Paradigms of Dynamic Control of Thyroid Hormone Signaling

Abstract

Thyroid hormone (TH) molecules enter cells via membrane transporters and, depending on the cell type, can be activated (i.e., T4 to T3 conversion) or inactivated (i.e., T3 to 3,3'-diiodo-l-thyronine or T4 to reverse T3 conversion). These reactions are catalyzed by the deiodinases. The biologically active hormone, T3, eventually binds to intracellular TH receptors (TRs), TRα and TRβ, and initiate TH signaling, that is, regulation of target genes and other metabolic pathways. At least three families of transmembrane transporters, MCT, OATP, and LAT, facilitate the entry of TH into cells, which follow the gradient of free hormone between the extracellular fluid and the cytoplasm. Inactivation or marked downregulation of TH transporters can dampen TH signaling. At the same time, dynamic modifications in the expression or activity of TRs and transcriptional coregulators can affect positively or negatively the intensity of TH signaling. However, the deiodinases are the element that provides greatest amplitude in dynamic control of TH signaling. Cells that express the activating deiodinase DIO2 can rapidly enhance TH signaling due to intracellular buildup of T3. In contrast, TH signaling is dampened in cells that express the inactivating deiodinase DIO3. This explains how THs can regulate pathways in development, metabolism, and growth, despite rather stable levels in the circulation. As a consequence, TH signaling is unique for each cell (tissue or organ), depending on circulating TH levels and on the exclusive blend of transporters, deiodinases, and TRs present in each cell. In this review we explore the key mechanisms underlying customization of TH signaling during development, in health and in disease states.

Figure 1.

Deiodinases modify local TH signaling. T4 and T3 enter virtually all cells through membrane transporters. Once inside the cells, T3 diffuses to the nucleus and interacts with TRs to modulate gene expression. T3–TR complexes control specific sets of T3-responsive genes, thus promoting T3-dependent biological effects. While inside the cells, TH molecules can be modified through the deiodinase group of enzymes. Deiodinases modify the biological activity of TH molecules either activating T4 (D2) or inactivating T4 and T3 (D3). As a result, the flow of T3 molecules diffusing from the cell membrane to the nucleus can be enhanced with additional T3 supplied by the D2 pathway, which locally converts T4 to T3. In contrast, the D3 pathway decreases the flow of T3 to the nucleus because it terminally inactivates T3 to T2. D2 is an ER-resident protein, a cell compartment that is adjacent to the nucleus. This explains why D2 activity results in higher TR occupancy with locally generated T3. In contrast, D3 sorts to the plasma membrane, where it undergoes endocytosis and recycling via early endosomes. Notably, under hypoxic and/or ischemic conditions, D3 is redirected to the nuclear envelope, where it inactivates T3 and slows down cellular metabolism. See reviews for more details (1, 4, 17). [Adapted with permission from “Hypothyroidism, thyroid hormones and deiodinases.” www.BiancoLab.org.]

Figure 2.

Molecular structure of deiodinases. Deiodinases are homodimeric type I integral membrane selenoproteins composed of a single N-terminal transmembrane segment connected to a larger globular domain with a selenocysteine-containing active center embedded in a thioredoxin-like fold (18). The structure of the three deiodinases is similar as modeled through hydrophobic cluster analysis in combination with position-specific iterated BLAST. Their extramembrane portion belongs to the thioredoxin-fold superfamily (18). The crystal structure of an inactive catalytic domain of one of the deiodinases (mouse D3) was solved and confirmed most aspects revealed with the three-dimensional modeling (19). It also revealed a close structural similarity to 2-Cys peroxiredoxin(s) (Prx), which suggests a route for transferring protons to the substrate during deiodination and a mechanism for subsequent recycling of the transiently oxidized enzyme (19). [Adapted with permission from “Hypothyroidism, thyroid hormones and deiodinases.” www.BiancoLab.org.]

Figure 3.

Sources and clearance mechanisms of circulating T3 in humans. The daily T3 production in a 70-kg adult individual is ∼30 μg/d. The thyroid gland contributes with ∼5 μg/d and the rest is produced outside of the thyroid parenchyma via two deiodinase-mediated pathways, D1 and D2; the latter is the most important source of circulating T3 in humans. Even though the thyroid contributes with a small fraction of the circulating T3, thyroidal T3 secretion is upregulated in response to TSH stimulation. This occurs through an increase in the T3/T4 ratio in the thyroglobulin and through increased thyroidal conversion of T4 to T3. Through this mechanism and the homeostatic changes in deiodinase activity, circulating levels of T3 are maintained fairly stable throughout the day. T3 is cleared from the circulation by deiodination via the D3 pathway that converts T3 to T2, as well as hepatic glucuronidation and sulfation, the latter followed by deiodination via the D1 pathway. In cells expressing D1, the T3 residence time inside the cells is relatively short, that is, ∼30 minutes, whereas in D2-expressing cells the residence time is several hours. This is probably the result of distinct subcellular localization of D1 vs D2, plasma membrane vs ER, respectively. Additionally, T3 produced in D2-expressing cells finds its way to the cell nucleus and binds to TRs, triggering biological effects. See reviews for more details (5). [Adapted with permission from “Hypothyroidism, thyroid hormones and deiodinases.” www.BiancoLab.org.]

