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RESEARCH COMMUNICATION |
a Laboratoire de Physiologie Générale et Comparée, Muséum National d'Histoire Naturelle, URA CNRS 90, 75231 Paris, cedex 5, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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were expressed with TRH-luciferase, all forms of TRß gave T3-dependent regulation of TRH transcription, whereas transcription was T3 insensitive with each TR tested. Moreover, chicken TR increased TRH transcription sixfold, whereas mouse TR decreased transcription. These transcriptional effects had correlated physiological consequences: expression of the chicken TR in the hypothalamus of newborn mice raised circulating T4 levels by fourfold, whereas mouse TR had opposite effects. Thus, TR subtypes have distinct, physiologically relevant effects on TRH transcription.—Guissouma, H., Ghorbel, M. T., Seugnet, I., Ouatas, T., Demeneix, B. A. Physiological regulation of hypothalamic TRH transcription in vivo is T3 receptor isoform specific. FASEB J. 12, 1755–1764 (1998)
Key Words: nonviral gene transfer • polyethylenimine • thyroid hormone • mouse • central nervous system
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1, TRß1, and TRß2—produced from two TR genes, also called c-erbA and c-erbAß (1). In combination with their heterodimeric partners, these TRs may determine recognition of different core motifs (thyroid response elements, TREs). Further complexity is brought about by interactions between receptors and other transcription factors either directly or through coactivator or corepressor proteins (for reviews, see refs 2, 3). As these TR-dependent interactions will have cell-specific and developmentally dependent dimensions, we chose to analyze T3-dependent, cell-specific responses by following regulation of a key target gene, TRH (thyrotropin-releasing hormone), within the hypothalamus. Although this gene is expressed both within the hypothalamus and in extrahypothalamic brain areas (4), we chose to examine hypothalamic transcription because the hypothalamic neurons expressing TRH are responsible for controlling the hypothalamo/hypophysio/thyroid (HHT) axis and are most sensitive to negative feedback by T3 (5). Indeed, the model of TRH transcription has the advantage of showing clear-cut responses that allow the contribution of different players to be dissected. In particular, the specific role of TRß in mediating T3-dependent regulation of this gene has been addressed in primary hypothalamic neuronal cultures (6) and in cell lines (7–9).
The distinct effects of TR subtypes on neuronal TRH transcription seen in chick hypothalamic cultures (6) reflect the spatial expression patterns of TRs in the chick (10, 11) and rat brain (12), with particularly high levels of expression of TRß in the hypothalamus. To take the physiological analysis a step further, into an in vivo context, we used the newborn mouse. In this model, not only could we examine TR subtype effects on TRH promoter activity in the hypothalamus in situ, but also the end point of the HHT axis output, namely, circulating thyroid hormones.
Only in vivo models can be used for this type of study. Modulation of TR status can be achieved in vivo by germinal transgenosis or somatic gene transfer. Transgenic mice have been generated that are homozygous for targeted inactivation of either the TR (13) or the TRß genes (14). These powerful approaches have shown differential effects of TR subtypes, producing distinct phenotypes. However, eliminating gene expression from the begining of development will affect processes downstream of early TR action. So to analyze TR effects at a defined, physiologically relevant moment, somatic gene transfer can have its advantages. Moreover, such methods allow one to study physiological responses in both genetically intact animals and modified genetic backgrounds.
Synthetic gene transfer methods, and polyethylenimine (PEI) in particular, can be adapted to these ends (15). By using PEI-based gene transfer in the hypothalamus, we show that all TRß isoforms tested mediated T3-dependent inhibition of transcription from the TRH promoter, whereas ligand binding TR isoforms did not provide T3-dependent inhibition. Moreover, introducing chicken TR or mouse TR into the hypothalamus respectively increased or decreased basal TRH-luc transcription, and these transcriptional effects were correlated with changes in circulating T4. In contrast, expressing TRß, the TR subtype most represented in the hypothalamus, did not disturb HHT equilibrium. These results bolster the hypothesis that TR subtypes have distinct transactivating properties that hold physiological relevance.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TheTRH-LUC construct contains a rat TRH gene 5' fragment (16) extending from -554 to +84 base pairs cloned upstream of the firefly luciferase-coding region (17).
