Download Role of PTEN/PI3K pathway in endothelial cells

172
Biochemical Society Transactions (2007) Volume 35, part 2
Role of PTEN/PI3K pathway in endothelial cells
A. Suzuki*1 , K. Hamada*, T. Sasaki†, T.W. Mak‡§ and T. Nakano¶
*Department of Molecular Biology, Akita University School of Medicine, Akita 010-8543, Japan, †Department of Microbiology, Akita University School of
Medicine, Akita 010-8543, Japan, ‡The Campbell Family Institute for Breast Cancer Research, Princess Margaret Hospital, Toronto, ON, Canada M5G 2M9,
§Departments of Immunology and Medical Biophysics, University of Toronto, Toronto, ON, Canada M5G 2C1, and ¶Department of Pathology, Medical School
and Graduate School of Frontier Biosciences, Osaka University, Suita 565-0871, Japan
Abstract
PTEN (phosphatase and tensin homologue deleted on chromosome 10) is an important tumour-suppressor
gene that encodes a 3-phosphatase. The major substrate of PTEN is PIP3 (phosphatidylinositol 3,4,5trisphosphate) generated by the action of PI3Ks (phosphoinositide 3-kinases). Hereditary mutation of PTEN
causes tumour-susceptibility diseases such as Cowden disease. We used the Cre-loxP system to generate
an endothelial cell-specific mutation of PTEN in mice. Heterozygous mutation of PTEN in endothelial cells
enhances postnatal neovascularization, including tumour angiogenesis necessary for tumour growth. This
observation suggests that Cowden disease patients are not only at risk for additional tumorigenic mutations
due to complete loss of PTEN function, but may also experience accelerated growth of incipient tumours due
to enhanced angiogenesis. Homozygous mutation of Pten in murine endothelial cells impairs cardiovascular
morphogenesis and is embryonic lethal due to endothelial cell hyperproliferation and impaired vascular remodelling. Additional homozygous mutation of p85α, the regulatory subunit of class IA PI3Ks, or p110γ ,
the catalytic subunit of the sole class IB PI3K, led to a partial rescue of all phenotypes in our PTEN-deficient
mice. Thus inhibition of the PI3K pathway, including the targeting of PI3Kγ , may be an attractive therapeutic
strategy for the treatment of various malignancies.
Introduction
PTEN (phosphatase and tensin homologue deleted from
chromosome 10) is a ubiquitously expressed tumour–suppressor gene [1]. PTEN is mutated in human sporadic cancers
of various tissues, and in hereditary cancer disorders such as
Cowden disease, Bannayan–Zonana syndrome, Lhermitte–
Duclos disease and Proteus syndrome [2,3]. Patients with
these latter disorders suffer from multiple hamartomas and
an increased risk of cancer.
PTEN is a multifunctional phosphatase whose lipid phosphatase activity is associated with tumour suppression [4].
The major substrate of PTEN is PIP3 (phosphatidylinositol
3,4,5-trisphosphate) [5], a lipid molecule generated by the
action of PI3Ks (phosphoinositide 3-kinases). The PI3Ks
are evolutionarily conserved lipid kinases [6]. To date, eight
distinct PI3K isoforms have been reported in mammals, four
of which belong to the class I PI3Ks. PI3Kα, PI3Kβ and
PI3Kδ constitute the class IA PI3Ks that are mainly activated by RTK (receptor tyrosine kinase) engagement, while
the single class IB PI3K, PI3Kγ , is activated by the βγ
Key words: cardiovasculogenesis, endothelial cell, p85α, phosphatase and tensin homologue
deleted on chromosome 10 (PTEN), phosphoinositide-3-kinase (PI3K), tumour angiogenesis.
Abbreviations used: bFGF, basic fibroblast growth factor; E9.5 (etc.), embryonic day 9.5 (etc.);
Foxo1, forkhead box O 1; GPCR, G-protein-coupled receptor; LOH, loss of heterozygosity; PC,
pericyte; PKB, protein kinase B; PI3K, phosphoinositide-3-kinase; PIP3 , phosphatidylinositol 3,4,5trisphosphate; PTEN, phosphatase and tensin homologue deleted on chromosome 10; RT, reverse
transcriptase; RTK, receptor tyrosine kinase; αSMA, anti-smooth-muscle antigen antibody; VCAM1, vascular cell adhesion molecule-1; VEGF, vascular endothelial growth factor; VEGFR, VEGF
receptor; VGF, vascular growth factor; VSMC, vascular smooth-muscle cell.
