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From the Departments of Pathology,* Endocrinology, and Pharmacology, University of Turin, Turin; and the Department of Pathology, University of Genoa, Genoa, Italy
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Abstract |
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In the stomach, ghrelin is localized in the neuroendocrine cell compartment of the oxyntic mucosa, produced by X-like cells that appear to be the main source of circulating ghrelin.9 The gastric antrum and small intestine contain very few ghrelin-producing cells and the large intestine is virtually devoid of ghrelin.9,14 This apparent foregut-restricted ghrelin expression prompted an analysis of other foregut endodermal derivatives. The thyroid gland is of endodermal origin and therefore a possible source of ghrelin, although no ghrelin immunoreactivity has been reported so far in normal rat and human thyroid. Recently, Kanamoto and colleagues22 reported substantial production of ghrelin in a medullary thyroid carcinoma cell line. In this study, reverse transcriptase-polymerase chain reaction (RT-PCR) analyses demonstrated prepro-ghrelin gene transcripts in a medullary thyroid carcinoma cell line and in total normal human thyroid tissue. The latter finding was not paralleled by ghrelin immunoreactivity of follicular cells and suggested that probably the minor C cell population of the thyroid is responsible for the RT-PCR signal.
As for the ghrelin receptor, by means of a binding assay, we previously demonstrated GH secretagogue binding sites in the thyroid gland, in thyroid carcinomas of follicular origin, and in several thyroid carcinoma cell lines.16,18 In these cell lines, synthetic GH secretagogues determined a biological effect, resulting in a significant inhibition of cell proliferation. However, no data were available on the possible expression and biological function of the natural GHS-receptor ligand, ghrelin, in normal thyroid, follicular-derived thyroid carcinomas, and related cell lines.
The aim of the present study was therefore to investigate ghrelin and GHS receptor expression in the human fetal and adult thyroid and in a series of thyroid carcinomas of follicular origin, by means of immunohistochemistry (IHC), in situ hybridization and RT-PCR analysis. In addition, ghrelin activity on cell proliferation was investigated in vitro using cell lines originating from follicular-derived thyroid tumors. We here show that fetal (but not adult) thyrocytes and a percentage of follicular-derived tumors contain large amounts of immunoreactive ghrelin and its specific mRNA. Unexpectedly, RT-PCR receptor analysis showed that mRNAs for both ghrelin/GHS receptor types 1a and 1b were absent in all of the thyroid tumors and cell lines investigated as well as in all but one normal adult thyroid. However, biotinylated ghrelin bound to the majority of both normal and neoplastic tissues, and cell lines. The presence of functional ghrelin receptors was also supported by in vitro studies in which ghrelin had a specific, dose-dependent anti-proliferative effect in follicular-derived thyroid carcinoma cell lines.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Fifteen fetal thyroids (from 8 to 38 weeks of gestation, from voluntary or elective abortions, or autopsies for intrauterine fetal deaths), and 4 infant and 10 adult thyroid glands (age of patients from 10 to 83 years, resected for hyperplastic goiter or papillary carcinoma) were collected from the files of the Departments of Pathology, Universities of Turin and Genoa. In addition, we collected 54 follicular-derived tumors of the thyroid for which formalin-fixed, paraffin-embedded tissue blocks were available for both conventional histology and IHC. Of these, in 33 cases fresh frozen material was available for mRNA studies. All cases were classified according to the World Health Organization or Armed Forces Institute of Pathology classification schemes of thyroid tumors.23,24 The follicular origin was confirmed by a negative calcitonin reaction and positive thyroglobulin immunostaining in all cases, except for anaplastic carcinoma.
Cell Lines and Cell Growth Studies
N-PAP and ARO, two cell lines of human thyroid carcinoma of follicular origin, were used. N-PAP was obtained from a papillary carcinoma and ARO from an undifferentiated carcinoma of the thyroid (kind gifts of Professor A. Fusco, University of Naples). The two cell lines were grown as a monolayer in RPMI medium (Gibco, Paisley, Scotland) with 10% fetal calf serum (Gibco) at 37°C in a 5% CO2-humidified atmosphere.
