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First published online 12 November 2008
doi: 10.1242/dev.027151
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Laboratory for Embryonic Induction, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
* Author for correspondence (e-mail: sasaki{at}cdb.riken.jp)
Accepted 10 October 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Hippo signaling, TEF, Tead, Yap1, Cell proliferation, Contact inhibition
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Pan, 2007;
Reddy and Irvine, 2008;
Saucedo and Edgar, 2007). The
core components of this pathway are an Ste20-like kinase Hippo (Hpo)
(Harvey et al., 2003;
Jia et al., 2003;
Pantalacci et al., 2003;
Udan et al., 2003;
Wu et al., 2003) and its
regulatory protein Salvador (Tapon et al.,
2002), NDR family kinase Warts (Wts)
(Justice et al., 1995;
Xu et al., 1995) and a
Mob1-related regulatory protein Mats (Lai
et al., 2005). Wts phosphorylates a transcriptional co-activator
Yorkie (Yki) and suppresses its nuclear accumulation
(Dong et al., 2007;
Zhao et al., 2007). Yki
promotes organ growth by stimulating cell proliferation and inhibiting
apoptosis; this is achieved by activating Cyclin E, the apoptosis inhibitor
Diap1 and the bantam microRNA
(Huang et al., 2005;
Nolo et al., 2006;
Thompson and Cohen, 2006).
Recent studies have revealed that the TEAD/TEF family transcription factor
Scalloped (Sd) interacts with Yki and mediates Hippo signaling
(Goulev et al., 2008;
Wu et al., 2008;
Zhang et al., 2008). Upstream
of the Hpo kinase cascade, the FERM domain-containing family of proteins,
Merlin (Mer) and Expanded, and atypical cadherin Fat are involved
(Bennett and Harvey, 2006;
Cho et al., 2006;
Hamaratoglu et al., 2006;
Silva et al., 2006;
Willecke et al., 2006),
suggesting that Hippo signaling is linked to extracellular spaces through
these proteins, although the underlying mechanisms are not known.
The Hippo signaling pathway appears to be conserved in mammals. Mammals
have multiple Hippo pathway-component counterparts, and some of them rescued
fly mutants and/or showed similar activities in flies
(Lai et al., 2005;
Tao et al., 1999;
Wu et al., 2003). The human
ortholog of Mer is encoded by a tumor suppressor gene, neurofibromatosis 2
(NF2), mutations of which lead to neurofibromatosis
(McClatchey and Giovannini,
2005). Mice that are mutant for a Wts homolog,
Lats1, develop soft-tissue sarcomas and ovarian tumors
(St John et al., 1999). The
Yki homolog yes-associated protein 1 (Yap1) is involved in cancer. A
genomic region containing Yap1 and cIAP2/Birc3 is amplified
in mouse models of liver cancer and human cancers, and these genes contribute
to tumorigenesis (Overholtzer et al.,
2006; Zender et al.,
2006). Yap1 overexpression in liver reversibly increases liver
size and prolonged overexpression causes liver tumor
(Camargo et al., 2007;
Dong et al., 2007). In cultured
cells, Yap1 alters subcellular localization depending on cell density and
Hippo signaling, and mediates the cell contact inhibition of proliferation
(Zhao et al., 2007). A
Yap1-related protein Wwtr1/TAZ also has similar functions
(Lei et al., 2008). Therefore,
the framework of the Hippo signaling cascade appears to be conserved in
mammals to regulate cell proliferation and to control organ size.
In Drosophila, the Hippo signal converges with the activity of
TEAD/TEF family transcription factor Sd through interaction with Yki
(Goulev et al., 2008;
Wu et al., 2008;
Zhang et al., 2008). In
mammals, Tead proteins also interact with Yap1
(Vassilev et al., 2001). Mice
have four Tead genes (Tead1-Tead4), and they are expressed
widely during development. Tead proteins regulate development of various
tissues, including heart, skeletal muscles, neural crest, notochord and
trophoectoderm (Chen et al.,
1994; Maeda et al.,
2002; Milewski et al.,
2004; Nishioka et al.,
2008; Sawada et al.,
2008; Sawada et al.,
2005; Yagi et al.,
2007). Our recent study on Tead1;Tead2 double-mutant
embryos revealed the genetic interactions between Tead1/2 and
Yap1 during embryogenesis, and their necessity in cell proliferation
and apoptosis (Sawada et al.,
2008). This observation supports the hypothesis of Tead1/2
involvement in the regulation of cell proliferation and Hippo signaling.
In this study, we first examined the role of Tead proteins in Hippo
signaling using cell culture systems. Although Zhao et al.
