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Published in final edited form as: J Endocrinol. 2011 Jul 25;211(1):73–78. doi: 10.1530/JOE-11-0248

Thyroxine-induced induction of pyroglutamyl peptidase II and inhibition of TSH release precedes suppression of TRH mRNA and requires type 2 deiodinase

Alessandro Marsili 1, Edith Sanchez 2,*, Praful Singru 2,#, John W Harney 1, Ann Marie Zavacki 1, Ronald M Lechan 2, P Reed Larsen 1
PMCID: PMC3558748  NIHMSID: NIHMS435192  PMID: 21788297

Abstract

Suppression of TSH release from the hypothyroid thyrotrophs is one of the most rapid effects of T3 or T4. It is initiated within an hour, and precedes the decrease in TSHβ mRNA inhibition and is blocked by inhibitors of mRNA or protein synthesis. TSH elevation in primary hypothyroidism requires both the loss of feedback inhibition by thyroid hormone in the thyrotrophs and the positive effects of TRH. Another event in this feed back regulation may be the thyroid hormone induction of the TRH-inactivating pyroglutamyl peptidase II (PPII) in the hypothalamic tanycytes. This study compared the chronology of the acute effects of T3 or T4 on TSH suppression, TRH mRNA in the hypothalamic paraventricular nucleus (PVN), and the induction of tanycyte PPII. In wild type mice, T3 or T4 caused a 50% decrease in serum TSH in hypothyroid mice by 5 hours. There was no change in TRH mRNA in PVN over this interval, but there was a significant increase in PPII mRNA in the tanycytes. In mice with genetic inactivation of the type 2 iodothyronine deiodinase, T3 decreased serum TSH and increased PPII mRNA levels, while T4-treatment was ineffective. We conclude that the rapid suppression of TSH in the hypothyroid mouse by T3 occurs prior to a decrease in TRH mRNA though TRH inactivation may be occurring in the median eminence through the rapid induction of tanycyte PPII. The effect of T4, but not T3, requires the type 2 iodothyronine deiodinase.

Keywords: type 2 deiodinase, feed-back, thyrotroph, TSH, hypothalamus, TRH, PPII, hypothyroid, thyroxine, triiodothyronine

Introduction

The feedback regulation of TSH secretion by thyroid hormones is mediated by the binding of 3,3', 5' T3 to the TRβ2 receptor in the pituitary and hypothalamus (Chiamolera and Wondisford 2009). The acute suppression of TSH release from the pituitary is initiated within 30 minutes of the injection of T4 or T3 to the hypothyroid rat and the degree of suppression parallels that of the saturation of the nuclear T3 receptors in the pituitary (Larsen, et al. 1981; Silva and Larsen 1977). This response is blocked by inhibitors of mRNA or protein synthesis, suggesting that there may be induction or suppression of specific proteins which are required for this effect (Bowers, et al. 1968a, b; Vale 1968). The decrease in circulating TSH in these models occurs well before suppression of the synthesis of TSHβ mRNA and, in fact, studies have shown that in the early phases of the response there is an increase in pituitary TSH content, suggesting that synthesis proceeds for some period of time in association with inhibition of release of the hormone (Shupnik, et al. 1989; Silva and Larsen 1978). While most studies have focused on events in the pituitary, it is clear that positive regulation of TSH by TRH is also required for TSH elevation during hypothyroidism, indicating that this is not simply due to the absence of negative feedback regulation on the thyrotroph TSH (Nikrodhanond, et al. 2006).

