Liver X receptor regulation of thyrotropin-releasing hormone transcription in mouse hypothalamus is dependent on thyroid status.

Reversing the escalating rate of obesity requires increased knowledge of the molecular mechanisms controlling energy balance. Liver X receptors (LXRs) and thyroid hormone receptors (TRs) are key physiological regulators of energetic metabolism. Analysing interactions between these receptors in the periphery has led to a better understanding of the mechanisms involved in metabolic diseases. However, no data is available on such interactions in the brain. We tested the hypothesis that hypothalamic LXR/TR interactions could co-regulate signalling pathways involved in the central regulation of metabolism. Using in vivo gene transfer we show that LXR activation by its synthetic agonist GW3965 represses the transcriptional activity of two key metabolic genes, Thyrotropin-releasing hormone (Trh) and Melanocortin receptor type 4 (Mc4r) in the hypothalamus of euthyroid mice. Interestingly, this repression did not occur in hypothyroid mice but was restored in the case of Trh by thyroid hormone (TH) treatment, highlighting the role of the triiodothyronine (T3) and TRs in this dialogue. Using shLXR to knock-down LXRs in vivo in euthyroid newborn mice, not only abrogated Trh repression but actually increased Trh transcription, revealing a potential inhibitory effect of LXR on the Hypothalamic-Pituitary-Thyroid axis. In vivo chromatin immunoprecipitation (ChIP) revealed LXR to be present on the Trh promoter region in the presence of T3 and that Retinoid X Receptor (RXR), a heterodimerization partner for both TR and LXR, was never recruited simultaneously with LXR. Interactions between the TR and LXR pathways were confirmed by qPCR experiments. T3 treatment of newborn mice induced hypothalamic expression of certain key LXR target genes implicated in metabolism and inflammation. Taken together the results indicate that the crosstalk between LXR and TR signalling in the hypothalamus centres on metabolic and inflammatory pathways

T₄ treatment of hypothyroid dams restores the GW3965-dependent repression of <i>Trh</i> promoter of newborn offspring.

<p><b>A.</b> One-day-old hypothyroid pups, from dams treated with T₄ at 12 µg/ml in drinking water 24 h before giving birth, were transfected in the hypothalamus with a solution of PEI-complexed TRH-f.luc (0.2 µg/pup)/MC4R-r.luc (0.8 µg/pup) with GW3965 at 10⁻⁶ M in the transfection mix. Firefly and Renilla luciferase activities were measured 24 h later. A representative experiment is shown. n = 10 per group. Non-parametric Mann-Whitney test was used to assess statistical significance. *, p<0.05. <b>B.</b> One-day-old hypothyroid pups, from dams treated with T₄ at 12 µg/ml in drinking water 48 h before giving birth, were co-transfected in the hypothalamus with a solution of PEI-complexed TRH-f.luc (0.2 µg/pup) MC4R-r.luc (0.8 µg/pup) with GW3965 at 10⁻⁶ M in the transfection mix. Firefly and Renilla luciferase activities were measured 24 h later. A representative experiment is shown. n = 10 per group. Non-parametric permutation test was used to assess statistical significance. **, p<0.01.</p

GW3965 represses hypothalamic <i>Trh</i> and <i>Mc4r</i> transcriptional activity only in euthyroid newborn mice.

<p><b>A</b> and <b>B</b>. Transcriptional repression of <i>Trh</i> (A) and <i>Mc4r</i> (B) promoters via LXR activation by subcutaneous injection of GW3965 (12.5 or 25 mg/Kg) in euthyroid newborn mice. <b>C</b> and <b>D</b>. Lack of effect of subcutaneous injection of GW3965 (12.5 or 25 mg/Kg) on the transcription from <i>Trh</i> (C) and <i>Mc4r</i> (D) promoters in hypothyroid newborn mice. <b>A–B/C–D</b>. <i>In vivo</i> gene reporter assays: one-day-old euthyroid (A and B) or hypothyroid (C and D) pups were subcutaneously injected by GW3965 at 12.5 or 25 mg/Kg of body weight, and co-transfected 24 h after in the hypothalamic region of the brain (Paraventricular nuclei, PVN) with 4 µl of a solution of PEI–complexed TRH-f.luc (0.2 µg/pup)/MC4R-r.luc (0.8 µg/pup). Firefly and Renilla luciferase activities were measured 24 h later. Representative experiments are shown. n = 10 per group. Non-parametric permutation test was used to assess statistical significance. *, p<0.05, **, p<0.01. <b>E</b> and <b>F</b>. Transcriptional repression of <i>Trh</i> (E) and <i>Mc4r</i> (F) promoters via LXR activation by ICV injection of GW3965 (10⁻⁶ or 10⁻⁷ M) in euthyroid newborn mice. <b>G</b> and <b>H</b>. Lack of effect of I.C.V injection of GW3965 (10⁻⁶ or 10⁻⁷ M) on the <i>Trh</i> (G) and <i>Mc4r</i> (H) transcriptions in hypothyroid newborn mice. <b>E–F/G–H.</b> One-day-old euthyroid (E and F) or hypothyroid (G and H) pups were co-transfected in the hypothalamic region of the brain (PVN) with a solution of PEI–complexed TRH-f.luc (0.2 µg/pup)/MC4R-r.luc (0.8 µg/pup) with GW3965 at 10⁻⁷ or 10⁻⁶ M in the transfection mix. Firefly and Renilla Luciferase activities were measured 24 h later. n = 10 per group, pools of three independent experiments are represented. Non-parametric permutation test was used to assess statistical significance. *, p<0.05, **, p<0.01; ***, p<0.001.</p