Figure 4.

D2 is inactivated by ubiquitination. ER stress rapidly reduces D2 activity via activation of eIF2a, which inhibits translation of Dio2 mRNA. D2 ubiquitination is the molecular mechanism underlying changes in D2 half-life, that is, the covalent attachment of multiple ubiquitin molecules to D2, which both inactivates the enzyme and targets it to degradation in the proteasomes. D2 is structured as a homodimer, D2:D2, and monomers are inactive. Ubiquitination is thought to inactivate D2 by disrupting the conformation of the D2:D2 dimer, critical for enzyme activity. A unique 18–amino acid loop confers intrinsic metabolic instability to D2, facilitating binding to proteins involved in the ubiquitination process. UBC6 and UBC7 are critical in the process of D2 ubiquitination, as well as two ubiquitin ligases, the hedgehog-inducible WSB1, and TEB4, a ligase involved in the degradation of proteins in the ER. The WD-40 propeller of WSB-1 recognizes an 18–amino acid loop in D2 that confers metabolic instability, whereas the SOCS box domain mediates its interaction with an ubiquitinating catalytic core complex, modeled as Elongin BC–Cul5–Rbx1. Ubiquitinated D2 (UbD2) can be reactivated by deubiquitination, a process catalyzed by two USP class D2–interacting deubiquitinases, USP20 and USP33. D2 ubiquitination occurs via K48-linked ubiquitin chains and exposure to its natural substrate, T4, accelerates UbD2 formation. UbD2 is retrotranslocated to the cytoplasm via interaction with the p97–ATPase complex. D2 retrotranslocation also includes deubiquitination by the p97-associated deubiquitinase Ataxin-3. Once in the cytosol, D2 is delivery to the proteasomes as evidenced by coprecipitation with 19S proteasome subunit S5a and increased colocalization with the 20S proteasome. See reviews for more details (86, 87). [Adapted with permission from “Hypothyroidism, thyroid hormones and deiodinases.” www.BiancoLab.org.]

Figure 5.

Nutrient availability and activation of TH signaling. Leptin is a key molecule signaling food intake and the availability of energy substrates to the hypothalamus, where it activates the HPT axis by stimulating secretion of TRH and TSH, and hence thyroidal activity. There is a drop in serum T3 levels with fasting, which reflects decreased thyroidal secretion and decreased extrathyroidal conversion of T4 to T3. The mechanism regulating DIO2 expression in skeletal muscle in this setting was modeled by shifting cells to media containing only 0.1% fetal bovine serum, which reduces DIO2 expression via FOXO1-mediated transcriptional repression (62). There is a FOXO1 binding site within the DIO2 promoter, close to the transcription start site. Binding of FOXO1 to this site suppresses DIO2 gene expression. In contrast, shifting cells back to a media containing 10% fetal bovine serum (after 24 h of fasting) increases DIO2 expression and D2 activity through a mechanism initiated by insulin and mediated by a series of kinases (PI3K–mTORC2–AKT) that end up phosphorylating FOXO1, hence relieving DIO2 repression. These findings are relevant for hypothyroid patients maintained on L-T4 that depend on D2 for >80% of all their T3 needs; thus, they are at greater risk to develop low serum T3 during caloric restriction (62). [Adapted with permission from “Hypothyroidism, thyroid hormones and deiodinases.” www.BiancoLab.org.]

Figure 6.

Developmental control of TH signaling via timed expression of deiodinases. Profiles of DIO2 (red line) and DIO3 (blue line) expression in (a) placenta, (b) retina, (c) cochlea, (d) SKM, (e) BAT, and (f) bone at the indicated periods of life. In most cases DIO2 and DIO3 exhibit a reciprocal inverse relationship. In general, D3 activity is high at early embryonic stages. Its expression drop is followed by an elevation in DIO2. See reviews for more details (7, 8, 48, 68, 132).

Figure 7.