The TREpal-LUC construct contains a synthetic oligodeoxynucleotide sequence encoding a palindromic TRE in SV-luciferase (18). The CMV-LUC expression vector was from Promega, Madison, Wis. The pCMVß-gal plasmid was from Clontech (Palo Alto, Calif.). Plasmids expressing mouse (m) TR1, mTRß1, and mTRß2 receptors were generously provided by Dr. W. Wood (University of Colorado). Each cDNA is in pRSVL (19) vector, with the Roux sarcoma virus (RSV) promoter. A plasmid expressing full-length wild-type chicken TR1 (c)TR1 was generously provided by Dr. H. H. Samuels (New York University Medical Center). This vector has the RSV promoter in pEXPRESS vector (20). The cTRß2 used was pSG5-TRß2 (11).
Treatment of animals: production of hypothyroid newborn mice
Female OF1 mice (Iffa Credo, l'Abresle, France) were mated. To induce fetal and neonatal hypothyroidism, dams were given 0.05% 6-n-propyl-2-thiouracil (PTU) in drinking water at day 14 of pregnancy. PTU administration was continued throughout the lactation period. For evaluating T3 effects on TRH mRNA levels or transcription, hypothyroid or normal pups were injected with 250 µg of T3 /100 g of body weight (in 9
saline). Controls received saline (9) injections.
Preparation of PEI/DNA complexes
DNA was diluted in 5% glucose. After vortexing, 25 kDa PEI (Sigma, France) was added (nine equivalents, PEI amine/DNA phosphate) and the solution was revortexed. The required amount of PEI, according to DNA concentration and number of equivalents needed, is calculated by taking into account that 1 µg DNA is 3 nmol of phosphate and that 1 µl 0.1 M PEI is 100 nmol of amine nitrogen.
In vivo gene transfer
Pups were injected on postnatal day 1 (P1). Pups were anesthetized by hypothermia on ice. The pups heads were held by hand and a small incision was made with iris scissors through the skin overlying the sagittal suture to expose the skull. A small hole was made through the skull approximately 1 mm lateral to the sagittal suture. A glass micropipette was lowered 2 mm through the incision and 2 µl of a 5% glucose solution containing plasmid/PEI complexes was slowly injected bilaterally into the hypothalamus. Pups were kept under an infrared heat lamp until active.
After 18 h, mice were anesthetized and decapitated. Hypothalami were dissected out for luciferase analyzes or nuclear receptor extraction. Animals treated with T3 received subcutaneous injections immediately after gene transfer. Controls received saline at the same time point.
Luciferase assay
Hypothalami were homogenized in 150 µl assay buffer, then luciferase assay was performed according to the standard protocol (luciferase assay system, Promega) in a single-well luminometer (ILA911, MGM Instruments, Hamden, Conn.). Luciferase was estimated during 10 s with 20 µl brain extract. Light was expressed as relative light units (RLU).
LacZ histochemistry
Pups were decapitated 24 h after transfection and brains were dissected out. In toto LacZ histochemistry was performed as described (21) and vibratome sections (100 µm) were prepared.
Nuclear receptor extraction
Pools of hypothalami from 12 P1 mice were used in each experiment. Hypothalami were removed, weighed, homogenized at 2°C (0.25 weight/volume) in 250 mM sucrose, 2 M MgCl2, 20 M Tris-HCl (pH 7.6) using a glass-teflon homogenizer, and filtered through nylon Bultex 48 µm. Filtrates were centrifuged (1500xg, 10 min). Pellets were washed twice in the homogenizing buffer supplemented with 0.5% triton X-100 and centrifuged (1500xg, 10 min). Pellets were resuspended in binding buffer, rehomogenized (50 mM NaCl, 10% glycerol, 2 mM EDTA, 5 M 2-mercaptoethanol, 20 mM tris-HCl, pH 7.6), and centrifuged (1500xg, 10 min). This operation was repeated three times. The final pellets were resuspended in 3 ml of 0.4 M KCl, 1 mM MgCl2, 20 mM tris-HCl (pH 7.9), stored for 30 min on ice, and centrifuged at 35,000xg for 30 min. Supernatants were dialyzed three times in binding buffer and centrifuged (5000xg, 20 min). Fifty microliters of the supernatants were used to dose protein content with a commercial kit (Bio-Rad, Richmond, Calif.).
