Distinct Tissue-Specific Roles for Thyroid Hormone Receptors ß and {alpha}1 in Regulation of Type 1 Deiodinase Expression

Lori L. Amma, Angel Campos-Barros, Zhendong Wang, Björn Vennström and Douglas Forrest

Department of Human Genetics (L.L.A., A.C.-B., Z.W., D.F.) Mount Sinai School of Medicine New York, New York 10029
Laboratory of Developmental Biology (B.V.) CMB, Karolinska Institute Stockholm, S-17 177, Sweden


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Type 1 deiodinase (D1) metabolizes different forms of thyroid hormones to control levels of T3, the active ligand for thyroid hormone receptors (TR). The D1 gene is itself T3-inducible and here, the regulation of D1 expression by TR{alpha}1 and TRß, which act as T3-dependent transcription factors, was investigated in receptor-deficient mice. Liver and kidney D1 mRNA and activity levels were reduced in TRß-/- but not TR{alpha}1-/- mice. Liver D1 remained weakly T3 inducible in TRß–/– mice whereas induction was abolished in double mutant TR{alpha}1–/–TRß–/– mice. This indicates that TRß is primarily responsible for regulating D1 expression whereas TR{alpha}1 has only a minor role. In kidney, despite the expression of both TR{alpha}1 and TRß, regulation relied solely on TRß, thus revealing a marked tissue restriction in TR isotype utilization. Although TRß and TR{alpha}1 mediate similar functions in vitro, these results demonstrate differential roles in regulating D1 expression in vivo and suggest that tissue-specific factors and structural distinctions between TR isotypes contribute to functional specificity. Remarkably, there was an obligatory requirement for a TR, whether TRß or TR{alpha}1, for any detectable D1 expression in liver. This suggests a novel paradigm of gene regulation in which the TR sets both basal expression and the spectrum of induced states. Physiologically, these findings suggest a critical role for TRß in regulating the thyroid hormone status through D1-mediated metabolism.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Type 1 iodothyronine deiodinase (D1) in peripheral tissues converts the main product of the thyroid gland, T4 into T3, the ligand of the thyroid hormone receptor (TR) (1, 2, 3). Although the thyroid gland secretes T3, up to 70% of circulating T3 may be derived from 5'-deiodination of T4 by D1 (4, 5, 6, 7). D1 also has an inactivating 5-deiodination role that converts T4 to rT3 and T3 to T2 (3,3'-diiodothyronine), products that do not activate the TR (8). D1 is abundant in liver and kidney, where it is itself induced by T3 and suppressed by hypothyroidism in a form of autoregulation that adapts to changes in the thyroid hormone status (2, 9, 10). T3 induces D1 expression at the transcriptional level, and T3 response elements (T3REs) have been identified upstream of the human D1 gene (11, 12, 13). Despite the physiological importance of D1, the TR pathways that control D1 expression are unknown.

TRs are T3-dependent transcription factors and belong to the family of nuclear receptors (14, 15). Distinct genes encode the TR{alpha}1 and TRß receptors, which are closely related in their central DNA binding and C-terminal T3-binding domains but which diverge in their N termini. In vitro, TRs can mediate both T3-dependent and T3-independent transcriptional control (16, 17, 18). TR{alpha}1 and TRß can transactivate through similar T3REs, although cotransfection assays also suggest that they differ in their regulation of certain genes including the TRH and pcp-2 genes (19, 20). Such distinctions may reflect TR structural differences that confer preferences in DNA binding or transactivation in T3RE-specific fashion (21, 22).

TR{alpha}1 and TRß have both specific and common roles in vivo, as revealed in TR-deficient mouse strains. TR{alpha}1–/– mice have a reduced heart rate (23) whereas TRß–/– mice exhibit deafness and a hyperactive pituitary-thyroid axis (24). TRß–/– mice provide a recessive model of human resistance to thyroid hormone (RTH), which is associated with TRß mutations (25). TR{alpha}1–/–TRß–/– double mutant mice are runted and have an array of exacerbated phenotypes, indicating the existence of common pathways in which TR{alpha}1 and TRß cooperate with or can substitute for each other (26, 27, 28).

