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Department of Endocrinology and Metabolism (A.A., C.L.V., O.B., W.M.W., E.F.), Academic Medical Center of the University of Amsterdam, 1100 DE Amsterdam, The Netherlands; Netherlands Institute for Brain Research (A.A., C.L.V., U.A.U., D.F.S., E.F.), 1105 AZ Amsterdam, The Netherlands; and Laboratory of Developmental Biology (B.V.), Department of Cell and Molecular Biology, Karolinska Institute, Stockholm S-17177, Sweden
Address all correspondence and requests for reprints to: A. Alkemade, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands. E-mail: A.Alkemade{at}nih.knaw.nl.
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Abstract |
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1, 2, ß1, and ß2 was studied in consecutive sections of six hypothalami and pituitaries. Staining intensity showed strong interindividual variation but was consistently present in the infundibular nucleus, paraventricular nucleus, and supraoptic nucleus. In addition, strong TR immunoreactivity was observed in the anterior pituitary. Neuropeptide Y and proopiomelanocortin mRNA-positive cells in the infundibular nucleus, which were studied in three other hypothalami, appeared not to express TRs, and thus, the neurons expressing TRs in the human mediobasal hypothalamus remain to be characterized. |
Introduction |
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1, 2, ß1, and ß2 has been demonstrated in a large number of brain areas in the adult rat, including the hippocampus, cerebral cortex, and cerebellum (1, 2, 3). TRs are also expressed in the rat hypothalamus and anterior pituitary, both at the peptide and mRNA level. TRH neurons in the paraventricular nucleus (PVN) express all four TR isoforms (4). Hypothalamic TRß2 expression is also prominent in the rat arcuate nucleus (ARC) and in the median eminence (5), whereas the supraoptic nucleus expresses more TR than TRß mRNA (3).
A functional role for hypothalamic TR in the endocrine feedback regulation of thyroid hormone has been suggested by a number of studies. TRß2 expression in thyrotropic cells of the pituitary increases in hypothyroid rats (6). In the hypothalamus, thyroidectomy induces increased TR2 expression in the PVN (7) and increased TRß1 expression throughout the rat brain, including the hypothalamus (8).
An essential role for TRß2 for thyroid hormone-negative feedback in TRH neurons in the PVN has been described in TR isoform-specific knockout (KO) mice (9). TR1 is essential for glucose utilization during brain development (10). In addition, a role for TR1 in thyroid hormone regulation is supported by studies in KO mice. TR1 KO mice show a mild hypothyroidism, which is more severe in animals with a combined deletion of TR1 and TRß (11, 12).
Only a few studies are available on TR isoform expression in the human pituitary. Yen et al. (2) reported immunocytochemical staining of TR 1, 2, ß1, and ß2 in anterior pituitary cells, and TR expression was reported to be lower in nonfunctioning tumors of the anterior pituitary compared with normal tissue (13). Remarkably, so far, no reports are available on TR isoform expression in the human hypothalamus. In the present study, we investigated the distribution of TR isoforms in the human hypothalamus and anterior pituitary by immunocytochemistry using polyclonal antisera directed against synthetic isoform-specific peptides derived from the human TR amino acid sequence (14, 15).
In a second series of experiments, we attempted to determine the cell type expressing TRs in the infundibular nucleus (IFN), which is the human homolog of the rat ARC (16). The IFN contains neurons expressing both neuropeptide Y (NPY) and agouti-related protein (17). These neurons project to the hypothalamic PVN and establish synaptic contacts with TRH neurons (18). Neurons containing proopiomelanocortin (POMC)-derived peptides are present more laterally in the IFN and also establish synapses with TRH neurons in the PVN (18). We examined TR expression in NPY and POMC cells in the IFN by TR immunocytochemistry combined with either NPY or POMC mRNA in situ hybridization.
