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Thyroid Section (M.A.C., R.R., W.S.d.S., J.W.H., P.R.L., A.C.B.), Division of Endocrinology, Diabetes, and Hypertension, Brigham and Women’s Hospital and Harvard Medical School, and Division of Endocrinology (S.A.H., A.C.), Children’s Hospital Boston, Boston, Massachusetts 02115; Tupper Research Institute and Department of Medicine (P.S., C.F., R.M.L.), Division of Endocrinology, Diabetes, and Metabolism, New England Medical Center, Boston, Massachusetts 02111; Department of Endocrine Neurobiology (C.F.), Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1083 Budapest, Hungary; and Department of Medicine/Endocrinology (D.F.G., E.C.R.), University of Colorado Health Sciences Center at Fitzsimons, Aurora, Colorado 80045
Address all correspondence and requests for reprints to: Antonio C. Bianco, M.D., Ph.D., Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, HIM Building 643, Boston, Massachusetts 02115. E-mail: abianco{at}partners.org.
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T1, demonstrate high expression of D2 in thyrotrophs and confirm its sensitivity to negative regulation by T4-induced proteasomal degradation of this enzyme. Despite this, expression of the Dio2 gene in TT1 cells is higher than their T4-induced D2 ubiquitinating capacity. As a result, D2 activity and net T3 production in these cells are sustained, even at free T4 concentrations that are severalfold above the physiological range. In this system, free T4 concentrations and net D2-mediated T3 production correlated negatively with TSHß gene expression. These results resolve the apparent paradox between the homeostatic regulation of D2 and its role in mediating the critical mechanism by which T4 triggers the TSH-negative feedback. |
Introduction |
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The free T4 concentration in serum is a key player mediating the TSH feedback mechanism (2). This assumption is derived from data obtained during iodine deficiency or mild primary hypothyroidism (3). In both conditions, a declining serum T4 concentration, in the presence of normal serum T3, promotes dramatic increases in TSH secretion. The effects of T4 on TSH secretion are possible only because of the presence of type 2 iodothyronine deiodinase (D2) in the pituitary, which rapidly converts T4 to the biologically active T3. Although serum T3 can also reach pituitary thyroid hormone receptors (TRs) and by itself plays a significant role in repressing TSHß gene transcription (4), most TR-bound T3 present in the pituitary gland originates from the local conversion of T4 to T3 (5, 6, 7, 8). In fact, both serum T4 and TSH levels were found to be significantly elevated in mice with targeted disruption of the Dio2 gene, confirming that in the absence of D2, the thyrotrophic cell is relatively resistant to the feedback effect of plasma T4 (9). Furthermore, whereas serum TSH levels in wild-type mice are suppressed by administration of either T4 or T3, only T3 was effective in the mouse with targeted disruption of the Dio2 gene (9).
Nonetheless, the intrinsic homeostatic nature of D2 weakens the argument for its proposed major role in the TSH feedback mechanism (10). It is well known that low-serum T4 increases D2 activity and that high T4 concentrations do the opposite (11). This has been understood as an adaptive mechanism in brain and other D2-expressing tissues to minimize changes in the intracellular concentration of T3 during iodine deficiency and hypothyroidism (12). However, such homeostatic behavior at the thyrotroph, if operational, would impair the efficient transduction of changes in serum T4, leaving TSH levels unchanged. This rationale raises questions about the role played by D2 in the TSH feedback mechanism. In fact, T4-to-T3 production has never been demonstrated in pure thyrotrophic cell lines, and data obtained from studies of human TSH-producing tumors are controversial, with one study (13) suggesting that the inactivating type 3 deiodinase (D3) is the predominant deiodinase in TSH-secreting tumor cells and another study (14) finding both type 1 iodothyronine deiodinase and D2.