Here, the receptor mechanisms that regulate D1 expression in vivo were investigated using TR-deficient mice. The results show that TRß has the major role in regulating D1 expression in liver and kidney. Remarkably, the deletion of all known TRs not only abolished T3-inducibility but also abrogated basal expression of D1 in liver. Thus, the D1 gene illustrates a novel paradigm of regulation where, rather than modulating expression around a basal level determined by other factors, TRs set basal as well as T3-inducible expression. Physiologically, the findings suggest that D1 deficiency may contribute to the hormonal imbalances caused by TRß mutations in mice or in human RTH syndrome.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
D1 Abnormalities in TR-Deficient Mice
In TRß–/– mice, liver D1 activity and mRNA levels were reduced to <=30% of wild type (wt) levels (P < 0.001), demonstrating that TRß was required for the maintenance of basal D1 expression (Fig. 1AGo). The D1 deficiency occurred despite the approximately 3-fold elevated thyroid hormone levels (total T4, TT4, and total T3, TT3), reported previously (24) (Table 1Go). This emphasized the need for TRß for sustaining D1 expression as T3 increases would normally induce D1. A similar trend occurred in kidney, where D1 mRNA and activity levels were reduced approximately 50% (P < 0.05) below normal.



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Figure 1. Abnormalities in D1 Activity and mRNA Expression in Liver and Kidney in TR-Deficient Mice

TRß–/– (panel A), TR{alpha}1–/– (panel B), and TR{alpha}1–/–TRß–/– (panel C) mice. Liver and kidney D1 activity was significantly reduced in TRß–/– mice (***, P < 0.001; *, P < 0.01, respectively, compared with wt) and in TR{alpha}1–/–TRß–/– mice (***, P < 0.001). In TR{alpha}1–/– mice, D1 activity increased slightly in liver (***, P < 0.001) and kidney (**, P < 0.01). Sample groups contained tissue from four to nine mice; mRNA levels were determined on pooled samples from three to five mice. Relative levels of mRNA, indicated numerically below Northern lanes, were normalized to G3PDH. G3PDH bands detected are shown below each D1 Northern. UD, Undetectable.

 

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Table 1. Thyroid Hormone Levels in TR-Deficient Mice under Different Treatments

 
In contrast, in TR{alpha}1–/– mice, liver D1 expression was not reduced but was 2-fold elevated (P < 0.01, Fig. 1BGo). In accord with the above indicated role for TRß, this probably represents a TRß-mediated response to the slightly elevated TT3 levels present in TR{alpha}1–/– mice (see below) (Table 1Go). Kidney D1 activity was slightly (1.49-fold) elevated in TR{alpha}1–/– mice (P < 0.01), although this was not accompanied by corresponding increases in mRNA levels (see Fig. 3Go, C and D).



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Figure 3. Kidney D1 Activity and mRNA Expression in TR-Deficient Mice under ND, Hypo- (LID), or Hyperthyroid (LID + 0.5 or 5.0 µg/ml T3, Respectively) Conditions

A and B, D1 induction was abolished in TRß–/– mice. C and D, Normal dose-dependent increase in D1 expression is present in TR{alpha}1–/– mice (**, P < 0.01). E and F, Neither D1 activity nor mRNA expression was induced in TR{alpha}1–/–TRß–/– mice. Activity was determined for groups of four to eight mice and mRNA levels with pools of three to five mice. The y axis is broken to accommodate the low levels of activity measured in most samples.