T3 binding assay
Samples (350 µl) of nuclear receptor extracts were incubated with [125I]T3 (2.6 nM, specific activity >1200 mCi/mg; Amersham, Buckinghamshire England) for 45 min at 25°C. After incubation, 3.5 ml binding buffer were added to each sample, then solutions were filtered at 2°C under vacuum through 0.45 µm Millipore nitrocellulose (HAWP) filters in order to separate bound and free [125I]T3. Filters were washed with 3 x 5 ml binding buffer. Radioactivity was measured in a gamma counter. Nonspecific binding (NSB) was determined in samples containing the tracer and an excess of (2.6 µM) stable T3. NSB was substracted from total binding to calculate the specific binding.
Radioimmunoassay of T4 and T3 hormones
Radioimmunoassay of plasmatic T4 and T3 hormones was carried out as previously described (22).
PCR analysis
Hypothalami of mice transfected with a given TR vector were homogenized in 150 µl luciferase buffer and centrifuged (10 min, 12,000xg). Phenol/chloroform extraction was carried out on 120 µl supernatant. After sodium acetate (3 M) precipitation, DNA was resuspended in TE buffer, pH8 (TE is 10 mM Tris-HCl, 1 mM EDTA).
Polymerase chain reaction (PCR) amplifications were performed in a final volume of 50 µl containing 1x reaction buffer, 5 µl template DNA, 2 µM of primers, 200 µM deoxynucleotides (dATP, dCTP, dGTP, dTTP), and 1 unit of Taq polymerase. The following primers were used to detect ampicillin cDNA sequence present in all the plasmids used in our experiments: sense, 5'-CCAATGCTTAATCAGTGAGGCACC-3'; antisense, 5'-ATTCAACATTTCCGTGTCGCCC-3'. Internal controls were run on the same samples using mouse ß-actin primers: sense, 5'-GCTGAGAGGGAAATCGTGCG-3'; antisense, 5'-CCAGGGAGGAAGAGGATGCG-3'. Amplification occurred during 40 cycles (1 min, 94°C; 1 min, 60°C; and 1 min, 72°C). PCR products were separated on 2.8% agarose gels containing 0.1 µg/ml ethidium bromide.
Statistical analysis of results
In vivo gene transfer results are expressed as means ± SEM per group. Student's t test was used to analyze differences between groups. Where appropriate, one-way analysis of variance (ANOVA) with post hoc multiple comparison analysis by the Bonferroni test for independent variables was applied. Differences were considered significant at P < 0.05. In all cases typical experiments are shown, each experiment having been repeated at least three times and providing the same result.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 1A
shows a section of a newborn mouse brain transfected with CMVß-gal. Transgene expression was seen around the third ventricule in numerous neuron-like cells. Expression was also found in some other sites close to ventricular spaces, such as regions of the hippocampus closely bordering the lateral ventricles ( Fig. 1B). This is to be expected as complexes reaching the third ventricule will diffuse through the ventricules to other brain areas (21). We quantified transgene distribution by comparing levels ofCMV-luc expression in the hypothalamus and extrahypothalamic tissue. Luciferase was detected within the hypothalamus, but also in the cortex, striatum, and hippocampus ( Fig. 1C). Generally, levels were lower in the extrahypothalamic areas, but differences were not significant. This was in sharp contrast to the result obtained with TRH-luc. As shown in Fig. 1D, expression was fivefold higher in the hypothalamus than in any other region (P<0.001) and, most important, only in the hypothalamus was transcription from this physiological promoter sensitive to T3 treatment.