To assay the effect of ghrelin on cell proliferation, cells were seeded in quadruplicate in 48-multiwell plates (104 cells/well) in standard culture medium and allowed to adhere for 24 hours. Ghrelin (10 nmol/L to 1 µmol/L) was then added to the medium and cells were treated for up to 96 hours. Experiments were repeated three times. After 48 and 96 hours of treatment, respectively, cells were fixed in 2.5% glutaraldehyde, stained with 0.1% crystal violet in 20% methanol and solubilized in 10% acetic acid. Cell growth was evaluated by measuring absorbance at 590 nm in a microplate reader (Multiskan Bichromatic; Labsystem Oy, Helsinki, Finland). A calibration curve was set up with known numbers of cells and a linear correlation between absorbance and cell counts was established up to 1 x 105 cells. Statistical analysis was performed by analysis of variance (cutoff for significance, P = 0.05).
To test for peptide toxicity in the different cell lines, cell viability was tested in control and treated cells by means of trypan blue staining.
To evaluate ghrelin secretion by N-PAP and ARO cell lines, ghrelin levels (in ng/L) were measured in the culture media of both cell lines in duplicate, after extraction in reverse-phase C18 columns, by radioimmunometric assay (Phoenix Pharmaceutical, Inc, Belmont, CA). Sensitivity of the assay was 30 pg/tube. Intra-assay coefficients of variation range was 0.3 to 10.7%.
Immunohistochemistry (IHC)
Sections serial to those used for conventional histology were collected onto poly-L-lysine-coated slides and stained for ghrelin using a standard manual immunoperoxidase procedure with streptavidin-peroxidase (LSAB2 kit; DAKO, Glostrup, Denmark) and diaminobenzidine as the final reaction product. Ghrelin antibody was an anti-human ghrelin polyclonal serum (amino acids 24 to 51, code H-031-30; Phoenix Pharmaceuticals, Inc.), diluted 1/15,000, incubated for 1 hour at room temperature, with no previous antigen retrieval and revealed applying catalyzed reporter deposition (CARD) amplification with slight modifications.25 Negative control reactions forghrelin included omission of the primary antibody and preabsorption with a 100-fold excess of the antigen, as described elsewhere.14 The positive control for ghrelin were the endocrine cells of the oxyntic mucosa of the stomach. The follicular origin of the tumors was evaluated by IHC with polyclonal anti-calcitonin (diluted 1/80, DAKO) and anti-thyroglobulin (diluted 1/3000; Biogenex, San Ramon CA) antibodies. Selected cases were also stained with a double-immunohistochemical procedure using the same antibodies that react with ghrelin and thyroglobulin. This procedure was based on an immunoalkaline phosphatase (LSAB plus kit, DAKO)/fast red (BioGenex) reaction for ghrelin, followed by immunoperoxidase staining of thyroglobulin, using an anti-rabbit secondary antibody and the EnVision kit (DAKO). Ghrelin protein expression was also evaluated in the two cell lines applying the same ghrelin antibody at a working dilution of 1/400, and visualized in immunofluorescence with an anti-rabbit fluorescein isothiocyanate-conjugated antibody (diluted 1/100; Sigma Aldrich, Steinheim, Germany).
