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1812 Research Article
IntroductionVessel lumenization is a crucial step in developing a functional
vascular system during vascular morphogenesis (Adams and Alitalo,
2007; Davis and Bayless, 2003; Davis et al., 2007; Egginton and
Gerritsen, 2003; Holderfield and Hughes, 2008; Horowitz and
Simons, 2008; Iruela-Arispe and Davis, 2009; Parker et al., 2004).
Our previous work has shown that endothelial cell (EC) lumen
formation in three-dimensional (3D) collagen matrices is regulated
by the formation and coalescence of EC intracellular vacuoles, a
process that is dependent on Cdc42 and Rac1 GTPases in response
to α2β1-integrin–collagen-type-I interactions (Bayless and Davis,
2002; Bayless et al., 2000; Davis and Bayless, 2003; Davis et al.,
2002; Davis and Camarillo, 1996; Davis and Senger, 2005; Koh et
al., 2008). Regulation of EC intracellular vacuole formation and
coalescence by Cdc42 GTPase has also been shown to be a major
mechanism of vascular development in zebrafish, suggesting that
Rho GTPases play a key role in EC vascular lumen formation in
vivo (Kamei et al., 2006). Rho GTPases are well-known to control
cytoskeletal structures and, thus, influence various cellular functions
that are necessary for EC vascular morphogenic events (Davis and
Bayless, 2003; Fryer and Field, 2005; Hall, 1998; Hall, 2005; Ridley,
2001; Schwartz, 2004). Among the diverse spectrum of Rho-
GTPase targets, p21-activated kinase (Pak) proteins are known as
key downstream effectors that are involved in the regulation of
cytoskeletal function (Bokoch, 2003), and we have shown that two
members of the Pak family, Pak2 and Pak4, are required during EC
lumen formation in 3D collagen matrices (Davis et al., 2007; Koh
et al., 2008).
Because Rho GTPases are activated downstream of integrins,
growth-factor receptors, cytokines and hormones, their activation
can be regulated by Src (Robles et al., 2005; Tatin et al., 2006;
Timpson et al., 2001). Src is a member of Src-family nonreceptor
protein tyrosine kinases (SFKs). SFKs influence a broad range of
cellular functions downstream of growth-factor receptors, integrins
and other adhesion molecules (Abu-Ghazaleh et al., 2001; Eliceiri
et al., 1999; Eliceiri et al., 2002; Kilarski et al., 2003; Parsons and
Parsons, 2004; Playford and Schaller, 2004; Thomas and Brugge,
1997; Tsuda et al., 2002; Werdich and Penn, 2005; Werdich and
Penn, 2006). SFKs are also known to be involved in protein kinase
C (PKC)-mediated signaling pathways to regulate actin-cytoskeletal
structures as well as cell invasion (Bruce-Staskal and Bouton, 2001;
Nomura et al., 2007). We have previously shown that PKC plays
a key role in EC lumen formation in 3D collagen matrices in
response to phorbol ester (TPA) (Davis et al., 2007; Koh et al.,
2008), raising a possible signaling mechanism involving both PKC
and SFKs to regulate this process. Src has also previously been
shown to control capillary-cord formation in 3D collagen matrices
(Liu and Senger, 2004).
Previous studies have shown that SFKs regulate the activation
of Pak2 along with Cdc42 or Rac1 (Renkema et al., 2002). It is
In this study, we present data showing that Cdc42-dependent
lumen formation by endothelial cells (ECs) in three-dimensional
(3D) collagen matrices involves coordinated signaling by PKCεin conjunction with the Src-family kinases (SFKs) Src and Yes.
Activated SFKs interact with Cdc42 in multiprotein signaling
complexes that require PKCε during this process. Src and Yes
are differentially expressed during EC lumen formation and
siRNA suppression of either kinase, but not Fyn or Lyn, results
in significant inhibition of EC lumen formation. Concurrent
with Cdc42 activation, PKCε- and SFK-dependent signaling
converge to activate p21-activated kinase (Pak)2 and Pak4 in
steps that are also required for EC lumen formation. Pak2 and
Pak4 further activate two Raf kinases, B-Raf and C-Raf,
leading to ERK1 and ERK2 (ERK1/2) activation, which all seem
to be necessary for EC lumen formation. This work reveals a
multicomponent kinase signaling pathway downstream of
integrin-matrix interactions and Cdc42 activation involving
PKCε, Src, Yes, Pak2, Pak4, B-Raf, C-Raf and ERK1/2 to
control EC lumen formation in 3D collagen matrices.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/122/11/1812/DC1
Key words: Rho GTPases, PKCε, Raf, Lumen formation, Src, Cdc42,
Extracellular matrix, Pak
Summary
Formation of endothelial lumens requires acoordinated PKCε-, Src-, Pak- and Raf-kinase-dependent signaling cascade downstream ofCdc42 activationWonshill Koh1, Kamakshi Sachidanandam1, Amber N. Stratman1, Anastasia Sacharidou1, Anne M. Mayo1,Eric A. Murphy2, David A. Cheresh2 and George E. Davis1,3,*1Department of Medical Pharmacology and Physiology, School of Medicine, University of Missouri, Columbia, MO 65212, USA2Department of Pathology, Moores UCSD Cancer Center, La Jolla, CA 92093, USA3Department of Pathology and Anatomical Sciences, School of Medicine, Dalton Cardiovascular Research Center, University of Missouri,Columbia, MO 65212, USA*Author for correspondence (e-mail: [email protected])
Accepted 26 February 2009Journal of Cell Science 122, 1812-1822 Published by The Company of Biologists 2009doi:10.1242/jcs.045799
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1813Kinase cascades and EC lumenogenesis
important to examine Pak-dependent signaling pathways in more
detail and their relationships to the formation of EC lumens and
tubes (Koh et al., 2008). Previously, it was shown that Pak1 regulates
angiogenesis and EC survival by activating C-Raf (Hood et al.,
2003). C-Raf is a member of the Raf kinase family of
serine/threonine kinases; this family is comprised of three isoforms,
A-Raf, B-Raf and C-Raf. B-Raf and C-Raf can be directly activated
by PKC, SFKs and other kinases (Fabian et al., 1993; Kolch et al.,
1993; Marais et al., 1995; Ueffing et al., 1997). Raf kinases are a
key component of the Raf-MEK-ERK mitogen-activated protein
kinase (MAPK) pathway that regulates many cellular functions
(Chong et al., 2003; Morrison and Cutler, 1997; Wellbrock et al.,
2004). They are also activated by SFKs downstream of various
angiogenic factors (Eliceiri et al., 1999; Eliceiri et al., 2002; Hood
et al., 2003). However, the role of Raf kinases in EC lumen
formation and the associated signaling pathways in relation to Rho
GTPases, PKC and SFKs have not been elucidated.