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Materials and Methods |
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Nine formalin-fixed and paraffin-embedded hypothalami and six pituitaries were obtained from The Netherlands Brain Bank at The Netherlands Institute for Brain Research in accordance with the formal permission for a brain autopsy and the use of human brain material and clinical information for research purposes. Patients with a short postmortem delay were selected. Clinicopathological data of the patients are summarized in Table 1
. Serial coronal sections (6 µm) were made from the level of the lamina terminalis to the mamillary bodies and mounted on Superfrost Plus Plus slides (Menzel Gläser, Germany). Sections were dried for at least two nights at 37 C.
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A set of four polyclonal antisera was obtained by immunization with peptides derived from the rat TR isoform sequences and homologous to human TRs [TR1 (antibody no. 314), TR2 (no. 317), and TRß1 (no. 319) are 100% homologous to the human and mouse sequence, and TRß2 (no. 321; EKPFPQVRSPPHSHK) is 85 and 93% homologous to the human and mouse sequence, respectively]. The polyclonal antisera were obtained by immunization with these peptides as conjugated by carbodiamide to keyhole limpet hemocyanin (Eurogentec, Seraing, Belgium) in the presence of Freund’s complete adjuvant. We used the third bleeding (3 months after immunization) of these antisera. Characteristics and specificity on Western blots of the antisera against TR1, TR2, and TRß1 have been described previously (14, 15).
Specificity tests
Specificity of the antisera sets was supported by staining of sections with the preimmune sera, assessment of cross-reactivity with preadsorption of heterologous peptides of TR isoforms, preadsorption tests with homologous peptides, and Western blotting. Western blots with the TR and TRß1 antisera performed on isoform-specific KO mice have been published elsewhere (14, 15). In short, no bands were observed at the expected molecular weight for TRs in KO mice in contrast to wild-type littermates.
Preimmune staining. Staining was performed with the preimmune sera of all four rabbits on human hypothalamus and pituitary sections. The staining procedure was identical to the immunocytochemical staining described later in Immunocytochemistry.
Cross-reactivity. To investigate cross-reaction between TR isoforms, all antisera were used for spot stainings of each individual synthetic TR peptide after fixation on nitrocellulose membranes. Peptides were dissolved (500 ng/µl) in a medium containing 10% glycerol, 10% dimethylformamide, and 2.5% Nonidet P-40 (Sigma, St. Louis, MO). Subsequently, the peptides were spotted on 0.2% gelatin-coated nitrocellulose transfer membranes (0.1 µm; Schleicher & Schuell, Dassel, Germany) in a concentration series (100–0.05 ng/µl medium), followed by overnight fixation with 4% paraformaldehyde filter paper using a press block (19). Arginine vasopressin (AVP; Sigma V9879 lot no. 35F5810; Sigma) was spotted (22 ng/µl) as a control. Aldehyde groups were inactivated by rinsing in distilled water (3 x 10 min), Tris-buffered saline (TBS; 0.05 M Tris and 0.15 M NaCl, pH 7.6; 3 x 10 min), and supermix (SUMI) (0.05 M Tris, 0.15 M NaCl, 0.25% gelatin, and 0.5% Triton, pH 7.6; 3 x 10 min). Subsequently, the spotted peptides were incubated with TR antisera (concentrations as used for immunocytochemistry) or with AVP antiserum (Truus 29.01.86, 1:1000) for 1 h at room temperature (RT) and overnight at 4 C. The staining procedure was performed using the avidin-biotin complex method, as described in Immunocytochemistry.
Preadsorptions. Ten micrograms of each synthetic peptide were spotted (20 x 1 µl spots; 500 ng/µl) on nitrocellulose, with AVP as a control. Fixation and inactivation of the aldehyde groups were performed as described earlier. The spotted homologous peptides were incubated with the first antiserum (TR antisera concentrations as used for immunocytochemistry, Truus 1:1000) for 3–4 h at RT. The antisera were adsorbed for a second time overnight at 4 C, followed by a third preadsorption for 3–4 h at RT.
This procedure was repeated one to three times until complete preadsorption was obtained, as confirmed by negative staining on the nitrocellulose membrane and negative staining in hypothalamus and pituitary sections.
