Requirement for Thyroid Hormone Receptor ß in T3 Regulation of Cholesterol Metabolism in Mice
Hjalmar Gullberg,
Mats Rudling,
Carmen Saltó,
Douglas Forrest,
Bo Angelin and
Björn Vennström
Department of Cell and Molecular Biology (H.G., C.S., B.V.), Karolinska Institute, S-171 77 Stockholm, Sweden; Centers for Metabolism and Endocrinology and Nutrition and Toxicology (M.R., B.A.), Department of Medicine, Karolinska Institute at Huddinge University Hospital, S-141 86 Stockholm, Sweden; and Department of Human Genetics (D.F.), Mount Sinai School of Medicine, New York, New York 10029
Address all correspondence and requests for reprints to: Dr. Björn Vennström, Department of Cell and Molecular Biology, Karolinska Institute, Room D427 Doktorsringen 2D, Solna, Sweden S-171 77. E-mail: bjorn.vennstrom{at}cmb.ki.se.
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ABSTRACT
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T3 potently influences cholesterol metabolism through the nuclear thyroid hormone receptor ß (TRß), the most abundant TR isoform in rodent liver. Here, we have tested if TR
1, when expressed at increased levels from its normal locus, can replace TRß in regulation of cholesterol metabolism. By the use of TR
2-/-ß-/- animals that overexpress hepatic TR
1 6-fold, a near normalization of the total amount of T3 binding receptors was achieved. These mice are similar to TRß-/- and TR
1-/-ß-/- mice in that they fail to regulate cholesterol 7
-hydroxylase expression properly, and that their serum cholesterol levels are unaffected by T3. Thus, hepatic overexpression of TR
1 cannot substitute for absence of TRß, suggesting that the TRß gene has a unique role in T3 regulation of cholesterol metabolism in mice. However, examination of T3 regulation of hepatic target genes revealed that dependence on TRß is not general: T3 regulation of type I iodothyronine deiodinase and the low density lipoprotein receptor were partially rescued by TR
1 overexpression. These in vivo data show that TRß is necessary for the effects of T3 on cholesterol metabolism. That TR
1 only in some instances can substitute for TRß indicates that T3 regulation of physiological and molecular processes in the liver occurs in an isoform-specific fashion.
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INTRODUCTION
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THYROID HORMONE (T3) is important for normal regulation of development and physiology. T3 deficiency leads to elevated cholesterol levels in blood plasma that can be normalized, however, by T3 substitution (1). The liver is central in cholesterol metabolism, balancing hepatic cholesterol synthesis and hepatic uptake of plasma lipoproteins from the circulation against the excretion of hepatic cholesterol and bile acids in the bile (2, 3, 4, 5). T3 can influence the metabolism of cholesterol at several critical steps in the liver (6, 7, 8, 9, 10, 11): the low-density lipoprotein (LDL) receptor (LDL-R), which mediates cholesterol uptake from the circulation, 3-hydroxy-3-methylglutaryl coenzyme A reductase, controlling cholesterol biosynthesis, and cholesterol 7
-hydroxylase (CYP7A1), the rate-limiting enzyme in the synthesis of bile acids where cholesterol is used as substrate.
Thyroid hormone receptors (TRs) are ligand-dependent transcription factors belonging to the nuclear receptor superfamily (12). TRs have a dual function in gene regulation: the ligand-receptor complex up-regulates many target genes, whereas the unliganded receptor often suppresses the basal level of transcription in a gene-specific manner (13, 14). The importance of the unliganded receptor is indicated by the relatively mild phenotype, as compared with congenital hypothyroidism, in TR
1-/-ß-/- mice, which lack all known TRs (15). The resistance to develop hypercholesterolemia in hypothyroid TRß-/- mice also suggests that unliganded TR can potently modulate serum cholesterol metabolism (16). TRs are encoded by the
and ß genes, which give rise to the ligand-binding proteins TRß1, TRß2, TRß3, and TR
1, respectively (17, 18, 19). The
gene also encodes the TR
2 protein that does not bind T3 although it binds to DNA with reduced affinity (20, 21). Its function in physiology is unclear (22). Both TR genes also encode truncated forms of receptor isoforms (19, 23). The TR
1 and TR
2 mRNAs are coexpressed in most tissues, with the latter typically being more abundant (24). Targeted mutations in mice indicate that TR
1 and TRß mediate distinct physiological effects (25, 26, 27, 28, 29, 30, 31).
