J. Endocrinol. Invest. 1.' 79, 1978

REVIEW ARTICLE

Nuclear receptors for thyroid hormone 1 L.J. DeGroot, S. Refetoff, J. Bernal, P.A. Rue, and A.H. Coleoni

.

Thyroid Study Unit, Department of Medicine, University of Chicago, Chicago, IllinoIs, USA hours after administration of a tracer was present in the nuclei, and that 90% of this was bound to semi-specific receptors of limited capacity. Not surprisingly, when similar studies are done on hypothyroid rats, a much higher fraction of the isotope was accumulated in the liver nuclei, reaching in some experiments at least 27% of the liverT3.ln both categories of animals, most of the remainder of the isotope in liver is bound less firmly to cytoplasmic binding proteins. The T3 accumulated in the liver nuclei is in equilibrium with triiodothyronine in blood and in cytoplasm, and falls in parallel with the removal of T3 from these compartments through excretion, deiodination, and other metabolic routes. We estimated that the total binding capacity of rat liver nuclear receptors was 2.4 ngl g liver (Fig. 1). With normal blood levels ofT30f approximately .8 -1 nglml, we found about 0.6 ng T3/g liver in the nuclei, suggesting that the receptors were approximately one-quarter saturated under normal conditions. Carrying these calculations to the extreme, one can estimate that about 10,000 molecules of T3 could be present on these alleged receptors per liver cell nucleus. Triiodothyronine bound to nuclear receptor in vivo was released during incubation in vitro at 20 C or 37 C, suggesting both release and inactivation of receptors (5). The nuclear receptors could be extracted from the nuclei by high salt solutions such as 0.4 M KCI-(5), and were found to sediment on sucrose gradients with a peak between 3.5 and 4.5 S, depending upon the KCI concentration in which the gradients were performed (7). The binding peak was digested by pronase but not by DNAse or RNAse, indicating its protein nature. Griswold and co-workers (2) and Oppenheimer and co-workers (8) found that the receptors were associated with chromatin and confirmed the extraction with high salt solutions. Although it was initially suggested that these binding sites were specific for T3, it rapidly became apparent that this was not the case. Labelled nuclear T3 could readily be displaced in our studies by triac, thyroxine, or even D-thyroxine given in vivo. The binding affinity of a series of thyroid hormone analogs given in vivo to rats, relative to the binding affinity of L-T3 taken as "1 ", was found to be 1 for 13, D-T3 was 0.7, L-T4 was 0.1, tetrac was 0.05, and reverse T3 was 0 (9) (Table 1). Relative binding affinities in heart nuclear receptors were essentially the same. That triac, which has onethird the metabolic activity of triiodothyronine, and 0-

INTRODUCTION The concept that thyroid hormone might exert its stimulatory metabolic effects through an action on the cell nucleus was implied clearly by the work of Tata and Widnell in 1966 (1). They observed, 12 hours after administration of thyroid hormoneto a hypothyroid rat, a stimulation of magnesium-dependent ribosomal RNA polymerase. Early studies seeking evidence for selective accumulation of thyroid hormone in nuclear or other subfractions of cells were without success. However, spurred by the observations of specific nuclear receptors for estrogen, efforts were continued, and in 1972 Griswold, Fisher, and Cohen (2) reported the presence of saturable thyroxine binding sites in tadpole liver nuclei. Oppenheimer, Koerner, Schwartz, and Surks reported the presence of saturable binding sites for triiodothyronine in liver, kidney, and rat pituitary (3, 4). Observations in this field have rapidly accumulated, and several laboratories have made important contributions. In this review, we shall attempt to include important observations from all groups, but will stress studies with wich we are most familiar from our own investigations. Most of the investigations have been performed in laboratory animals or cell culture systems; it is believed but not proved that they apply to man.

In vitro studies of nuclear thyroid hormone binding proteins When isotopic labelled triiodothyronine is injected. into a rat, it rapidly accumulates in the liver, reaching an equilibrium distribution with endogenous triiodothyronine within half an hour (5, 6). The triiodothyronine is concentrated in liver. The ratio of T3 per gram of liver IT 3 per .ml serum varies with time, averaging around 4 one hour after administration of tracer, and declining to 2 by 24 hours (5). When liver is homog~ni­ zed and nuclei are recovered by a sucrose gradient technique, about 60% of the DNA is recovered. Accounting for recovery of nuclei, we showed that about 15% of the liver triiodothyronine present two

1 Supported by United States Public Health Service Grants AM-13377. CA19266. AM-15070. and TW-02155 Key-words: Thyroid hormone receptors, thyroid hormone action, nuclear receptors

Correspondence: Leslie J DeGroot. MD - The University of Chicago. Department of MediCine - 950 East 59th Street - Box t 38. Chicago. Illinois 60637. USA

79

L.J. DeGroot, S. Refetoff, J. Bernal. PA Rue, and A.H. Co/eoni Table 2 - In vivo T3 binding capacity of nuclei from various rat tissues (reference 61). Tissue

Binding capacity ng T3 / mg DNA

Liver Kidney Heart Brain Anterior pituitary Spleen Testis

Fig. 1 - Triiodothyronine receptor capacity measured in vivo. Thyroidectomized rats were given [ ' 25 1]_ T3 plus increasing doses of unlabelled T3. The proportion of isotope bound in the

Table 1 - flelative potency of binding of thyroid hormone analogs in vivo to T3 receptors in rat liver and hea rt nuclei (reference 9) . Liver

Heart

1

1 0.04 0.02 0 1.0 0.3 2 .5

1 1

0

0.02 0.002

°

T 3, which has 5 or1 0% of T3potency should have surh relatively high binding affinities is at first glance disturbing. More will be said about this in subsequent sections. The relative binding capacity of various tissues for triiodothyronine has been investigated by Oppenheimer and colleagues, and the data are provided in Table 2. It is of considerable interest that binding capacity is highest in liver, heart, kidney, and anterior pituitary, and lower in brain , spleen , and testis, the latter three organs representing allegedly T3 nonresponsive tissues. Relative saturation of the receptors was essentially similar in the seven tested organs, suggesting that total quantity of occupied receptor, rather than relative saturation, may determine tissue responsivity to thyroid hormone.

