Activation and inhibition of transient receptor potential TRPM3-induced gene transcription

Abstract

Background and purpose: Transient receptor potential-3 (TRPM3) channels function as Ca2+ permeable cation channels. While the natural ligands for these channels are still unknown, several compounds have been described that either activate or inhibit TRPM3 channel activity. experimental approach: We assessed TRPM3-mediated gene transcription, which relies on the induction of intracellular signalling to the nucleus following activation of TRPM3 channels. Activator protein-1 (AP-1) and Egr-1-responsive reporter genes were integrated into the chromatin of the cells. This strategy enabled us to analyse gene transcription of the AP-1 and Egr-1-responsive reporter genes that were packed into an ordered chromatin structure.

Key results: The neurosteroid pregnenolone sulfate strikingly up-regulated AP-1 and Egr-1 transcriptional activity, while nifedipine and D-erythro-sphingosine, also putative activators of TRPM3 channels, exhibited either no or TRPM3-independent effects on gene transcription. In addition, pregnenolone sulfate robustly enhanced the transcriptional activation potential of the ternary complex factor Elk-1. Pregnenolone sulfate-induced activation of gene transcription was blocked by treatment with mefenamic acid and, to a lesser extent, by the polyphenol naringenin. In contrast, progesterone, pregnenolone and rosiglitazone reduced AP-1 activity in the cells, but had no inhibitory effect on Egr-1 activity in pregnenolone sulfate-stimulated cells.

Conclusion and implications: Pregnenolone sulfate is a powerful activator of TRPM3-mediated gene transcription, while transcription is completely inhibited by mefenamic acid in cells expressing activated TRPM3 channels. Both compounds are valuable tools for further investigating the biological functions of TRPM3 channels.

Figure 1

Regulation of AP-1 and Egr-1 activity in HEK293 cells. (A) Tetracycline-regulated expression of TRPM3 in HEK293/TRPM3 cells. The cells were cultured for 24 h in medium containing 0.05% serum. Stimulation with tetracycline (1 μg mL⁻¹) was performed with medium containing 0.05% serum. RNA was prepared at the indicated time points and analysed by PCR using TRPM3 and GAPDH-specific primers. (B) Schematic representation of the integrated proviruses encoding either an AP-1-responsive collagenase promoter/luciferase reporter gene (Coll.luc) or an Egr-1-responsive reporter gene (EBS2⁴.luc). The location and sequence of the phorbol TRE within the collagenase promoter is shown, as well as the sequence of the Egr-1 binding site within the regulatory region of the EBS2⁴.luc reporter gene. The location of the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and the HIV flap element are shown. (C) HEK293 cells containing a tetracycline-inducible TRPM3 expression unit were infected with a recombinant lentivirus expressing an Rαq-coupled designer receptor. In addition, the cells were infected with lentiviruses encoding either the collagenase promoter/luciferase reporter gene (Coll.luc), or the Egr-1-sensitive reporter gene EBS2⁴.luc. The cells were cultured for 24 h in medium containing 0.05% serum in the absence of tetracycline. Stimulation with CNO (1 μM) was performed with medium containing 0.05% serum. (D) HEK293 cells containing a tetracycline-inducible TRPM3 expression unit were infected with lentiviruses encoding either the collagenase promoter/luciferase reporter gene (Coll.luc), or the Egr-1-sensitive reporter gene EBS2⁴.luc. The infected cells were stimulated with TPA (10 ng mL⁻¹) for 24 h. Cell extracts were prepared and analysed for luciferase activities. Luciferase activity was normalized to the protein concentration. Data shown are mean ± SD, *, P < 0.05; **P < 0.01; ***, P < 0.001, significantly different as indicated; n = 4.