In Situ Hybridization
Selected cases of fetal thyroid (12 cases), infant and adult normal thyroid (9 cases), and tumors (38 cases) were studied by means of a nonradioactive in situ hybridization procedure to confirm immunohistochemical findings and to analyze ghrelin mRNA localization. Silane-coated slides were hybridized overnight at a working dilution of 33 nmol/L of an equimolar mixture of two 45-mer anti-sense probes1 corresponding to nucleotides 90 to 134 and 421 to 465 of prepro-ghrelin sequence. Probes were digoxigenin-labeled with the Boehringer labeling kit (Mannheim, Germany) following the manufacturer’s instructions. Prehybridization treatments included a microwave passage (5 minutes at 800 W in citrate buffer pH 6.0) and proteinase K digestion (1 µg/ml) for 4 minutes. Hybridization products were revealed applying the DAKO GenPoint kit (DAKO) as described elsewhere26 with minor modifications, including 1/5 dilution of tyramide solution and hot washing in phosphate-buffered saline (PBS) after tyramide incubation.25 Positive controls were represented by sections of normal oxyntic mucosa of the stomach. Hybridization with an unrelated probe, as well as omission of the specific probe and RNase digestion, served as negative controls, to test both the specificity of the anti-sense probes and possible tyramide-based background.
RT-PCR for Ghrelin and GHS-R
In 33 cases of follicular-derived thyroid tumors, as well as in 5 adult total thyroid tissue, RT-PCR was performed to reveal mRNAs for both ghrelin and its receptor. GHS-R type 1a and 1b mRNA expression was also evaluated in N-PAP and ARO cell lines. Total RNA extraction and complementary DNA transcription was performed as described elsewhere.26 The primers for ghrelin were synthesized according to the sequence reported by Gualillo and colleagues,11 and the sequences were 5'-TGAGCCCTGAACACCAGAGAG-3' for the forward primer and 5'-AAAGCCAGATGAGCGCTTCTA-3' for the reverse primer. Those for GHS-receptor 1a and 1b were synthesized according to Korbonits and colleagues13 and used for RT-PCR using the same conditions described by these authors. The following sequences were used: 5'-TCGTGGGTGCCTCGCT-3' as the forward primer for both GHS-R 1a and GHS-R 1b, 5'-CACCACTACAGCCAGCATTTTC-3' for the GHS-R 1a reverse primer and 5'-GCTGAGACCCACCCAGCA-3' for the GHS-R 1b reverse primer. The expected size of the amplicons was 327 bp, 65 bp, and 66 bp for ghrelin, GHS-R 1a and GHS-R 1b, respectively. ß2-microglobulin amplification served as a control of the RNA quality (see details in Papotti and colleagues26 ). Positive controls included normal gastric mucosa for ghrelin and pituitary tissue for GHS-R 1a and 1b amplifications. Negative controls consisted of omission of cDNA in the PCR mixture and of the reverse transcriptase enzyme during retrotranscription.
To further test the RT-PCR product specificity, Southern blot analysis was performed using the probe sequence previously published by Korbonits and colleagues.13 The following sequences were used: 5'-AGGGACCAGAACCACAAGCAAACCG-3' for ghrelin and 5'-TCCGGTTCAACGCCCCCTTTG-3' for GHSR-1 probes. Membranes were hybridized overnight at 42°C with 25 pmol of digoxigenin-labeled ghrelin and GHS-R oligonucleotide probes. The membranes were then washed with 2x standard saline citrate-0.1% sodium dodecyl sulfate for 5 minutes at 42°C and 0.5x standard saline citrate-0.1% sodium dodecyl sulfate at 42°C for 10 minutes. Digoxigenin-labeled specific hybridization was visualized using an immunological detection system (Boehringer Mannheim, Mannheim, Germany) using anti-digoxigenin antibodies conjugated with alkaline phosphatase. Detection was performed using the chemiluminescent substrate disodium 3-(4-methxyspiro-1,2-dioxetane-3,2-(5-chloro) tricyclo (3.3.1.) decan-4-yl) phenylphosphatase (Boehringer Mannheim), according to the manufacturer’s instructions. All blots were exposed to X-ray films with intensifying screens at room temperature for 3 or 5 hours.