(Zhao et al., 2008) recently
reported involvement of Tead in Yap1-dependent gene expression in cultured
cells, we took complementary approaches and further extended our analyses to
mouse embryos. Cell density and Hippo signaling regulates nuclear Yap1 and
endogenous Tead activity. Modulation of Tead activity altered cell
proliferation and cell death. The diverse effects of Yap1 overexpression were
mimicked by Tead2-VP16, and Tead and Yap1 regulated common sets of genes in
NIH3T3 cells. Thus, Tead is a key mediator of Hippo signaling in mouse.
However, the Tead/Yap1-regulated genes varied between experimental systems,
suggesting the complexity of Hippo signaling. Protein distribution in embryos
suggests that Tead, Yap1 and Hippo signaling may regulate both proliferation
and differentiation of cells.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), pam212 (Yuspa et al.,
1980) and HEK293T cells were cultured in Dulbecco's Modified Eagle
Medium containing 10% fetal calf serum (DMEM + 10% FCS). PLAT-E cells were
maintained as described previously (Morita
et al., 2000).
Mouse mutants
Tead1 and Tead2 mutant mice have been described
previously (Sawada et al.,
2008). Yap1tmlSmil mice
(Morin-Kensicki et al., 2006)
were crossed with Actb:Cre transgenic mice to remove the neomycin
cassette flanked by loxP sites. The resulting mice
(Yap1
tm1) are referred to as
Yap1 mutant mice in this paper. Mice were housed in environmentally
controlled rooms in the Laboratory Animal Housing Facility of the RIKEN Center
for Developmental Biology (CDB), under the institutional guidelines for animal
and recombinant DNA experiments.
Antibody staining
Rabbit anti-Yap1 antibody (No. 1) was raised by T. K. Craft (Gunma, Japan)
and affinity purified by Qiagen using the following peptide as the antigen:
CKLDKESFLTWL. Cells were cultured in LAB-TEK II chamber slides (Nunc) coated
with 0.1% gelatin. Cryosections (15 µm) of embryos were prepared.
Immunofluorescent staining was performed according to standard procedures.
Briefly, slides were incubated with rabbit anti-Yap1 antibody (1:300) or
rabbit anti-Tead1 antibody (1:430)
(Nishioka et al., 2008) at
4°C for 16 hours, followed by detection with anti-rabbit IgG-Alexa 594
(1:2000) (Invitrogen). Nuclei were counterstained with 1 µg/ml of 4',
6'-diamidino-2-phenylindole (DAPI).
Quantification of fluorescent signals
Average intensities of the Yap1, Tead1 and DAPI signals in the nucleus area
were measured with MetaMorph software (Molecular Devices). The Yap1/Tead1
signals were normalized to the DAPI signal.
Luciferase assay
8xGT-IIC-Luc was constructed by cloning eight copies of the following
oligonucleotides straddling the GT-IIC motif of SV40 enhancer
(Davidson et al., 1988) into
p
51-LucII (Kamachi and Kondoh,
1993): 5'-CCAGCTGTGGAATGTGTGTcc-3' and
3'-ggGGTCGACACCTTACACACA-5' (underlines indicate
GT-IIC, the additional c or g was added to facilitate directional cloning).
NIH3T3 cells were plated into 12-well plates at a density of 0.25 x
105 cells/well 24 hours before transfection. A DNA mixture
consisting of effector (0.1 µg), reporter (0.1 µg), reference
(pCS2-β-gal, 0.1 µg) and pBluescript (Stratagene) (0.5 µg) were
transfected for 24 hours using 2 µl of lipofectamine2000 (Invitrogen).
Preparation of lysates, luciferase and β-galactosidase assays were as
described (Sasaki et al.,
1999). Luciferase activities were normalized to
β-galactosidase activities. Results were shown by averaging two samples
with standard errors.
Transfection assay
pcDNA-HA-Lats2 was constructed by cloning the coding sequence of mouse
Lats2 cDNA (GenBank: BC053028) into pcDNA3-HA (a gift from Dr A. Shimono).
Cells were seeded into LAB-TEK II chamber slide (Nunc 154461) coated with 0.1%
gelatin at a density of 0.5 x 105 cells/well 24 hours before
transfection. DNA mixture consisting of pCMV-EGFP (0.2 µg), pcDNA3-HA-Lats2
or pcDNA3.1-MST2 (a gift from Dr Georg Halder) (0.2 µg) and pBluescript
(0.4 µg) were transfected for 24 hours with 2 µl of lipofectamine2000,
and were stained with antibodies as described above.
Bromodeoxyuridine (BrdU)-labeling
NIH3T3 cells were plated at the density of 0.5x105 (low
density) or 2x105 (high density) cells/well in gelatin-coated
LAB-TEK II chamber slides and were cultured for 48 hours. Cells were incubated
with 10 µg/ml BrdU for 30 minutes, followed by fixation, and incubation
with 2 M HCl for 30 minutes and 0.1 M sodium borate (pH 8.9) for 10 minutes.
