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First published online June 6, 2008
doi: 10.1242/10.1242/dev.017038
Genome-Scale Biology Program and Institute of Biomedicine, Biomedicum Helsinki, 00014 University of Helsinki, Finland.
Author for correspondence (e-mail:
tomi.makela{at}helsinki.fi)
Accepted 6 May 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: LKB1, TGFβ, Vascular smooth muscle cell, Angiogenesis, Endothelium, Differentiation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To
investigate the physiological functions of Lkb1 (Stk11 -
Mouse Genome Informatics), we, and others, have analyzed mice with homozygous
null mutations of Lkb1 (Bardeesy
et al., 2002; Jishage et al.,
2002; Ylikorkala et al.,
2001). While heterozygous Lkb1+/- mice develop
polyps similar to those seen in PJS patients
(Bardeesy et al., 2002;
Jishage et al., 2002;
Miyoshi et al., 2002;
Rossi et al., 2002), targeted
disruption of both Lkb1 alleles results in midgestational lethality
between embryonic days 9 and 11 (E9-E11)
(Bardeesy et al., 2002;
Jishage et al., 2002;
Ylikorkala et al., 2001).
Lkb1-/- mouse embryos manifest multiple developmental
abnormalities, such as deficient neural tube closure and somitogenesis, and
vascular abnormalities (Ylikorkala et al.,
2001). Formation of the primary blood vessel plexus
(vasculogenesis) proceeds normally in Lkb1-/- embryos but
further vascular development (angiogenesis), including remodeling, sprouting
and maturation of blood vessels, is severely defective, and the expression of
vascular endothelial growth factor (VEGF) is upregulated in mesenchymal cells
(Ylikorkala et al., 2001).
Blood vessel growth and differentiation is regulated by interactions
between the endothelium and the surrounding cells and extracellular matrix.
Signaling molecules involved in this crosstalk include growth factors,
integrins, chemokines, oxygen sensors and extracellular matrix molecules
(Carmeliet, 2003). VEGF has an
essential role in vessel formation
(Carmeliet et al., 1996) and
stabilization (Darland et al.,
2003), whereas TGFβ, angiopoietin 1 (ANG1; ANGPT1 - Mouse
Genome Informatics), and platelet-derived growth factor B (PDGFB) are key
regulators of subsequent blood vessel maturation
(Lindahl et al., 1997;
Pepper, 1997;
Suri et al., 1996).
Lkb1 encodes an intracellular serine-threonine kinase that
phosphorylates and activates a number of kinases, including PAR-1/MARK
(microtubule-affinity-regulating kinase) and AMPK (AMP-activated protein
kinase), implicating it in multiple signaling pathways regulating, for
example, cell metabolism and polarity
(Katajisto et al., 2006). Of
these, AMPK has been shown to regulate endothelial migration and tube
formation in culture under hypoxic conditions
(Nagata et al., 2003), whereas
no specific role in vascular development has been reported for the other LKB1
substrate kinases. LKB1 has also been reported to associate with a number of
other proteins, including LIP1 (Smith et
al., 2001) and the PTEN tumor suppressor
(Mehenni et al., 2005). The
latter association may be linked to vascular function, as LKB1 was reported to
phosphorylate and activate PTEN in human umbilical vein endothelial cells
following peroxynitrite or high glucose treatment
(Song et al., 2007).
Considering the ubiquitous expression pattern of Lkb1 during
embryogenesis (Luukko et al.,
1999) and the broad spectrum of implicated signaling pathways, it
has been unclear which cell types and signaling defects underlie the vascular
abnormalities in Lkb1-deficient embryos. Here, we have used
conditional mutagenesis to address this question and to investigate the
cellular functions of LKB1 during mammalian development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and conditional (Bardeesy
et al., 2002) alleles, as well as the Mox2-Cre
(Tallquist and Soriano, 2000)
and Tie1-Cre (Gustafsson et al.,
2001) mouse strains have been previously described. Mox2-Cre and
Tie1-Cre mice were first bred onto a Lkb1+/- background and then
mated with Lkb1lox/lox mice.
