0013-7227/97/$03.00/0
Endocrinology
Copyright © 1997 by The Endocrine Society
Vol. 138, No. 12
Printed in U.S.A.
Transforming Growth Factor-b1 Induces a MesenchymeLike Cell Shape without Epithelial Polarization in
Thyrocytes and Inhibits Thyroid Folliculogenesis in
Collagen Gel Culture
SHUJI TODA, SUEO MATSUMURA*, NOBORU FUJITANI, TOMOHISA NISHIMURA,
NOBUHISA YONEMITSU, AND HAJIME SUGIHARA
Departments of Pathology (S.T., T.N., N.Y., H.S.) and Biochemistry (S.M.), Saga Medical School, Saga
849; and the Department of Forensic Medicine and Human Genetics, Kurume University School of
Medicine (N.F.), Kurume 830, Japan
ABSTRACT
Transforming growth factor-b1 (TGFb1) induces a mesenchymelike cell shape in some epithelial cell types. To clarify the role of
TGFb1 in the morphological regulation of thyrocytes, we performed
collagen gel culture of porcine thyrocytes with serum-free medium.
TGFb1-nontreated cells organized follicles. In contrast, the cells
treated with 10 ng/ml TGFb1 became spindle shaped, i.e. they resembled mesenchymal fibroblasts, and did not form follicles. To characterize the spindle-shaped cells, we examined the fine structures and
expression of thyroglobulin (Tg) and cytoskeletal proteins using electron microscopy, immunohistochemistry, and immunoblotting.
TGFb1-nontreated cells had microvilli at the apical side facing follicle
lumen and had basal lamina at the basal side in contact with collagen
T
HYROID follicles, an essential unit of the thyroid, are
embedded in extracellular matrix (ECM) (1). In threedimensional collagen gel culture, thyrocytes (follicular epithelial cells) easily and stably organize follicle structures with
physiological cellular polarity of their component cells; the
apical pole with microvilli faces the follicle lumen and the
basal pole with basal lamina confronts ECM (2–7). This culture system is, therefore, suitable for studying the proliferation and differentiation of thyrocytes.
The multifunctional polypeptide transforming growth factor-b (TGFb) plays crucial roles in morphogenesis at the
embryonic and adult stages, wound healing, and immune
functions through its regulations of growth, differentiation,
apoptosis, ECM formation, and cytokine cross-talk in many
cell types (8 –11). In the morphogenesis of the breast, lung,
and kidney, several studies suggest that TGFb induces a
mesenchyme-like cell shape in epithelial cell types of these
organs and results in modulating the formation of their luminal structures (12–15). In fact, ample exogenous or endogenous TGFb inhibits the formation of ductal structures in the
breast (16 –18). Furthermore, thyrocytes as well as the epiReceived April 28, 1997.
Address all correspondence and requests for reprints to: Dr. Shuji
Toda, Department of Pathology, Saga Medical School, Nabeshima 5–1-1,
Saga 849, Japan. E-mail: [email protected].
* Current address: Department of Basic Allied Medicine, Faculty of
Health Science, Kobe University School of Medicine, Suma, Kobe 654 –
01, Japan.
gel. TGFb1-treated cells showed both microvilli and basal lamina at
the basal side. TGFb1-nontreated cells expressed Tg, whereas
TGFb1-treated cells showed no expression. TGFb1-nontreated cells
barely expressed vimentin, but they expressed enough cytokeratin.
TGFb1-treated cells extensively displayed vimentin along with the
change in shape to become spindle-like and retained a decreased
expression of cytokeratin. TSH (10 mU/ml) did not essentially influence any TGFb1 effects on the cells. These results indicate that
TGFb1 induces a mesenchyme-like cell shape accompanied by cytoskeletal molecular change and the loss of both epithelial polarization and a function in thyrocytes, and that it results in inhibiting
thyroid folliculogenesis with or without TSH. (Endocrinology 138:
5561–5575, 1997)
thelial cells of these organs organize luminal structures both
in vivo and in vitro, especially in collagen gel culture (5,
19 –21). We have, therefore, hypothesized about the possibility that TGFb may regulate the morphology of thyrocytes
and affect thyroid follicle formation. To address this hypothesis, we performed three-dimensional collagen gel culture of
isolated porcine thyrocytes with or without TGFb1 as a representative of the TGFb family (9). We also examined the
effects of TSH, a main regulator for thyrocytes, on the cells,
either alone or in combination with TGFb1.
In this study, TGFb1-treated thyrocytes drastically became
spindle shaped and did not form follicles. We also characterized these spindle-shaped cells using electron microscopy,
immunohistochemistry, and immunoblotting. We herein describe for the first time that TGFb1 induces a mesenchymelike cell shape without epithelial polarization in thyrocytes
and that it results in inhibiting thyroid folliculogenesis with
or without TSH.
Materials and Methods
Preparation of thyrocytes without follicle structures
Single thyrocytes without follicle structures were prepared from porcine thyroid as described previously (5, 22). Briefly, the cells dissociated
with dispase I solution (bacterial neutral protease; 1000 protease U/ml
MEM; Goudoh-Shusei Co., Tokyo, Japan) were first cultured in monolayer for 2– 4 days in Ham’s F-12 medium supplemented with 10% FCS
and 50 mg/ml gentamicin. Single cells without follicle structures were
obtained from the confluent monolayer with 0.15% trypsin treatment.
Most of the thyrocytes expressed cytokeratin and were clearly distin-
5561
5562
EFFECTS OF TGFb1 ON THYROCYTES
guished from fibroblasts or endothelial cells, of which a small population
might contaminate the primary isolated thyrocytes. Because fibroblasts
and endothelial cells did not display cytokeratin (23, 24), the isolated
single thyrocytes were embedded in collagen gel.
