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First published online January 11, 2008
doi: 10.1242/10.1242/dev.007047
1 James Graham Brown Cancer Center, Department of Ophthalmology and Visual
Sciences, University of Louisville Health Sciences Center, Louisville, KY
40202, USA.
2 Departments of Peiodontics, Endodontics and Dental Hygiene, Center for Oral
Health and Systemic Disease, University of Louisville School of Dentistry,
Louisville, KY 40292, USA.
3 Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
* Author for correspondence (e-mail: dcdean01{at}louisville.edu)
Accepted 16 October 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Zeb1, Epithelial-mesenchymal transition, Senescence, Transcription
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Thiery, 2003). This transition
involves repression of key epithelial genes, highlighted by the cell-cell
contact modulator, E-cadherin (also known as cadherin 1). Downregulation of
E-cadherin, and the resulting release of cell-cell adhesion in tumors, is
thought to contribute a motile phenotype that facilitates metastasis.
Conversely, mesenchymal genes including vimentin and smooth muscle actin as
well as various matrix and matrix-degrading enzymes are induced during EMT.
The resulting alterations in cell shape, matrix expression and ability to
digest and move through different tissues are thought to work in conjunction
with loss of cell-cell contact to generate the metastatic phenotype. However,
EMT is not simply a cancer-driven process, it is crucial early in
embryogenesis for delamination of neural crest from the neural tube, and
defining an ectodermal-mesodermal boundary
(Hay and Zuk, 1995;
Thiery, 2003;
Cheung et al., 2005). Later in
gestation, EMT is important for proper kidney and heart formation.
Furthermore, EMT is also a crucial feature of the pathologic fibrotic response
to injury (e.g. wound healing and fibrotic organ diseases)
(Hay and Zuk, 1995;
Thiery, 2003;
Zavadil and Bottinger, 2005).
EMT during embryogenesis, in cancer metastasis and in fibrotic responses, is
thought to be driven by TGF-β family members (reviewed by
Zavadil and Bottinger,
2005).
Zinc finger E-box binding homeobox 1 (Zeb1; also known as Zfhx1A,
EF1, Tcf8 and Zfhep) binds a set of E-box-like elements that overlap
with those bound by Zeb2 (also known as Sip1) and the Snail family
(Genetta et al., 1994;
Sekido et al., 1994;
Postigo and Dean, 2000). Each
of these E-box-binding proteins can act as a transcriptional repressor through
recruitment of the co-repressor, C-terminal binding protein (CtBP; Ctbp1)
(Postigo and Dean, 1999;
Grooteclase and Frisch, 2000; Chinnadurai,
2002; Hemavathy et al.,
2005). CtBP is a part of a larger complex including polycomb
proteins and CoREST (also known as Rcor2) that causes epigenetic modification
of DNA and histones leading to heterochromatin assembly and, thereby,
transcriptional silencing [see Ringrose et al.
(Ringrose et al., 2004) and
references therein]. A CtBP-CoREST repressor complex is targeted, via
interaction with Zeb1, to genes crucial for late-stage pituitary organogenesis
(Wang et al., 2007).
Overexpression of Zeb1 in cancer is associated with repression of
E-cadherin and EMT (Guaita 2002; Eger et
al., 2005; Pena et al.,
2005; Spoelstra et al.,
2006; Witta et al.,
2006; Peinado et al.,
2007). Zeb1 can also serve as a transcriptional activator, and
this seems to be directed at least in part toward mesenchymal genes such as
collagens, smooth muscle actin and myosin, vimentin, and genes in the vitamin
D signaling pathway, which is important in mesenchymal differentiation
(Chamberlain and Sanders, 1999;
Lazarova et al., 2001;
Dillner and Sanders, 2002;
Postigo, 2003;
Postigo et al., 2003;
van Grunsven et al., 2006;
Nishimura et al., 2006).
Heterozygous mutation of Zeb1 leads to impaired smooth muscle actin
and myosin expression and TGF-β-dependent smooth muscle cell
differentiation following vascular injury
(Nishimura et al., 2006).
Thus, Zeb1 can contribute to repression of epithelial genes as well as to
activation of mesenchymal genes.
