IMPORT AND EXPORT

Thyroid hormone entry into cells is mediated by specific transporters (see Fig. 76-2).^11–13 While early models suggested that lipophilic thyroid hormone molecules enter cells by diffusion across the plasma membrane, and this may occur to some extent, it is now clear that T₄ and T₃ entry mostly involves facilitated transport, a form of passive diffusion that requires membrane transport proteins.

Several proteins involved in thyroid hormone import and efflux across the plasma membrane of target cells have been identified. However, the quantitative contributions of these and possibly other transporters to overall thyroid hormone entry and export have not been established, and concentrations of various transporters in different tissues may vary. Potential transporters include members of several different multigene families: monocarboxylate transporters (MCTs), organic anion transporters (OATP), l-amino acid transporters, multidrug resistance–associated proteins, fatty acid translocase, and Na⁺/taurocholate-cotransporting polypeptide.

Presently, the best-characterized thyroid hormone transporter is the X-linked MCT8 isoform, which is important for thyroid hormone import into the brain. Expression of the MCT8 gene in cultured cells and frog oocytes enhances thyroid hormone transport into cells. Inactivating human MCT8 mutations are associated with severe neurologic defects in affected males, suggesting that MCT8-dependent thyroid hormone transport into neurons is essential for proper brain development. There are also changes in circulating thyroid hormones (low T₄ and high T₃). Analysis of MCT8 mutants in cultured cells reveals that the mutations abolish T₃ transport, likely explaining elevated circulating T₃ levels. Targeted deletion of the mouse MCT8 gene leads to alterations in thyroid hormone levels that are similar to those of affected humans, although mice are devoid of the neurologic defects. Here, liver thyroid hormone levels are normal, suggesting that other transporters are more important in this tissue.

Of other transporters, recent observations show that a closely related protein, MCT10, can also mediate T₄ and T₃ import into cells. MCT10 is a more effective transporter than MCT8 and displays a preference for T₃. It is widely expressed and particularly abundant in the liver, intestine, kidney, and placenta. Finally, OATP1C1-expressing cells exhibit preferential import of T₄ and rT₃. The protein is distributed widely in the brain, with locations consistent with a specific role in thyroid hormone transport across the blood-brain barrier. It is not clear whether there are other high-affinity thyroid hormone transporters that are yet to be identified or whether apparently low-affinity transporters may play a physiologically important role in some tissues.

Less is known about mechanisms involved in thyroid hormone export from cells. It is clear that MCT8 and MCT10 enhance both thyroid hormone uptake and export, but it is not clear whether these proteins are physiologically relevant hormone exporters or whether other, undescribed, exporters may alsoplay a role.

In some tissues—brown fat in the mouse is an example—hormone response is dependent on T₃ that is generated from intracellular T₄. The implication here is that free T₄ mostly penetrates these cells, where it constitutes a reservoir for T₃ generation, and that tissue uptake of T₃ is much lower. Regulated conversion of T₄ to T₃ provides an important control point; induction of the type 2 deiodinase via induction of second messenger pathways regulates T₃ levels and therefore the extent of the thyroid hormone response (also discussed later).

ACCUMULATION OF THYROID HORMONE IN CELLS

While free thyroid hormones are present at low concentrations in the circulation, levels of hormone can be much higher in target tissues. Although the extent to which this pool of hormone is active is not known, this observation suggests that there are mechanisms which permit thyroid hormone accumulation in target cells.

T₃ binds with nanomolar affinity to an intracellular protein that was originally called cytoplasmic T₃-binding protein (CTBP).¹⁴ This protein is found at high abundance in the kidney and, to a lesser extent, in liver. Because CTBP was later found to be homologous to mu-crystallin, a protein abundant in the lens of the kangaroo eye, the gene is now often called CRYM. Overexpression of CRYM in stable cell lines increases maximal cellular T₃ binding capacity and decreases T₃ efflux rates. However, these effects are coupled to reduced transcriptional responses to T₃, suggesting that CRYM sequesters T₃ in an inactive cytoplasmic complex, away from nuclear TRs.