In this work, using an in vitro EC-lumen-formation model in 3D
collagen matrices, we analyzed how Cdc42, PKCε, SFKs, Pak2,
Pak4 and Raf kinases coordinately regulate EC lumen formation
in 3D collagen matrices, and evaluate their individual functions
during this process. We show that SFKs play a key role in EC lumen
formation in response to PKCε as well as through their association
with Cdc42-dependent signaling. We identify Src and Yes as two
key SFKs, which play critical roles during EC lumen formation and
Pak2 and Pak4 which act downstream of PKCε, SFKs, as well as
Cdc42. Paks in conjunction with SFKs lead to activation of B-Raf
and C-Raf and in conjunction with downstream ERK1 and ERK2
(ERK1/2) phosphorylation control EC lumen formation in 3D
collagen matrices. Thus, a coordinated signaling pathway involving
Cdc42, PKCε, Src, Yes, B-Raf, C-Raf and ERK1/2 are required for
ECs to form lumen and tube structures in a 3D-collagen-matrix
environment.
ResultsPKCε stimulates EC lumen formation in 3D collagen matricesPrevious work using chemical inhibitors and siRNA knockdown
studies have identified a novel PKC isoform, PKCε, as a regulator
of EC lumen formation (Koh et al., 2008). To further evaluate its
functional relevance, we used recombinant adenoviruses carrying
either wild-type (WT) PKCε or dominant-negative (DN) PKCε,
which were confirmed to be expressed by western blotting (data
not shown). ECs infected with control GFP, WT-PKCε or DN-PKCεvirus were allowed to undergo lumen and tube morphogenesis. EC
lumenogenesis was markedly impaired using ECs expressing DN
PKCε, whereas ECs expressing WT PKCε showed significantly
stimulated lumen formation compared with ECs infected with
control GFP virus (Fig. 1A,B). These data suggest that activation
of PKCε strongly enhances EC lumen formation in 3D collagen
matrices.
We have previously shown that regulation of EC lumen formation
by PKC downstream of phorbol-ester treatment is mediated by
activation of two p21-activated kinases, Pak2 and Pak4 (Koh et al.,
2008). Therefore, we investigated whether the influence of PKCεon EC lumen formation correlates with Pak2 and Pak4 activation.
WT PKCε markedly induced Pak4 phosphorylation (Fig. 1C). Pak2
phosphorylation was also increased by WT PKCε, with an
appearance of a protein band whose size is consistent with the
expected size of dimeric Pak2 (Fig. 1C). It has been reported that
phosphorylated Pak2 can dimerize through its kinase domain and
that this dimerization process mediates trans-autophosphorylation
of Pak2 to induce its full activation (Pirruccello et al., 2006).
Expression of DN PKCε diminished phosphorylation of both Pak2
and Pak4 (Fig. 1C), indicating that PKCε regulates EC lumen
formation by activating both Pak2 and Pak4.
SFKs are involved in PKCε-induced EC lumen formation in 3Dcollagen matricesIn an attempt to identify additional kinase targets that are involved
in EC lumen formation, we examined the activation of SFKs and
their functional importance during the process of lumen formation.
Activity of SFKs is regulated by phosphorylation or
dephosphorylation at different residues (Thomas and Brugge,
1997). The Y416 residue in the activation loop is known to be a
key phosphorylation site that leads to full activation of SFKs
(Roskoski, 2004; Roskoski, 2005; Thomas and Brugge, 1997).
Expression of WT PKCε, which stimulates EC lumen formation,
showed higher SFK phosphorylation compared with control GFP
(Fig. 1C). By contrast, SFK phosphorylation was markedly
diminished when DN PKCε was expressed (Fig. 1C). Moreover,
when ECs were suspended in 3D collagen matrices in the presence
of TPA to induce EC lumen formation by activating PKCε,
phosphorylation of SFKs was elevated as well (Fig. 2A). PKC
inhibitors that target PKCε (and which inhibit EC lumen formation)
strongly reduce SFK phosphorylation, providing additional support
Fig. 1. PKCε stimulates EC lumen formation in 3D collagen matrices. (A) ECsinfected with adenoviruses (Ad) expressing GFP, WT-PKCε or DN-PKCεwere suspended within 3D collagen matrices for 24 hours. Scale bar: 50 μm.(B) Quantification of EC lumen formation at 24 hours. Data are shown asmean EC lumenal area ± s.d. (n=3). *P<0.05 compared with GFP control.(C) EC extracts were prepared at 24 hours for western blot analysis and probedfor phospho-Pak2, phospho-Pak4, phospho-Src or Pak2, Pak4, Src and actincontrols. The actin control blot was derived from cut lanes of the same gel anda single exposure.Jo
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for a collaborative role for these kinases in the molecular control
of EC lumenogenesis (Fig. 2A). Furthermore, these data argue that
PKCε is upstream of SFKs in this signaling cascade.
To examine whether SFKs are activated during EC lumen and
tube formation, we examined their phosphorylation levels over a
time-course of this process. SFK phosphorylation was highly
induced during EC lumen and tube formation (Fig. 2B), indicating
that SFK activation is required for these events. Also, the stimulation
of lumen formation that was observed following increased
expression of PKCε was markedly blocked by the SFK inhibitor
PP2 (Fig. 2C,D).
SFKs interact with Cdc42 in a PKCε-dependent manner toregulate EC lumen formation in 3D collagen matricesBecause it has been shown that SFKs act downstream of integrins,
and that SFKs can regulate activation of Rho GTPases by influencing
RhoGDIs (DerMardirossian et al., 2006; Playford and Schaller,
2004), we next analyzed the relationship between SFKs and Cdc42
during EC lumen formation. To examine whether SFKs directly
interact with Cdc42, we used a recombinant virus containing Cdc42
tagged with both GFP and S-tag, S-GFP-Cdc42 (Koh et al., 2008).
ECs were infected with S-GFP-Cdc42 virus 24 hours before they
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were suspended within 3D collagen matrices. EC culture extracts
were prepared at 16 hours during EC morphogenesis and lysates
were incubated with S-protein agarose beads to capture the
recombinant Cdc42 protein (Koh et al., 2008). Specificity of protein
interactions through S-tag/S-protein agarose beads have been
described previously in studies in which GFP-Cdc42 was used as a
control (Koh et al., 2008). As shown in Fig. 3A, there was a strong
association between Cdc42 and SFKs during EC lumen formation
in 3D collagen matrices, and this interaction was dependent on PKCε.