Western blotting. Western blotting was performed on extracts of human pituitary. Protein was run on a 10% SDS-PAGE gel and electroblotted onto nitrocellulose membrane (BA45; Schleicher & Schuell). After blocking with PBS, containing 5% milk, the blots were incubated with the TR antisera (1:500) overnight at RT. The blots were then washed and incubated in the second antibody for 1 h at RT (porcine-antirabbit horseradish peroxidase, 1:1000 in PBS-milk; Dako, Glostrup, Denmark). The bands were detected using chemiluminescence. The TRß2 preimmune serum was used as a negative control. To further characterize the polyclonal TRß2 antibody, additional Western blotting was performed on hypothalamic tissues of 3-month-old male TRß KO mice on a 129 SV/C57Bl background and wild-type littermates, according to the same protocol (20).
Immunocytochemistry
Six patients were used for studying the distribution of TR isoforms (Table 1
). For anatomical orientation, Nissl staining was performed on every 100th section. At each level, adjacent sections were stained with the four different antisera using microwave treatment (21), preincubation with milk (22), and the avidin-biotin complex method (23), according to the following protocol. Sections were deparaffinized in xylene and rehydrated through graded ethanol series. After rinsing in distilled water, the sections were microwave treated for 12 min at full power (700 W) in TBS (pH 7.6). After adjustment to RT, sections were preincubated in 5% milk-TBS (Elk Milk powder; Campina, Eindhoven, The Netherlands) for 1 h at RT. Subsequently, sections were incubated with the first antibody, diluted in SUMI-milk (TR1, 1:1000; TR2, 1:2000; TRß1, 1:1000; and TRß2, 1:1000), for 1 h at RT followed by incubation overnight at 4 C. The slides were rinsed in TBS (2 x 10 min) and incubated for 1 h at RT in biotinylated goat-antirabbit serum (1:400) in SUMI. After rinsing in TBS (2 x 10 min), the sections were incubated in avidin-biotin complex (1:800 in SUMI; Vector Laboratories, Burlingame, CA) for 1 h at RT and subsequently rinsed in TBS (2 x 10 min). Finally, sections were incubated in 0.5 mg/ml 3,3'-diaminobenzidine (Sigma) in TBS containing 0.2% ammonium nickel sulfate (BDH; Brunschwig, Amsterdam, The Netherlands) and 0.01% H2O2 (Merck, Darmstadt, Germany) for approximately 15 min. The reaction was stopped in distilled water. The sections were dehydrated in graded ethanol series, cleared in xylene, and coverslipped using Entellan (Merck).
Double-labeling by immunocytochemistry and in situ hybridization
To investigate TR isoform colocalization with NPY or POMC in the IFN, in situ hybridization was performed after immunocytochemical staining with TR antisera. Three hypothalami were studied (from subjects 93061, 98006, and 98024; Table 1). Both sense and antisense oligonucleotides for NPY and POMC were used to support specificity. The NPY probe was complementary to 99–146 bp of the human NPY mRNA sequence (24), and the POMC probe was complementary to 7106–7153 bp of the human corticotropin-ß-lipotropin precursor gene (25). Sections were mounted on sterile 2% aminoalkylsilane-coated (Sigma) slides and dried at 37 C for 2 nights. Immunocytochemical staining for TR isoforms was performed, using 3,3'-diaminobenzidine as a chromogen, followed by in situ hybridization. Methods for probe labeling using 35S-dATP, the composition of hybridization buffer, and tissue pretreatments have been described previously (26, 27) Each section was incubated in 70 µl of hybridization mix, containing 1 x 106 cpm of the probe. After a short rinse in 2x standard saline citrate (SSC) at 37 C, sections were subsequently washed for 30 min in 1x SSC at 50 C, twice for 15 min in 0.1x SSC at 50 C, and twice for 15 min in 0.1x SSC at RT. Sections were dehydrated in 300 mM ammonium acetate (pH 5.5)-ethanol 100% at volume ratios of 1:1, 3:7, 1:9, and 0:1, and dried in a stream of cool air.
