Dual regulation of skin sensitizer-induced HMOX1 expression by Bach1 and Nrf2: Comparison to regulation of the AKR1C2-ARE element in the KeratinoSens cell line

• Published in: Toxicology and applied pharmacology Vol. 288; no. 3; pp. 281 - 288
• Main Authors: Emter, Roger, Natsch, Andreas
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-11-2015
• Subjects: 60 APPLIED LIFE SCIENCES; Acrolein - analogs & derivatives; Acrolein - toxicity; Allergens - toxicity; Antioxidant Response Elements - drug effects; Bach1; Basic-Leucine Zipper Transcription Factors - genetics; Basic-Leucine Zipper Transcription Factors - metabolism; CALVES; Cell Line; Cloning, Molecular; CONCENTRATION RATIO; DIAGRAMS; Dose-Response Relationship, Drug; ECR HEATING; Epigenetic Repression; Fanconi Anemia Complementation Group Proteins - genetics; Fanconi Anemia Complementation Group Proteins - metabolism; Gene expression; Gene Knockdown Techniques; HEME; Heme Oxygenase-1 - genetics; Heme Oxygenase-1 - metabolism; HMOX1; Humans; Hydroxysteroid Dehydrogenases - genetics; Hydroxysteroid Dehydrogenases - metabolism; IN VITRO; In vitro testing; Keratinocytes - drug effects; Keratinocytes - metabolism; LUCIFERASE; Luciferases - genetics; Luciferases - metabolism; LYMPH; LYMPH NODES; NF-E2-Related Factor 2 - genetics; NF-E2-Related Factor 2 - metabolism; Nrf2; OXYGENASES; POLYMERASES; Promoter Regions, Genetic; RNA, Small Interfering - genetics; RNA, Small Interfering - metabolism; SENSITIZERS; SKIN; Skin - metabolism; Skin sensitization; Up-Regulation; VALIDATION; Bach1; NFκB; DMSO; Skin sensitization; EC1.5/EC2/EC3; qRT-PCR; ECVAM; AKR1C2; DNCB; siRNA; In vitro testing; Gene expression; AP-1; LLNA; REACH; DMEM; ARE; Nrf2; FCS; Keap1; HMOX1; Imax; HSF
• ISSN: 0041-008X; 1096-0333
• DOI: 10.1016/j.taap.2015.07.027

Heme oxygenase (decycling) 1 (HMOX1) is the most consistently found genetic marker induced by skin sensitizers. HMOX1 is often referred to as typical gene regulated by nuclear factor erythroid 2-related factor 2 (Nrf2), however, it is also regulated by other DNA-binding factors, including BTB and CNC homolog 1 (Bach1). The KeratinoSens™ assay is the first validated in vitro assay for sensitizers that measures gene induction. It is based on luciferase expression regulated by the antioxidant response element (ARE) of the aldoketoreductase 1C2 (AKR1C2) gene. Luciferase upregulation is dependent on Nrf2, while HMOX1 upregulation is only partially Nrf2-dependent. Thus, sensitizer-dependent activation of HMOX1 may integrate multiple signals thereby providing additional information. We constructed reporter cell lines containing the full HMOX1 regulatory region or the HMOX1-ARE sequence and compared them with the construct containing the AKR1C2-ARE sequence. Induction of the AKR1C2-ARE depends on Nrf2, but not on the repressor Bach1. Results obtained with HMOX1-ARE and the full HMOX1 promoter indicate that, within the HMOX1 promoter, the HMOX1-ARE is sufficient to explain the induction by sensitizers and that (i) inhibiting Bach1 leads to strong basal expression, (ii) fold-induction by sensitizers above this level is reduced in the absence of Bach1 and (iii) these constructs are less dependent on Nrf2 as compared to the AKR1C2-ARE. Nevertheless, congruent dose response curves for luciferase activity were obtained with all constructs. Thus, while sensitizer-induced HMOX1 activation is dependent on Nrf2 and Bach1, all constructs give identical information for the in vitro prediction of the sensitization potential.
•HMOX1 is a key genetic marker up-regulated by skin sensitizers.•HMOX1-, but not AKR1C2-upregulation, is dependent on both Nrf2 and Bach1.•AKR1C2 and HMOX1-dependent reporter constructs yield congruent dose response curves.•Combining both constructs offers no advantage over either construct used alone.