Overexpression of TRα1, but not TRα2, in the hypothalamus modulates circulating T₄ levels.

<p>pSG5-TRα1 (TRα1) or pSG5-TRα2 (TRα2) were transfected (100 ng/pup) into the hypothalamus of hypothyroid 2-day old mice. Serum was collected at the ages shown (3, 5 or 7 days old) and pooled (four individual samples per pool). At 5 days post-transfection (7 days old mice), T₄ circulating levels are significantly decreased when TRα1 is overexpressed and not modified by TRα2 overexpression. Means ± SEM of pooled samples are given, n≥4 for each point. **, p<0.01.</p

Subcutaneous T₃ treatment of hypothyroid newborn mice restores the GW3965-dependent repression of the <i>Trh</i> promoter.

<p>One-day-old hypothyroid pups were subcutaneously injected by T₃ (2.5 µg/g bw) and co-transfected 24 h after in the hypothalamic region of the brain (Paraventricular nuclei, PVN) with a solution of PEI-complexed TRH-f.luc (0.2 µg/pup)/MC4R-r.luc (0.8 µg/pup) with GW3965 at 10⁻⁶ M in the transfection mix. Firefly and Renilla luciferase activities were measured 24 h later. n = 10 pups per group. A representative experiment is shown. Non-parametric permutation test was used to assess statistical significance, **, p<0.01.</p

LXR and RXR are not detected simultaneously on <i>Trh</i> promoter.

<p>PCR quantification of Chromatin Immunoprecipitation (ChIP) assays carried out on hypothalami from hypothyroid newborn mice treated or not with T₃ (2.5 µg/g bw) 20 h before sacrifice. Samples were immunoprecipitated with LXR- or RXR- specific antibodies and amplified with TRH site 4 primer or its irrelevant control primer (TRH -2000). For negative controls, samples were processed through immunoprecipitation without antibody (Ab⁻). Results represent the occupancy of LXR and RXR at the TRE-site 4 in <i>Trh</i> promoter. Data are presented as percentage of input (starting sonicated DNA used for ChIP). The threshold value for a positive signal was set at 0.1% of input (dashed-line). The results are presented as percentage of input of (Ab+) minus (Ab–). The graph represents means of three independent experiments. LXR isoform is present at the TRE site 4 in the <i>Trh</i> promoter in hypothyroid animals only after T₃ treatment. RXR isoform is present at the TRE site 4 in the <i>Trh</i> promoter in hypothyroid animals but absent after T₃ treatment. There is no significant simultaneous recruitment of LXR and RXR to the site 4 of <i>Trh</i>.</p

TRα2 or TRα1 gain or loss of function held endogenous <i>Trh</i> levels constant.