Figure 2

Up-regulation of AP-1 and Egr-1 activities and Egr-1, c-Jun and c-Fos expression in pregnenolone sulfate-stimulated HEK293 cells expressing TRPM3 channels. (A) Pregnenolone sulfate. (B, D) HEK293 cells containing a tetracycline-inducible TRPM3 transcription unit were infected with recombinant lentiviruses encoding either the collagenase promoter/luciferase reporter gene or the Egr-1-responsive EBS2⁴.luc reporter gene. The cells were serum-starved for 24 h in the presence of tetracycline (1 μg mL⁻¹) and then stimulated with pregnenolone sulfate (PregS, 20 μM) for 24 h. (C, E) HEK293 cells expressing TRPM3 channels were stimulated with pregnenolone sulfate (20 μM) in medium containing 0.05% serum. Nuclear extracts were prepared and subjected to Western blot analysis using antibodies directed against either c-Fos, c-Jun (C), or Egr-1 (E). The antibody directed against HDAC1 was used as a loading control. (F) HEK293 cells containing a tetracycline-inducible TRPM3 transcription unit were infected with recombinant lentiviruses encoding either the collagenase promoter/luciferase reporter gene or the Egr-1-responsive EBS2⁴.luc reporter gene. The cells were serum-starved for 24 h in the absence of tetracycline and then stimulated with pregnenolone sulfate (20 μM) for 24 h. (G) Schematic representation of the integrated provirus encoding a Nrf2-responsive luciferase reporter gene (StRE.luc) The sequence of the stress–response element is in bold. The regulatory region contains three Nrf2 binding sites, derived from the haem oxygenase gene, upstream of a minimal haem oxygenase promoter. (H) HEK293 cells containing a tetracycline-inducible TRPM3 expression unit were infected with a recombinant lentivirus encoding the Nrf2-regulated reporter gene StRE.luc. The cells were serum-starved for 24 h in the presence of tetracycline (1 μg mL⁻¹) and then stimulated with pregnenolone sulfate (20 μM) for 24 h. Cell extracts were prepared and analysed for luciferase activities. Luciferase activity was normalized to the protein concentration. Data shown are mean ± SD, *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant, as indicated; n = 4.

Figure 3

Nifedipine and d-erythro-sphingosine as activators of TRPM3-mediated gene transcription. (A, E) Nifedipine (Nif) and D-erythro-sphingosine (SPH). (B, C, F, G) HEK293 cells containing a tetracycline-inducible TRPM3 transcription unit were infected with recombinant lentiviruses encoding either the collagenase promoter/luciferase reporter gene (B, F) or the EBS2⁴.luc reporter gene (C, G). The cells were serum-starved for 24 h in the presence of tetracycline (1 μg mL⁻¹) and then stimulated with either nifedipine (20 μM) (B, C), or D-erythro-sphingosine (20 μM) (F, G) for 24 h. (D, H) HEK293 cells containing a tetracycline-inducible TRPM3 transcription unit were infected with recombinant lentiviruses encoding either the collagenase promoter/luciferase reporter gene or the EBS2⁴.luc reporter gene. The cells were serum-starved for 24 h and then stimulated with either nifedipine (20 μM) (D), or D-erythro-sphingosine (20 μM) (H) for 24 h. Cell extracts were prepared and analysed for luciferase activities. Luciferase activity was normalized to the protein concentration. Data shown are mean ± SD, *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant, as indicated; n = 4.

Figure 4

Pregnenolone and progesterone inhibit pregnenolone sulfate-induced up-regulation of AP-1 activity. (A, D) Pregnenolone, progesterone. (B, E) HEK293 cells containing a tetracycline-inducible TRPM3 transcription unit were infected with recombinant lentiviruses encoding either the collagenase promoter/luciferase reporter gene or the EBS2⁴.luc reporter gene. The cells were serum-starved for 24 h in the presence of tetracycline (1 μg mL⁻¹) and then stimulated with pregnenolone sulfate (PregS, 20 μM) in the presence or absence of either pregnenolone (Preg, 10 μM) (B) or progesterone (Prog, 10 μM; E) for 24 h. Cell extracts were prepared and analysed for luciferase activities. Luciferase activity was normalized to the protein concentration. (C, F) The experiments were repeated in the absence of tetracycline. Data shown are mean ± SD, *, P < 0.05; n.s., not significant, as indicated; n = 4.

Figure 5

Mefenamic acid inhibits pregnenolone sulfate (PregS)-induced up-regulation of AP-1 and Egr-1 activity. (A) Mefenamic acid (Mef). (B, C) HEK293 cells containing a tetracycline-inducible TRPM3 transcription unit were infected with recombinant lentiviruses encoding either the collagenase promoter/luciferase reporter gene or the EBS2⁴.luc reporter gene. The cells were serum-starved for 24 h in the presence of tetracycline (1 μg mL⁻¹) and then stimulated with pregnenolone sulfate (20 μM) in the presence or absence of mefenamic acid (30 μM) for 24 h. Cell extracts were prepared and analysed for luciferase activities. Luciferase activity was normalized to the protein concentration. (D) The experiments were repeated in the absence of TRPM3 expression. Data shown are mean ± SD, ***, P < 0.001 significantly different as indicated; n = 4.