The pretreated cells were immunostained as described above using mouse
monoclonal anti-BrdU antibody (1:500 dilution, Sigma) and anti-mouse
IgG-Alexa488 as primary and secondary antibodies, respectively. For embryos,
pregnant mice were injected intra-peritoneally with BrdU equivalent to 200
µg/g of body weight, 2 hours prior to dissection. Immunostaining was
performed on paraffin-embedded sections as described previously
(Liu et al., 2000;
Megason and McMahon,
2002).
Preparation of Tead/Yap1 virus infected cells
Retroviral vectors, pMYs-Tead/Yap1-IRES-EGFPs, were generated by cloning
the coding sequences of full-length or modified Tead1, Tead2, Tead4 or Yap1
cDNAs into pMYs-IRES-EGFP (Kitamura et
al., 2003). Identities of the PCR-amplified cDNAs were verified
with DNA sequencing. Tead/Yap1-viruses were produced by transfecting
pMYs-Tead/Yap1-IRES-EGFP plasmids into PLAT-E packaging cells as described
(Morita et al., 2000).
Forty-eight hours after infection of Tead/Yap1-viruses into NIH3T3 cells,
EGFP-positive cells were selected with FACSAria cell sorter (BD Biosciences).
For growth curve analysis, EGFP-positive cells (0.5x105) were
seeded into 35 mm dishes coated with gelatin, and the total cell numbers in
each dish were counted. Results are shown as the average of two samples with
standard errors.
Leishman stain
Virus-infected NIH3T3 cells were plated in 35 mm dishes as growth curve
analysis. On the 18th day, plates were stained with 0.2% Leishman's stain
(Sigma).
Three-dimensional (3D) culture
pMYs-IRES-puro was constructed by cloning the IRES-puro fragment into pMYs
vector (Kitamura et al.,
2003). pMYs-Tead/Yap1-IRES-puros were generated by cloning
full-length or modified Tead2/Yap1 cDNA. Tead/Yap1-viruses were prepared and
infected into MTD1A cells as above, and were selected with 2 µg/ml
puromycin for 7 days from 48 hours post-infection. The 3D culture was
performed as described (Debnath et al.,
2003), with a change to the culture medium to DMEM supplemented
with 5% FCS and 10 ng/ml EGF (Peprotech).
Immunoprecipitation assay
The immunoprecipitation assay was performed as previously described
(Yamamoto et al., 2008). To
prepare the lysates, the DNA mixture consisting of 0.45 µg of
pcDNA-HA-Yap1-polyA83 or pcDNA-HA-Yap1-TeadBD-polyA83 and 0.45 µg of
pCMV-Flag-Tead1 or pCMV-Flag-Tead2 were transfected into HEK293T cells with
FuGENE HD (Roche).
Tumorigenesis assay
BALB/cAJc1 nude mice (8-week-old male) were obtained from CLEA Japan
(Japan). Virus-infected NIH3T3 cells (100 µl, 1x106) were
injected subcutaneously through 23-gauge needles into the dorsal flank
area.
Microarray analysis
Proliferating Yap1/Tead2-VP16-expressing cells were harvested at a density
that slightly exceeds the confluency of normal cells. For low- and
high-density cultures, control virus-infected cells were harvested at 30% or
complete confluency. Tead2-EnR-expressing cells were harvested at 30%
confluency of these cells. RNA was extracted with RNeasy kit (Qiagen) followed
by further purification. Biotinylated cRNAs were prepared according to the
Affymetrix standard labeling protocol, followed by fragmentation and
hybridization to the Affymetrix GeneChip Mouse Genome 430 2.0 Array. Chips
were washed and stained with Streptavidin R-phycoerythrin (Invitrogen). After
scanning the chips, expression values of probe sets were summarized with the
RMA algorithm (Irizarry et al.,
2003). Differently expressing probe sets were identified with the
eBayes method (FDR<0.1) (Smyth,
2004). The results of two independent experiments were used for
analysis. The microarray analysis was carried out at the Functional Genomics
Unit of RIKEN CDB. Genes that show significant differences
(P<0.0001) were used for analysis. Yap1/Tead2-VP16 cells were
compared with high-density cells. Tead2-EnR cells were compared with
low-density cells. All of the microarray data have been submitted to the Gene
Expression Omnibus (Accession Number GSE12498).