1
To whom correspondence should be addressed (email [email protected]).
C 2007
Biochemical Society
subunit of G-proteins and acts downstream of GPCRs (Gprotein-coupled receptors) [7,8]. PI3K activation occurs in
response to the binding of numerous kinds of growth factors,
cytokines, chemokines and hormones to their receptors, as
well as antigen receptor engagement. PIP3 generated by
PI3K activation in turn activates PKB (protein kinase B)/
Akt, a serine/threonine kinase involved in anti-apoptosis,
proliferation and oncogenesis. By dephosphorylating the
D3 position of PIP3 , PTEN negatively regulates the PI3K
pathway and PKB/Akt activation and thus tumorigenesis.
To investigate the functions of PTEN in vivo, we and
others generated null mutations of Pten in mice [9–13]. Animals heterozygous for a null Pten mutation develop a broad
range of tumours, including cancers of the breast, thyroid,
endometrium and prostate as well as T-cell lymphomas.
This spectrum of neoplasias closely resembles that seen in
humans with PTEN mutations. Pten+/− mice also develop
signs of auto-immune disease [14]. Homozygosity for a
null Pten mutation in mice leads to embryonic lethality
at approx. E9.5 (embryonic day 9.5) [9,10], indicating that
PTEN is indispensable for embryogenesis. Because this early
embryonic lethality precludes the analysis of PTEN functions
in adult organs, we and others created conditional mutant
mouse strains in which the Pten gene is ‘floxed’ (Ptenflox mice).
To determine the role of PTEN in a particular cell type, the
Ptenflox mice are crossed with transgenic mice expressing Cre
recombinase under the control of a tissue-specific promoter.
Analyses of these various conditional mutant mice have
revealed that tissue-specific deletion of PTEN causes a wide
range of malignancies, including T-cell lymphoma [15], skin
3rd Focused Meeting on PI3K Signalling and Disease
cancer [16], teratoma [17], hepatoma [18], breast cancer
[19], pancreatic cancer [20], prostatic cancer [21–23], bladder
cancer [24] and acute myeloid leukaemia [25,26]. These
mutant mice also develop various non-cancerous ailments
such as auto-immune disease [15], cardiac failure [27] and
non-alcoholic steatohepatitis [18].
Increased angiogenesis and accelerated
tumour growth in Tie2CrePtenflox/+ mice
Embryonic cardiovascular development and postnatal neovascularization (including tumour angiogenesis) are complex
processes that share a dependence on shear stress and particular signalling molecules [28]. Co-ordinated interactions
between endothelial VGFs [vascular growth factors; e.g.
VEGF (vascular endothelial growth factor), Ang-1, Ang-2,
bFGF (basic fibroblast growth factor), PDGF-B (plateletderived growth factor-B), ephrin-B2 and TGF-β (transforming growth factor-β) superfamily members], intracellular
signalling molecules [e.g. Notch1 and COUP-TFII (chicken
ovalbumin upstream promoter-transcription factor 2)], and
intercellular contacts [e.g. VCAM-1 (vascular cell adhesion
molecule-1)] are required for both prenatal and postnatal
vasculogenesis, and mutations of these molecules cause
defects in cardiovascular development [29–33]. Significantly,
all of the above VGFs also activate the PI3K–PKB/Akt
pathway.
PTEN is important for normal cardiovascular homoeostasis. In vitro, PTEN inhibits vascular sprouting and endothelial tube formation induced by VEGF, and dominantnegative mutation of PTEN abolishes these effects [34].
In vivo, Su et al. [35] have shown that tumour angiogenesis
and tumour growth in mice are blocked by PTEN overexpression in tumour cells or by administration of PI3K
inhibitors. However, it was not clear in this study whether the
inhibition of tumour growth was caused by the angiogenesis
defect or by a direct effect on the tumour cells. To resolve this
issue and to investigate the function of PTEN in endothelial
cells in vivo, we generated endothelial cell-specific PTENdeficient mice by mating Ptenflox mice and Tie2Cre transgenic
mice [36].