0.7

0.27 0.79

47 35 44 39

48

50 90

Triiodothyronine and other analogs bind efficiently to nuclear receptor molecules during incubation in vitro with intact washed nuclei or with KCI extracted receptor molecules (10, 16). The kinetics vary with temperature, with maximum binding being reached over many hours at C, during 2 - 3 hours at 20 C, and within five to ten minutes at 37C. Binding is also affected by ionic composition, is maximum between pH 7.5 and 8.7, is saturable by increasing doses of 13. and apparently is associated with one significant class of binding sites. A very minor class of binding sites has been detected by some workers , but this may represent a degradation product of the original binding molecules (17). Binding in vitro does not depend upon an energy source, is inhibited by proteins which compete for binding of thyroxine, is not affected by RNAse or DNAse, is destroyed by protease, and the affinity of the receptors is highest when studied in the presence of reducing compounds such as dithiothreitol (1 0). There is no requirement for a cytosol protein in exchange in vitro of T3 with receptor bound T3 in the nucleus (16) . Binding to soluble extracts is inhibited by heat or by exposure to 0.1 mM parahydroxymercurobenzoate, again indicating the importance of a sulfhydryl group in the binding reaction (11). Binding affinity for T3 in the system we have employed is 0.2 x 1O'°M -', and capaCity was 508 pg T3/ g wet tissue, or 53 x 1O -15 M T3/ 100 I1g DNA (10). We have subsequently shown that during in vitro incubations of nuclei, at 20 C, there is leakage of approximately half the receptor molecules to the incubation medium (7) . These receptors molecules are essentially the same as those remaining in the nuclei, and their binding capacity must be taken into account if total recovery is to be estimated. By accounting for 60% recovery of nuclei during homogenization and preparation of tissue, leakage of half of the receptors from the nuclei to the incubation soluble components during in vitro assay at 20 C, and about 60% exchange of labelled T 3 with endogenous T3 during the two hour incubation period, it can be shown that the receptor capacity measured in

nuclei at each dose was measured and expressed as a ratio to the total isotope content of an equivalent volume of liver. With increasing doses of T3 there is progressive saturation of specific receptors demonstrated in this Scatchard plot as a single set of binding sites with a capacity of approximately 2.4 ng/g tissue. In addition, there is evidence for a low level of nonspecific binding.

01 0.05 0

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Binding of thyroid hormone analogs to receptors in vitro

v ,nq Iq

L-T3 L-T4 Tetrac Reverse T3 Triac O-T3 Isopropyl T3 MIT

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0

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Nuclear receptors for thyroid hormone vitro is essentially equal to that found in vivo. The binding affinity and capacities recorded during in vitro studies have varied considerably depending upon the pH and ionic conditions of the incubation system employed. However, all studies have uniformly shown one major class of binding sites, to which it appears that all of the analogs tested bind. Binding affinities of various hormone analogs have been tested in isolated nuclei in a number of laboratories with fairly good agreement. In our hands triac binds with four times the affinity of thriiodothyronine, Lthyroxine with one-fourth the affinity, D-T3 with similar affinity in vitro, and MITand DITwith markedly reduced binding affinity (1 0) (Fig. 2). The relative binding affinity of the in vitro systems has been analyzed extensively by Koerner and co-workers (17) and by Jorgensen and co-workers (18). Throughout a wide range of analog potencies, there is fairly good correlation between binding affinity and analog metabolic activity. Obvious discrepancies include the equal or higher affinity of binding of triiodothyroacetic acid as compared to T3, and the equal affinity of the D-amino acid analogs to the binding of the L-amino acid analogs. Gosling and co-workers (19) have proposed that the two-fold greater metabolic clearance rate of triac may serve to explain how triac can bind to nuclear receptors in vitro with equal, or as we have observed, higher affinity than 13, but have one-third the apparent potency when given in vivo. This is certainly a sensible, although not totally satisfying explanation. It is also possible that the strong binding of triac to plasma proteins or cytosol proteins may retard its entry into the nucleus. A similar explanation for the discrepancy in binding and metabolic potency of D-analogs can be proposed, although it has not been experimentally proven.

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Fig. 2 - Saturation of triiodothyronine receptors in liver cell nuclei in vitro. Liver nuclei were incubated in vitro with [ '25 /]_ T3 plus increasing concentrations of unlabelled triiodothyronine triiodothyroacetic acid, or dextrotriiodothyronine. The molar ratio of D- T3 required to displace 50% of the binding of labelled L- T3 was nearly equivalent to that of L- T3, whereas triac was essentially four times as efficient in displacing labelled TJ, indicating a higher affinity of triac for the nuclear .receptors than the binding affinity of T3 itself.

nuclei (22). More recently these workers have reported that addition of L-triiodothyronine to GH-1 cells causes a dose dependent depletion of nuclear receptors with a maximum of 20 - 50% of the receptors being released from the nuclei (23). They reported that the dose response curve for receptor depletion is very similar to the dose response curve for stimulation of growth hormone synthesis and suggest that depletion of nuclear receptors by T3 may be the mechanism by which the hormone acts. As will be indicated below, this concept appears to be discordant with the reports from a number of laboratories which have studied liver receptors in vivo and in vitro. I n this tissue the phenomenon of receptor depletion has not been recognized. It must be noted that in most of the studies done in our laboratory, and possibly in others, it would be difficult to reliably detect a 20% depletion of receptors except by experiments conducted on a large number of animals. Thus receptor depletion has not been totally excluded in the action of hormone in the liver cell system.