Histology and immunohistochemistry
For histological and immunohistochemical analyses, tissues were fixed in 4%
paraformaldehyde overnight at 4°C, embedded in paraffin, and sectioned and
stained according to standard protocols. Whole-mount immunohistochemistry was
done as previously described (Ylikorkala
et al., 2001). Primary antibodies used were anti-phospho-SMAD2
ser465/467 (Chemicon AB3849), anti-VE-cadherin (BD Pharmingen 555289),
anti-smooth muscle actin (clone 1A4; Sigma A5691, A2547 and C6198),
anti-PECAM1 (Pharmingen 553369) and anti-PDGFRβ (eBioscience 14-1402).
Primary antibodies were detected by Alexa549-conjugated (Invitrogen),
FITC-conjugated (Jackson ImmunoResearch) or HRP-conjugated (Dako) secondary
antibodies. Alkaline phosphatase-conjugated anti-smooth muscle actin antibody
(Sigma C6198) was visualized with the Vector Black Alkaline Phosphatase
Substrate Kit (Vector SK-5200). For quantification of pSMAD2-positive cells,
serial sections were examined through the yolk sac, and over 1000 endothelial
cells and 2500 mesenchymal cells were scored.
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Treatment of yolk sacs with exogenous TGFβ1
E11.5 and E12.5 yolk sacs were divided in half, cultured in Dulbecco's
minimum essential medium (DMEM) supplemented with 0.3% fetal calf serum (FCS)
with or without 1 ng/ml TGFβ1 for 1 or 8 hours at 37°C, and then
fixed with 2% paraformaldehyde for 30 minutes and embedded in paraffin.
RNA interference
hTERT-immortalized human umbilical vein endothelial cells (HUVEC) (a kind
gift from Sonja Koopal, University of Helsinki) were plated into 12-well
plates, transfected with 100 nmol siRNA diluted in OptiMEM (Gibco) and
incubated with oligofectamine (Invitrogen), according to manufacturers'
instructions. Cells were collected for RNA extraction 72 hours after
transfection. The sequences for LKB1 siRNA were 5'-UAG UUG AAU UUC CUU
CUU CUU-3' (LKB1-si1; Dharmicon) and 5'-UAC UCA AGC AUC CUU UCA
UU-3' (LKB1-si2; Dharmicon). A non-targeting siRNA (Dharmicon catalog
number D-001210-04-20) was used as a control.
RNA extraction and real-time RT-PCR analysis
RNA from yolk sacs and immortalized HUVECs was isolated using an RNeasy
isolation kit (Qiagen), according to the manufacturer's instructions. Total
RNA was reverse transcribed using Taqman Reverse Transcription reagents
(Applied Biosystems). Real-time PCR was performed on an ABI Prism 7500 using
Power SYBR Green PCR Master Mix (Applied Biosystems). Relative mRNA amounts
were assayed by using the 7500 Fast Real-Time PCR System Software. Primers
used were:
mouse TGFβ1, 5'-CAC CAT CCA TGA CAT GAA CC-3' (forward) and 5'-ACT TCC AAC CCA GGT CCT TC-3' (reverse);
mouse GAPDH, 5'-TCA ACG ACC CCT TCA TTG AC-3' (forward) and 5'-ATG CAG GGA TGA TGT TCT GG-3' (reverse);
human TGFβ1, 5'-CCC TGG ACA CCA ACT ATT GC-3' (forward) and 5'-GTC CAG GCT CCA AAT GTA GG-3' (reverse);
human GAPDH, 5'-CGA CCA CTT TGT CAA GCT CA-3' (forward) and 5'-AGG GGA GAT TCA GTG TGG TG-3' (reverse); and
human PAI1, 5'-CAG GAA GCC CCT AGA GAA CC-3' (forward) and 5'-ATG CGG GCT GAG ACT ATG AC-3' (reverse).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) We have previously
shown that Lkb1-/- yolk sac vascularization fails to
develop beyond primary plexus stage and that allantoic blood vessels of
Lkb1-/- embryos are unable to invade the placenta
(Ylikorkala et al., 2001). To
determine whether the defects seen in Lkb1-/- embryos are
caused by extraembryonic dysfunction, we used an experimental approach to
supply Lkb1-deficient embryos with wild-type placentas and yolk sac
endoderm by using the Mox2-Cre (MORE) mouse strain in which Cre
expression is restricted to epiblast-derived cells
(Tallquist and Soriano, 2000).
Crossing mice with floxed Lkb1 alleles
(Bardeesy et al., 2002) with
Mox2-Cre mice deletes Lkb1 in the embryo, allantois and yolk
sac mesoderm, while Lkb1 in trophoblast cells of the placenta and
visceral endoderm of the yolk sac remains intact.
