Fig 1: Response to exogenous T3 suppression in WT and THRB K146Q mice and pituitary mRNA profile.Mice (n = 10/genotype) were made to have hypothyroidism with a low iodine/PTU diet. Mice were then given T3 (i.p.) daily for a total of 3 weeks, with an increasing dose each week. Blood was collected at the end of each week, and TSH was assayed using ELISA kit (see details in Methods). (A) TSH levels before and after mice were rendered hypothyroid (hypo). TSH is shown on a log10 scale because of the wide differences in the TSH levels in WT and THRB K146Q mice. (B) TSH level shown as percentage baseline at week 0 of T3 treatment. Insert shows the actual TSH level at the end of 3 weeks of T3 suppression. Statistical analysis was performed using paired Student’s t test (A and B). (C) The anterior pituitary gene expression analyzed by RNA-Seq is shown as log2 fold change (logFC). P log2FC is shown in parallel graph for upregulated and downregulated genes. TSHb, TSHß; CGa, common glycoprotein a subunit; Gh, growth hormone; Prl, Prolactin; Trhr, TRH receptor; Dio2, type 2 5'-deiodinase; Ghsr, growth hormone secretagogue receptor; T3, triiodothyronine; TSH, thyrotropin-stimulating hormone; PTU, propylthiouracil.
Fig 2: Exogenous TRH and TSH stimulation, hypothalamic gene expression, and TSH bioactivity in WT and THRB K146Q mice.(A and B) The mean (±SD) mRNA expression of hypothalamic TRH-associated genes by RNA-Seq are shown in bar figures and a heat map using 3 biological replicates (replicates 1, 2, 3) of WT and THRB K146Q mice. The scale of the intensity bar shows normalized CPM. The statistical analysis embedded in RNA-Seq data analysis software showed no significant differences. (C and D) Serum TSH and T3 in WT and THRB K146Q mice after TRH stimulation. A single dose of bovine TRH (5.0 µg/kg body weight) was injected (i.p.). Blood samples were collected at 0 and 30 minutes for TSH measurement and at 0 and 2 hours for T3 measurement. Individual values and mean ± SD are shown. Statistical analysis was performed with 2-way ANOVA to compare time points. (E) TSH bioactivity of serum from WT and THRB K146Q mice. CHO cells were transfected with TSHß expression vector with/without ß3-ADR expression vector. Sera of THRB K146Q mice were serially diluted with TSH-depleted sera. Mouse sera were added to the cells and incubated for 1 hour, and production of cAMP in CHO cells was determined. Statistical analysis was performed using 1-way ANOVA, *P < 0.01 compared with WT. (F) Mice were given a T3 injection (4 µg/100 g body weight/day) for 7 days to suppress TSH, and an i.p. injection of bovine TSH (200 mIU/100 g body weight) 20 hours after the last T3 injection. Serum T4 was analyzed at baseline and then 3 and 5 hours after bovine TSH injection. Statistical analysis was performed using paired Student’s t test. TRH, thyroid-releasing hormone; TRHr-1, TRH receptor-1; TRHr-2, TRH receptor-2; Dio2, Type 2 5'-deiodinase; THRB, thyroid hormone receptor ß; K146, lysine 146; TSH, thyrotropin-stimulating hormone; TRH, thyroid-releasing hormone; T3, triiodothyronine; T4, thyroxine, CPM, counts per million.