In the presence of PKC inhibitors that target PKCε (e.g. Go6983),
and that block lumen formation, the association of Cdc42 with SFKs
was strongly diminished (Fig. 3B,C). The addition of Go6976, which
shows blocking selectivity for conventional PKC isoforms and not
for novel isoforms such as PKCε, maintained the interaction between
Cdc42 and SFKs, as this inhibitor did not show any inhibitory effect
on this process (Fig. 3B,C). These data suggest that Cdc42 interacts
with multi-protein complexes containing SFKs to regulate EC
lumen formation in 3D collagen matrices and that the association
of Cdc42 with these complexes is dependent on PKCε.
SFKs are required for EC lumen formation in 3D collagenmatrices and are activated downstream of PKCεTo further show the requirement of SFKs for EC lumen formation
in 3D collagen matrices, general SFK activity was inhibited either
Fig. 2. SFKs regulate EC lumen formation in 3D collagen matricesdownstream of PKCε. (A) ECs were cultured in collagen matrices for 24 hoursin the absence or presence of TPA and/or the PKC inhibitors GF109203X(2.5 μM), Go6983 (5 μM), Ro-32-0432 (5 μM) or Go6976 (5 μM). Lysateswere prepared for western blot analysis and probed for phospho-Src or actincontrol. (B) Extracts of EC cultures in 3D collagen matrices were prepared atthe indicated time points and probed for phospho-Src, actin and total Src.(C,D) ECs were infected with adenoviruses (Ad) expressing GFP or WT-PKCεand were suspended within 3D collagen matrices. Culture media containedeither no additives or the Src inhibitor PP2 at 10 μM. Cultures were fixed after24 hours and photographed (C) or quantitated for lumen formation (D). Scalebar: 50 μm. Data are shown as mean EC lumenal area ± s.d. (n=3). *P<0.05compared with GFP control; **P<0.05 compared with PKCε-WT control.
Fig. 3. SFKs interact with Cdc42 in a PKCε-dependent manner to regulate EClumen formation in 3D collagen matrices. ECs were treated with S-GFP-Cdc42 recombinant adenovirus prior to suspension in 3D collagen matrices inthe absence or presence of TPA and/or GF109203X (5 μM), Go6983 (10 μM),Ro-32-0432 (10 μM) or Go6976 (10 μM). (A) Extracts were prepared at 16hours and equal amounts of extracts were incubated with S-protein agarosebeads and probed for phosphorylated SFKs to detect binding interactions.(B) Representative fields of EC cultures in the presence of the indicatedchemical inhibitors. Scale bar: 50 μm. (C) Quantification of EC lumenformation at 24 hours. Data are shown as mean EC lumenal area ± s.d. (n=3).*P<0.01 compared with TPA control.Jo
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by adding the chemical inhibitor PP2 or by expressing C-terminal
Src kinase, Csk, which is known to negatively regulate SFKs
(Howell and Cooper, 1994). The addition of PP2 to the EC-lumen-
formation assay had a strong inhibitory effect on this process (Fig.
2; Fig. 4A,C). ECs expressing increased Csk levels were also
strongly blocked in their ability to undergo lumen formation
compared with ECs expressing control GFP protein (Fig. 4D,F).
The presence of PP2 or expression of Csk also strongly reduced
SFK activation as detected through phosphorylation (Fig. 4B,E),
suggesting that SFK activation is required for EC lumen formation
in 3D collagen matrices.
Additional experiments addressed the role of SFKs and their
relationship to PKCε signaling in the lumen-formation cascade.
Suppression of Csk by using siRNA caused significant increases
in EC lumen formation (Fig. 4G), as did expression of a DN Csk
mutant by using an adenoviral vector (Fig. 4H). Increased expression
of WT Src or PKCε led to increases in EC lumenogenesis as well.
By contrast, expression of DN PKCε or WT Csk blocked lumen
formation (Fig. 4H). Interestingly, the inhibitory influence of DN
PKCε was rescued and, thus, reversed by coexpression of either
WT Src or DN Csk (Fig. 4H). These data suggest the crucial
involvement of both PKCε and SFKs during EC lumen formation
and that SFKs act downstream of PKCε in this signaling cascade.
Src and Yes play a key role in EC lumen formation in 3Dcollagen matricesThere are nine different SFKs in mammals – Src, Fyn, Yes, Yrk,
Lyn, Hck, Fgr, Blk and Lck – each of which exhibits a wide range
of expression patterns (Thomas and Brugge, 1997). To identify the
relevant SFKs that are involved in lumen formation in collagen
matrices, we examined the differential expression pattern of each
SFK member during this morphogenic process. Our screening
revealed that prominently expressed members of SFKs in human
ECs include Src, Yes, Fyn and Lyn; this was determined using
reverse transcriptase (RT)-PCR analysis (Fig. 5A) as well as
western blot analysis (data not shown). The other SFKs were either
not expressed or expressed at minimal levels in ECs undergoing
lumen and tube formation (data not shown). The expression of Srcand Yes mRNA was induced during the EC morphogenic process,
whereas Fyn and Lyn showed a constant expression pattern
throughout the process (Fig. 5A). Src and Yes have been previously
shown to regulate vascular permeability in response to VEGF
(Eliceiri et al., 1999), whereas Fyn and Lyn have been targeted for
anti-angiogenic treatment on the basis of their role in apoptotic
signaling pathways (Tang et al., 2007).
To further examine the role of Src, Yes, Fyn and Lyn in EC lumen
formation in 3D collagen matrices, we used siRNA suppression
analysis. Suppression of Src or Yes, two members whose
expression was differentially regulated during the EC
morphogenic process, resulted in a significant reduction
of EC lumen formation in 3D collagen matrices (Fig.
5B,C). siRNA suppression of Fyn or Lyn did not have any
significant effect on EC lumen formation compared with
control luciferase (Fig. 5B,C). Specificity of each siRNA
and its ability to knock down corresponding Src-family
members was confirmed by semi-quantitative RT-PCR
(Fig. 5D). These data suggest that Src and Yes play
important roles in EC lumen formation in 3D collagen
matrices.
