RhoA Activation Promotes Transformation and Loss of Thyroid Cell Differentiation Interfering with Thyroid Transcription Factor-1 Activity
Diego L. Medina1,
Marcos Rivas1,
Patricia Cruz,
Isabel Barroso,
Javier Regadera and
Pilar Santisteban
Instituto de Investigaciones Biomédicas "Alberto Sols"
(D.L.M., M.R., P.C., I.B., P.S.), Consejo Superior de Investigaciones
Científicas, Universidad Autónoma de Madrid, and the
Departamento de Morfología (J.R.), Facultad de Medicina,
Universidad Autónoma de Madrid, E-28029 Madrid, Spain
Address all correspondence and requests for reprints to: Dr. Pilar Santisteban, Instituto de Investigaciones Biomédicas, CSIC/UAM, Arturo Duperier, 4, E-28029 Madrid, Spain. E-mail:
psantisteban{at}iib.uam.es
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ABSTRACT
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Highly specialized cells, the thyrocytes, express a
thyroid-specific set of genes for thyroglobulin (Tg), thyroperoxidase,
and the transcription factors TTF-1, TTF-2, and Pax-8. The implication
of the small GTPase RhoA in TSH-mediated proliferation of FRTL-5 rat
thyroid cells has been previously demonstrated. To further analyze RhoA
function in thyroid cell proliferation and differentiation patterns, we
combined transient and stable transfection assays to express different
mutant RhoA forms in FRTL-5 cells. Constitutively active RhoA
(FRTL-5-RhoA QL cells) exhibited a fibroblast-like phenotype with
organized actin fibers, whereas cells expressing the RhoA negative
dominant phenotype (FRTL-5-RhoA N19 cells) present a rounded morphology
and lose normal cytoskeletal architecture. In addition, expression of
the constitutively active form of RhoA results in TSH-independent
proliferation and anchorage-independent growth and induces tumors when
inoculated in nude mice. Interestingly, FRTL-5-RhoA QL cells express
less Tg and TTF-1 than wild-type FRTL-5 (FRTL-5- vector) or
FRTL-5-RhoA N19, suggesting a loss at the differentiation stage. This
effect is mediated, at least in part, by a decrease in TTF-1 activity,
since transient or stable expression of RhoA QL results in a reduction
in the activity of the wild-type Tg promoter as well as an artificial
promoter the activation of which depends exclusively on TTF-1. The
similarity between RhoA effects and thyroid transformation by Ras
suggests that RhoA may act as a downstream effector of Ras; in fact,
the dominant negative RhoA N19 abolished the down- regulatory
effect of Ras V12 over the Tg promoter. Taken together, these results
show for the first time that active RhoA is able to transform FRTL-5
cells and that this effect is coupled to a loss of thyroid
differentiation due to impaired TTF-1 activity.
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INTRODUCTION
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THE RAS PROTEINS are a group of guanine
nucleotide-binding proteins that function as molecular switches in many
cellular signaling pathways, interacting with a wide spectrum of
regulators and downstream effectors; they produce a broad range of
cellular responses such as proliferation, differentiation, or apoptosis
(1, 2). The expression of transforming Ras oncogenes
interacts with the establishment and maintenance of cellular
differentiation in different tissues (3, 4, 5, 6), including the
thyroid. In this tissue, Ras inhibits the expression of
thyroid-specific genes and confers a proliferative advantage over
normal thyroid cells (7, 8, 9, 10, 11). Ras transformation in the
specialized epithelial thyroid cell line FRTL-5 suppresses the
expression of thyroid differentiation markers such as thyroglobulin
(Tg), thyroperoxidase (TPO), and iodine uptake (12). In
parallel, the transcription factors controlling thyroid gene
expression, such as TTF-1, TTF-2, or Pax-8, are either not present or
inactive (10, 13, 14). In K-ras-transformed thyroid cells
(FRTL-5-K-ras), both TTF-1 and Pax-8 mRNA are undetectable, whereas in
H-ras (FRTL-5-H-ras), TTF-1 is present at normal levels and maintains
its DNA binding properties, although the cells lack the ability to
express Tg and TPO (11). Several proteins have been
identified as potential effectors of Ras signaling, including Raf/MAPK
kinase/ERK, RalGDS, and PI3K, although the mechanism of
Ras-mediated inhibition of thyroid cell differentiation remains
essentially unclear.
Another Ras protein family that includes RhoA, Rac1, and Cdc42 plays a
pivotal role in controlling many cellular functions, such as
cytokinesis, motility, proliferation, and apoptosis (15).
These three proteins cooperate with Raf in cell transformation, and the
dominant negative forms of RhoA and Rac1 can inhibit Ras-induced
transformation, indicating an essential function in this process
(16, 17, 18, 19, 20). Moreover, Rac1 and RhoA have been implicated in
the morphogenic and mitogenic responses to transformation by oncogenic
Ras (21, 22). In the context of thyroid cells, the
positive effects of RhoA in thyroid cell proliferation have been
related to its role in p27Kip1 degradation
(23). Among other functions,
p27Kip1 has been implicated in
G1 arrest induced by inhibitors of
3-hydroxy-3-methylglutaryl-coenzyme-A reductase (23).
These inhibitors interfere with cell cycle progression by suppressing
the isoprenylation of proteins (24), and RhoA is a class
of isoprenylated small GTPases proposed to be involved in
G1/S transition in FRTL-5 cells (23, 24). We recently confirmed these results and demonstrated that
overexpression of either the dominant negative form RhoA N19 or the
specific inhibitor of RhoA activity, the exoenzyme C3, thus inhibits
FRTL-5 cell proliferation, causing G1 arrest
(25).