<p><b>A</b>: <b>TRα2 or TRα1 overexpression does not interfere with endogenous </b><b><i>Trh</i></b><b> mRNA levels.</b> Euthyroid 2-days old mice were transfected with 100 ng of empty pSG5 (ct), pSG5-TRα1 (TRα1) or pSG5-TRα2 (TRα2). Hypothalami were dissected at either 3 (3 days old) or 5 (5 days old) days of age (corresponding to 1 day and 3 days post transfection, respectively). Two µg of totRNA were reverse-transcribed and qPCR were performed. Endogenous <i>Trh</i> mRNA levels are not significantly modified by TRα2 or TRα1 overexpression in either 3 or 5-day old mice as compared to controls (Ct) <i>18S</i> mRNA were used as endogenous control. The whole experiment was repeated twice. <b>B</b>: <b>TRα2 or TRα1 knockdown does not interfere with endogenous </b><b><i>Trh</i></b><b> mRNA levels.</b> Euthyroid 2-days old mice were transfected with 400 ng of empty pCMV-H1 (Ct), pCMV-H1-TRα1 (shTRα1) or a mixture of 200 ng sh1TRα2 and 200 ng sh2TRα2 vectors (shTRα2). Hypothalami were dissected 1.5 days post transfection (3.5 days old mice). Endogenous <i>Trh</i> mRNA levels are not significantly modified by TRα2 or TRα1 knockdown as compared to control. <i>Gapdh</i> mRNA was used as an endogenous control. The whole experiment was repeated twice.</p

The repression of hypothalamic <i>Trh</i> activity by LXR agonist in euthyroid newborn mice is dependent on the presence of LXR.

<p>A: LXRs are obligatory for transcriptional repression of <i>Trh</i> promoter by GW3965 (10⁻⁶ M) in euthyroid newborn mice. B: Transcriptional activation of <i>Trh</i> promoter when LXR is knocked-down by ICV injection of shLXR in euthyroid newborn mice. One-day-old euthyroid pups were transfected in the hypothalamic region of the brain (PVN) with 4 µl of a solution of a transfection mix containing PEI-complexed TRH-f.luc (0.2 µg/pup) alone (first two columns in A) or with empty CMV-H1 (0.4 µg/pup) (following two columns in A, first two columns in B) or a mix of CMV-H1shLXRα (0.2 µg/pup) and CMV-H1shLXRβ (0.2 µg/pup) (last column in A, and last two columns in B) with (+) or without (−) GW3965 at 10⁻⁶ M. Firefly Luciferase activity was measured 24 h later. n = 10 or 12 per group, A: pools of two (first part of the graph) or three (second part of the graph) independent experiments and B: one representative experiment. Non-parametric permutation test was used to assess statistical significance. *, p<0.05, **, p<0.01; ***, p<0.001.</p

T₃ regulation of LXR target genes involved in metabolism in the PVN of newborn mice.

<p>Eu (controls) - and hypothyroid (from dams treated by PTU the last week of gestation) newborn mice are subcutaneously treated by T₃ (2.5 µg/g bw) or vehicle 6 h and 18 h before dissection. Hypothalami (PVN) were recovered and PCR quantification of some LXR target gene mRNA from these PVN was done. Gene expression was normalized with <i>Gapdh</i>. n = 8 per group, a pool of two independent experiments is shown. Non-parametric ANOVA followed by permutation test with strata was used to assess statistical significance. *, p<0.05, **, p<0.01; ***, p<0.001.</p

TRα2 shows dominant negative activity on positively and negatively regulated T₃ target genes <i>in vivo</i>.

<p><b>A</b>: <b>TRα2 exerts dominant negative activity on positively </b><b><i>ME-tk-luc</i></b><b> transcription.. </b><i>ME-tk-luc</i> transcription was measured in hypothyroid (PTU) 2 days old mice treated with T₃ (2.5 µg/g b.w.) (PTU+T3) or saline (PTU), 18 h after hypothalamic injection of 1 µg reporter construct and 100 ng expression vector (empty pSG5 (Ct) or pSG5-TRα1 (TRα1) or pSG5-TRα2 (TRα2)). Transcription from <i>ME-tk-luc</i> is significantly increased in the presence of T₃ when TRα1 is overexpressed (as compared to Ct) (p<0.001). In contrast TRα2 overexpression significantly increases basal, T₃-independent <i>ME</i> transcription as compared to Ct and TRα1 (p<0.001), but addition of T₃ does not modify transcription further. <b>B</b>: <b>TRα2 exerts dominant negative activity on negatively </b><b><i>Trh-luc</i></b><b> transcription.. </b><i>Trh-luc</i> transcription was measured in hypothyroid (PTU) 2 days old mice as described above (100 ng expression vector and 1 µg reporter gene, <i>Trh-luc</i> per pup). Transcription from a <i>Trh-luc</i> construct is significantly decreased both in absence (PTU) and presence of T₃ (PTU+T3) when TRα1 is overexpressed (as compared with Ct). In contrast, overexpression of TRα2 has no effect on T₃-independent <i>Trh</i> transcription, but blocks its T₃-dependent repression. SEMs are given, n≥10 per point. In each case, the whole experiment was repeated twice giving similar results. *, p<0.05; **, p<0.01; ***, p<0.001.</p