Pak2 and Pak4 serve as downstream targets of SFKsto regulate EC lumen formation in 3D collagenmatricesWe next examined whether SFK-dependent EC lumen
formation in 3D collagen matrices involves Pak2 and Pak4,
two downstream targets of Cdc42 and PKCε that play a
Fig. 4. SFKs are required for EC lumen formation in 3D collagenmatrices and act downstream of PKC activation during this process.(A-C) ECs were suspended in collagen matrices for 24 hours in theabsence or presence of the chemical inhibitor PP2 (10 μM).(A) Cultures were fixed for photography. Scale bar: 50 μm. Arrowsindicate EC lumenal structures. (B) Extracts were made for westernblot analysis and probed for phospho-Src or actin.(C) Quantification of EC lumen formation at 24 hours. (D-F) ECsinfected with GFP- or Csk-expressing adenoviruses (Ad) weresuspended in collagen matrices for 24 hours. (D) Cultures werefixed for photography. Scale bar: 50 μm. Arrows indicate EClumenal structures. (E) Extracts were made for western blot analysisand probed for phospho-Src or actin. (F) Quantification of EClumen formation at 24 hours. (G) ECs were treated with controlluciferase versus a siRNA to Csk and then suspended in collagenmatrices to undergo lumen formation. Cultures were fixed at 24hours and quantitated for lumen formation. (H) ECs weretransfected with the indicated adenoviral vectors and thensuspended in collagen matrices. After fixation at 24 hours, cultureswere quantitated for EC lumen formation. Data are shown as meanEC lumenal area ± s.d. (n=3). *P<0.01 compared with controls.
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key role in EC lumen formation (Koh et al., 2008). It has been
shown that Pak2 can be activated both by Cdc42 and SFKs
(Renkema et al., 2002). Given that Cdc42 and SFKs are both
required for EC lumen formation, it was vital to examine Pak2 and
Pak4 activation by Cdc42 (Koh et al., 2008) and SFKs in the context
of their regulatory roles on EC lumen formation. When ECs were
treated with the Src-kinase inhibitor PP2, phosphorylation of both
Pak2 and Pak4 were markedly reduced (Fig. 6A). Expression of
Csk also diminished Pak2 and Pak4 phosphorylation (Fig. 6B).
Overall phosphorylation levels of both Pak2 and Pak4 were also
reduced with siRNA suppression of Src and Yes, but not of Fyn and
Lyn, confirming the role of Src and Yes in the EC lumen formation
process (Fig. 6C). These data suggest that SFKs regulate EC lumen
formation in 3D collagen matrices by controlling Pak2 and Pak4
activation in conjunction with PKCε.
Raf kinases act downstream of Pak2 and Pak4 to regulate EClumen formation in 3D collagen matricesPak2 and Pak4 appear to serve as key targets at which signals
mediated by Cdc42, PKCε and SFKs converge to regulate EC lumen
formation in 3D collagen matrices. To further dissect their signaling
pathways, we examined downstream targets of Pak2 and Pak4
during EC lumen formation in 3D collagen matrices. C-Raf, a
member of the Raf-kinase family, has been previously implicated
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in the regulation of angiogenesis and EC survival downstream of
Pak1 and Src (Alavi et al., 2003; Eliceiri et al., 2002; Hood et al.,
2003). B-Raf is also shown to play a crucial role in VEGF-induced
angiogenesis and it is often mutated in various human cancers (Wan
et al., 2004; Wellbrock et al., 2004). To examine whether both B-
Raf and C-Raf are regulated by Pak2 and Pak4 to control EC lumen
formation, we used antibodies that recognize a residue that is known
to be phosphorylated by Pak proteins and which increases their
activity. We have previously shown that suppression of Pak2 and
Pak4 either by siRNA or expression of DN mutants significantly
blocks EC lumen formation in 3D collagen matrices (Koh et al.,
2008). To analyze whether these inhibitory effects are modulated
by B-Raf and C-Raf activation, lysates were made 24 hours after
ECs were suspended in 3D collagen matrices. Treatment of ECs
with either Pak2 or Pak4 siRNA significantly reduced
phosphorylation of both B-Raf and C-Raf (Fig. 6D). Expression of
DN Pak2 (T402A) or Pak4 (K350M), which block lumen formation
(Koh et al., 2008), also diminished B-Raf and C-Raf
Fig. 5. Src and Yes, play a key role in EC lumen formation in 3D collagenmatrices. (A) Extracts of EC cultures in 3D collagen matrices were prepared atthe indicated time points for RNA isolation. Semi-quantitative RT-PCR wasperformed for Src, Yes, Fyn, Lyn or G3PDH-1 control. (B) ECs were treatedwith the indicated siRNAs and were suspended within collagen matrices for24 hours before fixation for photography. Scale bar: 50 μm. (C) Quantificationof the EC-lumen-formation assay at 24 hours. Data are shown as the mean EClumenal area ± s.d. (n=6). *P<0.01 compared with luciferase (Luc) control.(D) siRNA-transfected ECs were prepared for RNA isolation. Semi-quantitative RT-PCR was performed for Src, Yes, Fyn, Lyn or G3PDH-1control.
Fig. 6. Pak2 and Pak4 act downstream of SFKs to coactivate Raf kinases thatare involved in EC lumen formation in 3D collagen matrices. (A) ECs wereresuspended in 3D collagen matrices in the absence or presence of PP2(10 μM). Extracts were prepared at 24 hours for western blot analysis andprobed for phospho-Pak2, phospho-Pak4 or actin. (B) ECs containing GFP- orCsk-expressing adenoviruses (Ad) were resuspended in 3D collagen matrices.Extracts were made at 24 hours for western blot analysis and probed forphospho-Pak2, phospho-Pak4 or actin. (C) ECs treated with the indicatedsiRNAs were resuspended in 3D collagen matrices. Extracts were made at 24hours for western blot analysis and probed for phospho- Pak2, phospho-Pak4or actin. (D) ECs treated with the indicated siRNAs or adenoviruses [GFP, DNPak2 (T402A) or DN Pak4 (K350M)] were resuspended in 3D collagenmatrices. Extracts were made at 24 hours for western blot analysis and probedfor phospho-B-Raf, phospho-C-Raf or actin. (E) Extracts of EC cultures in 3Dcollagen matrices were prepared at the indicated time points and probed forphospho-B-Raf or phospho-C-Raf. Actin, B-Raf and C-Raf were used asloading controls.
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phosphorylation (Fig. 6D). Moreover, both B-Raf and C-Raf were
highly activated during the EC-lumen-formation process in 3D
collagen matrices (Fig. 6E). Interestingly, when total B-Raf and C-
Raf proteins were examined, both of these proteins were induced
during these events. Together, these data suggest that B-Raf and C-
Raf represent key downstream targets of Pak2 and Pak4 that regulate
EC lumen formation in 3D collagen matrices.
B-Raf and C-Raf are required for EC lumen formation in 3Dcollagen matricesTo examine the function of Raf kinases in EC lumen formation, we
used a known Raf-kinase inhibitor (GW5074) (Lackey et al., 2000).
