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Original Studies |
Division of Endocrinology, Department of Medicine, St. Hedwig Hospital, 10115 Berlin, Germany; Humboldt University Berlin, Berlin, Germany; and Faculty of Biology, Aston University (E.A.), B4 7ET Birmingham, United Kingdom
Address all correspondence and requests for reprints to: Dr. Michael Derwahl, St. Hedwig Hospital, Grosse Hamburger Strasse 5-11, 10115 Berlin, Germany. E-mail: m.derwahl{at}alexius.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß in thyroid cells derived from human
goiter nodules and in human thyroid carcinoma cell line HTC-TSHr was
demonstrated. There was no difference between the expression levels of
estrogen receptor in males and females, but there was a significant
increase in expression levels in response to 17ß-estradiol.
Stimulation of benign and malignant thyroid cells with 17ß-estradiol
resulted in an increased proliferation rate and an enhanced expression
of cyclin D1 protein, which plays a key role in the regulation of
G1/S transition in the cell cycle. In malignant tumor cells
maximal cyclin D1 expression was observed after 3 h, whereas in
benign cells the effect of 17ß-estradiol was delayed. ICI 182780, a
pure estrogen antagonist, prevented the effects of 17ß-estradiol. In
addition, 17ß-estradiol was found to modulate activation of
mitogen-activated protein (MAP) kinase, whose activity is mainly
regulated by growth factors in thyroid carcinoma cells. In response to
17ß-estradiol, both MAP kinase isozymes, extracellular
signal-regulated protein kinases 1 and 2, were strongly phosphorylated
in benign and malignant thyroid cells. Treatment of the cells with
17ß-estradiol and MAP kinase kinase 1 inhibitor, PD 098059, prevented
the accumulation of cyclin D1 and estrogen-mediated mitogenesis. Our
data indicate that 17ß-estradiol is a potent mitogen for benign and
malignant thyroid tumor cells and that it exerts a growth-promoting
effect not only by binding to nuclear estrogen receptors, but also by
activation of the MAP kinase pathway. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The presence of estrogen receptors (ERs) in thyroid tissues has been demonstrated by immunohistochemistry and binding assays (6, 7). In some studies higher levels of ERs were found in neoplastic than in normal thyroid tissues (7, 8). Evidence for a direct stimulatory effect of 17ß-estradiol on thyroid cell growth comes from a very recent study in FRTL-5 rat thyroid cells that express functional ERs (9).
Classically, ERs are intracellular receptors that serve as transcription factors. The ligand-bound dimer ER can interact with an estrogen-responsive element, resulting in transcriptional activation of the target gene (10, 11). However, various studies have provided evidence that estrogen may also affect growth factor-dependent signaling pathways (12, 13, 14). One of the targets of estrogen action is the mitogen-activated protein (MAP) kinase (MAPK) whose activity is regulated by growth factors (14, 15).
Recently, the molecular mechanisms by which estrogen affects the cell cycle regulatory apparatus to induce cellular proliferation have been elucidated (16, 17, 18). 17ß-Estradiol stimulates cell cycle progression early in G1 phase by induction of cyclin D1 gene expression (18, 19, 20). In different cell lines the induction of cell growth was found to correlate with increased expression of cyclin D1 protein levels (18, 19).
In the present work we analyzed the effect of 17ß-estradiol on ER messenger ribonucleic acid (mRNA) and protein expression levels in human thyroid cells derived from thyroid nodules and in human thyroid carcinoma cell lines HTC-TSHr and XTC 133 and compared the growth stimulatory effects of 17ß-estradiol on benign and malignant thyroid cells. In addition, we investigated the influence of 17ß- estradiol on the kinetics of cyclin D protein expression and on the activation of growth factor-dependent MAPKs extracellular signal-regulated protein kinase 1 (ERK1) and ERK2 in the same tumor cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monolayer cultures of the human thyroid carcinoma cell line HTC-TSHr (21) were grown in Coon’s modified Ham’s F-12 medium (Life Technologies, Inc., Karlsruhe, Germany) supplemented with 10% FBS (Life Technologies, Inc.), 1% (vol/vol) MEM (Life Technologies, Inc.), five hormones or growth factors [H5-mix: 10 ng/mL glycyl-histidyl-lysine (Sigma, Deisenhofer, Germany), 10 µg/mL insulin (Hoechst, Frankfurt am Main, Germany), 10 ng/mL somatostatin (Sigma), 5 µg/mL transferrin (Sigma); 3.2 ng/mL hydrocortisone (Sigma), 100 U/mL penicillin and 100 µg/mL streptomycin (Roche, Mannheim, Germany), and 2.5 µg/mL amphotericin (Bristol-Myers Squibb Co., Munich, Germany)].