In the present study, we combined transient and stable expression of
different mutant RhoA forms as tools to study the role of this protein
in the differentiation of FRTL-5 thyroid cells. We found that
activation of RhoA induces TSH-independent proliferation, morphological
transformation, anchorage-independent growth, and tumorigenesis when
cells are injected into nude mice. These effects could be mediated by
the activation of c-fos and c-jun, the expression
of which is probably central in cell proliferation control and is also
necessary for neoplastic transformation by a variety of oncogenes
(26, 27). In addition, RhoA activation results in a less
differentiated thyroid phenotype, decreasing Tg and TTF-1 gene
expression by blocking transactivation of the Tg promoter through
inhibition of TTF-1 transcriptional activity. These results demonstrate
for the first time that RhoA induces transformation of FRTL-5 thyroid
cells.
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RESULTS
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Generation and Analysis of Stable FRTL-5 Cells Expressing Different
Mutant Forms of RhoA Protein
The rat thyroid follicular cell line FRTL-5 provides a useful
model with which to study growth and differentiation of specialized
epithelial cells. To analyze the role of RhoA protein in the
differentiation pattern of thyroid cells, FRTL-5 cells were stably
transfected with either an AU5-tagged dominant positive form of RhoA
(RhoA QL) (28), an AU5-tagged dominant negative form of
RhoA (RhoA N19) (29), or with AU5-RhoAQL together with an
expression vector for C3 toxin which ribosylates and inactivates RhoA
(28, 30). Transfected FRTL-5 clones were tested for
exogenous RhoA expression by immunoblotting analysis of total cell
extracts using anti-AU5 or -RhoA antibodies. Three representative
neomycin-resistant clones referred as FRTL-5-RhoA QL, RhoA N19, and
RhoA QL-C3 were selected for further study (Fig. 1A
). As has been described extensively,
the small GTP-binding protein RhoA regulates the assembly of focal
adhesion and actin stress fibers in response to growth factors
(15). We thus analyzed the possible morphological changes
due to overexpression of the different RhoA mutant forms. Phase
contrast photomicroscopy of early passage FRTL-5 clones indicated that,
in the presence of 6H complete medium, FRTL-5-RhoA QL cells present a
fibroblast-like appearance compared with neomycin-resistant FRTL-5
cells carrying control vector (FRTL-5-vector) (Fig. 1B
). In addition,
the cellular limits within the colonies are clearly distinguished.
Conversely, FRTL-5-RhoA N19 cells present a rounded shape and diffuse
cell-to-cell limits within colonies. Interestingly, no fibroblast-like
morphology was observed when the RhoA inhibitor, the exoenzyme C3, was
expressed together with RhoA QL. In fact, these cells show a rounded
morphology similar to those expressing the dominant negative form N19
(Fig. 1B
).

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Figure 1. Expression of RhoA Mutants in Thyroid Follicular
Cells
A, FRTL-5 cells were stably transfected with vector alone or harboring
the AU5-tagged-RhoA QL, AU5-tagged RhoA N19, or exoenzyme C3 plus the
AU5-tagged RhoA QL. Neomycin-resistant colonies were isolated and
analyzed by immunoblotting, using anti-AU5 or anti-RhoA antibodies. A
30-kDa band corresponding to exogenous RhoA protein was detected in
several independent clones. FRTL-5 positive clones studied
(vector, RhoA QL, RhoA N19, and RhoA QL-C3) were selected for
further studies. B, General morphology of the above clones obtained by
phase contrast photomicroscopy, of early-passage cells, 2 d after
plating. Magnification, x100. C, Cells were stained with fluorescein
isothiocyanate-phalloidin to visualize the actin cytoskeleton.
Magnification, x100.
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We support these results by immunofluorescence using
fluorescein-conjugated phalloidin to visualize the actin cytoskeleton.
FRTL-5-RhoA QL cells present a more protrusive appearance compared with
FRTL-5-vector cells, whereas FRTL-5-RhoA N19 and RhoA QL-C3 cells show
a redistribution of the actin fibers, lose their normal cytoskeleton
organization and round up (Fig. 1C
).
RhoA Activation Induces a Proliferative Advantage and
TSH-Independent Growth
In addition to the morphological changes induced by RhoA, results
from several laboratories demonstrate that this protein is involved in
TSH-mediated thyroid cell proliferation (23, 24, 25).
Inhibition of RhoA activity by the C3 exoenzyme or by transient
expression of dominant negative RhoA N19 thus decreases FRTL-5 cell
proliferation, causing G1 arrest in the cell
cycle. To confirm the role of RhoA in proliferation, growth curves were
performed in stably transfected cells cultured alone or in the presence
of TSH (see Materials and Methods).
In the presence of TSH (6H medium), overexpression of constitutively
active RhoA (FRTL-5-RhoA QL cells) induces a proliferative advantage
compared with controls (FRTL-5-vector cells), whereas expression of the
dominant negative mutant form of RhoA (FRTL-5-RhoA N19 cells) decreases
cell proliferation (Fig. 2B
).
Interestingly, FRTL-5-RhoA QL cells grow in the absence of TSH with a
growth rate similar to that seen in the presence of the hormone, while
the FRTL-5-vector or -RhoA N19 cells barely grow in the absence of TSH
(Fig. 2A
). These data confirm that RhoA activation induces
TSH-independent growth in FRTL-5 cells. Cytometric analysis of
FRTL-5-RhoA N19 clones demonstrated a decrease in the S phase of the
cell cycle as a consequence of G1 arrest (not
shown), confirming previous results (25).

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Figure 2. Proliferative Activity of Thyroid Follicular Cells
Carrying Constitutively Active RhoA QL or Inactive RhoA N19
FRTL-5 clones were maintained in the absence of TSH (5H medium) for
3 d. From then on, cells were cultured either in the same medium
(panel A) or in a medium with TSH (6H medium) (panel B).
