Noncanonical thyroid hormone signaling mediates cardiometabolic effects in vivo

Abstract

Thyroid hormone (TH) and TH receptors (TRs) α and β act by binding to TH response elements (TREs) in regulatory regions of target genes. This nuclear signaling is established as the canonical or type 1 pathway for TH action. Nevertheless, TRs also rapidly activate intracellular second-messenger signaling pathways independently of gene expression (noncanonical or type 3 TR signaling). To test the physiological relevance of noncanonical TR signaling, we generated knockin mice with a mutation in the TR DNA-binding domain that abrogates binding to DNA and leads to complete loss of canonical TH action. We show that several important physiological TH effects are preserved despite the disruption of DNA binding of TRα and TRβ, most notably heart rate, body temperature, blood glucose, and triglyceride concentration, all of which were regulated by noncanonical TR signaling. Additionally, we confirm that TRE-binding-defective TRβ leads to disruption of the hypothalamic-pituitary-thyroid axis with resistance to TH, while mutation of TRα causes a severe delay in skeletal development, thus demonstrating tissue- and TR isoform-specific canonical signaling. These findings provide in vivo evidence that noncanonical TR signaling exerts physiologically important cardiometabolic effects that are distinct from canonical actions. These data challenge the current paradigm that in vivo physiological TH action is mediated exclusively via regulation of gene transcription at the nuclear level.

Keywords: cardiometabolic effects; noncanonical signaling; skeleton; thyroid hormone action; thyroid hormone receptor.

Fig. 1.

Canonical and noncanonical TR signaling. (A) Canonical TR signaling requires binding of TR to regulatory DNA sequences, the TREs, mostly as a heterodimer with RXR. Binding of T₃ leads to an exchange of cofactors that initiates or represses the transcription of the target genes. (B) Noncanonical action of TRs involves rapid activation of signaling pathways without DNA binding. (C) Present (+) and absent (−) TR signaling in mouse models. In TR-WT mice, the TR can mediate both canonical and noncanonical signaling. In TR-KO mice, both effects are absent. In mice with TRE-binding–deficient TRs, canonical signaling is abolished, and only noncanonical signaling is preserved. Conversely, in mice with selective abrogation of PI3K signaling, this noncanonical TH effect is missing, while canonical signaling is preserved. Thus, a comparison of these mice can determine whether the signaling mechanism responsible for TH effects is canonical or noncanonical and PI3K mediated.

Fig. 2.

The GS mutation abolishes TR binding to DNA and canonical TR signaling in vitro. (A) Fluorescent EMSA was performed with 4 µL of reticulocyte-translated TRαWT, TRα71GS, TRβWT, TRβ125GS, and TRβY147F and 2 µL of RXRα on a 5′-Cy5–labeled DR+4 TRE probe. Arrows indicate bands of TRα and TRβ binding to probe; #, free probe. (B) HEK293 cells transfected with plasmids encoding for TRβ, TRβ125GS, TRβY147F, TRα, and TRα71GS and a DR+4-luciferase reporter plasmid. Empty vector (EV) and TR mutants without T₃ binding (TRαG291R and TRβG345R) served as negative controls. Cells were treated with vehicle (open bars) or with 10 nM T₃ for 48 h to induce luciferase expression via canonical TR/TRE-mediated action for TRα and TRβ variants (black bars); n = 3. Data are shown as mean ± SEM; ANOVA and Tukey’s post hoc test; *P < 0.05; ***P < 0.001.

Fig. 3.

