|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 25 February 2009
doi: 10.1242/dev.029538
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Department of Molecular and Cellular Biology, University of Arizona, Tucson,
AZ 85724, USA.
2 Department of Cell Biology and Anatomy, University of Arizona, Tucson, AZ
85724, USA.
Author for correspondence (e-mail:
pkrieg{at}email.arizona.edu)
Accepted 2 February 2009
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Tie2, VE-cadherin, VEGFR2, Xenopus, Endothelial lineage, Vascular development, KLF2
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
Ferrara et al., 1996).
Similarly, homozygous Flk1-null embryos die at E8.5-9.5 with severe
reduction in endothelial cell number and complete disruption of subsequent
vascular development (Shalaby et al.,
1995). Since FLK1 is amongst the earliest markers of the
endothelial lineage, characterization of transcription mechanisms regulating
Flk1 gene expression will help to advance our understanding of the
regulatory pathways controlling vascular development.
Mouse transgenic studies have shown that endothelial-specific expression of
Flk1 is regulated by an enhancer in the first intron, which contains
binding elements for the transcription factors TAL1 (SCL) and members of the
GATA and ETS families (Kappel et al.,
1999). Mutation of the GATA or ETS motifs abolishes reporter
expression in endothelial cells of transgenic mice, whereas mutation of the
TAL1 site results in reduced expression levels
(Kappel et al., 2000).
Additional ETS motifs are located in the promoter region of the mouse
Flk1 gene and these have been shown to function together with
HIF-2
(EPAS1 - Mouse Genome Informatics) to regulate Flk1
transcription (Elvert et al.,
2003). The requirement for ETS binding sites in Flk1
regulatory regions is consistent with the established function of ETS
transcription factors in the regulation of vascular development
(Dejana et al., 2007).
Approximately 30 ETS factors are known in mammals and all members share a
conserved DNA-binding domain that recognizes the core recognition sequence
GGA(A/T) (Lelievre et al.,
2001; Sharrocks,
2001). ETS proteins frequently interact with partners to influence
tissue-specific gene regulation (Lelievre
et al., 2001; Sharrocks,
2001; Oikawa and Yamada,
2003), but very little is known about possible ETS partners in
endothelial cells. Transgenic analysis has demonstrated the importance of ETS
motifs for expression of several vascular genes in addition to Flk1,
including Tie2 (Schlaeger et al.,
1997) and VE-cadherin (cadherin 5)
(Gory et al., 1999).
Gain-of-function experiments have shown that ETS factors can upregulate
endothelial gene expression in cultured cells
(Birdsey et al., 2008;
Hasegawa et al., 2004;
Schwachtgen et al., 1997;
Wakiya et al., 1996).
Overexpression of the ETS factor ERG in Xenopus embryos is sufficient
to activate ectopic transcription of the vascular marker X-msr
(Baltzinger et al., 1999).
At least four ETS genes, Ets1, Erg, Fli1 and Er71
(Etv2), are expressed in mouse embryonic endothelial cells
(Lelievre et al., 2001;
Lee et al., 2008). Owing to
functional redundancy between family members, most loss-of-function studies of
individual ETS factors have not revealed early vascular phenotypes. A striking
exception is the knockout of the mouse Er71 gene, which shows greatly
reduced angioblast cell numbers and severe disruption of vascular development
(Lee et al., 2008). Zebrafish
studies have shown that knockdown of four vascular ETS genes results in a near
complete loss of endothelial cells, whereas single knockdowns of individual
genes exhibit less severe phenotypes (Pham
et al., 2007).
The Krüppel-like factor (KLF) family of transcription regulators is
also involved in the regulation of vascular gene expression
(Atkins and Jain, 2007). KLFs
bind a consensus recognition sequence of CACCC
(Bieker, 2001; Dang et al.,
2001), and three of the 17 family members, KLF2, KLF4 and KLF6, are expressed
in the mouse embryonic vasculature (Kuo et
al., 1997; Yet et al.,
1998; Kojima et al.,
2000; Botella et al.,
2002; Lee et al.,
2006). KLF proteins can act as either transcriptional activators
or repressors and domain mapping of KLF2 has identified transactivating and
transrepression domains within the protein
(Conkright et al., 2001).
Numerous endothelial genes have KLF binding sites in their promoter regions
and cell culture studies have shown that KLF2 activates the expression of
vascular genes including thrombomodulin
(Lin et al., 2005) and
eNOS (Nos3) (Parmar et
al., 2006; Dekker et al.,
2005), but inhibits expression of other vascular genes including
endothelin and adrenomedullin (Dekker et
al., 2006). Mice lacking either KLF2 or KLF4 activity are not
viable; however, early vascular development in both knockouts is normal
(Kuo et al., 1997;
Lee et al., 2006;
Segre et al., 1999). Mice
lacking KLF6 function exhibit vascular assembly defects in the yolk sac, but
endothelial gene expression and development of blood vessels in the embryo
itself are apparently normal (Matsumoto et
al., 2006). It is interesting to note that embryonic stem (ES)
cells lacking KLF6 activity show reduced levels of FLK1 protein after
differentiation into embryoid bodies
(Matsumoto et al., 2006),
suggesting that KLF6 might function as an activator of Flk1
expression. Like the ETS proteins, it is possible that redundant expression of
different KLF family members can exert a rescuing function and, indeed,
redundancy has been demonstrated in studies using ES cells in which any one of
three specific KLFs (KLF2, KLF4 or KLF5) could substitute for the other two in
maintaining ES cells in an undifferentiated state
(Jiang et al., 2008). Of
particular relevance to the study of Flk1 transcriptional regulation,
both cell culture and microarray studies using adult endothelial cells have
suggested that KLF2 functions as a repressor of Flk1 expression
(Bhattacharya et al., 2005;
Dekker et al., 2006).
|
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
Baltzinger et al., 1999;
Devic et al., 1996). The KLF2
coding region was PCR amplified from BC043732 with Pfu polymerase, subcloned
into pT7TS and the sequence verified. For synthesis of Klf2 mRNA,
Klf2-pT7TS was linearized with XbaI and transcribed with T7
RNA polymerase (Message Machine Kit, Ambion). A dominant-repressor form of
KLF2 (DR-KLF2) lacking the first 87 amino acids that make up the
transactivation domain was generated by inverse PCR from Klf2-pT7TS
template using Pfu polymerase and the sequence verified. The ERG coding
sequence (AJ224126) was PCR amplified using Pfu polymerase, cloned into pT7TS,
sequence verified, linearized with XbaI and mRNA synthesized using T7
RNA polymerase.
