J.Cell.Mol.Med. Vol 7, No 3, 2003 pp. 297-306
Somatotroph to thyrotroph cell transdifferentiation during experimental hypothyroidism - a light and electron-microscopy study S. Radian a *, M. Coculescu a, J. F. Morris b a Department
of Endocrinology, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania, b Cellular Endocrinology Laboratory, Department of Human Anatomy and Genetics, Oxford University, United Kingdom
Received: August 31, 2003; Accepted: September 15, 2003
Abstract Somatotroph and thyrotroph pituitary cells share a common precursor cell expressing the transcription factor Pit1 in ontogeny. Cells expressing both thyrotropin (TSH) and growth-hormone (GH) are found in adult rat pituitary and in human pituitary adenomas in acromegaly, and these tumors contain both thyrotropin-releasing hormone (TRH) and the TRH receptors (TRHR). It has been shown that stimulation of TSH expression in primary hypothyroidism promotes changes suggestive of somatotroph to thyrotroph cell transdifferentiation. We tested this hypothesis and the role of TRH in experimental primary hypothyroidism in rats. Adult female Long-Evans rats, 6 months old, were administered the antithyroid drug methimazole (0,1% w/v) in the drinking water for 42 days. Animals were sacrificed by perfusion fixation under anaesthesia at weekly intervals and pituitary tissue processed in acrylic resin for immunofluorescence and immuno-electronmicroscopy for TSH, GH and TRHR. In the hypothyroid rat pituitary immunofluorescent somatotrophs were greatly reduced in number and gradually replaced by thyrotrophs during methimazole administration. Colocalization of GH and TSH in the same cell was noted. Immunoelectronmicroscopy demonstrated the development of enlarged thyrotrophs with dilated rough endoplasmic reticulum containing an electron-dense material and intracisternal granules, both of which are immunoreactive for TSH ('thyroidectomy cells'). The somatotrophs showed reduced GH immunoreactivity and also the presence of TSH-type, small-size secretory granules. This suggests that the greatly increased number of TSH-cells in methimazole-induced-hypothyroidism is due, at least partially, to the transdifferentiation of somatotroph into thyrotroph cells. TRHR immunofluorescence was expressed in many somatotrophs in normal rat pituitary and unlike immunoreactive GH, its expression was enhanced during hypothyroidism. The number of TRHR-immunoreactive cells increased in parallel with the number of TSH-immunoreactive cells. This indicates a role for TRH stimulation in the transdifferentiation process. Taken together, these data suggest that, in addition to the cell mutation mechanism involving an early totipotential progenitor cell, transdifferentiation of existing somatotroph cells also plays a part in the pathogenesis of multihormonal GH-secreting adenomas.
Keywords: pituitary • ontogenesis • transdifferentiation • hypothyroidism • TSH • GH • methimazole • TRH receptor
* Correspondence to: Serban RADIAN, “Carol Davila” University of Medicine and Pharmacy, Department of Endocrinology,
34-36 Bd. Aviatorilor, Bucharest 011863, Romania. Tel.: +40 21 2307705, Fax: +40 21 2307705 E-mail:
[email protected]
Introduction The development of pituitary cells in mammals follows an ontogenetic tree, with partially known relationships between cell lineages [1]. Thyrotrophs are derived through a pathway depending on the transcription factors Prop1 and Pit1, which links them to somatotrophs and lactotrophs. Snell (Pit1dw) and Ames (Prop1df) dwarf mice and humans with Pit-1 mutations fail to develop these three cell types [2,3]. In adult rat pituitary, bi-hormonal thyro-somatotroph cells have been noted [4]. Furthermore, cells with colocalization of GH and TSH are frequent in multihormonal pituitary adenomas [5]. This suggests a possible dedifferentiation of pituitary cells during tumor formation, enabling them to secrete multiple hormones, e.g. GH and TSH. However, the ontogenesis of either of these cell types is not dependant on the other, as shown by targeted cell type disruption in transgenic mice [6,7]. Another relationship between these two cell types was shown by demonstrating somatotroph to thyrotroph cell transformation as a result of high functional demand in hypothyroidism, a process called by Horvath et al. transdifferentiation [8]. Enhanced TRH secretion and pituitary TSH response to TRH is characteristic of primary hypothyroidism. Furthermore, abnormal release of GH by TRH is seen in hypothyroidism and acromegaly but not in euthyroid subjects [9]. Endogenous TRH was shown to be present in normal and adenoma pituitary cells both at the peptide and mRNA level [10-12]. The TRH receptor is expressed not only by thyrotroph but also somatotroph cells in normal pituitary and various pituitary adenomas including GH adenomas [13,14]. In order to verify the transdifferentiation hypothesis and to investigate the possible role of TRH stimulation, we performed pituitary double-labeling immuno-fluorescence for GH, TSH and thyrotropinreleasing hormone receptor (TRHR) and immunoelectronmicroscopy for GH and TSH in a model of chemically-induced hypothyroidism in rat.
