From the Unité de Recombinaison et Expression Génétique, Institut Pasteur, INSERM U163, 28 rue du Dr. Roux, 75015 Paris, France
Received for publication, July 18, 2002, and in revised form, December 2, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The tight regulation of gene expression by Wnt signaling guarantees a
stringent spatiotemporal coordination of downstream gene expression in
response to developmental and physiological cues. It has been shown
that deregulation of Initially cloned by its abundant expression in human heart and its
down-regulated expression in rhabdomyosarcoma cells (17-19), FHL2
(also known as down-regulated in rhabdomyosarcoma LIM protein and
skeletal muscle LIM protein 3) belongs to the family of LIM proteins.
The LIM domain is a specialized double zinc finger protein motif, and
LIM proteins play multiple roles as adapters and functional modifiers
in protein interactions (20). Containing exclusively four and a half of
LIM domains, FHL proteins display a high degree of homology between
family members and tissue-specific expression (21, 22).
The activity of FHL2 in transcriptional regulation has been evidenced
in recent reports demonstrating that FHL2 is a coactivator of the
androgen receptor (AR) and the cAMP response element-binding protein
(CREB) (21, 23). FHL2 activates gene expression through interaction
with DNA-binding transcriptional factors (21, 23). A link between FHL2
and Plasmids--
cDNA sequences encoding Yeast Two-hybrid Analysis--
The two-hybrid screen was
performed using the mating protocol described by Fromont-Racine
et al. (27). CG1945 cells transformed with Gal4
DBD- In Vitro Binding Assay--
35S-Labeled proteins
were produced in vitro using TNT-coupled
reticulocyte lysate system (Promega). The GST- Cell Culture, Transfection, and Luciferase Assay--
293,
SW480, CV1 and HeLa cells were maintained in Dulbecco's modified Eagle
medium with 10% fetal bovine serum. Transient transfection of 293 cells was carried out by calcium phosphate precipitation with 50 ng of
Antibodies, Coimmunoprecipitation, and Immunofluorescence--
A
polyclonal anti-FHL2 antibody was generated by injection of
GST-FHL2N-term (amino acids 1-126) into rabbits. Anti-RGSH, anti-
Immunofluorescence staining was carried out as described previously
(29). HeLa cells grown on coverslips were transfected with 2 µg of
RT-PCR Analysis--
Frozen tumor tissues were obtained after
surgery from different French hospitals. Total RNA was isolated from
tumor and liver tissues using RNA-Plus RNA extraction solution (Quantum
Biotechnologies). Up to 2 µg of total RNA was reverse transcribed
using Superscript II RT RNase H-reverse transcriptase (Invitrogen) and
oligo(dT) primer. PCR was carried out as follows: 94 °C for 3 min
followed by 32 cycles at 94 °C for 30 s; 57 °C for 30 s; 72 °C for 1 min, and a final extension of 6 min. PCR products
were analyzed in 1.5% agarose gels. The 18 S ribosomal RNA was
amplified as control. The primer sequences are as follows: FHL2-F,
5'-GCCAAGAAGTGTGCTGGG-3'; FHL2-R, 5'-GCAACGGGAGGTTACAGAG-3'; 18S-F,
5'-GTAACCCGTTGAACCCCATT-3'; and 18S-R, 5'-CCATCCAATCGGTAGTAGCG-3'.
Interaction of FHL2 with
FHL2 and
To test the binding specificity between Mapping of Interaction Domains in FHL2 and
To map the FHL2 domain mediating the interaction with FHL2 Stimulates
In SW480 colon carcinoma cells, endogenous wild type
We then assessed the synergistic function of FHL2 and
We next examined cellular localization of Increased FHL2 Expression in Liver Tumors--
To assess the
implication of FHL2 in activated expression of cancer-related
Wnt-responsive genes, we examined its expression in hepatoblastoma in
which a high rate of genetic mutations is associated with nuclear
accumulation of Enhancement of AR Transcriptional Activity by FHL2 and
The association of Taken together, our data support the idea that FHL2 might be recruited
by In previous studies, FHL2 has been shown to bind different
transcription factors acting either as a transcriptional coactivator or
as a corepressor (21, 23, 38). Interestingly, FHL2 has been shown to
activate The impact of FHL2- Whether the interaction of FHL2 with -Catenin is a key mediator of the Wnt pathway,
which plays a critical role in embryogenesis and oncogenesis. As a
transcriptional activator,
-catenin binds the transcription
factors, T-cell factor and lymphoid enhancer factor, and
regulates gene expression in response to Wnt signaling. Abnormal
activation of
-catenin has been linked to various types of cancer.
In a yeast two-hybrid screen, we identified the four and a half of
LIM-only protein 2 (FHL2) as a novel
-catenin-interacting protein.
