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Department of Genetics and Pathology (Å.F., B.W., N-E.H.), Unit of Pathology, University Hospital, SE-751 85 Uppsala, Sweden; and Ludwig Institute for Cancer Research (E.P., P.t.D.), Biomedical Center, Box 595, SE-751 24 Uppsala, Sweden
Address all correspondence and requests for reprints to: Åsa Franzén, Department of Genetics and Pathology, Unit of Pathology, University Hospital, SE-751 85 Uppsala, Sweden. E-mail: asa.franzen{at}genpat.uu.se
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Introduction |
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During recent years, Smad proteins have been identified with pivotal roles in the intracellular signal transduction of the TGF-ß family members (1, 2). The Smads can be divided into three groups according to their function, the pathway-restricted Smads (Smad1, Smad2, Smad3, Smad5, and Smad8), a common-mediator Smad (Smad4), and inhibitory Smads (Smad6 and Smad7) (1, 2). TGF-ß and activin signal via Smad2 and Smad3; and BMPs through Smad1, Smad5, and Smad8. The pathway-restricted Smads are phosphorylated by the type I receptor upon ligand binding, and the activated Smad proteins form complexes with Smad4. Subsequently, these complexes translocate to the nucleus, where they bind to regulatory regions of responsive genes, by themselves and/or in combination with DNA-binding proteins (1, 2).
The importance of different TGF-ß superfamily members in the regulation of thyroid growth and function is not fully understood. TGF-ß has been shown to inhibit thyrocyte growth and function (4, 5, 6). The functional inhibition could be explained, in part, by a down-regulation of thyroid specific gene expression, e.g. decreased synthesis of sodium iodide symporter and thyroglobulin (Tg) has been observed after treatment with TGF-ß1 (7, 8). Normal thyrocytes have also been demonstrated to produce and secrete TGF-ß1 (4, 9, 10, 11). Interestingly, increased production of TGF-ß has been implicated in the development of nodular goiter (12). Activin A has been reported to be expressed in thyroid epithelial cells (13) and to have an inhibitory effect on thyrocyte function and stimulatory effect on thyroid cell growth (14). Recently, Huang et al. (15) showed that Tg could induce TGF-ß-like responses in mink lung epithelial (Mv1Lu) and rat thyroid follicle (FRTL-5) cells.
In the present study, we have examined expression of the TGF-ß family members, TGF-ß1, activin A, and BMP-7, in porcine thyroids in vivo and examined their role and mechanism of action in porcine thyroid follicle cells in suspension cultures. TGF-ß1 and activin A were found to have inhibitory effects on thyrocyte growth and function. Stimulation with BMP-7 had an inhibitory effect on cell function but seemed to increase the growth response induced by epidermal growth factor (EGF). Our results suggest an important role for members of the TGF-ß family in negative regulation of the thyroid.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the blocking experiments, the TGF-ß1 antibody was incubated at room temperature for 2 h with an excess of the immunizing peptide (according to protocol from the manufacturer; Santa Cruz Biotechnology, Inc.). The activin A antibody was incubated with 100-fold excess of activin A for 2 h at room temperature. A control staining, omitting the primary antibody, was also performed.
Cell cultures
Porcine thyroids were collected from the local slaughterhouse.
The thyroids were cleaned from surrounding tissue and minced using a
scalpel, then digested for 1 h at 37 C in a solution containing 1
mg/ml collagenase A, 1.5 mg/ml dispase (both from Roche Molecular Biochemicals, Mannheim, Germany), 3 mM
CaCl2, 50 µg/ml deoxyribonuclease I (Sigma Chemical Co.), 0.1 M Tris-HCl (pH 7.4), 0.1 M
maleic acid, 0.78 M NaCl, 27 mM KCl, 1 mg/ml
glucose, and 1x nonessential amino acids (Life Technologies, Ltd., Paisley, Scotland, UK). Follicle cells were
enriched by passing the digest over a 5% Percoll gradient
(Amersham Pharmacia Biotech, Uppsala, Sweden) and then
washed several times in PBS to remove single cells and red blood cells.
