Regulation of endothelial cell-cell junctions and
vascular permeability by Rho GEFs and GAPs
Master thesis Karin Prummel (Student number: 3257975)
Master program: Cancer Genomics and Developmental Biology, Utrecht University
Supervisor: Stephan Huveneers, PhD
Examiner UU: Fried Zwartkruis, PhD
Date: 11/03/2013
1
Abstract
The endothelial monolayer covers the luminal side of blood and lymphatic vessels and functions
as a physical barrier. The cell-cell junctions between the endothelial cells are important to
regulate and maintain the barrier function. Adherens and tight junctions, the main adhesion
molecules found in endothelial cells, are regulated by Rho GTPases RhoA, Rac1 and Cdc42. All
three Rho GTPases can either induce endothelial permeability as protect the barrier, depending
on their spatiotemporal activation and upstream regulators. Rho GTPases are regulated by
several guanine exchange factors (GEFs) and GTPase activating proteins (GAPs), which switch
the GTPases between the active GTP-bound state and the inactive GDP-bound state respectively.
In turn, GEFs and GEFs are under control of inflammatory cytokines and vascular growth factors.
In this review is outlined which GEFs and GAPs are associated with the regulation of cellular
junctions. For some GEFs/GAPs is already established that they control the vascular
permeability in vivo. A specific attention is given to GEFs/GAPs involved in the regulation of
junctions in other cell types, but known to be expressed in endothelium. It is important to
unravel how GEFs/GAPs are activated and what their subcellular localization is, because this will
give more insight in the complex regulation of Rho GTPases.
2
Summary for layman
All organs and tissues are provided by oxygen and nutrients, and get rid of waste via a dense
network of blood vessels and lymphatic vessels. The blood facing side of these vessels is covered
by a layer of single cells, called endothelial cells. This layer of cells forms a barrier between the
lumen of the vessel and the surrounding tissues. The endothelial cells in the barrier are
connected to each other via protein complexes on the plasma membrane, called cell-cell
junctions. Adherens junctions, containing VE-cadherin, and tight junctions are the main
endothelial junctions contributing to cell-cell contacts. These protein complexes are
intracellulary connected to the skeleton of the cell: the actin filaments. Less cell-cell junctions
between endothelial cells will increase the permeability of the barrier. Control of these junctions
is important for several biological processes, such as the formation of new blood vessels and the
transport of immune cells over the blood vessel wall after an infection. Dysfunction in cell-cell
junction control can cause for example leakage of the vessels, fluid buildup (edema) in tissues
and a declined response to inflammation.
Several permeability inducing or barrier protecting factors circulate in the blood or are secreted
by neighboring cells and can regulate Rho GTPases. Rho GTPases are enzymes known to regulate
cell-cell junctions. The main Rho GTPases in this review are RhoA, Rac1 and Cdc42. Their activity
is regulated by guanine exchange factors (GEFs) and GTPase activating proteins (GAPs): Rho
GTPases switch between an inactive and active state. In this review, an overview is made of the
Rho GEFs and GAPs that might be involved in the control of cell-cell junctions and consequently
can regulate the permeability of the endothelial monolayer.
Firstly, we elucidated how six different inflammatory mediators and growth factors can activate
or inactive the three Rho GTPases and what is the eventual effect on the endothelial barrier. It
becomes clear that each GTPase can either protect or disrupt the barrier function. This indicates
that their activity is tightly regulation and will depend on the level of activation, the timing of
activating and its location in the cell. For example, RhoA can be located at different sites in the
cell: around VE-cadherin, but also near actin filaments attached to the cell-cell junctions. GEFs
and GAPs play a role in this complex (in)activation of Rho GTPases.
Secondly, we looked into the GEFs and GAPs known to regulate cell-cell contacts in all types of
cells. For some Rho GEFs and GAPs is already proven via in experiments in mice that they can
control the endothelial barrier function. However, the majority of the GEFs/GAPs is studied in
cultured endothelial, epithelial or cancer cells. Many GEFs/GAPs known to regulate cell-cell
junctions in culture are studied in epithelial cells. Epithelial cells can also form barriers in for
example the intestine. Almost all GEFs/GAPs found to regulate cell junctions in epithelial cells
are also expressed in endothelial cells, however, not for all these GEFs/GAPs is yet established
whether they cause (the same) effects in endothelial monolayers. Of note, tight junctions can be
more organized in epithelial cells than in endothelial cells, so it might be possible that a GEF or
GAP can have a different effect on the tight junctions in endothelial than in epithelial cells. To
give more understanding of the signaling networks controlling the activity of Rho GTPases in
endothelial cells, more experiments should be performed in mice. Moreover, the biological
relevance must be taken in account as well. For most GEFs/GAPs is described which
inflammatory mediators can regulate them in culture, however, can the same factors regulate
them also in a living animal? These kinds of experiments can lead to the discovery of interesting
therapeutic targets to for example reduce vascular leakage in patients with atherosclerosis.
3
Introduction
The vascular barrier: regulation of endothelial cell-cell junctions
The endothelium is a monolayer of cells covering the luminal side of blood and lymphatic vessels
and acts as a physical barrier between the intravascular fluid compartment and the surrounding
tissues. The ability of monolayer cells to form barriers between tissues is also occurring in
epithelial tissues, for example in play a crucial role in the formation and maintenance of the
barrier function. These cell-cell adhesions are not simply static adhesion structures; in contrast,
dynamic regulation of cell-cell junctions is required during inflammation and the formation of
new blood vessels (angiogenesis). The regulation of these junctions is also involved in immune
surveillance when immune cells migrate from the blood stream across the endothelial layer
towards tissues and vice versa (Vestweber et al., 2009). Upon certain stimuli, leukocytes,
macromolecules and fluids in the blood can enter the tissue through intercellular spaces in the
endothelium layer, caused by destabilized cell-cell junctions. The migration of leukocytes over
the endothelial layer is referred to as paracellular transendothelial migration (Bazzoni, 2006;
Vestweber et al., 2009). Without the ability to destabilize endothelial cell-cell junctions such
leukocyte transmigration events are strongly inhibited (Schulte et al., 2011) and will prevent
sprouting during angiogenesis (Abraham et al., 2009). When endothelial permeability is induced
inappropriately, for example during chronic infections or autoimmune disease,
pathophysiological effects may occur, including prolonged vascular leakage, acute lung injury,
tissue edema, and atherosclerosis (Wojciak-Stothard and Ridley, 2002).
