Research ArticleSteroid Hormones

Transcriptional activation of elephant shark mineralocorticoid receptor by corticosteroids, progesterone, and spironolactone

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Science Signaling  04 Jun 2019:
Vol. 12, Issue 584, eaar2668
DOI: 10.1126/scisignal.aar2668

Figures

  • Fig. 1 Comparison of the domains in elephant shark MR with those in vertebrate MRs.

    MRs from elephant shark (shark), zebrafish, coelacanth, Xenopus, chicken, and human were compared. The functional A/B domain through to the E domain is schematically represented with the numbers of amino acid residues. The percentage of amino acid identity is depicted. GenBank accession numbers are as follows: elephant shark MR (XP_007902220), zebrafish MR (NP_001093873), coelacanth MR (XP_014348128), Xenopus MR (NP_001084074), chicken MR (ACO37437), and human MR (NP_000892).

  • Fig. 2 Structures of steroids that are ligands for the MR.

    Aldo, 11-deoxycorticosterone, and 11-deoxycortisol are physiological mineralocorticoids in terrestrial vertebrates (4, 6, 12, 76). 11-deoxycortisol is both a mineralocorticoid and a glucocorticoid in lamprey (4, 77), whereas functions as a glucocorticoid in ray-finned fish (78). Cortisol is a physiological glucocorticoid in terrestrial vertebrates and ray-finned fish (4, 10, 7981). Corticosterone is a glucocorticoid in rats and mice (4). Aldo, 11-deoxycorticosterone, cortisol, corticosterone, and Prog have a similar high affinity for human MR (3, 8284). Prog, 19norProg, 17OH-Prog, and Spiron are antagonists for human MR (25, 48, 82) and rat MR (85, 86). Prog, 19norProg, and Spiron are agonists for fish MRs (25, 37, 45), whereas 19norProg is a weak agonist for rat MR (54, 55). All of the ligands shown here have a ketone at C3 and, thus, are called 3-ketosteroids.

  • Fig. 3 Transcriptional activation of elephant shark MR by 3-ketosteroids.

    (A and B) Full-length (A) and truncated (B) elephant shark MR were expressed in human embryonic kidney (HEK) 293 cells with an MMTV-luciferase reporter for full-length MR or a GAL4-binding site (GAL4-BS)–luciferase reporter for truncated MR. Cells were treated with dimethyl sulfoxide (DMSO) as a negative control or with the indicated ligands at either 0.1 or 1.0 nM. The y axis indicates the fold activation of luciferase compared to the luciferase activity of cell expressing the control vector that were treated with vehicle (DMSO) alone, which was set at 1. Results are means ± SEM of three independent experiments (36, 73).

  • Fig. 4 Concentration-dependent transcriptional activation of full-length and truncated elephant shark MR by 3-ketosteroids.

    (A to D) Full-length (A and C) and truncated (B and D) elephant shark MR were expressed in HEK 293 cells with an MMTV-luciferase reporter for full-length MR or a GAL4-BS–luciferase reporter for truncated MR. (A and B) Cells were treated with DMSO (vehicle control) or with the indicated concentrations of Aldo, cortisol, corticosterone, 11-deoxycorticosterone, or 11-deoxycortisol. (C and D) Cells were treated with DMSO (vehicle control) or with the indicated concentrations of Aldo, Prog, 17OH-Prog, 19norProg, or Spiron. The y axis indicates the fold activation of the luciferase activity compared to the luciferase activity of cells expressing the control vector and treated with vehicle (DMSO) alone, which was set at 1. Results are means ± SEM of three independent experiments (36, 73).

  • Fig. 5 Concentration-dependent transcriptional activation of full-length and truncated chicken and zebrafish MR by Prog, 19norProg, and Spiron.

    (A to D) HEK 293 cells were cotransfected with an MMTV-luciferase reporter or a GAL4-BS–luciferase reporter and plasmids expressing full-length chicken MR (A), full-length zebrafish MR (B), truncated chicken MR (C), or truncated zebrafish MR (D). Cells were treated with DMSO (vehicle control) or with the indicated concentrations of Aldo, Prog, 17OH-Prog, 19norProg, or Spiron. The y axis indicates the fold activation of luciferase compared to the luciferase activity of cells expressing the control vector and treated with vehicle (DMSO) alone, which was set at 1. Results are means ± SEM of three independent experiments.

  • Fig. 6 Expression of elephant shark and human MRs based on RNA-seq data.

    (A) Relative expression of the elephant shark NR3C2 based on RNA-seq data. Transcript abundances are shown in terms of normalized counts called fragments per kilobase of exon per million fragments mapped (FPKM) (71). FPKM values were estimated by normalizing gene length, followed by normalizing for sequencing depth. (B) Relative expression of the human MR based on RNA-seq data. Transcript abundances are shown in terms of normalized counts called reads per kilobase of transcript per million mapped reads (RPKM) (50).