Microinjection and embryological manipulation
For embryonic expression experiments, mRNAs encoding ERG, KLF2, DR-KLF2 and
EGFP were injected into a single vegetal blastomere of a four-cell stage
embryo in 0.4x MMR containing 6% Ficoll and cultured thereafter in
0.2x MMR until assay (stages 34-36). Embryos were staged according to
Nieuwkoop and Faber (Nieuwkoop and Faber,
1994). Whole-mount in situ hybridization was carried out using
digoxigenin-labeled probes and standard conditions
(Harland, 1991). Preparation
and microinjection of morpholino oligomers (MOs) was performed as described
previously (Garriock et al.,
2005). Klf2 antisense MO (Klf2 MO,
5'-ATCCGAATCAGATTGTCAGCAAAAC-3') was targeted to the 5'
untranslated region (UTR) of Klf2 transcripts. Klf2 MO
effectively blocked translation of Klf2 test transcripts containing
the 5' UTR plus a portion of the coding region sequences of
Klf2 fused to the coding region of EGFP (see
Fig. 3D,E). For in vivo
experiments, 12.5, 25 or 50 ng of Klf2 MO or control antisense MO
(5'-GGTAGTAATAGATGCTGTGATCTAT-3') was microinjected into the
mediolateral region of one cell of two-cell staged embryos and later assayed
at stage 34 for Flk1 transcripts by whole-mount in situ
hybridization. For measuring Flk1 transcript levels, Klf2 or
control MO was injected at the one-cell stage.
Xenopus transgenics and transient assays
A Flk1 genomic fragment comprising 
2.5 kb of sequence from
upstream of the transcriptional start site to within exon 2 was isolated. For
transgenic analysis, the EGFP coding region was inserted into exon 1 of a
Flk1 construction containing 2.5 kb of 5' flanking sequences
plus 1.5 kb of first intron sequences. Transgenic mutant constructs for the
ETS and KLF sites were generated by inverse PCR using Pfu polymerase and the
sequence verified. The ETS site 5'-GGAT-3' was mutated to
5'-GTAT-3' and the KLF site 5'-CACCCT-3' was mutated
to 5'-CGGTCG-3'. Xenopus transgenic embryos were
generated as described (Kroll and Amaya,
1996; Sparrow et al.,
2000).
For luciferase assays, mouse bEnd.3 endothelial cells (ATCC# CRL-2299) were transfected with 0.5 µg of reporter plasmid using Fugene 6 (Roche). Flk1 reporter constructions (wild-type, ETS mutant and KLF mutant) were identical to transgenic constructions except the luciferase coding region was substituted in place of EGFP. For assay of the ETS/KLF module alone, three copies of the ETS/KLF sequence were inserted in tandem upstream of the minimal SV40 promoter in the luciferase reporter construction pGL3-Promoter vector (Promega). For both sets of experiments, cells were co-transfected with 0.1 µg of plasmid containing a CMV promoter driving β-galactosidase for normalization of transfection efficiency. Transfected cells were incubated in a 12-well plate for 20 hours and luciferase and β-galactosidase activity was measured using a luminometer. Experimental transfections were performed in quadruplicate.
Electrophoretic mobility shift assay (EMSA)
Nuclear protein extracts from mouse EOMA (ATCC# CRL-2586) and L cell
fibroblast (CCL-1) cell lines were isolated according to standard procedures.
HA-tagged mouse KLF2 was generated by transfection of a HA-KLF2 expression
plasmid into COS-7 cells using Fugene 6, and protein extracts were isolated in
15 mM Tris-HCl (pH 7.5) containing 1% Triton X-100, 120 mM NaCl, 25 mM KCl and
protease inhibitors. Radiolabeled probes included: WT used in
Fig. 2A
(5'-GTACTCTCCACCCTGGTGC-3') and WT used in
Fig. 2B
(5'-TAAGACTCCACCCTGGCC-3'). Competition oligonucleotides included
a mutated KLF site (Mut, 5'-GTACTTTAGATGCAGGTGC-3') and a
serum-response element (SRE, 5'-CTAGGTTTCAGGGTCCTGCCATAAAAG-3').
EMSA was performed using standard techniques.
|
).
Co-immunoprecipitation experiments
Co-immunoprecipitation experiments were conducted as described
(Meadows et al., 2008).
Briefly, HA-tagged mouse KLF2 was generated by transfection into COS-7 cells
and verified by protein blotting with detection using chemiluminescent
solution (Supersignal, West Dura). ERG and EGFP proteins were radiolabeled
with 35S methionine by translation in the Wheat Germ Cell-Free
Translation System (Promega). COS-7 cell extracts containing HA-KLF2 were
mixed with in vitro translated products and immunoprecipitation was carried
out using standard protocols and anti-HA antibody (Roche). Bound proteins were
fractionated on a 10% SDS-PAGE gel and visualized by autoradiography. Human
ERG was modified with a FLAG epitope, inserted into the expression vector
pcDNA3.1+ (Promega) and co-transfected into COS-7 cells with HA-KLF2.
Immunoprecipitation was carried out using anti-FLAG monoclonal antibody
(Sigma) and proteins detected as described above.