Three-dimensional collagen gel culture
This culture was carried out as described previously (5, 22). A total
of 5 3 105 cells were embedded in 0.5 ml type I collagen gel (Nitta Gelatin
Co., Osaka, Japan). To avoid the effects of serum-containing factors on
culture cells, we used the following serum-free medium for culture of
thyrocytes (25, 26). The cells were cultured in a 1-ml 24-well plastic dish
of defined serum-free Ham’s F-12 medium supplemented with ITS premix (5 mg/ml insulin, 5 mg/ml transferrin, and 5 ng/ml selenious acid;
Becton Dickinson Labware, Bedford, MA), 10 mg/ml hydrocortisone, 10
ng/ml somatostatin (Peninsula Laboratories, Belmont, CA), 10 ng/ml
glycyl-l-histidyl-l-lysine acetate (Biomedical Technologies, Stoughton,
MD), 6 ng/ml NaI (Katayama Chemical, Osaka, Japan), and 50 mg/ml
gentamicin. Culture medium was exchanged for fresh medium every 2
days. In this serum-free medium, we used 6 ng/ml NaI, because our
previous studies on thyroid folliculogenesis (5–7) were performed in
10% FCS-added medium that contained about 6 ng/ml NaI.
FIG. 1. Immunohistochemistry for TGFb type I receptor. Thyrocytes
in vivo clearly express the receptor (A). An absorption test results in
negative staining for the receptor (B). The receptor is detected in
spherical cells just after being embedded in collagen gel (C). F, Follicle
lumen; *, gel.
Endo • 1997
Vol 138 • No 12
Stimulation of culture cells with TGFb1
Thyrocytes in collagen gel culture were stimulated by 10 ng/ml
purified TGFb1 (R&D Systems, Minneapolis, MN), either alone or in
combination with 10 mU/ml TSH (Sigma Chemcial Co., St. Louis, MO).
At the initiation of the culture, TGFb1 was added to the medium with
or without TSH; thereafter, the cells were stimulated with TGFb1 every
2 days. We also used recombinant TGFb1 (King Brewing Co., Kobe,
Japan) in the manner described above. No differences were found between purified and recombinant types of TGFb1 in their effects on
thyrocytes.
Immunohistochemistry
Deparaffinized sections of 4% formalin-fixed paraffin-embedded gel
or thyroid tissue were immunostained by the avidin-biotin complex
immunoperoxidase (ABC) method, as described previously (5). The
FIG. 2. Collagen gel culture of TGFb1-nontreated thyrocytes in the
absence (2) or presence (1) of TSH. The cells just after being embedded in collagen gel are spherical (A), and they organize small
follicle structures at 2 days in culture (B and C). The follicles do not
grow to larger entities even after 7 days in culture (F and G). TSH does
not appear to essentially affect follicle formation at 2 (D and E) and
7 days (H and I) in culture. A: TSH (2), 0 h; B and C: TSH (2), 2 days;
D and E: TSH (1), 2 days; F and G: TSH (2), 7 days; H and I: TSH
(1), 7 days. A, B, D, F, and H, Phase contrast microscopy; C, E, G, and
I, H-E staining. Arrow, Follicle lumen; *, gel.
EFFECTS OF TGFb1 ON THYROCYTES
5563
FIG. 3. Collagen gel culture of TGFb1-treated thyrocytes in the absence (2) or presence (1) of TSH. At 12 h in culture, spherical cells just after
being embedded in collagen gel are retracted in multipolar appearance (A and B), and they become spindle-shaped after 48 h in culture (E and
F). Even after 7 days in culture, the cells remain spindle shaped and do not form follicle structures (I). At 12 h (C and D), 48 h (G and H), and
7 days (J) in culture, TGFb1 induces the morphological changes described above in TSH-treated cells as well as in TSH-nontreated cells. A and
B: TSH (2), 12 h; C and D: TSH (1), 12 h; E and F: TSH (2), 48 h; G and H: TSH (1), 48 h; I: TSH (2), 7 days; J: TSH (1), 7 days. A, C, E,
G, I, and J, Phase contrast microscopy; B, D, F, and H, H-E staining. *, Gel.