The addition of this inhibitor resulted in significant blockade of
lumen formation (Fig. 7A,B). To further address their functional
role, we suppressed expression of B-Raf and C-Raf by siRNA
treatment (Fig. 7C). siRNA suppression of either B-Raf or C-Raf
significantly blocked EC lumen formation (Fig. 7D,E). Although
suppression of either B-Raf or C-Raf resulted in statistically
significant inhibition of EC lumen formation, B-Raf siRNA had a
more dramatic effect than C-Raf siRNA (Fig. 7D,E). Western blot
analysis showed that suppression of B-Raf led to a protein-level
reduction of not only B-Raf but also C-Raf, whereas suppression
of C-Raf did not have any effect on B-Raf protein levels, suggesting
that regulation of B-Raf is linked to subsequent C-Raf stability or
expression (Fig. 7C). Previous work has shown that B-Raf can
compensate for C-Raf in vivo and that C-Raf can serve as an effector
for B-Raf (Chong et al., 2003; Mikula et al., 2001; Wan et al., 2004).
Our data from inhibitor and siRNA analysis reveal that both Raf
isoforms play a role during the EC-lumen-formation process.
Rheb (Ras homolog enriched in brain) and RKIP (Raf kinase
inhibitory protein) are two endogenous inhibitors of Raf kinases
that act as negative modulators of Raf kinase signaling (Corbit et
al., 2003; Karbowniczek et al., 2006; Klysik et al., 2008). Both
Rheb and RKIP protein levels were examined during the lumen-
formation process and were found to be differentially expressed
(Fig. 8A). The level of both proteins decreased while ECs actively
underwent vacuole and lumen formation (i.e. 3-18 hours) (Fig. 8A)
(Koh et al., 2008). At a time when the EC-lumen-formation process
is substantially completed (i.e. 24 hours), there was an increase in
both Rheb and RKIP protein levels (Fig. 8A). Interestingly, these
levels decreased again as EC lumen and tube formation became
stabilized (48 hours) (Fig. 8A), indicating a complex expression
pattern for these two Raf inhibitors. To further examine the potential
role of Rheb and RKIP and their influence on Raf during these
events, we generated adenoviral recombinant constructs expressing
either Rheb or RKIP. ECs expressing increased Rheb or RKIP levels
were markedly blocked in their ability to undergo EC lumen
formation compared with ECs expressing control GFP protein (Fig.
8B,C). Expression of Rheb showed a greater inhibitory effect
compared with that of RKIP. Rheb is known to affect the activity
Fig. 7. Inhibition of Raf kinases blocks EC lumen formation in 3D collagenmatrices. (A,B) ECs were resuspended in 3D collagen matrices for 24 hours inthe absence or presence of Raf1 kinase inhibitor GW5074 (5 μM).(A) Representative fields of EC lumen formation assay. Scale bar: 50 μm.(B) Quantification of EC lumen formation. (C-E) siRNA suppression of B-Rafor C-Raf inhibits EC lumen formation in 3D collagen matrices. (C) Lysateswere prepared for western blot analysis and probed for phospho-B-Raf,phospho-C-Raf and actin control. ECs treated with the indicated siRNAs wereresuspended in 3D collagen matrices for 24 hours. (D) Representative fields ofEC-lumen-formation assay. Scale bar: 50 μm. (E) Quantification of EC-lumen-formation assay. Data are shown as the mean EC lumenal area ± s.d. (n=3).*P<0.01 compared with control.
Fig. 8. Increased expression of the Raf-kinase inhibitors Rheb or RKIP blockEC lumen formation in 3D collagen matrices. (A) Extracts of EC cultures wereprepared at the indicated time points and probed for Rheb, RKIP or actin.(B) Representative fields of the indicated cultures. Scale bar: 50 μm.(C) Quantification of EC lumen formation. Data are shown as the mean EClumenal area ± s.d. (n=6). *P<0.01 compared with GFP control.
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of both Raf isoforms because it disrupts heterodimerization of B-
Raf and C-Raf (Im et al., 2002; Karbowniczek et al., 2006), whereas
RKIP has been shown to selectively target the activity of C-Raf
(Trakul et al., 2005). These data, together with that from our siRNA
experiment, suggest that the activity of both B-Raf and C-Raf is
crucial for EC lumen formation in 3D collagen matrices.
B-Raf and C-Raf mediate EC lumen formation downstream ofPKCε, Paks and SFKs in 3D collagen matricesOur data indicate that B-Raf and C-Raf are activated downstream
of Pak2 and Pak4, which are regulated by PKCε and SFKs, as well
as Cdc42 and Rac1. To further show that B-Raf and C-Raf lie
downstream of Pak2 and Pak4 in response to PKCε and SFKs during
the process of EC lumen formation, we examined phosphorylation
of both B-Raf and C-Raf in the presence of PKC inhibitors or SFK
inhibitors. B-Raf and C-Raf can undergo phosphorylation on
various residues by different kinases (Chong et al., 2003; Thomas
and Brugge, 1997). However, we focused our analysis on a residue
that is phosphorylated by Paks, because Pak2 and Pak4 play a key
role in EC lumen formation and this residue is conserved in both
B-Raf and C-Raf (Li et al., 2001; Wellbrock et al., 2004). In the
absence of TPA or in the presence of PKC inhibitors that block EC
lumen formation by targeting PKCε, B-Raf and C-Raf
phosphorylation on these crucial residues were dramatically reduced
(Fig. 9A). Presence of the SFK inhibitor PP2 also diminished their
phosphorylation compared with TPA alone or PKCα inhibitor,
Go6976, which does not affect EC lumen formation in 3D collagen
matrices (Fig. 9A). These data suggest that there is a linear
Journal of Cell Science 122 (11)
signaling pathway involving PKCε–SFKs–Pak2–Pak4–B-Raf–C-
Raf to regulate EC lumen formation in 3D collagen matrices.
One issue that is raised by this data is how these different
signaling molecules temporally function in relation to the complex
processes of intracellular vacuolation and coalescence, lumen
expansion, EC process extension, and EC motility that characterize
EC tubulogenesis in 3D collagen matrices. To address this issue,
we performed time-lapse experiments over a 24-hour period in the
presence or absence of PKCε, Src and Raf inhibitors at different
doses. As shown in Fig. 10, both intracellular vacuolation and lumen
expansion were markedly suppressed by Go6983, PP2 and
GW5074, which block PKCε, Src and Raf kinases, respectively.
Interestingly, Go6976, which selectively blocks PKCα and PKC β(PKCα/β) isoforms, accelerates lumen expansion, suggesting that
these PKC isoforms might be inhibitory to EC lumen formation.
EC motility was increased by Src blockade and by novel and atypical
PKC-isoform blockade, whereas PKCα/β blockade decreased
motility (Fig. 10). In these cases, motility responses were inversely
correlated with lumen formation. Thus, although EC motility is
required for tube formation, there appears to be complex
relationships between motility and lumen formation that need to be
investigated further. EC process extension, which increases over
time, is stimulated by Src blockade and inhibited by novel and
atypical PKC blockade (Fig. 10). Overall, it appears that this
PKCε–SFKs–Pak2–Pak4–B-Raf–C-Raf-kinase cascade appears to
act proximally in the lumen-formation process such that both EC
vacuolation and lumen formation are strongly inhibited by the
blockade of each of the kinases in this pathway.