Cell number was monitored every 3 d for 12 consecutive days, and
viable cell number is represented. The data are the mean ±
SD of three independent experiments.
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RhoA Activation Induces Transformation in Vitro and
Increases the Tumorigenicity of FRTL-5 Thyroid Cells
FRTL-5 cells are considered a normal nontumorigenic cell line,
although contradictory results are found throughout the bibliography
(12, 31). The tumorigenicity of FRTL-5 cells is thus
conditioned by clonal variability, TSH levels in nude mice, and cell
passage number (12, 31, 32). Rho proteins have
transforming and oncogenic potential in a cell type-specific manner
(16, 17, 18, 19, 20). Cells expressing constitutively active mutants
of Rac and RhoA display enhanced growth in low serum, are anchorage
independent, and induce tumor formation when inoculated into nude mice
(20). In addition, Rac and Rho proteins are essential for
transformation by Ras (16, 18).
To assess RhoA-dependent FRTL-5 transformation, the ability of each
cell line was tested to form colonies in semisolid medium in the
presence or absence of TSH; only cells expressing the active mutant
form of RhoA (RhoA QL) were able to grow in soft agar (Table 1
). The number of colonies was similar in
the absence or presence of TSH, again demonstrating the TSH-independent
growth of cells expressing RhoA QL.
To test the role of RhoA protein in FRTL-5 tumorigenicity, we
inoculated FRTL-5-RhoA QL and FRTL-5-RhoA N19 cells, as well as
FRTL-5-vector cells, into nude mice (2 x
106 cells). Four weeks later, we observed tumors
in the FRTL-5-RhoA QL cell-inoculated group, whereas the groups
inoculated with FRTL-5-vector cells or FRTL-5-RhoA N19 cells remained
normal (Table 1
). These results indicate that RhoA induces FRTL-5 cell
transformation and tumorigenesis. Excised tumors were analyzed
histologically as described in Materials and Methods.
FRTL-5-RhoA QL-inoculated mice developed large tumors located in the
dermis (Fig. 3A
), whereas control or
FRTL-5-RhoA N19-inoculated mice remained normal. Tumors were highly
undifferentiated and infiltrative. Tumor cells were poorly
differentiated, showing small cytoplasm and large nuclei with abundant
mitosis (Fig. 3B
). They grew in a solid, diffuse pattern, with neither
follicles nor papillary structures. Infiltration was observed mainly as
satellite tumor nodules (Fig. 3C
). Bromodeoxyuridine (BrdU)
immunostaining showed that proliferative cells were located mainly in
peripheral regions of the tumor (Fig. 3D
); satellite tumor nodules were
highly proliferative, mainly in the peripheral area (Fig. 3E
). Detailed
necropsy of the mice revealed no other relevant effects.

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Figure 3. Histological Analysis of Tumors from FRTL-5-RhoA
QL-Inoculated Cells into Nude Mice
A, General view of hematoxylin-eosin-stained large nodular tumor
derived from FRTL-5-RhoA QL-inoculated cells (8x magnification). The
tumor is well defined, unencapsulated, and localized in the dermis. It
presents a solid growth pattern with moderate blood vessel
proliferation. B, Hematoxylin-eosin staining of the peripheral area of
an undifferentiated, solid tumor derived from FRTL-5-RhoA QL cells,
with anaplastic cells with small cytoplasm (125x magnification). C,
Anti-BrdU-hematoxylin staining of the peripheral area of the previous
figure showing abundant highly stained, BrdU- positive cells
(intense brown color) (125x magnification). D,
Anti-BrdU-hematoxylin staining of an infiltrative, satellite nodule
formed in the peripheral area of an undifferentiated tumor. The
arrow indicates the augmented area shown in Fig. 3E
(50x magnification). E, Anti-BrdU-hematoxylin staining of the
peripheral area of the infiltrative nodule shown in Fig. 3D . Note that
most of the nuclei are highly stained with BrdU (300x
magnification).
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Molecular mechanisms leading to transformation and tumorigenicity are
complex and involve the activation of different signaling pathways, as
well as the expression of a set of genes the induction of which may
play a role regulating these processes. In an attempt to understand
some of the mechanisms responsible for FRTL-5 RhoA QL transformation,
we analyzed the levels of cAMP as the main signaling pathway
controlling thyroid cell growth (33) and because most
thyroid tumors present increased levels of this second messenger
(34). The results obtained show similar levels of cAMP in
FRTL-5 vector (370 ± 25 fmol/well), RhoA QL (374 ± 20
fmol/well), and RhoA N19 (365 ± 19 fmol/well) cells maintained in
6H medium, suggesting that the upstream effector cAMP is not
responsible for either growth independence or cell transformation
induced by RhoA activation. To identify downstream genes that could
explain the above phenomena, we analyzed the levels of early response
genes such as c-fos and c-jun. The election of
these genes was based on previous reports demonstrating their
involvement in RhoA signaling (35, 36). Western blot
assays using nuclear protein extracts detect increased levels of c-Fos
and c-Jun in FRTL-5 cells expressing the constitutively active form of
RhoA (Fig. 4
); the active phosphorylated
form of c-Jun (P-Ser 63-c-Jun) is also increased after RhoA QL
overexpression. These data suggest that the activation of these
oncogenes is involved in RhoA transformation process in FRTL-5
cells.

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Figure 4. c-Fos and c-Jun Expression in Normal and RhoA
Expressing FRTL-5 Cells
Nuclear protein extracts (20 µg) from vector, RhoA QL, or RhoA N19
FRTL-5 clones were analyzed by Western blotting by use of anti-c-Fos,
anti-c-Jun, and anti-P-c-Jun antibodies. Anti-Sp1 antibody was used as
a loading control.