Nonradioactive in Situ Localization of Ghrelin Binding
Full-length ghrelin (Tocris Cookson Ltd., Bristol, UK) was labeled with biotin following a standard technique,27 with minor modifications. In particular, to minimize the steric hindrance between avidin (streptavidin) and the biotinylated protein, an aminocaproyl spacer arm (a modified activated biotin, caproylamidobiotin-N-hydroxy succimide ester) was introduced between the biotin and the activated carboxyl group of the protein (B002-6; Società Prodotti Antibiotici SpA, Milan, Italy). Ghrelin (0.5 mg/ml) was dialyzed against 0.1 mol/L of sodium bicarbonate buffer, pH 8.5, and subsequently incubated for 4 hours at room temperature under continuous slow rotation with B002-6 dissolved in dimethyl sulfoxide (1 mg/ml). The solution was dialyzed overnight at 4°C against PBS.
Frozen serial sections of 16 thyroid tumors and 3 normal thyroid tissues were collected onto poly-L-lysine-coated slides and stored for 1 week at -80°C. One section was stained with hematoxylin and eosin to assess the quality of tissue preservation. Then the sections were thawed at 37°C for 4 minutes and quenched in PBS. Endogenous peroxidase activity was blocked in 6% H2O2 for 8 minutes, after which the sections were incubated with biotinylated ghrelin at a concentration of 10-9 mol/L for 2 hours at room temperature. Sections were then fixed in -20°C methanol for 5 minutes and in -20°C acetone for 5 seconds. Bound biotinylated ghrelin was visualized after the streptavidin/tyramide procedure described for ghrelin IHC. Control experiments included competition assays in parallel sections with unlabeled full-length ghrelin at a concentration of 10-6 mol/L for 20 minutes at room temperature, before incubation with labeled ghrelin, as described, as well as omission of the biotinylated peptide. Pituitary tissue served as positive control. For in vitro binding experiments, N-PAP and ARO cell lines were incubated for 2 hours at 4°C with 10-9 mol/L biotinylated ghrelin, then fixed in -20°C methanol for 5 minutes and in -20°C acetone for 5 seconds, and the reaction visualized with the streptavidin/tyramide procedure described above. Control experiments included a competition assay using preincubation for 10 minutes at 4°C with 10-7 mol/L unlabeled ghrelin as well as with an unrelated peptide (10-7 mol/L oxytocin) (Sigma).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Fetal thyroid (from 8 to 38 weeks of gestational age) produced ghrelin (Table 1)
in the follicular cells, as shown by IHC using a specific antibody that recognizes the C-terminal portion of both acylated and nonacylated ghrelin (Figure 1)
. Immunostaining was completely abolished in parallel sections by pretreatment of the antibody with excess ghrelin. The extent of ghrelin protein expression was much lower in the late fetal period, in which ghrelin immunoreactivity was confined to the apical portion of the follicular cells.
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or in adult (Table 2)
thyroid follicular cells, or in the setting of multinodular goiter or in peritumoral follicles. Three cases of Hashimoto’s thyroiditis in adult patients were also tested for ghrelin protein expression, and showed no immunoreactivity (data not shown). Double-immunohistochemical reactions confirmed thyroglobulin expression in ghrelin-producing cells. No ghrelin immunoreactivity was ever observed in other structures of the thyroid, including blood vessel and the capsule.
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. No signal was detected in gastric mucosa or fetal thyroid tissue in the absence of the tyramide amplification step. The more sensitive RT-PCR technique revealed ghrelin transcripts detectable in three of five cases of adult normal thyroid (Table 2
, Figure 2
).
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Moderate to strong ghrelin immunoreactivity was observed in 42 of 54 cases, having either a focal (15 cases, <25% of tumor cells) or a diffuse (27 cases) pattern of reactivity (Figure 3)
. The focal pattern was mainly recognized in conventional follicular adenomas (six of eight cases). Interestingly, all five cases of benign tumors with oxyphilic features (oncocytic adenomas) were positive for ghrelin. Malignant tumors expressed ghrelin in 9 of 19 follicular, 9 of 12 papillary, 8 of 8 poorly differentiated, and 3 of 6 anaplastic carcinomas (Table 2)
. Ghrelin protein expression was also demonstrated by immunofluorescence in both N-PAP and ARO cell lines (Figure 4)
. Moreover, ghrelin protein was demonstrated by radioimmunometric assay in N-PAP (49 ng/L) but not in ARO cell culture media.
