title: Differential regulation of the rainbow trout (Oncorhynchus mykiss) MT-A gene by nuclear factor interleukin-6 and activator protein-1.
abstract: BACKGROUND
  Previously we have identified a distal region of the rainbow trout (Oncorhynchus mykiss) metallothionein-A (rtMT-A) enhancer region, being essential for free radical activation of the rtMT-A gene. The distal promoter region included four activator protein 1 (AP1) cis-acting elements and a single nuclear factor interleukin-6 (NF-IL6) element. In the present study we used the rainbow trout hepatoma (RTH-149) cell line to further examine the involvement of NF-IL6 and AP1 in rtMT-A gene expression following exposure to oxidative stress and tumour promotion.
  
  
  RESULTS
  Using enhancer deletion studies we observed strong paraquat (PQ)-induced rtMT-A activation via NF-IL6 while the AP1 cis-elements showed a weak but significant activation. In contrast to mammals the metal responsive elements were not activated by oxidative stress. Electrophoretic mobility shift assay (EMSA) mutation analysis revealed that the two most proximal AP1 elements, AP11,2, exhibited strong binding to the AP1 consensus sequence, while the more distal AP1 elements, AP13,4 were ineffective. Phorbol-12-myristate-13-acetate (PMA), a known tumor promoter, resulted in a robust induction of rtMT-A via the AP1 elements alone. To determine the conservation of regulatory functions we transfected human Hep G2 cells with the rtMT-A enhancer constructs and were able to demonstrate that the cis-elements were functionally conserved. The importance of NF-IL6 in regulation of teleost MT is supported by the conservation of these elements in MT genes from different teleosts. In addition, PMA and PQ injection of rainbow trout resulted in increased hepatic rtMT-A mRNA levels.
  
  
  CONCLUSIONS
  These studies suggest that AP1 primarily is involved in PMA regulation of the rtMT-A gene while NF-IL6 is involved in free radical regulation. Taken together this study demonstrates the functionality of the NF-IL6 and AP-1 elements and suggests an involvement of MT in protection during pathological processes such as inflammation and cancer.
authors.forename: Peter
authors.initials: P
authors.lastname: Kling
authors.forename: Carina
authors.initials: C
authors.lastname: Modig
authors.forename: Huthayfa
authors.initials: H
authors.lastname: Mujahed
authors.forename: Hazem
authors.initials: H
authors.lastname: Khalaf
authors.forename: Jonas
authors.initials: J
authors.lastname: von Hofsten
authors.affiliation: Örebro Life Science Center, School of Science and Technology, Örebro University, Örebro SE-701 82, Sweden. per-erik.olsson@oru.se.
authors.forename: Per-Erik
authors.initials: PE
authors.lastname: Olsson
mesh_terms: D000818:Animals; D001665:Binding Sites; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004742:Enhancer Elements, Genetic; D015972:Gene Expression Regulation, Neoplastic; D056945:Hep G2 Cells; D006801:Humans; D008668:Metallothionein / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D017686:Oncorhynchus mykiss / Q000235:genetics* / Q000378:metabolism; D018384:Oxidative Stress; D010269:Paraquat / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D022782:CCAAT-Enhancer-Binding Protein-beta; D010703:Phorbol Esters; D018808:Transcription Factor AP-1; C119222:phorbol-12-myristate; D008668:Metallothionein; D010269:Paraquat