The follicle cells were seeded in Ham’s F-10 medium (Sigma Chemical Co.), supplemented with 1% FCS and antibiotics (100 U
penicillin and 50 µg streptomycin per ml), and grown in suspension
cultures on agarose-coated cell culture dishes, or grown on coverslips
for the immunofluorescence experiments.
Cell proliferation assay
Follicle cells in thymidine-free Ham’s F-10 medium were grown
in 12-well plates and treated with different concentrations of
TGF-ß1, activin A, or BMP-7, in the absence or presence of 10 ng/ml
hepatocyte growth factor (HGF) or 10 ng/ml EGF, for 46 h. The
final 4 h, [methyl-3H]thymidine (0.25 µCi/ml;
Amersham Pharmacia Biotech) was added. The cells were
harvested on glass microfiber filters, GF/C (Whatman
International Ltd., Maidstone, England, UK), and were fixed with 10%
trichloroacetic acid (TCA) and rinsed with PBS. The filters were
treated with NaOH/SDS (0.3 M/1%) for 15 min, and the
amount of radioactivity incorporated was determined by liquid
scintillation counting.
cAMP assay
Porcine thyroid follicle cells grown in 12-well plates were
stimulated with TGF-ß1 or activin A for 2 or 24 h. During the
last 30 min, the cells received 1 mU/ml bovine TSH
(Intergen, Purchase, NY) in the presence of 1
mM 3-isobutyl-1-methylxantine (IBMX). The cells were
treated with 5% TCA for 15 min, followed by extractions with
water-saturated diethylether to remove the TCA and cell debris. The
amount of cAMP was determined using a cAMP assay system (TRK 432;
Amersham Pharmacia Biotech).
Northern blot analysis
Total RNA was extracted from cells stimulated with 10 ng/ml
TGF-ß1 or 50 ng/ml activin A, for different times, using a LiCl/urea
method (18). RNA samples (15 µg/lane) were size-fractionated by
electrophoresis on a 0.8% agarose gel under denaturing conditions and
were transferred to a nylon filter (Duralon UV;
Stratagene, La Jolla, CA). Hybridizations were performed
using QuikHyb solution (Stratagene), according to the
protocol supplied by the manufacturer. To analyze expression of activin
and BMP receptors (BMPRs) in thyroid follicle cells, 5 µg/lane of
poly (A)+ enriched RNA was electrophoresed, transferred to
a nitrocellulose filter (Hybond-C; Amersham Pharmacia Biotech), and hybridized as described previously (19). As a
control in the hybridizations, poly (A)+ -RNA
extracted from human thyroid tissue obtained from a patient operated
for toxic goiter was included. The fragments used were a 0.6-kb
EcoRI/AvaI fragment of human ActR-I (20), 0.8-kb
EcoRI/PvuII fragment of human BMPR-IA (20),
0.8-kb PstI fragment of BMPR-II (21), and the 2.6-kb coding
part of human TSH receptor (TSHR) (22) complementary DNAs (cDNAs).
Radiolabeling of ligands and affinity cross-linking studies
Human recombinant TGF-ß1 was obtained from Dr. N. Ferrara at
Genentech, Inc. (San Francisco, CA), activin A was a gift
from Dr. Y. Eto at the Ajinomoto Company, and BMP-7 was provided by Dr.
K. Sampath at Creative Biomolecules, Inc. The growth factors were
iodinated using the chloramine-T method (23). Affinity-binding of
TGF-ß1, activin A, and BMP-7 to cell surface binding proteins on
thyroid follicle cells, followed by immunoprecipitation of cell lysates
with receptor-specific antisera (TßR-I, VPN-antiserum; TßR-II,
DRL-4-antiserum; TßR-III, GET-antiserum; endoglin, KRE-antiserum;
ActR-I, RRN-2-antiserum; ActR-IB, RVY-antiserum; ActR-II,
ARC-2-antiserum; ActR-IIB, RKD-antiserum; BMPR-IA, KSI-antiserum;
BMPR-IB, DET-1-antiserum; and a mixture 1:1 of SMN- and NRR-antisera
for BMPR-II), was performed, basically as previously described
(24).