It is well-known that the conversion of T4 to T3 by the type 2 deiodinase (D2) is required for suppression of TSH at the pituitary level (Larsen, et al. 1979). The requisite degree of saturation of the thyrotroph nuclear T3 receptors requires both circulating T3 and D2-mediated conversion of intracellular T4. The normal feedback regulation of TRH mRNA synthesis similarly requires both circulating T3 and that derived from D2-mediated T4 to T3 conversion (Kakucska, et al. 1992; Lechan and Fekete 2005). Since there is no D2 mRNA in the parvocellular region of the hypothalamic paraventricular nucleus (PVN) where hypophysiotropic TRH is synthesized, it has been proposed that the D2 highly expressed in tanycytes of the medio-basal hypothalamus may provide T3 to TRH-producing neurons through extensive cell to cell interactions (Fekete and Lechan 2007; Riskind, et al. 1987; Tu, et al. 1997). Recent evidence has suggested that inactivation of TRH in the median eminence by pyroglutamyl peptidase II (PPII), a membrane-bound, highly specific TRH peptidase expressed in the tanycytes (Charli, et al. 1998; Heuer, et al. 2000), contributes to central regulation of TSH secretion (Sanchez, et al. 2009). PPII is also expressed in lactotrophs and somatotrophs in the rat pituitary but not thyrotrophs (Cruz, et al. 2008; Heuer, et al. 1998) indicating that pituitary PPII may not be involved in TSH regulation. As PPII synthesis is a T3-dependent process, with PPII mRNA being induced in the median eminence within a few hours of exposure to T3 or T4 (Sanchez et al. 2009), it suggests that its acute effects to inactivate TRH are localized in this region.

The D2 knockout mouse is resistant to the feedback effects of T4 on pituitary TSH release in both the euthyroid and the hypothyroid states (Christoffolete, et al. 2007; Schneider, et al. 2001). In the present study, we used this model to analyze the chronological correlations between the inhibition of TSH release in response to T4 or T3 in intact animals with the induction of PPII mRNA in the tanycytes as opposed to preproTRH mRNA in the PVN. Our results indicate that the increase in PPII mRNA occurs before any reduction in intact preproTRH mRNA, suggesting that an increase in PPII with its consequent inactivation of TRH could play a role in the acute suppression of TSH release by T3 or T4.

Materials and methods

Animals

All animal experimental protocols were approved by the Animal Research Committee of Harvard Medical School. C57BL/6J mice were purchased from The Jackson Laboratories (Bar Harbor, ME). D2KO/C57Bl/6J are as previously described (Schneider et al. 2001). All animals were maintained under 12 hour light/dark cycle and the standard animal facility temperature and humidity.

Hypothyroidism induction and experimental protocol

Mice were made hypothyroid by placing them on drinking water containing 0.1% MMI (Sigma) and 1% KClO4 (Fisher) (MMI/ClO4), as previously described (Marsili, et al. 2010). The day of the experiment, a serum sample was obtained from the tail for TSH measurement. Immediately after, 6n-propylthiouracil (PTU) (2 mg/animal) was administered intraperitoneally to block D1 activity. One hour later, D2KO and WT mice were subdivided into 3 groups, each mouse receiving a single intraperitoneal injection of T4 (3 µg/100 g body weight), T3 (1.2 µg/100 g body weight), or vehicle (PBS). Five hours after the hormone/vehicle administration, the mice were euthanized with an isofluorane overdose and blood collected via cardiac puncture.

Tissue processing

Animals were overdosed with pentobarbital (50 mg/kg; Ovation Pharmaceuticals, Inc., Deerfield, IL), and perfused transcardially with 0.01 M PBS (pH 7.4) containing 15,000 U/liter heparin sulfate, followed by 4% paraformaldehyde in PBS. The brains were removed and postfixed by immersion in the same fixative for 2 h at room temperature. Tissue blocks containing the hypothalamus were cryoprotected in 25% sucrose/PBS at 4 °C overnight and then snap frozen on dry ice. Serial 18 µm-thick coronal sections through the rostrocaudal extent of PVN and median eminence were cut on a cryostat (Leica CM3050 S; Leica Microsystems, Nussloch GmbH, Germany) and adhered to Superfrost/Plus glass slides (Fisher Scientific Co., Pittsburgh, PA) to obtain four sets of slides, each set containing every fourth section through the PVN or median eminence. The tissue sections were desiccated overnight at 42 °C and stored at −80 °C until prepared for in situ hybridization histochemistry.

In situ hybridization histochemistry

Every fourth section through the PVN or median eminence was hybridized with an 800-bp single stranded [35S] uridine 5-triphosphate (UTP)-labeled cRNA probe complementary to the entire coding region of the mouse TRH gene, or 644 bp single stranded [35S]-UTP-labeled cRNA probe complementary to the coding region of rat pyroglutamyl peptidase II (nucleotides 129–773), respectively, as previously described (Kadar, et al. 2010; Sanchez et al. 2009).