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Ghrelin Effect on Thyroid Carcinoma Cell Line Proliferation in Vitro
The proliferation of N-PAP and ARO cells was evaluated under both basal conditions and ghrelin treatment.
Ghrelin determined a dose-dependent, although modest, inhibition of cell growth in both cell lines tested (Figure 5)
. Reduction of cell growth was significant at any time point in N-PAP cells using 1 µmol/L of ghrelin (P = 0.005, treated cells versus controls), with a maximum 25% decrease in cell number. At 100 nmol/L, ghrelin still determined a significant inhibition in N-PAP cells after 96 hours of treatment (-22%, P = 0.01), whereas 10 nmol/L of ghrelin did not affect cell proliferation at any time point. In ARO cells, the growth inhibition was significant using 1 µmol/L of ghrelin only (P = 0.01 at 48 hours and P = 0.005 at 96 hours of treatment, in treated cells versus controls).
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GHS Receptor Expression and Ghrelin Binding Sites in Normal and Neoplastic Thyroid Tissues
RT-PCR was used to analyze ghrelin receptor (GHS-R 1a and 1b) in adult normal thyroid tissues and in 33 tumors, as well as in the two thyroid carcinoma cell lines. None of the tumors or cell lines expressed the specific mRNA. Nontumoral thyroid specimens tested in parallel were also negative with the single exception of GHS-R 1a and 1b mRNA expression in a single case of adult thyroid gland specimen (incidentally, a peritumoral sample of a papillary carcinoma, which was negative as stated above).
As opposed to GHS-R mRNA findings, a large percentage of both normal (2 of 3 cases) and neoplastic (14 of 16 cases) thyroid specimens (Figure 6)
, as well as the two cell lines (Figure 4)
, bound ghrelin according to a newly designed nonradioactive in situ technique. Specificity of the signal was confirmed using an excess of unlabeled ghrelin as a competitor, which abolished any staining determined by the labeled peptide. In cell culture experiments, additional negative controls included competition with an unrelated peptide (oxytocin), which did not affect biotinylated ghrelin-binding capacity.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Immunoreactive ghrelin was present in most follicular cells and co-localized with thyroglobulin in primordial cords of the embryonic thyroid, as early as week 8 of gestation, and was detected in decreasing amounts from week 20 of pregnancy to the late fetal period. The intensity of ghrelin immunoreactivity was similar to that of the oxyntic mucosa of the stomach, indicating that, in fetal life, the thyroid may be an additional important source of circulating ghrelin. The cells producing ghrelin in the fetal thyroid were follicular cells, as confirmed by the co-localization of ghrelin with thyroglobulin. In infant and adult thyroid glands, no immunoreactivity was observed in the follicular compartment. These data are in agreement with the previous study of Kanamoto and colleagues22 on ghrelin expression in parafollicular cells and tumors, in which immunohistochemical evaluation of normal thyroid tissue showed no reactivity. The finding of ghrelin mRNA in whole thyroid tissue mRNA extracts observed in RT-PCR experiments by Kanamoto and colleagues22 and confirmed by the present study, could be explained by a residual minimal transcription of ghrelin gene in follicular cells, although this hypothesis could not be confirmed by in situ techniques such as in situ hybridization, that are known to be less sensitive than PCR. In addition, the minor C cell population could be responsible for ghrelin mRNA PCR detection in whole thyroid tissue, as already originally suggested by Kanamoto and colleagues,22 but we have no evidence to confirm this hypothesis in the current study.