The present study was undertaken to test the paradigm that D2 is a major player in the TSH feedback mechanism in light of the substantial progress that has been made in our understanding of posttranslational regulation of D2 (15). Here we show that D2 is highly expressed in rat thyrotrophs and is up-regulated during hypothyroidism. This is the result of posttranslational mechanisms, as demonstrated in two mouse thyrotroph-derived cells, TtT-97, a transplantable thyrotrophic tumor, and TT1, an immortalized simian virus-40 T-antigen-expressing pituitary cell line. However, using the TT1 mouse tumor cell line, we find that the absolute rate of T4-induced loss of D2 activity in these cells is offset by the combined effect of D2 reactivation and a high rate of D2 synthesis. As a result, an increase in T4 rapidly translates into an increase in thyrotrophic D2-mediated T3 production and suppression of TSHß gene expression, thus explaining the T4-mediated TSH feedback mechanism.
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Materials and Methods |
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Animals
Adult, male Sprague Dawley rats weighing between 150 and 200 g were acclimated to a 12-h light, 12-h dark cycle (lights between 0600 and 1800 h) and controlled temperature (22 ± 1 C). The studies were approved by the Institutional Animal Care and Use Committee at Tufts-New England Medical Center and Tufts University School of Medicine. Rat chow and tap water were provided ad libitum. Animals were made hypothyroid by the addition of 0.05% methimazole and 0.5% sodium perchlorate to their drinking water for 3 wk. Nontreated euthyroid animals of the same age were used as controls.
Studies on LAF1 mice bearing TtT-97 thyrotropic tumors were conducted with the highest standards of humane animal care in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. The animal protocols were approved by the Committee on Animal Care and Use of the University of Colorado Health Sciences Center (Denver/Aurora, CO). LAF1 mice were radiothyroidectomized 2 months before being injected with a mince of TtT-97 thyrotrophic tumor, and the resulting tumors were allowed to propagate in vivo for 5 months (16). One tumor-bearing mouse received T4 (Sigma, St. Louis, MO), 5 mg/liter, in 0.75% ethanol in the drinking water for 3 wk, whereas a control hypothyroid mouse received the ethanol vehicle. The T4 treatment resulted in a serum level of 573 nmol/liter in the treated mouse, as compared with a level of 3 nmol/liter in the hypothyroid control. Tumors were excised and homogenized in 10 volumes of 4 M guanidinium thiocyanate solution supplemented with 5% 2-mercaptoethanol.
Animal and tissue preparation for in situ hybridization histochemistry
Thyroid hormone production was inhibited by treating adult, male Sprague Dawley rats with 0.02% methimazole in the drinking water for 3 wk. Control rats received regular drinking water. At the end of the treatments, the animals were anesthetized with sodium pentobarbital (50 mg/kg body weight ip); blood was taken from the inferior vena cava, and the animals were immediately perfused transcardially with 20 ml of 0.01 M PBS (pH 7.4), containing 15,000 U/liter heparin sulfate followed by 150 ml of 4% paraformaldehyde in PBS. The pituitary glands were removed, postfixed by immersion in the same fixative for 2 h at room temperature and then cryoprotected in 20% sucrose in PBS at 4 C overnight. The pituitaries were then placed in a cryo mold, covered with OCT (Tissue-Tek, Torrance, CA), and snap frozen on dry ice. Serial 14-µm-thick coronal sections were cut on a cryostat (Leica CM3050 S, Leica Microsystems GmbH, Nussloch, Germany) and adhered to SuperFrost/Plus glass slides (Fisher Scientific, Pittsburgh, PA). The tissue sections were desiccated overnight at 42 C and stored at –80 C until prepared for in situ hybridization histochemistry.
Cell culture
TT1 cells, kindly provided by Dr. Pamela L. Mellon (University of California, San Diego, San Diego, CA), were grown till confluence in DMEM supplemented with L-glutamine (2 mM), antibiotics, and 10% fetal bovine serum (FBS) (growth medium) as detailed described previously (17). GH4C1, MSTO-211, and HEK cells were cultivated as described previously (18, 19). Experiments were carried out in experimental medium: DMEM with 0.1% BSA (T4 experiments) or 0.5% BSA (T3 experiments). The free T4 fraction in 0.1% BSA is 2.7% of total T4, and the free T3 fraction is 3% of total T3 in 0.5% BSA (20).
