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Klinische Forschergruppe der Medizinischen Poliklinik der Universität Würzburg (A.B., J.K.), D-97070 Würzburg, Germany; Max-Planck-Institut für Experimentelle Endokrinologie (K.B.), D-30625 Hannover, Germany; and Universitäts-Frauenklinik (H.J.), D-37075 Göttingen, Germany
Address all correspondence and requests for reprints to: J. Köhrle, Medizinische Poliklinik, Röntgenring 11, D-97070 Würzburg, Germany.
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Abstract |
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Introduction |
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Recently, it was reported that 3,5-diiodothyronine (3, 5-T2), a naturally occurring metabolite of T3, not only exerts rapid, short-term effects on mitochondrial respiration (8, 9, 10, 11), but also acts as a specific agonist in the feedback mechanism on TSH-secretion at the pituitary level without other apparent thyromimetic effects (12).
Transcription of the TSH genes is suppressed by T3 bound to the pituitary-specific thyroid hormone receptor TRß2 (13) via negative T3 response elements in the promoters of these genes. TRß2 knock-out mice show phenotypes compatible with a predominant function of the TRß2 receptor at the brain and the pituitary level (14, 15). Alterations of pituitary-deiodinase activities, which themselves are regulated also by thyroid hormones (16, 17), are involved in this negative TSH feedback mechanism. As 3,5-T2 exerts obvious T3-like effects on TSH secretion at the pituitary level, we evaluated whether 3,5-T2 also modulates pituitary deiodinase activities in vivo and in vitro using euthyroid male rats, rat AP reaggregates, and the somatomammotroph rat cell line GH3. For examination of pituitary functions, the reaggregated AP cells, which are cultured in serum-free, hormonally defined media, provide a more physiological model than monolayers, because the 3-dimensional, tissue-like organization favors cell-to-cell communication and para/autocrine interactions between the different cell types of the AP (18).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animal treatment
Male adult Sprague-Dawley rats (6 months) were kept under
standard housing conditions (12-h day/night cycle). They received a
single injection of T3 (100 µg/kg BW), 3,5-T2 (800
µg/kg or 1600 µg/kg BW), or vehicle (15% dimethyl sulfoxide in
0.9% saline) ip between 0800 h and 0900 h and were killed by
decapitation at the indicated time points. Blood was collected from
every single animal for serum preparation; serum samples were stored at
-80 C until use.
Preparation of pituitary homogenates
After decapitation, the pituitaries were removed, AP and
posterior pituitary (including intermediate lobe) were separated,
immediately frozen on dry ice blocks, and stored at -80 C until use.
Single APs were homogenized by sonication (10 times, 0.5 sec, 300
watts) in 80 µl ice-cold homogenization buffer (250 mM
sucrose, 20 mM HEPES, 1 mM EDTA, 1
mM dithiothreitol, pH 7).
Reaggregate cultures of AP cells
Anterior pituitaries were removed from 20 adult male Cara rats
and processed according to the protocol of Denef et al.
(18). After 2 days in culture, reaggregates from two 35-mm petri dishes
were transferred to one 60-mm petri dish, and half of the serum-free
medium was replaced by 2 vol fresh medium. Three days later, half of
the medium was removed and substituted by fresh medium containing
3,5-T2 (final concentration 3 nM or 30 nM) or
solvent (final concentration 1.25 µM NaOH). After 48
h, the reaggregates were harvested, washed once with ice-cold PBS, and
sonicated, as described above. The homogenates were stored at -20 C
until further use.
Cell culture
GH3 cells (ATCC No. CCL. 82.1) were cultured in Ham’s F10
medium supplemented with 15% horse serum, 2.5% FCS, and antibiotics
(penicillin 10 U/ml, streptomycin 0.1 mg/ml). Three days after plating
the cells on 25-cm2 culture flasks, the medium was removed
and replaced by fresh medium containing T3 (final
concentration 3 nM), 3,5-T2 (final concentration 3, 30, or
300 nM) or vehicle (final concentration 1.25
µM NaOH). After 48 h incubation, the cells were
harvested and sonicated, as described above.