Molecular adhesion complexes at cell-cell junctions
Adherens junctions (AJs), nectin-based junctions and tight junctions (TJs) are the main cell-cell
adhesion complexes involved in the formation of cell-cell adhesions and in the maintenance of
barrier function (Meng and Takeichi, 2009). All junction types are intracellulary linked to the
actin cytoskeleton and may function as signaling scaffolds, locally regulating signal transduction
cascades in control of cytoskeletal remodeling, cell growth and differentiation. The major
function of AJs is to physically connect neighboring cells and to maintain these contacts. Loss of
cell-cell junctions will loosen the cell-cell contacts, resulting in disrupted tissue organization.
Therefore, they are crucial for cell-cell adhesion (Meng and Takeichi, 2009).
TJs tightly link the membranes of neighboring cells, forming a barrier that even prevents small
molecules and fluid to pass. They consist of transmembrane and intracellular proteins:
occludins, claudins and small junctional adhesion molecules (JAMs). Occludin and claudin bind
to zona occludens proteins (ZO), which can interact with actin filaments (Bazzoni and Dejana,
2004; Shin et al., 2006). In epithelial cells, TJs divide the membrane in an apical (lumen facing)
and basolateral region. In contrast, in endothelial cells TJs do not divide the plasma membrane in
such distinct regions in vitro and are more evenly distributed over the plasma membrane than in
epithelial cells (Bazzoni and Dejana, 2004). Interestingly, in the vasculature is found that the
density of TJs differs between the various segments of the vascular network, what may relate to
the variability in permeability of these segments (Bazzoni and Dejana, 2004). Variance occurs
due to the molecular composition of TJs and cellular expression levels of occludins and claudins.
The membrane of post-capillary venules contains many receptors for permeability factors and
less TJs. This is also the location for leukocyte transmigration. In veins, the organization of the
4
TJs less organized as well. Additionally, in the blood-brain-barrier (BBB), the endothelial cells
contain many and complex TJs (Bazzoni and Dejana, 2004).
In addition, vascular endothelial (VE-) cadherin is the most important component of AJs in
endothelium (Vestweber et al., 2009), contributing to adhesion between endothelial cells,
whereas epithelial cells mainly form epithelial (E-) cadherin-based adherens junctions. VEcadherin knockout studies have shown that VE-cadherin is a crucial protein for the formation of
endothelial cell-cell adhesions (Carmeliet et al., 1999; Gory-Fauré et al., 1999). Also neuronal (N) and placental (P-) cadherin can be found in endothelial cells (Bazzoni and Dejana, 2004).
Homophilic interactions between the extracellular domains of these cadherins mediate adhesion
between cells. The cytoplasmatic domain of cadherins associate with catenin proteins, linking
the AJs to the actin cytoskeleton and consequently stabilizing the cadherins at cell-cell contacts.
The proteins p120-catenin and β-catenin bind directly to cadherin’s cytoplasmatic domain. In
turn, β-catenin binds to α-catenin, which in turn associated to actin filaments via actin binding
proteins. Consequently, α- and β-catenin link cadherin complexes to the actin cytoskeleton
(Vestweber, 2008; Meng and Takeichi, 2009; Gottardi and Gumbiner, 2001; Yamada et al., 2005;
Nelson, 2008).
Control of endothelial cell-cell adhesion complexes by permeability regulators
Several inflammatory mediators and growth factors, circulating in the blood or locally secreted
by cells, tightly regulate the balance between the opening and closure of endothelial cell-cell
junctions to facilitate paracellular migration or avoid aberrant leakage of the vessels
respectively (Vestweber et al., 2009). Thrombin, histamine, tumor necrosis factor α (TNFα) and
vascular endothelial growth factor (VEGF) are well known factors that increase the permeability
of the endothelium by destabilizing endothelial cell-cell junctions. On the other hand,
sphingosine 1-phosphate (S1P) and angiopoietin 1 (Ang-1) enhance the endothelial barrier
function through stabilization of cell-cell junctions (Vestweber et al., 2009; Gavard et al., 2008;
Spindler et al., 2010). Thus, several growth factors and cytokines can control the endothelial
barrier function. Next, we will focus on how they can regulate the vascular permeability.
Vascular growth factors and inflammatory cytokines can increase the permeability of blood
vessels in several ways. First, for example VEGF may induce phosphorylation of components of
the VE-cadherin complex and stimulate recruitment of β-arrestin towards VE-cadherin,
enhancing endocytosis of the VE-cadherin-complex (Bazzoni and Dejana, 2004; Gavard and
Gutkind, 2006). Increased internalization of the VE-cadherin complex destabilizes AJs and
consequently results in loss of cell-cell contacts. Second, permeability regulators like thrombin,
VEGF and TNFα induce increased contraction of the actin/myosin II complex (actomyosin)
(Bryan et al., 2010; Mckenzie, 2007).
Contraction of actomyosin produces physical tension on adhesion structures like AJs to which
the actin bundles are attached to (De Rooij et al., 2005). These actomyosin dependent physical
forces destabilize endothelial AJs by pulling them apart (Huveneers et al., 2012) (Figure 1).