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tm1/tm1
and Tead1-/-; Tead2-/- embryos were used for
cDNA synthesis using Ready-To-Go You-Prime First-Strand Beads (GE Healthcare)
and random hexamer (Invitrogen) following the manufacturer's instructions. The
resultant cDNA was diluted at 1:100 (for cells) or 1:10 (for embryos) for
quantitative PCR. Primers used were as follows: Acta2,
5'-AGGGCTGTTTTCCCATCCATCG-3' and
5'-TCTCTTGCTCTGGGCTTCATCC-3'; Ctgf,
5'-CAAGGACCGCACAGCAGTT-3' and
5'-AGAACAGGCGCTCCACTCTG-3'; Etv5,
5'-TAGCGGAGACTTTGGAAGCACC-3' and
5'-AATCAAAGGTCGCCCTCGACAG-3'; Gapdh,
5'-ACCACAGTCCATGCCATCAC-3' and
5'-TCCACCACCCTGTTGCTGTA-3'; Hmga2,
5'-CAGCAAAAACAAGAGCCCC-3' and
5'-AGCAGGCTTCTTCTGAACG-3'; Il1rl1,
5'-TGGGCTTTGGCAATTCTGACAC-3' and
5'-TAAGTCGAGCGTCCTCTTTGGG-3'; Klhdc8a,
5'-TCCAAGATCTATGTGCTGGGGG-3' and
5'-GGAATGTTGGGGAACTTGGTCC-3'; Serpine1,
5'-GCGGCAGATCCAAGATGCTATG-3' and
5'-TCTCATTCTTGTTCCACGGCCC-3'; Tagln,
5'-TGGATTGTAGTGCAGTGTGGC-3' and
5'-TTCGATCCCTCAGGATACAGGC-3'; Tnfrsf1b,
5'-CGCCTGCACTAAACAGCAGAAC-3' and
5'-TTGCTCAGCCTCATGCACTGTC-3'; Vcl,
5'-TCCTATCCACAGTGAAGGCCAC-3' and
5'-CACAGACTGCATGAGGTTCTGG-3'. For qPCR, 1.6 µl of diluted cDNAs
were amplified using the SYBR Premix Ex Taq (Takara Bio, Japan) in a total
volume of 20 µl on a ABI PRISM 7900HT (Applied Biosystems). The PCR
conditions were 95°C for 10 seconds, and 40 cycles of 95°C for 5
seconds and 60°C for 30 seconds. qPCR was carried out in duplicate, using
Gapdh as a housekeeping control.
Cell death assay
Virus-infected NIH3T3 cells (5x105) were cultured in
six-well plates for 24 hours, followed by treatment with 500 nM Taxol (Sigma)
for 24 hours. Both floating cells and attached cells were collected and
combined, stained with propidium iodide following the procedure of Flow
Cytometry Core Facility of University of Michigan
(http://www.med.umich.edu/flowcytometry/PDF%20files/HYPOpi.pdf),
and were analyzed on a FACSCantoII (BD Bioscience) to determine percentage of
cells with sub-G1 DNA content.
Statistics
Statistical analyses, with the exception of microarray analysis, were
performed with Prism5 statistical software (GraphPad) using an unpaired,
two-tailed t-test or a one-way ANOVA followed by Tukey's multiple
comparison test.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). At a low
cell density, the cells proliferated actively and Yap1 showed strong nuclear
localization (Fig.
1A,A'). At a high cell density, cell proliferation was
suppressed, and nuclear levels of Yap1 were reduced
(Fig. 1B,B',E). Similar
behavior of Yap1 was also observed in two mouse epithelial cell lines, pam212
and MTD1A, indicating the generality of this observation (see Fig. S1 in the
supplementary material). Although Tead1 proteins constantly localized to the
nuclei, the level of nuclear Tead1 was reduced at a high cell density
(Fig. 1C,D,F). To monitor the
transcriptional activity of endogenous Tead proteins, we exploited a Tead
reporter (8xGT-IIC-Luc) containing an 8-mer of a Tead-binding motif,
GT-IIC (Davidson et al., 1988)
(Fig. 1G). As a similar
GT-IIC-containing reporter (pGT4Tluc) is activated by all four Tead
proteins (Vassilev et al.,
2001), the reporter should monitor the total activity of four Tead
proteins. The Tead reporter showed high activity at low cell density, whereas
the activity levels decreased with an increase in cell density
(Fig. 1H). Activity of the
reporter without the GT-IIC motif remained relatively unaffected. There is,
therefore, a good correlation between nuclear localization of Yap1, cell
proliferation, transcriptional activity of Tead proteins and cell density.
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). Based on
the identity of surrounding sequences, we introduced a similar point mutation
into mouse Yap1 (Yap1-S112A). Overexpression of Yap1-S112A increased nuclear
Tead1 (see Fig. S2 in the supplementary material), suggesting that the amount
of nuclear Yap1 has an effect on levels of nuclear Tead1. These results
suggest that the Hippo signal negatively regulates Tead activity by inhibiting
the nuclear localization of Yap1 and accumulation of Tead proteins.