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Activation of RhoA Is Sufficient to Affect Thyroid Cell
Differentiation
Cell transformation is usually coupled to a loss of differentiated
phenotype. Transformation of FRTL-5 cells with two oncogenic Ras forms,
Harvey (FRTL-5-H-Ras) and Kirnstein Ras (FRTL-5-K-Ras), has been
studied extensively. The work of several groups has showed that Ras
transformation of FRTL-5 cells results in loss of iodine uptake, as
well as in Tg and TPO gene expression (7, 8, 9, 10, 11). To study
further the effect of RhoA activation in FRTL-5 cells, we measured
expression of thyroid-specific mRNA for one of the critical
thyroid-specific markers, Tg, and for its transcription factor TTF-1,
in FRTL-5 clones carrying the constitutively active RhoA QL or the
dominant negative RhoA N19. Tg and TTF-1 mRNA levels were not
significantly affected by inactivation of RhoA protein (FRTL-5-RhoA N19
cells), whereas activation of RhoA (FRTL-5-RhoA QL cells) clearly
decreased both Tg and TTF-1 mRNA levels (Fig. 5A
). To test whether the RhoA-mediated
decrease in Tg and TTF-1 mRNA levels correlates with a loss of Tg and
TTF-1 protein levels, we performed Western blot assays using total or
nuclear protein extracts to detect Tg or TTF-1 protein levels,
respectively (Fig. 5B
). Total protein extracts from FRTL-5-RhoA QL
cells express less Tg than FRTL-5-vector cells or FRTL-5-RhoA N19
cells. Western blot assays using nuclear extracts from FRTL-5 cell
clones showed a decrease in TTF-1 protein levels in FRTL-5-RhoA QL
cells (Fig. 5B
). Figure 5
shows representative Northern and Western
blots. Similar results were obtained in all stable clones
generated.

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Figure 5. Tg and TTF-1 Levels in Normal and RhoA-Expressing
FRTL-5 Cells
A, Total RNA (30 µg) from stable FRTL-5 clones were blotted onto
nytran membranes and sequentially hybridized with specific cDNA probes
indicated at the right of the panels. As a loading
control, membranes were hybridized with an actin-labeled probe. B,
Total protein extracts (30 µg) from stable FRTL-5 clones were blotted
onto protran membranes and incubated with specific anti-Tg antibody.
Antiactin antibody was used as a loading control. Nuclear protein
extracts (20 µg) from different FRTL-5 clones were immunobloted with
anti-TTF-1 antibody. Anti-Sp1 antibody was used as a loading control.
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Transient Active RhoA Expression Represses the Activity of the Tg
Promoter
Several studies have implicated the protein RhoA in the
morphogenic and mitogenic responses to transformation by oncogenic Ras
(16, 18). In addition, previous studies of Ras-mediated
transformation have shown its negative effect in the activity of
thyroid-specific promoters (11, 37, 38). Transient
expression of Ras V12 in FRTL-5 cells thus decreases the activity of
TPO, Tg, and sodium iodide symporter gene promoter
(38). Our results show that RhoA activation decreases Tg
and TTF-1 mRNA levels, suggesting transcriptional inhibition of thyroid
differentiation. To test this hypothesis, FRTL-5 cells were transiently
transfected with the Tg promoter (TACAT-3) (39, 40), along
with expression vectors encoding for the different RhoA mutant forms or
for C3. Plasmid cytomegalovirus-luciferase (CMV-Luc) was transfected to
correct for transfection efficiency. Cells were lysed 72 h after
transfection, and chloramphenicol acetyltransferase (CAT) and
luciferase activity were measured. Expression of the dominant positive
RhoA QL resulted in approximately 75% inhibition of Tg promoter
activity. The effect was due to RhoA activation, as it was abolished
when cotransfected with its negative dominant form RhoA N19 or with the
exoenzyme C3 (Fig. 6A
). As described by
Alberts et al. (41), we confirmed activity of
transfected RhoA mutants by testing the activity of a construct
containing wild-type serum response elements (not shown).

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Figure 6. Effects of RhoA Expression on Tg Promoter Activity
FRTL-5 cells were transiently transfected with a construct containing
the wild-type Tg promoter (TACAT-3) (panel A), or a
construct containing five binding sites for TTF-1 (5C-CAT) (panel B),
and with vectors encoding the different mutant RhoA forms or the C3
exoenzyme. Plasmid CMV-Luc was transfected to correct for transfection
efficiency. At 72 h after transfection, cells were lysed, and CAT
and luciferase activities were measured. Promoter activity is
calculated as arbitrary units relative to the cells transfected with
vector alone (=100). The data are the mean ±
SD of three independent experiments.
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Tg promoter (TACAT-3) contains three binding sites for the
homeodomain-containing protein TTF-1, which has been reported as its
main transcriptional activator, although it is also regulated by other
transcription factors (13, 39, 40). To verify that TTF-1
is involved in the RhoA-induced reduction of the Tg promoter activity,
we used a 5C-CAT reporter gene containing an artificial promoter
carrying five binding sites for TTF-1 placed in tandem upstream of the
TATA box (42). This promoter is regulated exclusively by
TTF-1, showing strong activity in FRTL-5 thyroid cells. In transient
transfection assays, 5C-CAT transcription was suppressed by RhoA QL
expression to an extent similar to that of the wild-type (TACAT-3) Tg
promoter. Again, RhoA N19 or C3 expression reverts RhoA QL inhibition
of 5C-CAT promoter (Fig. 6B
). As a control for the specificity of the
transfected plasmids on the Tg promoter, we used the promoterless
TATA-CAT vector (14), the activity of which remained
unaffected (not shown).