RNA isolation, Northern blot, and real-time PCR (RT-PCR)
For all samples used in RT-PCR, the total RNA was isolated with Trizol reagent (Invitrogen, Carlsbad, CA) according to manufacturer’s protocol. For the Northern blot analysis of Dio2 mRNA in mouse TtT-97 thyrotrophic tumors, total RNA was isolated by sedimentation through a 5.7 M cesium chloride cushion and polyA+ RNA purified on oligo-(dT) cellulose as described (21). Five micrograms of polyA+ mRNA were separated by electrophoresis through a 0.8% agarose/6% formaldehyde denaturing gel and transferred to a nylon membrane (Schleicher and Schuell, Keene, NH). The mRNA separated on the filters was hybridized at 42 C with a cDNA probe for rat D2 (22) and subsequently probed with a nearly full-length cDNA for mouse TSHß or ß-actin (21).
RT-PCR was performed as described elsewhere using cyclophilin A as a housekeeping internal control (23, 24). For the reverse transcriptase reaction, 2–4 µg total RNA were used in the SuperScript first-strand synthesis system for RT-PCR (Invitrogen) on a Robocycler thermocycler (Stratagene, La Jolla, CA). RT-PCR was performed using IQ SYBR Green PCR kit (Bio-Rad, Hercules, CA). The cycle conditions were 5 min at 94 C (Hot Start); 30 sec at 94 C, 30 sec at 58 C, and 45 sec at 72 C for 40 cycles followed by the melting curve protocol to verify the specificity of amplicon generation. Standard curves consisting of four points serial dilution (factor of 5) of mixed experimental and control groups cDNA were performed in each assay and used as calibrators. Comparable efficiency was observed presenting r2 greater than 0.99. The following primers were used: TSHß (sense, 5'-CTCGGGTTGTTCAAAGCATGAGTG-3', antisense, 5'-TGGTGTTGATGGTCAGGCAGTAG-3'); and cyclophilin A (sense, 5'-GCCGATGACGAGCCCTTG-3', antisense 5'-TGCCGCCAGTGCCATTATG-3').
Double-labeling in situ hybridization and immunocytochemistry
Methods for double-labeling in situ hybridization and immunocytochemistry have been described in detail (25, 26, 27, 28). Pituitary sections were hybridized with an 800-bp single-stranded [35S]UTP-labeled cRNA probe complementary to the entire coding region of the rat D2 gene (29). Briefly, the hybridizations were performed under plastic coverslips in a buffer containing 50% formamide, 2x standard sodium citrate (SSC), 10% dextran sulfate, 0.5% sodium dodecyl sulfate, 250 µg/ml denatured salmon sperm DNA, and 6 x 105 cpm of radiolabeled probe for 16 h at 56 C. The slides were washed in 1x SSC for 15 min and then treated with RNase (25 µg/ml) for 1 h at 37 C. After additional washes in 0.1x SSC (2 x 30 min) at 65 C, the slides were washed in PBS and treated in 0.5% Triton X-100 and 0.5% H2O2 for 15 min and with 1% BSA in PBS. Then the sections were incubated with a rabbit antiserum to rat TSH (NIH, National Pituitary Hormone Program, Torrance, CA) diluted at 1:900 in PBS containing 1% BSA in a humidified chamber for 2 d at 4 C. After several rinses in PBS, the sections were incubated in biotinylated donkey antirabbit IgG (1:200; Jackson ImmunoResearch, West Grove, PA) for 2 h, followed by ABC Elite (1:100; Vector Laboratories, Burlingame, CA) in PBS for 2 h at room temperature. The immunoreaction product was developed with 0.025% 3,3 diamino benzidine/0.0036% H2O2 in 0.05 M Tris buffer (pH 7.6). Slides were dehydrated in a graded series of ethanol containing 0.3 M ammonium acetate and were dipped into NTB2 autoradiography emulsion (Eastman Kodak, Rochester, NY). The autoradiograms were developed after 3 d of exposure at 4 C.