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TRH-luciferase transcription in the hypothalamus is T3 dependent and regulated in a physiologically meaningful manner
We next pursued the analysis of physiological regulation of transgenes in the hypothalamus. As seen in Fig. 2A,
transcription from the TRH promoter was regulated in accordance with the thyroid status of the animals. In hypothyroid animals, the mean level of transcription from the TRH-luc construct (3.7±0.37 105 RLU/ hypothalamus, n=10) was twice that of normal animals (2.2±0.36 105 RLU/ hypothalamus, n=10,P<0.05). Treating normal pups with 250 µg of T3 /100 g body weight further decreased TRH promoter activity by half (1.1±0.27 105 RLU/ hypothalamus, n=10, P<0.05).
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In contrast, expression of luciferase from a constitutive promoter (CMV) was unaffected by T3 in the hypothalamus (
Fig. 2B).
Inter- and intraassay transfection variability
Intraassay variability in the transfection experiments was particularly low; SEMs were routinely 
10% of the mean with n 
10. This underlines the consistency of this gene transfer method in the hypothalamus. This low intraassay variability obviates the need to add a constitutively expressed gene to normalize for transfection efficiency as is often the case for most in vitro work and some in vivo work (23). Interassay variability could be higher, with different plasmid preparations providing higher luciferase readings. However, the same physiological regulations are always observed whatever the basal level of expression (compare Fig. 2A and Fig. 3A).
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Only TRßs, and not TR1, mediate T3-dependent regulation of TRH transcription
The effects of different T3 binding TR isoforms on TRH promoter activity were examined next. We tested both chicken (c) and mouse (m) forms of TR and TRß. As seen in Fig. 3A, cotransfecting a control plasmid containing the RSV promoter and no coding sequence had no effect on in vivo transcription from the TRH promoter, significant T3-dependent inhibition of transcription being found. When TRH-luc was expressed with ligand binding forms of mammalian or avian TR subtypes, there were clear-cut distinctions in TR activities.
First, both mTR1 and cTR1 abrogated the negative transcriptional effect of T3. Indeed, no statistical difference in TRH transcription was found in controls or T3-injected animals ( Fig. 3B) in which either TR1 was expressed. Moreover, the effects of mTR1 and cTR1 on basal transcription were different, with mTR1 halving basal TRH-luc transcription and cTR increasing transcription by sixfold (P<0.001 in each case).
Second, all forms of TRß isoforms mediated negative transcriptional effects of T3 on TRH transcription equivalent to that seen in controls. For each TRß isoform tested (cTRß2, mTRß1, and mTRß2), statistically significant decreases in transcription were seen in T3-injected animals as compared to controls ( Fig. 3C). Again, as for the subtypes, the TRß isoforms differentially affected basalTRH-luc transcription: cTRß2 decreased basal transcription by fivefold (P<0.001), mTRß1 did not modify basal transcription, whereas mTRß2 decreased it by threefold (P<0.001).
Both mTR1 and mTRß2 activate transcription from a positive TRE-containing promoter in the hypothalamus
To determine whether these isoform effects were specific to the TRH promoter, we carried out transfection experiments with the same TRs and a positively regulated TRE-pal construct injected into the hypothalamus. No T3-dependent regulation could be revealed in normal animals (data not shown), probably because normal levels of T3 maximally activated the promoter. However, in hypothyroid animals significant T3-dependent activation of TRE-transcription was seen, and this was equivalent for each TR isoform tested, whether from mice ( Fig. 4)
or chicken (data not shown).
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Each TR isoform increases specific T3 binding in the hypothalamus to an equivalent extent
To verify that differences in TR isoform effects on TRH transcription were not due to variations in the efficiency of expression and protein production, we carried out T3 binding studies on hypothalamic nuclear extracts from control and transfected mice. As shown in Fig. 5,
we found that the [125I]T3 binding capacity of nuclear extracts prepared from animals transfected with any one of the TR constructs used was approximately 2.5 x the capacity of control, nontransfected mice or mice transfected with an empty plasmid. Using ANOVA analysis, we found the values in TR transfected animals to be significantly higher (P<0.05) from those in the two control groups. There were no differences in T3 binding between TR groups.