Here we show specific interaction of FHL2 with
-catenin, which
requires the intact structure of FHL2 and armadillo repeats 1-9 of
-catenin. FHL2 cooperated with
-catenin to activate T-cell
factor/lymphoid enhancer factor-dependent transcription
from a synthetic reporter and the cyclin D1 and interleukin-8 promoters
in kidney and colon cell lines. In contrast, coexpression of
-catenin and FHL2 had no synergistic effect on androgen
receptor-mediated transcription, whereas each of these two coactivators
independently stimulated AR transcriptional activity. Thus, the ability
of FHL2 to stimulate the trans-activating function of
-catenin might
be dependent on the promoter context. The detection of increased FHL2
expression in hepatoblastoma, a liver tumor harboring frequent
-catenin mutations, suggests that FHL2 might enforce
-catenin
transactivation activity in cancer cells. These findings reveal a new
function of the LIM coactivator FHL2 in transcriptional activation of
Wnt-responsive genes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Catenin is a binding partner of E-cadherin in cell-cell
adherens junctions and a key effector in the Wnt signaling pathway, which plays a critical role in development and homeostasis (1, 2).
-Catenin is composed of three domains: a regulatory N-terminal region followed by 12 armadillo
(arm)1 repeats and a
C-terminal transactivation domain. The N-terminal region contains
serine and threonine residues whose phosphorylation signals
ubiquitin-dependent degradation of cytosolic
-catenin. Phosphorylation of
-catenin is controlled by a multiprotein complex composed of tumor suppressor adenomatous polyposis coli, Axin, glycogen
synthase kinase-3, and casein kinase I
. The arm repeats in the core
region mediate
-catenin interactions with a majority of partners
such as E-cadherin and the transcriptional factors, T-cell factor (TCF)
and lymphoid enhancer factor (LEF). Cytosolic accumulation of
-catenin leads to trans-location of the protein into the nucleus
where it forms a complex with DNA-binding factors of the TCF/LEF family
(3). This bipartite transcription factor complex recruits multiple
transcriptional coactivators and activates TCF/LEF-dependent transcription through the C-terminal
trans-activation domain of
-catenin (4).
-catenin, which leads to its nuclear
accumulation and activation of gene expression, is implicated in the
development of cancer (reviewed in Ref. 5). Among several candidate
downstream target genes,
-catenin activates transcription from the
promoters of c-myc, cyclin D1, the matrix metalloproteinase-7, neuronal cell adhesion molecule, and
interleukin-8 (IL-8), which are frequently overexpressed in human colon
carcinoma (6-11). Different mechanisms by which
-catenin promotes
target gene activation have been proposed. It has been shown that
-catenin can interact directly with the TATA-binding protein
in vitro (12) and with the transcription coactivator
CBP/p300, which is able to bind to TATA-binding protein and
transcription factor IIB, thus linking
-catenin to the RNA
polymerase II machinery (13-15). Moreover,
-catenin-interacting
proteins, such as the chromatin-remodeling factor Brg-1 and CBP/p300,
can be involved in altering chromatin structure to allow access of RNA
polymerase II (16). Here, we report the identification of four and a
half of LIM-only protein 2 (FHL2) as a novel
-catenin-binding
protein that possesses intrinsic trans-activation activity.
-catenin was first suggested by the findings that both FHL2 and
-catenin interact with AR and are capable of enhancing AR function
in androgen-dependent transcription (23-26). In this
study, we show that FHL2 binds
-catenin in vitro and in vivo. Although FHL2 alone had no effect on
TCF/LEF-dependent transcription, it potentiated the
trans-activating activity of
-catenin on the transcription of the
Wnt-responsive cyclin D1 and IL-8 promoters. FHL2 and
-catenin independently enhanced AR activity in a
hormone-dependent manner, but in this context, the combined
action of both proteins had only additive effects. Evidence of
up-regulated FHL2 expression in primary tumors suggests that
FHL2-activating function on
-catenin may be implicated in oncogenesis by further enhancing the expression of Wnt target genes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin arm
repeats 1-10 (amino acids 132-554) used as the bait in a yeast
two-hybrid screen as well as fragments encompassing different arm
repeats were inserted in-frame with Gal4 DNA-binding domain (DBD) into
the pAS2
vector (a gift of Dr. P. Legrain). FHL2 full-length
cDNA was isolated from a HeLa cDNA library after a two-hybrid
screen with the
-catenin bait. FHL2 deletion mutants were
constructed by inserting PCR-amplified fragments in-frame with the Gal4
activation domain into pACT2 (Clontech). ACT
full-length cDNA cloned into pACT2 was provided by Dr. M. Morgan.
pGEX-
-catenin, pGEX-FHL2, and pGEX-FHL2N-term were constructed by
inserting full-length
-catenin and FHL2 cDNAs and FHL2 sequences
coding for amino acids 1-126 in-frame with glutathione
S-transferase (GST) into pGEX-5X-1 (Amersham Biosciences). pSGHis-
-catenin and pSGHis-arm1-12 (amino acids 132-702) were constructed by inserting the RGSH6 sequence at the
N-terminal end of
-catenin into pSG5 (Stratagene). Expression
vectors for full-length FHL2 (pcDNAFHL2),
-catenin
(pcDNA
-cat), and
-catenin T41A (pcDNA
-catT41A) were
used in transient transfection assays. The IL-8 promoter-luciferase
construct used in this study contains 193 bp upstream of the
transcription start site (193-IL-8-Luc) as described previously (11).
