Absence of 11-keto reduction of cortisone and 11-ketotestosterone in the model organism zebrafish

  1. Alex Odermatt1
  1. 1Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland
  2. 2AstraZeneca AG, Zug, Switzerland
  3. 3Karlsruhe Institute of Technology (KIT), Institute of Toxicology and Genetics, Eggenstein-Leopoldshafen, Germany
  4. 4Nestlé Institute of Health Sciences SA, EPFL Innovation Park, Lausanne, Switzerland
  5. 5Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Experimental Genetics, Genome Analysis Center, Neuherberg, Germany
  6. 6Biozentrum, University of Basel, Basel, Switzerland
  1. Correspondence should be addressed to A Odermatt; Email: alex.odermatt{at}unibas.ch
  1. Figure 1

    Glucocorticoid and androgen metabolism pathways that involve the function of 11β-HSD1, 11β-HSD2 and 17β-HSD3. The main glucocorticoid cortisol is produced from 11-deoxycortisol by CYP11B1. Cortisol can be oxidized to the inactive steroid cortisone by 11β-HSD2, and cortisone can be re-activated through the reductive activity of 11β-HSD1. Upon synthesis, the androgen ∆4-androstene-3,17-dione (A4) can be converted to testosterone by 17β-HSD3. The reverse reaction is catalyzed by 17β-HSD2. A fraction of A4 and testosterone can be 11β-hydroxylated by CYP11B1. These metabolites can be further converted by 11β-HSD2 to their 11-keto forms 11KA4 and 11KT. The opposite reaction is catalyzed by 11β-HSD1. 11KA4 is converted to 11KT by 17β-HSD3. It remained controversial whether the same enzyme can accept 11OHA4 as a substrate to produce 11OHT. The dotted rectangles depict pathways involving 11β-HSD1 or 17β-HSD2 activities, which seem to be absent from zebrafish. Production of DHT, 11OHDHT and 11KDHT is catalyzed by 5α-reductase activity.

  2. Figure 2

    Verification of protein expression after transfection of different constructs. ΗΕΚ-293 cells were transfected with pcDNA3, Hs11β-HSD3 (coding for a protein of 286 residues), Dr11β-HSD3b (336 residues) or Dr11β-HSD3a (287 residues) (A), and with pcDNA3, Hs11β-HSD2 (405 residues) or Dr11β-HSD2 (414 residues) (B). All expressed proteins contain a C-terminal FLAG epitope. Western blots of cell extracts were analyzed using the anti-FLAG antibody M2. In (B) due to the high intensity of the signal produced by Hs11β-HSD2, two different exposure times (2 s and 2 min) are shown for the same blot. Membranes were reprobed for PPIA as a loading control.

  3. Figure 3

    GRIZLY assay for the evaluation of GRE activation by glucocorticoids in zebrafish larvae. GRE:luc larvae (n = 48) were treated with the glucocorticoids indicated, and the bioluminescence response was monitored over time. The relative reporter activity, based on luminescence measurements, is shown over time (left) or after 24 h (right). The steroids used to treat larvae were cortisol or cortisone (A), and prednisone or dexamethasone (B) at the indicated final concentrations.

  4. Figure 4

    Effect of various glucocorticoids on GR target gene expression. 3 dpf zebrafish larvae were treated for 24 h with the steroids indicated, and the expression of three GR target genes was measured with qPCR from cDNA generated from 15 larvae per sample. The genes tested were GILZ, FKBP5 and 11β-HSD2. The relative quantification and fold change calculation were performed in relation to the RPL13a house keeping gene expression using the ddCt method. The results represent mean ± s.d. from three independent experiments.

  5. Figure 5

    Androgen metabolism by 11β-HSD2 and 17β-HSD3. (A) HEK-293 cells were transfected with plasmids for Hs11β-HSD2 or Dr11β-HSD2. At 24 h after transfection, cells were incubated with 11OHT or 11OHA4 for 1 h and formation of 11KT and 11KA4, respectively, was measured by LC–MS/MS. (B) HEK-293 cells were transfected with a plasmid for Hs17β-HSD3, and ZF4 cells with a plasmid for Dr17β-HSD3. At 24 h after transfection, cells were incubated for 4 h with A4, 11OHA4 and 11KA4, followed by the determination of the generation of testosterone, 11OHT and 11KT, respectively, by LC–MS/MS. Results are presented as mean of three independent experiments, and error bars represent s.d.

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