We have previously ablated TR
1, TR
2, TRß1, and TRß2 in mice, individually or in combinations (15, 22, 25, 30, 32, 33). Deletion of the TR
1 isoform results in cardiac and thermoregulatory defects (27, 30, 34). Ablation of TR
2 expression has been achieved by insertion of a transcriptional termination signal that redirects the previously alternate splicing event to produce a single mRNA that encodes only TR
1, at levels that equal that in wild-type (wt) mice of TR
1 and TR
2 combined (22). This also results in an overexpression of TR
1 in TR
2-/-ß-/- mice (33). The TR
2-/- mice exhibit a mixed hypo- and hyperthyroid phenotype (22). This involves slightly low serum levels of T3 and T4 and inappropriately normal serum TSH, thyroid gland dysfunction, and elevated heart rate and body temperature. Lack of TRß results in auditory impairment and dysregulation of the pituitary-thyroid axis (25). Our previous experiments (16 0) identified TRß as the primary mediator of T3 action on CYP7A1 and as a major regulator of cholesterol metabolism in vivo: T3-deficient TRß-/- mice are resistant to challenge with dietary cholesterol and do not develop hypercholesterolemia to the same extent as their wt controls.
The present studies were undertaken to determine the receptor isoform specificity in cholesterol metabolism. First, we wanted to corroborate that TRs are necessary for the ability of T3 to decrease serum cholesterol, by studying mice devoid of all known TRs (TR
1-/-ß-/- mice) (15). CYP7A1 activity and serum cholesterol levels were found not to change in TR
1-/-ß-/- mice upon T3 treatment, suggesting that TRs are indeed necessary for the effects of T3 on cholesterol metabolism. Second, we tested whether the abundance of TRß in rodent liver (80% of all T3 binding activity) is the reason for the isoform specificity in cholesterol metabolism (35, 36). We therefore examined if an increased level of TR
1 could compensate for the absence of TRß by using the TR
2-/-ß-/- mice that overexpress TR
1 approximately 6-fold in their livers. These mice were similar to TRß-/- mice with regard to CYP7A1 and serum cholesterol: T3 failed to reduce serum cholesterol and did not stimulate CYP7A1 activity. Thus, the hepatic overexpression of TR
1 in the TR
2-/-ß-/- mice cannot substitute for the absence of TRß. The results highlight the specific importance of the TRß gene for regulation of cholesterol metabolism by T3.
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RESULTS
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Overexpression of TR
1 in TR
2-/-ß-/- Mice
To determine whether TR
1 can compensate for the absence of TRß in T3 regulation of serum cholesterol and CYP7A1 activity in liver, we crossed TRß-deficient mice (denoted Thrbtm1/tm1 in Ref. 33) with the TR
1 overexpressing TR
2-/- mice (Thratm2), thus creating a TR
2-/-ß-/- mouse (Thratm2/tm2Thrbtm1/tm1) (22, 33). To verify that overexpression of TR
1 also occurs in the liver as it does in cochlea and other organs of TR
2-/-ß-/- mice (33), we directly determined the hepatic expression of TR
1 by ribonuclease protection assay (RPA) (Fig. 1A
). When the protected TR
1 band in the TR
2-/-ß-/- mice was quantified, we found that the mice expressed about 6-fold higher levels of TR
1 mRNA as compared with wt controls, i.e. a near normalization of the total TR mRNA levels in liver. To confirm that this results in elevated receptor protein levels, hepatic nuclear T3 binding capacity in TR
2-/-, TR
2-/-ß-/-, TRß-/-, and wt mice was determined. TR
2-/-ß-/- mice displayed a near normalization of the binding capacity as compared with wt mice, TR
2-/- mice had a higher capacity, and TRß-/- mice had a severely decreased capacity (Fig. 1B
). This is consistent with the previous demonstration of a 3- to 4-fold increased T3 binding capacity in brain of TR
2-/- mice (22) and confirms that 80% of all T3 binding activity in rodent liver is mediated by TRß (35, 36). Taken together, the results indicate that TR
2-/-ß-/- mice have a near normalization of both hepatic TR mRNA content and nuclear T3 binding capacity.
Generation of Hypo- and Hyperthyroid Mice
The experimental setup has recently been described in detail (16) and is also described in Fig. 2
. To achieve maximal responses to T3 treatment, TR
1-/-ß-/-, TR
2-/-ß-/-, and their respective wt control mice were made hypothyroid before injection of the hormone. For this purpose the mice received a synthetic low iodine diet (LID), and 0.05% methimazole (MMI) and 1% potassium perchlorate (LID+MMI) was added to the drinking water. This regimen lowered free T4 and T3 in serum in all groups to hypothyroid levels (P < 0.01, compared with normal diet), without inducing severe hypothyroidism (Tables 1
and 2
). To induce hyperthyroidism, 5 µg T3 were injected daily for the last 5 d (Tables 1
and 2
; MMI+T3); this reduced serum T4 levels significantly (P < 0.01, compared with normal diet, Table 1
) in all mice and resulted in an increase in serum T3 (Table 2
). The small serum increase in free T3 after hormone injections in all mice except the TR
1-/-ß-/- mice was because the sampling was performed 24 h after the last injection. However, these mice do become functionally hyperthyroid by the T3 injections as indicated by their low serum cholesterol (Fig. 3
) and the elevated mRNA levels of the highly T3 responsive 5'deiodinase gene (Fig. 4
). The very high T3 levels resulting in TR
1-/-ß-/- mice suggest a defect in T3 metabolism and turnover in these mice (37).