visualization of each antigen was performed for 5 min with aminoethylcarbazole (AEC substrate kit, Nichirei Co., Tokyo, Japan). To estimate
a differentiating property of thyrocytes, thyroglobulin (Tg; polyclonal
antibody, Medac Gesellschaft fur Klinishe Spezialpraparate, Munich,
Germany) was immunostained. To elucidate the expression of cytoskeletal proteins in the cells, we also immunostained cytokeratin (monoclonal antibody, which covered a spectrum of molecular masses of 40,
45, 46, and 56 kDa; Nichirei Co., Tokyo, Japan) and vimentin (monoclonal antibody; Dako Japan Co., Tokyo, Japan). Cytokeratin and vimentin are well expressed in epithelial and mesenchymal cell types,
respectively, of thyroid tissue (23, 24). To examine an expression of TGFb
type I receptor that plays a central role in the signal transduction of TGFb
(27), the receptor (polyclonal antibody; Santa Cruz Biotechnology, Santa
Cruz, CA) was immunostained. As a positive control for Tg, cytokeratin,
or vimentin, immunohistochemistry was performed on thyroid tissue
(23, 24, 28). Formalin-fixed paraffin-embedded skin tissue was immunostained as a positive control for TGFb type I receptor in the manner
described above (27). These controls always gave positive results. As a
negative control for Tg, cytokeratin, or vimentin, PBS was used instead
of each primary antibody, and normal rabbit and mouse IgGs were used
in place of the primary antibodies for Tg and cytokeratin or vimentin,
respectively. As a negative control for TGFb type I receptor, the receptor
antibody (1 mg) neutralized with the TGFb receptor protein (10 mg; Santa
Cruz Biotechnology) was used. These controls always gave negative
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EFFECTS OF TGFb1 ON THYROCYTES
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FIG. 4. Immunohistochemistry for Tg (A–E), cytokeratin (F–J), and vimentin (K–O) in thyroid tissue and thyrocytes at 4 days in culture. Tg
is detected mainly in the lumens of thyroid follicles in vivo (A). In TGFb1-nontreated thyrocytes with TSH (C) or without TSH (B), Tg is strongly
stained in lumens of organized follicles. In TGFb1-treated cells with TSH (E) or without TSH (D), Tg is not detected. Cytokeratin is clearly
detected in thyrocytes in vivo (F). In TGFb1-nontreated thyrocytes with TSH (H) or without TSH (G), cytokeratin is expressed in the component
cells of organized follicles, although the expression seems to be better in the cells without TSH (G) than in those with TSH (H). TGFb1-treated
cells with TSH (J) or without TSH (I) retain the expression of cytokeratin. Vimentin is detected in endothelial cells of blood vessels (K), although
it is not expressed in thyrocytes in vivo. In TGFb1-nontreated thyrocytes with TSH (M) or without TSH (L), vimentin is weakly stained in
component cells of organized follicles. In contrast, TGFb1-treated cells with TSH (O) or without TSH (N) are strongly stained with vimentin
along with the spindle-shaped change. A, F, and K, Thyroid tissue; B, G, and L, without TSH or TGFb1; C, H, and M, with TSH, without TGFb1;
D, I, and N, without TSH, with TGFb1; E, J, and O, with TSH and TGFb1. Large F, Follicle lumen; *, gel.
EFFECTS OF TGFb1 ON THYROCYTES
results. In addition, to obtain the rate of cytokeratin- or vimentin-positive staining in culture thyrocytes, 1000 cells were counted, and the
percentage of positive cells was calculated. To confirm colocalization of
cytokeratin and vimentin in the cells, we performed double immunostaining: cytokeratin was immunostained by the ABC method and was
visualized using the AEC kit, whereas vimentin was immunostained by
the avidin-biotin complex immunoalkaline phosphatase and was visualized using the fast blue substrate kit (Nichirei).
Western blotting of cytokeratin and vimentin
To examine the effects of TGFb1 on the expression of intermediate
filaments in thyrocytes, 70 3 105 cells were embedded in 7 ml collagen
gel in 100-mm diameter plastic dishes and cultured for 7 days in 14 ml
serum-free medium under various conditions as described above. After
the media were aspirated, cell layer gels were washed three times with
5 ml cold PBS and scraped from the dishes. Each cell layer gel was
homogenized in 7 ml 0.1 m Tris-HCl (pH 6.8) containing 0.2% SDS and
5% 2-mercaptoethanol and then centrifuged for 30 min at 20,000 3 g. The
supernatants were made 1% with respect to SDS and boiled for 15 min
The samples were lyophilized and thereafter dissolved in 1 ml distilled
water. Ten microliters of each sample were subjected to 10% SDS-PAGE
and then transferred to a nitrocellulose membrane sheet (Bio-Rad, Richmond, CA). The sheet was incubated with anticytokeratin or antivimentin antibody. The antigen on the membrane was visualized by the
ABC method described in the manual supplied by Bio-Rad. The density
of the bands was assessed by densitometry. The results were presented
as a percentage of the control values derived from cultures with neither
TSH nor TGFb1 stimulation.
Detection of Tg in culture supernatant
To estimate the effects of TGFb1 on Tg synthesis and secretion of
thyrocytes, we tried to detect Tg in culture supernatants at 7 days under
various conditions as described above, using dot blotting with the same
anti-Tg antibody as that used for immunohistochemistry. Ten milliliters
of culture supernatants were lyophilized and thereafter dissolved in 0.1
ml distilled water. Two microliters of each sample were dotted onto a
nitrocellulose membrane. The antigenicity was detected by the ABC
method. As a standard of Tg, 0.001–100 mg/ml porcine Tg (Fluka Chemie, Buchs, Switzerland) were spotted onto the sheet. As a negative
control, culture medium in which cells were not cultured was used.
Cell proliferation
At 3 and 7 days in collagen gel culture, cell proliferation was examined by immunohistochemistry for bromodeoxyuridine (BrdU; Cell Proliferation Kit, Amersham, Arlington Heights, IL) after 24-h incubation
with 30 mg/ml BrdU, as described previously (5). To obtain the rate of
5565
nuclear BrdU intake, 1000 cells were counted, and percentage of BrdUpositive nuclei was calculated.
Morphology and morphometric analysis
Culture cells were observed by phase contrast microscopy. We further examined the cells with hematoxylin-eosin (H-E) staining, using
deparaffinized sections of the cell layer gel that were fixed with 4%
formalin, routinely processed, and embedded in paraffin (5). To examine
fine structures of the cells, we also performed transmission electron
microscopy by the standard method, using materials fixed with 2.5%
glutaraldehyde (5).