The slides were dipped in photographic emulsion (NTB2-Kodak; 1:1 diluted in distilled water at 42 C; Kodak, Rochester, NY), exposed for 1 wk at 4 C, developed for 2 min in Kodak Dektol Developer at 15 C, and fixed in Kodak Fixer for 8 min. Sections were washed in running tap water for 5 min to remove the fixative, dehydrated, cleared in xylene, and coverslipped using Entellan.
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Results |
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Specificity was supported by negative staining with preimmune sera and antisera preadsorbed with the homologous synthetic TR peptides in both hypothalamus and pituitary sections. No cross-reactivity between the TR isoform antisera was found on spot stainings.
Staining of Western blots (Fig. 1) of human pituitary with TR antisera resulted in a band of the expected 47 kDa for TR1, 58 kDa for TR2, 55 kDa for TRß1, and 58 kDa for TRß2. The preimmune serum did not show any bands. Additional experiments for TRß2 using TRß KO mice did not show a band for TRß2 at 58 kDa, whereas it was present in wild-type littermates (Fig. 2).
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All pituitaries studied showed staining for TR and TRß isoforms in a large proportion of cells of the anterior pituitary (Fig. 3). TR1, TRß1, and TRß2 staining showed an inhomogeneous distribution in the anterior pituitary, with more positive cells in the lateral regions. TR2 staining was more uniformly distributed over the anterior pituitary. Staining was mainly cytoplasmic, although subject 95101 showed predominantly nuclear TRß2 staining. Staining intensities for TR1, TR2, and TRß1 were quite uniform among the patients, whereas TRß2 showed a marked interindividual variation (Table 2). Patient 96009, who had been treated with thyroxine for primary hypothyroidism, showed only TR2 staining but no TRß or TR1 staining.
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Both parvocellular and magnocellular neurons in the PVN were positive for all four isoforms (Fig. 4). In addition, magnocellular neurons in the supraoptic nucleus showed TR immunoreactivity (Fig. 5). Staining in the IFN is illustrated in Fig. 6. No staining was present in the bed nucleus of the stria terminalis or the suprachiasmatic nucleus, whereas occasional TR-positive cells were found in the nucleus tuberalis lateralis and the tuberomammillary nucleus.
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. The marked interindividual variation is shown in Table 3.
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Discussion |
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1, TR2, TRß1, and TRß2 amino acid sequences. Specificity of the antisera was supported by negative preimmune staining, negative staining using preadsorbed antisera, and the absence of cross-reactivity using heterologous TR peptides. Earlier, Western blotting was performed on isoform-specific KO mice, supporting specificity of the antisera (14, 15). Western blotting on hypothalamic extracts from TRß KO mice in this study also supports specificity. In addition, we performed Western blotting on human pituitary tissue, and all antibodies directed at the different isoforms showed bands of the expected molecular weight. Using these antisera, we found staining for all TR isoforms in both the anterior pituitary and hypothalamus. TRs have been assumed to be largely confined to the nuclear compartment (28), but in our study, the intracellular distribution of the TRs varied between patients. Subject 95101 showed predominantly nuclear TRß2 staining, whereas other subjects showed mainly cytoplasmic staining in the pituitary. In Purkinje cells of rat cerebellum, TRß staining was either restricted to the nucleus, or staining was localized in the perinuclear region and the cytoplasm (29). Cytoplasmic localization of TRs has also been described in rats with the antisera that were used in this study (14, 15). More recent studies have shown that TRs may shuttle rapidly between the nuclear and cytoplasmic compartment (30). Translocation of the TR is suggested to be ligand dependent (31) and to require multiple protein interactions with various cofactors (32). Cytoplasmic localization, in addition to nuclear localization, appears to be a general phenomenon in the human brain for steroid hormone receptors (30, 33).