Quantitative analysis
Bright-field and dark-field microscopy were used to determine the percentage of immunostained thyrotrophs containing D2 mRNA and the mean number of silver grains denoting D2 mRNA per thyrotroph. Sections were visualized at x200 magnification, pictures taken at x120 and x630 magnification. All cells immunolabeled for TSH lying within a 1 mm x 1 mm area were counted from three different regions of the pituitary with the use of an ocular reticule. Cells that contained 2 or more times the number of silver grains per unit area were considered positive. A total of five to seven sections through the pituitary and an average of 70–80 cells were counted in each animal.
The number of silver grains per TSH cell were counted at x400 magnification under dark-field microscopy and adjusted for background grain counts. An average of approximately 30 TSH cells were counted from each pituitary section for a total of three sections per animal.
T4-to-T3 conversion in cultured cells
The production of 125I from outer ring-labeled T4 (NEN Life Science Products, Boston, MA), specific activity of 5692 µCi/µg, in intact cells can be analyzed by measuring the level of 125I in the medium as described and validated elsewhere (30, 31) with the following modifications: at the end of experiment, 300 µl of medium were removed, 200 µl of horse serum was added, and protein was precipitated by the addition of 100 µl 50% trichloroacetic acid followed by centrifugation at 12,000 x g for 3 min; 360 µl of the supernatant containing 125I– generated were counted in a 
-counter (Cobra II; Packard, Meriden CT) and expressed as the fraction of the total T4 counts minus the nonspecific deiodination in HEK cell lysate (<5% of the total 125I– T4 counts) and corrected for the volume counted (60%) and the 50% reduction in the specific activity relative to T4. The remaining medium was discarded, the cell pellet was sonicated in 0.1 M potassium phosphate-1 mM EDTA (pH 6.9) (PE buffer), and total protein was assayed for activity normalization. Net T3 production is calculated by multiplying the fractional conversion by the free T4 concentration in the media and expressed as fmol/h/mg protein.
Outer ring (5') deiodinase activity assay in cell sonicates
At the end of each experiment, cells were harvested in PBS and centrifuged at 10,000 g for 3 min; the pellet was sonicated briefly in PE buffer containing 10 mM DTT and 0.25 M sucrose. Protein determinations were by the Bradford method using BSA as standard. The assay was performed in the presence of 0.1–2 nM [125I]5'-T4, 20 mM DTT, in the presence and absence of 1 mM PTU, during different incubation periods, depending on cell type. Specific T4-to-T3 conversion was calculated by subtracting nonspecific deiodination using either a saturating concentration of T4 (100 nM) or the same amount of protein obtained from a HEK cell lysate. Deiodinase activity was expressed as femtomoles T4 per minute per milligram protein. The assays consumed less than 70% of the substrate.
Inner ring (5) deiodinase activity assay in cell sonicates
D3 activity was assayed by incubating 100–150 µg cellular protein, about 200,000 cpm of 3,5,[125I]3'-triiodothyronine (NEN Life Science Products), specific activity of 3390 µCi/µg, 1 mM (PTU), 10 mM DTT, and 0.1 nM unlabeled T3 for variable times. Reactions were stopped by the addition of methanol, and the products of deiodination were resolved and quantified by reverse-phase HPLC as described earlier (32). D3 velocities are expressed as fmol of T3 inner-ring deiodinated per milligram of sonicate protein per minute (fmol/min/mg protein).
Statistical analysis
All data were analyzed using PRISM software (GraphPad Software, Inc., San Diego, CA) and are expressed as mean ± SEM. ANOVA followed by Newman-Keuls multiple comparison test were used for statistical analysis in all experiments, except for in situ hybridization and immunocytochemistry studies, in which the mean percentage and SEs of positive and negative TSH cells and the means and medians of the total number of silver grains per TSH cell were calculated for each animal and for each group and compared using the Student’s t test. P < 0.05 was considered statistically significant.