Hybridizations were performed under plastic coverslips in a buffer containing 50% formamide, a 2-fold concentration of standard sodium citrate (2× saline sodium citrate), 10% dextran sulfate, 0.25% BSA, 0.25% Ficoll 400, 0.25% polyvinylpyrolidone 360, 250 mM Tris (pH 8.0), 0.5% sodium dodecyl sulfate, 250 µg/ml denatured salmon sperm DNA, and 5 × 105 cpm of the radiolabeled probe for 16 h at 55 C. Slides were dipped into Kodak NTB autoradiography emulsion (Eastman Kodak, Rochester, NY) diluted 1:1 in distilled water and the autoradiograms developed after 3 d of exposure for TRH mRNA or 30 d of exposure for pyroglutamyl peptidase II mRNA at 4 C. The specificity of hybridization was confirmed using sense probes, which resulted in the total absence of specific hybridization signal in the hypothalamus.

Image analysis

Slides were visualized with an Axioplan 2 imaging microscope (Carl Zeiss Microimaging Inc., Thornwood, NY) under dark-field illumination using a COHU 4912 video camera (COHU, Inc., San Diego, CA), and the images analyzed with a Macintosh G4 computer using Scion Image software (National Institutes of Health, Bethesda, MD). Background was removed by thresholding the image, and integrated density values (density × area) of the hybridized regions were measured in rostrocaudal serial sections through the PVN or median eminence in one set of slides for each animal. Nonlinearity of radioactivity in the emulsion was evaluated by comparing density values with a calibration curve created from autoradiograms of known dilutions of the radiolabeled probes, immobilized on glass slides in 1.5% gelatin, fixed with 4% paraformaldehyde, and exposed and developed simultaneously with the in situ hybridization autoradiograms.

Serum T4, T3, TSH measurement

All hormones were measured by RIA after collecting blood from the tail vein. Serum T4 and T3 were measured using the COAT-A-COUNT total T4 and T3 kit (DPC, Los Angeles, CA), following the manufacturer’s instructions, with mouse standard curves prepared in charcoal-stripped (T4 and T3 deficient) mouse serum as previously described (Christoffolete et al. 2007; Marsili et al. 2010). TSH was determined using the rat TSH RIA from Alpco Diagnostic (Salem, NH). All values fell within the linear range of a curve generated by the serial dilution of sample dilution buffer, according to the manufacturer’s instructions. The normal range for T4 was 1.61 ± 0.17 and 2.79 ± 0.32 µg/dl for WT and D2KO, respectively. The normal range for T3 was 0.76 ± 0.07 and 0.77 ± 0.06 ng/ml for WT and D2KO, respectively (Christoffolete et al. 2007). TSH concentrations (ng/ml) were determined by extrapolating from the intercept of the high TSH mouse serum with the purified rat TSH standard curve supplied by the manufacturer, after correction for the difference of the nonspecific binding obtained with serum vs. the nonspecific binding obtained with the assay buffer (Pohlenz, et al. 1999). TSH concentrations were 4.04±0.67 (range from 3.32 to 4.93) and 35.7.2±5.2 (range from 27.5 to 47.8) ng rat equivalent/ml of rat equivalent serum in euthyroid and hypothyroid male mice, respectively.

Statistical analysis

Results are presented as means ± SEM. When only two groups were analyzed, statistical significance was determined using an unpaired Student's t-test. Two-way ANOVA followed by Bonferroni correction using Prism 4 software (GraphPad Software, Inc., San Diego, CA) was used to compare the effects of three different treatment on two genotypes (WT and D2KO). Values of p < 0.05 were considered statistically significant.

Results

Both wild type and D2KO mice were markedly hypothyroid after five weeks of treatment with antithyroid drugs, as confirmed by measurements of T3, T4, and TSH (Table 1 and Fig. 1). Serum TSH values were markedly elevated in vehicle-treated mice in both groups in the range of 30–40 ng/ml, about 10-fold the normal value (Table 1). T3 caused a marked increase in serum T3 five hours after injection in both WT and D2KO mice (Fig. 1). Serum T4 concentrations were also 3- to 4-fold the normal level in both wild type and D2KO after the T4 injection, with no difference between the two genotypes. Since all animals had received PTU to block D1-mediated T4 to T3 conversion, it was not surprising that the serum T3 in the T4-treated mice was not significantly different from vehicle-treated mice. Injection of vehicle and manipulation of the animals caused no change in the serum TSH values (Fig. 1). T4 caused an approximately 60% decrease in TSH in the wild type animals (p<0.001), but had no effect in the D2KO mice (Fig. 1). On the other hand, both WT and D2KO animals had an approximately 70–80% decrease in TSH after injection of T3 (p<0.001).