Monolayer cultures of the differentiated human thyroid cancer cell line XTC 133, derived from a Hurthle cell carcinoma (22), were grown in DMEM (Life Technologies, Inc.) supplemented with 10% FBS (Life Technologies, Inc.), 1% (vol/vol) MEM (Life Technologies, Inc.), 100 U/mL penicillin and 100 µg/mL streptomycin (Roche), and 2.5 µg/mL amphotericin (Bristol-Myers Squibb Co.).
Primary cultures of human thyrocytes isolated from nodules and adenomas of 16 patients with nodular goiters undergoing surgery were established as described previously (23). The histological diagnosis of tissue samples was made at the Institute of Pathology (Wilhelmshaven, Germany; Prof. Gösta Fischer). For this part of the study informed consent of patients was obtained. The study was approved by the ethical committee of Ruhr University (Bochum, Germany). Primary cells were cultured in the same medium as HTC-TSHr cells, but supplemented with 2 mU/mL TSH (Sigma). All cells were kept in a humidified incubator at 37 C in 5% CO2, with a medium change each 3–4 days.
At 70–80% confluence of HTC-TSHr cells, the medium was switched to hormone-, growth factor-, and phenol red-free medium with only 0.5% FBS for 48 h before stimulation with 10 nmol/L 17ß-estradiol (E2; Sigma) in the same medium. A concentration of 10 nmol/L E2 is on the order of magnitude of serum concentrations detectable in women during the follicular phase and even lower than those during pregnancy (24).
XTC 133 cells and the primary cells were grown for 48 h before stimulation with 10 nmol/L E2 in the above-described medium.
When used, ICI 182780 (1 µmol/L; Zeneca Pharmaceuticals, Macclesfield, UK) and PD 098059 (50 µmol/L; Calbiochem, Bad Soden, Germany) were added 1 h before stimulation with E2.
RNA extraction and RT-PCR
Total RNA was extracted using RNeasy (QIAGEN, Hilden, Germany) according to the manufacturer’s specifications. RT-PCR was performed as described previously (25). Briefly, 1 µg RNA was added to 25 µL reaction mixture containing 5 µL 5 x myeloblastosis virus RT buffer, 2 µL 10 mmol/L deoxy (d)-NTPs, 1 µL RNasin, 500 ng random hexamers, and 1 µL myeloblastosis virus reverse transcriptase (Promega Corp., Mannheim, Germany). RT was carried out at 20 C for 10 min, 42 C for 60 min, and 95 C for 10 min and was terminated at 4 C.