Ghrelin expression was also observed in a high proportion of follicular-derived tumors of the thyroid. Medullary carcinomas were not tested here, because they have already been reported to express ghrelin22 as mentioned above. We demonstrated that neoplastic transformation enables follicular cells to (re-) express ghrelin, from the earliest phases, because adenomas contain ghrelin in three-quarters of cases. Malignant tumors retain the capacity to produce ghrelin irrespective of their grade of differentiation. Similarly, ghrelin was present in the thyroid follicular-derived tumor cell lines, and the anti-proliferative effect of the peptide was evident in both undifferentiated (ARO) and differentiated (N-PAP) tumor cell lines. No apparent differences in ghrelin expression were observed in thyroid tumors, despite tumor grade and differentiation, thus suggesting the absence of a prominent role for ghrelin in tumor progression mechanisms.
It is not known whether the mechanism mediating ghrelin activities in fetal thyroid and tumors is the result of a direct effect on specific receptors, or if it follows a more complex pathway via GH release. To address this point, the demonstration of specific receptors for ghrelin in thyroid cells is a crucial step. In the present case series, GHS-R types 1a and 1b mRNAs were not detected in any thyroid tumor, although specific binding sites for synthetic ghrelin analogs, such as hexarelin, were reported in a percentage of thyroid tumors in a previous report,18 and specific ghrelin-binding sites have been demonstrated using either radioactive (G Muccioli and M Papotti, unpublished observation) or nonradioactive methods, as in the current study. It can be speculated that, at the peripheral level, GHS/ghrelin receptors other than the known GHS receptor identified in central organs (pituitary and hypothalamus) by Howard and co-workers15 exist, representing either subtypes of the GHS-R itself or novel receptors, yet to be cloned. Discrepancies between the presence of binding sites for ghrelin and the identification of the specific GHS-R mRNA have already been reported in other tumors.19 Unfortunately, no fresh frozen material of fetal thyroid origin was available to evaluate both GHS-R mRNA and ghrelin-binding site status during gland development.
The effect of ghrelin on cell proliferation of thyroid neoplastic cells confirms the data previously reported in thyroid carcinoma cell lines using synthetic GHS.18 Similarly, in neoplastic cells from tissues other than the thyroid, both ghrelin and GHS were able to inhibit cell proliferation in vitro.19-21 The relatively high concentration of ghrelin (100 nmol/L to 1 µmol/L) required to exert an anti-proliferative effect in the studied thyroid carcinoma cell lines is in agreement with what was previously observed in lung carcinoma or breast cancer cell lines using either ghrelin or synthetic GHS.19,21 These data are at variance with recent findings on lower dose-dependent ghrelin effects on cell growth in prostate cancer cell lines,20 even if, however, different cancer cell lines were analyzed, and possible cell line-dependent or different culture conditions could be responsible for these discrepancies.
Because the anaplastic thyroid carcinoma cell line (ARO) required higher ghrelin concentrations (1 µmol/L) for inhibition of cell proliferation compared to the papillary carcinoma-derived cell line (N-PAP), we speculate that more undifferentiated cells may express a lower-affinity or modified GHS receptor. These in vitro evidences allow us to suggest that, besides their endocrine effect, synthetic and natural GH secretagogues may act as negative growth factors in neoplastic conditions, possibly through a receptor different from GHS-R1a. Therefore, such alternative binding sites for ghrelin could depend either on the central/peripheral location (as suggested above) or on the normal/neoplastic cell phenotype.
In conclusion, we have shown that: 1) follicular cells of the developing thyroid are an additional source of ghrelin in fetal life; 2) the adult thyroid is devoid of immunoreactive ghrelin, but most thyroid tumors of follicular origin express ghrelin mRNA and protein; 3) hypothetical receptor types other than GHS-R, the only currently known ghrelin receptor, could mediate ghrelin functions in thyroid tumors; and 4) ghrelin possesses anti-proliferative properties in thyroid carcinoma cell lines suggesting that autocrine circuits may be operating in the growth control of follicular tumors.
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Acknowledgements |
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Footnotes |
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Partially supported by grants from the Italian Ministry of University and Research (Rome), Associazione Italiana per la Ricerca sul Cancro (Milan), and the Fondazione per lo Studio delle Malattie e Endocrine e Metaboliche (Turin).
Accepted for publication October 17, 2002.
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