Increased Tead activity mimics the effects of Yap1 overexpression
If Tead is a downstream effector of Hippo signaling, altering its
transcriptional activity would modulate cell proliferation. As
Tead1/2 double-mutants display growth defects
(Sawada et al., 2008), we
expressed modified forms of Tead2 in NIH3T3 cells and compared their effects
with those of Yap1. We used a bi-cistronic retrovirus vector, which also
expresses EGFP, and selected the Tead- or
Yap1-overexpressing cells with a cell sorter by EGFP expression. To
avoid clonal-selection effects, all experiments were performed with short-term
culture of EGFP-selected and uncloned pools of cells. Overexpression of Yap1
increased Tead activity (Fig.
2E), and such cells continued to proliferate even after reaching
the density of normal confluency and resulted in a higher saturation density
than control virus-infected cells, as previously reported
(Zhao et al., 2007)
(Fig. 2A, day 9-14;
Fig. 2F, day 9). Similarly,
increasing Tead activity by expressing the activator-modified Tead2, a fusion
protein of the N-terminal region of Tead2 containing the TEA domain and the
activation domain of herpes simplex virus VP16 (Tead2-VP16)
(Fig. 2B,E), promoted cell
proliferation beyond normal confluency and resulted in a higher saturation
density (Fig. 2C, day 9-14;
Fig. 2F, day 9). As other Tead
proteins (Tead1-VP16 and Tead4-VP16) also promoted cell proliferation
(Fig. 2B,D), the
growth-promoting activity of Tead2-VP16 may represent the general activity of
Tead family proteins as a whole. By contrast, suppression of Tead activity by
the repressor-modified Tead2, a fusion protein of the TEA domain of Tead2 and
the repression domain of Drosophila Engrailed (Tead2-EnR), resulted
in slower cell proliferation and reached a lower saturation density than that
of control cells (Fig. 2B,C,E).
Morphologically, Tead2-EnR-expressing cells tend to have longer processes than
control cells at day 2, and stopped proliferation at day 9, leaving open
spaces between cells (Fig. 2F).
Expression of full-length Tead2 had no effect on Tead activity, cell
proliferation and saturation density (Fig.
2C,E). In summary, increased Tead activity mimicked effects of
Yap1 overexpression on cell proliferation and saturation density, and reduced
Tead activity displayed opposite effects. As Tead-modulated cells stopped
proliferation at different cell densities, the cell contact inhibition system
is likely to be operating in these cells with a change in sensitivity to the
inhibition signal.
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60% of
diameter, i.e. 20% in volume) than control cells
(Fig. 2G,H). The colony size of
full-length Tead2-expressing cells was comparable with that of control cells
(Fig. 2H). As colony sizes
reflect proliferation rates, the results suggest that Tead and Yap1 also
regulate cell proliferation rate. Yap1-overexpressing mammary epithelial
cells, MCF10A, often form invasive colonies in matrigel culture, an indication
of epithelial-to-mesenchymal transition (EMT)
(Overholtzer et al., 2006).
Some of the Yap1-overexpressing MTD1A cells and Tead2-VP16-overexpressing
cells also formed similar invasive colonies, whereas such colonies were not
observed with vector control or full-length Tead2-overexpressing cells
(Fig. 2G; data not shown).
Therefore, EMT-inducing activity of Yap1 was also mimicked by Tead2-VP16.
Although Yap1 promotes cell proliferation, it also suppresses apoptosis.
Yap1-overexpressing NIH3T3 cells showed reduced apoptosis after treatment with
a pro-apoptotic reagent, Taxol, as previously reported
(Overholtzer et al., 2006); a
similar anti-apoptotic effect was also observed with Tead2-VP16-expressing
cells (Fig. 2I,J). In summary,
Yap1 controls cell proliferation rate, contact inhibition, EMT and apoptosis,
and all of these activities were mimicked when Tead activity was
increased.
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TeadBD) abolished
interaction with Tead1/2 (Fig.
3A,B). Yap1-TeadBD had no effect on Tead activity or cell
proliferation (Fig. 3C,D). Yap1
interacts with other transcription factors through two WW domains. A Yap1
mutant lacking WW domains (Yap1-WW) showed weaker co-activator activity
for Tead proteins with unknown reasons, and also displayed weaker enhancement
of cell proliferation (Fig.