CRYM is important for thyroid hormone action in vivo. Mice with a targeted CRYM gene deletion appear normal but exhibit moderate decreases in circulating T₄ (25%) and T₃ (13%) levels and perhaps more importantly, also exhibit extremely rapid rates of T₃ entry into and escape from target tissues, presumably because there is no CRYM to sequester cytoplasmic hormone from export mechanisms. Human patients with a natural CRYM mutation that blocks its ability to bind T₃ (K314T) are deaf, a known consequence of defective thyroid hormone signaling during development. Interestingly, CRYM requires NADPH for dimerization and T₃ binding (Fig. 76-3). As NADPH levels are reflective of levels of cellular reducing power and anabolic capacity, it is conceivable that CRYM may sequester or release T₃ in response to alterations in cellular metabolic status. This notion is attractive but not proven.

FIGURE 76-3. Thyroid hormone interactions with CTBP. Intracellular T₃ can bind CTBP dimers and become sequestered in the cytoplasm. CTBP dimer formation is sensitive to intracellular NADPH/NADP⁺ concentrations such that free hormone will probably be released in response to reductions of NADPH levels.

There could be other T₃-binding proteins in addition to CRYM and the nuclear TRs (which are described later). Radioisotope-labeled T₃ interacts with proteins in the endoplasmic reticulum, mitochondrion, and nuclear envelope.² These putative T₃-binding proteins have not been identified, and the functional significance of these interactions is unknown.

DEIODINASES IN THYROID HORMONE ACTIVATION AND INACTIVATION

As discussed earlier and in Chapter 73, the thyroid gland produces mostly thyroxine. Limited amounts of T₃ and rT₃ are secreted by the gland, but more than 80% of both these forms of thyroid hormone are produced in the periphery via actions of specific deiodinases, D1, D2, and D3.^15,16 D1 and D2 remove the iodine group from the 5′ position of the outer thyronine ring (Chapter 74). As T₃ binds to TRs with higher affinity than T₄, the actions of D1 and D2 deiodinases serve to increase thyroid hormone activity through generation of T₃, and this represents a step-up in activity. Conversely, the action of D3 decreases hormone activity and represents a step down by removing the iodine group from the 5 position of the inner thyronine ring and converting T₄ and T₃ to rT₃ and 3,3′-T₂, respectively, neither of which interacts substantially with nuclear TRs at physiologic concentrations. Changes in deiodinase expression and activity are important control points in thyroid hormone signaling; they influence the production and availability of the biologically active form of thyroid hormone, T₃.

D2 activity generates most plasma T₃ in euthyroid (defined as levels of thyroid hormone within normal range) conditions via conversion of T₄ to T₃ in peripheral tissues. Comparisons of rates of T₃ production by D2 and D1 indicate that D2 is probably more important for T₄ to T₃ conversion at physiologic T₄ concentrations; it displays a higher K_m value for substrate than D1 and is likely to be sensitive to alterations in T₄ concentrations and more active than D1 at physiologic hormone concentrations. In addition, environmental stimuli regulate D2 activity catalyzing local T₃ production from T₄ in certain tissues. For example, signals such as cold exposure increase D2 expression in brown fat (discussed earlier), and the resulting increase in intracellular T₃ promotes induction of uncoupling protein with consequent heat generation. These lines of evidence suggest that D2 is important in generating T₃ that activates TRs. Accordingly, D2 is located in the perinuclear region where it can deliver T₃ efficiently to TRs.

D1 acts in a similar way to D2, but its functions are not as clear as with D2, because it displays a relatively high K_m value for T₄ to T₃ conversion (i.e., the enzyme binds the T₄ substrate with low affinity). D1 is highly expressed in liver and kidney, and its expression is induced by thyroid hormone. While previous models suggested that D1 is important for regulation of circulating T₃, more recent models suggest that D1 may only contribute substantially to plasma T₃ in conditions of thyroid hormone excess. D1 may also be involved in 5′ deiodination of other forms of thyroid hormone, including rT₃.

D3 is important for clearance of plasma T₃.¹⁷ It is highly expressed in human placenta, where it is thought to be important for protection of the developing fetus from effects of maternal thyroid hormones. D3 is also highly expressed in the brain and skin. Increased D3 levels may account for reductions of circulating thyroid hormone levels that are often observed in critically ill patients and states of inflammation. Finally, D3 is often overexpressed in vascular tumors, and this in turn can result in marked reductions in circulating thyroid hormone levels.