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Figure 2. Experimental Setup
Altogether, 109 male mice aged 23.5 months (29 TR 1-/-ß-/-, 32 TR 1/ß wt mice, 25 TR 2-/-ß-/-, and 23 TR 2/ß wt mice) were used. Before the experiment, 23 animals (six TR 1-/-ß-/-, six TR 1/ß wt mice, five TR 2-/-ß-/-, and six TR 2/ß wt mice) were killed to obtain basal liver samples, T3, and serum cholesterol levels. Thereafter, four to ten KO and three to six of the respective wt animals were assigned to five groups as shown. LID, Low iodine diet; MMI, inclusion of both 0.05% methimazole and 1% potassium perchlorate in the drinking water; T3, daily injections of T3 for the last 5 d; , animals were killed for collection of serum and tissue.
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Figure 3. Total Serum Cholesterol (mmol/liter) in TR 2-/-ß-/-, TR 1-/-ß-/-, and Corresponding wt Mice
Determinations were done on serum obtained before treatment (normal diet) and after a LID fed for 14 d followed by the additional inclusion of both 0.05% methimazole and 1% potassium perchlorate in the drinking water for a further 21 d (MMI), resulting in hypothyroidism. Hyperthyroidism was induced by daily injections of T3 for the last 5 d of the regimen (MMI+T3). The two last animal groups (MMI+Cholesterol) and (MMI+T3+Cholesterol) were treated the same way as above, except that their food was supplemented with 2% cholesterol from d 14. Results are expressed as mean ± SEM. Number of animals in each group is indicated to the left of each bar. *, Statistical differences between wt and KO mice within each treatment. €, Statistical differences in total serum cholesterol between hypothyroid mice (MMI and MMI+Cholesterol) and hyperthyroid (MMI+T3 and MMI+T3+Cholesterol) mice. * And €, P < 0.05; ** and €€, P < 0.01; *** and €€€, P < 0.001.
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Figure 4. Hepatic mRNA Levels in TR 2-/-ß-/-, TR 1-/-ß-/-, TR 2-/-, and Corresponding wt Mice
Effect of T3 deprivation (MMI) and T3 treatment (MMI+T3), in the presence or absence of 2% cholesterol in the diet, of TR 2-/-ß-/- and corresponding wt mice (A), or TR 1-/-ß-/- and corresponding wt mice (B), on the abundance of mRNA for 5'DI-1, CYP7A1, LDL-R, and CYP8B1. Effect of T3 deprivation and T3 treatment of TR 2-/- and corresponding wt mice (C) on the abundance of mRNA for CYP7A1. Levels of mRNA expression were normalized to that of G3PDH, and results are expressed as mean correlated to the expression in wt mice on normal diet, which was set to 1.0.
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Abnormal Regulation Of Serum Total Cholesterol in TRß-Deficient Mice Fails to Be Rescued by TR
1 Overexpression
Because we previously showed that TRß is important for T3 to reduce serum cholesterol (16), we first tested the hypothesis that an increased level of TR
1 in liver may compensate for the absence of TRß. For this, TR
2-/-ß-/- mice that overexpress TR
1 were used. On normal diet, the serum total cholesterol level in TR
2-/-ß-/- mice was slightly higher as compared with that in wt mice (Fig. 3A
), a finding in accordance with the somewhat lower serum T3 and T4 levels in TR
2-/-ß-/- mice (Tables 1
and 2
). However, upon T3 deprivation, total serum cholesterol levels were only marginally increased (1.2-fold; P < 0.01) in these mice as compared with the 2.3-fold elevation in wt animals (P < 0.001 for wt vs. KO, MMI; Fig. 3A
). In addition, in the TR
2-/-ß-/- mice, the serum total cholesterol level was unaltered after T3 treatment, whereas a clear 4-fold reduction (P < 0.001) was seen in the controls. The differences between the TR
2-/-ß-/- and the wt mice were more pronounced when cholesterol was included in the diet during T3 deprivation (MMI+Cholesterol). Here, the TR
2-/-ß-/- and the control mice increased serum cholesterol levels 1.3-fold and 2.8-fold (P < 0.01), respectively, as compared with what was seen on a normal diet. T3 injections (MMI+T3+Cholesterol, Fig. 3A
) reduced the cholesterol levels only marginally in the TR
2-/-ß-/- mice, in contrast to the 3-fold reduction to below baseline values in the control mice. These findings were confirmed by analysis of lipoprotein cholesterol by fast performance liquid chromatography (FPLC) (not shown). T3 regulation of serum cholesterol levels in TR
2-/- was similar to that in wt mice (not shown).