The promoter with mutated TCF site at position
186 to
177
(193mt-IL-8-Luc) was used as control (11). pTOPFLASH, pFOPFLASH and
p
NTCF4 were provided by Dr. H. Clevers; pA3Luc (cyclin D1 promoter)
was provided by Dr. R. Pestell; pMMTV-Luc was provided by Dr. P. Chambon; and pSG5-hAR (human androgen receptor) was provided by Dr. G. Castoria. Standard recombinant DNA techniques including PCR followed by
sequencing were used to construct all of the plasmids.
-catenin-arm1-10 were mixed with Y187 cells
transformed with a HeLa MATCHMAKER cDNA library (HL4048AH,
Clontech). Transformants were selected by their
ability to grow on minimal medium lacking tryptophan, leucine, and
histidine. Positive clones were then confirmed in
-galactosidase
overlay assay. Prey plasmids with high
-galactosidase activity were
rescued in Escherichia coli, and their sequences were
subsequently analyzed. To map interaction domains in
-catenin and
FHL2, deletion constructs were cotransformed into the diploid strain
CG1945/Y187 and quantitative
-galactosidase assay was performed
as described previously (28).
-catenin and GST-FHL2 fusion proteins were purified from E. coli according to the
manufacturer's instructions (Amersham Biosciences) and then linked to
glutathione-Sepharose beads. Incubation with in vitro
translated proteins was carried out for 4 h at 4 °C in binding
buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl,
10% glycerol, 0.05% Triton X-100, and protease inhibitor mixture
(Roche Molecular Biochemicals)). Beads were washed with the binding
buffer, and bound proteins were eluted with Laemmli buffer at 95 °C
and subjected to SDS-PAGE.
-catenin T41A, 0.25-1 µg of FHL2, 250 ng of
NTcf4, and 0.5 µg of pTOPFLASH or pFOPFLASH or with 250 ng of
-catenin T41A,
0.25-1 µg of FHL2, and 0.5 µg of pCyclinD1-Luc or 0.25 µg of
pIL-8-Luc. SW480 cell transfection was performed using calcium
phosphate precipitation with 0.25 or 1 µg of FHL2, 250 ng of
NTcf4, and 0.5 µg of pTOPFLASH or pFOPFLASH. CV1 cells were
cotransfected with 0.5 µg of pMMTV-Luc, 50 ng of pSG5-hAR, 0.5 µg
of
-catenin, and 0.25 µg of FHL2 using LipofectAMINE PLUS (Invitrogen). For AR transcriptional activity assays, CV1 cells were
washed 3 h after transfection and cultured in Dulbecco's modified
Eagle medium supplemented with 10% stripped fetal calf serum in the
presence or absence of 10 nM dihydrotestosterone (DHT)
(Sigma). The total amount of transfected DNA was kept constant by
adding pcDNA3. Each transfection was performed in duplicate and
repeated at least three times. A thymidine
kinase-
-galactosidase plasmid was cotransfected to normalize
luciferase activity for transfection efficiency. However, because FHL2
was found to activate transcription of this reporter, it could not be
used for normalization; the results were confirmed by multiple
independent assays.
-catenin, and anti-FLAG antibodies were purchased from Qiagen, Transduction Laboratories, and Sigma, respectively. For
coimmunoprecipitation assays, HeLa cells were transfected with
His-
-catenin or His-arm1-12 using LipofectAMINE. Cells were lysed
in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5%
Nonidet P-40, and protease inhibitor mixture. Cell lysates were
incubated with polyclonal anti-FHL2 antibody. Bound proteins were
eluted and analyzed by immunoblotting with anti-RGSH monoclonal
antibodies at 1:1000 dilution.
-catenin T41A and FLAG-FHL2 with calcium phosphate. 24 h later,
cells were washed in phosphate-buffered saline, fixed with 3.7%
paraformaldehyde, and permeabilized with 0.5% Triton X-100 in
phosphate-buffered saline. Cells were then incubated with monoclonal
mouse anti-
-catenin and polyclonal rabbit anti-FLAG antibodies
followed by incubation with the corresponding Texas Red- and
fluorescein isothiocyanate-coupled secondary antibodies. Images were
obtained on a Leica DMRB microscope equipped with a Princeton
CoolSnapFX CCD camera controlled by MetaVue software.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Catenin--
To identify
-catenin-binding proteins, a yeast two-hybrid system was used to
screen a HeLa cDNA expression library with
-catenin arm repeats
1-10 (amino acids 132-554) as a bait (Fig.
1A). One clone interacting
specifically with
-catenin contained the coding sequence for
full-length FHL2 (279 amino acids) (Fig. 1B). The interaction of FHL2 with
-catenin was further confirmed by GST pull
down. Using GST-
-catenin bound to Sepharose beads and in vitro expressed 35S-labeled FHL2 or conversely with
GST-FHL2 and 35S-labeled
-catenin, we found specific
binding of FHL2 to
-catenin, since GST alone was not able to pull
down the binding partners (Fig.
2A).
View larger version (27K):
[in a new window]
Fig. 1.