TGF-β expression by tumors and surrounding stroma drives EMT in
cancer, and TGF-β family members are also crucial for EMT during
development (Zavadil and Bottinger,
2005). Accordingly, TGF-β represses epithelial genes such as
E-cadherin and induces mesenchymal genes such as vimentin. In vivo, Zeb1 is
important for TGF-β-dependent smooth muscle cell differentiation, and
TGF-β-dependent expression of smooth muscle actin and myosin genes
(Nishimura et al., 2006). Zeb1
binds activated Smads as well as the histone acetyl transferase p300 (also
known as pCaf), which is an essential Smad co-activator, and this binding
facilitates assembly of a Smad-p300 complex while leading to dissociation of
Zeb1 from its co-repressor, CtBP (Zhang et
al., 2000; Postigo et al.,
2003; Postigo,
2003; van Grunsven et al.,
2006). Thus, in response to TGF-β and in the presence of
activated Smads and p300, Zeb1 is switched from a repressor to a
co-activator.
Zeb1 is expressed in proliferating mesenchymal and neural progenitors, and
mutation of the Zeb1 leads to cleft secondary palate, defective nasal
formation and other craniofacial abnormalities
(Takagi et al., 1998). Forming
cartilage at these sites appears hypoplastic. In addition to craniofacial
defects, the mice have skeletal abnormalities including shortened limbs and
digits as well as fusion and curvatures in the skeleton and tail. A subset of
the embryos exhibits severe CNS defects, including failure of neural tube
closure at both cranial and caudal ends, and exencephaly
(Takagi et al., 1998). The
molecular basis for these various defects is unknown.
As opposed to EMT seen when Zeb1 is overexpressed in rapidly proliferating cancer cells, we present evidence here that mutation of Zeb1 causes mesenchymal-epithelial transition in gene expression and diminished proliferation in progenitor cells at sites of developmental defects in mouse embryos. This phenotype extends to mouse embryo fibroblasts (MEFs) derived from mutant mice. These cells ectopically express E-cadherin, which is associated with an abnormal epithelial-like morphology. Additionally, they undergo premature replicative senescence in culture, which is associated with ectopic expression of cell cycle inhibitory cyclin-dependent kinase inhibitors (CDKIs), p15Ink4b and p21Cdkn1a (also known as Cdkn2b and Cdkn1a, respectively - Mouse Genome Informatics). This ectopic expression of p15Ink4b is also seen in vivo at sites of diminished proliferation of progenitor cells (and at sites of developmental defects) in Zeb1 mutant embryos.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Cells
of different genotypes were cultured in DMEM with 10% fetal bovine serum (FBS)
at 10% CO2, and split 1:3 as soon as confluent. For experiments
with TGF-β, 1 day after splitting, TGF-β1 (BioSource, Camarillo, CA)
was added to the cultures at final concentrations of 5 pM, 25 pM and 75 pM,
and RNA was isolated 24 hours later.
RNA extraction and real-time PCR
RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA). cDNA was
synthesized using the Invitrogen RT Kit according to the manufacturer's
protocol (Invitrogen). SYBR Green real-time quantitative PCR was performed
using the Mx3000P Real-Time PCR System (Stratagene, Cedar Creek, TX) according
to the manufacturer's instructions. The RT-PCR primers and annealing
temperature are shown in Table
1. Three independent samples were analyzed for each condition
and/or cell type, and each sample was compared in at least three independent
RT-PCR amplifications.
|
), histone
H3 and histone H4 (Santa Cruz Biotechnology, Santa Cruz, CA) were used for
immunoprecipitation. Equal amounts of anti-IgG or preimmune serum were used as
controls. ChIP PCR reactions were similar to those described above for
real-time PCR using primer sets (Table
2) to amplify promoter sequences of E-cadherin and Gapdh
genes.
|
Analysis of cell proliferation in vivo
Two hours before collection of E15.5 embryos, mothers received an
intraperitioneal injection of 40 mg/kg 5'-bromodeoxyuridine (BrdU) in
PBS. Embryos were fixed in 10% buffered formalin, embedded in paraffin and
sectioned at 5 µm. Sections were incubated with 0.1% Tween 20, 4% goat (for
Zeb1 antibody) (Darling et al.,
2003) or sheep (for BrdU antibody) serum, and 2% bovine serum
albumin (BSA) (Sigma) for 1 hour. Polyclonal primary antibodies against Zeb1
and BrdU (raised in rabbit and mouse, respectively) were applied to the
sections at 1:50, and incubated at 4°C overnight. Slides were then
incubated at 1:300 either with anti-rabbit IgG conjugated with Alexa Fluor 488
or Cy3 for 1 hour. The slides were viewed with an Olympus confocal
microscope.
|
). For
these experiments, sections (5 µm) from corresponding regions of wild-type
and null littermate embryos were immunostained for BrdU and observed using a
Zeiss LSM 510 confocal laser-scanning microscope. Optical sections (1 µm)
were incorporated into a z-stack using a 40x objective, and
LSM510 software was used to create a 3-D image. Three adjacent sections were
analyzed from each embryo. The percentage of BrdU-positive cells within
similar fields was determined. Similar results were also obtained simply by
counting the number of BrdU-positive cells in the field. Averages from two
wild-type and three null embryos (and three adjacent sections from each
embryo) are presented.