Lkb1lox/-, Mox2-Cre conceptuses were analyzed at E9.5,
which is the stage when Lkb1-/- embryos show severe
developmental defects (Ylikorkala et al.,
2001). Out of 133 conceptuses, 27 were genotyped as
Lkb1lox/-, Mox2-Cre. Morphologically, six of these were
indistinguishable from wild-type littermates; the remaining 21 embryos had
variable phenotypes resembling those of Lkb1-/- embryos
(Fig. 1D), with 15 being less
severely affected (Fig. 1B) and
six being similar although bigger (Fig.
1C). Yolk sac vasculature of all 21 Lkb1lox/-,
Mox2-Cre embryos remained at the primary vascular plexus stage
(Fig. 1F,G,J,K). The phenotypic
variation seen in Lkb1lox/-, Mox2-Cre embryos could be due
to incomplete recombination, as Mox2-Cre has been reported to be
mosaic in activity (Hayashi et al.,
2002). Consistent with this, efficiency of Cre-mediated deletion
of the floxed Lkb1 allele correlated with phenotype severity in
individual Lkb1lox/-, Mox2-Cre embryos, as assessed by
semi-quantitative PCR (data not shown).
The analysis of placentas of severely affected Lkb1lox/-,
Mox2-Cre embryos revealed that the wild-type trophoblasts had formed a
thick labyrinthine layer with prominent maternal blood lacunas (see Fig. S1B
in the supplementary material), which was distinct from that of
Lkb1-/- trophoblasts
(Ylikorkala et al., 2001).
Invasion of Lkb1-/- allantoic blood vessels into the
trophoblast layer was, however, poor compared with wild type.
Taken together, these data demonstrate that wild-type trophoblast and visceral endoderm cells are not sufficient to restore extraembryonic vascularization and development of the embryo proper, and indicate that the defective development of Lkb1-/- mutant embryos is primarily due to defective function of embryonic cell lineages rather than to impaired placental or yolk sac function.
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). Both
undifferentiated mesenchymal cells and differentiated vSMCs and pericytes
produce VEGF, which stimulates the migration, proliferation and survival of
ECs (Darland et al., 2003).
Although VEGF expression is upregulated in mesenchymal cells in
Lkb1-/- embryos
(Ylikorkala et al., 2001),
this is unlikely to underlie the severe vascular phenotype, as mice
overexpressing VEGF show normal development of the vasculature up to E14
(Miquerol et al., 2000).
In order to determine whether the disruption of Lkb1 leads to
cell-autonomous defects in ECs, we generated mice with an
endothelium-restricted deletion of Lkb1 by using Tie1-Cre
transgenic mice (Gustafsson et al.,
2001). The Tie1-Cre mouse strain has been widely used and
efficiently inactivates target genes in ECs starting at E8.5
(Carvalho et al., 2004;
Gustafsson et al., 2001). In
accordance with this, immunohistochemical staining of yolk sacs from
Lkb1+/-, Tie1-Cre and Lkb1lox/lox
intercrosses using antibodies against Cre and the endothelial marker
VE-cadherin showed efficient and EC-restricted expression of Cre
(Fig. 2A).
Out of 64 offspring from Lkb1+/-, Tie1-Cre and Lkb1lox/lox intercrosses, no Lkb1lox/-, Tie1-Cre mice were recovered, demonstrating the embryonic lethality of this genotype. Analysis of the embryos revealed that, up to E10.5, Lkb1lox/-, Tie1-Cre embryos were indistinguishable from wild-type littermates. However, at E11.5, almost half (21/46) of the Lkb1lox/-, Tie1-Cre embryos had pale, bloodless yolk sacs (Fig. 2C) and pericardial swelling (Fig. 2E, arrow), indicative of embryonic circulatory defects. Blood vessels within these embryos appeared dilated and showed congested blood (Fig. 2E, arrowheads). By E12.5, all analyzed Lkb1lox/-, Tie1-Cre embryos (n=25) were anemic and exhibited large areas of hemorrhages (Fig. 2G), demonstrating disruption of the vasculature. No live Lkb1lox/-, Tie1-Cre embryos were recovered after E12.5.