Homologous recombination disrupting the Pten gene occurred in approx. 95% of endothelial and endocardial cells
in Tie2CrePtenflox/+ mice but not in the PCs (pericytes) or
VSMCs (vascular smooth-muscle cells) of these mutants.
Histological analyses of systemic vessels and the heart
revealed no significant structural differences between
Tie2CrePten+/+ and Tie2CrePtenflox/+ mice. We then subcutaneously implanted Matrigels impregnated with bFGF,
bFGF+VEGF, bFGF+Ang-1, or PBS into Tie2CrePten+/+
and Tie2CrePtenflox/+ mice and quantified blood vessel infiltration of the implants by immunostaining with anti-CD31
antibody. In contrast with implanted Tie2CrePten+/+ mice,
implanted Tie2CrePtenflox/+ mice showed a significant increase in vascularization in response to VGF administration.
To determine whether heterozygous Pten deficiency
in endothelial cells affected adult tumour angiogenesis,
Tie2CrePtenflox/+ and Tie2CrePten+/+ mice were injected
subcutaneously with either melanoma (B16BL6) or LLC
(Lewis lung carcinoma) cells. Both cell lines induced significantly larger tumours in Tie2CrePtenflox/+ mice than in
Tie2CrePten+/+ mice, and microvessels were more abundant
and of larger size in the Tie2CrePtenflox/+ mice. In vitro
experiments revealed that total numbers of PCNA+ (proliferating-cell nuclear antigen-positive) cells and their migration in response to stimulation with Ang-1 or VEGF-A
were significantly increased in Tie2CrePtenflox/+ cultures
compared with controls.
Our study was the first to demonstrate that the loss
of PTEN specifically in mouse endothelial cells makes
them hypersensitive to VGFs and thus is responsible for
enhanced angiogenesis leading to accelerated tumour growth.
An identical mechanism may be operating in humans with
Cowden disease, since this cancer susceptibility disorder is
caused by heterozygous mutation of PTEN. Our results
suggest that an individual who inherits a mutated PTEN allele
is not only at risk for additional tumorigenic mutations due
to the LOH (loss of heterozygosity) of PTEN, but may also
experience accelerated growth of incipient tumours due to
enhanced angiogenesis (Figure 1A).
Death of Tie2CrePtenflox/flox mice by E11.5
due to endothelial cell hyperproliferation
and impaired vascular remodelling
The intercrossing of Tie2CrePtenflox/+ and Ptenflox/+ mice
generated homozygous mutant embryos that were present
at the expected Mendelian frequency up to E9.5. However,
resorption of Tie2CrePtenflox/flox embryos commenced at
E10.5 and embryonic loss occurred at E11.5. Histologically,
E8.25 Tie2CrePtenflox/flox embryos were essentially normal
with respect to gross appearance of the central vascular
tree, including the rostral-caudal aorta and the anterior and
posterior cardiac veins. PTEN is thus dispensable for the
differentiation of angioblasts from the ventral mesoderm,
their appropriate migration within the embryo, and their
alignment to form major vessels. However, E9.5 homozygous
mutants exhibited an enlarged capillary plexus featuring
a meshwork of interconnected, oversized endothelial celllined tubes that showed increased staining for Ki67. Distinct
branches of large vessels such as the anterior cardinal vein
in the embryo proper and vitelline vessels in the yolk sac
were not formed because of a failure in primary vascular
plexus remodelling. By E10.5, most of the mutants showed
profound growth retardation as well as pericardial cavity
enlargement and frequent bleeding into the pericardial
cavity or large trunk vessels.
Cross-talk between endothelial cells and PCs/VSMCs is
critical for vascular remodelling and maturation. We therefore
examined the recruitment of PCs and VSMCs to developing
vascular channels using immunostaining with αSMA (antismooth-muscle antigen antibody). At E10.0, αSMA+ cells
were observed in perivascular regions of the Tie2CrePten+/+
yolk sac. In contrast, no αSMA+ PCs/VSMCs were present
C 2007
Biochemical Society
173
174
Biochemical Society Transactions (2007) Volume 35, part 2
Figure 1 Parallels between Cowden disease in humans and endothelial-cell-specific mutation of Pten in mice
(A) Cowden disease patients are heterozygous for a germline mutation of PTEN. Such individuals are not only at risk for
additional tumorigenic mutations due to the LOH of PTEN, but may also experience accelerated growth of incipient tumours
due to enhanced angiogenesis. (B) Mutant mice homozygous for an endothelial cell-specific mutation of Pten are embryonic
lethal at E11.5. The loss of PTEN results in constitutive activation of PKB/Akt, and positively regulates eNOS (endothelial
nitric oxide synthase) and negatively regulates Foxo1. Moreover, in the absence of PTEN, endothelial cells experience up- or
down-regulation of the indicated molecules (Ang-1, Ang-2 etc.). This mass dysregulation leads to the indicated failures in
cardiovasculogenesis that most likely underlie the lethality of Tie2CrePtenflox/flox mice.