 
In TR{alpha}1–/–TRß–/– mice, a distinct phenotype arose that represented an extreme form of the defect in TRß–/– mice (Fig. 1CGo). Liver D1 activity fell to the detection limit and was <= 0.03% of normal, while mRNA levels were undetectable despite very high TT3 levels that would normally induce D1 (Table 1Go) (27). The sensitivity of the Northern assay was high as poly(A)-selected mRNA was analyzed, and prolonged exposures failed to detect signals (see Materials and Methods), suggesting that any putative, residual D1 mRNA expression was at very low levels. The results indicate a requirement for either TR{alpha}1 or TRß to sustain any detectable D1 expression in liver. In contrast, kidney D1 expression was only 50–60% reduced, resembling the kidney D1 deficiency in TRß–/– mice (see Fig. 3Go). Thus, unlike in liver, the additional loss of TR{alpha}1 did not significantly worsen the phenotype in TR{alpha}1–/–TRß–/– mice, indicating a unique dependence on TRß with no detectable role for TR{alpha}1 in kidney.

D1 expression levels varied according to the genetic background of the strains that carried the TR mutations, which differed due to the gene targeting approaches used (see Materials and Methods). Liver D1 activity was 3- to 4-fold greater in wild-type (wt) mice on the 129/Sv x C57BL/6J (TRß+/+) than on the 129/OlaHsd x BALB/c (TR{alpha}1+/+) mixed backgrounds (Fig. 1, A and B, compare left hand columns in activity graphs). In kidney, a similar but less pronounced trend occurred. These data are consistent with reports that D1 activity is relatively high in the C57BL/6J strain, intermediate in a 129 substrain (129/J), and low in the BALB/c strain (29).

Liver D1 Regulation by T3 in TR-Deficient Mice
To investigate the TR specificity in the adaptive response of D1 expression to changes in T3 levels, TR-deficient mice were studied under normal, hypo-, and hyperthyroid conditions. Groups received a normal diet (ND) or were made hypothyroid using methimazole and a low iodine diet (LID), or were made hyperthyroid with graded doses of T3. The diet treatments produced the expected hypo- and hyperthyroid conditions in all wt and mutant strains (Table 1Go). Under ND, TT3 and TT4 were each elevated approximately 3-fold in TRß–/– mice and about 7- and 33-fold, respectively, in TR{alpha}1–/–TRß–/– mice. TT4 was normal and TT3 slightly increased in TR{alpha}1–/– mice. This varied somewhat from free T4 and T3 levels that were previously shown to be marginally low and normal, respectively, in TR{alpha}1–/– mice (23), possibly suggesting abnormalities in serum hormone binding in TR{alpha}1–/– mice.

In wt, TRß–/–, and TR{alpha}1–/– mice, hypothyroidism suppressed liver D1 activity and mRNA levels to and below the detection limit, respectively (Fig. 2Go). In TRß+/+ mice, intermediate hyperthyroid conditions (serum TT3 levels 10-fold above wt normal levels) increased D1 activity 4.2-fold and mRNA 6.5-fold above normal (Fig. 2Go, A and B). Higher T3 levels did not induce further increases. In TRß–/– mice, even high TT3 levels (84-fold above wt normal levels) only induced D1 activity 7-fold above the very low basal levels in TRß–/– mice (P < 0.001). This corresponded to an absolute activity level that was only approximately 1.5-fold above normal levels in wt mice. Thus, liver D1 induction was severely blunted but not abolished in TRß–/– mice, suggesting that TRß has a major role while TR{alpha}1 has a limited role or can partially substitute for TRß.



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Figure 2. Liver D1 expression in TR-Deficient Mice under ND or Hypo- (LID) or Hyperthyroid (LID + 0.5 or 5.0 µg/ml T3, Respectively) Conditions

A and B, Blunted induction of D1 by T3 in TRß–/– mice. The residual induction in TRß–/– mice was significant in response to both T3 doses (a and b, P < 0.001 compared with TRß–/– mice on ND). C and D, Normal T3-responsiveness of D1 expression in TR{alpha}1–/– mice. E and F, In TR{alpha}1–/–TRß–/– mice on T3 treatment (5.0 µg/ml T3), D1 activity showed no detectable increase and D1 mRNA only a diminutive increase compared with ND (c, P < 0.05). The lower range of the y axis is amplified to show the very low levels of activity being measured in some samples. There was no detectable increase in D1 mRNA. Activity was determined for groups of four to nine mice and mRNA levels with samples of pooled from three to five mice. Relative levels of mRNA were normalized to G3PDH. UD, Undetectable.