ALTERNATE METABOLIC FATES OF THYROID HORMONE: INACTIVE AND ACTIVE DERIVATIVES

In addition to 5′ deiodination, T₄ undergoes other modifications which are generally believed to inactivate the hormone.¹⁸ About 45% of circulating T₄ is converted to rT₃ and 35% to T₃. T₃ and rT₃ undergo further deiodinations to create T₄ derivatives with all possible combinations of iodinated and deiodinated inner and outer rings (3,3′-T₂, 3,5-T₂, 3′,5-T₂, 3′-T₁, 3-T1 and T₀), none of which bind significantly to nuclear TRs. T₄ and T₃ can also be inactivated by glucuronidation in liver, followed by secretion into the bile or by sulfation in liver and kidney and excretion in the urine.

The fact that most secreted T₄ is rapidly converted to apparently inactive forms of the hormone has led investigators to question whether some of these T₄ derivatives have unappreciated functions in activation of alternate receptors or as metabolic precursors of different active forms of the hormone. As mentioned earlier, one of the most prominent, rT₃, is a very weak thyroid hormone partial agonist that is unlikely to exert significant actions through the TRs. However, there are two cases in which alternate metabolic modification of T₄ does produce biologically active forms of hormone:

BASAL METABOLIC RATE

One of the most important effects of thyroid hormone involves changes in basal metabolic rate (BMR) in multiple tissues.^21–24 Thyroid hormones stimulate oxygen consumption (indicative of enhanced metabolism) in multiple locations, including skeletal muscle, liver, kidney, and intestine. Increases in BMR are probably partly related to increased mitochondrial activity and number. Importantly, thyroid hormones induce expression of uncoupling proteins (UCPs), mitochondrial membrane proteins that dissipate the proton gradient in the absence of ATP synthesis, thereby converting potential energy to heat. This effect is probably a contributor to thyroid hormone–dependent increases in BMR.

Thyroid hormone does not always enhance BMR. For example, BMR is not enhanced by thyroid hormone in most regions of the brain, and the hormone actually suppresses metabolic activity in the pituitary. The receptors in these tissues are functional, suggesting that key mediators of thyroid hormone regulation of BMR are blocked or absent.

TISSUE EFFECTS

Thyroid hormone exerts specific effects on growth, development, and metabolism in a variety of tissues. Some of these effects are summarized briefly here:

SYSTEMATIC ANALYSIS OF THYROID HORMONE TARGET GENES

Many thyroid hormone–responsive genes involved in the responses described earlier are known.² For example, in liver, thyroid hormone induces carnitine palmitoyl transferase 1a (that mediates the rate-limiting step in fatty acid oxidation); glucose-6-phosphatase and phosphoenolpyruvate carboxykinase (rate-limiting steps in gluconeogenesis); the NR coregulator PGC-1α, which is an important transcriptional coregulator that stimulates genes important for mitochondrial biogenesis, gluconeogenesis, and fat oxidation; CYP-7a1 (cholesterol-to–bile acid conversion); fatty acid synthetase; acetyl-CoA carboxylase; malic enzyme (increases fat synthesis); and spot 14, which is required for fat synthesis in some tissues and up-regulated in contexts in which carbohydrates are converted to fat. Many other hormone-responsive genes are probably still unknown.

The earliest systematic attempt to define thyroid hormone response was performed in the late 1970s. Use of two-dimensional (2D) gels to analyze extracts from radiolabeled methionine–pulsed cells revealed that about 1% of 1000 rat liver proteins change in response to altered thyroid hormone.^36,37 There were inductions and repressions, and the studies began to define the “domain” of the thyroid hormone response. Recent advances in gene-profiling technology have permitted much more detailed descriptions of hormone-dependent alterations in gene profile. Presently there have been relatively few systematic genome-wide descriptions of thyroid hormone responses.^25,26,38,39 An early study of thyroid hormone action in mouse liver revealed that about 1% of mouse genes change in response to altered thyroid hormone (hypothyroid versus hyperthyroid),²⁵ confirming estimates from 2D gels. This study revealed target genes involved in carbohydrate, fat, and amino acid metabolism and unexpected thyroid hormone–regulated pathways; for example, genes involved in apoptosis are induced. The experiment also provided insights into patterns of thyroid hormone–responsive gene expression. Most regulated genes (>65%) in liver were repressed by thyroid hormone. This preponderance of negatively regulated genes is not seen in all tissues. Target genes are generally up-regulated by hormones in human muscle primary culture. Later studies of thyroid hormone patterns of gene expression in TR knockout mice provided insights into TR-specific effects and subtle differences in thyroid hormone–responsive gene expression and are discussed in more detail later in the chapter. Systematic screening of thyroid hormone–responsive genes should provide detailed descriptions of novel target genes and pathways in different tissues.