Table 1.

Serum thyroid hormone concentrations in hypothyroid WT and D2KO mice 5 hours after hormone or vehicle injection (mean ± SEM; n=5–7 per group)

WT D2KO
Serum T3
(ng/ml)		 Serum T4
(µg/dl)		 Serum T3
(ng/ml)		 Serum T4
(µg/dl)
Treatment
Vehicle 0.3 ± 0.1 1.1 ± 0.2 0.2 ± 0.1 0.9 ± 0.1
T3
(1.2 µg i.p./100 g bw)		 19.4 ± 0.4*** 1.2 ± 0.2 16.3 ± 3.0*** 0.9 ± 0.1
T4
(3 µg i.p./ 100 g bw)		 0.6 ± 0.1 13.8 ± 2.5*** 0.4 ± 0.2 11.5 ± 2.5***
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***

= p<0.001 vs. vehicle.

Fig. 1. Effect of T3 and T4 on serum TSH in hypothyroid WT and D2KO mice.

Fig. 1

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TSH values were measured before and 5 hours after ip administration of vehicle, T4 (3 µg/100 g body weight) or T3 1.2 µg/100 g body weight. All animals received 2 mg PTU ip 1h before hormone or vehicle administration. (A) wild type mice; (B) D2KO mice. Data are mean ± SEM of 5–7 animals per group; ***, p <0.001 for difference from pre-injection.

To determine whether the T3- or T4-induced acute decrease in TSH involved suppression of TRH mRNA, we performed in situ hybridization examination of preproTRH mRNA expression in the PVN of the same mice. In all animals, the TRH mRNA was markedly increased over that in euthyroid mice, as a consequence of their hypothyroidism (Kadar et al. 2010). Despite the significant decrease in TSH in all mice receiving T3 and in the WT mice receiving T4, there was no significant change in the integrated density values of the TRH mRNA in the thyroid hormone-treated groups (Fig. 2).

Fig. 2. Absence of an acute response of TRH mRNA 5 hours after T3 and T4 treatment in hypothyroid WT and D2KO mice.

Fig. 2

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A) Dark-field illumination photomicrographs of TRH mRNA in the medial parvocellular subdivision of the the hypothalamic paraventricular nucleus (PVN) in hypothyroid WT (A–C) and D2KO mice (A1–C1) 5 hours after ip administration of vehicle, T4 (3 µg/100 g body weight) or T3 (1.2 µg/100g body weight). (D) Graph represents the densitometric analyses of the in situ hybridization autoradiograms. Data are analyzed by Two-Way ANOVA after Bonferroni correction. NS=not significant. Data are mean ± SEM of 5–7 mice per group. III = third ventricle.

Because it has been previously shown that there is an increase in the PPII mRNA in hypothalamus 4–7 hours after a single dose of T3 or T4 (Sanchez et al. 2009), we examined the mediobasal hypothalamus for changes in PPII mRNA. In vehicle-treated WT animals, PPII mRNA was barely detectable in tanycytes lining the floor of the third ventricle, but increased significantly in WT mice treated with T4 (Fig. 3). No change was observed in the T4-treated D2KO animals (Fig. 3). Thus, the tanycytes in the median eminence require D2-mediated T4 to T3 conversion for induction of PPII transcription, and this occurs within a 5-hour interval. As expected, PPII mRNA in both WT and D2KO mice responded modestly to T3 (Fig. 3) with a significant increase in PPII (p<0.05), which was, however, less that that in the T4-treated WT animals (p<0.01).

Fig. 3. The acute increase in PPII mRNA induced by T4, but not T3, is absent in D2KO mice.

Fig. 3

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Dark-field illumination photomicrographs of PPII mRNA (arrows) in tanycytes lining the floor of the third ventricle (III) at the level of the median eminence (ME) in hypothyroid WT (A–C) and D2KO (A1–C1) 5 hours after administration of vehicle, T4 (3 µg/100 g body weight) or T3 (1.2 µg/100 g body weight. (D) Graph represents the densitometric analyses of the in situ hybridization autoradiograms. Data were analyzed by Two-Way ANOVA after Bonferroni correction. ***, p<0.001; ns=not significant. Data are mean ± SEM of 5–7 mice per group.