Cellular senescence assays
Senescent β-galactosidase activity was analyzed using the X-Gal-based
Cell Senescence Staining Kit (Sigma) following the manufacturer's protocol.
Stained cells were photographed, and randomly selected fields were counted to
determine the percentage of positive cells.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As noted above, a previous report suggested from histological evidence that at least some sites of forming cartilage are hyoplastic in Zeb1-null mice. We found that Zeb1 is expressed in the perichondrium surrounding forming cartilage at E15.5, which consists of mesenchymal progenitors that contribute to the forming cartilage (Fig. 2A). As in the palate and nasal region, vimentin was co-expressed with Zeb1 in the perichondrium (Fig. 2B), and E-cadherin was not expressed in these mesenchymal cells (it was, however, expressed in the forming skin) (Fig. 2C). The perichondrium is known to be highly proliferative, and indeed these cells were labeled when pregnant mice were injected with BrdU, as were embryos harvested 2 hours later (Fig. 2D). Double immunolabeling for Zeb1 and BrdU showed that most BrdU-positive cells in the perichondrium expressed Zeb1 (Fig. 2E-G). Next, we assessed whether mutation of Zeb1 would have an effect on expression of vimentin or E-cadherin in the perichondrium, or on proliferation of the cells. As in the palate and nasal mesenchyme, vimentin expression was lost in the perichondrium with Zeb1 mutation (Fig. 2H), and whereas E-cadherin expression was maintained on the forming skin, it became ectopically expressed on the perichondrium (Fig. 2G). Concomitant with this change in E-cadherin and vimentin expression, the perichondrium showed diminished proliferation (Fig. 2J).
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|
). As
with vimentin, we found that GFAP was expressed in the forming retina, optic
nerve and surrounding eye muscles (Fig.
3D). Again, mutation of Zeb1 led to ectopic expression of
E-cadherin in the forming retina, optic nerve and eye muscles
(Fig. 3E), whereas expression
of both vimentin and GFAP in the retina, optic nerve and eye muscle was
diminished (Fig. 4F).
In addition to neuroectodermally derived progenitors in the eye, Zeb1 was
also evident in progenitor cells in the brain, including cells in the
ventricular zone of the lateral ventricles
(Fig. 4A), the olfactory bulb
and other regions of the brain (see Fig. S3 in the supplementary material).
Cells in these regions also expressed both vimentin and GFAP
(Fig. 4B,C). And as in the
retina and optic nerve, vimentin and GFAP expression was diminished in these
cells in Zeb1-null mice (Fig.
4D). E-cadherin was not detected in the embryonic brain late in
gestation (E15.5-17.5) (Fig.
4E; see Fig. S3 in the supplementary material). However, in the
null mice, it appeared on cells in the ventricular zone of the lateral
ventricles and in the olfactory bulb, which is populated by progenitors from
the ventricular zone (Curtis et al.,
2007) (Fig. 4F,G;
see Fig. S3 in the supplementary material). Additionally, E-cadherin also
appeared in proliferative regions of the third ventricle, the telencephalic
vesicle, the thalamus and the hypothalamus
(Fig. 4H; data not shown). This
ectopic expression of E-cadherin was confined to sites in the brain that
normally express Zeb1, and which are known to be sites of proliferating
progenitor cells late in gestation.