3A,C,D). The Yap1-S112A mutant is supposedly insensitive to
inhibition by Hippo signaling. At low cell density or under conditions where
Hippo signaling is assumed to be weak, Yap1-S112A was not significantly
different from Yap1 in its Tead co-activator activity and cell proliferation
(Fig. 3A,C,E). However, once
the cells reach 100% confluency or under conditions where Hippo signaling is
assumed to be strong, Yap1-S112A-expressing cells continued to proliferate far
beyond the confluence reached by Yap1-expressing cells
(Fig. 3E), demonstrating the
importance of S112 for regulation of Yap1 by Hippo signaling or cell-cell
contact. Deletion of the activation domain of Yap1 (Yap1-AD) or
replacement of activation domain of Yap1-S112A with EnR (dnYap1) converted
Yap1 into weak or strong co-repressors of Tead, respectively
(Fig. 3A,C). Yap1-AD
slightly reduced saturation density, whereas dnYap1 strongly reduced cell
proliferation rate and saturation density
(Fig. 3F). Correlation of Tead
co-activator activity of various mutant forms of Yap1 and their effects on
cell proliferation further supports the hypothesis that Yap1 regulates cell
proliferation by modulating the transcriptional activity of Tead proteins.
Increased Tead activity transforms cells
Yap1 has oncogenic activities and its persistent overexpression in mouse
liver results in tumorigenesis (Dong et
al., 2007). Therefore, we next asked whether increased Tead
activity is sufficient for cell transformation. When Yap1-overexpressing cells
were cultured for an extended period, they stopped proliferating at one point
when they reached saturation density, and then re-initiated cell proliferation
(Fig. 2A, day 14-18). At this
time, some cells form nodules, in which cells become piled up (compare
Fig. 4A-A'' with
Fig. 4D-D''), indicating
anchorage-independent growth of these cells; this is an indication of
transformation. Similar late-onset proliferation and nodule formation was also
observed with Tead2-VP16-expressing cells, but not with control virus-infected
or Tead2-expressing cells (Fig.
2A,C; Fig.
4B-B'',C-C''). Therefore, the nodule-forming activity of
Yap1 was mimicked by increasing Tead activity. To further corroborate the
oncogenic activities of Tead, we examined tumorigenic activities of Tead2-VP16
expressing NIH3T3 cells by subcutaneously transplanting them into nude mice.
Control virus-infected cells formed no tumor, whereas Yap1-overexpressing and
Tead2-VP16-expressing cells formed tumors
(Fig. 4E-H and data not shown).
Taken together, increased Tead activity also mimicked transforming activity of
Yap1.
Tead, Yap1 and cell density regulate common sets of target genes
To compare the genes that are regulated by Yap1, Tead2-VP16 and cell
density, we examined gene expression profiles with microarrays. Consistent
with previous observations (Zhao et al.,
2007), the set of genes induced by Yap1 overlapped with the set of
genes repressed by high cell density (Fig.
5A). Similarly, the majority of the genes repressed by Yap1
overlapped with the genes induced by high cell density
(Fig. 5A). By contrast, the set
of genes induced or repressed by Yap1 did not significantly overlap with the
set of genes induced or repressed by high cell density, respectively
(Fig. 5A). Similar results were
also obtained with the genes regulated by Tead2-VP16 and cell density. Namely,
the majority of the genes induced or repressed by Tead2-VP16 overlapped with
the genes repressed or induced by high cell density, respectively
(Fig. 5B). No significant
overlap was observed between the set of genes induced or repressed by
Tead2-VP16 and the set of genes induced or repressed by high cell density,
respectively (Fig. 5B).
Furthermore, the majority of the set of genes induced or repressed by
Tead2-VP16 was also induced or repressed by Yap1, respectively
(Fig. 5C). There is no overlap
between the set of genes induced or repressed by Tead2-VP16 and repressed or
induced by Yap1, respectively (Fig.
5C). These results suggest that Tead2-VP16 mimics the effects of
Yap1 overexpression at the transcriptional level.
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) and
Yap1-/-
(Morin-Kensicki et al., 2006)
embryos at E8.0. qRT-PCR analysis of the ten representative genes commonly
induced by Tead2-VP16 and Yap1 in NIH3T3 cells confirmed their induction in
NIH3T3 cells (Fig. 5D). By
contrast, among the seven genes expressed in E8.0 embryos, only two
(Tagln and Acta2) showed a significant decrease in both
mutants (Fig. 5E; see Fig.
S3A,B in the supplementary material). Although the levels of Ctgf
were decreased in Yap1 mutants, they were increased in
Tead1/2 mutants (see Fig. S3A,B in the supplementary material).