Fig 3: Thyroid status, thyroid gland, and pituitary findings in THRB K146Q mice.(A) Serum T4, T3, and TSH concentrations in WT and THRB K146Q mice (n = 13/ genotype) are shown as mean (±SD) and paired t test for statistical analysis. TSH is shown in log10 scale because of the wide differences in the levels in WT and K146Q mice. (B) Thyroid was rinsed with saline, patted dry, and weighed. The weight is shown as wet weight per mouse (n = 18/genotype). (C) Representative histology of thyroid gland stained with H&E. Transverse section of thyroid gland (top panel) and thyroid follicles (lower panel). (D) Dissected pituitaries were rinsed with saline, patted dry, and weighed, and values are shown as wet weight of pituitary from each mouse (n = 13/genotype). (E) Pituitaries are shown from WT and THRB K146Q mice. (F) Image of representative pituitary tissue histology with H&E stain from WT and THRB K146Q mutant mice. (G) Immunofluorescence staining for TSHß (green) and for nuclei (DAPI blue). Frozen sections of the pituitaries were incubated with anti-TSHß antibody at 1:50 dilution and conjugated with Alexa Fluor 488. (H) The TSHß-expressing cells and total cell numbers were counted using green and blue filters. (I) Western blot detection of TSHß and common glycoprotein a subunit (CGa) proteins. Pituitaries (n = 3) were lysed in RIPA buffer, and 30 µg of protein was loaded on an 8% SDS gel. Membranes were Ponceau S–stained (Supplemental Figure 2) prior to blot with anti-TSHß and anti-CGa. (J) Quantification of TSHß and CGa protein band in Western blot using LI-COR Image Studio Lite. (K and L) Western blot detection of THRB protein in the thyroid and pituitary. Protein (30 µg) was loaded onto a 10% SDS gel, transferred to a PVDF membrane, and blotted with anti-THRB antibody. The protein loading is shown (Supplemental Figure 3). Statistical analysis was performed using paired t test (A, B, D, and H). THRB, thyroid hormone receptor ß; K146, lysine 146; TSH, thyrotropin-stimulating hormone, T4, thyroxine; T3, triiodothyronine.
Fig 4: Location of sumoylation site K146 in the THRB1 protein and functional assay of the THRB1 K146Q mutant receptor.(A) Diagram of 3 sumoylation sites (K50, K146, K438) in the THRB1 protein indicating the amino terminus (A/B domain), DBD, and LBD. (B) Residue K146 is located within the Zn 2 of the THRB DBD (mutated K shown in red and sumoylation motif highlighted). (C) Ribbon diagram based on crystallographic data showing the THRB and RXR heterodimer bound to DNA DR4-TRE and the location of the mutated K146, outside of the region of direct DNA contact. (D) The THRB K146Q mutant was analyzed for its T3-mediated gene transcription in a reporter assay. The reporter contained 3 copies of the consensus DR4-TRE upstream of a luciferase gene. JEG3 cells were cotransfected with reporter and plasmids expressing THRB1 or THRB K146Q or a combination of THRB and K146Q. The amount of DNA in each transfection was kept constant. The luciferase activity was determined 12 hours after transfection using a multifunction plate reader. Results are presented as luciferase expression relative to the maximal THRB control transfection induction (shown as 100%). (E) The THRB point mutation in the mutant mice, from A to C, resulting in substitution of glutamine for lysine (K146Q), was confirmed by direct DNA sequencing. The THRB K146Q gene targeting strategy is shown in Supplemental Figure 1. Statistical analysis was performed for the reporter assay (D), using multiple paired t test in Prism statistical software. THRB, thyroid hormone receptor ß; K146, lysine 146; DBD, DNA-binding domain; LBD, ligand-binding domain; Zn 2, second zinc finger; RXR, retinoid X receptor; DR4-TRE, direct repeat, 4-base pair gap, thyroid hormone response element.
Fig 5: Metabolic phenotype of THRB K146Q mice.Mice (10 weeks old) were maintained at a normal light cycle on a regular chow diet and fasted for 6 hours before blood collection. Results are shown for WT (blue) and THRB K146Q mice (orange). Dots show individual values and bars indicate average ± SD. (A) BW is shown, and body composition, fat, and lean body mass are shown as a percentage of BW, as determined by Echo-MRI. (B) Serum concentrations of cholesterol, triglycerides, and free fatty acids. (C) Fasting glucose and serum insulin levels. (D) Transcriptome sequencing data show the mRNA expression level of known T3 target genes in the liver of THRB K146Q and WT mice. One-way ANOVA was used for statistical analysis, except transcriptome sequencing data (P < 0.04 is shown). Paired Student’s t test with normal distribution was used in statistical analysis. The statistical analysis for data shown in all panels was performed using Student’s t test for paired analysis. Thrsp/Spot14, thyroid hormone responsive protein; Cyp7a1, cytochrome P450 member 7a1; Slc2a2/Glut2, solute carrier family 2 member a2; G6pc, glucose-6-phosphatase catalytic subunit; Hmgcr, 3-hydroxy-3-methylglutaryl-CoA reductase; Ldlr, low density lipoprotein receptor; Acaca/ACC1, acetyl-CoA carboxylase 1; Cpt1a, carnitine palmitoyltransferase 1A; GSK3b, glycogen synthase kinase 3 ß; Pdk2, pyruvate dehydrogenase kinase 2; THRB, thyroid hormone receptor ß; K146, lysine 146; T3, triiodothyronine.
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