Discussion

The present studies confirm a previous report that administration of both T4 and T3 results in >50% suppression of TSH in hypothyroid mice within 5 hours and that the response to T4, but not to T3, is absent in D2KO mice (Schneider et al. 2001). This pattern is identical to the results found earlier in hypothyroid rats in which the degree of acute suppression of TSH release was shown to parallel the occupancy of the pituitary nuclear T3 receptors (Silva and Larsen 1977, 1978), effect blocked by inhibition of D2 with iopanoic acid (Larsen et al. 1979). A decrease in TSH secretion from TαT1 mouse thyrotroph cells by T4 also requires D2-mediated T4 to T3 conversion but does not require TRH (Christoffolete et al. 2007). Thus, some or all of the acute effects of T4 may be due to a direct interaction of the intracellular T3 produced by D2 in the thyrotrophs.

In addition, TRH synthesis is also negatively regulated by T3 (Segerson, et al. 1987). While a decrease in preproTRH gene transcription by T3 has been demonstrated within 5 hours using an intronic probe, we found no change in the TRH mRNA content of the hypothalamus over this time period (Fig. 2) (Sugrue, et al. 2010). This argues that a decrease in TRH synthesis is not required for the rapid inhibition of TSH release by thyroid hormone in the hypothyroid mouse, but does not eliminate the possibility that an acute suppression of TRH also play a role in this response.

Recent studies have identified high expression of the TRH-inactivating PPII in the tanycytes in the floor of the third ventricle. This metalloprotease is encoded by a positively T3-responsive gene, and its mRNA increases as early as 4 hours within the cell bodies of the tanycyte population (Sanchez et al. 2009). There is a close association between tanycyte end foot processes and preproTRH-containing axon terminals in the median eminence, suggesting a potential inactivation of TRH at this site. Our experiments revealed a significant increase in PPII mRNA within 5 hours of T3 treatment, with an even more potent effect of T4 at this dosage in WT mice, while PPII expression was unchanged after T4 administration in D2KO mice. These results indicate that D2-mediated T4 to T3 conversion, presumably in tanycytes, is required for the rapid induction by T4 of PPII mRNA in hypothyroid mice. Of particular importance, however, is the observation that PPII inhibition increases the TSH response to cold exposure or TRH (Sanchez et al. 2009), indicating that PPII plays a role in increasing endogenous TSH release. It is conceivable, therefore, that the acute fall of TSH in hypothyroid animals treated with thyroid hormone could also involve a reduction in the TRH supply to thyrotrophs as a result of its degradation in the median eminence by the rapid T3-mediated rapid induction of tanycyte PPII. This may supplement the effect of T3-induced suppression of TSH release in the thyrotroph per se through an as yet undefined mechanism.

We cannot exclude the possibility that increase in somatostatin or dopamine could be involved in this acute response. These agents are known to inhibit TSH release and could be stimulated by T3. However, in vitro effects of these agents require several days rather than hours of exposure (Foord, et al. 1984; Levy, et al. 1992; Tam, et al. 1996). Given the previously demonstrated blockade of this T3 effect by synthetic protein or gene transcriptional inhibitors (Bowers et al. 1968a, b; Vale 1968), and the correlation between T3 receptor occupancy and the magnitude of the effect in earlier studies (Silva and Larsen 1977, 1978), a rapid gene transcriptional effect in thyrotrophs and perhaps in the tanycytes seems a more attractive hypothesis for the acute action of T3 in this system.

Acknowledgments

This work was supported by the National Institutes of Health Grants DK36256 and T32DK007529 to PRL and DK37021 to RML. A.M. was partially supported by a fellowship stipend from Department of Endocrinology and Kidney, University Hospital of Pisa, Italy.

Footnotes

Publisher's Disclaimer: Disclaimer. This is not the definitive version of record of this article. This manuscript has been accepted for publication in Journal of Endocrinology, but the version presented here has not yet been copy edited, formatted or proofed. Consequently, the Society for Endocrinology accepts no responsibility for any errors or omissions it may contain. The definitive version is now freely available at doi: 10.1530/JOE-11-0248, 2011, Society for Endocrinology

Disclosure Statement

No competing financial interests exist.

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