Thyroid hormone receptor β sumoylation is required for thyrotropin regulation and thyroid hormone production
Sujie Ke, … , Anna Milanesi, Gregory A. Brent
Sujie Ke, … , Anna Milanesi, Gregory A. Brent
Published July 8, 2021
Citation Information: JCI Insight. 2021;6(16):e149425. https://doi.org/10.1172/jci.insight.149425.
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Research Article Endocrinology

Thyroid hormone receptor β sumoylation is required for thyrotropin regulation and thyroid hormone production

Abstract

Thyroid hormone receptor β (THRB) is posttranslationally modified by small ubiquitin-like modifier (SUMO). We generated a mouse model with a mutation that disrupted sumoylation at lysine 146 (K146Q) and resulted in desumoylated THRB as the predominant form in tissues. The THRB K146Q mutant mice had normal serum thyroxine (T4), markedly elevated serum thyrotropin-stimulating hormone (TSH; 81-fold above control), and enlargement of both the pituitary and the thyroid gland. The marked elevation in TSH, despite a normal serum T4, indicated blunted feedback regulation of TSH. The THRB K146Q mutation altered the recruitment of transcription factors to the TSHβ gene promoter, compared with WT, in hyperthyroidism and hypothyroidism. Thyroid hormone content (T4, T3, and rT3) in the thyroid gland of the THRB K146Q mice was 10-fold lower (per gram tissue) than control, despite normal TSH bioactivity. The expression of thyroglobulin and dual oxidase 2 genes in the thyroid was reduced and associated with modifications of cAMP response element–binding protein DNA binding and cofactor interactions in the presence of the desumoylated THRB. Therefore, thyroid hormone production had both TSH-dependent and TSH-independent components. We conclude that THRB sumoylation at K146 was required for normal TSH feedback regulation and TH synthesis in the thyroid gland, by a TSH-independent pathway.

Authors

Sujie Ke, Yan-Yun Liu, Rajendiran Karthikraj, Kurunthachalam Kannan, Jingjing Jiang, Kiyomi Abe, Anna Milanesi, Gregory A. Brent

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Figure 1

Location of sumoylation site K146 in the THRB1 protein and functional assay of the THRB1 K146Q mutant receptor.

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Location of sumoylation site K146 in the THRB1 protein and functional as...
(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 β; 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.