title: Cross-regulations among NRFs and KEAP1 and effects of their silencing on arsenic-induced antioxidant response and cytotoxicity in human keratinocytes.
abstract: BACKGROUND
  Nuclear factor E2-related factors (NRFs), including NRF2 and NRF1, play critical roles in mediating the cellular adaptive response to oxidative stress. Human exposure to inorganic arsenic, a potent oxidative stressor, causes various dermal disorders, including hyperkeratosis and skin cancer.
  
  
  OBJECTIVE
  We investigated the cross-regulations among NRF2, NRF1, and KEAP1, a cullin-3-adapter protein that allows NRF2 to be ubiquinated and degraded by the proteasome complex, in arsenic-induced antioxidant responses.
  
  
  RESULTS
  In human keratinocyte HaCaT cells, selective knockdown (KD) of NRF2 by lentiviral short hairpin RNAs (shRNAs) significantly reduced the expression of many antioxidant enzymes and sensitized the cells to acute cytotoxicity of inorganic arsenite (iAs(3+)). In contrast, silencing KEAP1 led to a dramatic resistance to iAs(3+)-induced apoptosis. Pretreatment of HaCaT cells with NRF2 activators, such as tert-butylhydroquinone, protects the cells against acute iAs(3+) toxicity in an NRF2-dependent fashion. Consistent with the negative regulatory role of KEAP1 in NRF2 activation, KEAP1-KD cells exhibited enhanced transcriptional activity of NRF2 under nonstressed conditions. However, deficiency in KEAP1 did not facilitate induction of NRF2-target genes by iAs(3+). In addition, NRF2 silencing reduced the expression of KEAP1 at transcription and protein levels but increased the protein expression of NRF1 under the iAs(3+)-exposed condition. In contrast, silencing KEAP1 augmented protein accumulation of NRF2 under basal and iAs3+-exposed conditions, whereas the iAs(3+)-induced protein accumulation of NRF1 was attenuated in KEAP1-KD cells.
  
  
  CONCLUSIONS
  Our studies suggest that NRF2, KEAP1, and NRF1 are coordinately involved in the regulation of the cellular adaptive response to iAs(3+)-induced oxidative stress.
authors.affiliation: School of Forensic Medicine, China Medical University, Shenyang, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhao
authors.forename: Yongyong
authors.initials: Y
authors.lastname: Hou
authors.forename: Qiang
authors.initials: Q
authors.lastname: Zhang
authors.forename: Courtney G
authors.initials: CG
authors.lastname: Woods
authors.forename: Peng
authors.initials: P
authors.lastname: Xue
authors.forename: Jingqi
authors.initials: J
authors.lastname: Fu
authors.forename: Kathy
authors.initials: K
authors.lastname: Yarborough
authors.forename: Dawei
authors.initials: D
authors.lastname: Guan
authors.forename: Melvin E
authors.initials: ME
authors.lastname: Andersen
authors.forename: Jingbo
authors.initials: J
authors.lastname: Pi
mesh_terms: D000975:Antioxidants / Q000378:metabolism*; D017209:Apoptosis; D001151:Arsenic / Q000494:pharmacology*; D018053:Arsenites / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004785:Environmental Pollutants / Q000633:toxicity*; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D000072019:Kelch-Like ECH-Associated Protein 1; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D016086:Lentivirus; D051265:NF-E2-Related Factor 1 / Q000235:genetics / Q000378:metabolism*; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D034741:RNA, Small Interfering / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012871:Skin Diseases / Q000139:chemically induced / Q000503:physiopathology; D017670:Sodium Compounds / Q000494:pharmacology
chemical_list: D000975:Antioxidants; D018053:Arsenites; D004785:Environmental Pollutants; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D051265:NF-E2-Related Factor 1; D051267:NF-E2-Related Factor 2; D034741:RNA, Small Interfering; D017670:Sodium Compounds; C017947:sodium arsenite; D001151:Arsenic