To investigate blood vessel structure, Lkb1lox/-,
Tie1-Cre conceptuses were stained with antibodies against VE-cadherin and
PECAM1. Whole-mount staining of the adherens junction protein VE-cadherin did
not show abnormalities in endothelial junctions
(Fig. 3B), and PECAM1 staining
revealed normal capillary density and formation of arteries and veins,
demonstrating that vascular remodeling and maturation had commenced in yolk
sacs (Fig. 3D) and embryos
(data not shown). Moreover, vascular sprouting and branching proceeded
normally in Lkb1lox/-, Tie1-Cre embryos, contrary to
Lkb1-null embryos, where intersomitic branches terminated prematurely
in the mesenchyme (Ylikorkala et al.,
2001). Lkb1lox/-, Tie1-Cre yolk sac vessels
were, however, distorted and fragile, with small local dilations and irregular
vessel wall structure (Fig.
3B,D). In the E11.5 Lkb1lox/-, Tie1-Cre embryo
proper, defects in superficial microvessels were less pronounced, but
histological analysis revealed dramatic dilation of the large trunk vessels
(Fig. 3F) and blood was
congested in these vessels and in the liver, indicating insufficient
circulation (Fig. 3F). This is
likely to be caused by defects in the blood vessels, as no obvious alterations
in heart morphology were noted (data not shown).
|
; Lai et al.,
2003). These include the EC-specific knockout of the PTEN
tumor suppressor, which results in enhanced proliferation of ECs, cardiac
failure and fused capillaries, leading to embryonic death at E11.5
(Hamada et al., 2005). To evaluate the proliferation of ECs in Lkb1lox/-, Tie1-Cre conceptuses, yolk sacs were stained for the proliferation marker Ki-67. No significant differences were observed between wild-type and Lkb1lox/-, Tie1-Cre yolk sacs in the percentage of Ki-67-positive ECs (27±7% and 26±12%, respectively) at E11.5 (see Fig. S2A-D in the supplementary material). Similarly, TUNEL assays revealed very low numbers of apoptotic ECs, with no difference between wild-type and Lkb1lox/-, Tie1-Cre yolk sacs (see Fig. S2E,F in the supplementary material). These observations suggest that alterations in EC proliferation or death rates do not contribute to the vascular defects following loss of endothelial Lkb1, and further imply that the mechanisms underlying the Lkb1lox/-, Tie1-Cre vascular phenotype are distinct from those in PTEN mutants, which involve the PTEN/PI3K pathway.
Loss of vascular smooth muscle cells in Lkb1lox/-, Tie1-Cre conceptuses
The stabilization and maturation of blood vessels requires the formation of
vascular smooth muscle cells (vSMCs) around the nascent endothelial tube, a
process regulated by signaling from the endothelium to the surrounding
mesenchymal cells and differentiating vSMCs
(Armulik et al., 2005).
Staining wild-type E11.5 yolk sacs with antibodies against smooth muscle
cell-specific 
-smooth muscle actin (SMA) identified vSMCs around the
blood vessels as expected (Fig.
4C). Interestingly, SMA staining was strongly reduced or absent in
Lkb1lox/-, Tie1-Cre yolk sacs
(Fig. 4D), although PECAM1
staining revealed an intact endothelium
(Fig. 4B), as noted before.
Similarly, staining with another vSMC marker, platelet-derived growth factor
receptor β (PDGFRβ), identified vSMCs in wild-type yolk sacs
(Fig. 4G) but not in
Lkb1lox/-, Tie1-Cre yolk sacs
(Fig. 4H). Staining of vSMCs in
the embryo proper also revealed a significant reduction of vSMCs at E11.5 in
Lkb1lox/-, Tie1-Cre embryos compared with wild-type
littermates, as is shown in the aorta wall
(Fig. 4I,J).
These results show that EC-specific deletion of Lkb1 results in a
marked reduction of vSMCs, suggesting that Lkb1-/- ECs are
defective in signaling to adjacent vSMCs. The observed loss of vSMCs is likely
to cause vessel fragility and vascular disruption of Lkb1lox/-,
Tie1-Cre mutants, as has been shown in previous studies describing
similar phenotypes (Carvalho et al.,
2004; Jadrich et al.,
2006; Li et al.,
1999).