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
120
bp segment that is highly conserved from human to frog. This conserved element
is contained within a region of the mouse Flk1 intron that has
previously been demonstrated to possess enhancer activity
(Kappel et al., 1999;
Kappel et al., 2000). As shown
in Fig. 1A, a module within the
larger conserved region contains consensus binding sites for ETS and KLF
proteins. This ETS binding site in the mouse Flk1 gene is known to be
essential for expression in transgenic animals
(Kappel et al., 2000), but the
KLF element has not previously been reported. The conservation of the putative
KLF binding site in evolutionarily distant organisms suggests that it might
also be important for regulation of Flk1 expression.
KLF2 binds to the consensus KLF element in the Flk1 enhancer
To determine the possible functional relevance of the KLF site, we used the
electrophoretic mobility shift assay (EMSA) to determine whether nuclear
proteins from endothelial cells associate with the KLF element. Nuclear
extracts were prepared from the mouse EOMA endothelial cell line, which
expresses the Flk1 gene (data not shown). A single EMSA band was
observed when protein extracts were incubated with an oligonucleotide
containing the KLF binding sequence found in the enhancer
(Fig. 1B, lane 2). This shift
could be competed with wild-type KLF sequences
(Fig. 1B, lane 3), but not with
a mutated KLF sequence or with other binding site sequences
(Fig. 1B, lanes 4 and 5).
Nuclear proteins from a fibroblast cell line (CCL-1) failed to form a complex
with the KLF binding sequence (data not shown). These experiments indicate
that a binding activity specific for the KLF site in the Flk1
enhancer is present in endothelial cell nuclear extracts.
|
). Extracts from non-transfected COS-7 cells yielded a shifted
band (Fig. 1C, lane 1). Since
KLF proteins are expressed in a wide range of cell types
(Kaczynski et al., 2003), we
cannot exclude the possibility that this band results from the binding of one
or more KLF proteins to the probe sequence. However, an additional, prominent
complex was detected when nuclear extracts expressing HA-tagged KLF2 were
incubated with the probe (Fig.
1C, lane 2). This band could be supershifted using an antibody
directed against the HA-tag on the KLF2 protein
(Fig. 1C, lane 3). These
experiments demonstrate that KLF2, a representative endothelially expressed
member of the KLF protein family, specifically binds to the KLF motif located
in the Flk1 enhancer.
|
;
Sparrow et al., 2000). A
construction containing 2.5 kb of Xenopus Flk1 5' flanking
sequences plus first intron sequences is sufficient to drive GFP expression
throughout the blood vessels of transgenic embryos
(Fig. 2A-C)
(Warkman et al., 2004;
Doherty et al., 2007).
Expression is evident in major vessels including the posterior cardinal veins,
intersomitic vessels and the vascular plexus, and is equivalent to endogenous
Flk1 expression at a comparable stage (compare
Fig. 2A with
Fig. 3A). Reporter expression
persists during subsequent development, when GFP fluorescence is observed
throughout the smallest vessels of the trunk and tail
(Fig. 2B,C)
(Doherty et al., 2007).
To determine whether the conserved ETS and KLF binding sites were required
for embryonic expression of the Flk1 gene, the individual sites were
mutated in the context of the GFP reporter construction. In these experiments,
the 
-crystallin promoter driving GFP expression in the eye was used as
a marker for transgenesis. As expected from previous mouse studies
(Kappel et al., 2000),
mutation of the core ETS sequence reduced transgene expression to almost
undetectable levels (n=28) (Fig.
2D,E). To assess the role of the KLF binding site, the consensus
sequence in the Flk1-GFP reporter construction was altered from CACCC
to CGGTC and transgenic embryos were generated. Transgenic embryos showed a
dramatic reduction in GFP reporter expression compared with the wild-type
construction (n=31) (compare Fig.
2F,G with
2B), but any detectable
expression remained endothelial-specific. We conclude from these experiments
that the KLF site is required for efficient expression of the Flk1
gene during embryonic development and that the normal function of the site is
to activate transcription.
Previous studies have suggested that KLF2 acts as an inhibitor of
Flk1 expression (Bhattacharya et
al., 2005; Dekker et al.,
2006). Therefore, we wished to confirm that the KLF site in the
enhancer indeed functioned as a positive regulatory element. First, the coding
region of luciferase was substituted for GFP in the wild-type and ETS and KLF
mutant Flk1 reporter constructions. These constructions were
transfected into a mouse endothelial cell line (bEnd.3) and luciferase
activity was measured. The results indicated that mutation of either the ETS
or KLF elements results in a significant reduction in transcriptional activity
compared with the wild-type construction
(Fig. 2H). Second, in order to
investigate the activity of the ETS and KLF binding sites independent of other
potential regulatory sequences in the Flk1 gene, we inserted three
tandem copies of the ETS/KLF module upstream of a minimal promoter driving a
luciferase reporter. Reporter expression was then assayed in transfected
bEnd.3 cells. As shown in Fig.
2I, mutation of either the ETS or the KLF binding site resulted in
a reduction in reporter expression in endothelial cells. Taken together, the
transgenesis and cell culture studies strongly suggest that the ETS and KLF
sites in the Flk1 enhancer function as positive regulators of
Flk1 transcription.
Klf2 is expressed in endothelial cells in the Xenopus embryo
Several ETS family genes, including Ets1, Fli and Erg,
are known to be expressed in Xenopus embryonic endothelial cells
(Stiegler et al., 1990;
Stiegler et al., 1993;
Meyer et al., 1993;
Meyer et al., 1995;
Meyer et al., 1997;
Baltzinger et al., 1999). For
example, whole-mount in situ hybridization for Flk1 and Erg
transcripts (Fig. 3A,B) show
very similar expression patterns, with transcripts detected in endothelial
cells of the posterior cardinal veins, intersomitic vessels, aortic arches and
the ventral vascular plexus. Previous reports have shown that Klf2 is
expressed in the developing vasculature of mouse and zebrafish embryos
(Kuo et al., 1997;
Oates et al., 2001;
Lee et al., 2006), but
expression of Klf2 in Xenopus has not been reported. As
shown in Fig. 3C, Klf2
is expressed in all major developing blood vessels of the Xenopus
embryo, in a pattern very similar to Flk1 and Erg. The
expression of Erg and Klf2 in early endothelial structures
indicates that these factors are present in the correct place at the correct
time to regulate embryonic transcription of Flk1.