Interaction of FHL2 with
-catenin and mapping of the interaction domains by
the yeast two-hybrid system. A, schematic
representation of
-catenin and arm repeat constructs used as baits
in fusion with GAL4 DBD.
-Galactosidase activity in yeast cells
transformed with
-catenin arm repeats 1-10 and full-length FHL2 was
arbitrarily set to 100. B, schematic representation of FHL2
and the LIM domains used as preys in fusion with GAL4 activation domain
to map the interaction domain with
-catenin arm repeats 1-10.
View larger version (16K):
[in a new window]
Fig. 2.
Interaction of FHL2 with
-catenin in vitro and in
vivo. A, 35S-labeled FHL2 or
-catenin proteins were incubated with GST-
-catenin or GST-FHL2,
respectively. GST was used as control. 50% of input was loaded on the
gels. B, the FHL2 antibody specifically recognizes the
N-terminal domain of FHL2. Plasmids expressing LIM domains 3 and 4 (L3-4), full-length FHL2, and the N-terminal LIM domains
(L1/2-2) were transfected in HeLa cells. Cells
lysates were analyzed by immunoblotting with FHL2 antibody.
C,
-catenin coimmunoprecipitates with FHL2. HeLa cells
were transfected with His-tagged
-catenin and arm repeats 1-12.
Cell lysates were immunoprecipitated with FHL2 antibody, and
-catenin in the immune complexes was revealed by immunoblotting with anti-His antibody
(panel I). Expression of
-catenin (panel II)
and FHL2 (panel III) in HeLa cells was revealed by
immunoblotting with His and FHL2 antibodies. The arrow in
panel I indicates His-tagged arm repeats 1-12
immunoprecipitated by FHL2. IP, immunoprecipitation;
IB, immunoblotting.
-catenin interaction was tested next by
coimmunoprecipitation experiments. We generated a polyclonal antibody
against the N-terminal domain of FHL2, which recognized specifically
the FHL2 protein (Fig. 2B). His-tagged full-length
-catenin and His-tagged arm repeats 1-12 were transiently
transfected in HeLa cells, and cell lysates were precipitated by FHL2
antibody followed by immunoblotting analysis with anti-His antibody.
Both full-length and arm1-12
-catenin proteins were revealed in the
immune complexes (Fig. 2C, panel I). Specific
coimmunoprecipitation of
-catenin with FHL2 was verified using
preimmune sera that failed to precipitate
-catenin (Fig.
2C, panel IV). Thus, the interaction of FHL2 with
-catenin observed in the two-hybrid system also occurs in mammalian cells.
-catenin and FHL2, we next
tested whether ACT, a related member of the FHL family (30), can
interact with
-catenin in the yeast two-hybrid assay. When
full-length ACT in the pACT2 vector was cotransformed with the
-catenin bait arm1-10 into yeast cells, no interaction was observed
between
-catenin and ACT (data not shown).
-Catenin--
Serial
deletion mutants of
-catenin carrying various arm repeat sequences
in-frame with GAL4 DBD (Fig. 1A) were first tested for their
trans-activation activity in yeast, and constructs devoid of intrinsic
activity were used to map the region mediating the binding to FHL2.
Stable expression of
-catenin fragments in yeast was confirmed by
immunoblotting with anti-GAL4 antibody (data not shown). Our results
show that the arm repeats 3-8, which mediate
-catenin binding to
LEF1 (31), were required but not sufficient for interaction with FHL2
(see Fig. 1A). Deletion of arm repeat 10 resulted in a
4-fold increase of binding activity, designating
-catenin arm
repeats 1-9 as the optimal domain responsible for FHL2 binding.
-catenin,
overlapping constructs containing different LIM domains in fusion with
GAL4 activation domain were tested for interaction with
-catenin arm
repeats 1-10 in the yeast (see Fig. 1B). Although truncated
FHL2 proteins were stably expressed in yeast (data not shown), all of
the four and half of LIM domains were required for interaction with
-catenin, consistent with the finding that only full-length FHL2 was
pulled out from the initial two-hybrid screen.
-Catenin-activated Transcription from
TCF-responsive Promoters--
The
-catenin-TCF complex
activates gene expression in response to Wnt signaling. Therefore, we
assessed the effect of FHL2 on
-catenin trans-activating function in
293 cells using the TOPFLASH luciferase reporter, which contains
TCF/LEF consensus-binding sites. As expected, the TOPFLASH reporter
gene activity was enhanced by 20-fold by the constitutively active
-catenin T41A in which threonine 41 was changed to alanine, as
frequently found in tumors (Fig.
3A) (32). Importantly, when
FHL2 was coexpressed with
-catenin, the reporter gene activity was
further enhanced up to 4.5-fold in a dose-dependent manner
(Fig. 3A), indicating synergistic cooperation between FHL2
and
-catenin on trans-activation of the reporter gene. FHL2
trans-activation activity was dependent on
-catenin, because
expression of the dominant negative TCF4 (
NTCF4), which retains DNA
binding activity but fails to interact with
-catenin, completely
abolished this effect (Fig. 3A). Furthermore, coexpression
of FHL2 and
-catenin failed to trans-activate the luciferase gene
under the control of mutant TCF/LEF-binding elements (FOPFLASH),
demonstrating that the synergistic function of FHL2 and
-catenin was
dependent on TCF/LEF. However, FHL2 alone had a weak
dose-dependent effect on TOPFLASH and FOPFLASH reporter gene activity (Fig. 3A, lane 10), suggesting that
it might also activate transcription through
-catenin-independent
mechanisms.