Hormone receptors in cells studied in vitro In vitro cultured rat pituitary tumor cells of the GH-1 line

have proven most interesting in the study of hormone receptors in the laboratories of Samuels and Refetoff. Samuels et al. reported that the GH-1 cells contained receptors essentially similar to those found in liver nuclei (20). Growth of these cells was stimulated by thyroxine or triiodothyronine (21). Half maximal stimulation was produced at a level of 6.5 x 10-9M 13, which was estimated to be equivalent to 8 x 10-12M free 13 Dissociation constants obtained during studies of intact cells in vitro are apparently lower than those recognized during in vitro incubations of isolated nuclei or isolated receptor. In the GH-1 cell sytem the dissociation constant for T3 was 29 pM and for T4 was 260 pM. Thyroid hormone was found to stimulate growth hormone production by these cells and to inhibit prolactin secretion. Samuels and Tsai initially reported that preincubation of GH-1 cells with increasing concentrations of nonradioactive T3 resulted in subsequent increase in binding when radioactive T3 was incubated directly with the isolated

Nature of receptors Most of the information relevant to the nature of the receptors has been described in other sections. The receptor molecules appear to be proteins with a negative charge under alkaline conditions, a sedimentation constant between 3.5 and 4.5, depending on ionic conditions, and according to some workers a nonspherical configuration (24). Molecular weight is 81

L.J. DeGroot, S. Retetoff, J. Bernal, PA Rue, and A.H. Coleoni occupancy of the receptor by T 3, rather than the ability of the receptor-T3 complex to bind to chromatin. The latter appears to be relatively nonspecific, and there appear to be a large number of available binding sites on chromatin. We have, in unpublished studies, shown that binding of the T3 receptor complex to chromatin depends upon the presence of DNA, histones, and acidic nuclear proteins, rather than anyone component of chromatin.

estimated to be approximately 40,000 - 50,000. The receptors contain a required sulfhydryl group (25). They are thermolabile, especially at temperatures above 37 C, but very stable at low temperatures. The receptors tend to aggregate, especially in low ionic strength media. Purification as been attempted in a number of laboratories using standard column chromatography methods and affinity chromatography, but to date there has not been overwhelming success in this field. Thus the exact characteristics of totally purified receptor cannot be reported. The receptor has not been identified specifically with any enzymatic activity. Some receptor appear to exist in a soluble or loosely bound form in the cell since a large proportion can be extracted simply by incubating nuclei in 0.14 M NaCI containing solutions. Another portion is more tightly bound to chromatin and is dissociated only with increasing ionic strength solutions.

Table 3 - Binding in vitro of liver nuclear receptor- T3 complex to chromatin. Chromatin

% Specific binding of receptor-T3 complex

Liver Heart Kidney Testis Spleen Brain

9.8 14.8 10.5 12.7 9.7 8.0

± ± ± ± ± ±

4.6 5 2.6 4.3 4 2.6

Association of receptors with chromatin As previously indicated, a significant proportion of the receptor is found associated with chromatin, but at least 90% of receptor which can be labelled in vivo is extractable from chromatin by 0.4 M KCI, and receptor-T3 complex can reassociate with chromatin in vitro (25). Gardner has reported that thyroid hormone T3 receptors are associated specifically with nucleolar chromatin (26). Charles et al. reported in 1975 (27) that triiodothyronine was bound specifically to slowly sedimenting chromatin fractions which contained the highest active template capacity for RNA synthesis. These workers reported subsequently (28) that there was relatively nonspecific distribution of receptors through chromatin fractions when chromatin was fractionated by a less vigorous technique utilizing DNAse rather than mechanical shearing. Macleod and Baxter have also reported binding of isolated receptors to free DNA (29). Surprisingly, there was similar binding to native and denatured DNA, or even to prokaryotic DNA. Binding was less extensive to cytoplasmic RNA or synthetic polynucleotides (30). We have investigated the binding of receptor complexes to chromatin prepared from a variety of rat tissues (31 ). Binding was most extensive to liver, heart, kidney, and testis, and somewhat reduced in spleen and brain. However, there seemed to be no good correlation between metabolic responsiveness and ability of the T3 receptor complex to bind to chromatin (Table 3). Binding was also nonsaturable under the conditions we employed in any of the chromatins studied. Final interpretation of these data must be guarded, since the studies were conducted with impure receptor complexes and may need to be

Physiology of receptors Relatively little is known so far about normal or abnormal physiology of the receptor complexes. It has been reported from three laboratories that the ability to form receptor complexes in nuclei is not altered by induced hypothyroidism or T3 administration in vivo (15, 16) This suggests that the receptor content and affinity are not altered by prior exposure of the animal to hormone, and further that there is no necessary translocation of receptor from cytosol to nucleus. According to this hypothesis, metabolic responsiveness would depend upon proportional occupancy of a constant level of receptors present in the cell nucleus. We have studies the receptors during maturation of male rats and found that receptor capacity is approximately one-quarter the adult level per milligram of DNA in neonatal rats, reaching the normal level at 30-50 days (32) (Table 4). Interestingly, the relative occupancy of the receptors by T3 is approximately the same or even a little higher in the neonatal animals in comparison to adult liver nuclei. Further, alphaglycerophosphate dehydrogenase activity in the