|
|
). Since at least three KLF proteins are expressed in
mouse embryonic endothelial cells, we reasoned that a dominant-repressor form
of KLF2 might inhibit the function of possibly as yet uncharacterized KLF
proteins that are also expressed in the frog vasculature. Xenopus
embryos were injected with mRNA encoding DR-KLF2 and assayed at the tailbud
stage for Flk1 expression. Similar to the results obtained with
Klf2 MO, DR-KLF2-expressing embryos displayed a significant reduction
in Flk1 transcripts throughout the embryonic vasculature (compare
Fig. 3J,K with
3G,H). Again, the effect was
dose-dependent, and at the highest levels of DR-KLF2 transcript injected (250
pg), 87% of embryos exhibited a reduction in Flk1 expression
(Table 1). The observation that
general inhibition of KLF function using DR-KLF2 was slightly more inhibitory
than with Klf2 MO, raises the possibility that additional KLF
proteins might also be involved in the regulation of Flk1 expression
in the frog embryo. Finally, we examined whether knockdown of KLF2 activity
inhibited formation of vascular structures in the embryo, as would be expected
when FLK1 receptor function is reduced. In histological sections, vascular
tubes were absent on the injected side of a Klf2 MO-treated embryo
(Fig. 3M-M''). Taken
together, these inhibition studies support a role for KLF proteins in the
activation of Flk1 expression during embryonic development.
KLF2 and ERG activate Flk1 expression in vivo
Previous studies have shown that expression of the ETS factor ERG in the
Xenopus embryo is sufficient to induce ectopic expression of the
endothelial marker X-msr
(Baltzinger et al., 1999). This
activation of an endothelial marker is a remarkable observation and has not
been replicated with any other class of transcription factor. In order to
determine whether expression of ERG is also sufficient to activate ectopic
expression of Flk1, we injected Erg mRNA into the frog
embryo. Injections were targeted to ventral blastomeres that contribute to
posterior ventral regions of the embryo, which are largely free of endothelial
cells. In all mRNA injection experiments, the test mRNA was co-injected with
EGFP mRNA, which served as a tracer. Injection of Erg mRNA
resulted in strong, ectopic expression of Flk1 in a dose-dependent
manner (Fig. 4D-F,
Table 2), whereas embryos
injected with EGFP mRNA alone showed no ectopic expression
(Fig. 4A-C,
Table 2). Significantly,
injection of Klf2 mRNA was also sufficient to activate ectopic
expression of the Flk1 gene (Fig.
4G-I). At the highest doses of Klf2 examined (1 ng of
mRNA), 79% of embryos showed the presence of ectopic Flk1 transcripts
(Table 2). Ectopic expression
was observed in both mesodermal and endodermal tissue but apparently not in
the ectoderm. These experiments demonstrate that ERG and KLF2 are
independently sufficient to activate ectopic Flk1 transcription in
the frog embryo.
|
|
;
Oikawa and Yamada, 2003).
However, much less is known concerning possible regulatory partners for the
KLF family proteins (Atkins and Jain,
2007; Turner and Crossley,
1999). Since our results (Fig.
4) show that ERG and KLF2 are independently capable of activating
Flk1 transcription, and because the ETS and KLF sites are adjacent in
the Flk1 enhancer region (Fig.
1A), we considered the possibility that ETS and KLF proteins might
cooperate to activate transcription of Flk1. To test this, we first
titrated the individual doses of Erg and Kfl2 mRNA to
amounts that resulted in little to no ectopic activation of Flk1
(Fig. 5B,C,
Table 3). However, when these
limiting amounts of Klf2 and Erg mRNA were co-expressed,
robust ectopic expression of Flk1 was observed in a large proportion
of embryos (Fig. 5D,
Table 3). The large increase in
expression observed when both Klf2 and Erg sequences were
present suggests possible synergistic activation of Flk1
transcription.
|
25-fold above background levels. Strong activation was also observed for
the vascular-specific angiopoietin receptor gene Tie2, but not for
the PECAM gene. These experiments demonstrate that KLF2 and ERG
synergize to activate embryonic expression of Flk1. Although
cooperation was also observed for Tie2 expression, failure to
activate PECAM indicates that not all endothelial genes respond to
KLF/ERG co-regulation.
ERG and KLF2 physically associate
The functional cooperation between KLF2 and ERG in the regulation of
Flk1 raised the possibility that the two proteins might physically
associate. To test this, we carried out co-immunoprecipitation (Co-IP)
experiments using HA-tagged KLF2 protein (HA-KLF2) produced in COS-7 cells,
and radiolabeled in vitro translated ERG protein
(Fig. 6A, lanes 1 and 2). ERG
co-precipitated with HA-KLF2 (Fig.
6A, lanes 4 and 5), whereas control GFP protein
(Fig. 6A, lane 3) failed to
associate with HA-KLF2 (Fig.
6A, lane 5). In reciprocal experiments, FLAG-tagged ERG and
HA-tagged KLF2 proteins were co-expressed in COS-7 cells. Using anti-FLAG
antibody, KLF2 co-precipitated with ERG protein, but no significant
precipitation of GFP control protein was observed under the same conditions
(Fig. 6B, lanes 4 and 5). These
studies indicate that ERG and KLF2 are components of a single physical complex
and very likely associate through direct protein-protein interactions.
|
|
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
), but the KLF site has not previously been recognized.
Using a Flk1 transgenic reporter construction in Xenopus, we
confirmed the importance of the ETS binding site
(Fig. 2D,E) and also
demonstrated that the KLF site is essential for efficient Flk1
expression (Fig. 2F,G). These
results demonstrate that both the conserved ETS and KLF motifs in the intronic
enhancer play an important role in the developmental regulation of the
Flk1 gene.