 
In both wt and TR{alpha}1–/– mice, T3 stimulated normal increases in liver D1 expression, indicating that TR{alpha}1 was nonessential for D1 induction, in contrast to the requirement for TRß (Fig. 2Go, C and D). Although the degree of increased expression was greater in TR{alpha}1+/+ than in TRß+/+ mice, the absolute levels attained were comparable, suggesting that the greater degree of increase in TR{alpha}1+/+ mice was due to the lower initial D1 levels on this genetic background.

To ascertain whether the residual T3 inducibility of D1 in TRß–/– mice was mediated by TR{alpha}1, responses to changes in T3 levels were determined in TR{alpha}1–/–TRß–/– mice (Fig. 2Go, E and F). Liver D1 mRNA was undetectable and D1 activity fell to the detection limit in TR{alpha}1–/–TRß–/– mice under ND, a phenotype that was similar to the suppression of D1 caused by hypothyroidism in wt mice. Even high T3 doses failed to induce any detectable D1 mRNA. This indicated that TR{alpha}1 accounts for the residual basal and T3-inducible expression of liver D1 in TRß–/– mice.

A marginal increase in D1 activity (P < 0.05) above the almost undetectable levels in TR{alpha}1–/–TRß–/– mice (Fig. 4Go, A and B) was observed in the presence of very high (>180-fold above normal wt levels) TT3 levels. Although non-TR-mediated responses to T3 have been suggested in other systems (30), this is not likely to explain the present data given the failure of such high TT3 levels to induce a more significant D1 increase. The diminutive increase may reflect variability in the D1 assay at the detection limit.



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Figure 4. TR Expression in TR-Deficient Mouse Strains

A, EMSA of TR proteins detected in nuclear extracts from liver and kidney from wt (+/++/+) TR{alpha}1–/–, TRß–/–, and TR{alpha}1–/–TRß–/– mice using the DR4 response element as a probe. EMSA was performed in the absence or presence of TR and RXR antibodies to identify specific bands representing TR{alpha}1 and TRß (*). The bands that were shifted by antibodies against RXR but remained intact in the presence of antibodies against TR were likely to represent other RXR complexes, bound with other nuclear receptors. A ß-fibrinogen probe was used as a control for the integrity of nuclear proteins in samples. B, TR{alpha}1 mRNA expression was similar in wt and TRß–/– mice. Expression remained at basal levels in response to hypothyroidism (LID), but was slightly reduced under hyperthyroidism (T3 treated) in all samples except TRß–/– kidney. C, TRß mRNA expression was similar in wt and TR{alpha}1–/– mice and remained constant under T3 treatment.

 
Kidney D1 Regulation by T3 in TR-Deficient Mice
Hypothyroidism suppressed kidney D1 expression in wt and TRß–/– mice (Fig. 3Go, A and B). Unlike in liver, however, significant activity and mRNA levels were still detectable, indicating that D1 was less sensitive to T3-dependent regulation in kidney than in liver. In wt mice, T3 induced dose-dependent increases in D1 expression whereas in TRß–/– mice, no significant increase occurred, even with high T3 doses. This total block of induction indicated an absolute requirement for TRß for induction of kidney D1 expression. Kidney D1 expression responded normally in wt and TR{alpha}1–/– mice to changes in T3 levels (Fig. 3Go, C and D). Thus, TRß but not TR{alpha}1 was essential for regulation of D1 expression in kidney.

Kidney D1 expression in TR{alpha}1–/–TRß–/– mice under ND was reduced by approximately 50% (P < 0.001, Fig. 3Go, E and F), which was markedly less severe than in liver where D1 was almost abolished (Fig. 2Go, E and F). Thus, in the absence of TRs, basal expression of D1 is set at a higher level in kidney than in liver. In wt mice under hypothyroid conditions, the suppressed D1 activity was not significantly different from the deficient values in TR{alpha}1–/–TRß–/– mice (P > 0.78). The reduced D1 activity in TR{alpha}1–/–TRß–/– mice did not vary significantly under any condition (P > 0.20). In TR{alpha}1–/–TRß–/– mice, as in TRß–/– mice, kidney D1 expression was noninducible by T3 (Fig. 3Go, E and F). The absence of TR{alpha}1 functions in D1 induction indicated that TRß was solely responsible for the T3-dependent expression of D1 in kidney.