Table 4 - Nuclear T3 binding during maturation. Days

Ka (x 10 '0

M-')

0-6 9-13 20-29 50-80 80-120 120-210

revised when purified receptor is available. However,

at face values the studies suggest that the responselimiting factor in thyroid hormone action may be in 82

0.12 0.06 0.27 0.25 0.18 0.18

Binding Serum Nuclear % aGTDH T3 T3 capacity Satura- activity pg T3/g ng/ml ng/ml tion liver 232 528 831 1085 1177 788

0.1 0.25 077 0.76 078

0.14 0.32 0.4 0.46 0.5 0.24

30 30 25 22 22 16

077 0.63 1.0 0.84 0.43 0.59

Nuclear receptors for thyroid hormone neonatal rat liver mitochondria is similar to that adult animals. These studied raised the hypothesis that, in certain circumstances, hormone response may be regulated by relative saturation of receptor rather than total occupied receptor concentration. Thus one could visualize a system in which occupied receptors had a positive effect and unoccupied receptors a negative effect. In this model the observed response would depend upon the relative concentration of the two forms of receptor. It must be noted that there is absolutely no experimental verification for this hypothesis. An alternate explanation of the changes in neonatal animals could be that growth, and function of enzymes such as a-glycerophosphate dehydrogenase, may be under controls other than thyroid hormone in the neonatal animals. We have examined in immature animals the effect of injections of estrogen, testosterone, and glucocorticoid, and have noted no alterations in receptor content or affinity (32) . Recent studies have shown that starvation of rats is associated with a dramatic reduction of serum TSH , thyroxine, and triiodothyronine (33) . Associated with this we found a marked reduction in liver nuclear triiodothyronine receptor capacity which reached 18 48% below the baseline level by 48 hours of fasting, in four sets of experiments. There was also a reduction in nuclear T3 content and in nuclear DNA-dependent RNA polymerase activity. Primacy among these alterations is not yet known but the data suggest a physiologic response during starvation associated with diminished tissue action of thyroid hormone, possibly in an effort to stem the catabolic effect of these hormones. Bernal has studied certain characteristics of receptors from normal and thyroidectomized animals (34) . Empty receptors can be recognized by incubation at 0 C and filled receptors can be quantitated by incubations at 20 C. Using this technique, he found no difference in the sedimentation rate of empty and filled receptors , their differential KCI extractability from liver tissue, or in their ability to bind to chromatin .

Relationship of receptor metabolic response

saturation of nuclear receptor sites is associated with the maximal rate of synthesis of a-glycerophosphate dehydrogenase and that higher T3 doses simply prolong the period during which maximal synthe~is occurs without further augmenting the rate of synthesis. We have studied the effect of chronic exposure of hypothyroid rats to T 3 in doses of 1 -15 Jig / day (36) (Fig. 3) . As one would expect, increasing doses of the hormone were associated with increasing quantities of hormone in nuclei and relative saturation of the receptors. There appear to be discordant responses to receptor occupancy, depending upon the response measured. Serum and nuclear T3 , aglycerophosphate dehydrogenase activity, DNAdependent RNA polymerase activity, and mRNA content were returned essentially to the normal range by doses of 0.3 - 1 Jig/day. At doses of 5 - 15 Jig T 3/ day, serum and nuclear levels of T 3 continue to climb, as did a-glycerophosphate dehydrogenase activity, but polymerase activity, liver weight, and weight gain were reduced (Fig. 4). These data suggest that total occupancy of the receptors is associated with the catabolic state of hyperthyroidism. They also suggest that different tissue metabolic activities may respond in a different manner, as indicated by the disparity between polymerase function and aglycerophosphate dehydrogenase activity.

32

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I n our initial studies we found receptors in rat liver nuclei to be approximately 25 - 30% occupied under normal conditions, suggesting that augmented occupancy could correlate with thyrotoxicosis . Initial reports from the Oppenheimer group suggested that the receptors were normally at least 75% occupied (6), but subsequent data reduced this to an estimated 35 47% occupancy for most tissues (10).

3 5

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Fig. 3 - Thyroidectomized rats were given increasing doses of T3 each day for one week, and the body weight gain, liver weight, and nuclear RNA polymerase activities were measured. It can be seen that there is a progressive and nearly linear increase in all parameters, with doses up to approximately 3 I1g/ day. Above this level all parameters decrease. These data strongly suggest that progressive saturation of receptors is associated with a progressive increment in T3response, but that complete occupancy of the nuclear receptors is associated with the catabolic response recognized as thyrotoxicosis.

Oppenheimer and co-workers (35) have investigated the relationship between nuclear receptor occupancy and metabolic response measured by a-glycerophosphate dehydrogenase activity. They suggest that total

83

L.J DeGroot, S. Refetoff, J Bernal, PA Rue, and AH Coleoni of RNA synthesis or other actions. The progesterone receptor has been shown to consist of two proteins with possibly different functions , each of which binds one molecule of progesterone. In contrast, the thyroid hormone receptors appear to exist as a single protein, and to function primarily in the nucleus. There is no evidence for a required binding of hormone in cytosol with subsequent transport to the nucleus. It also may be noted, and will be subsequently discussed, that there is much less evidence that T3 has a very early effect on nuclear function than is the case for the steroid hormone systems.