Deletion of Lkb1 from ECs leads to defective TGFβ signaling
The PDGFB/PDGFRβ, sphingosine-1-phosphate (S1P)/S1P1 and
TGFβ pathways are well-known mediators of signaling from ECs to adjacent
mesenchymal cells and differentiating vSMCs
(Armulik et al., 2005). Based
on mouse knockout studies, the de novo formation of vSMCs from mesenchymal
cells around blood vessels is dependent upon endothelial TGFβ signaling
(Carvalho et al., 2007;
Carvalho et al., 2004;
Larsson et al., 2001;
Li et al., 1999;
Oh et al., 2000;
Urness et al., 2000). PDGFB
and S1P are required at later embryonic stages (E12-E18), promoting vSMC
migration along new blood vessel sprouts
(Hellstrom et al., 1999;
Liu et al., 2000), and are
therefore unlikely to underlie the phenotype in the Lkb1lox/-,
Tie1-Cre mice noted at an earlier developmental stage. Loss-of-function
mutants of several central TGFβ pathway components (ALK1, ALK5, endoglin,
SMAD5) (Larsson et al., 2001;
Li et al., 1999;
Oh et al., 2000;
Yang et al., 1999), as well as
of the TGFβ signaling modulator TAK1
(Jadrich et al., 2006), are
embryonic lethal at E10-E11, with vascular abnormalities and defective vSMC
formation. The necessity of intact endothelial TGFβ signaling for
vascular development has been demonstrated by EC-specific knockouts of ALK5
and TGFβRII, and by the knockout of endoglin, an endothelial TGFβ
co-receptor, all of which lead to vascular disruption and midgestational
lethality (Carvalho et al.,
2007; Carvalho et al.,
2004).
The relevance of endothelial TGFβ signaling for vascular smooth muscle
formation and the similarities in phenotypes of the mouse knockouts of
TGFβ signaling components with that of Lkb1lox/-,
Tie1-Cre suggested that loss of endothelial Lkb1 might lead to
defective TGFβ signaling in ECs. This hypothesis was supported by the
recent observation that loss of Lkb1 in mouse embryonic fibroblasts
(MEFs) decreases the amount of active TGFβ in culture supernatants
(Katajisto et al., 2008).
The effect of LKB1 deficiency on endothelial TGFβ signaling was
investigated in hTERT-immortalized HUVEC cells, by using siRNA-mediated
knockdown of LKB1 with two siRNAs, which led to a 56% (LKB1-si1) and
a 67% (LKB1-si2) decrease in LKB1 mRNA levels
(Fig. 5A). Subsequently, mRNA
expression of TGFβ1 and plasminogen activator inhibitor
1 (PAI1; SERPINE1 - Human Gene Nomenclature Database), a
known downstream target of the TGFβ
(Goumans et al., 2002;
Hackett and Campochiaro,
1993), was analyzed. Interestingly, both
TGFβ1 and PAI1 mRNAs were decreased following
LKB1 knockdown, and levels correlated with the efficiency of the
knockdown (Fig. 5A). As both
PAI1 and TGFβ1
(Letarte et al., 2005;
Van Obberghen-Schilling et al.,
1988) are regulated by TGFβ pathway activity, this result
suggests that LKB1 regulates TGFβ signaling in endothelial cells.
Importantly, a decrease in Tgfβ1 mRNA levels was also
noted in Lkb1lox/-, Tie1-Cre yolk sacs compared with the
yolk sacs of wild-type littermates (Fig.
5A).
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Defects caused by LKB1 deficiency are partially restored by exogenous TGFβ
To investigate whether the defects resulting from endothelial LKB1
loss in immortalized HUVECs could be rescued by exogenous TGFβ1, mRNA
levels of TGFβ1 and PAI1 were analyzed before
and after TGFβ treatment of LKB1-siRNA or control-siRNA transfected
HUVECs. A significant increase in the levels of both mRNAs was noted following
TGFβ treatment of LKB1-siRNA treated HUVECs, resulting in 81%
TGFβ1 and 87% PAI1 mRNA levels in LKB1-siRNA
compared with controls (Fig.
6A), and demonstrating that exogenous TGFβ1 can partly rescue
the TGFβ signaling defects of LKB1-deficient endothelial cells.