For PCR amplification of ER and ERß complementary DNAs, the
following sense and antisense primers were used: for ER, 5'-GGG TGA
AGT GGG GTC TGC TG-3' and 5'-TGC CTC CCC CGT GAT GTA AT-3'; and for
ERß, 5'-CCC TGC TGT GAT GAA TTA CAG-3' and 5'-CTT CTC TGT CTC CGC ACA
AG-3'. For PCR amplification, 4 µL complementary DNA were added to a
50-µL reaction containing 5 µL 10 x reaction buffer, 50
mmol/L MgCl2, 1 µL dNTPs, 50 pmol sense and
antisense primers, and 0.5 µL Taq DNA polymerase (5
U/µL) (Life Technologies, Inc.). Reactions were carried
out at 95 C for 10 min; 35 cycles of 95 C for 1 min, 65 C for 1 min for
ER and 61 C for ERß, 72 C for 1 min; and then 72 C for 10 min and
4 C to terminate.
Western blot analysis
Total protein was isolated from the cells, and aliquots of 50 µg protein were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Aylesbury, UK). The membranes were incubated with 5% nonfat dry milk in 1 x TBS-T (0.1 mol/L Tris base, 0.15 mol/L sodium chloride, and 0.05% Tween-20, pH 7.4) for 1 h. They were then incubated with a 1:1000 dilution of polyclonal rabbit anti-ERK antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 1 µg/mL monoclonal mouse anti-pERK antibody (Santa Cruz Biotechnology, Inc.), and 2 µg/mL polyclonal rabbit anticyclin D1 antibody (Santa Cruz Biotechnology, Inc.) in 5% nonfat dry milk in 1 x TBS-T for 1 h at room temperature. After washing three times with 1 x TBS-T, the membranes were incubated with secondary antibody (peroxidase- conjugated donkey antirabbit/anti-mouse IgG, Amersham Pharmacia Biotech) at a dilution of 1:500 for 45 min at room temperature. After washing three times with 1 x TBS-T and once with TBS, enhanced chemiluminescence (Amersham Pharmacia Biotech) was used to detect immunopositive protein bands.
The membranes for ER and ERß were initially blocked in 3% nonfat
dry milk in PBS (9.1 mmol/L dibasic sodium phosphate, 1.7 mmol/L
monobasic sodium phosphate, and 150 mmol/L sodium chloride, pH 7.4) for
3 h at room temperature and were then incubated with 2 µg/mL
monoclonal mouse anti-ER antibody (Upstate Biotechnology, Inc., Lake Placid, NY) and 2 µg/mL polyclonal rabbit
anti-ERß antibody (Upstate Biotechnology, Inc.) in 3%
nonfat dry milk in PBS overnight at 4 C. After washing twice with
distilled water, the membranes were incubated with secondary antibody
(peroxidase-conjugated donkey antirabbit/anti-mouse IgG, Amersham Pharmacia Biotech) at a dilution of 1:2000 for 2 h at room
temperature. After washing three times with distilled water and once
with PBS, enhanced chemiluminescence (Amersham Pharmacia Biotech) was used to detect immunopositive protein bands.
Growth assays
The primary and the HTC-TSHr cells were maintained for 2 days before stimulation in hormone-, growth factor-, and phenol red-free medium with only 0.5% FBS. For stimulation the described media were replaced with the media containing 10 nmol/L E2 for 2 days and 1 day, respectively. Bromodeoxyuridine (BrdU; 100 µmol/L) was added to the media 4 h before detection. The labeled cells were stained using a BrdU kit (Roche) according to the manufacturer’s specifications and were detected microscopically. Growth rates were estimated by counting the percentage of BrdU-labeled cells. For each experiment at least 200–300 cells were counted.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß in thyrocytes derived from thyroid
nodules and in HTC-TSHr cells
The presence of both ER and ERß was established by RT-PCR and
Western blot analysis. Total RNA from HTC-TSHr and primary cells was
isolated, and RT-PCR for ER and ERß was performed with ß-actin
as a control. ER and ERß with the expected sizes of 935 bp for
ER, 872 and 762 bp for ER isoforms, and 552 bp for ERß were
detected in HTC-TSHr cells (Fig. 1A
) and
primary cells from seven different nodular tissues [two males (no. 3
and 6) and five females; Fig. 1B]. There was no difference between the
expression levels in males and females. Validation of RT-PCR products
was carried out by restriction endonuclease experiments (data not
shown).
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and ERß was confirmed by Western blot analysis
using a monoclonal anti-ER and a polyclonal anti-ERß antibody.
Both HTC-TSHr cells (Fig. 2A) and
thyrocytes derived from thyroid nodules (Fig. 2B) showed the expected
size of 67 kDa for ER. For ERß, whose function in thyroid cells is
unknown, the expected 65-kDa band was detected. Analysis of seven
different nodular tissues (two males and five females) revealed no
significant difference in the expression of ER and ERß protein
levels.
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A range of E2 concentrations (0.1, 1, and 10
nmol/L) was added to the HTC-TSHr cells for 12, 24, and 48 h to
assess its effect on the quantity of ER and ERß. The Western blot
analysis showed an increase in the level of ER in a time-dependent
manner, with a maximum at 48 h for all three concentrations tested
(Fig. 2A
). In primary cells stimulated with 10 nmol/L
E2, an increase in the protein levels of ER
was also observed in a time-dependent manner (Fig. 2B). ERß protein
levels remained constant after stimulation with
E2 in both HTC-TSHr cells (Fig. 2A) and cells
derived from thyroid nodular goiter (Fig. 2B).