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Evidence for the importance of thyroid hormone binding proteins, called "receptors" in this review, comes from four sources. As indicated above, the work of Oppenheimer and co-workers suggests that the capacity of the receptors for T3 binding varies from tissue to tissue, but correlates with the responsivity of these tissues to in vivo administered thyroid hormone. A second argument is that the relative affinity of the receptors for hormone analogs correlates with the metabolic effect of the analogs. Surely this is true over the extreme range of analog activities, but there are important discrepancies, as previously indicated, including the higher affinity of triac than L-T3, and equal binding affinity of the dextro-isomers. Probably these discrepancies can be largely " explained away" by altered absorption , distribution, or metabolism, of the compounds in vivo, factors which might tend to reduce their metabolic potency in comparison to L -T 3. A third line of reasoning supporting physiologic importance is the correlation, at least so far as determined, between occupancy and metabolic response. However, this is a most indirect feature, since a similar relationship could obviously be shown for plasma hormone binding proteins and administered T3, and no one would argue that plasma transport proteins are involved in the specific metabolic response to thyroid hormone. A fourth, and probably important argument , has to do with variat ions in receptors in certain patients, which will be described below. All in all it can be said that the arguments strongly support the role of the receptors in the expression of thyroid hormone metabolic effects, and that so far this remains an interesting but unproven hypotheSiS.

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Fig. 4 - Enzyme activities of liver were studied in vitro in specimens derived from normal rats (N), thyroidectomized rats (H), or thyroidectomized (T) rats given 15 /-Ig triiodothyronine / l00 g body weight at from 1 - 24 hours before sacrifice. Enzyme activities are expressed as a ratio of the activity found in a normal or T3 treated group to the enzyme activity found in preparations from a cOincidentally studied hypothyroid group. There is a dramatic reduction in total polymerase, polymerase I, and polimerase II enzyme activity (measured as a-amanitin sensitive enzyme), and in mithocondrial a-glycerophosphate dehydrogenase. Within 8 - 10 hours after administration of T3, these is clear evidence of stimulation of all enzyme activities, which are returned essentially to normal by 24 hours after this single dose of T3.

Comparison of thyroid receptors to steroid hormone receptors It may briefly be noted that there are several obvious discrepancies between the receptors for estrogen, progesterone, testosterone, and glucocorticoids, and the system functioning with thyroid hormone analogs (37, 38). Current theory indicates that the steroid receptors exist in the cytosol , and that steroid hormones bind to them in this compartment, inducing some sort of conformational change in the receptor. After this the receptor.-hormone complex moves to the nucleus and binds to chromatin , causing a stimulation

Clinical studies in patients with thyroid hormone resistance A syndrome comprising retarded growth, deaf mutism, stippled epiphyses, goiter and elevated thyroid hormone production and serum levels was reported by Refetoff co-workers (39, 40) and has been extensively studied by our group. The crucial findings are that three children in this sibship produce a fivefold excess of normal thyroid hormones per day, but are, even with

84

Nuclear receptors for thyroid hormone

high levels of circulating free hormone, in a eumetabolic state. Fetal hypothyroidism is believed to account for deaf mutism, and variable tissue responsivity is believed to account for features such as stippled epiphyses, even with elevated levels of thyroid hormone. We have studied nuclear receptors for triiodothyronine in lymphocytes from these patients. The striking observation made was that receptor content is either absent or reduced by a factor of tenfold in lymphocytes from one of the children (41). Further, when the receptor material present is extracted with KCI, it binds abnormally to chromatin. Thus, in this sibship, thyroid hormone resistance appears to correlate with a specific abnormality in lymphocyte receptors. The in vitro assay of lymphocyte receptors is a difficult task, and the findings are restricted so far to one patient. Another patient, with alleged thyroid hormone resistance, but of milder severity, has been reported to have essentially normal lymphocyte receptors (42). Possibly hormone resistance is caused by a variety of mechanisms.

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Fig. 5 - Tritium labelled leucine incorporation into proteins of normal (N), thyroidectomized (H), and thyroidectomized animals given 15 J1 T3 per 100 g body weight, was studied in vivo. Within five ,hours of administration of T3, there was definite evidence of increased synthesis of cytosol proteins, nuclear globulins, and nonhistone nuclear proteins. The low protein synthetic activity characteristic of the hypothyroid animals was returned to normal by 24 hours after administration of this dose of triiodothyronine.

Metabolic response to thyroid hormone The list of metabolic responses to thyroid hormone, including biomedical and clinical parameters, is too extensive to detail in this review dedicated to consideration of nuclear receptors. However, the overwhelmingly important question of the moment is how nuclear receptors may relate to the metabolic response to thyroid hormone. Thus renewed interest has been given to studies on the effects of thyroid hormone on nuclear physiology initiated twelve years ago by Tata and co-workers. We and other groups of workers have reported thatT3 given to hypothyroid rats in vivo stimulates nuclear RNA synthesis (43, 46). We find an increase in polymerase I and II activity within 8 10 hours after administration of the hormone (Fig. 4), and also find that mRNA content of the liver tissue is increased at this time (46). Similar stimulation of RNA polymerase is seen in rat brain and in tadpole liver (47, 48). The response iri rat liver was blocked by administration of a-amanitin which may prevent messenger RNA synthesis, or cycloheximide which may prevent protein synthesis. Thus it seemed natural to examine the effect of thyroid hormone on protein synthesis, which was done by administering labelled leucine to normal and hypothyroid animals (49). We found that increased labelling of cytosol and nuclear proteins was stimulated within five hours, earlier than detectable changes in RNA synthesis (Fig. 5). We have also