Western blot analysis
Suspension cultures of porcine thyroid follicle cells were
stimulated with TGF-ß1 (10 ng/ml), activin A (100 ng/ml), or BMP-7
(500 ng/ml) for 1 h at 37 C; centrifuged; and lysed in a buffer
containing 1% Triton X-100, 150 mM NaCl, 10 mM
Tris-HCl (pH 7.4), 1 mM EGTA, 1 mM EDTA, and
0.5% NP-40 supplemented with protease and phosphatase inhibitors (35
ng/ml phenylmethylsulfonyl fluoride, 1.4 µg/ml aprotinin, 1
mM Na3VO4, 10 mM NaF, 1
mM ZnCl2, 50 mM
Na2MbO4, 10 mM
Na2P2O7) on ice for 40 min. Cell
debris was removed by a centrifugation at 14,000 x g
for 10 min. Protein content was determined using a commercial kit
(Pierce Chemical Co., Rockford, IL). Samples (12
µg/lane) were separated on a 4–12% Bis-Tris NuPAGE gel
(Novex, San Diego, CA) and transferred to a Hybond-ECL
filter (Amersham Pharmacia Biotech). The filters were
blocked for 1 h with 5% BSA in 10 mM Tris-HCl (pH
7.7), 150 mM NaCl, and 0.1% Tween-20 (TBS-T) and were
incubated overnight at 4 C with antibodies against Smad2 or
phosphorylated Smad2 (25). After several washes in TBS-T, the filters
were incubated for 2 h with a secondary horseradish peroxidase
conjugated antirabbit IgG (Amersham Pharmacia Biotech)
diluted 1:5,000. Enhanced chemiluminiscence was performed using
SuperSignal Chemiluminescence Substrate (Pierce Chemical Co.). The filters were stripped for 30 min at 50 C in a solution
containing 100 mM ß-mercaptoethanol, 2% SDS, and 62.5
mM Tris-HCl (pH 6.7) and were rinsed thoroughly before
being used again.
Immunofluorescence staining
Cells were grown on coverslips in Ham’s F-10 medium
containing 10% FCS. Before the experiment, the cells were kept in low
serum (1%) for 24 h. TGF-ß1 (10 ng/ml) or activin A (50 ng/ml)
were added for 1 h at 37 C. Cells were fixed in 1.5%
paraformaldehyde for 5 min and permealized with 0.1% Triton X-100 for
3 min. The nonspecific binding was blocked with 0.1% BSA in PBS for 20
min and incubated with antisera to Smad 2, Smad3, and Smad4 [Smad2,
DQQ-antiserum; Smad3, DHQ-antiserum; or Smad4, HPP-antiserum (26)].
The coverslips were washed three times in PBS and incubated with a
fluoresceine isothiocyanate swine antirabbit antiserum (diluted 1:20;
DAKO Corp. A/S) for 1 h. The slides were mounted
(Vectashield; Vector Laboratories, Inc., Burlingame, CA)
and photographed using a UV-microscope (Leica Mikroskopie und Systeme GmbH, Wetzlar, Germany).
Statistical analyses
Results presented are from multiple experiments in either
duplicate or triplicate samples and are expressed as percent of control
attributable to the interexperimental variation in the level of
stimulation, as a result of using primary cultures of thyroid follicle
cells. Statistical analyses were performed using Student’s
t test. The different levels of significance are indicated
in the figures (1, P values < 0.05; **, P
values < 0.005; ***, P values < 0.001).
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Results |
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) and activin A (Fig. 1C) was localized
to the cytoplasm of the epithelial cells of the thyroid. There appeared
to be a variation in the intensity of expression between different
follicles. Stromal and endothelial cells between the follicles did not
stain for the ligands. Preincubation of the TGF-ß1 antibody with the
immunizing peptide, and the activin A antibody with a 100-fold excess
of ligand, nearly completely abolished the staining demonstrating the
specificity of the antibodies (Fig. 1, B and D, respectively). We also
observed a staining for BMP-7 in the thyroid (Fig. 1E), which (similar
to staining for activin A and TGF-ß1) was localized to the follicle
cells. There appeared to be a higher degree of heterogeneity between
different cells within the same follicle in the case of BMP-7 staining,
compared with those of TGF-ß1 and activin A.