KLF2 function is required for embryonic expression of Flk1
Numerous studies have demonstrated the importance of ETS proteins for
developmental regulation of vascular endothelial gene expression
(Dejana et al., 2007;
Lelievre et al., 2001;
Pham et al., 2007). However,
less attention has been directed towards the role of KLF family proteins
during early vascular development. At least three KLF genes (Klf2,
Klf4 and Klf6) are expressed in the endothelium during mouse
development (Kuo et al., 1997;
Yet et al., 1998;
Kojima et al., 2000;
Botella et al., 2002;
Lee et al., 2006). Similarly,
a Klf2 gene (klf2a) is expressed in endothelial cells of the
zebrafish embryo (Lee et al.,
2006; Oates et al.,
2001). Mouse embryos lacking KLF2 or KLF6 function die as embryos
owing to defects in the ability to recruit smooth muscle cells to blood
vessels and disorganization of the yolk sac vasculature, respectively
(Kuo et al., 1997;
Matsumoto et al., 2006), but
early formation of the embryonic endothelium is normal in these animals.
Knockout of Klf4, which is also endothelially expressed, results in
perinatal death owing to loss of skin barrier function
(Segre et al., 1999), but
vascular development is again normal. The presence of multiple KLF proteins in
the endothelium raises the possibility of functional redundancy. An argument
for functional equivalence of at least some KLF proteins is supported by
studies carried out using ES cells, which suggested that any one of three
specific KLF proteins (KLF2, KLF4 or KLF5) could substitute for the other two
in maintaining ES cells in an undifferentiated state
(Jiang et al., 2008). In
agreement with observations from other species, we determined that
Klf2 is expressed in early endothelial cells of the Xenopus
embryo (Fig. 3C). Inhibition of
KLF2 function, using either a sequence-specific MO
(Fig. 3F-H,
Table 1) or more general
interference with KLF function using a dominant-repressor construction
(Fig. 3I-K,
Table 1), results in
significant downregulation of Flk1 expression in the Xenopus
embryo. We note, however, that inhibition of KLF function never resulted in
complete loss of Flk1 expression. This is consistent with the results
of the transgenic studies and cell transfection experiments
(Fig. 2F-I), in which mutation
of the KLF site resulted in a reduction, but not elimination, of reporter gene
expression. Taken together, these results indicate that KLF2 is required for
normal regulation of Flk1 expression in the frog embryo. We cannot
exclude the possibility that other KLF genes are expressed in frog embryonic
endothelial cells, but the KLF2-specific knockdown studies indicate that any
such proteins are insufficient to completely rescue the loss of KLF2
function.
KLF2 is an activator of Flk1 expression during vascular development
Our studies consistently indicate that KLF proteins function as activators
of Flk1 expression. First, we have shown that mutation of the KLF
site within the first intron of the Flk1-EGFP reporter construction
results in a major reduction of reporter expression in transgenic embryos
(Fig. 2). Second, expression of
KLF2 in the frog embryo has the remarkable ability to activate ectopic
transcription of Flk1 (Fig.
4G-I). Third, inhibition of KLF2 activity using MOs or a
dominant-repressor protein reduces embryonic expression of the endogenous
Flk1 gene (Fig. 3,
Table 1). These observations
stand in apparent contradiction to previous studies showing that KLF2 may act
as an inhibitor of Flk1 expression
(Bhattacharya et al., 2005;
Dekker et al., 2006). When
KLF2 was introduced into human primary endothelial cells using an adenoviral
vector, levels of both FLK1 mRNA and protein were reduced
(Bhattacharya et al., 2005).
Using a reporter construction containing the human FLK1 promoter, but
not the conserved first intron sequences, KLF2 inhibitory activity was found
to be mediated through SP1 binding sites close to the transcription start
site. Another study suggesting KLF2 inhibition involved microarrays, where
FLK1 was included in a population of genes downregulated following
expression of KLF2 in HUVECs (Dekker et
al., 2006). In these experiments, gene expression was analyzed
after 7 days of forced KLF2 expression, raising the possibility that the
effect on FLK1 transcript levels was indirect. Our results, however,
are in broad agreement with studies of mouse KLF6 which indicated that FLK1
expression was reduced in embryoid bodies derived from
Klf6-/- ES cells
(Matsumoto et al., 2006). The
most likely explanation for these contrasting observations is that the studies
were carried out over different time periods and using different sources of
endothelial cells. KLF proteins exhibit both activator and repressor
properties (Conkright et al.,
2001; Yet et al.,
1998) and different regulatory activities are likely to be
revealed in distinct cellular contexts, probably through interactions with
different partner proteins.
KLF2 and ERG cooperate to activate expression of Flk1: a general vascular mechanism?
ETS family proteins frequently interact with partner proteins to regulate
gene expression (Lelievre et al.,
2001; Sharrocks,
2001; Oikawa and Yamada,
2003). These binding partners act to modulate ETS DNA-binding
affinity and transcriptional activity (Li
et al., 2000). However, despite many documented interacting
proteins, no partner that functions specifically in endothelial cells has been
characterized. Our studies suggest that endothelially expressed KLF and ETS
family members might function together to regulate transcription of
Flk1. First, both ERG and KLF2 are expressed in embryonic angioblasts
during early vascular development (Fig.
3A-C) and are therefore in the right place at the right time to
activate or maintain Flk1 transcription. Second, we have shown that
ERG and KLF2 regulate Flk1 expression synergistically. When low
amounts of Klf2 and Erg mRNA were expressed in the frog
embryo, we never observed high levels of ectopic Flk1 expression
(Fig. 5B,C,
Table 3). However, when the
same low doses were co-expressed, approximately one-third of embryos showed
strong ectopic Flk1 expression
(Fig. 5D,
Table 3). Quantitation of
equivalent experiments using real-time PCR showed that co-expression of ERG
and KLF2 resulted in a 25-fold increase in Flk1 transcript
levels over either ERG or KLF2 alone, strongly suggesting synergistic
activation of Flk1 expression. Finally, we demonstrated that ERG and
KLF2 are able to physically associate with each other
(Fig. 6A,B). Our experiments do
not exclude the possibility that other proteins are present in the complex,
and do not demonstrate direct physical interaction between ERG and KLF2, but
they do place the two proteins in the same complex. Additional studies will be
required to determine whether the ability to form a physical complex is
specific for ERG and KLF2 or whether this property is shared by additional
family members.