Due to the parallelism observed in the inhibition of TTF-1
transcriptional activity mediated by Ras (11, 13, 38) and
RhoA (present study), we asked whether the dominant negative form of
RhoA, the N19 mutant, is able to revert Ras-induced Tg promoter
repression. FRTL-5 cells were transiently transfected, as described
above, with the wild-type Tg promoter (TACAT-3), along with
expression vectors encoding Ras V12, Ras N17, and RhoA N19. Expression
of Ras V12 resulted in 8090% inhibition of the Tg promoter activity,
and its dominant negative form Ras N17 reverted the effect. The
dominant negative form RhoA N19 also reverted the inhibitory effect of
Ras V12 on the Tg promoter (Fig. 7
).
These results clearly demonstrate that the Ras effect on the Tg
promoter is RhoA dependent and suggest that RhoA is downstream of Ras
in the control of thyroid differentiated phenotype.

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Figure 7. RhoA N19 Reverts Ras-Mediated Inhibition of Tg
Promoter Activity
FRTL-5 cells were transiently transfected with a construct containing
the wild-type Tg promoter (TACAT-3) and with expression vectors for Ras
V12 alone or together with Ras N17 or RhoA N19. Plasmid CMV-Luc was
transfected to correct for transfection efficiency. At 72 h after
transfection, cells were lysed, and CAT and luciferase activities were
measured. Promoter activity is calculated as arbitrary units relative
to the cells transfected with vector alone (=100). The data are the
mean ± SD of three independent experiments.
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Stable RhoA QL Expression Represses TTF-1 Transcriptional Activity
in FRTL-5 Cells
To confirm the RhoA-mediated decrease in TTF-1 transcriptional
activity in FRTL-5 cells, we performed similar assays using FRTL-5 cell
clones stably expressing RhoA N19 or RhoA QL. The cells were
transfected with 5C-CAT and CMV-Luc; after 72 h cells were lysed
and CAT and luciferase activities measured. Expression of the
constitutively active RhoA QL clearly inhibits 5C-CAT reporter gene
activation, whereas stable expression of the dominant negative RhoA N19
form had no effect on the TTF-1 transcriptional activity (Fig. 8A
). As a control, we tested 5C-CAT
reporter gene activity in two stable FRTL-5 cell lines expressing the
oncogenic Ras forms, H-ras and K-ras (12). As extensively
described (13, 14), Ras transformation inhibits TTF-1
transcriptional activity in these cell lines (Fig. 8A
).

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Figure 8. Effect of Stable RhoA Expression on TTF-1
Transcriptional Activity
A, FRTL-5 cells expressing the different mutant RhoA forms were
transiently transfected with the 5C-CAT construct containing five
specific binding sites for TTF-1. Plasmid CMV-Luc was transfected to
correct for transfection efficiency. As a control, we transfected the
5C-CAT construct in FRTL-5 cells stably expressing K-ras and H-ras. B,
FRTL-5 cells expressing the different mutant RhoA forms were
transiently transfected with 5C-CAT, CMV-Luc, and a TTF-1 expression
vector. As a control, we transfected FRTL-5-H-ras. In both panels (A
and B), cells were lysed at 72 h after transfection, and CAT and
luciferase activities were measured. The promoter activity is
calculated as arbitrary units relative to the cells transfected with
vector alone (=100). The data are the mean ± SD of
three independent experiments.
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TTF-1 transcriptional activity is regulated mainly by posttranslational
modifications such as site-specific phosphorylation and redox
mechanisms (43, 44, 45). In Ras-transformed FRTL-5 cells
(FRTL-5-H-ras), Tg expression is absent due to expression of an
inactive form of TTF-1 (11, 14). To understand the
mechanism of RhoA-mediated dedifferentiation of thyroid cells, we
determined whether RhoA activation affects TTF-1 transcriptional
activity. The 5C-CAT reporter was transiently transfected with a vector
harboring the TTF-1 cDNA into stable FRTL-5 cell clones expressing
either RhoA QL or RhoA N19. 5C-CAT activity was not affected either in
FRTL-5-vector cells or in FRTL-5 N19 cells when TTF-1 was overexpressed
(Fig. 8B
). This may be due to the existence of saturated levels of
endogenous active TTF-1, as previously reported in FRTL-5 cells
(14, 43). Interestingly, overexpression of this
transcription factor in FRTL-5-RhoA QL had no effect in reverting
RhoA-mediated inhibition of TTF-1 transcriptional activity, supporting
the idea that RhoA may decrease Tg expression through some
posttranslational modifications of TTF-1 protein (Fig. 8B
). A similar
phenomenon has been described in FRTL-5-H-ras cells overexpressing
TTF-1 (11, 14).
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DISCUSSION
|
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We show that constitutively active RhoA QL transforms FRTL-5
thyroid cells, rendering them more proliferative, tumorigenic, and
undifferentiated. As has been previously demonstrated, RhoA is required
for TSH-mediated thyroid cell proliferation (23, 24).
Transient expression of dominant negative RhoA N19 or the inhibition of
RhoA activity by choleric exoenzyme C3 decreases thyroid cell
proliferation as a consequence of G1 arrest
(25). Here we present evidence that stable expression of
the dominant positive form RhoA QL induces a proliferative advantage in
FRTL-5 thyroid cells. Conversely, expression of the dominant negative
RhoA N19 decreases FRTL-5 thyroid cell proliferation.