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Expression of mouse or chicken TR in the hypothalamus modifies thyroid status
The end point of the HHT regulatory system is the secretion of thyroid hormone, principally T4 in mammals (24). To determine the effects of TR subtype expression within the hypothalamus on thyroid hormone secretion, we expressed various TRs and measured circulating T4 and T3 over the following week. As seen in Fig. 6,
expressing cTR1 in the hypothalamus resulted in a significant, nearly fourfold, increase in circulating T4 at P4, whereas mTR1 decreased serum T4 at the same time point. Both these modifications were highly significant (P<0.001 in each case). No changes in circulating T4 were seen in controls (injected with RSV control plasmid) nor in animals transfected with equivalent amounts of mTRß1. In cTR1-injected animals, circulating levels of T4 returned to control levels by P6. This lack of cTR1 effect at this time point was correlated with loss of plasmid from the hypothalamus. As seen in Fig. 7,
PCR showed plasmid to be present in the hypothalamus of transfected pups for up to P4, but not P6. Levels of T3, produced principally at the periphery by desiodation (24), were not significantly different in any group at any time point examined (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fig. 1C, D). Second, only the TRH-luc construct is regulated by T3 and regulation is seen only in the hypothalamus ( Fig. 1C). That low levels of transcription from the TRH promoter were found in each of the different brain regions dissected out (cortex and thalamus/striatum) fits with published data. First, pro-TRH mRNA is found in numerous extrahypothalamic sites (4); second, in transgenic mice expressing luciferase downstream from a sequence containing several hundred base pairs of the 5' region and exon 1 of the rat TRH gene, luciferase expression is found both in the forebrain and in the hindbrain (25).
The finding that it is only in the hypothalamus that T3 down-regulates transcription from TRH promoter corroborates data obtained by in situ hybridization in the adult rat brain by Koller et al. (5). These authors showed that modulating thyroid status changed TRH mRNA production in the hypothalamus, but not in the thalamus. Thus, although injection into the hypothalamus results in diffusion of PEI/DNA into the third ventricle and from thence into other brain regions (21), expression from the TRH promoter reflects endogenous gene expression and, most important, T3-dependent negative feedback occurs only in the hypothalamus. Moreover, feeding PTU to the dams rendered the pups hypothyroid (T3 levels were undetectable in the pups, data not shown), and this treatment significantly raised TRH transcription. Thus, modulation of thyroid status resulted in transcription from the TRH-luc construct expressed in the hypothalamus that faithfully reflected that recorded for the endogenous gene followed by in situ hybridization (5).
The negative feedback effect of T3 on TRH transcription is mediated by the ß receptor
We next analyzed the differential effects of TR isoforms on transcription. Only TRßs, but not cTR or mTR, mediated T3-dependent inhibition of transcription in this in vivo context. These differential effects were not due to variations in TR expression, since each TR used produced equivalent increases in functional T3 binding. This finding of differential effects of TRß vs. TR in regulating TRH transcription agrees with previous data from our laboratory (6) obtained using the same TRH-luc in primary cultures of chick hypothalamic neurons. In this in vitro work, we tested cTRß and cTR1, and observed that only cTRß down-regulated TRH transcription whereas both isoforms were equipotent on transcription from a TRE construct (6). Similarly, it has been shown that human TRß1, but not TR1, down-regulates transcription from the human TRH promoter in transient transfection assays in the CV-1 cell line (7).
The specific action of TRß in regulating the TRH gene correlates well with the high expression of this receptor in the hypothalamus (12). However, an additional level of complexity must be taken into account, the production of two isoforms from the c-erbAß locus: TRß1 and TRß2 in rodents and humans (26, 27), and TRß0 and TRß2 in chicken (11). In mammals, it is the TRß2 isoform that has a particularly restricted expression, with the mRNA found almost exclusively in hypothalamus in the rat (28). Recently, Langlois et al. (9) ascribed a unique role to TRß2 in negative regulation by T3 when they showed hTRß2 to be much more efficient than hTRß1 in ligand-dependent repression of transcription from the hTRH promoter introduced into CV1 cells. This finding differs from our present data showing the two mouse isoforms to be equipotent in repressing ligand-dependent TRH transcription in the mouse hypothalamus in vivo. Clearly, the relative contributions of these isoforms need more detailed analysis.