The data indicate that the overexpressed TR
1 gene cannot compensate for the absent TRß in regulation of serum cholesterol by T3. To confirm that the ability of T3 to decrease serum cholesterol is mediated by TRs, we examined serum total cholesterol changes in response to alterations in T3 status in the TR
1-/-ß-/- mice, which lack all known T3 binding TRs (15). Despite the fact that these mice have grossly elevated T3 and T4 levels, their serum cholesterol level were similar to those in wt controls while on normal diet (Fig. 3B
). T3 deprivation (MMI) or T3 injections (MMI+T3), with or without dietary cholesterol, did not alter serum cholesterol levels in the TR
1-/-ß-/- mice, in contrast to what was observed in control mice. Similar results were obtained using FPLC analysis for separation of individual lipoproteins (not shown). This demonstrates that the presence of TRs is necessary for T3 to modulate serum cholesterol levels.
Altered Expression of Putative Hepatic Target Genes
To elucidate whether the failure of T3 to reduce serum cholesterol in TR
2-/-ß-/- mice was reflected by a general dependence on TRß for liver gene regulation by T3, the expression of putative target genes for TRs was surveyed in TR
2-/-ß-/-, TR
1-/-ß-/-, and corresponding wt mice under eu-, hypo- and hyperthyroid conditions (Fig. 4
). A classical T3 responsive gene, that for type 1 iodothyronine deiodinase (5'DI-1), was used to verify the responses to hypo- and hyperthyroidism. Figure 4
shows that 5'DI-1 mRNA levels were strongly decreased under hypothyroid conditions (MMI and MMI+Cholesterol) and increased by T3 (MMI+T3 and MMI+T3+Cholesterol) in both wt and TR
2-/-ß-/- mice, although the response in the mutant mice was somewhat lower as compared with the control mice. The mRNA data on 5'DI-1 was confirmed by 5'DI-1 enzyme activity measurements (data not shown). 5'DI-1 in the TR
1-/-ß-/- mice was totally unresponsive to T3 (Fig. 4B
) as also reported elsewhere (37). The responses in 5'DI-1 expression to changes in T3 status confirm that the experimental setup was appropriate and that the treatment made the animals functionally hypo- and hyperthyroid.
CYP7A1 is the rate-limiting enzyme in bile acid synthesis via the classical (neutral) pathway and is regulated in part by cholesterol and T3. CYP7A1 mRNA levels were reduced both in hypothyroid (MMI) wt and TR
2-/-ß-/- mice (Fig. 4A
), which also was reflected by reduced enzyme activity in the wt mice (Fig. 5A
). In the TR
2-/-ß-/- mice, T3 failed to elevate CYP7A1 mRNA levels (MMI+T3, Fig. 4A
) or enzyme activity (Fig. 5A
), contrasting the 4-fold increase seen in the wt mice (P < 0.05, MMI+T3, Fig. 5A
). Addition of cholesterol to the diet (MMI+Cholesterol) caused only a limited increase in CYP7A1 mRNA and activity in the wt mice, whereas in the TR
2-/-ß-/- mice both CYP7A1 mRNA and enzyme activity increased to a level 2 to 3 times higher than in the wt mice (P < 0.05 for wt vs. KO, MMI+Cholesterol, Fig. 5A
). This indicates that TRß is obligatory for T3 to increase CYP7A1 activity.

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Figure 5. Quantitation of Liver CYP7A1 Enzyme Activity in TR 2-/-ß-/-, TR 1-/-ß-/-, and Corresponding wt Mice
The enzymatic activity of CYP7A1 was determined in liver microsomes as described in Materials and Methods. Three separate pools of liver were prepared from each animal group containing four to six animals. Bars indicate means and error bars indicate SEMs. *, Statistical differences in CYP7A1 activity between wt and KO mice within each treatment. €, Statistical differences between hypothyroid mice (MMI and MMI+Cholesterol) and hyperthyroid (MMI+T3 and MMI+T3+ Cholesterol) mice. #, Statistical differences between hypothyroid mice before and after addition of 2% cholesterol in the diet. *, , €, and #, P < 0.05; **, €€, and ##, P < 0.01; ***, €€€, and ###, P < 0.001.