adjacent to endothelial cells in the vessels or yolk sacs
of Tie2CrePtenflox/flox mice. In the embryo proper, the
αSMA staining seen in the vessels, heart and dorsal aorta
of Tie2CrePten+/+ embryos was dramatically decreased in
Tie2CrePtenflox/flox embryos. The bleeding from the large
trunk vessels noted in the mutants may have resulted from
the impaired recruitment of PCs/VSMCs to these tissues.
A reduction in the size of the developing intraventricular
septum and cardiac trabeculae, and a thinning of the cardiac
wall, were also observed in homozygous mutants. The thinnest cardiac walls resembled membranes such that bleeding
in the mutant pericardial cavity could have resulted from
cardiac wall rupture or leakage of blood cells from the cardiac
lumen.
RT (reverse transcriptase)–PCR analyses of gene expression in whole yolk sacs from E8.5 Tie2CrePten+/+
and Tie2CrePtenflox/flox embryos showed that a lack of
PTEN significantly decreased expression of Ang-1, ephrinB2
and VCAM-1 but increased expression of Ang-2, VEGFA, VEGFR1(Flt-1) (where VEGFR is VEGF receptor) and
VEGFR2(Flk-1). These differences were confirmed by RT–
PCR analyses of VEGFR2+ cells from E9.5 Tie2CrePten+/+
and Tie2CrePtenflox/flox embryos, and by RT–PCR and protein analyses of HUVEC (human umbilical vein endothelial
cells) in which PTEN expression was decreased by siRNA
(small interfering RNA) treatment. These results suggest that
C 2007
Biochemical Society
PTEN deficiency leads directly to an altered VGF profile
that may be responsible for the cardiovascular defects of
Tie2CrePtenflox/flox mice.
Several lines of evidence support our contention that dysregulation of downstream targets of PKB/Akt due to loss of
PTEN function is responsible for the lethal phenotype
of Tie2CrePtenflox/flox mice (Figure 1B). Constitutively active
PKB/Akt (myrAkt) expression in vivo causes fatal vascular
malformations and bleeding due to a failure in vascular remodelling [37]. Fatal vascular remodelling and cardiac
defects are also present in mice deficient in Foxo1 (forkhead
box O 1), a transcription factor negatively regulated by PKB/
Akt [38]. Mice deficient in the ephrinB2, which is downregulated by PTEN deficiency, die at E10.5 due to defects
in vascular remodelling and sprouting that lead to pericardial
effusion or bleeding [39]. Similarly, disruption of Ang-1 or its
receptor Tie2 in knockout mice causes vascular defects due
to impaired recruitment of PCs/VSMCs [29,40]. In contrast,
administration of Ang-2 (whose action opposes Ang-1)
causes a dose-dependent pericyte ‘dropout’ in the normal
retina [41]. VEGF expression, and increased VEGF during
lung organogenesis, overstimulates endothelial cell growth
that leads to abnormally large capillaries [42]. Consistent
with this latter observation, mice overexpressing VEGF die
at E14 due to cardiac abnormalities [43]. Finally, VCAM-1
deficiency in mice causes pericardial bleeding and impaired
3rd Focused Meeting on PI3K Signalling and Disease
cardiomyocyte development [44]. Taken together, these
results highlight the vital role PTEN plays in maintaining
normal vascular biology.
Partial rescue of Tie2CrePtenflox/+ and
Tie2CrePtenflox/flox mice by null mutation
of p110γ or p85α
p85α is the most abundantly expressed regulatory subunit of
the class IA PI3Ks that are activated following RTK engagement by VGFs. p110γ is the catalytic subunit of the sole
class IB PI3K that is activated by the βγ subunit of Gproteins and acts downstream of activated GPCRs [7,45].