Elevated tension on AJs, induced by growth factors or cytokines, contributes to permeability of
the endothelial monolayer (Fischer et al., 2010; Mckenzie, 2007; Bryan et al., 2010). Adhesion of
cells to the extracellular matrix influences the cytoskeletal tension as well: thrombin induces
larger intercellular gaps in endothelial monolayers grown on a stiff substrate compared to cells
grown on less rigid cellular microenvironments in vitro (Krishnan et al., 2011; de Rooij et al.,
5
2005). In drosophila, it is recognized that the generated force on AJs, induced by actomyosin
contraction, is crucial to direct epithelial morphogenesis (Rauzi et al., 2010). On the other hand,
increased tension on AJs can also induce stabilization and growth of junctions in endothelial
monolayers not exposed to growth factors and hormones (Liu et al., 2010), corresponding with
the notion that the formation of E-cadherin based junctions depends on myosin II activity
(Yamada and Nelson, 2007). Thus, inflammatory mediators can disrupt the endothelial barrier
via stimulation of endocytosis of adhesion complexes and via induction of actomyosindependent tension on adhesion structures.
Figure 1. Regulation of adherens junctions by inflammatory mediators. Cadherin binds
homophylically to the extracellular domain of cadherin on neighboring cells (left). Permeability
inducing factors such as thrombin and VEGF induce contraction of the actin/myosin II complex
(actomyosin). Contraction of actomyosin creates strong actin bundles (stress fibers), associating to αcatenin via actin binding proteins (right). Pulling by these stress fibers provides tension on the
adherens junctions, leading to destabilization of the junctions. This induces weakening of cell-cell
contacts, consequently resulting in more permeability of the endothelial monolayer.
6
Aim of the thesis
The regulation of vascular permeability is important for angiogenesis and inflammatory
responses. Adherens and tight junctions are the cell-cell adhesion structures underlying the
endothelial barrier function, and they are, in turn, controlled by the actin cytoskeleton.
Inflammatory cytokines and vascular growth factors control the actin-connected cell-cell
junctions through several signaling routes, and in this thesis I will address the role of activating
Rho GTPases. Rho GTPases are molecular switches that can control the actin cytoskeleton and
cell-cell junctions. The activity of Rho GTPases is controlled by guanine nucleotide exchange
factors (GEFs) and GTPase-activating proteins (GAPs), activating and inactivating Rho GTPases
respectively. I will focus on which GEFs and GAPs are known or proposed to regulate Rho
GTPases in control of endothelial cell-cell junctions. This thesis gives an overview of the Rho
GTPase signaling networks that lie in between the by endothelial growth factors/cytokines
induced signaling and cell-cell junction regulation.
After briefly introducing the Rho GTPases and their regulators GEFs and GAPs, I will first
summarize the effects of six key inflammatory mediators and growth factors on the endothelial
barrier function. Also, I will explain how these effects relate to the activity of three main Rho
GTPases Rac1, Cdc42 and RhoA. Second, I will explain in detail which GEFs and GAPs are
associated with regulation of cellular junctions and I will point out the upstream regulators of
GEFs/GAPs if known. Finally, I will define which GEFs/GAPs are shown to control endothelial
junctions and are likely to play a role in inflammation or angiogenesis by regulating endothelial
permeability.
7
Rho GTPases
The actin cytoskeleton and the organization of AJs and TJs can be tightly regulated by Ras
homologous GTPases (Rho GTPases). Rho GTPases, consisting of 20 family members, transduce
signals from receptors on the plasma membrane to specific intracellular effector proteins
(Wennerberg et al., 2005). Besides regulation of junctions and the cytoskeleton, they are also
implicated in regulating the cell-cycle and gene transcription. Rho GTPases can stimulate a
variety of cell processes, such as adhesion, migration, vesicle transport, cellular remodeling,
phagocytosis and cancer (proliferation) (Heasman and Ridley, 2008; Rossman et al., 2005; Sahai
and Marshall, 2002a). Rho GTPases are often activated via G-protein coupled receptors (GPCRs),
which can activate heterotrimeric G-protein complexes, made up of an α-subunit and tightly
associated β/γ-subunits. Many classes of Gα-subunit are known; Gα12/13 and Gα11/q are the main
subunits involved in direct or indirect activation of Rho GTPases respectively (Rossman et al.,
2005; Oldham and Hamm, 2008).
The best-studied members of the Rho family in regard to actin cytoskeletal organization are
RhoA, Rac1 and Cdc42. Although all three Rho GTPases are intracellular proteins, their specific
location in the cell and especially their spatiotemporal activation pattern differ. All can localize
at the plasma membrane once activated (Ridley, 2006). In general, activated RhoA is involved in
actomyosin contractions and the formation of stress fibers, whereas activated Cdc42 and Rac1
are involved in the formation of lamellipodial protrusions by producing branched actin at the
leading edge of cells. Cdc42 is also the main activator of filopodia formation, which are actin-rich
protrusions of the plasma membrane beyond lamellipodia (Ridley, 2006).
Figure 2. Rho GTPases signaling pathways with their downstream effectors. In these potential
pathways, Cdc42, Rac1 and RhoA (highlighted) influence the actin cytoskeleton and the organization of
cell-cell junctions (TJs and AJs) in non-muscle cells. The dotted arrows indicate indirect activation.
Activated Rho GTPases interact with multiple downstream effectors involved in contractility and
polymerization of the actin cytoskeleton. Cdc42 and Rac1 stimulate branching of actin filaments.
RhoA stimulates the formation and polarization of non-branching actin filaments. Downstream
of active RhoA and upon activation of myosin II, actin filaments slide over each other, leading to
contraction of the actin cytoskeleton. Important downstream effectors of RhoA are Rho8
associated kinase (ROCK) and Diaphanous-related formins (Dia). ROCK can activate the Ca2+dependent myosin light chain kinase (MLCK) and can also directly phosphorylate the myosin
light chain (MLC) (Amano et al., 2010; Citi et al., 2011). It also phosphorylates the MLC
phosphatase (MLCP), inhibiting MLCP’s phosphatase activity (Figure 1) (Amano et al., 2010; Citi
et al., 2011). Dia1 stimulates the formation and polarization of non-branching actin filaments
(Ridley, 2006; Citi et al., 2011; Lammers et al., 2008). To sum up, RhoA activates Dia1, resulting
in actin polymerization, and activates ROCK, leading to actomyosin contractions. An overview of
key effectors of Rho GTPases involved in actin remodeling is displayed in Figure 2.