QY

shown tha t thiS occurs, not due to alterations of cell leu·

cine pOOl, but due to clear stimulation of protein synthesis. The response occurs with doses of thyroid hormone that are physiologically relevant, 0.5 J,lg/1 00 g body weight. Further, it has been demonstrated that hypothyroid and normal animals have strikingly

NG

85

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Fig. 6 - Nuclear globulins were extracted from nuclei of normal and hypothyroid rats by incubation with 0.14 M NaCI in vitro, and electrophoresed in polyacrylamide gels. On the electrophoretogram it is apparent that one protein band, very evident in the normal nuclear globulins, is dramatically reduced in the hypothyroid animal preparation, and another nuclear globulin appears in much higher concentration in the hypothyroid specimen. The polyacrylamide gel electrophoretic patterns of cytosol proteins, his tones, and nonhistone proteins, from normal and hypothyroid animals, were not different.

L.J. DeGroot, S. Retetotf, J. Bernal, P.A. Rue, and A.H. Coleoni different patterns of liver nuclear globulins which can be detected on polyacrylamide gel electrophoresis (Fig. 6). The role of these different nuclear proteins is as yet unknown, but may suggest that synthesis of specific proteins may be involved in the subsequently observed stimulation of RNA synthesis occuring in response to thyroid hormone. Kim and Cohen (50) have shown in tadpole liver that the induction of metamorphosis by thyroxine associated both with alterations in polymerase function and with increased availability of tadpole liver chromatin. We have so far been unable to demonstrate similar changes in the hypothyroid rat, but Zoncheddu et al. (51) have found an increase in soluble nucleolar polymerase I ten hours after T3 administration. Using GH-I cells, Seo and co-workers (52) have shown that thyroxine specifically stimulates the production of messenger RNA for growth hormone. It has also been shown that thyroid hormone stimulates the production of messenger RNA for liver a2-microglobulin (53).

(59,60). Thus thyroid hormone may have multiple sites of action, including multiple intracellular receptors. While these data are damaging to the comfortable concept of a single set of nuclear receptors being responsible for all actions of thyroid hormone, our knowledge in the field is as yet too limited to rule out other possibilities.

REFERENCES 1. Tata J.R., Widnell C.C. Ribonucleic acid synthesis during the early action of thyroid hormones. Biochem. J. 98: 604, 1966. 2. Griswold M.D., Fischer M.S., Cohen P.P. Temperature-dependent intracellular distribution of thyroxine in amphibian liver. . Proc. Nail. Acad. Sci. USA 69: 1486, 1972.

3. Schadlow A.R., Surks M.I., Schwartz H.I., Oppenheimer J.H. Specific triiodothyronine binding sites in the anterior pituitary of the rat. Science 176: 1252, 1972.

Epilogue

4. Oppenheimer J.H., Koerner D., Schwartz H.L., Surks M.I. Specific nuclear triiodothyronine binding sites in rat liver and kidney. J. Clin. Endocrinol. Metab. 35: 330, 1972.

A host of questions remain unanswered and the actual verification that the triiodothyronine binding proteins discussed in this review are receptors for thyroid hormone and a conduit for the metabolic effect of thyroid hormone, remains unproven. It is not known exactly how receptor occupancy is coupled to metabolic response, and clearly we do not know how thyroid hormone activates RNA or protein synthesis in the cell. Further, it is not known whether thyroid hormone activates synthesis of specific individual proteins or mRNAs, or has a more general effect on cell responsivenes. Little is known about receptor characeristics from various tissues, and it remains possible that one analog of thyroid hormone may be more active in the metabolism of one tissue than it is for another. We know little about the physiology of the receptors; the question of receptor depletion as a metabolic response mechanism has been opened but not settled, and in fact it is obvious that the number of questions to be answered at this time is very large. This review has concentrated on nuclear receptors for thyroid hormone and possible actions of thyroid hormone on RNA and protein synthesis. This one sided approach is not meant in any way to depreciate the role of other putative thyroid hormone receptors or thyroid hormone actions. Nothing has been said about the important studies on cytosol binding proteins, or about the reported receptors of very high affinity in rat liver mitochondria (54). We have also omitted extensive consideration of studies showing stimulation of DNA synthesis in cultured cells (55), stimulation of mitochondrial RNA synthesis by T3 (56), effects of thyroid hormone on myocardial or rat liver protein kinases (57, 58), and the exciting studies reporting stimulation of membrane transport by thyroid hormone within an hour of exposure of tissues to the hormone

5. DeGroot L.J., Strausser J.L. Binding of T3 in rat liver nuclei. Endocrinology 95: 74, 1974. 6. Oppenheimer JH, Schawrtz H.L., Koerner D., Surks M.I. Limited binding capacity sites for L-triiodothyronine in rat liver nuclei. Nuclear-cytoplasmic interrelation, binding constants, and cross-reactivity with L-thyroxine. J. Clin. Invest. 53: 768, 1974. 7. Bernal J., DeGroot L.J. Thyroid hormone receptors. Release of receptor to the medium during in vitro incubation of isolated rat liver nuclei. Endocrinology, 100: 648, 1977. 8. Surks M.I., Koerner D., Dillman W., Oppenheimer J.H. Limited capacity binding sites for L-triiodothyronine in rat liver nuclei. J. BioI. Chem. 245: 7066, 1973. 9. Oppenheimer J.H., Schwartz H.L., Dillman W. Surks M.I. Effect of thyroid hormone analogues on the displacement of 1251-L-triiodothyronine from hepatic and heart nuclei in vivo: possible relationship to hormonal activity. Biochem. Biophys. Res. Commun. 55: 544, 1973. 10. DeGroot L.J., Torresani J. Triiodothyronine binding to isolated liver cell nuclei. Endocrinology 96: 357, 1975.