To clarify the contribution of class IA and class IB PI3Ks to
the phenotypes observed in Tie2CrePtenflox/+ and Tie2CrePtenflox/flox mice, we generated p85α −/− Tie2CrePtenflox/+ ,
p85α −/− Tie2CrePtenflox/flox , p110γ −/− Tie2CrePtenflox/+ and
p110γ −/− Tie2CrePtenflox/flox double mutant mice. Homozygous mutation of p110γ partially restored control of angiogenic responses to VGFs and reduced decreased angiogenesis
and tumour growth in Tie2CrePtenflox/+ mice. Moreover, loss
of p110γ led to a dramatic rescue of all Tie2CrePtenflox/flox
phenotypes such that these double mutant mice survived
until E18.5–19.5. Homozygous mutation of p85α restored
control of angiogenic responses in Tie2CrePtenflox/+ mice to
the same degree as mutation of p110γ . However, the defects
in cardiovascular morphogenesis seen in Tie2CrePtenflox/flox
mice were only partially rescued by p85α deficiency such that
these animals survived only until E14.5–15.5.
Because most VGF receptors have tyrosine kinase activity,
class IA PI3Ks likely play major roles in cardiovasculogenesis
and tumour angiogenesis. Consistent with this notion, loss
of p85α partially resolved the enhanced angiogenesis and
accelerated tumour growth observed in our Tie2CrePtenflox/+
mice, as well as the impaired cardiovascular morphogenesis
seen in Tie2CrePtenflox/flox mice. Intriguingly, the enhanced
angiogenesis in Tie2CrePtenflox/+ mice induced by RTK
agonists (e.g. VEGF and Ang-1) was partially rescued by
p110γ deficiency. This result was unexpected because p110γ
has been postulated to be activated downstream of GPCRs
but not of RTKs. It may be that, in vivo, an unknown VGF
(possibly a GPCR ligand) activates p110γ and influences
RTK signalling initiated by VEGF or Ang-1 that leads to
angiogenesis. Our generation of double mutant mice lacking
both PTEN and class IB PI3K functions has shed much
needed light on the potential role of class IB PI3K in
cardiovascular morphogenesis and postnatal angiogenesis.
Conclusion
Our study has demonstrated that normal function of
the PI3K–PTEN–PKB/Akt pathway in endothelial cells
is required to control cardiovascular morphogenesis and
postnatal neovascularization, including tumour angiogenesis
supporting tumour growth. Inhibition of the PI3K pathway,
including the targeting of PI3Kγ , may be an attractive
therapeutic strategy for the treatment of various malignancies.
References
1 Li, J., Yen, C., Liaw, D., Podsypanina, K., Bose, S., Wang, S.I., Puc, J.,
Miliaresis, C., Rodgers, L., McCombie, R. et al. (1997) Science 275,
1943–1947
2 Liaw, D., Marsh, D.J., Li, J., Dahia, P.L., Wang, S.I., Zheng, Z., Bose, S.,
Call, K.M., Tsou, H.C., Peacocke, M. et al. (1997) Nat. Genet. 16,
64–67
3 Marsh, D.J., Dahia, P.L., Zheng, Z., Liaw, D., Parsons, R., Gorlin, R.J. and
Eng, C. (1997) Nat. Genet. 16, 333–334
4 Myers, M.P., Pass, I., Batty, I.H., Van der Kaay, J., Stolarov, J.P.,
Hemmings, B.A., Wigler, M.H., Downes, C.P. and Tonks, N.K. (1998)
Proc. Natl. Acad. Sci. U.S.A. 95, 13513–13518
5 Maehama, T. and Dixon, J.E. (1998) J. Biol. Chem. 273, 13375–13378
6 Toker, A. and Cantley, L.C. (1997) Nature 387, 673–676
7 Stoyanov, B., Volinia, S., Hanck, T., Rubio, I., Loubtchenkov, M., Malek, D.,
Stoyanova, S., Vanhaesebroeck, B., Dhand, R., Nurnberg, B. et al. (1995)
Science 269, 690–693
8 Stephens, L.R., Eguinoa, A., Erdjument-Bromage, H., Lui, M., Cooke, F.,
Coadwell, J., Smrcka, A.S., Thelen, M., Cadwallader, K., Tempst, P. and
Hawkins, P.T. (1997) Cell 89, 105–114
9 Suzuki, A., de la Pompa, J.L., Stambolic, V., Elia, A.J., Sasaki, T.,
del Barco Barrantes, I., Ho, A., Wakeham, A., Itie, A., Khoo, W. et al.