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). TSH cells in euthyroid animals were small and angulated and contained a small nucleus. In contrast, TSH cells in hypothyroid rats were larger, less angulated, and had a larger nucleus. Accumulation of silver grains over TSH cells, denoting the presence of D2 mRNA, was seen in both euthyroid and hypothyroid animals. However, the percentage of TSH cells containing D2 mRNA was significantly greater in the hypothyroid animals (Fig. 2A). In addition, the number of silver grains accumulating over TSH-immunoreactive cells was significantly greater in the hypothyroid animals, with grain density ranging from 1 to 70 above background in the hypothyroid animals and from 1 to 25 in euthyroid animals. (Fig. 2B). Silver grains also accumulated over anterior pituitary cells that were not immunoreactive for TSH.
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T1) constitute the only experimental models available for the study of D2 regulation in thyrotrophs. TtT-97 cells have the highest endogenous D2 activity reported to date, 4.4 pmol/min·mg protein, and an apparent Michaelis constant (T4) of 1.5 nM, whereas TT1 cells have a lower endogenous activity, 150 fmol/min·mg protein and an apparent michaelis constant (T4) of 2.4 nM (Fig. 3A), but still higher than most D2-expressing cells (19). No type 1 iodothyronine deiodinase activity was detected in TtT-97 or TT1 cells (data not shown). However, by supplementing growth medium with 10–7 M selenium and using extrasensitive D3 activity assay conditions (low substrate concentration and extended incubation times), we detected some D3 activity in the TT1 cell line (Fig. 3B). Although significantly stimulated by TPA or the combination of TPA and TGFß or a cocktail containing aFGF, bFGF, EGF, TGFß1, TPA, and IL-1ß (P < 0.05), D3 activity remained at very low levels (Fig. 3B).
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). Despite this loss in D2, the remaining D2-mediated outer ring deiodinase capacity is still by far higher than in any other tissue. In similar studies, treatment of TT1 cells with T4 resulted in 65–80% loss of D2 activity but only a 20–35% decrease in D2 mRNA levels (Fig. 4A). Poor Dio2 gene responsiveness to thyroid hormone was further confirmed by treating TT1 cells with T3. Whereas incubation with T3 barely changed D2 mRNA levels, TSHß mRNA levels decreased by more than 50% (Fig. 4B). This confirms that posttranscriptional mechanisms are the major determinants of D2 activity in these two thyrotrophic cell models.
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T1 cells, we used 100 µM cycloheximide and observed a loss of approximately 50% D2 activity in the first 60 min (Fig. 4C). Next, we used different concentrations of the proteasome inhibitor MG132 to test how efficiently these cells degrade D2. GH4C1 cells, originally used to describe the effects of MG132 on D2 activity (18), were used as reference. Whereas a 5-h incubation of GH4C1 cells with MG132 resulted in an approximately 2-fold increase in D2 activity, in similarly treated TT1 cells D2 activity increased by almost 4-fold (Fig. 4D). These results indicate that TT1 cells actively process D2 in a fashion similar to that used by many other D2-expressing cells characterized to date (33). Thus, the remainder of the experiments was performed in TT1 cells.