The GS mutation abolishes TR binding to DNA and canonical TR signaling in vivo. (A) Relative expression of Myh6 and Myh7 in hearts of male WT (black bars), TRα⁰ (gray bars), and TRα^GS (open bars) mice; n = 6. Data are shown as mean ± SEM; ANOVA and Tukey’s post hoc test; ns, not significant, **P < 0.01, ***P < 0.001. (B) Response of TH target genes (Dio1 and Spot14) to T₃ in livers of hypothyroid WT, TRβ⁻, and TRβ^GS mice. Hypothyroid mice were injected either with vehicle (open bars) or with 50 ng/g BW T₃ (black bars) for four consecutive days; n = 6 mice per genotype. Data are shown as mean ± SEM; ANOVA and Tukey’s post hoc test; ns, not significant, **P < 0.01; ***P < 0.001. (C) ChIP of TRβ, RXRα, and H3K27Ac was followed by qPCR to determine H3K27 acetylation and recruitment of RXRα and TR to TR-binding sites located +7.0 kb and +22.4 kb from the transcriptional start site of Dio1 and NcoR2, respectively, in T₃-treated TRβ^147F, TRβ⁻, and TRβ^GS mice compared with WT mice; n = 4–6. Data are shown as mean ± SEM; Student’s t test; *P < 0.05; ^#P < 0.005; ^§P < 0.001; NC, negative control. (D) H3K27Ac ChIP-seq experiments were performed using livers from hypothyroid WT mice treated with PBS or T₃ for 6 h. A heatmap illustrates log2 fold change of H3K27Ac comparing T₃ treatment with PBS for all genotypes at 537 DNase-accessible regions with differential H3K27Ac calculated by DEseq2 (Bioconductor). (E) University of California, Santa Cruz Genome browser tracks of H3K27Ac enrichment profiles at the Dio1 gene locus for all four genotypes injected with PBS or T₃. Vertical blue bars mark TR-binding sites. (F) Hierarchical clustering of gene-expression data (microarray) from livers of hypothyroid WT, TRβ⁻, TRβ^GS, and TRβ^147F mice treated with either 200 ng/g BW T₃ or PBS for 6 h (n = 3).

Fig. 4.

Canonical TR action is required for TSH repression and normal growth. (A and B) TSH (A) and T₄ (B) in serum of 15-wk-old WT (black triangles; n = 11) TRβ⁻ (x; n = 9), TRβ^GS (open circles; n = 10), TRα⁰ (gray diamonds; n = 5), and TRα^GS (open squares; n = 6) male mice. Data are shown as mean ± SEM; ANOVA and Tukey’s post hoc test; *P < 0.05; ***P < 0.001; ns, not significant. (C) Tail length of 21-d-old WT (black bar), TRα⁰ (gray bar), and TRα^GS (open bar) mice. (D) Linear growth was recorded until the age of 70 d. (E and F) Bodyweight on P21 (E) and recorded until the age of 70 d (F); n = 6; mean ± SD for tail length and BW at P21 and follow-up; ANOVA and Tukey’s post hoc test; ns, not significant; *P < 0.05; ***P < 0.001.

Fig. 5.

Canonical TRα action is necessary for normal skeletal development of mice at P21. (A, Upper Left) Gray-scale images of caudal vertebrae from P21 WT (n = 8), TRα^GS (n = 5), and TRα⁰ (n = 3) mice were pseudocolored according to a 16-color palette in which low mineral content is blue and high mineral content is red. (Scale bar, 1,000 μm.) (Upper Right) The graph demonstrates caudal vertebra length in WT, TRα^GS, and TRα⁰ mice. Data are shown as mean ± SEM; ANOVA and Tukey’s post hoc test; ***P < 0.001. (Lower) Relative (Left) and cumulative (Right) frequency histograms display bone mineral content of vertebrae from TRα^GS and TRα⁰ mice vs. WT mice; Kolmogorov–Smirnov test, ***P < 0.001. (B, Upper) Proximal tibia growth plate sections stained with Alcian blue (cartilage) and van Gieson (bone) (magnification: 50× and 100×, respectively). HZ, hypertrophic zone; PZ, proliferative zone; RZ, reserve zone. (Scale bars, 500 μm.) (Lower) Growth plate chondrocyte zone measurements (Left) and relative proportions corrected for total growth plate height (Right) are shown for WT (n = 8), TRα^GS (n = 6), and TRα⁰ samples; n = 6. Data are shown as mean ± SEM; ANOVA and Tukey’s post hoc test; *P < 0.05; **P < 0.01; ***P < 0.001. (C) Micro-CT images of longitudinal femur midline sections demonstrate bone morphology. (Scale bar: 1,000 μm.) (D, Upper) Micro-CT images showing transverse sections of the distal metaphysis. (Scale bar: 1,000 μm.) (Lower) Graphs demonstrate trabecular number (Tb.N) (Upper) and trabecular spacing (Tb.Sp.) (Lower). Data are shown as mean ± SEM; ANOVA and Tukey’s post hoc test; **P < 0.01; ***P < 0.001.