11. Torresani J., DeGroot L.J. Triiodothyronine binding to liver nuclear solubilized proteins in vitro. Endocrinology 96: 1201, 1975. 12. Surks M.I., Koerner DH, Oppenheimer JH In vitro binding of L-triiodothyronine to receptors in rat live.r nuclei. Kinetics of binding. extraction properties. and lack of requirement for cytosol proteins. J. Clin. Invest. 55: 50, 1975. 13. Docter R., Visser T.J., Stinis J.T., Van Den Hout-Goemaat

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27. Charles M.A., Ryffel G.U., Obinata M., McCarthy B.J., Baxter J.D. Nuclear receptors for thyroid hormone: evidence for flonradom distribution within chromatin. Proc. Natl. Acad. Sci. USA 72: 1787, 1975.

14. Thomopoulos P., Dastugue B., Defer N. In vitro triiodothyronine binding to non-histone proteins from rat liver nuclei. Biochem. Biophys. Res. Commun. 58: 499, 1974.

28. Beatriz L.W., Baxter J.D. Distribution of thyroid and glucocorticoid hormone receptors in transcriptionally active and inactive chromatin. Biochem. Biophys. Res. Commun. 68: 1045 1976.

15. Spindler B.J., Macleod K.M., Ring J., Baxter J.D. Thyroid hormone receptors. Binding characteristics and lack of hormonal dependency for nuclear localization. J. BioI. Chem. 250: 4113,1975.

29. Macleod K.M., Baxter J.D.: DNA binding of thyroid hormone receptors. Biochem. Biophys. Res. Commun. 62:577, 1975.

16. DeGroot L.J., Torresani J., Carrayon P., Tirard A. Factors influecing triiodothyronine binding properties of liver nuclear receptors. Acta Endocrinol. 83: 293, 1976.

30. MacLeod K.M., Baxter J.D. Chromatin receptors for thyroid hormones. Interactions of the solubilized proteins with DNA. J. BioI. Chem. 251: 7380,1976.

17. Koerner D., Schwartz H.L., Surks M.I., Oppenheimer J.H. Binding of selected iodothyronine analogues to receptor sites of isolated rat hepatic nuclei. High correlation between structural requirements for nuclear binding and biological activity. J. BioI. Chem. 250: 6417,1975.

31. DeGroot L.J., Hill L., Rue P. Binding of nuclear triiodothyronine (T3) binding protein-T3 complex to chromatin. Endocrinology 99: 1605, 1976. 32. DeGroot L.J., Robertson M., Rue P.A. Triiodothyronine receptors during maturation. Endocrinology 100: 1511, 1977.

18. Jorgensen E.C., Bolger M.B., Dietrich SW. Thyroid hormone analogs: correlations between structure, nuclear binding and hormonal activity. Proc. V Inter. Cong. Endocrinol. Hamburg, Germany, 1976.

33. DeGroot L.J., Coleoni A.H., Rue P.A., Seo H., Martino E., Refetoff S. Reduced nuclear triiodothyronine receptors in starvation-induced hypothyroidism Biochem. Biophys. Res. Commun. 19. 113, 1977. 34. Bernal J., Coleonl A.H., DeGroot L.J. Thyroid hormone receptors: characteristics of receptor from normal, thyroidectomized, and triiodothyronine treated rats, and chromatin binding of empty and filled receptors. Submitted for publication.

19. Goslings B., Schwartz H.L., Dillmann W., Surks M.I., Oppenheimer J.H. Comparison of the metabolism and distribution of Ltriiodothyronine and triiodothyroacetic acid, in the rat: a possible explanation of differential hormonal potency. Endocrinology 98: 666, 1976. 20. Samuels H.H., Tsai J.S., Casanova J., Stanley F. Thyroid hormone action. In vitro characterization of solubilized nuclear receptors from rat liver and cultured GHl cells. J. Clin. Invest. 54: 853,1974.

35. Oppenheimer J.H., Schwartz H.L., Surks M.I. Nuclear binding capacity appears to limit the hepatic response to L-triiodothyronine (T3). Endocrin. Res. Commun. 2: 309, 1975.

21. Samuels H.H., Tsai J.S., Cintron R. Thyroid hormone activity: a cell culture system responsive to physiological concentrations of thyroid hormones. Science 181: 1253, 1973.

36. DeGroot L.J., Rue PA T3 receptor occupancy and metabolic responses. Submitted for publication. 37. Jensen EV, Suzuki T., Kawashima T., Stumpf W.E., Jungblut PW., Desombre E.R. A two-step mechanism for the interaction of estradiol with rat uterus. Proc. Natl. Acad. Sci. USA 59: 632, 1968. 38. O'Malley SW, Means A.R. Female steroid hormones and target cell nuclei. Science 183: 610, 1974.

22. Samuels H.H., Tsai J.S. Thyroid hormone action in cell culture: demonstration of nuclear receptors in intact cells and isolated nuclei. Proc. Natl. Acad. Sci. USA 70: 3488, 1973. 23. Samuels H.H., Stanley F., Shapiro L.E. Dose-dependent depletion of nuclear receptors by Ltriiodothyronine: evidence for a role in induction of growth hormone synthesis in cultured GHl cells. Proc. Natl. Acad. Sci. USA 73: 3877, 1976.