Effect of E2 on proliferation
The detection of BrdU-labeled cells was used to examine the effect
of E2 on proliferation of HTC-TSHr thyroid
carcinoma cells and thyrocytes derived from nodules. After stimulation
with 10 nmol/L E2 for 24 h (HTC-TSHr cells)
and 48 h (primary cells), the percentage of stained cells
significantly increased from 40% to 57% in HTC-TSHr cells
(P < 0.05; Fig. 3A) and
from 10.5% to 27% in thyrocytes derived from goiter nodules
(P < 0.05; Fig. 3). Addition of 1 µmol/L ICI 182780,
a pure estrogen antagonist, prevented the proliferative effect of
E2 in both types of cells. The percentage of
labeled cells remained approximately at the control level (Fig. 3). The
same inhibitory effect on E2-mediated mitogenesis
was observed when 50 µmol/L PD 098059, a specific inhibitor of
activation of MAPK kinase 1, was added (26).
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After stimulation with 10 nmol/L E2, cyclin
D1 protein expression levels significantly increased in HTC-TSHr cells,
with a maximum at 3 h (Fig. 4A). In
cells derived from nodular tissues and in differentiated Hurthle
carcinoma cells, XTC 133, a delayed increase in cyclin D1 expression
levels (maximum after 6–9 h) in response to E2
was observed (Fig. 4, B and C). In HTC-TSHr cells, application of the
estrogen antagonist, ICI 182780 (1 µmol/L), prevented an increase in
cyclin D1 protein levels in response to E2 (Fig. 4A). In both cells derived from nodular tissues and differentiated
Hurthle carcinoma cells, XTC 133, the addition of ICI 182780 prevented
an increase and, moreover, produced a delayed decrease in cyclin D1
protein level (Fig. 4, B and C). Application of 50 µmol/L PD 098059
to the HTC-TSHr cells stimulated with E2 lead to
a rapid transitory decrease in cyclin D1 levels (Fig. 4A). In response
to PD 098059 and stimulation with E2, cells
derived from nodular tissues and XTC 133 cells showed uniform
expression of cyclin D1 levels for 6–9 h, with a slight decrease
thereafter (Fig. 4, B and C).
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After stimulation with 10 nmol/L E2 ERK1 and
ERK2, expression levels remained constant in HTC-TSHr cells, whereas a
strong, rapid phosphorylation of ERKs was already observed at 30 min
and began to decrease after 3–9 h (Fig. 5A). Incubation of
E2-stimulated HTC-TSHr cells with ICI 182780
resulted in a strong inhibition of ERK phosphorylation, with pERK
levels lower than the control values, and, after 6 h of treatment,
in a delayed phosphorylation signal (Fig. 5A). The differentiated
Hurthle carcinoma cells, XTC 133, showed a sustained incremental
increase in phosphorylation of ERK1 and ERK2 with constant ERK levels
after stimulation with E2. Addition of ICI 182780
before stimulation with E2 led to a minimal
decrease in phosphorylation of ERK with a moderate increase after 6–9
h, followed by a return to the control level (Fig. 5B); the kinetics
fell in between those of the undifferentiated HTC-TSHr and those of the
cells derived from nodular tissues.
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, the ERKs
protein level remained fairly constant, and phosphorylation in response
to E2 occurred not earlier than 12 h.
Addition of ICI 182780 to the E2-stimulated
primary cells initially did not affect the phosphorylation signal and
later decreased it (Fig. 5C). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present studies confirm and extend these earlier findings. Thus, RT-PCR and Western blot analysis were used to demonstrate expression of ER mRNA and protein representing both subtypes in benign and malignant thyroid cells, partly confirming previous results obtained by immunohistochemical methods (6, 7). Moreover, our cell culture experiments showed growth stimulation of differentiated and undifferentiated thyroid tumor cells, and this effect was blocked by the pure antiestrogen, ICI 182780. ICI 182780 also reduced the proliferation rate when added alone to thyroid cell culture. As such, these results provide evidence that, at least in females, E2 may play a role in thyroid tumorigenesis.