THYROID HORMONE–REGULATED MICRORNAs

MicroRNAs (miRs) are small (18 to 25 nucleotides), noncoding RNAs that hybridize with coding mRNAs to inhibit translation and, in some cases, also promote mRNA degradation.⁴⁰ The enormous roles these miRs play in regulation is just beginning to be appreciated. MiRs are produced by cleavage and processing of large primary transcripts, which often code for proteins. Thus, many of the influences that regulate expression of parental primary transcripts also regulate expression of the associated miR. While effects of thyroid hormone on miR expression have not been studied extensively, one miR (miR-208) lies within a noncoding region of the thyroid hormone–responsive human αMHC transcript (and is therefore up-regulated by thyroid hormone itself). MiR-208 is implicated in up-regulation of stress-dependent cardiac responses. It is likely that other thyroid hormone–responsive miRs exist, and they may play important roles in hormone effects on protein translation.

DISCOVERY OF THYROID HORMONE RECEPTORS AND THEIR MECHANISM OF ACTION

TRs were identified in the 1970s.^42,43 Following the discovery of several nuclear receptors, including the estrogen and glucocorticoid receptors, investigators in the thyroid hormone field utilized similar approaches to look for TRs. Specific binding of radiolabeled T₃ to liver cells was initially discovered by Oppenheimer and colleagues using intact cells and later characterized further by Samuels and colleagues, who found that the mechanism of TR actions differed mechanistically from the steroid receptors, which translocate into the nucleus on ligand binding. For TRs, hormone could bind directly to nuclei, and the location of receptors did not alter with T₃.

In the late 1970s and early 1980s, multiple aspects of the receptors were revealed.^36,44 Analysis of properties of TRs revealed a single class of high-affinity binding sites with K_d values for T₃ in the 0.1 nM range. Specific hormone-binding sites were observed in extracts of several responsive tissues, including liver, anterior pituitary, brain, and heart. Estimates of the number of binding sites per cell suggested that the proteins are rare; highly responsive tissues only contained about 10,000 specific hormone-binding sites per cell. Evidence that the receptor preparations correspond to physiologic hormone targets came from observations that affinities for different thyroid hormones parallel their biological potencies (Triac>T₃>T₄>rT₃). Partial TR purification and photo-affinity labeling revealed two major nuclear hormone-binding proteins with molecular weights of 46 and 57 kD, which were later found to correspond to products of distinct genes—the TR α and β isoforms—described later in the chapter. TRs were also found to be associated with chromatin, and preferentially with active chromatin, suggesting that they influence gene expression through interactions with DNA.^45–48

DISCOVERY THAT THYROID HORMONES REGULATE SPECIFIC mRNAS AND GENE TRANSCRIPTION

As molecular biology techniques became available, it was possible to isolate specific mRNAs from tissues where they were abundant and to translate them. By the 1970s, regulation of specific mRNAs had been demonstrated for estrogens, progestins, and glucocorticoids. Growth hormone (GH) mRNA was abundant in rat pituitary cells, and Baxter and colleagues were able to demonstrate that thyroid hormones could specifically regulate GH mRNA, conclusively demonstrating for the first time that thyroid hormones alter the levels of specific mRNAs. The same group cloned rat GH gene sequences, and these permitted demonstrations that thyroid hormones increased mRNA hybridizable to a rat GH cDNA probe. The cloned GH sequences were also used in so-called “polymerase run-on experiments” that showed that hormones stimulate transcription rather than induce some posttranscriptional modification of the mRNA. These studies resolved the controversies prevalent at the time of experiments by Tata and colleagues, discussed previously. Thus the fundamental concept of thyroid hormone action whereby the receptor regulates the transcription of specific genes was established.

DISCOVERY THAT TRS BIND PREFERENTIALLY TO ACTIVE CHROMATIN AND SPECIFIC DNA SEQUENCES

Studies during the 1970s and 1980s also set the stage for understanding the nature of TR binding sites and their relationship to active chromatin. GH genomic DNA was used for interaction studies with partially purified TR preparations, demonstrating that TRs bound to specific DNA sequences. These experiments were forerunners of the now familiar concept that receptors act by binding to specific thyroid hormone response elements (TREs). That the receptors could affect DNA was further evidenced by the finding that they induce bending of rat or human TR DNA binding sites. Subcellular fractionation revealed that TRs were associated with chromatin, and preferentially with active chromatin, suggesting a relationship between the receptors and transcriptionally active chromatin.^45–48 These studies set the stage for definition of thyroid hormone response elements (TREs).