View larger version (17K):
[in a new window]
Fig. 3.
TCF/LEF-dependent activation
of -catenin by FHL2. A,
luciferase reporter assay with TOPFLASH/FOPFLASH. Plasmid amounts are
in micrograms. Luciferase activity in 293 cells transfected with the
TOPFLASH reporter and empty vector was arbitrarily determined as 1. B, luciferase assay with TOPFLASH/FOPFLASH reporters in
SW480 cells. The ratio of luciferase activity in cells transfected with
TOPFLASH versus FOPFLASH reporters is shown. C
and D, luciferase assays with a reporter gene under control
of the cyclin D1 promoter (C) and the IL-8
promoter (D) in 293 cells. The wild type IL-8 promoter
(193-IL8) as well as the promoter with mutated Tcf site
(193mt-IL8) was used in the assay. E, subcellular
localization of transfected
-catenin and FHL2 in HeLa cells. Cells
cultured on coverslips were transfected with
-catenin T41A and
FLAG-FHL2. 24 h after transfection, cells were immunostained with
monoclonal anti-
-catenin and polyclonal anti-FLAG antibodies
followed by Texas Red-conjugated anti-mouse and fluorescein
isothiocyanate-conjugated anti-rabbit secondary antibodies.
-catenin is
constitutively active because of defective adenomatous polyposis coli
in this cell line. Transient expression of FHL2 in SW480 cells resulted
in dose-dependent activation of the TOPFLASH reporter, which was inhibited by the dominant negative Tcf4 (Fig. 3B).
Thus, FHL2 is able to stimulate the trans-activating activity of both wild type and stabilized mutant
-catenin.
-catenin on
natural TCF-responsive promoters. 293 cells were transfected with a
luciferase reporter controlled by either the cyclin D1 promoter known to be regulated by the
-catenin-TCF complex (8) or
the IL-8 promoter, recently identified as a direct
-catenin-TCF target (11). As found for the synthetic TOPFLASH reporter, FHL2 increased the cyclin D1 promoter activity in association
with
-catenin in a dose-dependent manner (Fig.
3C). This cooperative effect was strongly inhibited by
NTcf4 and totally abolished in reporter assays using a cyclin D1
promoter carrying mutated TCF-binding sites (data not shown).
Similarly, coexpression of
-catenin and FHL2 synergistically
activated the wild type IL-8 promoter, and this effect was abolished
when the TCF-binding site in the promoter was mutated (Fig.
3D). These data suggest that the synergistic interaction of
FHL2 and
-catenin might function in vivo on
Wnt-responsive genes.
-catenin and FHL2 in
transfected cells by immunofluorescence analysis. When constitutively active
-catenin T41A and FLAG-tagged FHL2 were exogenously expressed in HeLa cells, the two proteins showed a predominant nuclear staining (Fig. 3E), suggesting that their interaction may occur in
the nucleus.
-catenin in a majority of cases (32, 33). FHL2
expression was detected by RT-PCR in all of the tumor samples (Fig.
4). Importantly, in 8 of 10 cases, FHL2
expression was markedly up-regulated in tumors compared with matched
nontumor livers. This finding suggests that FHL2 expression might play
a role in tumor cells by enhancing the trans-activation function of
-catenin.
View larger version (25K):
[in a new window]
Fig. 4.
Enhanced FHL2 expression in
hepatoblastoma. FHL2 expression was analyzed by RT-PCR in tumors
(T) and matched nontumor tissues (N). 18 S rRNA
was amplified as control.
-Catenin--
It has been shown that FHL2 as well as
-catenin
can individually interact with AR and activate AR-driven transcription
(23, 24). The binding of FHL2 to AR was confirmed by GST pull-down experiments using GST-FHL2 and in vitro translated
35S-labeled AR (Fig.
5A). To explore the potential
effect of the interaction between FHL2 and
-catenin coactivators on
AR function, we cotransfected CV1 cells with a luciferase reporter gene
under control of the mouse mammary tumor virus promoter (MMTV-Luc)
known to be regulated by steroid hormone receptors and vectors
expressing wild type
-catenin, FHL2, and human AR. In accordance
with previous reports, luciferase activity was enhanced ~2-fold by
either
-catenin or FHL2 in the presence of DHT (Fig. 5B).
When
-catenin and FHL2 were coexpressed with AR, they enhanced AR
transcriptional activity by ~4-fold (Fig. 5B), indicating
that the combined effect of both proteins was only additive. This
effect was dependent on the presence of DHT, indicating that
transcriptional activation of AR by
-catenin and FHL2 was not
attributed to nonspecific interactions of
-catenin or FHL2 with the
reporter. This result was further confirmed by the observation that
-catenin and FHL2 were not able to activate the MMTV promoter in the
absence of AR (Fig. 5B).