A subset of Zeb1-null embryos show severe CNS defects, with
failure of neural tube closure at both the cranial and caudal ends and
exencephaly. As with proliferating mesenchymal cells, Zeb1 expression
overlapped significantly with BrdU incorporation in the ventricular zone of
the lateral ventricles and in other proliferative regions of the brain at
E15.5 (Fig. 4I-L; data not
shown). Low-magnification views of head sections of embryos immunostained for
BrdU suggested that proliferation in the ventricular zone of the lateral
ventricles and other regions of the brain was diminished in Zeb1-null
mice (see Fig. S4 in the supplementary material). Therefore, we counted the
percentage of BrdU-positive cells at various sites in the brain (see Materials
and methods). Corresponding regions from three null and two wild-type
littermates were analyzed. Three adjacent sections were counted for each
embryo. A significant decrease in BrdU incorporation was seen in each of the
sites in the null embryos, as compared with the wild-type embryos
(Fig. 4M). This diminished
proliferation in progenitors in the ventricular zone of the brain was similar
to, or greater than, that seen with mutation of Bmi1
(Molofsky et al., 2005). As a
control, no significant difference in BrdU incorporation was seen in the
tongue of wild-type versus Zeb1-null embryos
(Fig. 4M; see Fig. S4 in the
supplementary material). Thus, the switch in expression of the epithelial gene
E-cadherin and of the mesenchymal gene vimentin in both mesenchymal
progenitors in the forming cartilage and in neuroectodermally derived
progenitors in the brain of Zeb1-null mice is linked to diminished
proliferation of these populations and to developmental defects.
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|
We wondered whether the ectopic expression of E-cadherin seen with
Zeb1 mutation might result indirectly from the downregulation of one
of the other E-box-binding repressors. Therefore, we compared expression of
Zeb2, Snail1 and Snail2 (also known as Snai1 and
Snai2 - Mouse Genome Informatics) mRNA levels in wild-type and
Zeb1 mutant MEFs. No significant change was seen in Snail1
or Snail2 mRNA levels in heterozygous or null MEFs, and expression of
Zeb2 was induced with Zeb1 mutation
(Fig. 6B). This finding of
Zeb2 mRNA induction is consistent with a recent report showing
increased expression of Zeb2 in smooth muscle cells of Zeb1 mutant
mice (Nishimura et al., 2006).
Therefore, ectopic expression of E-cadherin in Zeb1 mutant MEFs is
not an indirect result of downregulation of Zeb2, Snail1 or
Snail2 mRNA. Indeed, E-cadherin is expressed in the cells despite the
fact that Zeb2 mRNA is upregulated (and Snail1/2 mRNA is
unchanged).
|
Zeb1 and TGF-β induction of vimentin and repression of E-cadherin
TGF-β drives EMT by inducing mesenchymal genes such as vimentin, and
repressing epithelial genes such as E-cadherin. Given the previously
documented linkage between Zeb1 and TGF-β signaling discussed above, we
wondered whether the effects of Zeb1 on repression of E-cadherin and induction
of vimentin might be related to TGF-β signaling. Initially, we asked
whether basal vimentin mRNA expression was affected by Zeb1 mutation
in MEFs. However, we found no significant difference in the level of vimentin
mRNA in wild-type versus null MEFs (Fig.
6C). Therefore, we asked whether Zeb1 might be important for
TGF-β-mediated induction of vimentin. Indeed, we found that vimentin mRNA
induction by TGF-β was lost in the null MEFs
(Fig. 6C). However,
Zeb1 mutation did not lead to a general block in TGF-β signaling
because we found that TGF-β-mediated induction of plasminogen activator
inhibitor 1 (also known as Serpine1 - Mouse Genome Informatics) mRNA
was not diminished in Zeb1-null MEFs
(Fig. 6D). We conclude that
Zeb1 is required for TGF-β-mediated induction of vimentin in the
MEFs.
Our results above show that Zeb1 is required to prevent ectopic
E-cadherin expression in MEFs. We then assessed whether TGF-β would be
able to repress E-cadherin expression in the null MEFs. Indeed, we found that
TGF-β was unable to repress E-cadherin mRNA in the absence of
Zeb1 (Fig. 6A). Zeb1
expression is known to be induced by TGF-β
(Nishimura et al., 2006).
Thus, taken together, the results raise the possibility that TGF-β might
repress E-cadherin through induction of Zeb1.
Mutation of Zeb1 leads to Ink4a-independent premature replicative senescence in MEFs
We then asked whether the proliferative defects in Zeb1-null mice
that we observed in ventricular zone and mesenchymal progenitor cells might
also be reflected in MEFs from mutant mice. Zeb1 mutant and wild-type
littermate-matched MEFs were compared for proliferation. We found that
Zeb1-null cells arrested by passage (P) 2, whereas heterozygous cells
stopped proliferating by P4 (Fig.
7A). Wild-type cells continued proliferating beyond P10
(Fig. 7A; data not shown). We
noticed that the arrested cells adopted a flattened morphology and they
remained non-proliferative but viable for months in culture
(Fig. 7B; data not shown).