In this study, follicle formation at 7 days in culture was estimated as
follows. On H-E-staining sections of cell layer gel obtained from 10
blocks in each of various conditions, we performed the morphological
analyses of culture cells by light microscopy. Structures that consisted
of 2 or more cells and had clearly luminal spaces were judged as reconstructed follicles (see Fig. 2C). A total of 1000 follicles were counted
in at least 20 randomly chosen noncontiguous and nonoverlapping fields
(at high power view, 320 objective) of the H-E-staining sections. The
sizes of the follicles were determined by measuring the largest diameter,
using an objective micrometer. The follicles were separated into less than
30, 30 –50, and more than 50 mm size groups, and the percentages of these
grouped sizes were calculated. In addition, to obtain the rate of Tgpositive staining in the follicles at 2 and 7 days in culture, 1000 follicles
were counted using the method described above, and the percentage of
Tg-positive follicles was calculated.
Statistical analyses
The data obtained through five independent experiments were statistically examined. The immunohistochemical data for Tg, cytokeratin,
vimentin, and BrdU were analyzed by two-way ANOVA. The data for
both follicle size and density of bands in electrophoresis and Western
blotting were analyzed by paired Student’s t test. Values represent the
mean 6 sd. P , 0.05 was considered significant.
Results
Expression of TGFb type I receptor
We examined an expression of TGFb type I receptor in
thyrocytes, using immunohistochemistry. In thyroid tissue,
many thyrocytes clearly expressed the receptor (Fig. 1A). An
absorption test resulted in negative staining for the receptor
(Fig. 1B). Thyrocytes just after being embedded in collagen
gel also displayed TGFb type I receptor (Fig. 1C). These
results indicated that thyrocytes would be expected to re-
FIG. 5. Detection of Tg in supernatants of collagen gel culture at 7 days by dot blotting. The 100-fold concentrated samples were subjected to
dot blotting as described in Materials and Methods. In TGFb1-nontreated culture, Tg is detected in both supernatants with and without TSH.
In contrast, Tg is not detected in TGFb1-treated culture supernatants with or without TSH. The concentrated supernatants of TGFb1nontreated culture appear to contain 0.1–1 mg/ml Tg on the basis of their color yields by comparison with those of standard porcine Tg (positive
control). NC, Negative control (culture medium with which cells are not cultured). Arrowhead, Faint staining of 0.1 mg/ml Tg.
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EFFECTS OF TGFb1 ON THYROCYTES
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FIG. 6. Electrophoresis and Western blotting of cytokeratin and vimentin. Thyrocytes cultured in collagen gel in the absence (lanes 1 and 2)
or presence (lanes 3 and 4) of TGFb1, either alone (lanes 1 and 3) or in combination with TSH (lanes 2 and 4), are extracted and subjected to
SDS-PAGE (A) and immunoblotting with anticytokeratin (B) or antivimentin (C) antibody. A, TGFb1-treated cells (lanes 3 and 4) show more
EFFECTS OF TGFb1 ON THYROCYTES
spond to TGFb1, although TGFb type II receptor remained
to be elucidated.
Three-dimensional collagen gel culture
Isolated single thyrocytes without follicle structures were
cultured in collagen gel with serum-free medium. Just after
being embedded in collagen gel, the cells were spherical in
shape, and singly and uniformly distributed (Fig. 2A).
TGFb1-nontreated cells clearly organized follicle structures
after 48 –72 h in culture (Fig. 2, B and C). The reconstructed
follicles thereafter did not grow to larger entities, and the size
of most follicles (87.5 6 5.5%) was less than 30 mm even after
7 days in culture (Fig. 2, F and G). The findings reported
above were not essentially affected by 10 mU/ml TSH (percentage of follicles ,30 mm, 92.2 6 3.0%; Fig. 2, D, E, H, and
I). In contrast, 10 ng/ml TGFb1-treated cells retracted and
showed a multipolar appearance after 6 –18 h in culture (Fig.
3, A and B). At 48 –72 h in culture, over 90% of the cells
became spindle shaped and did not organize follicle structure (Fig. 3, E and F). After 7 days in culture, the cells remained spindle shaped and failed to form follicles (Fig. 3I).
Some of the cells (30 –50%) had cellular linkage. This suggested that some TGFb1-affected cells retained an epithelial
nature. TSH did not essentially affect these morphological
changes in TGFb1-treated cells (Fig. 3, C, D, G, H, and J).
Finally, 1 ng/ml TGFb1 induced the morphological changes
described above in only a small population of the cells, although 0.1 ng/ml TGFb1 did not induce those changes (data
not shown). To clarify the effects of TGFb1 on the cells in
more detail, we, therefore, examined 10 ng/ml TGFb1-affected cells.
Effect of TGFb1 on Tg expression
To elucidate an effect of TGFb1 on functional differentiation of thyrocytes, we examined Tg expression as a representative of differentiating properties of the cells. Tg was
clearly detected in the lumen of follicles organized by TGFb1nontreated cells as well as in the lumen of follicles in vivo,
although the staining intensity was stronger in follicles in
vitro than in their counterparts in vivo (Fig. 4, A and B).
Tg-positive rates of the reconstructed follicles after 2 and 7
days in culture were 22.3 6 4.0% and 90.8 6 7.3%, respectively. TSH (10 mU/ml) did not affect the staining intensity
(Fig. 4C) or the positive rates on both culture days (day 2,
21.6 6 3.8%; day 7, 90.1 6 6.4%). In contrast, TGFb1-treated
cells with or without TSH did not organize follicles or express
Tg on the same culture days (Fig. 4, D and E). Data for Tg
detection in culture supernatants clearly supported all of the
immunohistochemical results reported above (Fig. 5).
5567
Effects of TGFb1 on the expression of cytokeratin and
vimentin
To evaluate the effects of TGFb1 on the cytoskeleton of
thyrocytes, we examined the expression of cytokeratin and
vimentin, which are easily detected in epithelial and mesenchymal cell types, respectively, of the thyroid (23, 24, 26).