View larger version (19K):
[in a new window]
Fig. 5.
Activation of AR transcriptional activity by
FHL2 and -catenin. A, in
vitro binding assay using 35S-labeled AR with GST-FHL2
or GST. 50% of input was loaded on the gel. B, pMMTV-Luc
reporter assay. Luciferase activity was measured in CV1 cells
transfected with the indicated expression vectors in the presence or
absence of DHT.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin with TCF is an essential step in
the transduction of the Wnt signal, and transcriptional activity of the
-catenin-TCF complex can be modulated by coactivators and
corepressors that interact with
-catenin or TCF. Here we report that
FHL2 is a novel partner and coactivator of
-catenin. The physical
interaction between FHL2 and
-catenin was demonstrated by in
vitro and in vivo assays, including yeast two-hybrid
screens, in vitro pull-down assays, and
coimmunoprecipitation. Further characterization of the interaction
using the yeast two-hybrid assay showed that
-catenin arm repeats
1-9 and the complete set of LIM domains in FHL2 were required for
optimal binding. By contrast, no interaction could be detected between
-catenin and ACT, a LIM domain protein closely related to FHL2,
ruling out the possibility that LIM domains might mediate nonspecific
association with
-catenin. Furthermore, we show that FHL2
potentiates
-catenin trans-activating function on
TCF/LEF-dependent transcription of Wnt-responsive genes
such as cyclin D1 and interleukin-8 in human kidney and colon cells. Importantly, FHL2 expression in the absence of nuclear
-catenin did not affect the activity of TCF target gene promoters, and cooperation between FHL2 and
-catenin was strictly dependent upon the presence of consensus TCF-binding sites in the cyclin D1 and
IL-8 promoters. Finally, ectopically expressed FHL2 and
-catenin
were found to colocalize in HeLa cell nuclei, suggesting that nuclear
interaction of these proteins might be involved in enhancing TCF transcription.
-catenin to TCF-dependent promoters, although no direct evidence has been provided so far for the interaction of FHL2 at
an endogenous
-catenin target promoters. While this paper was in
revision, similar conclusions were reported by Martin et al.
(34) who demonstrated that FHL2 specifically and functionally interacts
with endogenous
-catenin in vivo but not with LEF-1 and
that
-catenin is able to bind simultaneously FHL2 and LEF-1, forming
a ternary protein complex in vitro. The authors identified the N-terminal region and first arm repeat of
-catenin as the FHL2-binding region. However, in our study, the arm repeats 1 and 2 of
-catenin were required for the interaction with FHL2, but optimal
binding efficiency was observed for repeats 1-9. This apparent
discrepancy might be explained by different strategies that have been
used to localize the interacting regions. Alternatively, it is
conceivable that different
-catenin domains might be able to bind
FHL2. It is striking that different regions in
-catenin have been
implicated in the binding of several partners, including TATA-binding
protein (12), CBP/p300 (13-15, 35), and adenomatous polyposis coli
(36, 37).
-catenin-dependent transcription in epithelial cells (this study and Ref. 34) while it has opposite down-regulating effect in myoblasts, suggesting a cell type-specific regulation of
-catenin function by FHL2 (34). LIM domains function as molecular
adapters mediating the assembly of multiprotein complexes. Therefore,
FHL2 might target other cofactors to
-catenin to link the
-catenin-TCF complex to the RNA polymerase II machinery or to
chromatin-remodeling complexes.
-catenin interaction on AR transcriptional
activity remains unclear, since we did not observe a cooperative effect
of these AR coactivators. Furthermore, all of the FHL2 deletion mutants
analyzed had lost the ability to bind AR as well as
-catenin, and
the activity of FHL2 and
-catenin on AR transcriptional function did
not appear to be
dose-dependent.2
Therefore, our data might be interpreted as additive effects of each
individual protein in contrast with the results obtained for TCF/LEF,
which indicates that synergistic activity of
-catenin and FHL2 may
be dependent on the promoter context. Further studies using FHL2 null
cells or FHL2 mutants retaining only binding to either AR or
-catenin would shed light on the role of FHL2-
-catenin interaction on AR function.