Expression of TR in the hypothalamus abolishes the negative feedback effect of T3 on TRH transcription
There is consensus across the literature regarding the distinct actions of TR1 vs. TRß on TRH transcription, specifically, the lack of T3-dependent regulation of TRH in the presence of the TR isoform (6, 8). These observations are particularly intriguing given that in each experiment a ligand binding form of TR was used, and yet no change in transcriptional activation of the TRH promoter was seen in the presence or absence of ligand ( Fig. 3B; refs 6, 8). Yet we know from our experiments that ligand-dependent transcriptional modification can be elicited, as T3-dependent activation results from TR coexpressed with the TRE construct ( Fig. 4). Many of the differential effects of TR vs. TRß can be attributed to their distinct amino termini (7). Although the amino termini of mouse and chicken TR1 are different (65% isology), we found that both mTR1 and cTR1 had similar effects in this respect: expression of either one blocked T3-dependent repression of TRH transcription. However, the effects of these two TRs on basal levels of TRH-luc transcription were opposite, mTR1 and cTR1 respectively lowering and raising TRH transcription. This result was somewhat unexpected given the high conservation of sequence between the two isoforms (90% isology overall). Aligment of the two sequences shows a few conservative changes in the first 30 amino acids of the amino terminal and in the hinge region behind the DNA binding domain. It is possible that these conservative changes are sufficient to produce distinct interactions with the transcriptional machinery. Indeed, a 10 amino sequence (amino acids 21 to 30) in the amino terminal of the cTR has been shown to be essential for an interaction between the TR and TFIIB (29). Given the complexity of the interactions of TR with the transcriptional machinery and accessory proteins such as coactivators (for review, see ref 2) or corepressors (3) and that TRs show developmental and spatial regulations, it will be interesting to see whether exploring TR action in vivo will produce a better definition of the respective roles of receptor subtypes in modulating target gene transcription.
TR effects on TRH-luc transcription have correlated repercussions on thyroidal status
A final, striking observation resulting from these studies is that modulating TR expression in the hypothalamus results in correlated changes in thyroid hormone production. Indeed, expressing cTR increased basal transcription from the TRH promoter by sixfold and the effect on the HHT axis was a fourfold increase in circulating T4 at P4. In contrast, expression of mTR1 halved both TRH-luc transcription and T4 secretion. Thus, there would appear to be a direct link between the effects of the different receptor subtypes on basal levels of transcription from the exogenous TRH promoter and their effects on endogenous TRH gene (as deduced from modulation of the HHT axis end point: T4 production). No effect on circulating T3 was seen. This is probably because most T3 is not produced in the thyroid, but from peripheral deiodination (24). No doubt, the changes in T4 production produced by modulation of TR expression can be compensated for by changes in deiodination activity in the target tissues, thereby maintaining an euthyroid T3 status overall.
In conclusion, our results confirm that the restricted expression of TRß2 correlates with its action in mediating negative feedback effects of thyroid hormone on TRH transcription. Moreover, misexpression of TR1 in the hypothalamus results in up or down-regulation of TRH transcription according to the species origin of the TR1 used, with alterations in TRH transcription played out down the HHT axis. Finally, this versatile methodology opens up new possibilites for dissecting the physiological relevance of transcription factor function in integrated contexts.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: ANOVA, one-way analysis of variance; c, chicken; HHT, hypothalamo/hypophysio/thyroid; m, mouse; NSB, nonspecific binding; P, postnatal day; PCR, polymerase chain reaction; PEI, polyethylenimine; PTU, 6-n-propyl-2-thiouracil; RLU, relative light units; RSV, Roux sarcoma virus; T3; tri-iodo-thyronine; TRH, thyrotropin-releasing hormone; TR; T3 binding receptor; TREs, thyroid response elements.
Received for publication March 13, 1998.
Revision received July 8, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß thyroid hormone receptor genes. EMBO J. 9, 1516–1528
and N-terminal variant ß receptors. Development 114, 39–47[Abstract]
gene encoding a thyroid hormone receptor is essential for post-natal development and thyroid hormone production. EMBO J. 16, 4412–4420[Medline]
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