It is noteworthy that all of the T3-dependent regulation of D1 mRNA expression could be attributed to TRß and TR{alpha}1 in liver and to TRß in kidney. This supported the conclusion that TRß and TR{alpha}1 represent the entire complement of nuclear TRs and argued against the existence of other hypothetical TRs in these tissues (27, 28).

TR Gene Expression in TR{alpha}1–/– and TRß –/– Mice
To support a direct role for TRs in D1 gene regulation, the presence of TR{alpha}1 and TRß proteins in liver and kidney was demonstrated by electrophoretic mobility shift assay (EMSA) (27) (Fig. 4AGo). Using a DR4 T3RE as a probe, two specific shifted bands were detected in wt nuclear extracts. Antibodies against TR or retinoid X receptors (RXR) abolished or supershifted these bands, indicating that they represented TR-RXR heterodimeric complexes bound to the DNA. The lower TR-specific band was absent in TR{alpha}1–/– extracts, the upper band in TRß–/– extracts, and both bands in TR{alpha}1–/–TRß–/– extracts, consistent with the bands representing TR{alpha}1 and TRß, respectively. Although not quantitative, the EMSA suggested that in liver the presumptive TRß band was more abundant than that of TR{alpha}1, whereas in kidney, TR{alpha}1 was somewhat more abundant.

To determine whether changes in TR{alpha}1 expression in TRß–/– mice could explain the ability of TR{alpha}1 to substitute for TRß in liver but not in kidney, TR{alpha}1 mRNA levels were investigated (Fig. 4BGo). Over- or underexpression of TR{alpha}1 was excluded as TR{alpha}1 mRNA levels were similar in both wt and TRß–/– mice. T3 administration led to a slight decrease in TR{alpha}1 mRNA in liver in both wt and TRß–/– mice, agreeing with previous reports for rat liver (31). Conversely, TRß mRNA was not up-regulated in the absence of TR{alpha}1 (Fig. 4CGo), suggesting that the normal D1 regulation in TR{alpha}1–/– mice was achieved through normal levels of TRß. The lack of major changes in TR{alpha}1 expression in TRß–/– mice suggested that tissue-specific differences other than changes in TR expression levels account for the restriction of TR{alpha}1 function to liver.

Weight Changes in Hypo- and Hyperthyroidism
To rule out major differences in the general condition of TRß–/– and TR{alpha}1–/– mice as an influence over the D1 phenotypes, weight gain was assessed under the hypo- and hyperthyroid treatments (Fig. 5Go). For wt mice, hypothyroidism produced a decline in body weight, which was reversed with moderate T3 doses (compare Fig. 5AGo to 5B and 5C). Very high T3 doses, however, did not rescue weight gain but typically caused further loss of weight, presumably through hyperstimulated metabolism (Fig. 5Go, D, E, and F). Both TRß–/– and TR{alpha}1–/– mouse strains showed a similar response as wt mice, indicating that their overall responses were not grossly impaired. The sole exception was that very high T3 doses in TRß–/– mice resulted in a weight gain rather than loss (Fig. 5EGo). This suggested that TRß mediated the weight loss caused by T3 excesses in wt mice. In TR{alpha}1–/–TRß–/– mice, neither hypo- nor hyperthyroidism produced significant changes in weight (Fig. 5DGo), consistent with these mice lacking all nuclear TRs.