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Furthermore, in TR
2-/- mice, hormonal regulation of CYP7A1 mRNA is similar to that in wt mice (Fig. 4C
), supporting the hypothesis that TRß specifically regulates the CYP7A1 gene in vivo.
In mice devoid of all ligand-binding TRs (TR
1-/-ß-/- mice), T3 had no clear stimulatory effect on CYP7A1 mRNA levels (Fig. 4B
) or enzyme activity (Fig. 5B
). These mice had high CYP7A1 mRNA levels and enzyme activity regardless of serum T3 levels or cholesterol diet. The corresponding wt mice, on the other hand, had lower enzymatic activity under hypothyroid conditions (P < 0.001 for wt vs. KO, MMI, Fig. 5B
), which was stimulated 4-fold by T3 injections (P < 0.001, MMI+T3; Fig. 5B
), and 2-fold by dietary cholesterol (P < 0.05, MMI+Cholesterol; Fig. 5B
). A further 2-fold increase was seen after injecting T3 while on cholesterol diet (MMI+T3+Cholesterol, Fig. 5B
).
Hepatic LDL-Rs are important for the removal of LDL from plasma. LDL-R mRNA levels were induced by T3 in both TR
2-/-ß-/- and wt mice (Fig. 4
). In contrast, TR
1-/-ß-/- mice had higher LDL-R mRNA levels under euthyroid and hypothyroid conditions, whereas there was no effect of T3 treatment. The liver enzyme sterol 12
-hydroxylase (CYP8B1) is involved in cholesterol degradation to bile acids and is responsible for the synthesis of cholic acid (38). CYP8B1 mRNA levels in rat liver have previously been shown to be suppressed by T3 (39). Our data confirm this in the wt mice (Fig. 4
), but T3 failed to suppress CYP8B1 mRNA levels in TR
2-/-ß-/- or TR
1-/-ß-/- mice, indicating that TRß is responsible for the T3 regulation of this enzyme.
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DISCUSSION
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Previously we showed that TRß is a major mediator of T3 effects on serum cholesterol and that it is involved in the transcriptional regulation of the CYP7A1 gene (16). In the current study we show that this dependence on TRß is not simply due to its, in relation to TR
1, high hepatic expression, because overexpression of TR
1 in the TR
2-/-ß-/- mice does not substitute for the ablated TRß. This is in contrast to the rescue of deafness and normalization of serum T3 and T4 recently reported for the TRß-deficient TR
2-/-ß-/- mice (33).
The dependence on TRß for T3 regulation of serum cholesterol levels is supported by a recent report in which the TRß-selective agonist GC-1 was as efficient as T3 in decreasing serum cholesterol in hypothyroid mice (40).
A role for TR
2 in cholesterol metabolism cannot be completely ruled out. It was recently suggested that deletion of TR
2, in an ablation of all TR
products [TR
0/0 (34)], increases sensitivity to T3 in reduction of serum cholesterol. In such a scenario, TR
2 could be hypothesized to modulate the activity of TRß on cholesterol metabolism. Our present results do not address this issue directly, due to differences in experimental design. In the TR
2-/-ß-/- mice the overexpression of TR
1 does not rescue the abnormal serum cholesterol regulation in the absence of TRß. Moreover, T3 regulation of serum cholesterol levels in TR
2-/- mice was similar to that found in wt mice (data not shown), thus indicating that TR
2 does not have a profound role in these processes. It has also been shown that TR
2 is unable to bind T3 and binds DNA with reduced affinity (20, 21), thus excluding that TR
2 mediates T3 effects on serum cholesterol levels directly. Taken together, this suggests that the inability to substitute for TRß in cholesterol metabolism by overexpression of TR
1 is not caused by deletion of TR
2 per se. TR
2, however, may have other roles in modulating T3 receptor activity (22).
Rodents, which are normally resistant to high concentrations of dietary cholesterol, accumulate lipoproteins when made hypothyroid (41, 42). This hypercholesterolemic response is absent in mice lacking TRs (TR
1-/-ß-/- mice) (Fig. 3B
) and severely blunted in TRß-deficient mice even when TR
1 is overexpressed (TR
2-/-ß-/- mice) (Fig. 3A
). This resistance to develop hypercholesterolemia suggests that TRs are crucial for modulation of serum cholesterol levels, and that unliganded TR potently modulates serum cholesterol metabolism. The data also corroborate the importance of TRß in regulation of cholesterol metabolism.
Investigation of the expression of T3-regulated genes in liver revealed that the dependence on TRß is not a general finding for genes expressed in the liver. The classical T3-responsive gene, 5'DI-1 (43), has been shown previously to depend on TRß in the liver (37). We show that T3 responsiveness of 5'DI-1 could be partially rescued by overexpression of TR
1 in the TR
2-/-ß-/- mice (Fig. 4A
), suggesting that 5'DI-1 is not dependent on TRß per se.