Therefore, the genes regulated by Tead/Yap1 in NIH3T3 cells may be divergent
from those in vivo. Consistent with this hypothesis, none of the eight genes
previously identified as Yap1-regulated genes in mouse liver
(Zender et al., 2006) were
induced in NIH3T3 cells by Yap1 or Tead2-VP16 (see Fig. S3C in the
supplementary material, and data not shown). Therefore, it is likely that
expression of the Tead/Yap1-regulated genes varies depending on the cell type,
and the results obtained from overexpression of Tead2-VP16 or Yap1 in NIH3T3
cells has revealed only some of the Tead/Yap1-regulated genes. In further
support of this hypothesis, the four growth-related genes induced by Yap1 in
mouse liver (Myc, Birc2, Birc5 and Mcl1)
(Dong et al., 2007) were not
downregulated in Tead1-/-;Tead2-/- or
Yap1-/- embryos (see Fig. S3D,E in the supplementary
material). As the analysis with increased Tead activity represented only part of the roles of Tead/Yap1, we next asked whether decreasing Tead activity by expression of Tead2-EnR better represents these roles. Similar to the results of Tead2-VP16 and Yap1, the majority of the Tead2-EnR-regulated genes overlapped with the cell density-regulated genes; namely, the majority of the genes repressed or induced by Tead2-EnR overlapped with the genes repressed or induced by high cell density, respectively (Fig. 5F). Interestingly, however, only one-quarter of the Tead2-EnR-regulated genes were also regulated by Tead2-VP16 or Yap1 (see Fig. S3F,G in the supplementary material), suggesting that, even in a single cell type, growth regulations imposed by increasing or decreasing Tead activities are achieved through distinct target genes. Taken together, the results are consistent with the model that Tead and Yap1 regulate cell proliferation through diverse mechanisms that are dependent on cell types and/or conditions of cells.
|
The cell type-dependent accumulation of Tead1 and Yap1 was more evident at E10.5. Although Tead1 signal was widely detected in most nuclei, an especially strong signal was observed in the nuclei of the myocardium, notochord, floor plate of the neural tube and myotomes (Fig. 6C-F). Relatively strong signals were also observed in the endoderm and epidermis. Yap1 was also expressed widely and was excluded from the nuclei of the majority of cells (Fig. 6I). A particularly strong signal was observed in the notochord, and relatively strong signals were observed in the mesenchymal cells, including myotomes (Fig. 6I-K). In the myocardium, Yap1 was not clearly excluded from the nuclei, and some cells showed clear nuclear accumulation of Yap1 (Fig. 6L). Similar results were also obtained with a commercially available anti-Yap1 antibody (data not shown). Therefore, dynamic regulation of subcellular localization of Yap1 proteins also takes place in mouse embryos, and simultaneous increase of nuclear Yap1 and Tead1 levels in the myocardium and the notochord suggests that the Hippo signal is weak in these cells. Strong nuclear Tead1 levels in the floor plate and myotomes were not accompanied by strong nuclear Yap1 levels, suggesting that Tead1 is also regulated by mechanisms other than Hippo signaling.
As myocardium showed strong nuclear Yap1 and Tead1, an indication of low
Hippo signaling, we next examined whether Tead1 promotes cell proliferation in
these cells. Tead1-/- embryos die at E11.5 with severe
heart defects (Chen et al.,
1994; Sawada et al.,
2008). At E9.5, the Tead1 mutants are slightly smaller
than control littermates, and BrdU incorporation was slightly reduced
throughout embryos (Fig. 7A,B).
The myocardium showed strong reduction of BrdU labeling
(Fig. 7B,D), whereas BrdU
labeling of the endocardium, in which Tead1 was absent
(Fig. 7E), was not
significantly affected (Fig.
7B,D). These results are consistent with the hypothesis that
Tead1-Yap1 complex regulates cell proliferation as a mediator of Hippo
signaling in these cells.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Expanding on these observations, we first showed that cell density and Hippo
signaling also affect the level of nuclear Tead1 without altering its
subcellular distribution, and demonstrated that cell density and Hippo
signaling actually modulate transcriptional activity of endogenous Tead
proteins. Recently, others have also reported involvement of mammalian Tead
proteins in Hippo signaling (Zhao et al.,
2008). Whereas Zhao et al.
(Zhao et al., 2008) used
Tead1/3/4 knockdown and ChIP-on-chip in cultured cells to show requirement of
Tead proteins for Yap1 activities, we used gain-of-function and
dominant-negative approaches, and microarray analysis in NIH3T3 cells to
examine the effects of modulating Tead activity. These complementary studies
reached the same conclusion that mammalian Tead proteins mediate Yap1
activity. Both studies showed the involvement of Tead in growth promoting and
EMT-inducing activities of Yap1, and additionally, we showed the involvement
of Tead in two other Yap1 activities: suppression of apoptosis and cell
transformation/tumor formation. Therefore, information about cell density,
which likely originates from cell-cell contacts, ultimately converges toward
Tead activity by modulating nuclear accumulation of Tead and Yap1 through
Hippo signaling. Then, the Tead activity regulates cell proliferation,
apoptosis and EMT. Constitutive activation of Tead proteins was found to
overcome cell contact inhibition and to promote tumor formation. Although the
analysis by Zhao et al. (Zhao et al.,
2008) was restricted to cultured cells, we have extended our
analysis to mouse embryos, including Tead1/2 and Yap1 mutants, and have
revealed diversity of growth regulating mechanisms dependent on cell types,
and also showed that Tead, Yap1 and Hippo signaling may play multiple roles in
mouse embryos.