Materials and methods Animals Adult Long-Evans female rats bred at the Department of Human Anatomy and Genetics, Oxford University
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were used. The Principles of Laboratory Animal Care (NIH publication no. 85-23) was followed and the study was in accordance with the UK Animals (Scientific Procedures) Act 1986. Rats were housed in groups of 3-4 per cage in a room with 14 hours of light and 10 hours of darkness at a temperature of 21-22 oC. They were allowed food and drinking water ad libitum. Methimazole (Sigma) 0,1% (w/v) was administered in the drinking water of the test animals for a maximum of 42 days. All experiments were started between 09.00h and 10.00h. Animals (3 controls and 10 methimazole-treated) were sacrificed at the following intervals: 3 at 7 days, 3 (1 control) at 21 days, 3 (1 control) at 35 days and 4 (1 control) at 42 days of methimazole administration. Rats were anaesthetized by intraperitoneal injection of sodium pentobarbital (50mg/kg), the chest wall and abdomen was dissected, and a cannula inserted into the left ventricle. Blood was washed out with oxygenated, 37 oC, 7.4 pH phosphate buffered saline (PBS) and then fixative (4% formaldehyde, 0.25% glutaraldehyde in PBS pH 7.4) was perfused for four minutes. Pituitaries were removed, immersed in fixative overnight, vibratome sectioned and embedded in LR Gold resin (Agar Scientific, Stansted, UK) for microscopic studies [15].
Immunofluorescence Semithin sections (1µm) were mounted on gelatincoated slides. Sections were rinsed twice with PBS, non-specific antibody binding was blocked by preincubation with 1% (w/v) bovine serum albumin (BSA) in PBS, rinsed twice with PBS, incubated for 2 h at 25 oC with the primary antibody, rinsed twice with PBS then incubated for 30 min with a fluorescein-coupled (FITC) secondary antibody, rinsed with PBS and mounted in Vectashield fluorescence mounting medium with propidium iodide (Vector Laboratories, Burlingame, CA, USA). The primary antibodies used were rabbit anti-rat TSH at a dilution of 1:500, monkey anti-rat GH at a dilution of 1:1500 (National Hormone and Pituitary Program, Gaithersburg, MD, USA) and mouse monoclonal IgM anti-rat TRH receptor (TRHR01; gift from Dr. M. Mellado, Centro Nacional de Biotecnologia, Madrid, Spain) at 1:200 dilution. For GH-TSH double labeling experiments, we sequentially applied the above protocol for each antigen using monkey anti-rat GH (dilution 1:1500) primary antibodies and guinea-pig anti-human IgG cou-
J. Cell. Mol. Med. Vol 7, No 3, 2003 pled with FITC (for GH) and rabbit anti-rat TSH (dilution 1:500) primary antibody and biotinylated guinea pig anti-rabbit IgG secondary antibody and Texas Red Avidin D (for TSH). For TRHR-GH double labeling we used the same reagents for GH labeling as above and mouse anti-rat TRH receptor antibody (dilution 1:100) with a goat anti-mouse mu chain secondary biotinylated antibody (dilution 1:100) and Texas Red Avidin D (dilution 1:300) (Vector Laboratories, Burlingame, CA, USA) for the second antigen (TRH receptor). Nuclei were stained with the nuclear fluorescent stain TO-PRO-3 (Molecular Probes, Eugene, OR, USA) (1:1000 in water) and sections were mounted in Vectashield mounting medium without propidium iodide. For control sections, the primary antibody was omitted and replaced by 4% (w/v) BSA in PBS. The sections were examined on a Leica laser confocal microscope and images acquired at 1024x1024 pixels resolution.