Regulation of Rho GTPases by GEFs and GAPs
Rho GTPases cycle, like other small GTPases, between the inactive GDP-bound state and the
active GTP-bound state. These switches are controlled by several proteins. Guanine nucleotide
exchange factors (GEFs) stimulate the replacement of the nucleotide GDP for GTP in inactive
GTPase. GTPase-activating proteins (GAPs) accelerate the GTPase’s intrinsic GTP hydrolysis
reaction to become in the GDP-bound conformation. Lastly, guanine nucleotide dissociation
inhibitors (GDIs) bind inactive GTPases to keep them in their inactive state in the cytoplasm
(Figure 2) (Bos et al., 2007).
Figure 3. Regulation of Rho GTPases.
GDI sequesters the inactive Rho GTPase
(GDP-bound). When released from the
Rho GDI, Rho GEFs can induce the
replacement of GDP for GTP. The GTPbound Rho GTPase is active and enhances
activation of several target and effector
proteins. Rho GAPs mediate hydrolysis of
GTP into GDP, inducing the Rho GTPase to
become in the inactive state.
The Rho GEF family, also called Dbl-family, contains approximately 69 members (Rossman et al.,
2005). GEFs consist of several domains, including a Dbl homology (DH) domain and a pleckstrin
homology (PH) domain. The PH domain is for most Rho GEFs necessary to localize at and attach
to the plasma membrane (Stam et al., 1997), however, some GEFs have other domains that
influence their cellular localization (Rossman et al., 2005). GTPases bind the nucleotides GTP
and GDP between two loops, called switch 1 and 2. GEFs interacts via their DH domain with
these switches and modify their conformation, resulting in release of GDP (Bos et al., 2007;
Rossman et al., 2005). Since small GTPases have a similar affinity for GDP and GTP, and the
cellular GTP concentration is 10x higher than the GDP concentration, there is an increased
likelihood that the GTPase binds GTP over GDP after modification by GEFs. Moreover, GEFs can
be regulated by for example posttranslational modifications. These regulations can result in
activation and/or translocation of the GEF towards a specific GTPase (Bos et al., 2007).
9
Additionally, GAPs are needed to enhance the slow intrinsic GTP hydrolysis of Rho GTPases.
Most Rho GAPs are ubiquitously expressed (Su et al., 2004) and stabilize the catalytic glutamine
of the GTPase by lowering the transition state energy. Moreover, they stabilize the switch
regions (Bos et al., 2007). GAPs can be regulated via similar mechanisms like GEFs (Bos et al.,
2007). In this review, we will focus on the GEFs and GAPs that may be involved in cell-cell
junction regulation and vascular permeability induced by inflammatory cytokines and growth
factors.
Rho GTPases and cell-cell contacts
As mentioned before, small Rho GTPases can regulate cell junctions. RhoA and Rac1 play a role
in the formation of cell-cell junctions: Rac1 can initiate the formation of AJs and RhoA is needed
for completion of the cell-cell contact (Yamada and Nelson, 2007). In epithelial cells, Rac1 can be
activated near E-cadherin-based adhesions, which is required for the association of the actin
cytoskeleton and the cadherin complex, supporting cell-cell adhesion (Braga et al., 1997;
Takaishi et al., 1997; Jou and Nelson, 1998). RhoA is involved in both the formation of AJs and
TJs (Takaishi et al., 1997; Braga et al., 1997). In more detail is shown that the activities of RhoA,
Rac1 and their effectors are restricted to specific regions of (growing) cell-cell contacts. First,
Rac1 is found active near the tips of lamellipodia in expanding cell-cell contacts (Yamada and
Nelson, 2007). Thereafter, RhoA and its effector ROCK become activated at the distal edges of
growing cell-cell contacts, what is in accordance with the high levels of activated myosin II found
at the edges. This drives the accumulation of E-cadherin-based adhesions (Yamada and Nelson,
2007; Shewan et al., 2005).
The role of RhoA in cell-cell junction regulation is complex. Whether RhoA stabilizes or
destabilizes AJs will depend on its spatiotemporal regulation and the level of activity (Van Nieuw
Amerongen et al., 2007; Yamada and Nelson, 2007). Enhanced ROCK and myosin II activity can
induce contractile forces, pulling neighboring cells apart. On the other hand, basal ROCK and
myosin II activity supports integrity of AJs. Moreover, via Dia1, RhoA promotes localization of αcatenin towards cadherin and clustering of AJs, resulting is more stable cadherin-based
junctions (Sahai and Marshall, 2002b; van Nieuw Amerongen et al., 2007).
Furthermore, Rac1 and Cdc42 play a role in the stabilization of AJs. They inhibit IQGAP, a
scaffolding protein that in its active form dissociates α-catenin from β-catenin. This disables the
actin cytoskeleton to bind to cadherin and will weaken the AJs. So, inhibition of IQGAP by
Rac1/Cdc42 results in strong cadherin-mediated cell junctions (Figure 1) (Kuroda, 1998;
Kaibuchi et al., 1999). Moreover, Cdc42 is also implicated in the transport of E-cadherin from the
Golgi to plasma membrane in epithelial cells to support the formation of cell-cell adhesions
(Wang et al., 2005).
10
Effect of inflammatory mediators and growth factors on Rho GTPases and
endothelial permeability
Growth factors and inflammatory mediators regulate endothelial cell-cell junctions. As
mentioned in the Introduction, thrombin, histamine, TNFα and VEGF are known factors that
increase the permeability of the endothelium. In contrast, S1P and Ang1 stabilize the cell-cell
junctions and restore the endothelial barrier. This chapter will focus on the known effects of
these factors on endothelial or, where relevant on, epithelial permeability. Moreover, I will
explain which Rho GTPases function downstream of the different factors. It will become clear
that each Rho GTPase has the ability to either induce or inhibit endothelial permeability,
depending on their spatiotemporal activation.