The molecular mechanisms controlling CYP7A1 regulation by bile acids and cholesterol metabolites have recently been unveiled (44, 45). Liver X receptor-
(LXR
) and farnesoid X receptor are two ligand-dependent transcription factors that are receptors for derivatives of cholesterol and bile acids in the control of CYP7A1 expression (46, 47, 48, 49). LXR
, an oxysterol-binding transcription factor, directly activates CYP7A1 transcription in response to challenge with dietary cholesterol to mice; thus LXR
-/-mice fed cholesterol-rich diets fail to induce enzyme activity and therefore accumulate toxic levels of cholesterol in the liver (50). CYP7A1 is induced by T3 at the transcriptional level (8, 10, 11, 51), but additionally, GH is also required for normal CYP7A1 regulation in rats and mice (52, 53). Previous experiments suggested that TRß is the major mediator of T3 effects on CYP7A1 expression (16). Our new data on the TR
2-/-ß-/- and TR
1-/-ß-/- mice clarify how TRs regulate CYP7A1: in the absence of TRs (TR
1-/-ß-/- mice), neither cholesterol nor T3 stimulated CYP7A1 expression and activity (Figs. 4B
and 5B
). CYP7A1 mRNA expression and enzymatic activity remained on a high level in these mice regardless of the T3 status and irrespective of whether cholesterol was added to the diet or not. The blunted CYP7A1 stimulation in response to T3 confirms the importance of TRß. The absence of up-regulation in response to dietary cholesterol was at first unexpected, but is likely due to the critical dependence of normal CYP7A1 regulation on GH (52, 53, 54) and to the fact that TR
1-/-ß-/- mice have severely reduced GH levels (15).
In wt animals T3 deprivation decreases CYP7A1 enzyme activity (MMI, Fig. 5A
) much more potently than in the TR
2-/-ß-/- mice (P < 0.05 for wt vs. KO). Addition of cholesterol in the diet causes an induction of CYP7A1 activity in TR
2-/-ß-/- mice (Fig. 5A
) to a level 2- to 3-fold higher than in the wt mice. This is compatible with the hypothesis that the absence of TRß relieves repression so that liganded LXR
can strongly induce CYP7A1 transcription. The lack of CYP7A1 mRNA and enzyme activity stimulation by T3 furthermore shows that TR
1 cannot substitute for TRß in the regulation of CYP7A1. The observation that overexpression of TR
1 in the presence of TRß, as in TR
2-/- mice, does not affect T3 regulation of CYP7A1 (Fig. 4C
) is consistent with this.
Hepatic LDL-Rs mediate removal of LDL from plasma, and LDL-R mRNA expression is stimulated by T3 (9, 55, 56). In both wt and TR
2-/-ß-/- mice, in contrast to TR
1-/-ß-/- animals, T3 induces LDL-R mRNA (Fig. 4
), which indicates that, in contrast to the regulation of CYP7A1, TR
1 can substitute for TRß in T3 regulation of LDL-R mRNA levels (Fig. 4A
) and that T3 is unable to stimulate LDL-R expression in the absence of TRs. CYP8B1 in the liver is also modulated by T3 (38, 39), and our data (Fig. 4
) indicate that, similar to what was found for CYP7A1, the suppressive action of T3 on CYP8B1 is dependent on TRß and cannot be rescued by TR
1 overexpression.
There are several possible models for explaining the molecular mechanism behind the dependence or independence on TRß for T3 regulation of target genes. One possibility is that the promoter context determines the TR isoform that regulates expression of the target gene. This implies that TR
1 and TRß bind certain T3 response elements with different affinities. However, both isoforms have previously been shown to bind the T3 response element in the human 5'DI-I gene with similar affinities (57), and to transactivate equally well through many elements. Moreover, these receptors, in the absence of ligand, inhibit LXR
equally efficiently from activating through LXR response element in reporter gene assays (Gullberg, H., and B. Vennström, unpublished). A second model is that the different TR isoforms utilize cofactors differently. TR
1 and TRß could use different cofactors for modulation of gene expression, e.g. due to their distinct N-terminal regions. The availability of specific cofactors in a particular cell type would therefore govern which TR regulates the gene.