|
;
Zhao et al., 2007). Only two
genes (Tagln and Acta2) out of the 10 genes induced by
Tead2-VP16/Yap1, and none of the previously identified Yap1-induced genes were
downregulated in both Tead1-/-;Tead2-/- and
Yap1 mutant embryos. Furthermore, only one-fifth of the
Tead2-VP16/Yap1-induced genes in NIH3T3 cells were suppressed by Tead2-EnR in
the same cell line. As Tagln and Acta2 were not suppressed
by Tead2-EnR, the commonly regulated genes from all of the assays have yet to
be determined. One interpretation of these complex observations is that
Tead/Yap1 has an ability to control a wide range of growth regulators, and
regulates different subsets of the targets, depending on the types and/or
conditions of cells. An alternative interpretation is that the small number of
genes commonly regulated in various types of cells and under various
conditions constitute core batteries of the growth regulators of Hippo
signaling, and variable genes are secondarily regulated by these core
regulators. Although we do not rule out the latter possibility, we prefer the
former model, because similar differential regulation of multiple cell-cycle
regulators by a single signaling pathway was observed when cell proliferation
was modulated by increasing or decreasing Sonic hedgehog signaling in chicken
limb buds (Towers et al.,
2008). In further support of this model, although Ctgf is
a direct target gene of Tead-Yap1, which is essential for proliferation of
cultured cells (Zhao et al.,
2008), it is not downregulated in
Tead1-/-;Tead2-/- embryos, and
Ctgf-/- embryos can develop to term
(Ivkovic et al., 2003),
suggesting that regulation of Ctgf expression is not a general
mechanism for growth regulation by Hippo/Tead-Yap1.
Potential roles of Tead, Yap1 and Hippo signaling in developing mouse embryos
In NIH3T3 cells, Tead and Yap1 mediate Hippo signaling and regulate cell
contact inhibition of proliferation. In mouse embryos, the majority of the
cells showed weak Yap1 signal in the nuclei, suggesting that Hippo signaling
is active and negatively modulates cell proliferation. Although the
Yap1-related protein Wwtr1 also regulates cell proliferation and is inhibited
by Hippo signaling in cultured cells (Lei
et al., 2008), Wwtr1 proteins are always localized in the nuclei
in mouse embryos (M.O. and H.S., unpublished). Therefore, the role of Wwtr1 in
Hippo signaling in embryos may differ from that of Yap1. The cell
proliferative role of Tead1 and Yap1 is most evident in the myocardium. Strong
levels of nuclear Yap1 and Tead1 indicate weak Hippo signaling in the
myocardium, and Tead1 is required for the proliferation of
myocardium. The role of Tead/Yap1, however, may not be restricted to growth
regulation. For example, strong Tead1 signal was also observed in the
notochord, the floor plate of the neural tube and the myotomes. In the
notochord, Tead1/2 activates enhancer of Foxa2, a key regulator of
notochord differentiation (Sawada et al.,
2008; Sawada et al.,
2005). As accumulation of Yap1 and Tead1 proteins indicates
suppression of Hippo signaling in the notochord, Hippo signaling may suppress
differentiation of the notochord. A similar cell fate specification role for
Hippo signaling is also present in Drosophila photoreceptor cells
(Mikeladze-Dvali et al.,
2005). In the myotomes, Tead1 accumulation was not accompanied by
clear increase in Yap1. Instead, another Tead co-factor protein, Vgll2, is
specifically expressed in the myotomes and promotes skeletal muscle
differentiation through Tead proteins (Chen
et al., 2004; Maeda et al.,
2002). Therefore, in the myotome, Tead regulates skeletal muscle
differentiation independently of Hippo signaling. In the floor plate, strong
Tead1 signal is not accompanied by increased Yap1 or Vgll. Although the role
of Tead proteins in the floor plate is currently unknown, considering that the
floor plate is a mitotically inactive tissue, it is tempting to speculate that
Tead1 suppresses its targets in order to suppress cell proliferation in this
region. In fact, overexpression of Tead proteins inhibits their activator
functions by `squelching' co-activator proteins in cultured cells
(Xiao et al., 1991).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/24/4059/DC1
|
ACKNOWLEDGMENTS |
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|
REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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