The growth stimulatory effect of E2 on benign and malignant thyroid cells was associated with an increased expression of cyclin D1. Cyclin D1 protein plays a key role in regulation of G1/S transition in the cell cycle. It functions as a regulatory subunit of a holoenzyme that phosphorylates and inactivates the tumor suppressor pRB, nuclear retinoblastoma protein, resulting in cellular proliferation (32, 33). Cyclin D1 gene possesses an estrogen-responsive regulatory region that has been mapped within the first 944 bp upstream of the transcriptional start site (19). In human breast cancer cells induction of cellular proliferation by estrogen was found to correlate with cyclin D1 expression (18, 19, 20). Moreover, in thyroid carcinoma tissues overexpression of cyclin D1 was described in about one third of tumor samples (34).
In HTC-TSHr thyroid carcinoma cells, the maximum of cyclin D1 expression was already observed after 3 h of E2 stimulation, whereas in differentiated Hurthle cell carcinoma cell line XTC 133 and in primary cells derived from goiter nodules the peak of cyclin D1 expression was delayed. This may be explained by a slower cell cycle progression in differentiated than in undifferentiated thyroid tumor cells and corresponds to the lower growth rate in differentiated cells.
Growth factors and their receptors play a major role in the control and regulation of thyroid tumor proliferation (35). Many tumor cells, including thyroid carcinoma cells, are stimulated by secreted growth factors, such as epidermal growth factor, in an autocrine manner (36). The pathophysiological importance of growth factors is also reflected by the fact that most oncogenes are derived from growth factors or their receptors or from signaling proteins that are part of a network of pathways that regulates cell growth (28).
For control of malignant thyroid growth, the Ras-Raf-MAPK pathway is of
major importance (37, 38, 39). The Ras-Raf-MAPK pathway may be
activated by overexpression of the tyrosine kinase receptor (such as
epidermal growth factor receptor) or by overexpression or mutational
activation of h-Ras (37, 40, 41). However, the
Ras-Raf-MAPK pathway may also be stimulated by estrogens with kinetics
similar to those of growth factors operating through tyrosine kinase
receptors, as first revealed in MCF-7 breast cancer cells
(15). These estrogen effects are mediated through putative
membrane ER, which can couple to different second messengers (reviewed
in Ref. 42). Data obtained in the present work indicate
that in thyroid carcinoma cells both MAK kinases, ERK1 and ERK2, are
strongly phosphorylated and thus activated in response to
E2. This finding indicates that in malignant
thyroid cells growth factor-dependent signaling may be augmented by
E2 stimulation, whereas in the XTC cell line and
cells derived from benign lesions, the effect was much weaker (compared
with the control) and delayed (Fig. 5, B and C).
It has been demonstrated that estrogen activates transcription of the immediate-early genes c-myc and c-fos, thus initiating mitogenesis (43, 44, 45, 46). In the present work we show that estrogen, in addition to promoting the c-myc/c-fos pathway, stimulates thyroid tumor growth via MAPK cytoplasmic signaling. Inhibition of this signaling by PD 098059 prevented E2-induced cyclin D1 accumulation and, subsequently, stimulation of cell growth. From these data the question arises of how the enhanced transcription of immediate-early genes interacts with the activated MAPK pathway in estrogen-mediated mitogenesis. Very recently, it has been shown that although PD 098059 prevents cyclin D1 accumulation in response to E2, it does not inhibit estrogen-stimulated c-myc and c-fos gene expression (47). These findings point to a predominant role of the MAPK cytoplasmic pathway in estrogen-dependent growth stimulation.
Taken together, the growth stimulatory effect of estrogen on benign and malignant human thyroid cells may provide insights into the molecular mechanism underlying the epidemiological data that show a prevalence of thyroid carcinomas 3 times higher in females than in males (1). From the clinical point of view, the finding that growth factor-dependent signaling is enhanced by E2 may be of relevance in females with occult thyroid tumors, because the use of estrogens may continuously stimulate cell growth.
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Acknowledgments |
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Footnotes |
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Received April 3, 2000.
Revised October 6, 2000.
Accepted December 2, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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