The Rho protein subfamily, including RhoA, Rac1, and Cdc42, are
involved in cell shape regulation and actin filament assembly
(46, 47). Lovastatin-induced inactivation of these
proteins causes cell rounding and actin filament disassembly
(48). In several cell types, spread cellular morphology
allowed DNA synthesis, whereas rounded cells did not proliferate or
undergo apoptosis (49). The analysis of FRTL-5-RhoA QL
clones with phalloidin shows that these cells spread in a
fibroblast-like fashion and display actin stress fibers. This
morphology disappears and becomes rounded, with a distinct actin
distribution, in clones generated after transfection of expression
vectors for C3 exoenzyme and RhoA QL, demonstrating that the changes
observed in the cell phenotype are due to the genes transfected and not
to an indirect action of the transgene used. FRTL-5-RhoA N19 cells also
presented a rounded cell shape and diffuse cell-to-cell limits. This
altered cell morphology was followed by a decrease in FRTL-5-RhoA N19
cell proliferation. In this regard, Zhu et al.
(50) reported that for cell cycle progression to occur,
the actin cytoskeleton must be assembled. These clones could thus be
useful to study in greater detail the links between RhoA-mediated
cytoskeletal rearrangement and its positive effects in thyroid cell
proliferation.
RhoA activation has transforming and oncogenic potential in some cell
lines (21, 22). Fully differentiated FRTL-5 thyroid cells
are considered nontumorigenic, although they can be rendered so in a
single transformation step by expression of retroviral oncogenes such
as H-ras or K-ras (12). As we show here, RhoA QL induces
in vitro transformation of FRTL-5 cells, assessed by
anchorage- and TSH-independent growth and tumors when inoculated into
nude mice. Excised tumors revealed highly proliferative infiltrated
carcinomas, suggesting that RhoA induces FRTL-5 thyroid transformation
in vivo. We show that RhoA transformation of FRTL-5 cells is
not due to increased cAMP levels, as has been reported in most thyroid
tumors (34). The expression of c-Fos and c-Jun is
increased in FRTL-5-RhoA QL cells, suggesting that these two oncogenes
may be involved in the cell transformation described here. As has
previously been reported, the expression of these two proteins is
likely to play a role in the control of cell proliferation (26, 51), as they are necessary for cell cycle progression in several
cellular systems and for neoplastic transformation by a variety of
oncogenes (27). Interestingly, phospho-c-Jun levels are
also increased in FRTL-5-RhoA QL transformed cells. These results open
new questions about whether RhoA activation increases JNK
activity in FRTL-5 cells as has been demonstrated in other systems
(28). All together, these data concur with the previous
observation of the regulation of early gene expression and cellular
transformation elicited by RhoA.
A general effect in thyroid cell transformation is the loss of cell
differentiation. Oncogenic Ras thus suppresses thyroid differentiation
marker expression, and Ras-transformed cells do not express Tg or TPO,
do not respond to TSH, and do not take up iodine (12). In
parallel, the transcription factors controlling the expression of
thyroid-specific genes are either not present or inactive (10, 11, 13, 14). In K-ras-transformed thyroid cells, both Pax-8 and
TTF-1 mRNA are undetectable, whereas in H-ras-transformed cells, TTF-1
is present at normal levels and maintains its binding capacity,
although the cells lack the ability to express Tg and TPO (11, 14). Our results show that expression of active RhoA decreases
Tg mRNA levels, whereas expression of dominant negative RhoA N19 shows
Tg mRNA levels comparable to those of controls (FRTL-5 cells). RhoA
expression also decreases Tg protein levels, confirming that RhoA
modifies the thyroid phenotype affecting Tg expression. The
RhoA-mediated decrease in Tg gene expression may be due to a lack of
activity of TTF-1, a specific transcription factor involved in Tg gene
activation. Our results clearly demonstrate that active RhoA decreases
TTF-1 expression when compared with FRTL-5-RhoA N19 and FRTL-5-vector
cells. Moreover, RhoA activation decreases TTF-1 protein levels in the
nucleus, where this factor activates thyroid-specific promoters. The
fact that overexpression of exogenous TTF-1 in stable FRTL-5-RhoA QL
cells has no effect on Tg promoter construct activity, together with
the observation that RhoA QL inhibited the effect of TTF-1 in transient
transfection experiments, suggests that RhoA QL affects TTF-1 involving
a posttranslational mechanism. This may account not only for the
reduction in Tg protein levels, but also for the reduction in TTF-1, as
this transcription factor is autoregulated (52).
Mechanisms involved in modulating the transcriptional potential of
TTF-1 include phosphorylation (43), control of the redox
state (44, 45), and interaction with other factors
(53). Ras repression of the Tg promoter involves changes
in TTF-1 phosphorylation (14). The fact that RhoA N19
reverts the effect of Ras V12 on the wild-type Tg promoter suggests
that RhoA is a downstream effector of Ras in this process. Thus, it
would be of interest to test the ability of RhoA to modify the
phosphorylation state of TTF-1. Interestingly, the protein kinases PKN,
MKK3/6, and ERK6 have been proposed as components of a novel signal
transduction pathway involved in the regulation of gene expression and
cellular transformation elicited by RhoA (36). TTF-1
contains several minimal consensus sequences for ERK phosphorylation
and has been reported to be phosphorylated by ERK2 (38).
Although ERK2 does not mediate the RhoA effect on NIH-3T3 fibroblasts
(36), the role of this family of kinases in response to
RhoA and its effect in TTF-1 phosphorylation could be addressed in
FRTL-5 cells. However, we cannot rule out the possibility that TTF-1
may be phosphorylated by other RhoA effectors.