Biochemical assays
Specific activities of type I and type II 5'D were determined in
parallel by the release of 125I- from
3,3',5'-[125I]-reverse T3 (DuPont, Bad
Homburg, Germany) in the absence or presence of 1 mM PTU
(6-N-propyl-2-thio-uracil) using 10 nM
nonradioactive rT3 and 20 mM dithiothreitol
(19, 17). The fraction of iodide release blocked by 1 mM
PTU was assigned to 5'DI; the residual activity not inhibited by PTU
was ascribed to 5'DII. 5'D-activities of each sample were determined in
triplicate and expressed as fmol 125I-
released per min per mg protein. The protein contents of the
homogenates were determined by a modified Bradford (20) protein assay
(Biorad, Munich, Germany) using 
-globulin as protein standard.
Electrophoretic mobility shift assays (EMSAs)
GH3 cells were grown in 25-cm2 culture flasks in
Ham’s F10 medium supplemented with 15% horse serum and 2.5% FCS for
3 days and then kept under serum-free conditions for 24 h before
stimulation with T3 (final concentration 3 nM)
or 3,5-T2 (final concentration 3 nM or 30 nM)
for 0.5 h, 1.5 h, or 24 h. At the indicated time points,
nuclear cell extracts were prepared as described by Grandison et
al. (21). Protein concentrations of the nuclear extracts were
determined as described above. Equal amounts of protein (6 µg) were
used for EMSAs. The oligonucleotide used was derived from the DR+4
element in the 5'regulatory region of human 5'DI gene (22), which
represents an ideal DR+4 thyroid hormone response element
(5'-CGGGTAGGTCATCTGAGGTCAGGAGT-3').
Oligonucleotides were labeled with [32P]-ATP using
T4 polynucleotide kinase. Binding reactions were performed
for 30 min at room temperature in reaction buffer described by
Grandison et al. (21). For each binding reaction, 20,000 cpm
of labeled oligonucleotide were used. Supershifting was performed with
anti-RXR antibody (kindly provided by Dr. P. Chambon).
Northern analysis
Total RNA was isolated and purified from pooled rat AP
homogenates by centrifugation through CsCl gradient, as described by
Chirgwin (23) (six pituitaries per data point). Fifteen micrograms of
total RNA were separated by electrophoresis in a denaturing agarose gel
(2.2 M formaldehyde, 1% agarose) and capillary transferred
to a nylon membrane (Hybond, Amersham, Braunschweig, Germany).
Hybridization was performed at high stringency in 50% formamide, 5x
sodium chloride, sodium dihydrogen phosphate, EDTA (0.9 M
NaCl, 75 mM NaH2PO4, 7.5
mM EDTA, pH 7.4), 0.5% SDS, 5x Denhardts (0.1% BSA,
0.1% ficoll, 0.1% polyvinylpyrrolidone), and 500 µg denatured
salmon sperm DNA in a final vol of 10 ml with a TSHß-complementary
RNA probe or 18S ribosomal RNA-complementary DNA (cDNA). Blots were
washed in 1% SDS and decreasing concentrations of SSC (0.15
M NaCl, 15 mM sodium citrate
Na3C6H5O7 x 2
H2O, pH 7.0). TSHß-complementary RNA was transcribed from
a 150-bp cDNA, cloned in Bluescript and linearized with
EcoRI, with T7 RNA polymerase in the presence of
[
32P]CTP. TSHß cDNA containing plasmid was kindly
provided by W. W. Chin (24).
Determination of TSH
TSH levels were determined by RIA according to the
manufacturer’s instructions (NIH, Bethesda, MD).
Statistical analysis
Data were analyzed by multiple ANOVA. When the main effect was
significant, the U test of Mann and Whitney was applied as
posthoc test to determine individual differences between
means.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). When the rats received a single
injection of 3,5-T2 (800 µg/kg BW), 5'DI activity also increased,
with maximum levels 24 h after application, decreased thereafter,
and reached basal levels after 48 h. No difference in the extent
of 5'DI stimulation was observed after injection of a higher dose
3,5-T2 (1600 µg/kg BW), but the time course was slightly delayed
(data not shown). Fig. 1B illustrates the dose-response relationship of
stimulation of 5'DI activity 24 h after injection of
T3 or 3,5-T2 when the maximum effect is observed for 3,5-T2
in vivo. 3,5-T2 seems to be, by a factor of 5–10, less
potent than T3 in 5'DI stimulation, but a trend for a dose
relationship is visible that, however, does not reach significant
levels.