Our studies have focused largely on transcriptional regulation of
Flk1 because this represents one of the earliest and most important
steps in the endothelial differentiation program. However, the observed
cooperation between ERG and KLF2 might have implications for regulation of
additional vascular endothelial genes. ETS binding sites are important for
regulation of many endothelial genes
(Dejana et al., 2007;
Schlaeger et al., 1997;
Gory et al., 1999) and recent
experiments have shown that a large number of endothelial genes are
potentially responsive to KLF2 regulation
(Dekker et al., 2006).
Although preliminary, we have obtained results showing that KLF2 is required
for the expression of other embryonic vascular markers including
X-msr and VE-cadherin and is capable of activating ectopic
transcription of these genes (Fig.
7, Table 4).
However, until functional studies are carried out, we do not know whether KLF2
is a direct transcriptional regulator of these genes. In summary, our studies
suggest that cooperation between ETS and KLF proteins is important for
activation of the vascular program in the embryo. Cooperation might also play
a role in the maintenance of normal vascular function and in vascular
disease.
|
Footnotes |
|---|
* Present address: Department of Molecular Biology, University of Texas
Southwestern Medical Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA 
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Atkins, G. B. and Jain, M. K. (2007). Role of
Kruppel-like transcription factors in endothelial biology. Circ.
Res. 100,1686
-1695.
Baltzinger, M., Mager-Heckel, A. and Remy, P.
(1999). Xl erg: Expression pattern and overexpression during
development plead for a role in endothelial cell differentiation.
Dev. Dyn. 216,420
-433.[CrossRef][Medline]
Bhattacharya, R., SenBanerjee, S., Lin, Z., Mir, S., Hamik, A.,
Wang, P., Mukherjee, P., Mukhopadhyay, D. and Jain, M. K.
(2005). Inhibition of vascular permeability factor/vascular
endothelial growth factor-mediated angiogenesis by the Kruppel-like factor
KLF2. J. Biol. Chem.
280,28848
-28851.
Bieker, J. J. (2001). Kruppel-like factors:
three fingers in many pies. J. Biol. Chem.
276,34355
-34358.
Birdsey, G. M., Dryden, N. H., Amsellem, V., Gebhardt, F.,
Sahnan, K., Haskard, D. O., Kdjana, E., Mason, J. C. and Randi, A. M.
(2008). Transcription factor Erg regulates angiogenesis and
endothelial apoptosis through VE-cadherin. Blood
111,3498
-3506.
Botella, L. M., Sanchez-Elsner, T., Sanz-Rodriguez, F., Kojima,
S., Shimada, J., Guerrero-Esteo, M., Cooreman, M. P., Ratziu, V., Langa, C.,
Var, C. P. et al. (2002). Transcriptional activation of
endoglin and transforming growth factor-beta signaling components by
cooperative interaction between Sp1 and KLF6: their potential role in the
response to vascular injury. Blood
100,4001
-4010.
Bouwmeester, T., Kim, S., Sasai, Y., Lu, B. and De Robertis, E.
M. (1996). Cerberus is ahead-inducing secreted factor
expressed in the anterior endoderm of Spemann's organizer.
Nature 382,595
-601.[CrossRef][Medline]
Carmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S.,
Kieckens, L., Gertsenstein, M., Fahrig, M., Vandenhoeck, A., Harpal, K.,
Eberhardt, C. et al. (1996). Abnormal blood vessel
development and lethality in embryos lacking a single VEGF allele.
Nature (London) 380,435
-439.[CrossRef][Medline]
Cleaver, O., Tonissen, K. F., Saha, M. S. and Krieg, P. A.
(1997). Neovascularization of the Xenopus embryo. Dev.
Dyn. 210,66
-77.[CrossRef][Medline]
Conkright, M. D., Wani, M. A. and Lingrel, J. B.
(2001). Lung Kruppel-like factor contains an autoinhibitory
domain that regulates its transcriptional activation by binding WWP1, an E3
ubiquitin ligase. J. Biol. Chem.
276,29299
-29306.
Dang, D. T., Pevsner, J. and Yang, V. W.
(2000). The biology of the mammalian Kruppel like family of
transcription factors. Int. J. Biochem. Cell Biol.
32,1103
-1121.[CrossRef][Medline]
Dejana, E., Taddei, A. and Randi, A. M. (2007).
Foxs and Ets in the transcriptional regulation of endothelial cell
differentiation and angiogenesis. Biochim. Biophys.
Acta 1775,298
-312.[Medline]
Dekker, R. J., van Thienen, J. V., Rohlena, J., de Jager, S. C.,
Elderkamp, Y. W., Seppen, J., de Vries, C. J., Biessen, E. A., van Berkel, T.
J., Pannekoek, H. et al. (2005). Endothelial KLF2 links local
arterial shear stress levels to the expression of vascular tone-regulating
genes. Am. J. Pathol.
167,609
-618.
Dekker, R. J., Boon, R. A., Rondaij, M. G., Kragt, A., Volger,
O. L., Elderkamp, Y. W., Meijers, J. C., Voorberg, J., Pannekeok, H. and
Hoorevoets, A. J. (2006). KLF2 provokes a gene expression
pattern that establishes functional quiescent differentiation of the
endothelium. Blood 107,4354
-4363.
Devic, E., Paquereau, L., Vernier, P., Knibiehler, B. and
Audigier, Y. (1996). Expression of a new G protein-coupled
receptor X-msr is associated with an endothelial lineage in Xenopus laevis.
Mech. Dev. 59,129
-140.[CrossRef][Medline]
Doherty, J. R., Johson-Hamlet, M. R., Kuliyev, E. and Mead, P.