Thrombin
Thrombin is serine protease, functioning as a blood coagulation factor (converting fibrogen into
fibrin) and as a remodeler of endothelial junctions. This inflammatory mediator increases
endothelial permeability. Thrombin can cleave the extracellular N-terminus of proteaseactivated receptor 1 (PAR-1) on endothelial cells, consequently activating this receptor
(McLaughlin et al., 2005). PAR-1 is a GPCR and activates heterotrimeric G-proteins. Thrombin
induces fast and slower responses in in vitro experiments. Subunits of these G-proteins, Gα11/q,
Gα12/13 and Gαi, can rapidly activate RhoA (Figure 4). During this phase of a thrombin response,
Rac1 is inhibited after PAR-1 activation. By contrast, Cdc42 and Rac1 are activated at later
phases of the response (1 hour past thrombin treatments), because they are needed for recovery
of the thrombin-mediated endothelial barrier
destabilization.
Quickly after thrombin stimulation, several
signaling mechanisms are initiating RhoA
activation and induce disruption of endothelial
cell-cell junctions. RhoA remains active for roughly
30 minutes (Van Nieuw Amerongen, 2002; Mehta
et al., 2003). One of the signaling routes induced by
thrombin activates Gα11/q, inducing phospholipase
C to convert phospholipids into inositol
triphosphate (IP3) and diacyl glycerol (DAG)
(Figure 4). IP3 and DAG can induce endothelial
permeability through RhoA activation dependent
and independent routes. IP3 increases cytosolic
Ca2+-concentration, activating calmodulin, which in
turn activates MLCK. The latter phosphorylates
MLC, thereby enhancing actomyosin contraction.
This destabilizes the cell-cell contacts and
consequently induces endothelial permeability
(Satpathy et al., 2004). DAG also elevates the
cytosolic Ca2+ levels. Moreover, DAG activates
Protein Kinase Cγ (PKCγ), promoting MLC
phosphorylation via two ways. First, PKCγ can
inactivate MLCP via phosphorylation (Satpathy et
Figure 4. Thrombin-mediated regulation
of RhoA, Rac1 and Cdc42. The G-protein
pathways are in more detailed discussed in
the text. The signaling downstream of PKCα
(green arrow) is depicted in Figure 5.
11
al., 2004). Second, PKCγ activates RhoA through inhibition of RhoGDI and stimulation of a RhoA
GEF (Mehta et al., 2001; Holinstat et al., 2003).
A second signaling route directly downstream of thrombin induced PAR1 activation occurs via
Gα12/13, activating RhoA via a GEF in a Ca2+-independent matter (Birukova et al., 2004). The third
route is via Gαi, a subunit that inhibits the production of cAMP through inhibition of adenylate
cyclase (Hung et al., 1992) (Figure 4). Consequently, Protein Kinase A (PKA) is not activated,
resulting in inhibition of RhoGDI. RhoA is released and free to be activated (Qiao et al., 2008).
Thus, thrombin can activate RhoA via all three signaling routes, resulting in increased
phosphorylation of MLC and enhances actomyosin contraction. The increased tension on cell-cell
adhesions, including cell-cell junctions, is the major cause of endothelial permeability induction
by thrombin.
In conjunction with the rapid RhoA activation, Rac1 is inactivated by thrombin for
approximately 1 hour, which is thought to weaken the cell-cell junctions. It is established that
Rac1 is inactivated after thrombin stimulation because Gαi reduces of cAMP levels in endothelial
cells (Baumer et al., 2008, 2009; Hung et al., 1992). Moreover, it is proposed that effectors of
RhoA can activate two Rac1 GAPs, leading to Rac1 inhibition as well (Sanz-Moreno et al., 2008;
Ohta et al., 2006). However, this mechanism to inactive Rac1 is only observed in melanoma cells
and not yet confirmed to take place in endothelial cells.
After approximately 1 hour, Rac1 and Cdc42 are (re)activate and recover the endothelial barrier,
an important repairing function, which in vivo is important to prevent e.g. severe leakage of the
blood vessel. Thrombin induces sphingosine-1-phosphate (S1P) activation via activation of the
sphingosine kinase (SPHK), consequently activating Rac1 (Tauseef et al., 2008). See the
paragraph below concerning the role of S1P for a detailed description of this signaling route
affecting cell-cell junctions. Also elevated cAMP levels increase Rac1 activity (Baumer et al.,
2009, 2008). Moreover, Cdc42 is activated 1 hour after thrombin treatments, preceding junction
formation. This is required for the barrier function of endothelial cells, including the as observed
in mice lung endothelium. Subsequently, association of the actin cytoskeleton with the VEcadherin complex is thought to stabilize the junction at this stage (Kouklis et al., 2004).
Furthermore, the disassembly event of AJs itself might activate Cdc42 as well, since
subconfluent endothelial monolayers have an increased Cdc42 activity compared to confluent
monolayers (Kouklis et al., 2004). These observations indicate that Cdc42 and Rac1 recover the
endothelial barrier function after thrombin by stabilizing cell-cell junctions.
In conclusion, activation of PAR-1 by thrombin activates three known pathways, initiated by
Gα11/q, Gα12/13 and Gαi that induces endothelial permeability which may occur with or without
RhoA activation. Effectors of RhoA subsequently induce actomyosin contraction. After an hour,
the inhibited Rac1 and Cdc42 become activated and inhibit RhoA. This enhances cell-cell
junction stability and repairs the endothelial barrier.
12
S1P
Sphingosine is a member of a class of plasma membrane
lipids and can be phosphorylated by sphingosine kinases
(SPHK), leading to the generation of S1P, a signaling
molecule (Strub et al., 2010). S1P can function as an
intracellular lipid second messenger or can be secreted
to subsequently function as an extracellular ligand for
the GPCR S1P1 till -5 in an autocrine or paracrine manner
(Hla, 2003; Alvarez et al., 2007; Strub et al., 2010). S1P
can be secreted by for example platelets (Mehta et al.,
2005). As observed in endothelial and rat pancreatic
islet cells, SPHK can be activated downstream of
thrombin, TNFα and histamine (Figure 5) (Tauseef et al.,
2008; Mastrandrea et al., 2005; Huwiler et al., 2006).