The new role for RhoA protein in FRTL-5 thyroid cells increases the
complexity of the signal transduction pathways implicated in the
control of thyroid cell proliferation and function. FRTL-5 thyroid
cells depend mainly on TSH for proliferation (33, 54, 55);
this hormone stimulates thyroid cell proliferation through both
PKA-dependent and -independent pathways (25, 56). After
TSH stimulation, downstream effectors such as Akt (57),
Rac1 (38), or RhoA (23, 24, 25) are involved in
thyroid cell growth and function. Activation of Akt, Rac1, or RhoA
increases thyroid cell proliferation, although Akt or Rac1 expression
has no effect on the differentiation status of thyroid cells. Here we
suggest, for the first time, a role for RhoA in thyroid
differentiation. The similarity between Ras-mediated thyroid cell
transformation, together with our observation that RhoA N19 reverses
the effect of Ras V12 on the Tg promoter, suggests the existence of
cross-talk involving both proteins, as described previously for other
cell systems (58). Further studies are needed to explain
in detail the role of RhoA in Ras-mediated thyroid transformation.
 |
MATERIALS AND METHODS
|
---|
Cell Culture
Rat thyroid follicular FRTL-5 cells (ATCC CRL 8305;
American Type Culture Collection, Manassas, VA) were
kindly provided by Dr. L. D. Kohn (Edison Biotech Institute,
Athens, OH). The cells had the properties previously described
(59, 60), were diploid, and their doubling time with TSH
was 2436 h. Cells were maintained in Coons modified Hams F-12
medium (Sigma, St. Louis, MO) supplemented with 5% calf
serum (Life Technologies, Inc., Gaithersburg, MD) and
six-growth factor (6H complete medium), including TSH (0.5 mU/ml) and
insulin (10 µg/ml) (49). FRTL-5-K-ras and FRTL-5-H-ras
cells were maintained as previously described (11, 12).
Plasmids and Expression Constructs
TACAT-3 corresponds to the wild-type Tg promoter (39, 40). As a negative control, we used the TATA-CAT plasmid
construct corresponding to the wild-type Tg promoter minus the
Sal/I-NheI fragment (39), resulting
in only the Tg TATA box linked to the CAT gene. For detection of TTF-1
transcriptional activity, the C5E1b-CAT construct (Ref. 42 ; referred to
here as 5C-CAT) was transiently transfected into FRTL-5 cells. The
5C-CAT construct contains five tandem repeats of the C binding site for
TTF-1 from the Tg promoter and is exclusively dependent on TTF-1 for
transactivation (42). The CMV-Luc plasmid was used to
correct for transfection efficiency (39). For TTF-1
overexpression assays, we used a vector containing wild-type TTF-1
(41). The role of RhoA was analyzed with expression
vectors encoding the dominant positive AU5-tagged-RhoA QL
(28), the dominant negative AU5-tagged-RhoA N19
(29), the dominant negative Ras N17 or positive Ras V12
(61), or the botulinum C3 exoenzyme (28).
Transfection Assays
FRTL-5 cells were stably transfected by the calcium phosphate
DNA precipitation method, as described (39, 11). Briefly,
calcium phosphate DNA precipitates were prepared with 10 µg of
plasmid DNA containing either constitutively active mutant AU5-tagged
RhoA (RhoA QL) (28) or the dominant negative form of
AU5-tagged RhoA (RhoA N19) (29). To abolish RhoA
activation, a stable cell line was generated transfecting together 5
µg of AU5-tagged RhoA QL and 5 µg of the C3 expression vector. In
all cases 1 µg of plasmid DNA containing the neomycin resistance gene
under the control of viral long terminal repeat promoter, and 40
µg of calf thymus genomic DNA as carrier (Roche Molecular Biochemicals) was also cotransfected. Cells were selected with
300 µg/ml G418 (Sigma). After 3 wk, G418-resistant
colonies were isolated and expanded.
For transient transfection assays, cells were plated at a density of
5 x 105/60 mm diameter tissue culture dish;
48 h later, TACAT-3, 5C-CAT, TATA-CAT (2.5 µg), or CMV-Luc (1
µg) reporter plasmids were transfected with the expression vectors as
indicated in the figure legends. After 72 h, cell extracts were
lysed in lysis buffer (10 mM HEPES, pH 7.9, 40
mM NaCl, 0.1 mM EGTA, 0.5 mM
dithiothreitol, 5% glycerol, and 0.5 mM
phenylmethylsulfonyl fluoride). Luciferase (Luc) and chloramphenicol
acetyltransferase (CAT) activity were measured as described (62, 63).
Immunoblotting and Immunofluorescence
Nuclear or total extracts were prepared in sample buffer, and
protein concentration was determined by the Bradford technique
(Bio-Rad Laboratories, Inc., Hercules, CA). Protein
samples were resolved in SDS-PAGE and transferred to Protran membranes
(Schleicher & Schuell, Inc., Keene, NH). Monoclonal
anti-AU5 antibody (0.5 µg/ml) used to detect AU5-tag was purchased
from Badco. Polyclonal anti-RhoA (1 µg/ml), anti-actin (1
µg/ml), anti-Sp1 (1 µg/ml), anti c-Fos (2 µg/ml), anti-c-Jun (1
µg/ml), and anti-P-Ser 63-c-Jun (2 µg/ml) antibodies were
purchased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). Anti-Tg antibody (0.5 µg/ml) was from DAKO Corp. (Carpinteria, CA), and anti-TTF-1 antibody (1 µg/ml)
from Biopat Immunotechnologies (Italy). Immune
complexes were detected with Luminol reagent as indicated by the
manufacturer (Santa Cruz Biotechnology, Inc.).
For immunofluorescence assays, FRTL-5-vector, FRTL-5-RhoA QL,
FRTL-5-RhoA N19, and FRTL-5-RhoA QL-C3 cells were fixed in 4%
formaldehyde for 30 min, and then permeabilized with 0.1% Triton X-100
in PBS. Samples were incubated with fluorescein
isothiocyanate-phalloidin (Sigma, 1:40 dilution in
PBS) for 1 h at 37 C. Cells were then washed twice with PBS and
mounted on microscope slides. Fluorescence was visualized in a
photomicroscope (Carl Zeiss, Thornwood, NY) equipped with
epifluorescence. Photographs were taken using Kodak 400
ASA film.