Figure 6

Effect of rosiglitazone (Rosi) on AP-1 and Egr-1 activities following activation of TRPM3. (A) Rosiglitazone (B, C) HEK293 cells containing a tetracycline-inducible TRPM3 transcription unit were infected with recombinant lentiviruses encoding either the collagenase promoter/luciferase reporter gene or the EBS2⁴.luc reporter gene. The cells were serum-starved for 24 h in the presence of tetracycline (1 μg mL⁻¹) and then stimulated with pregnenolone sulfate (PregS, 20 μM) in the presence or absence of rosiglitazone (10 μM) for 24 h. Cell extracts were prepared and analysed for luciferase activities. Luciferase activity was normalized to the protein concentration. (D) The experiments were repeated in the absence of TRPM3 expression. Data shown are mean ± SD, *, P < 0.05; n.s., not significant, as indicated; n = 4.

Figure 7

Regulation of AP-1 and Egr-1 activities by naringenin (Nar) and quercetin (Quer) following activation of TRPM3. (A) Naringenin, quercetin (B, C) HEK293 cells containing a tetracycline-inducible TRPM3 transcription unit were infected with recombinant lentiviruses encoding either the collagenase promoter/luciferase reporter gene or the EBS2⁴.luc reporter gene. The cells were serum-starved for 24 h in the presence of tetracycline (1 μg mL⁻¹) and then stimulated with pregnenolone sulfate (PregS, 20 μM) in the presence or absence of either naringenin (10 μM) or quercetin (10 μM) for 24 h. (D) The experiments were repeated in the absence of TRPM3 expression. Cell extracts were prepared and analysed for luciferase activities. Luciferase activity was normalized to the protein concentration. Data shown are mean ± SD, *, P < 0.05; **, P < 0.01, significantly different as indicated; n = 4.

Figure 8

Up-regulation of the transcriptional activation potential of Elk-1 in pregnenolone sulfate (PregS)-stimulated HEK293 cells expressing TRPM3 channels. (A) Schematic representation of the modular structure of Elk-1, GAL4-Elk-1, Sp1, and GAL4-Sp1. (B) Schematic representation of the GAL4-responsive transcription unit. (C, D) HEK293 cells containing a tetracycline regulated transcription unit to express TRPM3 channels were infected with a lentivirus encoding a GAL4-responsive luciferase reporter gene. In addition, cells were infected with a lentivirus encoding either GAL4-Elk-1 or GAL4-Sp1. The infected cells were stimulated with tetracycline for 24h to induce TRPM3 expression. Then, cells were treated with either pregnenolone sulfate (20 μM) (C) or D-erythro-sphingosine (SPH, 20 μM) (D). (E, F) The experiment was repeated in the presence of either mefenamic acid (E) or rosiglitazone (F) as indicated. Cell extracts were prepared and analysed for luciferase activities. Luciferase activity was normalized to the protein concentration. Data shown are mean ± SD, *, P < 0.05; **, P < 0.01; n.s., not significant, as indicated; n = 4.

Figure 9

A sigma receptor antagonist (BD1047) interferes with TRPM3-induced gene transcription. (A) Structure of BD1047. (B, C) HEK293 cells containing a tetracycline-inducible TRPM3 transcription unit were infected with recombinant lentiviruses encoding either the collagenase promoter/luciferase reporter gene (B) or the Egr-1-responsive EBS24.luc reporter gene (C). The cells were serum-starved for 24 h in the presence of tetracycline (1 μg mL⁻¹) and then stimulated with pregnenolone sulfate (PregS, 20 μM) in the presence or absence of BD1047 (100 μM) for 24 h. Cell extracts were prepared and analysed for luciferase activities. Luciferase activity was normalized to the protein concentration. Data shown are mean ± SD, *, P < 0.05; ***, P < 0.001, significantly different as indicated; n = 4.