E. (2007). A flk-1 promoter/enhancer reporter transgenic
Xenopus laevis generated using the Sleeping Beauty transposon system: an in
vivo model for vascular studies. Dev. Dyn.
236,2808
-2817.[CrossRef][Medline]
Elvert, G., Kappel, A., Heidenreich, R., Englmeier, U., Lanz,
S., Acker, T., Rauter, M., Plate, K., Sieweke, M., Breier, G. et al.
(2003). Cooperative interaction of hypoxia-inducible
factor-2alpha (HIF-2alpha) and Ets-1 in the transcriptional activation of
vascular endothelial growth factor receptor-2 (Flk-1). J. Biol.
Chem. 278,7520
-7530.
Ferrara, N., Carver-Moore, K., Chen, H., Dowd, M., Lu, L.,
O'Shea, K. S., Powell-Braxton, L., Hillan, K. J. and Moore, M. W.
(1996). Heterozygous embryonic lethality induced by targeted
inactivation of the VEGF gene. Nature (London)
380,439
-442.[CrossRef][Medline]
Garriock, R. J., D'Agostino, S. L., Pilcher, K. C. and Krieg, P.
A. (2005). Wnt11-R, a protein closely related to mammalian
Wnt11, is required for heart morphogenesis in Xenopus. Dev.
Biol. 279,179
-192.[CrossRef][Medline]
Gory, S., Vernet, M., Laurent, M., Dejana, E., Dalmon, J. and
Huber, P. (1999). The vascular endothelial-cadherin promoter
directs endothelial-specific expression intransgenic mice.
Blood 93,184
-192.
Harland, R. M. (1991). In situ hybridization:
an improved whole-mount method for Xenopus embryo. Methods Cell
Biol. 36,685
-695.[Medline]
Hasegawa, Y., Abe, M., Yamazaki, T., Niizeki, O., Shiiba, K.,
Sasaki, I. and Sato, Y. (2004). Transcriptional regulation of
human angiopoietin-2 by transcription factor Ets-1. Biochem.
Biophys. Res. Commun. 316,52
-58.[CrossRef][Medline]
Jiang, J., Chan, Y. S., Loh, Y. H., Cai, J., Tong, G. Q., Lim,
C. A., Robson, P., Zhong, S. and Ng, H. H. (2008). A core Klf
circuitry regulates self-renewal of embryonic stem cells. Nat. Cell
Biol. 10,353
-360.[CrossRef][Medline]
Kaczynski, J., Cook, T. and Urrutia, R. (2003).
Sp1- and Kruppel-like transcription factors. Genome
Biol. 4,206
.[CrossRef][Medline]
Kappel, A., Ronicke, V., Damert, A., Flamme, I., Risau, W. and
Breier, G. (1999). Identification of vascular endothelial
growth factor (VEGF) receptor-2 (Flk-1) promoter/enhancer sequences sufficient
for angioblast and endothelial cell-specifc transcription in transgenic mice.
Blood 93,4284
-4292.
Kappel, A., Schlaeger, T. M., Flamme, I., Orkin, S. H., Risau,
W. and Breier, G. (2000). Role of SCL/Tal, GATA and Ets
transcription factor binding sites for the regulation of Flk-1 expression
during murine vascular development. Blood
96,3078
-3085.
Kojima, S., Hayashi, S., Shimokado, K., Suzuki, Y., Shimada, J.,
Crippa, M. P. and Friedman, S. L. (2000). Transcriptional
activation of urokinase by Kruppel-like factor Zf9/COPEB activates latent
TGF-beta1 in vascular endothelial cells. Blood
95,1309
-1316.
Kroll, K. L. and Amaya, E. (1996). Transgenic
Xenopus embryos from sperm nuclear transplantations reveal FGF signaling
requirements during gastrulation. Development
122,3173
-3183.[Abstract]
Kuo, C. T., Veselits, M. L., Barton, K. P., Lu, M. M.,
Clendenin, C. and Leiden, J. M. (1997). The LKLF
transcription factor is required for normal tunica media formation and blood
vessel stabilization during murine embryogenesis. Genes
Dev. 11,2996
-3006.
Lee, D., Park, C., Lee, H., Lugus, J. J., Kim, S. H., Arntson,
E., Chjung, Y. S., Gomez, G., Kyba, M., Lin, S. et al.
(2008). ER71 acts downstream of BMP, Notch, and Wnt signaling in
blood vessel progenitor specification. Cell Stem Cell
2, 497-507.[CrossRef][Medline]
Lee, J. S., Yu, Q., Shin, J. T., Sebzda, E., Bertozzi, C., Chen,
M., Wericko, P., Stadtfeld, M., Zhou, D., Cheng, L. et al.
(2006). KLF2 is an essential regulator of vascular hemodynamic
forces in vivo. Dev. Cell
11,845
-857.[CrossRef][Medline]
Lelievre, E., lionneton, F., Soncin, F. and Vandenbunder, B.
(2001). The Ets family contains transcriptional activators and
repressors involved in angiogenesis. Int. J. Biochem. Cell
Biol. 33,391
-407.[CrossRef][Medline]
Li, R., Pei, H. and Watson, D. K. (2000).
Regulation of Ets function by protein-protein interactions.
Oncogene 19,6514
-6523.[CrossRef][Medline]
Lin, Z., Kumar, A., SenBanjeree, S., Staniszewski, K., Parmar,
K., Vaughan de Gimbrone, M. A., Jr, Balasubramanian, V., Garcia-Cardena, G.
and Jain, M. K. (2005). Kruppel-like factor 2 (KLF2)
regulates endothelial thrombotic function. Circ. Res.
96,e48
-e57.
Matsumoto, N., Kubo, A., Huixian, L., Akita, K., Laub, F.,
Ramirez, F., Keller, G. and Friedman, S. L. (2006).
Developmental regulation of yolk sac hematopoisis by Kruppel-like factor 6.
Blood 107,1357
-1365.
Meadows, S. M., Salanga, M. C. and Krieg, P. A.