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Effects of 3,5-T2 on 5'deiodinase activities in
reaggregates of male rat AP
In serum-free reaggregate cell cultures of male rat APs specific
5'DI activity was an order of magnitude higher than 5'DII activity.
Incubation with 3 nM T3 for 24 h lead to a
two-fold stimulation of 5'DI activity over basal levels, whereas 5'DII
activity was no longer detectable. A smaller, but significant, increase
in 5'DI activity was observed 24 h after incubation with 30
nM 3,5-T2. Again 5'DII activity was no longer detectable
(Fig. 2). Incubation with 30 nM 3,5-T2 for
48 h or 72 h lead to a further increase of 5'DI activity
(2.3-fold over basal levels 48 h and 6-fold over basal levels
72 h after 3, 5-T2 application) (Fig. 3).
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illustrates the stimulation of 5'DI in the
somatomammotroph rat cell line GH3 after 48 h incubation with
T3 (final concentration 3 nM) and T2 (final
concentrations 3, 30, or 300 nM). The activity of the 5'DI
in cells that were T3 treated was 1.7-fold higher than
control. 3,5-T2 in all concentrations was as potent as T3
in increasing 5'DI activity but did not show a dose response in this
concentration range.
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TSH-levels were transiently reduced by the application of 800 and 1600
µg/kg BW of 3,5-T2, with a nadir 24 h after application and
increased thereafter. Basal levels were reached 72 h after a
single injection. There was a trend for dose-dependent suppression of
TSH by 3,5-T2, which did not reach statistical signigificance, and
3,5-T2 was less potent than T3 (Fig. 5B).
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shows that nuclear extracts of GH3 cells
formed three specific protein-DNA complexes with the labeled DR+4
oligonucleotide corresponding to the functional TRE in the human 5'DI
promoter. The shifted bands disappeared completely by the addition of
100-fold excess of unlabeled DR+4 oligonucleotide (Fig. 7, lane 9).
This competition for protein-DNA complexes ensures specificity of the
interactions. Nuclear extracts of T3-treated GH3 cells (3
nM T3 for 30 min) lead to an increase in the
intensity of the retarded bands (Fig. 7, lane 2), compared with the
nuclear extracts of untreated control cells (Fig. 7, lane 1). A
comparable increase was observed using nuclear extracts of cells that
were treated with 3 or 30 nM 3,5-T2 (Fig. 7, lanes 3 and
4). Protein-DNA interactions that occurred using nuclear extracts of
untreated control cells, decreased when the cells were cultivated in
serum-free medium for another 24 h (Fig. 7, lanes 1 and 5).
Activation of DNA-binding was observed at all tested time points after
a single administration of T3 or 3,5-T2 in both
concentrations but also decreased during incubation up to 24 h.
The middle band presumably corresponds to a TRß-RXR heterodimer, as
it is supershifted by addition of an RXR antibody (Fig. 7, lane 10).
These binding experiments suggest that functional T3- and
3,5-T2-responsive TR-RXR complexes form on the ideal DR+4 TRE of the
5'DI gene, suggesting transcriptional modulation to be involved in
thyroid hormone-dependent expression of 5'DI in rat pituitary
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Horst et al. (12) recently reported that a long-term administration of 3,5-T2, the 5'-deiodination product of T3, exerts suppressive action on TSH secretion without other apparent thyromimetic effects, and Everts et al. (25) demonstrated that 3,5-T2, at concentrations as low as 1 nM, significantly reduced TSH-response to TRH in rat AP cells cultured in monolayer but was 100-fold less effective than T3. 3,3'-T2, the common metabolite of rT3-5'-and 5-deiodination, showed no inhibitory effect at that low concentration.