  • Fig. 7 Alignment of elephant shark MR to Ser810 and Ala773 in helices 3 to 5 in human MR.

    Elephant shark MR and skate MR each contain a methionine corresponding to Ser810 in the human MR and an alanine corresponding to Ala773. Lamprey CR and hagfish CR also contain a corresponding methionine, as well as a cysteine corresponding to Ala773. The residues Ser810 and Ala773 in the human MR are conserved in MRs from coelacanths, terrestrial vertebrates, and ray-finned fish. Amino acid residues that are identical to those in the human MR are denoted by (−).

Tables

  • Table 1 Activation of full-length MR and truncated MRs (LBD) by corticosteroids.

    Analysis of luciferase activity in the presence of corticosteroids as a percentage of the luciferase activity induced by Aldo alone. Statistical analysis was performed as described previously (36). DOC, 11-deoxycorticosterone.

    MRAldoCorticosteroneCortisolDOCDOC
    EC50 (M)EC50 (M)EC50 (M)EC50 (M)EC50 (M)
    Elephant shark full1.1 × 10−101.7 × 10−104.6 × 10−106.3 × 10−111.1 × 10−10
    100%101%114%83%83%
    Elephant shark LBD3.7 × 10−119.9 × 10−111.9 × 10−102.4 × 10−116.8 × 10−11
    100%90%79%81%77%
    Skate LBD*7 × 10−111 × 10−101 × 10−93 × 10−112.2 × 10−8
    Human full2.7 × 10−101.2 × 10−95.5 × 10−94.2 × 10−103.6 × 10−9
    100%119%133%74%42%
    Human LBD2.8 × 10−105.9 × 10−103.2 × 10−91.8 × 10−9
    100%95%74%44%8%§
    Chicken full6.2 × 10−115.1 × 10−112.8 × 10−103.4 × 10−116.7 × 10−10
    100%109%128%110%112%
    Chicken LBD1.3 × 10−101.6 × 10−106.9 × 10−101.7 × 10−104.7 × 10−9
    100%92%75%92%36%
    Alligator full2.8 × 10−103.6 × 10−106.9 × 10−92.3 × 10−102.7 × 10−9
    100%138%176%85%45%
    Alligator LBD3.5 × 10−103.8 × 10−102.3 × 10−95.2 × 10−10
    100%88%68%51%8%§
    Xenopus full4.6 × 10−106.2 × 10−101.1 × 10−87.6 × 10−109.1 × 10−9
    100%105%126%59%31%
    Xenopus LBD1.5 × 10−91.9 × 10−91.2 × 10−8
    100%74%37%10%§6%§
    Zebrafish Full8.2 × 10−113.0 × 10−104.4 × 10−106.3 × 10−114.0 × 10−10
    100%112%123%103%94%
    Zebrafish LBD2.7 × 10−111.5 × 10−103.1 × 10−101.0 × 10−109.1 × 10−10
    100%96%77%99%67%

    *Values obtained from Carroll et al. (49).

    †Values obtained from Katsu et al. (36).

    ‡Curve did not saturate.

    §Relative induction at 1 μM compared to Aldo.

    • Table 2 EC50 values for the activation by Prog and Spiron of full-length and truncated (LBD) constructs of elephant shark, zebrafish, and chicken MRs.

      Analysis of luciferase activity in the presence of Aldo, the indicated progestins, and Spiron and statistical analysis were performed as described previously (36). Relative induction is presented as a percentage of the luciferase activity induced by Aldo alone.

      MRAldoProg17OH-Prog19norProgSpiron
      Elephant shark full1.1 × 10−102.7 × 10−101.4 × 10−94.3 × 10−115.5 × 10−10
      100%43%25%84%45%
      Elephant shark LBD3.7 × 10−114.8 × 10−102.9 × 10−91.8 × 10−114.2 × 10−10
      100%40%26%98%53%
      Zebrafish full8.2 × 10−112.4 × 10−91.8 × 10−89.4 × 10−103.8 × 10−9
      100%77%44%83%54%
      Zebrafish LBD2.7 × 10−119.8 × 10−8*6.4 × 10−8*
      100%122%24%122%73%
      Chicken full6.2 × 10−117.1 × 10−102.9 × 10−86.8 × 10−105.1 × 10−9
      100%62%15%68%30%
      Chicken LBD1.3 × 10−10****
      100%21%29%

      *Curve did not saturate.

      †Relative induction at 1 μM compared to Aldo.

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