This results in activation of Rac1 and inhibition of RhoA,
consequently leading to mitigation of the monolayer
permeability (Mehta et al., 2005; Schaphorst et al.,
2003).
Knocking down SPHK in endothelial cells induces a
constant RhoA activation and lowers Rac1 activity
(Tauseef et al., 2008), inducing permeability of
endothelial monolayers. Additionally, it was shown in
vivo that S1P stimulates activation of endothelial Rac1.
Administration of S1P to lungs of rats indeed stabilized
their endothelial cell-cell adhesions in lung microvessels
(Adamson et al., 2010).
Figure 5. S1P-mediated regulation
of RhoA and Rac1. S1P can be
formed after thrombin and TNFα
stimulation, but can also be secreted
by platelets. The G-protein subunit
Gαi induces RhoA inhibition via two
pathways: one via Ca2+ and RhoGDI,
the other via Rac1 and p190RhoGAP
activation, which are more detailed
described in the text. The dashed
arrow indicates indirect activation.
The S1P1 receptor can activate the Gαi subunit via the PI3-kinase (PI3K)-Akt pathway,
subsequently activating Rac1 (Figure 5) (Adamson et al., 2010). In human umbilical vein
endothelial cells (HUVECs), S1P induces translocation of Rac1 and two Rac1 GEFs towards cellcell contacts, resulting in AJ assembly (Lee et al., 1999). Moreover, the G-protein subunit
increase the cytosolic levels of second messengers such as calcium and cAMP (Hla, 2003). This
elevated calcium level contributes to Rac1 activation as well. It has been shown that calcium
induces RhoGDI to release from Rac1, enabling GEFs to activate Rac1 (Mehta et al., 2005; Price et
al., 2003). Rac1 in its turn induces the formation of lamellipodia at the cell-cell contacts,
initiating accumulation of VE-cadherin and thereby promoting cell-cell adhesion. Moreover, S1P
can promote the formation of TJs via Rac1. Active Rac1 mediates the distribution of ZO-1
towards the cell-cell junctions in endothelial cells (Lee et al., 2006). Furthermore, RhoA might be
suppressed via RhoGDI and via Rac1-mediated p190RhoGAP activation (Figure 5)
(Papaharalambus et al., 2005). In addition, several studies showed that elevated levels of
intracellular S1P can decrease the endothelial permeability as well. However, the signaling
routes downstream of intracellular S1P are not yet elucidated (Strub et al., 2010).
To sum up, S1P formation can be induced by inflammatory mediators such as thrombin and
TNFα. S1P can activate in an autocrine/paracrine manner the S1P receptors. This active Rac1
and inactivate RhoA, provoking stabilization of AJs and TJs. This supports the endothelial barrier
13
and prevents vascular leakage. Also increased intracellular concentrations of S1P seem to
protect the barrier function.
Histamine
Histamine is a vasoactive mediator and is known to rapidly induce
vascular permeability after a trauma or allergy. This mediator exerts
its function via four different histamine receptors (HRH1-4), which
are all GPCRs. The HRH1 is particularly highly expressed on
endothelial cells (Clough et al., 1998). Activation of the receptor leads
to RhoA activation, inducing increased actomyosin contractions and
subsequently disrupting junctions (Figure 6).
HRH1 activates the G-protein subunit Gα11/q, mediating activation of a
RhoA specific GEF in endothelial cells (Pfreimer et al., 2012). More
details about the specific RhoA activation pathway remain unclear
and are not yet established in endothelium. Through Gα11/q signaling,
the cytosolic calcium levels elevate and active MLCK (Figure 6),
resulting in phosphorylation of MLC and consequently causing
instable cell-cell junctions through actomyosin contractions of the
connected actin filaments (Wei et al., 2011; Wojciak-Stothard and
Ridley, 2002; Guo et al., 2007; Wójciak-stothard et al., 2001).
Furthermore, histamine induces significant loss of TJs and AJs
between endothelial cells (Wójciak-stothard et al., 2001).
Figure 6. Histamineinduced activation of
RhoA. Via the Gα11/q
pathway, a RhoA GEF
and elevated Ca2+ levels
contribute to increased
RhoA activation.
However, these histamine effects on endothelial cell-cell junctions are transient. Histamine
reduces the endothelial barrier function for only 3-5 min, whereas e.g. thrombin induces a
prolonged effect of more than 40 min in HUVECs. There are several differences between the
responses of endothelial cells to histamine and thrombin. For example, less MLCs are
phosphorylated in histamine stimulated HUVECs compared to thrombin stimulated cells (Moy et
al., 1996). Moreover, thrombin induces bigger gaps between the individual endothelial cells in
vitro, whereas histamine induces smaller intercellular gaps (Wójciak-stothard et al., 2001).
Summarizing, histamine induces activation of RhoA, resulting in disruption of AJs and TJs. This
contributes to endothelial permeability.
VEGF
The permeability factor VEGF is strongly involved in angiogenesis during embryogenesis and
adult life. Endothelial cells express two VEGF receptors: VEGFR1 and VEGFR2. In adult
endothelial cells, VEGFR2 is the main receptor that recognizes and binds VEGF, whereas both
receptors are involved during embryogenesis. VEGFR2 is a tyrosine kinase receptor that
dimerizes after binding of VEGF, resulting in autophosphorylation of its intracellular domain.
Accordingly, several adaptor proteins bind. VEGFR2 mediates the activation of the MAPK
pathway and PKC via phospholipase Cγ (PLCγ). PLCγ also increases the cytosolic Ca2+
concentration. Moreover, VEGFR2 activates the PI3K pathway (Olsson et al., 2006). Intriguingly,
VEGF is able to induce activation of RhoA, Rac1 and Cdc42, although their timing of activation is
different.