To understand the relationship between T4 concentration and D2 activity in thyrotrophs, we exposed TT-1 cells overnight to variable free T4 concentrations and compared the results with those of two other D2-expressing cell lines, ie a rat pituitary tumor cell line (GH4C1) and a human mesothelioma cell line (MSTO-211H). Before the experiment, all cells were preincubated for 24 h in medium containing charcoal-stripped FBS. The next day, cells were exposed for 12 h to medium containing 0.1% BSA and known amounts of T4 to generate a defined range of free T4 concentrations of 0–400 pM (the physiological free T4 concentration in serum is 
20 pM). This resulted in a sharp drop in D2 activity in all three cell lines, with most of the loss occurring over the 0–50 pM T4 range (Fig. 5A). It is notable that the fall in D2 activity varied according to the initial D2 level. In GH4C1 cells, which had the lowest starting point, D2 activity dropped to undetectable levels at 40 pM T4, whereas the decrease was less pronounced in MSTO-211H and TT1 cells. In MSTO-211H cells, D2 activity continued to fall as the free T4 concentration increased, eventually disappearing at approximately 350 pM T4. Remarkably, D2 activity in TT1 cells stabilized at approximately 50 pM T4, despite increasing free T4 concentrations to 400 pM (Fig. 5A).
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T1 cells, we repeated the T4-induced loss of D2 activity experiment exposing cells to zero or 100 pM T4 for 16 h, except that 2 h before harvesting, we treated cells with cycloheximide to stop protein synthesis. Such treatment resulted in a sharp drop in D2 activity, at a fractional rate that was independent of the T4 content in the medium (Fig. 5, B and C). This indicates that the D2 turnover in TT1 cells remains high, despite a relatively constant level of D2 activity.
D2-mediated T3 production suppresses TSHß gene expression in TT1 cells
Next we wished to test the hypothesis that maintenance of D2 activity in the presence of increasing free T4 concentrations, as evidenced in Fig. 5A, would increase net T3 production and suppress TSH expression. To test this, we monitored TT1 whole-cell deiodination measuring the 12-h integrated T3 production (from added T4) and also processed cells for measurement of TSHß mRNA levels. T3 production was calculated by multiplying the T4-to-T3 conversion rate (125I-T3/125I-T4) by the gravimetric amounts of added T4 as described in detail elsewhere (34). As predicted, incubation of TT1 cells with increasing concentrations of T4 resulted in a progressive decrease in the fractional conversion of T4 to T3 (Fig. 6A, inset) but, because of the increasing free T4 concentration in the media, this results in a reciprocal increase in D2-mediated T3 production (Fig. 6A) and suppression of TSHß gene expression (Fig. 6B). This decrease in the fractional conversion (Fig. 6A, inset) also indicates that increasing T4 amounts are entering the cells, and thus, D2 stabilization is not likely to be due to decreased T4 transport.
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T1 cells with 50 nM rT3, a relatively high concentration of rT3 that not only will induce D2 inactivation but also will compete with T4 for binding to the residual active D2 molecules. Treatment with rT3 stopped T4-to-T3 conversion and prevented D2-mediated suppression of TSHß (Fig. 7A) but did not interfere with TSHß suppression mediated by exogenously added T3 (Fig. 7B).
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Discussion |
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), the key deiodinase that activates thyroid hormone and mediates the signal transduction between serum T4 and binding of T3 to TR. In fact, this mechanism is possible only because of the typical homeostatic behavior of D2 (inactivation by catalysis) is offset in the thyrotrophs by the high rate at which this enzyme is synthesized in these cells. This allows for sustained T3 production, even in the presence of elevated T4 concentrations, restraining and eventually turning off the expression of the TSHß gene, the basis for the TSH feedback mechanism.
The presence of D2 in the pituitary gland is generally accepted, but its presence in thyrotrophs is not well established. Based on the intrinsic role of serum T4 in the TSH feedback mechanism (2), D2 is expected to be present in thyrotrophs. However, this has been difficult to prove because of the lack of abundance of TSH-producing cells in the pituitary gland (35). One alternative strategy has been to screen pituitary tumors for the presence of D2. At least two reports (14, 36) indicate that D2 is present in TSH-secreting adenomas, but another (13) has found D3 as the predominant deiodinase in TSH-producing tumors. In the present investigation, the presence of D2 in thyrotrophs was clearly demonstrated in rat pituitary sections by in situ hybridization histochemistry (Figs. 1 and 2) and in two rodent thyrotrophic cell models (Fig. 3A). In the rat pituitary sections, the fact that only approximately 30% of the thyrotrophs are positive for D2 under euthyroid conditions (Fig. 2A) probably reflects the low abundance of D2 mRNA, which is below the detection level of this technique. This is supported by the increase in the presence of D2 mRNA to almost 80% of the thyrotrophs during hypothyroidism (Fig. 2B). However, it is not possible to exclude at this time the existence of a thyrotrophic subpopulation that does not express D2 in which TSH secretion would be controlled primarily by serum T3.