Because these are properties of senescence, we stained the cells for senescent
β-galactosidase activity. We found that essentially all of the arrested
heterozygous and null cells expressed senescent β-galactosidase, whereas
only 
30% of wild-type cells were positive at P9
(Fig. 7C). We conclude that the
mutant MEFs undergo premature replicative senescence in a Zeb1
dosage-dependent fashion.
|
). Bmi1 negatively regulates Ink4a, and in the absence of Bmi1
this locus expresses the CDKI, p16Ink4a, and the p53 regulator p19Arf (Arf)
(reviewed by Gil and Peters,
2006). Induction of Ink4a in Bmi1-null embryo fibroblasts
triggers senescence of MEFs (Valk-Lingbeek
et al., 2004), and a cross of the Bmi1-null mice to
Ink4a-null mice prevents senescence of the ventricular zone
progenitors in vivo (Jacobs et al.,
1999). It is of note that normal replicative senescence of
wild-type MEFs results from time-dependent induction of the Ink4a
locus in culture (usually leading to senescence around P10-12)
(Lowe and Sherr, 2003;
Gil and Peters, 2006).
Mutation of Bmi1 accelerates this Ink4a activation, leading to premature
replicative senescence.
To investigate whether activation of the Ink4a pathway was responsible for
premature senescence of the Zeb1 mutant MEFs, we used real-time PCR
to analyze expression of mRNAs from the Ink4a locus, and from genes
known to regulate this locus including Bmi1 (repressor),
Ets1 (activator), Tbx2 (repressor) and Tbx3
(repressor) (Jacobs et al.,
1999; Lessard and Sauvageau,
2003; Lingbeek et al.,
2002; Ohtani et al.,
2001). We failed to find upregulation of p16Ink4a or
Arf (or any of the Ink4a regulators) in senescent
Zeb1 mutant cells (Fig.
8A). Instead, expression of both mRNAs was downregulated in the
null and heterozygous cells compared with in wild-type cells. It is of note
that this downregulation of p16Ink4a and Arf mRNA correlates
with Zeb1 dosage-dependent downregulation of the Ink4a inducer
Ets1 (Fig. 8A). As a
positive control, we found that p16Ink4A expression was induced with passage
number in the wild-type MEFs, as reported previously by a number of groups
(Lowe and Sherr, 2003;
Gil and Peters, 2006;
Liu et al., 2007). We conclude
that the premature replicative senescence seen in the Zeb1 mutant
MEFs is not the result of activation of the classic Ink4a locus.
p15Ink4b and p21Cdkn1a are ectopically expressed in Zeb1 mutant MEFs
TGF-β treatment classically induces growth arrest, but this does not
involve induction of Ink4a; instead, two other CDKIs, p21Cdkn1A and p15Ink4b,
are induced to trigger the growth arrest
(Reynisdottir et al., 1995).
Because of the known linkage of Zeb1 to TGF-β signaling, we asked whether
these CDKIs might be ectopically expressed in the Zeb1 mutant MEFs.
Indeed, we found a gene dosage-dependent increase in both mRNAs, with the
increase in p15Ink4b mRNA being the most dramatic
(Fig. 8B). These results
demonstrate that Zeb1 is required to prevent ectopic expression of
these CDKIs in MEFs.
Next, we asked whether Zeb1 acts directly on the p21Cdkn1a and p15Ink4b gene promoters. In ChIP assays, Zeb1 was bound to both promoters, but no interaction was seen with the control Gapdh promoter (Fig. 8C-E). These results are consistent with the notion that Zeb1 is represses these CDKIs directly by binding to their promoters in vivo.
p15Ink4b is ectopically expressed in mesenchymal and ventricular zone progenitor cells in Zeb1-null embryos
p15Ink4b expression was dramatically induced in Zeb1 mutant MEFs
(Fig. 8B), and expression of
p15Ink4b is known to block cell proliferation
(Reynisdottir et al., 1995).
Therefore, we asked whether p15Ink4b might become ectopically expressed in
Zeb1-null mice at sites of diminished proliferation. We did not
detect p15Ink4a expression in mesenchymal cells or in the ventricular zone in
wild-type mice (Fig. 9A,B).
Indeed, the developing skin was the only tissue where we observed significant
expression (see Fig. S5 in the supplementary material). However, as in the
MEFs, p15Ink4b became ectopically expressed in the perichondrium in
Zeb1-null mice (Fig.
9C,D; see Fig. S5 in the supplementary material), and these cells
showed diminished proliferation (Fig.
2D,J).
p15Ink4b expression was also ectopically expressed in the ventricular zone
of Zeb1-null mice (Fig.