CLONING OF TRS

TRs were cloned in 1986.^49,50 They were identified as cellular homologs of a retroviral oncogene (cancer-causing gene), v-erbA,⁵¹ which causes erythroblastoma in chickens. Isolation of these cDNAs occurred after those for other members of the NR family, the glucocorticoid and estrogen receptors, which led to the realization that v-erbA was derived from an NR. Both of the v-erbA homologs bound thyroid hormone with high affinity and appropriate specificity. The Vennstrom group clone was most closely related to v-erbA and was originally designated as c-erbA, but it is now called thyroid hormone receptor α (TRα). As compared to TRα, v-erbA contains 17 different amino acid substitutions and a C-terminal truncation, which are now known to prevent the viral oncogene from binding hormone.⁴⁹ The Evans group clone encoded a highly homologous but distinct 57-kD protein that was closely related to TRα and v-erbA but was the product of the THRB gene.⁵⁰ The characterization of TR genes (THRA and THRB), which encode the two major TR isoforms (TRα1 and TRβ1) and several variants that arise from differential splicing (described later), helped to reveal that TRs belong to the NR family, which includes receptors for the steroid hormones, vitamins A and D, and other small lipophilic molecules.⁴

Identification of these receptor cDNAs paved the way for many studies of receptor structure and function. Although it was known at the time that NRs were composed of modular domains, the receptor structures provided the primary sequences in these domains and the abilities to express them and study their functions. In addition, the availability of TR cDNAs paved the way for functional studies, analog development, and improvements in understanding TR mechanisms. While these studies are not described in historical context, there have been rapid advancements in understanding of TR action, including the definition that unliganded TRs are transcriptionally active, leading to a new paradigm for thyroid hormone action; definition of TRE sequences; identification of retinoid X receptor (RXR) heterodimer partners and coregulators; x-ray structural analysis of DBDs and LBDs, which demonstrated a new and unexpected concept that the receptor folds around the ligands; improved understanding of TR function through generation of TR gene knockout animals; and development of new thyroid hormone analogs with selective actions.

HORMONE BINDING AND OCCUPANCY

Measurements of thyroid hormone affinity for its receptors reveal that T₃ binds to purified and recombinant TRs with high affinity. T₃ binds with about 10- to 15-fold greater affinity than T₄ (K_d values are around 2 nM for T₄ and 0.2 nM for T₃). These values appear higher than measured concentrations of circulating free hormones (0.02 nmol for T₄ and 0.06 nmol with T₃), implying that receptor occupancy could be minor in normal conditions. However, based on direct estimates of occupancy of nuclear TRs in different tissues after radiolabeled T₃ and T₄ injection into hypothyroid rats, about 25% to 35% of liver and kidney TRs and 58% of pituitary TRs become occupied with thyroid hormones in euthyroid conditions. Thus, there appear to be mechanisms to bring the hormone into the correct range to bind and activate receptors. One possibility was described earlier: intracellular generation of T₃ from T₄ leads to increased T₃ production in target cells. Facilitated transport alone would not be enough to concentrate T₃, but combinations of transport and cytoplasmic sequestration by CTBP/CRYM or similar proteins could play a role. Finally, TR complexes with stabilizing coactivators could display higher affinities for target hormones than isolated TRs tested in vitro or partially purified from cell extracts. In any case, it is clear that thyroid hormone concentrations are in the correct range to achieve reasonable receptor occupancy in target tissues under euthyroid conditions.

TR ACTION

Unlike classic models of hormone action in which unliganded receptor is inactive and hormone binding triggers its activity, TRs are transcriptionally active in the absence and in the presence of hormone (Fig. 76-5).⁵² Rather than activating the receptor, hormone binding alters the spectrum of its actions.

FIGURE 76-5. Thyroid hormone receptor action. Actions of unliganded and liganded TRs at positively regulated genes. The receptor binds DNA as a heterodimer with RXR in the absence and presence of the hormone. Unliganded TRs recruit corepressors, which repress gene transcription by condensing local chromatin structure and blocking coactivator binding. Hormone binding promotes a conformational change, which leads to exchange of corepressor for coactivators, which reverses effects of coactivators.

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