14
Activation of VEGFR2 by VEGF is followed by a fast Rac1 activation for approximately 15 min in
vitro (Figure 7A) (Beckers et al., 2010; Garrett et al., 2007). The rapid Rac1 activation is followed
by a more sustained Rac1 activation at 30 min (Garrett et al., 2007). This biphasic Rac1
activation by VEGF corresponds with the biphasic reaction of human pulmonary artery
endothelial cells after stimulation with VEGF in vitro. First, the endothelial barrier function
briefly increases (for 10 min), followed by a sustained decrease, indicating an increased
endothelial permeability (Figure 7B) (Mirzapoiazova et al., 2006).
A
B
Figure 7. Effect of VEGF addition on Rho GTPases in endothelial cells and on the resistance
of the endothelial monolayer. A) The activation of RhoA, Rac1 and Cdc42 after VEGF
stimulation is plotted against the time after VEGF addition (in minutes). The response of Rac1 is
biphasic. The figure is adapted from Beckers et al. (Beckers et al., 2010). B) The transendothelial
electrical resistance (TER) is measured after stimulating a monolayer of human pulmonary artery
endothelial cells with VEGF (at time point 0 min). The cells were stimulated with two different
concentrations of VEGF or a vehicle. The cells showed a biphasic response after treatment with
100ng/ml VEGF. The resistance across the monolayer first increased, followed by a decreased in
resistance. The figure is adapted from Mirzapoiazova et al. (Mirzapoiazova et al., 2006).
Several studies showed that VEGF-induced Rac1 activates PAK1. Dominant-negative Rac1 and
RNA interferences approaches for Rac1 reduce the endothelial permeability (Eriksson, 2003;
Gavard and Gutkind, 2006; Garrett et al., 2007). On the other hand, a constitutive active form of
Rac1 can disrupt VE-cadherin based cell junctions (Gavard and Gutkind, 2006). Gavard and
Gutkind showed that the serine/threonine kinase PAK1 phosphorylates VE-cadherin at Ser665,
inducing β-arrestin recruitment towards AJs and eventually inducing internalization of the total
VE-cadherin complex, destabilizing cell-cell junctions (Figure 8) (Gavard and Gutkind, 2006).
This function of Rac1 is in contrast with its function established downstream of e.g. thrombin,
where it enhances cell-cell junction stability. Of note, a recent study looking into the role of
phosphorylation of VE-cadherin in vivo could not corroborate the data that Ser665
phosphorylation is necessary for VE-cadherin internalization upon VEGF stimulation (Orsenigo
et al., 2012). This study showed that VE-cadherin can be (tyrosine) phosphorylated in venous
endothelial cells even in the absence of permeability inducing factors. Possibly, the
phosphorylation of VE-cadherin makes cell-cell junctions more dynamic and sensitive for
inflammatory mediators (Orsenigo et al., 2012).
Furthermore, VEGF-induced Rac1 activation stimulates the production of Reactive Oxygen
Species (ROS) via NADPH oxidase. The generation of ROS may have distinct effects on cell-cell
15
junctions; either destabilizing or stabilizing. First, ROS can induce phosphorylation of VEcadherin and β-catenin in microvascular endothelial cells, leading to a reduced affinity of βcatenin for VE-cadherin and consequently in decreased AJ integrity (Monaghan-Benson and
Burridge, 2009). Secondly, ROS production results in translocation of the RhoA GAP
p190RhoGAP towards the plasma membrane and AJs. P190RhoGAP interacts with p120-catenin
and has the ability to block RhoA activity at the AJs (Wildenberg et al., 2006; Nimnual et al.,
2003) (Figure 8). This inhibits internalization of N-cadherin and disrupts AJs in fibroblasts.
However, thus far a link between ROS-p190RhoGAP was only found in fibroblasts, although the
p190RhoGAP is expressed in endothelial cells as well. It still
needs to be clarified how ROS-production regulates RhoA’s
spatiotemporal activation.
In addition, VEGF activates the RhoA/ROCK pathway (Bryan
et al., 2010), suggesting that this pathway is involved in
VEGF-mediated permeability (Bryan et al., 2010). Moreover,
Cdc42 is activated after binding of VEGF to VEGFR2 in
endothelial cells, which could play a, as yet unclear, role in
the regulation of cell-cell junctions by VEGF as well
(Kusuhara et al., 2012; Lamalice et al., 2004).
Although thrombin and VEGF are both vascular permeability
factors, their downstream signaling pathways are very
different. The levels of activated RhoA are low at 1 min after
Figure
8.
VEGF-mediated
regulation of Rac1 and RhoA.
VEGFR,
a
RTK,
mediates
activation of Rac1, resulting in
phosphorylation of cell junction
proteins. RhoA is inactivated
after VEGF stimulation. The
pathways are in more detail
described in the text. Dotted
arrows
indicate
indirect
regulation.
stimulation VEGF compared to the major increase in
activated RhoA after thrombin stimulation (Van Nieuw
Amerongen, 2002). This is probably caused by the lack of
Gα12/13 activation in VEGF stimulated endothelial cells. This
G-protein subunit is in thrombin stimulated cells a crucial
mediator in RhoA activation. Where thrombin primarily
initiates actomyosin contraction, the by VEGF-mediated
RhoA activation seems more involved in stimulation of cell
migration which might be relevant for the induction of
sprouts during VEGF driven angiogenesis (Van Nieuw
Amerongen, 2002).