In thyroid tissue, cytokeratin was detected in most thyrocytes (Fig. 4F). In contrast, vimentin was not expressed in
thyrocytes, although it was detected in endothelial cells of
blood vessels (Fig. 4K). In collagen gel culture, cytokeratin
was expressed in both TGFb1-treated and -nontreated cells,
although its expression was slightly decreased in TGFb1treated cells (Fig. 4, G and I, and Fig. 6, B and D). Vimentin
was strongly detected in TGFb1-treated cells along with a
change in shape to become spindle-like, whereas TGFb1nontreated cells minimally expressed vimentin (Fig. 4, L and
N, and Fig. 6, C and D). TSH (10 mU/ml) did not essentially
affect the expression of cytokeratin and vimentin in TGFb1treated cells, whereas TSH decreased the expression of their
intermediate filament types in TGFb1-nontreated cells (Fig.
4, H, J, M, and O, and Fig. 6, B–D). Cytokeratin-positive rates
of the cells during the time tested under all conditions
showed no significant change, and cytokeratin was constantly detected in more than 85% of the cells. The vimentinpositive rates of the cells exhibited various changes in response to culture time or conditions (Fig. 7). Finally, these
results definitely confirmed that TGFb1-induced spindleshaped cells originated from epithelial thyrocytes themselves, but not from fibroblasts or endothelial cells, of which
a small population might contaminate the primary-isolated
thyrocytes, because TGFb1-induced spindle-shaped cells
displayed cytokeratin, whereas fibroblasts or endothelial
cells did not express cytokeratin (23, 24, 26).
Effects of TGFb1 on fine structures of thyrocytes
To examine the effects of TGFb1 on fine structures of
thyrocytes, we performed electron microscopy. In TGFb1nontreated culture, the component cells of organized follicles
had physiological cellular polarity; the apical pole with microvilli faces the follicle lumen, and the basal pole with basal
lamina confronts the ECM, although microvilli and basal
lamina were observed after 2 and 10 days of culture, respectively (Fig. 8, A, D, and F). The component cells of the reconstructed follicles had foot processes at the basal side (Fig.
8, A and G). Colloid substances were prominently and
densely seen in the follicle lumen (Fig. 8A). This finding was
clearly consistent with the immunohistochemical results for
Tg (Fig. 4B). The findings presented above were not essentially affected by 10 mU/ml TSH (Fig. 8, B, C, and E). In
increased expression of actin filaments (arrow; 42 kDa) with or without TSH than TGFb1-nontreated cells (lanes 1 and 2). B, TGFb1-nontreated
cells without TSH (lane 1) and with TSH (lane 2) express the highest and lowest levels, respectively, of a low molecular cytokeratin (40 kDa).
TGFb1-treated cells with TSH (lane 4) or without TSH (lane 3) express a lower level of the protein than TGFb1-nontreated cells without TSH
(lane 1). C, TGFb1-treated cells with TSH (lane 4) or without TSH (lane 3) express the highest level of vimentin (54 kDa), followed in order
by TGFb1-nontreated cells without TSH (lane 1) and with TSH (lane 2). D, Densitometric analysis of the density of the bands. In actin expression,
there is a statistical significance only between TGFb1-nontreated cells with or without TSH and TGFb1-treated cells with or without TSH (P ,
0.002). In cytokeratin expression, there is a statistical significance only between TGFb1-nontreated cells without TSH and TGFb1-nontreated
cells with TSH (P , 0.001) or between TGFb1-treated cells with and those without TSH (P , 0.002). In vimentin expression, there is a statistical
significance between TGFb1-nontreated and -treated cells (P , 0.0001). There is also a statistical significance between TGFb1-nontreated cells
with TSH and those without TSH (P , 0.0005).
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EFFECTS OF TGFb1 ON THYROCYTES
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FIG. 7. Time course of vimentin expression and colocalization of cytokeratin and vimentin in culture thyrocytes. Vimentin-positive rates of
TGFb1-nontreated cells with TSH and without TSH are increased to 8.4 6 2.9% and 15.2 6 3.3%, respectively, in a time-dependent manner,
although the rates show no significant change after 48 h in culture. There is a statistical significance between TSH-treated and TSH-nontreated
cells after 48 h in culture (P , 0.02). This indicates that TSH decreases vimentin expression of thyrocytes. In TGFb1-treated culture, the
vimentin-positive rate of the cells with or without TSH is extensively increased to over 90% in a time-dependent manner, although the rate
shows no significant change after 48 h in culture. There is a statistical significance between TGFb1-treated cells and TGFb1-nontreated cells
with or without TSH after 12 h in culture (P , 0.001). This indicates that TGFb1 prominently increases vimentin expression of thyrocytes with
or without TSH. Colocalization of cytokeratin (CK) and vimentin (V) in the same section is examined with double immunostaining (C). A, CK
is stained in light red with AEC. After the result was photographed (A), the staining color was removed with xylene. Then, anti-CK antibody
was inactivated in 0.01 M citrate buffer for 10 min at 90 C. B, V is stained in blue with fast blue. After the result was photographed (B), the
staining color was removed with 95% methanol. Anti-V antibody was inactivated with the method described above. Lastly, double staining of
CK and V was performed using the same procedures without the color removal of AEC and fast blue. C, Colocalization of CK and V is presented
in color mixed with light red and blue.
contrast, TGFb1-treated cells became spindle shaped and did
not organize follicles (Fig. 9A). Rough endoplasmic reticulae
(RER) and Golgi apparatuses were well developed, and there
was a spindle-shaped change in the cells at 2– 4 days in
culture (Fig. 9, A and B). Thereafter, RER and Golgi apparatuses gradually decreased along with the development of
actin and intermediate filaments (Fig. 10, A and B). The cells
formed fragmented basal lamina at the contact side with
collagen gel at 4 –7 days in culture (Fig. 10B). The cells also
had several microvilli at the basal side contacting with collagen gel and had no apical lumen with microvilli (Fig. 9, A
and B). Colloid droplets were not observed in the cells (Fig.