(1998) Curr. Biol. 8, 1169–1178
10 Di Cristofano, A., Pesce, B., Cordon-Cardo, C. and Pandolfi, P.P. (1998)
Nat. Genet. 19, 348–355
11 Podsypanina, K., Ellenson, L.H., Nemes, A., Gu, J., Tamura, M., Yamada,
K.M., Cordon-Cardo, C., Catoretti, G., Fisher, P.E. and Parsons, R. (1999)
Proc. Natl. Acad. Sci. U.S.A. 96, 1563–1568
12 Stambolic, V., Suzuki, A., de la Pompa, J.L., Brothers, G.M., Mirtsos, C.,
Sasaki, T., Ruland, J., Penninger, J.M., Siderovski, D.P. and Mak, T.W.
(1998) Cell 95, 29–39
13 Stambolic, V., Tsao, M.S., Macpherson, D., Suzuki, A., Chapman, W.B. and
Mak, T.W. (2000) Cancer Res. 60, 3605–3611
14 Di Cristofano, A., Kotsi, P., Peng, Y.F., Cordon-Cardo, C., Elkon, K.B. and
Pandolfi, P.P. (1999) Science 285, 2122–2125
15 Suzuki, A., Yamaguchi, M.T., Ohteki, T., Sasaki, T., Kaisho, T., Kimura, Y.,
Yoshida, R., Wakeham, A., Higuchi, T., Fukumoto, M. et al. (2001)
Immunity 14, 523–534
16 Suzuki, A., Itami, S., Ohishi, M., Hamada, K., Inoue, T., Komazawa, N.,
Senoo, H., Sasaki, T., Takeda, J., Manabe, M. et al. (2003) Cancer Res. 63,
674–681
17 Kimura, T., Suzuki, A., Fujita, Y., Yomogida, K., Lomeli, H., Asada, N.,
Ikeuchi, M., Nagy, A., Mak, T.W. and Nakano, T. (2003) Development
130, 1691–1700
18 Horie, Y., Suzuki, A., Kataoka, E., Sasaki, T., Hamada, K., Sasaki, J.,
Mizuno, K., Hasegawa, G., Kishimoto, H., Iizuka, M. et al. (2004)
J. Clin. Invest. 113, 1774–1783
19 Li, G., Robinson, G.W., Lesche, R., Martinez-Diaz, H., Jiang, Z.,
Rozengurt, N., Wagner, K.U., Wu, D.C., Lane, T.F., Liu, X. et al. (2002)
Development 129, 4159–4170
20 Stanger, B.Z., Stiles, B., Lauwers, G.Y., Bardeesy, N., Mendoza, M.,
Wang, Y., Greenwood, A., Cheng, K.H., McLaughlin, M., Brown, D. et al.
(2005) Cancer Cell 8, 185–195
21 Backman, S.A., Ghazarian, D., So, K., Sanchez, O., Wagner, K.U.,
Hennighausen, L., Suzuki, A., Tsao, M.S., Chapman, W.B., Stambolic, V.
and Mak, T.W. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 1725–1730
22 Wang, S., Gao, J., Lei, Q., Rozengurt, N., Pritchard, C., Jiao, J., Thomas,
G.V., Li, G., Roy-Burman, P., Nelson, P.S. et al. (2003) Cancer Cell 4,
209–221
23 Chen, Z., Trotman, L.C., Shaffer, D., Lin, H.K., Dotan, Z.A., Niki, M.,
Koutcher, J.A., Scher, H.I., Ludwig, T., Gerald, W. et al. (2005) Nature
436, 725–730
24 Tsuruta, H., Kishimoto, H., Sasaki, T., Horie, Y., Natsui, M., Shibata, Y.,
Hamada, K., Yajima, N., Kawahara, K., Sasaki, M. et al. (2006)
Cancer Res. 66, 8389–8396
25 Zhang, J., Grindley, J.C., Yin, T., Jayasinghe, S., He, X.C., Ross, J.T.,
Haug, J.S., Rupp, D., Porter-Westpfahl, K.S., Wiedemann, L.M. et al.