A third possibility is that the local availability of TR
1 and TRß, respectively, govern which TR regulates a target gene in a specific cell; this assumes that the spatial expression patterns are distinct for different TRs in the liver. This would be analogous to the metabolic zonation in the liver where different metabolic processes are spatially separated along the porto-central axis of the liver units (58, 59). In fact, several of the aforementioned genes are known to be zonally expressed in the liver. The CYP7A gene is expressed in a narrow zone around the central vein (60, 61), whereas 5'DI-1 has a somewhat wider distribution (62). In addition, Zandieh Doulabi et al. (62) reported recently that in the rat, TRß is expressed preferentially around the central vein. This supports the idea that hepatic target gene specificity by TRs may be preferentially governed by distinct zonal expression of TRs and their respective target genes, and less by promoter selection. Future experiments are clearly needed to decide the precise mechanism for TR isoform specificity in regulation of hepatic genes.
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MATERIALS AND METHODS
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Hormone Assays
Free T3 and T4 were measured using the Amerlex-MAB kit as described (30). Ligand binding experiments were performed as previously described (15) to determine the extent to which T3 binding was increased because of TR
1 overexpression. Nuclear extracts from liver tissue were prepared essentially as described (63). Binding assays with 125I-T3 were performed as described (64). Extracts were incubated with increasing concentrations of 125I-T3 in the presence or absence of a 1000-fold excess of unlabeled T3 until saturation of high-affinity binding was achieved. Saturation curves were established for two mice of each genotype tested except for wt, for which three mice were used.
Ribonuclease Protection Assay
RNA was prepared from frozen liver, and extraction of polyadenylated mRNA was done as described earlier (65). Relative mRNA levels of TR
1 in the TR
2-/-ß-/- compared with the corresponding wt mice were determined by RPA using the RPAIII kit (Ambion, Inc., Austin, TX). RNA antisense probes were generated by in vitro transcription using the MAXIscript kit (Ambion, Inc.) according to the manufacturers recommendations. Template for the in vitro transcription of the TR
1 antisense RNA probe was produced by PCR on a TR
1 cDNA clone with the PCR primers:
Forward primer: 5'-GAC AAG ATC GAG AAG AGT C
Reverse primer: 5'-GGG CAC TCG ACT TTC ATG T
The TR
1 PCR product was subcloned in the PCR2.1 vector using the Topo cloning kit (Invitrogen BV, Groningen, The Netherlands), and the sequence was verified by DNA sequencing. The pTRI-actin mouse supplied with the MAXIscript kit was used as template for generation of the actin RNA probe. Antisense RNA probe (2.5 x 105 cpm) was hybridized to 4 µg of mRNA in the RPA. After separation on a 5% acrylamide/8 M urea gel, TR
1 mRNA levels were determined using a Fuji Photo Film Co., Ltd. BAS2500 (Fujifilm, Tokyo, Japan) and normalized to the expression of actin in each sample. The expression of TR
1 in TR
2/ß wt was set to 1.0. The experiment was repeated three times with similar results.
Serum and Lipoprotein Cholesterol Determination
Plasma lipoproteins were size fractionated by FPLC, using a micro-FPLC column (30 x 0.32 cm Superose 6B) coupled to a system for on-line assay of cholesterol. Ten microliters of serum were injected on to the column from every animal at a flow rate of 40 µl/min. The separated lipoproteins were continuously mixed with the commercially available MPR 2 1 442 350 cholesterol assay kit (Roche Molecular Biochemicals, Indianapolis, IN) added at a flow rate of 40 µl/min. Absorbance was measured at 500 nM every 20th sec, and the data were collected using EZ Chrom software (Scientific Software, San Ramon, CA).
Serum total cholesterol was assayed with the above cholesterol assay, using a 5.2 mmol/liter cholesterol standard from Merck \|[amp ]\| Co., Inc. (Darmstadt, Germany; catalog no. 14164).
Determination of mRNA Levels in Liver
RNA was prepared from frozen liver. For the TR
1/ß animals, polyadenylated mRNA (65) was prepared from three separate liver pools from each animal group containing four to 10 animals, TR
2 mRNA was prepared by the same method from three animals in each group. For the TR
2/ß animals, total RNA was prepared using Ultraspec (Biotecx Laboratories, Houston, TX) solution from three to six animals in each group, an equal amount of RNA from each animal in the group was then used for the pool. Northern blots were hybridized with cDNA probes for mouse 5'DI-1, rat CYP7A1, rat LDL-R (LDL-R), and mouse CYP8B1. Hybridization with glyceraldehyde-3-phosphate dehydrogenase (G3PDH) served as a control for equivalent loading of mRNA. Levels of mRNA expression were normalized to that of G3PDH using a Fuji Photo Film Co., Ltd. BAS2500 for quantification.
Enzymatic Activity of CYP7A1
Hepatic microsomes were prepared by differential ultracentrifugation of liver homogenates as described previously (66). Three separate pools were prepared from each animal group. The activity of CYP7A1 was determined from the formation of 7
-hydroxycholesterol (picomoles/mg protein/min) from endogenous microsomal cholesterol by the use of isotope dilution-mass spectrometry as described previously (67). Assays were carried out in duplicate.