9, A and B). This finding clearly supported the immunohistochemical result for Tg (Fig. 4D). The cells also had several
lysosomes (Fig. 9B). Linked cells had junctional complexes at
the contact point of the cells (Fig. 11, A and B). This indicated
that some TGFb1-affected cells retained an epithelial nature
in their fine structure. TSH did not essentially affect any of
the TGFb1-induced fine structures in the cells (Figs. 9 and 10,
C and D, and Fig. 11C). These results indicated that TGFb1
drastically inhibited epithelial polarization of thyrocytes.
Effects of TGFb1 on proliferation of thyrocytes
Cell multiplication was evaluated by nuclear BrdU incorporation of thyrocytes after 3 and 7 days in culture (Fig. 12,
A and B). The BrdU uptake of the cells under all conditions
showed no significant differences between 3 and 7 days in
culture (Fig. 12). The rates of BrdU intake in TGFb1-nontreated and -treated cells were about 13% and 5%, respectively (Fig. 12). TSH (10 mU/ml) decreased BrdU intake of
the cells under all of the conditions described above, although there was no statistical significance between TGFb1treated cells with TSH and their counterparts without TSH
EFFECTS OF TGFb1 ON THYROCYTES
5569
FIG. 8. Electron micrograph of organized follicles in collagen gel culture of TGFb1-nontreated thyrocytes in the absence (A, D, F, and G) or
presence (B, C, and E) of TSH. Follicle structure at 2 days in culture with TSH (B) or without TSH (A) has dense colloid substances (*) in its
lumen. After 7 days in culture with TSH (C), dense colloid materials (*) are also observed in the lumen of organized follicle. Microvilli (MV)
that show filamentous appearance (F) are clearly observed at the apical surface of follicle lumen (A and C). Foot processes (FP) that have no
filamentous appearance (G) are also seen at the basal side contacting with collagen gel (A, B, and C). After 12 days in culture with TSH (E)
or without TSH (D), basal lamina (arrowhead) is demonstrated at the contact side with collagen gel. Arrow, Junctional complex; L, lysosome;
CD, colloid droplet.
EFFECTS OF TGFb1 ON THYROCYTES
5570
(Fig. 12). These results support the findings of other studies
that both TGFb1 and TSH inhibit the proliferation of porcine
thyrocytes (29 –34), although their coaction does not appear
to elicit prominent inhibition of cell proliferation.
Discussion
We have shown in this study that in collagen gel culture
TGFb1 drastically induces spindle cell shape in thyrocytes
and inhibits thyroid follicle formation. In contrast, thyrocytes
not treated with TGFb1 organize follicle structures. TGFb1affected thyrocytes have the following characteristics. 1) The
shape of the cells resembles that of mesenchymal fibroblasts
(35). 2) The cells acquire prominent expression of vimentin
and retain expression of cytokeratin that is epithelial cell
specific, although some smooth muscle cells exceptionally
display cytokeratin, although slightly, in development and
atherosclerosis of the aorta (36). 3) Fifty to 70% of the cells
have no cellular linkage, whereas the remnant linked cells
have junctional complexes that are generally observed in
epithelial cell types (1). 4) The cells do not express colloid
droplets or Tg. 5) The cells have both microvilli and basal
lamina at the basal side contacting with collagen gel and have
no apical lumen. That is, the cells lose the epithelial apicalbasal polarity specific for thyrocytes. 6) The cells well develop RER and Golgi apparatuses at an early culture stage,
and then they abundantly acquire both actin filaments along
with the cell membrane and intermediate filaments in the
cytoplasm. This means that the cells show mesenchymal
fibroblast-like reorganization of the fine structures (35). 7) All
of the findings above are unaffected by TSH. The TGFb1affected thyrocytes seem to be in a dedifferentiated state and
to undergo an epithelial to mesenchymal semitransdifferentiation, in that those cells coexpress some phenotypes of both
epithelial and mesenchymal fibroblast-like differentiation
(37). To our knowledge, this is the first instance where TGFb1
is a potent morphological regulator for thyrocytes and an
effective inhibitor of thyroid folliculogenesis.
Our results support an interesting finding by Greenburg
and Hay (38) that with 10% FCS-added medium, some thyrocytes can change into mesenchyme-like cells in type I collagen gel culture of follicles, but not of isolated single thyrocytes. It is unclear, however, whether TGFb1 is involved in
their phenomenon. In our previous (5, 7) and present studies,
isolated single thyrocytes reconstruct follicles and do not
change into mesenchyme-like cells in type I collagen gel
culture with 10% FCS-added or serum-free medium. In their
study, therefore, it seems to be essential that the cells with
follicle structures are embedded in collagen gel. Unknown
factors involved in the follicle structure itself may play a
crucial role in the mesenchyme-like transdifferentiation of
some thyrocytes in cooperation with collagen gel or serum
factors. Considering the structure of thyroid follicles that
consist of both thyrocytes and parafollicular cells (1) or may
contain their bipotential precursor cells in endodermal origin
(39 – 41), it is also conceivable that thyrocyte-parafollicular or
-precursor cell interaction in the isolated follicles may be
involved in the phenomenon reported by Greenburg and
Hay. Finally, in our unpublished data, TGFb1 induces mesenchyme-like cell shape in isolated porcine or human single
Endo • 1997
Vol 138 • No 12
thyrocytes cultured in Matrigel (Becton Dickinson Labware,
Bedford, MA) that consists mainly of type IV collagen, laminin, and fibronectin. This suggests that TGFb1-induced mesenchyme-like cell morphology is not inhibited by at least
these ECM types, although concentrations of these ECM
components remain to be elucidated.