TSH levels are normalized to slightly higher circulating T4/lower T3 in LT4-treated patients with hypothyroidism. TSH secretion is defined by the balance between the positive input provided by TRH secretion and the negative input provided by circulating T4 and T3 levels. In LT4-treated patients with hypothyroidism the negative input is based on a slightly higher circulating T4/T3 ratio when compared with normal individuals. This is because of an imbalance between D2 ubiquitination in the hypothalamus vs the rest of the body. While outside the hypothalamus T4-induced D2 ubiquitination limits T3 production; in the hypothalamus–pituitary axis this mechanism is less efficient, preserving D2-mediated T3 production even as circulating T4 rises with LT4 administration. A growing body of work suggests that the relatively lower circulating T3 levels in LT4-treated patients with hypothyroidism are clinically relevant. LT4-treated patients weigh ∼10 pounds (4.5 kg) more, exhibit higher serum cholesterol levels, are more likely to be on statin and antidepressive medications, and display a slower rate of energy expenditure. See reviews for more details (86, 87). [Adapted with permission from “Hypothyroidism, thyroid hormones and deiodinases.” www.BiancoLab.org.]

Figure 8.

Transport and metabolism modulate TH signaling in the brain. T4 crosses the BBB through Oatp1c1, reaching astrocytes where it is converted to T3 via D2. T3 exits astrocytes and is likely to enter neurons via MCT8. Circulating T3 can also reach neurons crossing the BBB through MCT8. The presence of D3 in neurons inactivates T3 to T2. It is not clear whether D3 preferentially targets incoming, outgoing, or both flows of T3 molecules. Shown are conditions known to affect TH signaling along with their main characteristics: (1) global-D2KO mouse; (2) Astro-D2KO mouse; (3) global-D3KO mouse; and (4) mutations of MCT8; also shown are conditions known to stimulate (5) Dio2 or (6) Dio3 pathways. See reviews for more details (10, 294, 299, 300). [Adapted with permission from “Depression due to deiodinase defect, despite normal thyroid hormone levels. PMID: 27501182 .” www.BiancoLab.org.]

Figure 9.

Local TH signaling accelerates BAT thermogenesis. BAT is a specialized organ that produces heat in response to cold exposure or excessive caloric intake (362). BAT expresses both TRα and TRβ (365). Heat is generated due to mitochondrial uncoupling triggered by the sympathetic nervous system [i.e., NE-induced adenylyl cyclase (AC) activation and cAMP production] that also stimulates Dio2, increases T3, and leads to the induction of T3-responsive thermogenic genes, including Ucp1. Moreover, D2-generated T3 also stimulates BAT lipogenesis, which generates fatty acids used to sustain accelerated mitochondrial activity. Hypothyroid animals have impaired the ability to thermoregulate in the cold due to decreased BAT function. Global-D2KO animals exhibit a reduction in the expression of genes that define the tissue thermogenic identity (i.e., Ucp1, Pgc1α, and Dio2) and exhibit impaired oxidative capacity. See reviews for more details (350, 362–364). [Adapted with permission from “Hypothyroidism, thyroid hormones and deiodinases.” www.BiancoLab.org.]

Figure 10.

Perinatal Dio2 liver expression defines future susceptibility to obesity and liver steatosis. A brief surge in Dio2 expression in the liver around the first day of life affects the methylation status [differentially methylated region (DMR)] of hundreds of genes, including Foxo1. The neonatal surge in TH signaling prevents methylation of three sites within the Foxo1 promoter, allowing the gene to be expressed and stimulate Zfp125, a liver transcription factor that suppresses the expression of 18 genes involved in the assembly and secretion of VLDL particles. As a result, normal mice develop steatosis when placed on an HFD. In contrast, mice in which liver Dio2 was inactivated exhibit three DMRs in the Foxo1 gene, reducing its expression to about half of that in control mice. Consequently, the expression of the Foxo1 downstream target Zfp125 is also greatly reduced in the absence of the perinatal surge in Dio2. The reduction in Zfp125 expression accelerates VLDL secretion, minimizing lipid deposition and steatosis when animals are fed with an HFD (72, 408).

Figure 11.

Physical exercise enhances TH signaling in skeletal muscle via induction of Dio2. One of the downstream targets of T3 is the thermogenic coactivator PGC1α that is key to mitochondrial function. Physical exercise accelerates cAMP production within skeletal myocytes, which induce the expression of both Dio2 and PGC1α. Dio2 expression accelerates local activation of T4 to T3, which enhances TH signaling and further stimulates PGC1α expression. This TH-mediated mechanism for induction of PGC1α is a component of the mitochondrial adaptation induced by exercise, which is lost in animals with skeletal muscle–specific Dio2 inactivation (130). [Adapted with permission from “Physical exercise activates thyroid hormone in skeletal muscle.” www.BiancoLab.org.]