Immunoelectronmicroscopy : Ultrathin sections (50-80 nm) were cut and immunogold labeled for growth-hormone (GH) and thyrotropin (TSH) [15]. Briefly, grids were soaked in water, rinsed twice in PBS, non-specific binding was blocked by incubation in 4% (w/v) BSA in PBS, rinsed twice in PBS, incubated for 2 h at 25 oC with the antibody, rinsed twice in PBS, incubated with protein A gold (15nm diameter) for 15 min, rinsed twice in PBS, contrasted with uranyl acetate and lead citrate, rinsed in PBS, then in distilled water and air dried. For GH-TSH double immunogold labeling experiments the protocol of Bendayan was used [16]. Briefly, grids were floated rough-side down on drops of 4% (w/v) BSA in PBS, twice in PBS, incubated for 2 h at 25 oC with the anti-GH antibody, rinsed twice in PBS, incubated with protein A gold (15nm diameter) for 15 min, rinsed twice in PBS, then in distilled water and air dried. The same steps were applied using the other side of the grid, replacing the GH antibody with TSH antibody and the protein A-gold 15nm with 10nm. Before drying, grids were contrasted with uranyl acetate and lead citrate, rinsed and air dried. Sections were viewed with a JEM-1010 transmission electron microscope (JEOL USA Inc., Peabody, MA, USA) and micrographs of selected fields prepared.
Results Somatotroph and thyrotroph cells The immunofluorescence studies confirmed that the majority of cells in control pituitary tissue are GH immunoreactive, of relatively small-size. The somatotrophs were evenly distributed in the tissue, representing approximately 50% of all cells. A small number of clustered TSH-immunoreactive cells of larger size were also identified (Fig. 1A). With administration of the antithyroid drug GH expression was gradually reduced, as reflected by a reduction in the number of somatotroph cells and the immuno-reactivity of the individual cells. This was evident at 7 days (not shown) and fully expressed at 21 (Fig. 1B) and 28 days (Fig. 1C) of antithyroid treatment, when immunoreactive somatotrophs have virtually disappeared. By contrast, TSH expression increased progressively as shown by an increase in the number and size of TSH-immunoreactive cells, evident at day 21 (Fig. 1B) and maximal at days 28-35 (Fig. 1C) (i.e. with a slower onset than the decrease in GH immunoreactivity). The TSH-immunoreactive cells occured in clusters throughout the tissue. GH and TSH colocalization was not observed in normal rat pituitary tissue, but numerous cells expressed both TSH and GH in the 21-days-hypothyroid rats (Fig. 2). These were small sized cells, suggesting a possible origin from somatotrophs. Ultrastructurally, somatotrophs in the normal rats demonstrated a typical appearance, round in shape and containing large, round, homogenous, electron-dense secretory vesicles immunoreactive for GH (Fig. 4A). A few exocytoses were seen. A decrease in GH cell number and GHimmunoreactivity was evident as early as seven days of antithyroid drug administration and there was progressive loss of typical somatotrophs with time of antithyroid drug administration. At 21 days of hypothyroidism, GH- immunoreactive cells were of small size (comparable to that of control somatotrophs) with euchromatic nuclei and visible nucleolus, well-developed Golgi network and endoplasmic reticulum, and fewer secretory vesicles than control somatotrophs. Secretory vesicles were heterogenous in size and density; the larger typical somatotroph vesicles were immunoreactive for GH, but only few of the smaller vesicles were positive 299
Fig. 1 GH and TSH double immunofluorescence labeling in control (A) and hypothyroid adult female rat pituitary, after 21 days (B) and 28 days (C) of methimazole administration. A gradual replacement of GH-cells by numerous, enlarged TSH-cells is seen along hypothyroidism. GH is stained with fluorescein (green), TSH with Texas Red (red) and nuclei with To-Pro-3 (blue). Detail images are magnified 2 times.
for GH (Fig. 4B). The presence of larger GH secretory vesicles and the intensity of GH immunostaining were correlated, with both diminishing progressively during antithyroid drug administration. Thyrotroph cells in the control animals were larger than somatotrophs, with cytoplasmic extensions filled with secretory vesicles immunoreactive for TSH. The secretory vesicles were round, more
heterogenous in size and density and smaller on average than those of somatotrophs (Fig. 5A). With hypothyroidism, there was a gradual increase in the number and size of the thyrotroph cells. Most of these cells (Fig. 5B), developed the appearance of the “thyroidectomy cells” described by Horvath et al. (8) i.e. large size cells with a well-developed Golgi complex and dilated rough endoplasmic
Fig. 2 Colocalization of GH and TSH in hypothyroid female rat pituitary (C), at 21 days of antithyroid drug administration. Double labelling immunofluorescence, GH labelled with fluorescein (green), TSH with Texas-Red (red) and nuclei with To-Pro-3 (blue). Arrows indicate cells co-expressing both hormones (C). Same section scanned for GH only (A), and TSH only (B).