39. Refetoff S., Dewind L.T., DeGroot L.J. Familial syndrome combining deafmutism, stippled epiphyses, goiter, and abnormally high PBI: possible target organ refractoriness to thyroid hormone. J. Clin. Endocrinol. Metab. 27: 279, 1967.

24. Latham K.R., Ring J.C., Baxter J.D. Solubilized nuclear "receptors" for thyroid hormones. J. BioI. Chem. 251.' 7388, 1976. 25. DeGroot L.J., Refetoff S., Strausser J., Barsano C. Nuclear triiodothyronine-binding protein: partial characterization and binding to chromatin. Proc. Natl. Acad. Sci. USA 71: 4042,1974.

40. Refetoff S., DeGroot L.J., Benard B., Dewind L.T. Studies of a sibship with apparent hereditary resistance to the intraceliular action of thyroid hormone. Metabolism 21: 723,1972.

26. Gardner R.S. Nuclear thyroid hormone receptors: evidence for association with nucleolar chromatin. Biochem. Biophys. Res. Commun. 67: 625, 1975.

41. Bernal J., DeGroot L.J., Refetoff S., Fang V.S., Barsano C. Absent nuclear thyroid hormone receptors and failure of T 3-induced TRH suppression in the syndrome of periphe-

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Increased RNA polymerase activity in isolated liver nucleoli from thyroidectomized rats treated with triiodothyronine. Endocrinology 101: 209, 1977. 52. Seo H., Vassart G., Brocas H., Refetoff S. Triiodothyronine stimulates specifically growth hormone mRNA in rat pituitary tumor cells. Proc. Natl. Acad. Sci. USA 74: 2054, 1977.

42. Liewendahl K. Triiodothyronine binding to lymphocytes from euthyroid subjects and a patient with peripheral resistance to thyroid hormone. Acta Endocrinol. (Kbh) 83: 64, 1976. 43. Smuckler EA, Tata J.R. Changes in hepatic nuclear DNA-dependent RNA polymerase caused by growth hormone and triiodothyronine. Nature 234: 37, 1971.

53. Kurtz D.T., Sippel A.E., Feigelson P. Effect of thyroid hormones on the level of the hepatic mRNA cl. 2/J globulin. Biochemistry 15. 1031, 1976. 54. Sterling K., Milch P.O The mitochondria as a site of thyroid hormone action. Excerpta Medica 361: 68, 1975.

44. Viarengo A., Zoncheddu A., Taningher M., Orunesu M. Sequential stimulation of nuclear RNA polymerase activities in livers from thyroidectomized rats treated with triiodothyronine. Endocrinology 97: 955, 1975.

55. Siegel E, Siegel EP. Triiodothyronine influences nuclear and extranuclear DNA in cultured human kidney cells. Nature 249: 139, 1974.

45. Jothy S., Bilodeau J.L., Champsaur H., Simpkins H. The early enhancement of rat liver deoxyribonucleic acid-dependent ribonucleic acid polymerase II activity by triiodothyronine. Biochem. J. 150: 133, 1975.

56. Barsano C.P., Degroot LJ., Getz G.S. The effect of thyroid hormone on in vitro rat liver mitochondrial RNA synthesis. Endocrinology 100: 52, 1977. 57. Gibson K., Tichonicky L., Kruh J. The effect of triiodothyronine on myocardial protein kinases. Mol. Cell. Biochem. 9: 79, 1975. 58. Kru J., Tichonicky L. Effect of triiodothyronine on rat liver chromatin protein kinase. Eur. J. Biochem. 62: 109, 1976.

46. DeGroot L.J., Rue PA, Robertson M., Bernal J., Scherberg N. Triiodothyronine stimulates nuclear RNA synthesis. Endocrinology 101: 1690, 1977. 47. Krawiec L., Montalbano CA, Gomez C.J. Influence of neonatal hypothyroidism upon transcription in isolated rat brain and liver nuclei. J. Neurochem. 26: 1181, 1976.

59 Goldfine 1.0., Simons C.G., Smith G.J., Ingbar S.H. Cycloleucine transport in isolated rat thymocytes: in vitro effects of triiodothyronine and thyroxine. Endocrinology 96: 1030, 1975.

48. Blatt L.M., Kim K-H, Cohen P.P. The effect of thyroxine on ribonucleic acid synthesis by premetamorphic tadpole liver cell suspensions. J. BioI. Chem. 244: 4801, 1969.

60. Goldfine 1.0., Smith G.J., Simons C.G., Ingbar S.H., Jorgensen EC. Activities of thyroid hormones and related compounds in an in vitro thymocyte assay. J. BioI. Chem. 251: 4233, 1976.

49. Bernal J., Coleoni AH, DeGroot L.J. Triiodothyronine stimulates synthesis of nuclear proteins. Endocrinology, in press. 50. Kim K-H, Cohen P.P. Modification of tadpole liver chromatin by thyroxine treatment. Proc. Natl. Acad. Sci. USA 55: 1251, 1966.

61. Oppenheimer J.H., Schwartz H.L., Surks M.1. Tissue differences in the concentration of triiodothyronine nuclear binding sites in the rat: Liver, kidney, pituitary, heart, brain, spleen, and testis. Endocrinology 95: 897, 1974.

51. Zoncheddu A., Viarengo A., Accomando R., F. 1assa E, Orunesu M.

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