Statistical Analysis
Data are presented as means ± SEM. P values were calculated by a two-sample Students t test (assuming equal variance) using Excel software (Microsoft Corp., Redmond, WA). P values < 0.05 were considered significant.
Animals and Experimental Setup
Thratm2Ven/tm2Ven mice (nomenclature according to Mouse Genetics Guidelines, The Jackson Laboratory, Bar Harbor, ME) designated as Thratm2/tm2 in Ref. 33 are here designated TR
2-/-, and Thrbtm1/tm1 in Ref. 33 are designated TRß-/- for readability. Similarly, in figures and tables, TR
1-/-ß-/- and TR
2-/-ß-/- are denoted TR
1/ß knockout (KO) and TR
2/ß KO, respectively.
Altogether, 109 male mice aged 23.5 months (29 TR
1-/-ß-/-, 32 TR
1/ß wt mice, 25 TR
2-/-ß-/-, and 23 TR
2/ß wt mice) were used. The studies were approved by the Institutional Animal Care and Use Committee. TR
1-, TR
2-, and TRß-deficient mice were genotyped by Southern blot or PCR analysis using PCR primers specific for the mutant TR
1, TR
2, and TRß alleles, as described previously (22, 25, 30, 33). The genetic background of the TRß-/- mice is a hybrid of 129/Sv x C57Bl/6J and that of TR
1-/- mice is a hybrid of 129/OlaHsd x BALB/c. The TR
1-/-ß-/- (15) and the TR
2-/-ß-/- mice and their housing conditions were recently described (33). The wt control and their respective compound KO strains used in this study were originally derived from crosses between heterozygotes for the respective single KO strains. The control strains, therefore, had the same, mixed genetic background as their respective receptor-deficient strain. To minimize the risk for genetic drift between the wt and the KO strains, no more than three generations of breeding was done until new intercrosses were set up for regeneration of the respective wt and compound KO strains as described previously (15, 33).
The mice were divided into five groups, each consisting of three to ten wt (TR
1/ß wt or TR
2/ß wt) and four to seven KO (TR
1-/-ß -/- or TR
2-/-ß -/-) animals. The experimental setup has recently been described in detail (16) and is summarized in Fig. 2
. In brief, the mice were made hypothyroid by feeding a LID and inclusion of 0.05% MMI and 1% potassium perchlorate in the drinking water (MMI) for 21 d when on LID diet. From day 35 one group of animals were injected daily with 5 µg T3 for an additional 5-d period to induce hyperthyroidism. Two other groups of mice were treated the same way as described above except that the chow was supplemented with 2% cholesterol from d 14. The TR
2 mice were treated as described above with the exception that 0.5 µg T3 was injected per animal and day. At the time of decapitation, trunk blood was collected. Livers were immediately frozen on solid CO2. All animals used were subject to determination of free T3 and free T4 levels.
 |
ACKNOWLEDGMENTS
|
---|
We thank Kristina Nordström for animal setup, handling, and analyses and Mrs. Ingela Arvidsson for technical assistance. We also thank Dr. K. I. Okuda for the cDNA probe for rat cholesterol 7
-hydroxylase, Dr. M. Gåfvels for the 12
-hydroxylase probe, Dr. P. R. Larsen for the mouse 5'DI-1 probe, and Dr. G. Ness for the rat LDL-R cDNA probe.
 |
FOOTNOTES
|
---|
This work was supported by grants from the Medical Research Council (03X-7137, 32X-14053, and 14GX-13571), Strategiska stiftelsen, the Swedish Society for Medical Research, the Ax:son Johnson, the Ruth and Richard Julin, and the Swedish Heart Lung Foundations, and by grants from the Karolinska Institute and the Nordic Insulin Fund (M.R. and B.A.), March of Dimes Birth Defects Foundation, and a Hirschl Award (D.F.), the Swedish Cancer Society, Hedlunds Stiftelse, and the Karolinska Institute (B.V. and H.G.).
Abbreviations: CYP7A1, Cholesterol 7
-hydroxylase; CYP8B1, sterol 12
-hydroxylase; 5'DI-1, type 1 iodothyronine deiodinase; FPLC, fast performance liquid chromatography; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; KO, knockout; LDL, low-density lipoprotein; LDL-R, LDL receptor; LID, low-iodine diet; LXR, liver X receptor; MMI, methimazole; RPA, ribonuclease protection assay; TR, thyroid hormone receptor; wt, wild-type.
Received for publication January 9, 2002.
Accepted for publication April 10, 2002.
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