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Fig. 3 TRH receptor and GH double immunofluorescence labeling in control (A) and hypothyroid adult female rat pituitary, after 21 days (B) and 28 days (C) of methimazole administration. TRHR immunoreactivity was mainly found in the cytoplasm of GH cells in control animals. There was increased TRHR immunoreactivity at 28 days of antithyroid drug administration and a shift from the cytoplasm to the membrane, where patches of immunoreactivity were seen. GH was stained with fluorescein (green), TRHR with Texas-Red (red) and nuclei with To-Pro-3 (blue). Detail images are magnified 2 times.
Fig. 4 Pituitary immunoelectronmicroscopy sections stained with monkey anti-rat GH antibody and protein A gold (15nm particles) in control (A) female rats and after 21 days of methimazole administration (B). Control pituitary displays electron-dense round secretion vesicles but hypothyroid pituitary shows smaller, more heteregenous in size and less dense secretory vesicles, with less intense GH-staining. Detail images are magnified 3 times.
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Fig. 5 Pituitary immunoelectronmicroscopy sections stained with rabbit anti-rat TSH antibody and protein A gold (15 nm particles) in control female rats and after 21 days of methimazole administration. Control pituitary (A) showed large-size thyrotrophs, with numerous small-size and a few large secretory vesicles. In the hypothyroid rat large thyrotroph cells were seen (B); their cytoplasm was occupied by abundant dilated rough endoplasmic reticulum cisternae, filled with an electron-dense, TSH-staining material. Occasional cisternae contained TSH-staining electron-dense granules (shown in detail). These features are characteristic of “thyroidectomy” cells. Detail images are magnified 3 times.
Fig. 6 Double immunogold labeling of GH (10 nm gold particles) and TSH (15 nm gold particles) in 28 days hypothyroid rat pituitaries. A thyroidectomy cell is shown in (A). Dilated rough endoplasmic reticulum profiles stained both for TSH and GH (B) - detail image.
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reticulum cisternae filled with electron-dense material immunoreactive for TSH. Some of the rough endoplasmic reticulum cisternae contain electrondense round granules, which were immunopositive for TSH and are characteristic of thyroidectomy cells. Intracisternal granules were noted beginning at 7 days of hypothyroidism and fully expressed by day 28. A few small-size, TSH-immunoreactive secretion vesicles were seen in the periphery of these cells, close to the membrane and exocytoses were also noted. Double labeling immunogold staining demonstrated co-localization of GH and TSH in the rough endoplasmic cisternae of a significant proportion of thyroidectomy cells at day 28 (Fig. 6).
TRH receptor in somatotroph cells Double immunofluorescence staining for GH and the pituitary TRH receptor was also performed. The TRH receptor antibody used detects both the intracellular pool of TRH receptor and the membrane-bound fraction [17]. Normal rat pituitary expressed TRH receptors mainly in the cytoplasm of somatotroph cells (Fig. 3A), with little membrane- bound immunoreactivity. The TRH receptor immunoreactivity was unchanged by day 21 but we noted a shift of immunoreactivity from the cytoplasm to the membrane, where it had a patchy appearance (Fig. 3B). By day 28 of hypothyroidism TRH receptor immunoreactivity was enhanced in terms of both the number of cells and their staining intensity with expression both on the cell membrane and in the cytoplasm (Fig. 3C). At 28 days of antithyroid drug administration TRHR immunoreactivity was seen in clusters of large size cells, most likely thyrotrophs.