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). Addition of TGF-ß1
significantly inhibited the growth stimulation of 10 ng/ml of EGF or
HGF (Fig. 2A). Activin A also inhibited DNA synthesis of thyrocyte
cells, as measured by thymidine incorporation (Fig. 2B). The growth
inhibitory effect of activin A was not as pronounced as that of
TGF-ß1; an effect comparable with the maximal effect of TGF-ß1 (a
70% inhibition) was observed at 100 ng/ml activin A (60% inhibition).
Activin A also inhibited EGF- and HGF-induced growth stimulation of the
thyrocytes (however, not as potently as TGF-ß1). Taken together, both
activin A and TGF-ß1 have an inhibitory effect on thyrocyte growth,
but TGF-ß1 is more potent than activin A. Addition of BMP-7 alone, in
concentrations up to 500 ng/ml, did not significantly inhibit cell
growth (Fig. 2C). However, in combination with EGF, BMP-7 significantly
increased the growth stimulation, observed already at 50 ng/ml of
BMP-7.
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, a preincubation for 24 h
with TGF-ß1 (Fig. 3A) or activin A (Fig. 3B) inhibited the
TSH-induced cAMP production. A maximal inhibitory effect of about
30–40% was obtained using 1 ng/ml TGF-ß or 10 ng/ml activin A (Fig. 3, A and B). The inhibitory effect of TGF-ß1 and activin A was
reduced when shortening the preincubation time. A 2-h preincubation
with the ligands resulted only in a 15% inhibition of the
intracellular level of cAMP. Interestingly, addition of BMP-7 inhibited
TSH-mediated increase in cAMP level; a 2-h preincubation of cells with
BMP-7 induced a 25–30% inhibition (Fig. 3C). The inhibitory effect of
BMP-7 was not observed after 24 h of stimulation. To further
elucidate the possible mechanism behind the inhibitory effect on
thyroid function of TGF-ß1 and activin A, we performed Northern blot
analysis on thyroid follicle cells treated with the two ligands, to
study the expression of thyroid specific proteins. Total RNA extracted
from the porcine thyroid follicle cells treated with TGF-ß1 or
activin A for 2 h, 12 h, or 24 h were hybridized with a
TSHR cDNA fragment. As seen in Fig. 4, 10
ng/ml TGF-ß1 induced a decrease in TSHR messenger RNA (mRNA) level,
observed already after 2 h. After 12 h of stimulation with
TGF-ß1, the TSHR mRNA level was almost undetectable. Treatment of
cells with 50 ng/ml activin A did not have any major effect on the TSHR
mRNA level (maybe a slight decrease at 2 h of stimulation) (Fig. 4).
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). As seen in Fig. 5A, we could detect
labeled protein complexes in samples precipitated with specific
antisera to TGF-ß type I (TßR-I) and type II (TßR-II) but not
with antisera to TßR-III or endoglin. In Northern blot analysis of
RNA from porcine thyrocytes, we also failed to detect TßR-III mRNA in
the porcine thyroid follicle cells (data not shown). In the affinity
cross-linking with 125I-activin A, the cells were found to
express activin type IB (ActR-IB), type II (ActR-II), and a lower level
of type I (ActR-I) receptor proteins but not activin type IIB
(ActR-IIB) (Fig. 5B). In a Northern blot analysis, we readily detected
the expression of ActR-I in mRNA obtained from thyroid follicle cells
(Fig. 6). The exposure time needed to
visualize the cross-linked 125I-activin A was much longer,
compared with the time needed for visualizing cross-linked
125I-TGF-ß1, which may indicate that activin receptors
are present at a much lower number. The observed coprecipitations of
type I and type II receptors in the cross-linking by the
receptor-specific antisera indicate that they form complexes upon
ligand binding. The high molecular band observed after cross-linking of
125I-TGF-ß1 was not recognized by the TßR-III
antiserum; its identity remains to be established. One possibility,
because a similar band was also seen after cross-linking of
125I-activin A (Fig. 5B), is that it represents iodinated
Tg. The 80-kDa band observed after cross-linking of
125I-TGF-ß1 is also of unknown origin. Finally, in an
affinity-cross-link experiment using 125I-BMP-7, followed
by immunoprecipitations with receptor specific antisera, we were unable
to detect any receptor complexes (data not shown). However, we found
expression of mRNA for BMP type IA (BMPR-IA) and type II (BMPR-II)
(Fig. 6), but not for BMP type IB (BMPR-IB) receptor (data not shown),
in both primary cultures of porcine thyroid follicle cells and human
toxic goiter tissue (Fig. 6).