-catenin plays a role during
oncogenesis is an interesting issue. In human cancers affecting epithelial tissues,
-catenin is frequently activated and the stimulating function of FHL2 on
-catenin might have impact on oncogenic processes. This notion is strongly supported by our finding
that the cyclin D1 promoter is markedly activated by FHL2 in
a
-catenin-dependent manner and that FHL2 expression is
up-regulated in hepatoblastoma. In this pediatric liver tumor,
-catenin was found to be mutated in >50% of cases (32, 33), and
overexpression of cyclin D1 was correlated with
-catenin
mutation (39). In recent studies of prostate cancer (40), nuclear
expression of FHL2 has been detected at higher levels in tumor cells
than in normal prostate epithelium, and nuclear trans-location and
transcriptional activity of FHL2 might be induced by Rho signaling. Rho
family members are frequently overexpressed in human tumors, and the activation of Rho signaling might be involved in the migration and
dissemination of tumor cells. Localized in both cytoplasm and nucleus,
FHL2 has been found to bind multiple partners including
- and
-integrins that are key regulators of cell adhesion and migration
(22). Thus, functional interactions between FHL2 and
-catenin might
have important consequences in multiples aspects of the activities of
both proteins.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. M. Fabre for providing tumor tissues, Drs. C. Transy and A. Reimann for insightful discussion, and Drs. F. Bergametti and O. Bischof for critical comments on the paper. We are grateful to Prof. P. Tiollais for constant interest in this work. We also thank Drs. R. Pestell, H. Clevers, P. Chambon, M. Morgan, P. Legrain, and G. Castoria for kindly providing constructs used in this study.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grant 4395 from the Association pour la Recherche contre le Cancer (ARC).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
33-1-40-61-33-07; Fax: 33-1-45-68-89-43; E-mail: ywei@pasteur.fr.
§ Supported by the Fondation pour la Recherche Médicale.
¶ Supported by the ARC.
Published, JBC Papers in Press, December 3, 2002, DOI 10.1074/jbc.M207216200
2 C. Labalette and Y. Wei, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: arm, armadillo; IL, interleukin; FHL2, four and a half of LIM-only protein 2; ACT, activator of CREM in testis; AR, androgen receptor; TCF, T-cell factor; LEF, lymphoid enhancer factor; CREB, cAMP response element-binding protein; CBP, CREB-binding protein; DHT, dihydrotestosterone; His, histidine epitope tag; RT, reverse transcription; MMTV, mouse mammary tumor virus; DBD, DNA-binding domain; GST, glutathione S-transferase; Luc, luciferase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Peifer, M.,
and Polakis, P.
(2000)
Science
287,
1606-1609 |
2. | Moon, R. T., Brown, J. D., and Torres, M. (1997) Trends Genet. 13, 157-162[CrossRef][Medline] [Order article via Infotrieve] |
3. | Behrens, J., von Kries, J. P., Kuhl, M., Bruhn, L., Wedlich, D., Grosschedl, R., and Birchmeier, W. (1996) Nature 382, 638-642[CrossRef][Medline] [Order article via Infotrieve] |
4. | Barker, N., Morin, P. J., and Clevers, H. (2000) Adv. Cancer Res. 77, 1-24[Medline] [Order article via Infotrieve] |
5. |
Polakis, P.
(2000)
Genes Dev.
14,
1837-1851 |
6. |
He, T. C.,
Sparks, A. B.,
Rago, C.,
Hermeking, H.,
Zawel, L.,
da Costa, L. T.,
Morin, P. J.,
Vogelstein, B.,
and Kinzler, K. W.
(1998)
Science
281,
1509-1512 |
7. | Tetsu, O., and McCormick, F. (1999) Nature 398, 422-426[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Shtutman, M.,
Zhurinsky, J.,
Simcha, I.,
Albanese, C.,
D'Amico, M.,
Pestell, R.,
and Ben-Ze'ev, A.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
5522-5527 |
9. |
Brabletz, T.,
Jung, A.,
Dag, S.,
Hlubek, F.,
and Kirchner, T.
(1999)
Am. J. Pathol.
155,
1033-1038 |
10. |
Conacci-Sorrell, M. E.,
Ben-Yedidia, T.,
Shtutman, M.,
Feinstein, E.,
Einat, P.,
and Ben-Ze'ev, A.
(2002)
Genes Dev.
16,
2058-2072 |
11. |
Levy, L.,
Neuveut, C.,
Renard, C. A.,
Charneau, P.,
Branchereau, S.,
Gauthier, F.,
Van Nhieu, J. T.,
Cherqui, D.,
Petit-Bertron, A. F.,
Mathieu, D.,
and Buendia, M. A.
(2002)
J. Biol. Chem.
277,
42386-42393 |
12. |
Hecht, A.,
Litterst, C. M.,
Huber, O.,
and Kemler, R.
(1999)
J. Biol. Chem.
274,
18017-18025 |
13. |
Hecht, A.,
Vleminckx, K.,
Stemmler, M. P.,
van Roy, F.,
and Kemler, R.
(2000)
EMBO J.
19,
1839-1850 |
14. |
Takemaru, K. I.,
and Moon, R. T.
(2000)
J. Cell Biol.
149,
249-254 |
15. |
Miyagishi, M.,
Fujii, R.,
Hatta, M.,
Yoshida, E.,
Araya, N.,
Nagafuchi, A.,
Ishihara, S.,
Nakajima, T.,
and Fukamizu, A.
(2000)
J. Biol. Chem.
275,
35170-35175 |
16. |
Barker, N.,
Hurlstone, A.,
Musisi, H.,
Miles, A.,
Bienz, M.,
and Clevers, H.
(2001)
EMBO J.