TT1 cells express WSB-1, the D2-specific ubiquitin ligase adaptor, and VDU-1 and VDU-2, two deubiquitinases involved in rescuing and reactivation of inactive ubiquitinated D2 (data not shown). The presence of these proteins involved in D2 ubiquitination and degradation explains the relatively short half-life of D2 in these cells (Fig. 4C) as well as its sensitivity to T4 (Fig. 4, A and B) and MG132 (Fig. 4D). However, when a range of concentrations of T4 is used, it is notable that at T4 concentrations greater than 50 pM the loss of D2 activity is impaired in TT1 cells as compared with that of two other D2-expressing cells under identical treatment conditions (Fig. 5A). This could indicate that the ubiquitinating/proteolytic machinery is exhausted at high T4 concentrations or that it has reached its maximal capacity. The experiment with cycloheximide strongly favors the second possibility because D2 activity was rapidly lost after D2 synthesis was stopped (Fig. 5, B and C). This indicates that the ubiquitinating/proteolytic machinery is not exhausted at high T4 concentrations. Rather, it supports the idea that the rate of D2 synthesis in these cells equals the maximal rate of T4-induced D2 ubiquitination. This is likely to explain the persistence of D2 activity in the presence of high T4 concentrations. A high rate of reactivation of inactive ubiquitinated D2 via VDU-1/2-mediated deubiquitination could also contribute to this phenomenon, given the high expression of VDU-1 in the pituitary gland, although levels in TT1 cells are not particularly high (data not shown).
The expression of D2 in thyrotrophs is at the core of the T4-mediated TSH feedback mechanism. D2-mediated net T3 production is low at lower T4 concentrations and high at high T4 concentrations (Fig. 6A), demonstrating that fluctuations in D2 activity caused by ubiquitination do not compensate for changes in T4. This, however, is not the case in GH4C1 and MSTO-211 cells, which express D2 at lower levels (Fig. 5A). In these cell lines, D2 activity decreases with the increase in T4 concentration so that T3 production eventually halts at a minimal level. Such a scenario would not be desirable in thyrotrophs because a major decline in D2 activity resulting from an increase in serum T4 would disrupt the transduction mechanism by which T4 controls TSHß gene expression. As a result, the feedback mechanism would lose its exquisite sensitivity to minor elevations in serum T4 concentrations that can normally result in profound TSH suppression.
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Footnotes |
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The authors have no conflict of interest.
First Published Online January 5, 2006
Abbreviations: aFGF, Acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; D2, type 2 iodothyronine deiodinase; D3, type 3 deiodinase; DTT, dithiothreitol; EGF, epidermal growth factor; FBS, fetal bovine serum; PTU, 6-n-propyl-2-thiouracil; RT-PCR, real-time PCR; SSC, standard sodium citrate; Se, sodium selenite; TPA, 12-O-tetradecanoylphorbol 13-acetate; TR, thyroid hormone receptor.
Received October 12, 2005.
Accepted for publication December 22, 2005.
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References |
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, and -subunits after thyroxine administration: comparison of mouse hypothyroid pituitary and thyrotropic tumors. Endocrinology 112:2050–2053[Abstract]T1 cells. Endocrinology 139:4476–4482 and ß thyroid hormone receptors from thyrotrope cells: the mouse pituitary-specific ß2 isoform differs at the amino terminus from the corresponding species from rat pituitary tumor cells. Mol Endocrinol 5:1049–1061[Abstract]This article has been cited by other articles:
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