B-Raf and C-Raf regulate EC lumen formation in 3D collagenmatrices through ERK1/2Raf kinases are key components of the MAPK pathway (Leicht et
al., 2007; Morrison and Cutler, 1997; Wellbrock et al., 2004). To
examine whether B-Raf and C-Raf regulate EC lumen formation
in 3D collagen matrices through the MAPK pathway, we analyzed
the phosphorylation and functions of ERK1/2 during this process.
Following an early initial induction in their phosphorylation level,
ERK1/2 activation levels remained fairly constant (Fig. 9B).
Because ERK1/2 is known as a major downstream target of Raf
kinases, its involvement in EC lumen formation downstream of Raf
kinases was examined. Inhibition of Raf-kinase activity by the Raf
inhibitor GW5074 resulted in a reduction of ERK1/2
phosphorylation (Fig. 9C), which accompanied its ability to block
EC lumen formation (Fig. 7A,B). Suppression of B-Raf or C-Raf
by siRNA also showed a modest reduction in ERK1/2
phosphorylation (Fig. 9D), indicating that ERK1/2 plays a role
downstream of B-Raf and C-Raf in EC lumen formation.
To further evaluate the function of the MAPK pathway in EC
lumen formation, we used recombinant viruses that express
constitutively active (CA) MEK1, DN MEK1, or MKP3, a
phosphatase that selectively dephosphorylates ERK1/2 (Arkell et
al., 2008; Keyse, 2008). Expression of DN MEK1 or MKP3
significantly impaired EC lumen formation and strongly inhibited
ERK1/2 phosphorylation compared with GFP control (Fig. 11A,B).
Expression of CA MEK1 induced the phosphorylation of ERK1/2
but did not show enhanced EC lumen formation (Fig. 11B). To
further analyze the MAP signaling pathway in EC lumen formation,
we next examined whether the expression of CA MEK1 could rescue
the EC-lumen-formation defect resulting from Raf-kinase-inhibitor
addition, because ERK1/2 acts downstream of Raf kinases. When
ECs expressing control GFP, CA MEK1, DN MEK1 or MKP3 virus
Fig. 9. B-Raf and C-Raf activation occur downstream of PKCε and SFKsduring EC-lumen-formation events in 3D collagen matrices. (A) ECs wereresuspended in 3D collagen matrices in the absence or presence of TPA,GF109203X (2.5 μM), Ro-32-0432 (5 μM), Go6983 (5 μM), Go6976 (5 μM)or PP2 (10 μM). Lysates were prepared at 24 hours for western blot analysisand probed for phospho-B-Raf, phospho-C-Raf, B-Raf, C-Raf or actin.(B-D) ERK1/2 proteins are phosphorylated during EC lumen formation in 3Dcollagen matrices. (B) Extracts of EC cultures were prepared at the indicatedtime points and probed for phospho-ERK1/2 or actin. (C) ECs wereresuspended in 3D collagen matrices for 24 hours in the absence or presenceof Raf-kinase inhibitor, GW5074 (5 μM). Lysates were prepared for westernblot analysis and probed for phospho-ERK1/2 or actin. (D) ECs treated withthe indicated siRNAs were resuspended in 3D collagen matrices. Lysates wereprepared for western blot analysis and probed for phospho-ERK1/2 or actin.
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1819Kinase cascades and EC lumenogenesis
were suspended in 3D collagen matrices in the presence of GW5074,
blockade of EC lumen formation was not rescued by the expression
of CA MEK1 (Fig. 11B). When the inhibitor was added to ECs
expressing DN MEK1 or MKP3 virus, we observed decreased EC
survival (supplementary material Fig. S1), showing the dual
involvement of ERK1/2 and Raf kinases in supporting EC survival
during morphogenic events. This data is consistent with the known
ability of Raf kinases to inactivate kinases such as the pro-apoptotic
kinase ASK-1 independently of ERK1/2 activation (Alavi et al.,
2003). These data together indicate that, although ERK1/2 is
required for EC lumen formation, it is not sufficient for these events
and, thus, works in conjunction with the other identified kinases in
the pathway (Fig. 11C).
DiscussionPKCε, Src and Yes control EC lumen formation in 3D collagenmatricesActivation of PKC has been implicated in the regulation of
angiogenesis both in vivo and in vitro (Davis and Camarillo, 1996;
Montesano and Orci, 1985; Montesano et al., 1987; Morris et al.,
1988). Our previous work identified PKCε as a key PKC isoform
mediating TPA-induced lumen formation in 3D collagen matrices
(Koh et al., 2008). Here we show that increased expression of PKCεmarkedly stimulates EC lumen formation. PKCε has been shown
to promote cell survival and anchorage-independent growth (Ding
et al., 2002; Okhrimenko et al., 2005). Various human cancers show
increased expression of PKCε, indicating that this PKC isoform
plays a key role in developing tumors as well as in other pathological
conditions that are often accompanied by angiogenesis (Basu and
Weixel, 1995; Gubina et al., 1998). PKCε has also been shown to
regulate the trafficking of integrins, thereby influencing cell motility
and adhesion (Ivaska et al., 2005; Ivaska et al., 2002), which are
necessary functions regulating EC vascular morphogenesis.
Increased PKCε-induced phosphorylation of SFKs suggests that
SFKs are regulated by PKCε to control EC lumen formation in 3D
collagen matrices. Studies have shown that SFKs act downstream
of various signal-transduction pathways such as integrins, growth-
factor receptors, Rho GTPases and PKC to regulate angiogenesis,
vascular permeability, actin-cytoskeleton organization, capillary
morphogenesis, cell proliferation and endothelial remodeling (Abu-
Ghazaleh et al., 2001; Amos et al., 2005; Basu and Weixel, 1995;
Bruce-Staskal and Bouton, 2001; Eliceiri et al., 1999; Eliceiri et
al., 2002; Friedlander et al., 1995; Liu and Senger, 2004; Nomura
et al., 2007; Robles et al., 2005; Tatin et al., 2006). Given that SFKs
exhibit such versatile functions in response to diverse cellular
factors, including PKC, Rho GTPases and integrins, it has led us
to examine their role during EC lumen formation in 3D collagen
matrices. Our study found that the expression of SFKs is highly
induced during EC morphogenesis and there is a strong association
between SFKs and Cdc42 in a PKCε-dependent manner during EC
lumen formation. Inhibition of SFK activity by either PP2 or
increased Csk expression impaired EC lumen formation and,
furthermore, we demonstrate that Src and Yes, but not Fyn and Lyn,
control this process. The opposite experiment was performed
whereby siRNA suppression of Csk or increased expression of a
DN Csk protein led to marked increases in lumen formation. The
inhibitory influence of DN-PKCε expression was overcome and
reversed by increased expression of either WT Src or DN Csk,
showing that SFKs are activated downstream of
PKCε. Downstream of the Cdc42, PKCε and SFK
signals, Pak2 and Pak4 represent common platforms
at which these signaling events converge to control
EC lumen formation in 3D collagen matrices.