(2008). The myocardin-related transcription factor, MASTR,
cooperates with MyoD to activate skeletal muscle gene expression.
Proc. Natl. Acad. Sci. USA
105,1545
-1550.
Meyer, D., Wolff, C.-M., Stiegler, P., Senan, F., Befort, N.,
Befort, J. J. and Remy, P. (1993). Xl-fli, the Xenopus
homologue of the fli-1 gene, is expressed during embryogenesis in a restricted
pattern evocative of neural crest cell distribution. Mech.
Dev. 44,109
-121.[CrossRef][Medline]
Meyer, D., Stiegler, P., Hindelang, C., Mager, A.-M. and Remy,
P. (1995). Whole-mount in situ hybridization reveals the
expression of Xl-Fli gene in several lineages of migrating cells in Xenopus
embryos. Int. J. Dev. Biol.
39,909
-919.[Medline]
Meyer, D., Durliat, M., Senan, F., Wolff, M., Andres, M.,
Hourdy, J. and Remy, P. (1997). Ets-1 and Ets-2
roto-oncogenes exhibit differential and restricted expression patterns during
Xenopus laevis oogenesis and embryogenesis. Int. J. Dev.
Biol. 41,607
-620.[Medline]
Nieuwkoop, P. D. and Faber, J. (1994).
Normal Table of Xenopus laevis (Daudin): A Systematical and
Chronological Survey of the Development from the Fertilized Egg til the End of
Metamorphosis. New York, NY: Garland Press.
Oates, A. C., Pratt, S. J., Vail, B., Yan, Y.-L., Ho, R. K.,
Johnson, S. L., Postlethwait, J. H. and Zon, L. I. (2001).
The zebrafish klf gene family. Blood
98,1792
-1801.
Oikawa, T. and Yamada, T. (2003). Molecular
biology of the Ets family of transcription factors.
Gene 303,11
-34.[CrossRef][Medline]
Parmar, K. M., Larman, H. B., Dai, G., Zhang, Y., Wang, E. T.,
Moorthy, S. N., Kratz, J., Lin, Z., Jain, M. K., Dimbrone, M. A. et al.
(2006). Integration of flow-dependent endothelial phenotypes by
Kruppel-like factor 2. J. Clin. Invest.
116, 49-58.[CrossRef][Medline]
Pham, V. N., Lawson, N. D., Mugford, J. W., Dye, L., Castranova,
D., Lo, B. and Weinstein, B. M. (2007). Combinatorial
function of Ets transcription factors in the developing vasculature.
Dev. Biol. 303,772
-783.[CrossRef][Medline]
Schlaeger, T. M., Bartunkova, S., Lawitts, J. A., Teichmann, G.,
Risau, W., Deutsch, U. and Sato, T. N. (1997). Uniform
vascular-endothelial-cell-specific gene expression in both embryonic and adult
transgenic mice. Proc. Natl. Acad. Sci. USA
94,3058
-3063.
Schwachtgen, J. L., Janel, N., Barek, L., Duterque-Coquillaud,
M., Ghysdael, J., Meyer, D. and Kerbiriou-Nabias, D. (1997).
Ets transcription factors bind and transactivate the core promoter of the von
Willebrand factor gene. Oncogene
15,3091
-3102.[CrossRef][Medline]
Segre, J. A., Bauer, C. and Fuchs, E. (1999).
Klf4 is a transcription factor required for establishing the barrier function
of the skin. Nat. Genet.
22,356
-360.[CrossRef][Medline]
Shalaby, F., Rossant, J., Yamaguchi, T. P., Gertenstein, M., Wu,
X.-F., Breitman, M. L. and Schuh, A. (1995). Failure of
blood-island formation and vasculogenesis in flk-1 deficient mice.
Nature (London) 376,62
-66.[CrossRef][Medline]
Sharrocks, A. D. (2001). The Ets-domain
transcription factor family. Nat. Rev. Mol. Cell Biol.
11,827
-837.
Sparrow, D. B., Latinkic, B. and Mohun, T. J.
(2000). A simplified method of generating transgenic Xenopus.
Nucleic Acids Res. 28,E12
.[CrossRef][Medline]
Stiegler, P., Wolff, C. M., Baltzinger, M., Hirtzlin, J., Senan,
F., Meyer, D., Ghysdael, J., Stehelin, D., Befort, N. and Remy, P.
(1990). Characterization of Xenopus laevis cDNA clones of the
c-Ets-1 proto-oncogene. Nucleic Acids Res.
18, 5298.
Stiegler, P., Wolff, C. M., Meyer, D., Senan, F., Durliat, M.,
Hourdry, J., Befort, N. and Remy, P. (1993). The c-Ets-1
proto-oncogenes in Xenopus laevis: expression during oogenesis and
embryogenesis. Mech. Dev.
41,163
-174.[CrossRef][Medline]
Turner, J. and Crossley, M. (1999). Basic
Kruppel-like factor functions within a network of interacting haematopoietic
transcription factors. Int. J. Biochem. Cell Biol.
31,1169
-1174.[CrossRef][Medline]
Wakiya, K., Begue, A., Stehelin, D. and Shibuya, M. A.
(1996). A cAMP response element and an Ets motif are involved in
the transcriptional regulation of flt-1 tyrosine kinase (vascular endothelial
growth factor receptor 1) gene. J. Biol. Chem.
271,30823
-30828.
Warkman, A. S., Meadows, S. M., Small, E. M., Cox, C. M. and
Krieg, P. A. (2004).Cardiovascular genomics: the promise of
Xenopus. Drug Discov. Today: Disease Models
3, 249-255.
Yet, S. F., McA'Nulty, M. M., Folta, S. C., Yen, H. W.,
Yoshizumi, M., Hseih, C. M., Layne, M. D., Chin, M. T., Wang, H., Perrella, M.
A. et al. (1998). Human EZF, a Kruppel-like zinc finger
protein, is expressed in vascular endothelial cells and contains
transcriptional activation and repression domains. J. Biol.
Chem. 273,1026
-1031.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in Development:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