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Figure 5. Weight Responses of wt, TRß–/–, TR{alpha}1–/–, and TR{alpha}1–/–TRß–/– Mice to Hypo- and Hyperthyroid Conditions

A, Adult wt, TR{alpha}1–/–, and TRß–/– strains under ND showed progressive weight gain with time. B, C, E, and F, Under LID, wt, TRß–/–, and TR{alpha}1–/– exhibited moderate decreases in body weight. Supplementation with 0.5 µg/ml T3 ameloriated this effect. Higher T3 doses (5.0 µg/ml) led to a further decrease in body weight in all but TRß–/– mice. D, TR{alpha}1–/–TRß–/– mice showed no response either to hypo- (LID) or hyperthyroidism (+T3) conditions. Groups contained 9–10 mice except TR{alpha}1–/–TRß–/– and wt controls which contained n = 4.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
This study reveals the critical role of TRs in the control of expression of the D1 gene in its natural context in vivo. The most fundamental role was the obligatory requirement for a TR, whether TR{alpha}1 or TRß, for any detectable D1 expression in liver. Thus, the D1 gene in liver illustrates a novel paradigm of regulation in which TRs set the basal expression as well as the spectrum of T3-induced states. This contrasts with the scenario suggested by many cotransfection and in vivo studies in which the TR modulates expression around a basal level set by other factors. There is less dependence on TRs in kidney where there is substantial basal expression of D1 in the absence of TRs. Thus, other transcription factors play a more prominent role in kidney than in liver. This reveals differential utilization of TR signaling in the regulation of the same gene in different tissues.

On certain T3REs in vitro the TR exhibits a bimodal function, as it not only mediates T3-dependent activation but in the absence of T3, actively represses expression below basal levels (16, 17, 18). These T3-independent actions of the TR provide a possible mechanism for repression of basal transcription in hypothyroidism. For D1 expression, however, the deletion of all TRs caused an equally strong suppression as did hypothyroidism, indicating that TRs and any T3-independent function of TRs are unnecessary for suppression. Thus, models of exchange between repression and activation by the TR (32, 33) may have limited applicability in vivo and would vary depending on the target gene. Rather, the combined requirement for both the TR and T3 for any level of liver D1 expression is consistent with expression in this case being set by a continuous gradient of T3-dependent, positive activation states of the TR.

The utilization of such a positive mode of regulation, even in the sub-basal range of expression in liver, could account for the greater sensitivity of D1 induction in liver than in kidney (Figs. 2Go and 3Go). This may also confer upon the liver D1 enzyme a greater importance in response to modest fluctuations in thyroid hormone levels. In its highly tuned sensitivity and activation-based mode of regulation, this role of TRs is reminiscent of that of the positively inducible signaling pathways that activate cellular immediate early genes (34). As these mutant mice lack TRs throughout life, it is not excluded that uncharacterized developmental defects or other indirect changes, involving for example, other transcription factors, contribute to the deficiency in D1 expression.

The results demonstrate a clear TR isotype specificity in the T3-inducible expression of D1, which is mediated predominantly by TRß in liver and exclusively by TRß in kidney. This cannot be explained solely by differential expression of TR isotypes since both TRß and TR{alpha}1 are expressed at levels that may be expected to allow regulation of D1. Kidney contains relatively abundant levels of TRß and TR{alpha}1 mRNA (Fig. 4Go) (31, 35, 36, 37) and T3 binding proteins, as suggested by immunoprecipitation with antibodies against TRs (38). D1 expression is, however, enriched in the kidney proximal tubules (39, 40). Thus, segregation of TR isotypes in different cell types might in theory contribute to differential regulation by TRß and TR{alpha}1, although this seems unlikely given the widespread distribution of TR{alpha}1 in many tissues. Alternatively, differences in the structure or conformation of TR{alpha}1 and TRß could result in differential recognition of the target T3RE, exposure of activation domains, or interaction with cofactors (33, 41). Indeed, TR{alpha}1 tends to form monomeric and TRß homodimeric DNA binding complexes (21, 22). Two T3REs have been identified upstream of the human D1 gene (11, 12), but these do not occur in the mouse, despite D1 being T3 inducible in both species (42). Clarification of the physiologically relevant T3REs may allow investigation of the TR specificity in control of D1 expression.