Discussion The reciprocal changes in the numbers of the somatotrophs and thyrotrophs in the course of methimazole-induced hypothyroidism in adult rat, together with the development of bihormonal GH-TSH cells, as shown by immunofluorescence and the altered ultrastructural appearance of somatotrophs which develop secretory vesicles of thyrotroph
type, strongly suggests the transformation of somatotrophs to thyrotrophs, by transdifferentiation. Transdifferentiation is the process of phenotypic transformation of one differentiated cell type to another [18–22], through activation of the differentiation factor systems. Examples of endocrine cell transdifferentiation include pituitary (GH to TSH [8, 23], GH to PRL [24–28] and GH to gonadotroph cells [29]), thyroid C cells [30] and adrenal chromaffin cells [31, 32]. The process of pituitary transdifferentiation [8] implies the existence of bi-hormonal cells as intermediates, it does not require cell division [24, 26, 27] and can be induced by stimulation of hormonal feedback systems [8, 23, 28] or by treatment with growth factors [24, 27]. Like Horvath et al., we were able to demonstrate by double immunogold labeling GH-TSH colocalization in a subpopulation of thyrotrophs in hypothyroid rat pituitary. We have also observed, by immunofluorescence, a second subset of bi-hormonal cells in the hypothyroid animals’ pituitaries. These were smallsize cells, representing most of the GH-immunofluorescent cells in hypothyroid pituitaries and corresponding to the small-vesicle type somatotrophs described by Horvath et al. We were able to demonstrate TSH-GH coexpression in these cells only by immunofluorescence and not by double immunogold labelling, due perhaps to the relatively poor antigen preservation of TSH during specimen processing and also to the lesser sensitivity of immunogold staining compared to that of immunofluorescence. We propose that these cells represent intermediate forms of somatotroph-derived thyrotrophs with ultrastructural features and a bi-hormonal expression pattern distinct from that of “mature” thyroidectomy cells. The corresponding condition in humans is thyrotroph hyperplasia in primary hypothyroidism and bihormonal thyro-somatotroph cells have been demonstrated in pituitary specimens from such patients [23]. TRH receptor was detected in virtually all somatotrophs of control animals by double immunofluorescence. This persisted during methimazoleinduced hypothyroidism, but was accompanied by a change in the distribution of the TRHR immunofluorescence from the cell cytoplasm to the cell membrane (day 21). TRH receptor mRNA synthesis and TRH binding to cell membrane are enhanced in hypothyroidism [33] and cell membrane- bound 303
receptor oligomerization occurs upon exposure to TRH, leading to immunoreactive patches formation on the cell membrane [34]. This suggests that somatotrophs are under TRH stimulation during primary hypothyroidism. Indeed, despite the reduced GH pituitary content and basal secretion during hypothyroidism, GH release can be stimulated by TRH administration, both in humans [9] and in rats [35]. An acquired abnormal secretory response to TRH is also present in patients with pituitary adenomas, in acromegaly where GH-release is stimulated [36] and in gonadotropinomas where LH release is stimulated [37]. TRH has been shown to be produced in normal pituitary and in pituitary adenomas, including acromegaly [11,12,38]. Our group has demonstrated pro-TRH peptide immunoreactivity in pituitary adenomas, in 1 of 8 acromegaly patients and 2 of 18 patients with non-functioning adenomas [39]. Pro-TRH mRNA was expressed more frequently, in 5 of 8 GH-producing pituitary adenomas and 11 of 18 non-functioning adenomas, 6 of which were also expressing beta-TSH [10]. TRHR receptor mRNA is also present in normal somatotrophs [11] and in pituitary GH adenomas in acromegaly, where TRHR mRNA levels correlate with the amplitude of serum GH abnormal release following TRH administration [13]. Pituitary adenomas containing bi-hormonal cells expressing GH and TSH within the same cell and secretion vesicles [40] have been described and there are also multihormonal adenomas containing these and other hormones. Many hormones present in the multi-hormonal pituitary adenomas are often clinically mute and do not alter the serum hormone levels [41]. For some of these tumors a monoclonal origin has been demonstrated [42]. Their multihormonal status has therefore been explained as the proliferation of a single pluripotential cell type, closer to pituitary stem cells [43]. It is tempting to propose the transdifferentiation of tumor cells as an alternative mechanism explaining the formation of multihormonal pituitary adenomas. The TRH stimulation pathway provides a link between the hypothyroid state and GH-TSH bihormonality and is also a good candidate in the pathogenesis of multi-hormonal pituitary adenomas, through autocrine/paracrine mechanisms. The cellular mechanisms by which TRH could act in transdifferentiation is yet to be clarified, though it seems likely that it changes the expression 304
of key transcription factors, like Pit-1 and Gata 2, that are known to be involved in pituitary endocrine cell differentiation [44,45].
Acknowledgements Dr. A.F. Parlow (National Hormone and Pituitary Program, Gaithersburg, MD, USA) for the kind supply of rat GH and TSH antibodies and Dr. M. Mellado (Centro Nacional de Biotecnologia, Madrid, Spain) for the TRHR01 antibody. Lynne Scott, Sara Rodgers and Ann Stanmore for their expert technical help.
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