To address the significance of reduced endothelial TGFβ production in vivo, wild-type and Lkb1lox/-, Tie1-Cre E11.5 or E12.5 yolk sacs were split following dissection and incubated in medium with or without added TGFβ1 (1 ng/ml). Interestingly, after only one hour of TGFβ1 treatment the number of pSMAD2-positive cells was already increased in the mutant yolk sacs (Fig. 6B-E). Moreover, the added TGFβ1 was able to induce SMA expression in mesenchymal cells of Lkb1lox/-, Tie1-Cre yolk sacs, noted at 8 hours (Fig. 6F-H).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mechanism by which downregulation of LKB1 attenuates TGFβ
signaling and subsequent TGFβ production in ECs warrants further studies.
Based on results from LKB1-deficient MEFs, where attenuated TGFβ
signaling is associated with a comparable drop in both active and total
TGFβ in culture supernatants
(Katajisto et al., 2008)
(K.V., Eeva Ventelä, M.T. and T.P.M., unpublished), it does not appear
likely that TGFβ signaling defects reflect an altered extracellular
activation of latent TGFβ. Another possible mechanism is suggested by the
reported ternary complexes between LKB1, LIP1 and SMAD4
(Smith et al., 2001), although
we have not been able to detect these complexes (data not shown). In this
case, loss of LKB1 would regulate pSMAD2 indirectly through the autocrine
regulation of TGFβ production. The mechanism involved may be shared with
other cell types in which LKB1 appears to regulate TGFβ signaling,
including fibroblasts (Katajisto et al.,
2008) (K.V., Eeva Ventelä, M.T. and T.P.M., unpublished), and
possibly epithelial cells, based on the observation that Lkb1
overexpression in HeLa cells increases expression of the TGFβ target
genes PAI1 and SM22 (Lin-Marq et al.,
2005).
Comparison of vascular phenotypes in Lkb1lox/-,
Tie1-Cre mice and Lkb1-/- mice
(Ylikorkala et al., 2001)
demonstrates significant similarities, suggesting that lack of endothelial
Lkb1 function contributes to the phenotype noted in
Lkb1-null embryos. The later onset of defects (E11.5) and lethality
(E12.5) in Lkb1lox/-, Tie1-Cre embryos compared with the
lethality of Lkb1-/- embryos (E9.5-E10.5) could be due to
the contribution of defects in other Lkb1-deficient cell types to the
null phenotype, or to the LKB1 protein half-life following Tie1-Cre-mediated
Lkb1 excision starting at E8.5
(Gustafsson et al., 2001).
Defective blood vessel maturation is a feature shared not only between
these genotypes, but also with mice lacking core TGFβ pathway components
TGFβ1, TGFβRII, ALK1, ALK5 and SMAD5
(Dickson et al., 1995;
Larsson et al., 2001;
Oh et al., 2000;
Oshima et al., 1996;
Yang et al., 1999), which also
display variation in the severity of vascular phenotypes, with lethality
between E10.5-E11.5. The most severe phenotypes include the inability of the
yolk sac vascular plexus to mature and the defective vascular smooth muscle
cell recruitment noted in Lkb1, Alk5 and Smad5 knockout
embryos (Larsson et al., 2001;
Yang et al., 1999;
Ylikorkala et al., 2001)
Lkb1lox/-, Tie1-Cre embryos were able to initiate
angiogenesis and defects were observed at a later developmental stage than in
endothelial knockouts of TGFβ receptors
(Carvalho et al., 2007;
Carvalho et al., 2004).
Interestingly, defects caused by deficiencies in genes with a regulatory role
in TGFβ signaling, such as endoglin
(Carvalho et al., 2004) and
Tak1 (Jadrich et al.,
2006) are similar but arise later, and share phenotypes with the
Lkb1lox/-, Tie1-Cre mice. Knockout of TGFβRII
specifically in smooth muscle cells leads to a very similar phenotype to that
of Lkb1lox/-, Tie1-Cre mice
(Carvalho et al., 2007),
supporting the notion that the major defect in Lkb1-deficient ECs is
decreased TGFβ production. This would also be consistent with the noted
progression of the initial stages of angiogenesis before vSMC recruitment, in
the absence of endothelial Lkb1. Taken together, these observations
suggest that regulation of TGFβ signaling in ECs represents a vital
function of the LKB1 kinase during mammalian development.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/13/2331/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Global Medical Writing, Bayer Schering Pharma Oy, 02151
Espoo, Finland
Present address: National Agency for Medicines, 00301 Helsinki, Finland
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