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). As seen in Fig. 7, stimulation of
cells with 100 ng/ml activin A also induced phosphorylation of Smad2,
although to a much lower level than induced by TGF-ß1. Activin A
might be less potent, compared with TGF-ß, because of a lower
receptor number present on the cell surface. The level of Smad2 was not
altered by the different treatments (Fig. 7). Stimulation with BMP-7
(500 ng/ml) did not induce phosphorylation of Smad2 in the thyrocytes
(Fig. 7).
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). The nuclear staining of the Smad2 and
Smad4 proteins was almost absent in unstimulated cells but increased
severalfold after TGF-ß1 stimulation. The nuclear staining of Smad3
was high already in unstimulated cells, but it increased further after
TGF-ß1 stimulation. An increased nuclear staining of Smad2, Smad3,
and Smad4 was also observed after addition of 50 ng/ml activin A to the
thyroid follicle cells (Fig. 8). Addition of activin A seemed to more
strongly activate and translocate Smad3 than Smad2. However, the
nuclear staining of Smad2 and Smad3 was stronger in TGF-ß1-stimulated
cells, compared with activin A-stimulated cells, but there was no
obvious difference in the nuclear translocation of Smad4 between
TGF-ß1- or activin A-stimulated cells (Fig. 8).
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Discussion |
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We examined the localization of TGF-ß1, activin A, and BMP-7 in porcine thyroid tissue by immunohistochemistry. A specific immunostaining of all three ligands was found. TGF-ß1 and activin A were widely expressed and localized to the epithelial cells. The staining for the two ligands was localized to the cytoplasm of the follicle cells, indicating an active synthesis. Furthermore, there was heterogeneity in the expression level, mainly between different follicles but also among different cells in the same follicle. The staining for BMP-7 showed a pronounced heterogeneity in expression levels between cells within the follicle, compared with the TGF-ß1 and activin A stainings. However, comparison of relative amounts between the ligands is difficult because affinity of antibodies may differ. The BMP-7 staining was localized to the cytoplasm; but in contrast to TGF-ß1 and activin A, it was also localized to the nuclei of the cells.
There is still some controversy regarding the production of TGF-ß1 in normal thyroid follicular epithelium. Several previous reports have failed to demonstrate TGF-ß1 in normal thyroid follicle cells in vivo (31, 32, 33); however, as mentioned previously, TGF-ß1 production has been demonstrated in many in vitro cell systems of normal thyrocytes (4, 9, 10) and in diseased (12, 31) and hyperplastic thyroid tissue (32, 33). Our results regarding the production of TGF-ß1 in thyrocytes are consistent with previous in vitro studies; however, we found an abundant expression of TGF-ß1 in porcine thyroids in vivo. An explanation for the discrepancy between the results might be that different antibodies were used. In addition, our activin A staining results are in agreement with Wada et al. (13), who also found activin A in thyroid follicular cells.
Previous reports have demonstrated TGF-ß-mediated inhibition of thyroid cell growth, in the absence or presence of growth factors, as mentioned above. We have extended these studies by comparing effects of multiple TGF-ß family members found to be localized within the thyroid. TGF-ß1 and activin A were found to inhibit, in part, the basal proliferation as well as EGF- or HGF-stimulated proliferation of thyrocytes. Our observations regarding the effects of activin A are in contrast with a previous report of Kotajima et al. (14), in which activin was found to stimulate thyroid cell growth. Activin A-induced growth inhibition has been reported using mink lung epithelial cells (34), human keratinocytes (HaCaT) (35), and B cells (36). In the present study, we also show a synergistic stimulatory effect of BMP-7 and EGF on the growth of our porcine thyroid follicle cells.