Fig. 6.

Noncanonical TRβ signaling influences blood glucose and hepatic triglyceride synthesis. (A) Under fasting conditions, WT (black triangles), TRβ^GS (open circles), TRβ⁻ (x), and TRβ^147F (open triangles) mice received a single injection of T₃ (7 ng/g BW), and blood glucose concentration was measured at indicated time points; n = 4. Data are shown as mean ± SEM; Student’s t test; *P < 0.05). (B) Serum triglyceride concentration in untreated WT (black bar), TRβ⁻ (dark gray bar), TRβ^GS (open bar), and TRβ^147F (light gray bar) mice. (C) Representative immunoblots against Fasn, Me1, and Scd1 from liver samples. Gapdh was used as loading control; n = 2. (D) Triglyceride concentration in liver tissue was assessed after lipid extraction; n = 4–6 mice per genotype. Horizontal bars in the box plots indicate mean values, and whiskers indicate minimum and maximum values; ANOVA and Bonferroni’s post hoc test for multiple comparison; *P < 0.05; **P < 0.01; ***P < 0.001. (E) Immunoblots against Scd1 (Left), Me1 (Center), and Fasn Right), with a group size of n = 4 were used for densitometric measurements. Data are shown as mean ± SEM; ANOVA with Tukey’s post hoc test; *P < 0.05; **P < 0.01; ***P < 0.001. (F) Oil red O staining of liver sections from untreated WT, TRβ⁻, TRβ^GS, and TRβ^147F mice was done to visualize triglycerides in liver tissue. (Scale bar: 50 µm.)

Fig. 7.

Noncanonical action of TRβ increases body temperature, VO₂, and locomotor activity. (A) Body temperature of male mice in a TRα⁺ (WT) or a TRα⁰ genetic background; n = 6. Box plots indicate mean values, and whiskers indicate minimum and maximum values; ANOVA and Bonferroni’s post hoc test for multiple comparison; ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. (B) Locomotor activity of WT, TRβ⁻, and TRβ^GS mice expressed as centimeters traveled in 15 min; n = 10–20 mice per genotype. Horizontal bars in the box plots indicate mean values, and whiskers indicate minimum and maximum values; ANOVA with Tukey’s post hoc test; **P < 0.01, ns, not significant. (C and D) Average VO₂ of TRβ^GS (C) and TRβ⁻ mice (D) and WT littermate controls was measured by indirect calorimetry and is plotted against body weight (n = 10).

Fig. 8.

Noncanonical TRα signaling maintains normal basal heart rate without altering cardiac pacemaker channel gene expression. (A) Heart rate of nonsedated male WT (black triangles; n = 7), TRα⁰ (gray diamonds; n = 6), and TRα^GS (open squares; n = 6) mice. Data are shown as mean ± SD; ANOVA followed by Bonferroni’s post hoc test for multiple comparisons; ***P < 0.0001; ns, not significant. (B) Relative expression of pacemaker channels Hcn2 and Hcn4 and of potassium channel subunits with importance for repolarization (Kcnd2, Kcne1, Kcnb1, and Kcnq1) in hearts of WT (black bars),TRα⁰ (gray bars), and TRα^GS (open bars) mice; n = 6. Data are shown as mean ± SEM; ANOVA and Tukey’s post hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. (C) Ex vivo heart rate measured in hearts isolated from untreated WT (black triangles; n = 15), TRα⁰ (gray diamonds; n = 8), or TRα^GS (open squares; n = 7) mice. Data are shown as mean ± SD; ANOVA followed by Tukey’s post hoc test; *P < 0.05; ***P < 0.0001; ns, not significant.