Growth Curve Profiles
To perform growth curves profiles, cells were seeded at a
confluence of 105/100 mm dish and maintained
3 d in the absence of TSH (5H medium). From then on, cells were
cultured either in medium with TSH, for the 6H curve, or maintained in
5H medium. Fresh medium (6H or 5H, respectively) was added every 3
d. The number of viable cells was determined by cell counting every
3 d for 12 consecutive days. The mean ± SD of
three independent experiments is represented.
Anchorage-Independent Growth
Approximately 9,000 cells were seeded in 60-mm petri dishes in
0.35% noble agar (Sigma) on a 0.5% agar underlayer.
Cells were tested to grow in soft agar containing 6H or 5H medium.
Plates were incubated for 3 wk, during which time fresh medium (6H or
5H, respectively) was added to the plates every 3 or 4 d. After
crystal violet staining, colonies larger than 50 µm diameter were
counted. The mean ± SD of three independent
experiments is represented.
Growth of Tumors in Nude Mice
Tumorigenicity was assayed by injecting 2 x
106 cells sc into nude mice, which were palpated
weekly for tumor development. After 4 wk, the animals were killed and
tumors excised for histological analysis under protocols approved by
the Human Research Committee (Vanderbilt University, Nashville, TN).
For histopathological analysis (64), tissues were fixed in
10% buffered formaldehyde for 48 h, embedded in paraffin, and cut
into 6-µm serial sections that were stained with hematoxylin-eosin
(Fig. 3
), Masons trichrome, and periodic acid Schiff methods
(not shown). BrdU staining was performed as follows: RhoA QL-inoculated
mice received 1% 5-bromo-2' deoxyuridine (Sigma) ip
2 h before being killed. Tumors were removed and sections prepared
as for hematoxylin-eosin staining. Before immunohistochemistry,
sections were deparaffined, hydrated, and washed with PBS. PBS was then
removed and endogenous peroxidase inhibited using 3% hydrogen
peroxidase (10 min, 37 C). Sections were blocked in goat serum
(Zymed Laboratories, Inc., South San Francisco, CA) and
incubated overnight with a mouse monoclonal anti-BrdU antibody
(Amersham Pharmacia Biotech, Piscataway, NJ). After
washing with PBS, sections were incubated with biotinylated goat
antimouse antibody (Biocell) in 20% human serum PBS buffer. After
washing, sections were incubated with streptavidin-biotin-peroxidase
complex (Zymed Laboratories, Inc.) and developed with
diaminobenzidine (Sigma). Sections were counterstained
with Harris hematoxylin, dehydrated in ethanol, and mounted in DePex
(Probus, Barcelona, Spain).
RNA Isolation and Analysis
Total RNA was isolated by the guanidinium-isothiocyanate-phenol
method (65). Total RNA samples were electrophoresed in 1%
agarose gels containing formaldehyde. RNA was transferred to Nytran
membranes (Schleicher & Schuell, Inc.), and RNA integrity
was verified by methylene blue staining of the blots. Hybridization and
washing were performed with probes specific for Tg (66),
TTF-1 (67), or ß-actin (68) labeled with
[
32P]-dCTP by random priming.
cAMP Assays
The Biotrak cAMP competitive enzyme immunoassay system
(Amersham Pharmacia Biotech) was used following
manufacturers instructions for the determination of intracellular
cAMP. Briefly, cells were cultured in 24-well plates
(105 cells per dish) and then lysed, moved to a
donkey antirabbit Ig-precoated microtiter plate and incubated with
anti-cAMP antiserum (2 h, 4 C). Samples were then incubated with a
cAMP-peroxidase-conjugated antibody (1 h, 4 C) and washed four times
with washing buffer. The enzyme substrate was added immediately
afterward to all wells and incubated (1 h, room temperature). Before
optical density determination in a plate reader at 450 nm, the reaction
was terminated by adding 0.1 M sulfuric acid to each well.
In parallel, a standard curve with cAMP concentrations from 12.53,200
fmol/well was prepared. Each value represents the mean ±
SD of three independent experiments. As control for assay
validation, FRTL-5 cells maintained in 5H and 6H medium were used,
being the cAMP levels 50 ± 7 fmol/well and 390 ± 30
fmol/well, respectively.
 |
ACKNOWLEDGMENTS
|
---|
We are indebted to Dr. Leonard D. Kohn (Edison Biotech
Institute, Athens, OH) for FRTL-5 cells, to Dr. Silvio Gutkind
(National Cancer Institute, NIH, Bethesda, MD) for the RhoA QL, RhoA
N19, C3, RasV12, and RasN17 expression vectors, and to Dr. Roberto Di
Lauro (Stazione Zoologica, A. Dohrn, Naples, Italy) for cDNA and
expression vectors of TTF-1 as well as the different Tg promoter
constructs. We thank Carmen Sánchez-Palomo for help with the
immunohistochemical techniques and Catherine Mark for her linguistic
assistance.
 |
FOOTNOTES
|
---|
This work was supported by DGICYT Grants PM97/0065 and BMC2001-2087,
and CAM Grant 08.1/0025/99. D.L.M. and M.R. are recipients of a
fellowship from the Spanish Ministerio de Educación y Cultura and
Ciencia y Tecnología, respectively.
1 D.L.M. and M.R. contributed equally to this work and both should be
considered first authors. 
Abbreviations: BrdU, Bromodeoxyuridine; CAT, chloramphenicol
acetyltransferase; CMV-Luc, cytomegalovirus-luciferase; Tg,
thyroglobulin; TPO, thyroperoxidase; TTF-1, thyroid transcription
factor.
Received for publication December 27, 2000.
Accepted for publication August 31, 2001.
 |
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