TGF-ß1 was found to attenuate the TSH-stimulated cAMP response in thyrocytes. The inhibitory effect was observed already after a 2-h pretreatment, but it increased upon prolonged TGF-ß1 treatment. These results are in contrast to previous reports that described no effect of TGF-ß in this assay (5, 6); however, they are in conformity with another study (4). Differences in assay and cell culture conditions are likely reasons for these discrepancies; in contrast to other reports, our cells are kept in the three-dimensional structure of a follicle and may thus reflect a more physiological situation. As previously also reported by Kotajima et al. (14), addition of activin A to our thyroid follicle cells for 24 h inhibited the TSH-induced cAMP production. When comparing TGF-ß1 and activin A, also in this assay, we found TGF-ß1 to be more potent than activin A. Interestingly, BMP-7, in contrast to TGF-ß1 and activin A, had a slight inhibitory effect on TSH-stimulated cAMP formation when cells were pretreated for 2 h, but not after 24 h. A possible mechanistic explanation for TGF-ß-mediated inhibition was provided by the observed down-regulation of TSHR mRNA level detectable after 2 h of treatment with TGF-ß1. A reduced binding of 125I-TSH was observed in FRTL-5 cells 12 h after addition of TGF-ß1 (4). The lack of effect of activin A in this assay may suggest mechanisms other than TSHR down-regulation that contribute to the inhibition by activin A on the TSH-stimulated cAMP response.
To obtain insight into the mechanism of action of TGF-ß family members in thyroid follicle cells, we examined ligand-induced receptor binding and Smad activation in these cells. Whereas TGF-ß1 was found to induce a heteromeric complex of TßR-I and TßR-II, activin induced a heteromeric complex predominantly consisting of ActR-IB and ActR-II. A weak binding of activin A to ActR-I was observed, whereas ActR-I expression was readily detected by mRNA analysis. This is also apparent from other studies; activin A binds much more efficiently to ActR-IB than to ActR-I (34). Furthermore, ActR-I has also recently been shown to bind BMP-7 rather than activin A (37). Moreover, Carcamo et al. (38) showed that TßR-I and ActR-IB, which are structurally related, mediate growth inhibition of Mv1Lu epithelial cells, whereas TSR-1 (ALK1) or ActR-I did not. We did not observe any expression of TßR-III in the porcine thyroid follicle cells, which differs from results from studies on rat and human thyrocytes (Ref. 15 ; and Heldin et al., unpublished results) that showed a high level of TßR-III. We were unable to detect BMP-7 receptor binding on these cells, as assessed by affinity cross-linking. However, Northern blot analysis of BMPR-IA and BMPR-II showed expression of these receptors in RNA from porcine thyrocytes grown in vitro, as well as in human thyroid tissue from a patient with toxic goiter. The lack of efficient binding of BMP-7 may be explained by an inefficient translation of BMPR mRNAs, or by a low affinity of BMP-7 to the BMPRs.
Recently, Shimizu et al. (35) reported that TGF-ß1 signaling through TßR-I activated Smad2 and Smad3, but activin signaling through ActR-IB activated predominantly Smad3 in human keratinocytes. In the present study, examination of the subcellular distribution of Smad2, Smad3, and Smad4, after TGF-ß1- or activin A stimulation, revealed that both ligands were able to induce nuclear translocation of these Smads. The nuclear translocation of Smad2 and Smad3 was more intense after stimulation with TGF-ß1, compared with activin A. In agreement with this finding, TGF-ß1 was found to induce the phosphorylation of Smad2 more efficiently than activin A. Thus, in porcine thyrocytes, there was no obvious difference in the activation of Smads by TGF-ß1 or activin A, although TGF-ß1 was more potent than activin A. As expected, we observed neither a nuclear translocation of Smad2 or Smad3 nor Smad2 phosphorylation upon BMP-7 challenge of thyroid cells.
Taken together, the potent negative effects of TGF-ß1 and activin A on thyroid follicle cell growth and function indicate an important regulatory role for these factors in thyroid tissue. The physiological significance of BMP-7 in the thyroid, if any, is presently unclear. Simultaneous expression of TGF-ß1, activin A, and their receptors, together with downstream Smad effectors in normal follicular epithelium, suggests an interesting autocrine role for these factors.
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Acknowledgments |
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Footnotes |
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Received December 23, 1998.
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