Stimulation and inhibition of lumen formation by WT
PKCε and DN PKCε, respectively, directly correlates
with the levels of activated Pak2 and Pak4.
Furthermore, activation of both Paks is controlled by
SFKs, suggesting that they work together during EC
lumen formation and appear to be activated
downstream of SFK activation.
Fig. 10. Temporal analysis of the influence of PKC, Src andRaf kinases on EC lumen and tube formation in 3D collagenmatrices. EC cultures were established in 3D collagenmatrices and the indicated kinase inhibitors were added ateither 10 μM (A panels) or 2.5 μM (B panels). Time-lapsemovies were made by acquiring images every 10 minutesover a 24-hour period as described (Koh et al., 2008). Fourindependent parameters were assessed, includingmeasurements of total EC lumen area per field (first row), thepercentage of ECs with intracellular vacuoles (second row),total EC process length per field (third row) and total ECmotility per field (fourth row). Fields were acquired at amagnification of 150�. Quantitation of lumen area, processlengths and EC motility used MetaMorph software asdescribed (Koh et al., 2008). Images were obtained from threeindependent cultures and from at least three different fieldsfor each indicated value at each time point. Statisticalsignificance relative to control cultures was set at P<0.05 andis indicated by an asterisk.
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1820
Accumulating data reveals how work using in vitro
morphogenesis systems directly correlate with findings
demonstrated in vivo with regards to lumen and tube formation
(Davis et al., 2007; Egginton and Gerritsen, 2003; Holderfield and
Hughes, 2008; Iruela-Arispe and Davis, 2009). Two key examples
are those showing the role of intracellular vacuole formation and
coalescence in vitro using human ECs, and in vivo lumenogenesis
during zebrafish vascular development (Kamei et al., 2006), as well
as the demonstration that the cerebral cavernous malformation
protein, CCM2, is required for EC lumen formation in vitro as well
as lumen formation and patency of the developing mouse
vasculature, including the first branchial arch artery and intersomitic
arteries (Whitehead et al., 2009).
Journal of Cell Science 122 (11)
Pak- and Src-dependent Raf activation regulates EC lumenformationFurther molecular examination downstream of Pak2 and Pak4
revealed B-Raf and C-Raf as targets, the activities of which are
regulated by Pak2 and Pak4 to mediate EC lumen formation in 3D
collagen matrices. Raf kinases have drawn much attention as a
promising target for anti-angiogenic therapy as they control many
crucial cellular functions by integrating signals that regulate vascular
events (Alavi et al., 2003; Hood et al., 2003; Leicht et al., 2007;
Morrison and Cutler, 1997; Wellbrock et al., 2004). The effect of
Raf kinases is often carried out by their involvement in a conserved
signaling pathway, the Raf-MEK-ERK MAPK pathway (Leicht et
al., 2007; Roberts and Der, 2007; Wan et al., 2004; Zebisch et al.,
2007). The MAPK pathway also regulates other crucial cellular
functions such as survival, cytoskeletal organization, proliferation
and transcriptional regulation (Chong et al., 2003; Lefloch et al.,
2008; Leicht et al., 2007; Wellbrock et al., 2004), as well as playing
a role during epithelial tube morphogenesis (O’Brien et al., 2004).
It is clear that overlapping signaling pathways control tubulogenesis
in endothelial and epithelial cells, although there are unique features
of each that are becoming increasingly apparent (Davis et al., 2007;
Iruela-Arispe and Davis, 2009; Lubarsky and Krasnow, 2003;
O’Brien et al., 2004).
Here, we show that both B-Raf and C-Raf play a role to regulate
EC lumen formation. Suppression of their activity either by siRNA,
chemical inhibitors or increased expression of negative regulators
(i.e. Rheb and Rkip) resulted in marked inhibition of EC lumen
formation. In support of these findings, studies using genetic
knockout mice have shown that, although knockout of either B-
Raf or C-Raf causes embryonic lethality (Wojnowski et al., 1997),
MAPK signaling in C-Raf-knockout mice can be rescued by B-Raf
(Chong et al., 2003). Because both B-Raf and C-Raf kinases appear
to be required for EC lumen formation, we then examined the role
of the MAPK signaling during these events. Suppression of MEK
activity either by expression of a DN mutant or by increased
expression of the ERK1/2 phosphatase MKP3 blocked EC lumen
formation in 3D collagen matrices, indicating that ERK1/2 activity
is also required. Interestingly, the Raf-ERK pathway is typically
associated with cell-proliferation signaling; however, there is no
evidence for EC proliferation during the tube-formation process in
3D collagen matrices in this system.
Protein-kinase cascades downstream of Cdc42-dependentsignaling control EC lumen formation in 3D collagen matricesData presented in this study provide new insights into signaling
pathways regulating the crucial EC-lumen-formation step during
vascular morphogenesis in a collagen-matrix environment (Fig.
10C). EC–collagen-matrix interactions result in integrin-dependent
signaling, leading to activation of Cdc42 as well as its downstream
effectors, Pak2 and Pak4 (Koh et al., 2008). Activation of Pak2 and
Pak4 are also regulated by SFKs, especially Src and Yes, as well
as PKCε. Similar to Pak2 and Pak4, activated SFKs associate with
Cdc42 in multiprotein signaling complexes (Koh et al., 2008) to
control the lumen-formation process. These data indicate that, during
EC lumen formation in 3D collagen matrices, Pak2 and Pak4 serve
as common targets that integrate signals from Cdc42, SFKs and
PKCε to induce Raf activation and EC lumenogenesis. Overall, it
appears that EC lumenogenesis requires coordinated signaling
events leading to cytoskeletal changes (Cdc42, SFKs, Paks), pro-
survival signals (Raf kinases) and transcriptional controls (ERK1/2)
that are necessary for EC lumen and tube formation. These latter
Fig. 11. Raf kinases regulate EC lumen formation in 3D collagen matricesthrough ERK1/2 and possibly other targets. ECs infected with GFP, CAMEK1, DN MEK1, or MKP-3 adenovirus were resuspended in 3D collagenmatrices in the absence or presence of the Raf kinase inhibitor GW5074(5 μM). (A) Extracts were made at 24 hours for western blot analysis andprobed for phospho-ERK1/2 or actin. (B) Quantification of EC lumenformation at 24 hours. Data are shown as the mean EC lumenal area ± s.d.(n=3). *P<0.01 compared with GFP control. (C) Schematic diagramillustrating the mechanisms underlying Cdc42-dependent EC lumen and tubeformation signaling pathways in 3D collagen matrices. Pak2 and Pak4 alsoserve as downstream effectors of PKCε, which activates SFKs and controlstheir interaction with Cdc42. Pak2 and Pak4 activation leads tophosphorylation of B-Raf and C-Raf as well as ERK1/2, which togethercontrol EC lumen formation. In addition, these kinases might directly activatenew effectors to regulate EC lumen formation (shown as broken lines). Theircoordinated influence appears to control events such as cytoskeletalrearrangements, EC survival and transcriptional events that are necessary toboth form and maintain tube structures.