The permissiveness of liver for some TR{alpha}1 function occurs despite TR{alpha}1 being expressed at relatively low levels in this tissue where it is approximately 4-fold less abundant than TRß (35, 38, 43). Several corepressors and coactivators have been shown to modify TR activity in vitro (32, 33, 41, 44, 45) and conceivably, tissue- or TR isotype-specific cofactors in liver could facilitate, or in kidney preclude, a role for TR{alpha}1. A precedent may be the differential interaction of the SMRT corepressor with retinoic acid receptors {alpha}, ß, or {gamma} (46). Thus, intrinsic differences between TR isotypes as well as tissue-specific factors are likely to extend the specificity of the functions of TRß and TR{alpha}1 in different physiological situations.

Interestingly, TRß has been suggested to have the primary role in other liver functions as well as in the control of D1 expression. Thus, TRß–/– mice show defective T3-dependent regulation of cholesterol metabolism (47) and impaired T3-inducibility of malic enzyme and spot 14 mRNAs (48).

TRß is known to have a critical role in the feedback control of the pituitary-thyroid axis and the thyroidal secretion of thyroid hormones, as TRß–/– mice or human RTH patients with TRß mutations have goiter and overproduce thyroid hormones (24, 25, 49). The present study extends the functions of TRß to regulating the thyroid hormone status at the level of the peripheral metabolism of thyroid hormones. This raises the possibility that the thyroid hormone excesses caused by TRß mutations are due, in part, to D1 deficiency. RTH typically results from heterozygous TRß mutations that generate dominant inhibitory proteins (25), and virally mediated expression of such proteins in mice impairs the T3-induction of liver D1 mRNA (50). Our results predict that in RTH, any D1 deficiency would be the result of inhibition of a TRß rather than TR{alpha}1 pathway.

The consequences of D1 deficiency may be complex given that D1 has alternative activities that either convert T4 into T3 or that inactivate T3 (8). Assuming that the induction of D1 by T3 serves to protect against excesses of active hormone through the inactivating role of D1, then D1 deficiency may reduce hormone clearance rates. This could cause the accumulation of serum T4 and T3. Partial D1 deficiency, attributed to changes in the 5'-region of the D1 gene, occurs in the C3H/HeJ mouse strain (29, 51), and these mice have also been suggested to have reduced T3 clearance rates (42). It has not been ruled out that changes in D1 activity in the pituitary and thyroid glands (10, 52) or changes in type 2 or type 3 deiodinases (2, 3) also contribute to the net hormone changes in TR-deficient mice.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mouse Strains
The TRß mutation, deleting both TRß1 and TRß2 products, was maintained on a mixed background of 129/Sv x C57BL/6J strains (24); the TR{alpha}1 mutation was on a 129/OlaHsd x BALB/c background (23). The strains were crossed to derive TR{alpha}1-/-TRß-/- double mutant mice that were devoid of detectable nuclear T3 binding capacity in brain and liver (27). wt mice were derived with the same genetic backgrounds as each mutant strain. Adult males, ranging in age at the start of experiments from 6–11 weeks (TR{alpha}1–/–TRß–/–) and 6–8 weeks (TR{alpha}1–/–, TRß–/–) were studied. Mice were housed with 12-h light/12-h dark cycles. All animal experiments followed approved institutional protocols.

Diet Treatments
ND-fed mice received defined pellets containing iodine at 5 mg/kg (ICN Biochemicals, Inc., Cleveland OH) and distilled drinking water for 4 weeks. Other groups were fed the same pellets with reduced iodine (0.05 mg/kg) and were provided with water containing 0.05% MMI and 1% potassium perchlorate (KClO4) (LID) to induce hypothyroidism, for 4 weeks. Subgroups under LID were made hyperthyroid by the addition of T3 at 0.5 µg/ml or 5.0 µg/ml in the drinking water containing MMI/KClO4 (LID + T3), for an additional 8–14 days. Given the limited numbers of TR{alpha}1–/–TRß–/– double mutant mice available (due to fertility problems and the complex breeding program) (27), these groups received ND, LID, or LID with only a single T3 dose (5.0 µg/ml).