9E,F). Although there was overlap between p15Ink4b and BrdU
incorporation, it is of note that there were a number of additional
p15Ink4b-positive cells in this region that were BrdU-negative
(Fig. 9G-I), and there was
overall diminished proliferation of cells in this region. These results are
consistent with the notion that ectopic expression of p15Ink4b in
Zeb1-null mice in both mesenchymal and ventricular zone progenitors
leads to diminished proliferation. Previous studies have found expression of
p15Ink4b in the chick embryo hindbrain, consistent with a role in controlling
cell proliferation in this region (Kim et
al., 2006).
|
|
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
The epithelial and mesenchymal genes as well as the CDKIs that are
deregulated in response to Zeb1 mutation, have in common their
regulation by TGF-β, effecting EMT and cell cycle arrest, respectively.
The linkage of each of these genes to TGF-β, together with the fact that
Zeb1 participates in TGF-β signaling via binding to activated Smads,
suggests that the function of Zeb1 in maintaining the balance between
mesenchymal and epithelial gene expression and in cell proliferation might be
associated with TGF-β superfamily signaling. In this regard, it is of
note that Zeb1 was required for TGF-β regulation of key epithelial and
mesenchymal genes (e.g. E-cadherin and vimentin). Furthermore, Zeb1 had
opposing effects on the two genes, in keeping with the mesenchymal-epithelial
gene expression transition seen in Zeb1 mutant mice. Mutation of
Zeb1 led to ectopic expression of E-cadherin, and TGF-β was no
longer able to repress the gene. Because TGF-β induces Zeb1
(Nishimura et al., 2006), we
suggest that induction of Zeb1 is a mechanism for TGF-β repression of
E-cadherin, and this is likely to occur via recruitment of a Zeb1-CtBP
repressor complex to the E-cadherin promoter. In contrast to E-cadherin,
vimentin expression is induced by TGF-β. Its expression was diminished in
Zeb1 mutants, and Zeb1 was required for TGF-β-mediated induction
of vimentin in MEFs. Gene induction by TGF-β is mediated by activation of
Smad transcription factors, and we suggest that Zeb1-dependent
TGF-β-mediated induction of genes such as vimentin and smooth muscle
actin and myosin might be the result of Zeb1 being required to mediate
efficient assembly of a Smad-p300 transcription complex at their promoters (a
complex which excludes CtBP) (Postigo,
2003).
It has been shown that overexpression of Zeb1 facilitates TGF-β
induction of the p15Ink4b promoter in transfection assays
(Postigo, 2003). Yet, here we
found that p15Ink4b is ectopically expressed in Zeb1 mutant cells.
These findings are not necessarily contradictory. Taken together, they suggest
that p15Ink4b is under repression by Zeb1. Because the
p15Ink4b gene is a known target of activated Smads, we propose that
recruitment of a Smad-Zeb1-p300 complex to the promoter in response to
TGF-β might serve to displace CtBP from Zeb1, leading to derepression of
the gene.
We suggest that Zeb1 is important for regulating the balance between
mesenchymal and epithelial gene expression and for maintaining the
proliferation of a subset of progenitor cells late in gestation. But, it is
interesting that this phenotype extends to premature replicative senescence in
cultured MEFs. Mutations in other genes such as Bmi1 show a similar
premature senescence phenotype in MEFs. Such a phenotype in MEFs is closely
linked to diminished proliferation and senescence of progenitor cells in the
CNS and bone marrow. Although these other mutations involve Ink4a regulators
and p16Ink4a itself, it is of note that we observe diminished CNS progenitor
proliferation in Zeb1-null mice, and this effect was similar to, or
even greater than, that observed with Bmi1 mutation
(Molofsky et al., 2005).
Interestingly, TGF-β and p15Ink4b have key roles in restricting
proliferation of T-cell progenitors, and mutation or epigenetic silencing of
p15Ink4b leads to lymphoproliferative disease
(Latres et al., 2000;
Wolff et al., 2003;
Lessard and Sauvageau, 2003;
Mishra et al., 2005), and is
common in leukemia (reviewed by Claus and
Lubbert, 2003). It is of note that Zeb1-null mice have a
diminished number of T-cell progenitors, which fail to populate the thymus
(Higashi et al., 1997;
Takagi et al., 1998). These
results raise the possibility of a proliferative defect in a subset of bone
marrow-derived progenitors with Zeb1 mutation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/3/579/DC1
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ACKNOWLEDGMENTS |
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