In this study, we present experimental evidence that 3,5-T2 exerts effects on both 5'deiodinase activities directly at the pituitary level. In vivo, as well as in vitro, 3,5-T2 stimulates 5'DI activity and transiently reduces 5'DII activity. The doses of 3,5-T2 for the in vivo experiments were chosen according to the experiments of Horst et al. (12). Our findings support the observation that doses of 3,5-T2 5- to 10-fold higher than those of T3 are needed in vivo to exert the same effects at the pituitary level. One possible explanation would be that, in the intact rat, 3,5-T2 is more rapidly and effectively inactivated than T3. This explanation is supported by the different time courses and the dose-dependent effects of 3,5-T2 and T3 in vivo in rat AP reaggregates and GH3 cells using either pituitary 5'DI and 5'DII, serum TSH, or TSHß-mRNA as endpoints, respectively. Whereas T3 and 3,5-T2 seem equipotent in 5'DI stimulation in the GH3 model, 3,5-T2 only transiently decreases TSHß mRNA steady-state levels and supresses serum TSH, independent of the dose administered in vivo. Similar potencies of T3 and 3,5-T2 in 5'DI stimulation in vitro in the GH3 model suggest at least a partial contribution of a mechanism of action mediated by nuclear T3-receptors, as supported by the rapid activation of binding of nuclear transcription factors to the DR+4 functional TRE of the human 5'DI promoter. The difference in transient 5'DII suppression and prolonged 5'DI stimulation, both by T3 and 3,5-T2, were expected to be caused by the different half-lifes and the divergent mechanism of modulation of both deiodinase enzymes (6, 26). Previous analyses of serum binding, production, and metabolic clearance rates of 3,5-T2, compared with T3, suggested a shorter half-life for 3,5-T2 in human serum (27, 28, 29, 30). An even more rapid metabolism of 3,5-T2 in the normal rat, which has very low circulating TBG levels compared with human serum, might prevent accumulation of nuclear T3 receptor saturating concentrations of 3,5-T2 required for prolonged and persistent TSH suppression, in contrast to T3. No information is yet available for kinetic parameters of 3,5-T2 in the rat. Early studies on comparisons of thyromimetic potencies of rather impure preparations of 3,5-T2 vs. T4 indicated that in euthyroid animal models, 3,5-T2 exhibits 1/10 to 1/40 of the activity of T4, whereas in thyroidectomized rats and human myxedema, 3,5-T2 is less potent than T4 by more than two orders of magnitude (31).
Currently, it remains unclear whether the effects of 3,5-T2 are mediated by nuclear T3 receptors or involve nonnuclear receptor-mediated direct mechanisms at the cell membrane, mitochondrial, or cytoskeletal level, as recently summarized (32). Some of our data presented here suggest at least a participation of activation of nuclear T3/RXR-receptor heterodimers in the mediation of 3,5-T2 effects at the pituitary level, but contribution of other mechanisms cannot be excluded. T3 stimulates 5'DI gene transcription in pituitary GH4C1 cells by a mechanism not requiring ongoing protein synthesis, whereas T3 effects on GH-mRNA synthesis are blocked by cycloheximide (33). The inhibitory effect of T3 on TSH synthesis is directly related to the saturation of nuclear T3 receptors (34), and for this response, no ongoing or de novo protein synthesis is needed (35). These results indicate, that T3 can modulate gene transcription in the AP by at least two different mechanisms. In GH3 cells, 3,5-T2 is capable of regulating GH and TRß2 gene expression and can bind to in vitro translated TRs (36). With EMSAs, we demonstrated, that 3,5-T2 rapidly activates proteins that can specifically bind to the DR+4 TRE that was identified in the promoter region of the human 5'DI gene (22). 3,5-T2 was as potent as T3 in stimulating DNA-protein interactions. One of the retarded bands presumably represents a TRß2-RXR heterodimer, because it is supershifted by the addition of a RXR antibody. The exact mechanism of T3 or 3,5-T2-dependent recruitment or increased DR+4-binding of nuclear transcription factors, such as RXRs or other DNA-binding proteins, remains to be analyzed. Ligand-dependent stabilization of protein-DNA interactions, removal of corepressors, and/or nuclear translocation of receptors might contribute to this increased binding. Whether 3,5-T2 exerts its effects on T3-dependent genes in the AP directly by binding and activating TRs and interacting with TREs in the promoter regions of these genes or indirectly by stimulation of 5'DI activity and consequently leading to higher intrapituitary T3 production, remains to be evaluated. The demonstration of both 5'DI and 5'DII activity in somatomammotroph GH3 cells, responsive to T3 and its potential 5'-deiodination product 3,5-T2, also raises the possibility that local auto- and paracrine interactions between the more abundant somatotrophes, generating T3 and/or 3,5-T2, and thyrotrophes, responsive to these thyroid hormones, in the TSH-feedback system seems possible and needs to be studied in more detail.
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Acknowledgments |
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Footnotes |
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Received December 26, 1996.
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