(2006) Nature 441, 518–522
26 Yilmaz, O.H., Valdez, R., Theisen, B.K., Guo, W., Ferguson, D.O., Wu, H.
and Morrison, S.J. (2006) Nature 441, 475–482
27 Crackower, M.A., Oudit, G.Y., Kozieradzki, I., Sarao, R., Sun, H., Sasaki, T.,
Hirsch, E., Suzuki, A., Shioi, T., Irie-Sasaki, J. et al. (2002) Cell 110,
737–749
28 Daniel, T.O. and Abrahamson, D. (2000) Annu. Rev. Physiol. 62,
649–671
C 2007
Biochemical Society
175
176
Biochemical Society Transactions (2007) Volume 35, part 2
29 Suri, C., Jones, P.F., Patan, S., Bartunkova, S., Maisonpierre, P.C., Davis, S.,
Sato, T.N. and Yancopoulos, G.D. (1996) Cell 87, 1171–1180
30 Lindahl, P., Johansson, B.R., Leveen, P. and Betsholtz, C. (1997) Science
277, 242–245
31 Lindblom, P., Gerhardt, H., Liebner, S., Abramsson, A., Enge, M.,
Hellstrom, M., Backstrom, G., Fredriksson, S., Landegren, U., Nystrom,
H.C. et al. (2003) Genes Dev. 17, 1835–1840
32 Dickson, M.C., Martin, J.S., Cousins, F.M., Kulkarni, A.B., Karlsson, S. and
Akhurst, R.J. (1995) Development 121, 1845–1854
33 Larsson, J., Goumans, M.J., Sjostrand, L.J., van Rooijen, M.A., Ward, D.,
Leveen, P., Xu, X., ten Dijke, P., Mummery, C.L. and Karlsson, S. (2001)
EMBO J. 20, 1663–1673
34 Huang, J. and Kontos, C.D. (2002) J. Biol. Chem. 277, 10760–10766
35 Su, J.D., Mayo, L.D., Donner, D.B. and Durden, D.L. (2003) Cancer Res.
63, 3585–3592
36 Hamada, K., Sasaki, T., Koni, P.A., Natsui, M., Kishimoto, H., Sasaki, J.,
Yajima, N., Horie, Y., Hasegawa, G., Naito, M. et al. (2005) Genes Dev.
19, 2054–2065
37 Sun, J.F., Phung, T., Shiojima, I., Felske, T., Upalakalin, J.N., Feng, D.,
Kornaga, T., Dor, T., Dvorak, A.M., Walsh, K. and Benjamin, L.E. (2005)
Proc. Natl. Acad. Sci. U.S.A. 102, 128–133
C 2007
Biochemical Society
38 Furuyama, T., Kitayama, K., Shimoda, Y., Ogawa, M., Sone, K.,
Yoshida-Araki, K., Hisatsune, H., Nishikawa, S., Nakayama, K., Ikeda, K.
et al. (2004) J. Biol. Chem. 279, 34741–34749
39 Wang, H.U., Chen, Z.F. and Anderson, D.J. (1998) Cell 93, 741–753
40 Dumont, D.J., Gradwohl, G., Fong, G.H., Puri, M.C., Gertsenstein, M.,
Auerbach, A. and Breitman, M.L. (1994) Genes Dev. 8, 1897–1909
41 Hammes, H.P., Lin, J., Wagner, P., Feng, Y., Vom Hagen, F., Krzizok, T.,
Renner, O., Breier, G., Brownlee, M. and Deutsch, U. (2004) Diabetes 53,
1104–1110
42 Zeng, X., Wert, S.E., Federici, R., Peters, K.G. and Whitsett, J.A. (1998)
Dev. Dyn. 211, 215–227
43 Miquerol, L., Langille, B.L. and Nagy, A. (2000) Development 127,
3941–3946
44 Kwee, L., Baldwin, H.S., Shen, H.M., Stewart, C.L., Buck, C., Buck, C.A. and
Labow, M.A. (1995) Development 121, 489–503
45 Dimmeler, S., Assmus, B., Hermann, C., Haendeler, J. and Zeiher, A.M.
(1998) Circ. Res. 83, 334–341
Received 28 November 2006