In thyroid folliculogenesis under collagen gel culture of
follicles, Westermark et al. (42, 43) have shown that epidermal growth factor (EGF) alone or EGF and TGFb1 (0.1–1
ng/ml) together promote both migration of thyrocytes from
mother (primarily embedded) follicles and rupture of the
follicle walls, and result in an increase in new microfollicle
formation. They also report that microfollicles from mother
follicles are formed even with a low dose of 0.1–1 ng/ml
TGFb1 alone (43). In our study, a high dose of 10 ng/ml
TGFb1 abolishes reorganization of follicle structures from
isolated single cells. These results suggest that TGFb1 may
have dose-dependent differential effects on folliculogenesis;
a high dose of TGFb1 inhibits follicle formation of thyrocytes,
whereas a low dose of TGFb1 does not inhibit it.
The mechanistic basis for TGFb1-induced inhibition of
thyroid folliculogenesis is unclear. In our present study,
TGFb1 drastically inhibits epithelial polarization of thyrocytes. Considering the role of E-cadherin, which regulates the
organization of cellular polarity in thyrocytes as well as other
epithelial cell types and results in modulating their organomorphogenesis or differentiated states (44 – 47), it is conceivable that TGFb1 may inhibit thyroid folliculogenesis through
down-regulation of E-cadherin expression of thyrocytes.
This possibility is supported by an interesting study by Nilsson et al. (43), which showed that down-regulation of Ecadherin expression of thyrocytes may be involved in follicle
disruption generated by cooperation of TGFb1 and EGF. In
addition, loss of cellular polarity of thyrocytes seems to
closely relate to their spindle-shaped change at least in collagen gel culture. In fact, in the transition of some thyrocytes
to mesenchyme-like cells, Greenburg and Hay (38) show that
the spindle-shaped change in the cells is accompanied by
their loss of epithelial polarization. In other epithelial cell
types of the breast, lung, and kidney, Miettinen et al. (15)
show that TGFb1-induced down-regulation of their E-cadherin expression is involved in the spindle-shaped change in
those cell types. These studies and ours suggest that the loss
of polarization through TGFb1-mediated down-regulation
of E-cadherin expression of thyrocytes or other epithelial
cells may play a crucial role in an induction of the mesenchyme-like cell shape in them.
Cytokeratin is constantly expressed in thyrocytes under in
vivo or in vitro conditions. In contrast, vimentin expression of
the cells seems to depend on various situations or the species
of the cell (23, 24, 26, 48, 49). Thus, coexpression of cytokeratin and vimentin is expected to be observed in normal or
abnormal thyrocytes under various conditions, although in
our current study coexpression of the two intermediate filament types is not seen, at least in porcine thyrocytes, in vivo.
In thyroid tumors, including hyalinizing trabecular adenoma
and papillary, follicular, or anaplastic carcinoma, the spindle
or nonspindle tumor cells coexpress both cytokeratin and
vimentin (23, 24, 26, 48). Also, in spindle cell carcinoma of
skin, esophagus, gallbladder, or larynx, the sarcomatoid tu-
EFFECTS OF TGFb1 ON THYROCYTES
5571
FIG. 9. Electron micrograph of TGFb1-treated thyrocytes in the absence (A and B) or presence (C and D) of TSH. A, TGFb1 clearly induces
the spindle-shaped phenotype of the cells after 3 days in culture without TSH. B, In a higher magnification of a part of the cell in A, many RER
(ER) and Golgi apparatuses (G) are clearly observed in the cytoplasm, in which lysosomes (*) are also seen. The cells have a few microvilli
(arrowheads) at the contact side with collagen gel. C and D, After 3 days in culture, TSH-treated cells show structures similar to those in
TSH-nontreated cells. Arrowheads, Microvilli.
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EFFECTS OF TGFb1 ON THYROCYTES
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Vol 138 • No 12
FIG. 10. Electron
micrograph
of
TGFb1-treated thyrocytes in the absence (A and B) or presence (C and D) of
TSH. A and B, After 7 days in culture
without TSH, the spindle-shaped cells
have well developed intermediate filaments (IF) in the cytoplasm, and the
filaments appear to be consistent with
vimentin. The cells also have many actin filaments (arrow) along with cell
membrane and clearly form basal lamina (arrowhead) at the contact side with
collagen gel. C and D, After 7 days in
culture with TSH, the cells are similar
to TSH-nontreated cells in the development of intermediate (IF) and actin filaments (arrow), and basal lamina (arrowhead).
mor cells coexpress cytokeratin and vimentin (23, 50, 51).
Furthermore, TGFb1 induces spindle cell shape with coexpression of cytokeratin and vimentin in thyrocytes, as described in our present study. These results suggest that
TGFb1 may be involved in the pathogenesis of coexpression
of cytokeratin and vimentin or spindle cell shape in epithelial
tumor cells of the thyroid or other organs. In addition, Coclet
et al. (49) show that EGF-treated cells thereafter cultured with
TSH can regain an epithelial morphology from EGF-induced
spindle cell shape despite the persistence of coexpression of
cytokeratin and vimentin (49). This suggests that the coex-
pression of cytokeratin and vimentin may not always be
associated with the dedifferentiated fibroblast-like cell
shape.