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1821Kinase cascades and EC lumenogenesis
controls through ERK1/2 are probably responsible in part for the
marked changes in gene expression that accompany EC lumen and
tube formation (Bell et al., 2001).
Materials and MethodsReagentsGF109203X, Go6983, Ro-32-0432, Go6976, PP2 and Raf-1-kinase inhibitor were
purchased from Calbiochem (La Jolla, CA). 12-O-tetradecanoyl-phorbol-13-acetate
(TPA) and a polyclonal antibody against phospho-Pak2 (Ser141) were obtained from
Sigma-Aldrich (St Louis, MO). Polyclonal antibodies targeting phospho-Pak4
(Ser474), B-Raf, phospho-B-Raf (Ser445), C-Raf, phospho-C-Raf (Ser338), phospho-
Src (Y416), Rheb, RKIP and phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204)
were obtained from Cell Signaling Technology (Danvers, MA). A monoclonal
antibody against actin (CP01) was obtained from Calbiochem. WT PKCε, DN PKCε,
CA MEK1, DN MEK1 and MKP3 adenoviruses were purchased from Seven Hills
Bioreagents (Cincinnati, OH), WT Src, WT Csk and DN Csk were purchased from
Cell Biolabs (San Diego, CA) and amplified as previously described (Bayless and
Davis, 2002).
EC lumen and tube formation in 3D collagen matricesHuman umbilical vein ECs (HUVECs) were purchased from Clonetics (San Diego,
CA) and were cultured (passage 2-5) as previously described (Davis and Camarillo,
1996). For the lumen-formation assay, ECs were suspended within 3.75 mg/ml of
collagen-type-I matrices and allowed to undergo EC morphogenesis as described
previously (Davis and Camarillo, 1996). Cultures were fixed at the indicated time
points with 3% glutaraldehyde for 30 minutes. In some cases, cultures were stained
with 0.1% Toluidine Blue in 30% methanol and destained prior to photography and
visualization. Some 3D collagen gels were also extracted to examine protein
expression. Extracts were run on SDS-PAGE gels, transferred to membranes, probed
and developed. Adenovirus infection of ECs was carried out as previously described
(Bayless and Davis, 2002).
Transfection of ECs with siRNAssiGENOME SMARTpool human Src, Yes, Fyn, Lyn, B-Raf and C-Raf were obtained
from Dharmacon (Lafayette, CO) and prepared as previously described (Saunders et
al., 2005). Luciferase GL2 duplex was used as a control. EC transfection with siRNAs
was carried out in growth media with 1% serum. Details of our siRNA-transfection
protocol have been described previously (Saunders et al., 2005).
EC-lumen-formation pulldown assayGeneration of S-GFP-Cdc42 adenovirus has been described previously (Koh et al.,
2008). ECs were infected with S-GFP-Cdc42 adenovirus and the EC-lumen-formation
assay was set up as described (Koh et al., 2008). EC cultures were extracted at the
indicated time points and bound Cdc42-associated proteins were detected by western
blot analysis.
RT-PCRTotal RNA was extracted from EC cultures at the indicated time points or from siRNA-
treated (luciferase, Src, Yes, Fyn and Lyn) ECs using the Totally RNA Isolation kit
obtained from Ambion (Austin, TX) according to the manufacturer’s instructions.
RNA (1 μg) was reverse transcribed using AccuScript High Fidelity 1st strand cDNA
synthesis kit (Stratagene). RT-PCR amplification was performed using the primers:
Src up (5�-TGTATTGCCAAGTACAACTTC-3�), Src dn (5�-CAAAGTACACCTC-
CTCGTC-3�), Yes up (5�-CAAGTGTGAGCCATTATG-3�), Yes dn (5�-AAATAC-
CATTCTTCTGCC-3�), Fyn up (5�-ACGAGAAGGAGGAACAGGAG-3�), Fyn dn
(5�-GTATCCACCATTGTCAAGTTTG-3�), Lyn up (5�-AGGCCAGTTCCA-
GAATCTC-3�), Lyn dn (5�-GCACAGGGTCAAAGTCTC-3�), G3PDH-1 up (5�-GC-
CAAAAGGGTCATCATCTC-3�) and G3PDH-1 dn (5�-GTAGAGGCAGGGAT-
GATGTTC-3�).
Generation of Rheb and RKIP adenovirusesRheb and RKIP were amplified from human cDNA clone (Origene) using the primers:
Rheb up (5�-AGCTCGAGGCCACCATGCCGCAGTCCAAGTCCCGGAAG-3�),Rheb dn (5�-AGTCTAGATCACATCACCGAGCATGAAGACTTGCC-3�), RKIP up
(5�-AGCTCGAGGCCACCATGCCGGTGGACCTCAGCAAG-3�) and RKIP dn
(5�-AGTCTAGACTACTTCCCAGACAGCTGCTC-3�) (Sigma Genosys, The
Woodlands, TX). Standard restriction digestion cloning was performed to clone Rheb
and RKIP into pAdTrack-CMV. Recombination and virus production were carried
out as previously described (Bayless and Davis, 2002).
Microscopy/imaging and statistical analysisVisualization and image acquisition of EC lumen and tube-formation assays were
done using an inverted microscope (CKX41; Olympus) as previously described
(Saunders et al., 2006). Image analysis was done using MetaMorph software.
Statistical analysis of EC lumen and tube formation was performed using SPSS
11.0 software (SPSS). Statistical significances were accessed by paired-samples t-test or a one-way ANOVA with a Dunnett’s test.
The authors would like to thank Kristine Malotte for excellenttechnical assistance. This work was supported by NIH grants HL59373and HL79460 to G.E.D. Deposited in PMC for release after 12 months.
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