite.5. There are crosstalks among NRFs and KEAP1:(1) NRF1 and NRF3 compensate the deficiency of NRF2, whereas NRF2 does not compensate the lack of NRF1; (2) Lack of NRF2 or NRF1 leads to reduced expression of KEAP1; (3) Knockdown of KEAP1 increases the protein accumulation of NRF2. (4) Lack of NRF3 has no effect on the expression of NRF1, NRF2 and KEAP1. These findings provide evidence of a primary molecular response in a target cell of arsenic exposure and, as such, may help further understanding of the mechanisms by which arsenic causes skin disorders. (5) The typical skin injuries due to arsenic exposure provide evidence to identify arsenic poisioning in forensic practice. In addition, NRF1 protein accumulation in human skin may be a valuable specific marker for idnetifying arsenic poisioning.