Thyrocytes express Tg in monolayer or collagen gel culture (2, 5). The cells in vitro have apical-basal polarity. The
cells in the monolayer have the apical side with microvilli
facing culture medium and the attachment (basal) side without basal lamina confronting plastic surface (2, 22), whereas
the cells in the collagen gel exhibit physiological polarity, as
explained in the introductory section above. However,
TGFb1-affected thyrocytes have no apical-basal polarity, as
EFFECTS OF TGFb1 ON THYROCYTES
5573
FIG. 11. Electron micrograph of TGFb1-treated thyrocytes in the absence (A and B) or presence (C) of TSH. Low magnification (A) or high
magnification of serial sections of the area indicated by an arrow in A (B) is shown. After 3 days in culture without TSH, a junctional complex
(arrow) is clearly formed at the contact points (arrowhead) between two linked cells. C, On the same day of culture with TSH, the structures
(arrow) are also observed. E, RER; *, lysosome.
described in this study. In addition, several studies using
collagen gel culture of thyrocytes show that single cells without polarization do not express Tg before reorganization of
follicle structures (5, 7, 38). These results suggest that polarization of thyrocytes may be a prerequisite for Tg expression
of the cells, although we cannot at present rule out the possibility that TGFb1 may directly inhibit Tg expression of the
cells, because TGFb1 inhibits iodide intake and its organization in thyrocytes (30, 31). Finally, in TGFb1-nontreated
cells we unexpectedly found that colloid substances and Tg
in the lumens of the organized follicles with or without TSH
are more prominently and densely detected in serum-free
culture than in 10% FCS-added culture (5–7), although both
of those culture media have almost the same concentration
of iodide. It is conceivable that some factors added to the
serum-free medium, although unnameable at present, may
play a crucial role in this phenomenon. It is also conceivable
that serum-containing factors inhibit Tg expression of the
cells.
Many studies show that TGFb extensively promotes ECM
production in some cell types (52). In thyrocytes, Garbi et al.
(53) have shown that TGFb1 accelerates the production of
fibronectin and laminin. Our present study also has shown
by electron microscopy that TGFb1-treated thyrocytes in collagen gel culture clearly form fragmented basal lamina even
at 4 –7 days in culture, although TGFb1-nontreated cells do
not organize basal lamina before 10 days in culture, as described herein and previously (5– 6). These results suggest
that TGFb1 accelerates ECM production in thyrocytes. Further studies are needed to clarify in more detail what kinds
of ECM components TGFb1-treated cells can produce and
what role each of the ECM molecules produced in the microenvironment plays in the biological behavior of the cells.
TSH is a main differentiating factor for thyrocytes. However, it has not yet been clearly decided whether thyroid
folliculogenesis is TSH independent (54, 55) or TSH dependent (56 57). In collagen gel culture of TGFb1-nontreated
thyrocytes with serum-free medium, we have shown in this
study that the cells can reconstruct follicle structures in TSHfree medium as well as in TSH-added medium. Furthermore,
our present and previous studies have demonstrated that the
growth of reorganized follicles is less extensive in serum-free
medium than in 10% FCS-added medium (5– 6). These results
suggest the following conclusions. 1) Thyroid folliculogenesis itself may be essentially TSH independent, at least in
collagen gel culture, although TSH receptor activity of the
cells in this culture system remains to be elucidated. 2) Many
known or unknown serum-containing growth factors may
cooperatively play crucial roles in growth of the follicles, as
suggested by Dumont et al. (58). Finally, our present study
EFFECTS OF TGFb1 ON THYROCYTES
5574
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FIG. 12. Effects of TGFb1 on proliferation of thyrocytes at 3 and 7 days in
culture. Nuclear BrdU intake (arrowhead) in TGFb1-treated (B) and -nontreated cells (A) is clearly detected. The
BrdU intakes of the cells in all conditions have no statistical significance between 3 and 7 days in culture. In
TGFb1-nontreated cultures, rates of
BrdU intake in the cells with and without TSH are about 7% and 13%, respectively (P , 0.02). In TGFb1-treated cultures, the rates in the cells with and
without TSH are about 4% and 5%, respectively, and there is no statistical
significance between the two conditions. There is statistical significance
between TGFb1-treated cells with or
without TSH and TGFb1-nontreated
cells without TSH (P , 0.01), although
there is no significance between
TGFb1-treated cells with or without
TSH and TGFb1-nontreated cells with
TSH. *, Gel.
also disclosed that TSH decreases the expression of cytokeratin and vimentin in TGFb1-nontreated thyrocytes. In our
current study, their significance remains to be elucidated,
and further studies are in order.
In conclusion, we have shown in collagen gel culture of
isolated single thyrocytes that TGFb1 induces a mesenchyme-like cell shape without epithelial polarization in the
cells and that it results in inhibiting both differentiation of the
cells and thyroid folliculogenesis in a TSH-independent
manner. This suggests that TGFb1 is a potent morphological
regulator for the cells and may be involved in development
and morphogenesis of the thyroid. Further studies using this
culture method will probably provide new clues to the mechanism of thyroid folliculogenesis that closely involves the
proliferation and differentiation of thyrocytes.
Acknowledgments
We thank Messrs. H. Ideguchi, S. Nakahara, F. Mutoh, K. Tomoda,
and S. Takuma for technical assistance, and Messrs. T. Tanamachi and
Y. Tateishi for photography. We also thank Prof. H. Kimura, Dr. T.
Hashiguchi, and Prof. R. Gärtner for their helpful support, and Prof. K.
Tohkaichi for helping to edit the English of the manuscript.
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