The regional distribution of the TRs in the anterior pituitary is in agreement with earlier studies in rat pituitary, showing TRß2 mRNA mostly in somatotropic and thyrotropic cells, which are located in the lateral part and anteromedial part of the anterior pituitary, respectively (34), and the regional distribution of TRß2 was later shown to overlap with TSH distribution (6). Whether the cell types expressing TR isoforms in the human anterior pituitary are thyrotropes is still unclear. Additional studies will be needed to further identify human anterior pituitary cells expressing TR isoforms.
In the hypothalamus, staining in the IFN and PVN was prominent. This is in agreement with rat studies, which showed all TR isoforms in TRH-expressing cells in the PVN (4) and in the ARC (35, 36). In contrast to animal studies (3), we did not find any suprachiasmatic staining, which may reflect a difference in expression levels or an interspecies difference.
TRs in the anterior pituitary and hypothalamus are involved in the neuroendocrine feedback of thyroid hormone as evident from animal experimental studies. The observation of extremely low TR and TRß1 expression in the anterior pituitary of subject 96009 (Table 2), who was treated with thyroxine for hypothyroidism, supports a similar role for TRs in the human pituitary. In the hypothalamic ARC, type II deiodinase (D2) is expressed in rats (37, 38). This enzyme plays a key role in the regulation of T3 availability in the brain because it is responsible for the conversion of T4 into T3. D2 expression responds to thyroid status and increases in hypothyroidism (37, 38). The presence of D2 in the ARC and projections of the NPY neurons in the ARC running to TRH cells in the PVN (39) suggest an important role for the ARC in thyroid hormone feedback in the rat hypothalamus. Our study supports a similar role for the IFN in the human hypothalamus. In an attempt to identify TR-expressing neurons in the PVN, we performed combined immunocytochemistry for TRs and in situ hybridization for NPY and POMC mRNA as candidate neurons for colocalization on the basis of observations in the rat (39). However, no abundant colocalization was observed, suggesting that other neurons than those expressing NPY and POMC are involved in thyroid hormone feedback by the IFN.
In the present study, we found a large interindividual variation in TR staining intensity, possibly related to the premortem duration and severity of the fatal illness. During severe illness, serum concentrations of T3 and T4 can decrease without giving rise to elevated TSH levels. This phenomenon is known as nonthyroidal illness (NTI) (40). The pathogenesis of NTI is still unclear, but decreased TRH expression in the PVN suggests a major change in thyroid hormone feedback regulation at the level of the hypothalamus, resulting in altered thyroid hormone serum levels (41). Whether altered hypothalamic TR expression plays a role in NTI is still unknown, but it certainly is an interesting possibility that will require further investigation.
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Footnotes |
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First Published Online November 23, 2004
Abbreviations: ARC, Arcuate nucleus; AVP, arginine vasopressin; D2, type II deiodinase; IFN, infundibular nucleus; KO, knockout; NPY, neuropeptide Y; NTI, nonthyroidal illness; POMC, proopiomelanocortin; PVN, paraventricular nucleus; RT, room temperature; SSC, standard saline citrate; SUMI, supermix; TBS, Tris-buffered saline; TR, thyroid hormone receptor.
Received March 10, 2004.
Accepted November 12, 2004.
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References |
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and ß thyroid hormone receptor genes in rat brain and pituitary. Proc Natl Acad Sci USA 86:7250–7254 2 mRNA in the parvocellular region of the paraventricular nucleus of the hypothalamus following thyroidectomy in the adult male rat. Neurosci Lett 164:159–162[CrossRef][Medline]
1. EMBO J 17:455–461[CrossRef][Medline]
isoforms in rodent liver. J Endocrinol 179:379–385[Abstract]
-melanocyte-stimulating hormone. J Clin Endocrinol Metab 85:2596–2603This article has been cited by other articles:
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 I Stoykov, B Zandieh-Doulabi, A F M Moorman, V Christoffels, W M Wiersinga, and O Bakker Expression pattern and ontogenesis of thyroid hormone receptor isoforms in the mouse heart. J. Endocrinol., May 1, 2006; 189(2): 231 - 245. [Abstract] [Full Text] [PDF]
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