20,
4935-4943 |
17. | Chan, K. K., Tsui, S. K., Lee, S. M., Luk, S. C., Liew, C. C., Fung, K. P., Waye, M. M., and Lee, C. Y. (1998) Gene (Amst.) 210, 345-350[CrossRef][Medline] [Order article via Infotrieve] |
18. | Genini, M., Schwalbe, P., Scholl, F. A., Remppis, A., Mattei, M. G., and Schafer, B. W. (1997) DNA Cell Biol. 16, 433-442[Medline] [Order article via Infotrieve] |
19. | Morgan, M. J., and Madgwick, A. J. (1996) Biochem. Biophys. Res. Commun. 225, 632-638[CrossRef][Medline] [Order article via Infotrieve] |
20. | Dawid, I. B., Breen, J. J., and Toyama, R. (1998) Trends Genet. 14, 156-162[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Fimia, G. M., De,
Cesare, D.,
and Sassone-Corsi, P.
(2000)
Mol. Cell. Biol.
20,
8613-8622 |
22. |
Wixler, V.,
Geerts, D.,
Laplantine, E.,
Westhoff, D.,
Smyth, N.,
Aumailley, M.,
Sonnenberg, A.,
and Paulsson, M.
(2000)
J. Biol. Chem.
275,
33669-33678 |
23. |
Muller, J. M.,
Isele, U.,
Metzger, E.,
Rempel, A.,
Moser, M.,
Pscherer, A.,
Breyer, T.,
Holubarsch, C.,
Buettner, R.,
and Schule, R.
(2000)
EMBO J.
19,
359-369 |
24. |
Truica, C. I.,
Byers, S.,
and Gelmann, E. P.
(2000)
Cancer Res.
60,
4709-4713 |
25. |
Yang, F., Li, X.,
Sharma, M.,
Sasaki, C. Y.,
Longo, D. L.,
Lim, B.,
and Sun, Z.
(2002)
J. Biol. Chem.
277,
11336-11344 |
26. |
Pawlowski, J. E.,
Ertel, J. R.,
Allen, M. P., Xu, M.,
Butler, C.,
Wilson, E. M.,
and Wierman, M. E.
(2002)
J. Biol. Chem.
277,
20702-20710 |
27. | Fromont-Racine, M., Rain, J., and Legrain, P. (1997) Nat. Genet. 16, 277-282[Medline] [Order article via Infotrieve] |
28. | Transy, C., and Legrain, P. (1995) Mol. Biol. Rep. 21, 119-127[Medline] [Order article via Infotrieve] |
29. | Carvalho, T., Seeler, J. S., Öhman, K., Jordan, P., Pettersson, U., Akusjärvi, G., Carmo-Fonseca, M., and Dejean, A. (1995) J. Cell Biol. 131, 45-56[Abstract] |
30. | Fimia, G. M., De, Cesare, D., and Sassone-Corsi, P. (1999) Nature 398, 165-169[CrossRef][Medline] [Order article via Infotrieve] |
31. | von Kries, J. P., Winbeck, G., Asbrand, C., Schwarz-Romond, T., Sochnikova, N., Dell'Oro, A., Behrens, J., and Birchmeier, W. (2000) Nat. Struct. Biol. 7, 800-807[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Koch, A.,
Denkhaus, D.,
Albrecht, S.,
Leuschner, I.,
von Schweinitz, D.,
and Pietsch, T.
(1999)
Cancer Res.
59,
269-273 |
33. | Wei, Y., Fabre, M., Branchereau, S., Gauthier, F., Perilongo, G., and Buendia, M. A. (2000) Oncogene 19, 498-504[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Martin, B.,
Schneider, R.,
Janetzky, S.,
Waibler, Z.,
Pandur, P.,
Kuhl, M.,
Behrens, J.,
von der Mark, K.,
Starzinski-Powitz, A.,
and Wixler, V.
(2002)
J. Cell Biol.
159,
113-122 |
35. |
Sun, Y.,
Kolligs, F. T.,
Hottiger, M. O.,
Mosavin, R.,
Fearon, E. R.,
and Nabel, G. J.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
12613-12618 |
36. | Rubinfeld, B., Souza, B., Albert, I., Müller, O., Chamberlain, S. H., Masiarz, F. R., Munemitsu, S., and Polakis, P. (1993) Science 262, 1731-1734[Medline] [Order article via Infotrieve] |
37. | Munemitsu, S., Albert, I., Souza, B., Rubinfeld, B., and Polakis, P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3046-3050[Abstract] |
38. |
McLoughlin, P.,
Ehler, E.,
Carlile, G.,
Licht, J. D.,
and Schafer, B. W.
(2002)
J. Biol. Chem.
277,
37045-37053 |
39. |
Takayasu, H.,
Horie, H.,
Hiyama, E.,
Matsunaga, T.,
Hayashi, Y.,
Watanabe, Y.,
Suita, S.,
Kaneko, M.,
Sasaki, F.,
Hashizume, K.,
Ozaki, T.,
Furuuchi, K.,
Tada, M.,
Ohnuma, N.,
and Nakagawara, A.
(2001)
Clin. Cancer Res.
7,
901-908 |
40. |
Muller, J. M.,
Metzger, E.,
Greschik, H.,
Bosserhoff, A. K.,
Mercep, L.,
Buettner, R.,
and Schule, R.
(2002)
EMBO J.
21,
736-748 |