RNA Analysis
Poly (A)-selected mRNA was prepared from pooled liver or kidney samples (TR{alpha}1–/– and TRß–/– strains, n = 5 each; TR{alpha}1–/–TRß–/– strain, n = 3–5). Northern blots were prepared as described (36) using as a D1 probe, a 357-bp fragment from the mouse D1 that was cloned by RT-PCR, using primers homologous to rat D1 sequence (5'-primer, 5'-CCGGTCGACGCTGAGATGGGGCTGCCCCAGCTATG-3'; 3'-primer, 5'-CAGCA-GGGGTCTGCTGCCTTGAATGAAATCCCAGACGTTGCA-CTT-3') (9). Sequence analysis verified the identity of the resulting clone. Northern lanes contained 5 µg of poly(A)-selected mRNA for all samples except TR{alpha}1–/–TRß–/– and their wt control samples which contained 7.5 µg. A mouse glyceraldehyde-3-phosphodehydrogenase (G3PDH) probe was used to ascertain integrity and quantity of RNA samples. Signals were recorded by autoradiography (1- to 2-day exposure) and were quantified using a Phosphorimager (Molecular Dynamics, Inc., Sunnyvale, CA). In D1-deficient samples (from liver in TR{alpha}1–/–TRß–/– mice and LID-treated groups) prolonged exposures (1–2 weeks) were used but failed to detect additional signals. Moreover, spacer lanes were used to avoid possible spillover between D1-expressing and D1-deficient sample lanes. Spacer lanes were cut out from final figures.

D1 Enzyme Assays
Liver and kidney (n = 4–9) samples were homogenized individually on ice in 5–6 vol of 25% (wt/vol) sucrose in 10 mM HEPES (pH 7.0) containing 10 mM dithiothreitol (DTT) and frozen immediately. 5'-D1 activity was determined in diluted aliquots of the homogenate by the release of radioiodide from 10 µM (5'-125I]rT3 (NEN Life Science Products-Dupont, Boston, MA) in the presence of 5 mM DTT, as described previously (53). The addition of 6-n-propyl-2-thiouracil (PTU, 1 mM) inhibited activity, thus confirming that the measured activity was that of D1 (as opposed to PTU-resistant D2). Reactions were adjusted so that the production of iodide was directly proportional to the incubation times (30–60 min; prolonged times for deiodinase-deficient samples enhanced detection at the lower limits of sensitivity). Heart, which does not normally express D1, served as a negative control.

EMSAs
The DR4 DNA probe sequence was: 5'-GGAGCTTCAGGTCACTTCAGGTCA-AGCT-3' and the ß-fibrinogen probe was as described (27). Nuclear protein extracts were prepared and EMSAs performed as described previously, using the F2 T3RE (27). Supershift assays were performed with antibodies against mouse RXR (4RX-1D12), which detect all three RXRs (a kind gift of Dr. P. Chambon), and against full-length TRß, which detects both TR{alpha} and TRß (27).

Hormone RIAs
Blood was collected when mice were killed, and serum was separated by centrifugation at 2000 x g and immediately frozen. RIAs were performed for total T3 and T4 using antibodies (Sigma, St. Louis, MO) and [5'-125I]-T4 and [3'-125I]T3 (NEN Life Science Products-Dupont) tracers, as described previously (54).


    ACKNOWLEDGMENTS
 
We are grateful to Dr. P. Chambon for antibodies against RXR. We thank Dr. L. Ng for comments on the manuscript and I. Lisoukov for technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Dr. Douglas Forrest, Department of Human Genetics, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, New York 10029.

This work was supported in part by the Human Frontiers Science Program, March of Dimes Birth Defects Foundation, NIH and the Hirschl Trust (D.F.), the Swedish Cancer Foundation (B.V.), and an NIH Predoctoral Training Grant (T32-HD-07105, L.L.A.). A.C.B. received support from the Spanish Ministry of Culture and Education Grant 97 PF 00679951.

Received for publication October 6, 2000. Revision received November 20, 2000. Accepted for publication November 21, 2000.


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 RESULTS
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 MATERIALS AND METHODS
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