TNFα
Tumor necrosis factor α (TNFα) is an inflammatory cytokine produced by monocytes and
macrophages. Within 30 min., increased stress fibers are formed, followed by sustained
formation of interendothelial gaps for many hours in vitro (Wójciak-Stothard et al., 1998). TNFα
can bind to its two plasma membrane receptors TNFR1 and TNFR2, activating the PI3K/Akt and
MAPK pathway, and several phospholipids (Wójciak-Stothard et al., 1998) (Figure 9). As
previously mentioned, TNFα can also increase the activity of SPHK, supporting increase in
intracellular S1P levels in pancreatic cells (Mastrandrea et al., 2005). It has been proposed that
RhoA, Rac1 and Cdc42 function downstream of TNFα in HUVECs and are responsible for the
induced changes of the actin cytoskeleton and destabilization of cell-cell junctions (WójciakStothard et al., 1998). However, it should be noted here that several follow-up studies could not
substantiate the finding that RhoA is essential for TNFα-induced permeability (Mckenzie, 2007;
16
Schlegel and Waschke, 2009). By contrast, Rac1 seems to be a major player; although also for
this GTPase conflicting data are published (Cain et al., 2010; Papaharalambus et al., 2005;
Schlegel and Waschke, 2009).
First, it has been shown that TNFα induces RhoA activation, resulting in activated ROCK and
Dia1. However, the level of phosphorylated MLC does not rise above basal levels when TNFα
stimulated cells are compared to unstimulated cells, suggesting that the RhoA/ROCK signaling
towards MLC is not involved in the endothelial response to TNFα (Mckenzie, 2007; Schlegel and
Waschke, 2009). It is a possibility that the improved Dia1 activation contributes to stress fiber
formation. On the other hand, it is possible that the RhoA/ROCK pathway indeed plays a role in
the TNFα reaction, but that the effects are local and no significant changes in activation can be
detected in total lysate of cells.
Within 30 min after TNFα, no significant increase in endothelial permeability can be observed,
whereas thrombin already induces strong permeability at this time point. In contrast, TNFα
induces endothelial permeability in the long run (8-24 hr). After sustained TNFα stimulation,
stress fibers are accumulated, occludin and JAM-A are removed from the TJs, and ZO-1 is not
located properly at the TJs. This results in weakened TJs and in increased permeability of the
HUVEC monolayer (Mckenzie, 2007). It is not clear if and how RhoA is involved in this long term
effect.
Several studies show, however, that TNFα induces activation of Rac1 in
endothelial and epithelial cells underlying its effect on permeability
(Cain et al., 2010; Papaharalambus et al., 2005; Wójciak-Stothard et al.,
1998). In HUVECs, this is initiated via activation of PI3K pathway (Figure
9). It is suggested that the p110α subunit stimulates recruitment of a
Rac1 GEF towards VE-cadherin. As a result, Rac1 is activated at cell-cell
contacts (Cain et al., 2010). However, conflicting data are published by
Schlegel and Waschke. They used human dermal microvascular
endothelial cells and found that Rac1 was inactivated after TNFα
stimulation as a consequence of decreased cAMP levels. This inhibition
of Rac1 disrupted the junctions and increased the endothelial
permeability (Schlegel and Waschke, 2009).
Figure 9. TNFαinduced activation
of RhoA and Rac1.
Via PI3K and Akt,
Rac1 is activated.
In conclusion, TNFα induces prolonged permeability compared to other
inflammatory mediators. Findings concerning the role of RhoA and Rac1
in the TNFα-induced response on endothelial cell-cell junctions are
contradictory. It is not yet established whether RhoA’s role is crucial.
Furthermore, a majority of the studies on the role of Rac1 show that Rac1 activation induces
endothelial and epithelial permeability, although again, opposite effects are reported.
Ang-1
Angiopoietin-1 (Ang-1) is a growth factor maintaining the vascular integrity. It is established
that Ang-1 attenuates the by inflammatory mediators induced vascular permeability (Van der
Heijden et al., 2011; Gavard et al., 2008). Ang-1 binds to the RTK Tie2 on endothelial cells,
activating MAPK (ERK) and the PI3K/Akt pathway, consequently leading to sustainability of the
junctions and prevents actomyosin contractions, thereby stabilizing the endothelial barrier
function (Figure 10) (Fukuhara et al., 2008; Saharinen et al., 2008). In confluent HUVECs, Tie2 is
17
located at AJs and binds to Tie2 on neighboring cells. This localization towards cell junctions is
induced by Ang-1 (Saharinen et al., 2008).
Ang-1 signals via Rho GTPases to enhance the endothelial
barrier function: Rac1 is activated after approximately 15
minutes and RhoA is inactivated (David et al., 2012).
PI3K has several downstream effects, such as activating
Akt, but also GEFs, subsequently targeting and activating
Rac1 at cell-cell junctions. It is suggested that IQGAP is
required for efficient and sustainable activation of Rac1
in endothelial cells (David et al., 2012). Subsequently,
Rac1 can mediate activation of p190RhoGAP (a finding
which was confirmed in vivo), inhibiting RhoA.
P190RhoGAP activation is essential for enhancing the
barrier function (Mammoto et al., 2007). However, it
remains remarkable that TNFα/PI3K-mediated Rac1
activation induces breakdown of the cell-cell contacts,
whereas S1P or Ang-1/PI3K-mediated Rac1 activation
supports restoration of cell adhesion. The effect of Rac1
activation on cell-cell junctions probably depends on its
specific location in the cell, and may depend on which
cytokine activated Rac1.
Figure
10.
Ang-1-mediated
regulation of Rac1 and RhoA. Near
the plasma membrane, Ang-1
stimulates activation of RhoA,
whereas signaling via active Rac1
inhibits RhoA in the cytosol. The
pathways
are more
detailed
explained in the text. The dashed
lines indicate indirect activation.
Surprisingly, Ang-1 can also stimulate RhoA close to
where VEGFRs localizes (Gavard et al., 2008). RhoA in
turn activates Dia. Active Dia will bind Src kinase and
prevents Src from binding to the VEGFR, leading to
reduced phosphorylation of VE-cadherin by Src (Figure
10). Consequently, this mitigates the disruption of cell-cell contacts induced by VEGF, thus
protecting the endothelial barrier (Gavard et al., 2008).
To conclude, Ang-1 can restore the endothelial barrier via initiating dephosphorylation of VEcadherin and via PI3K-mediated activation of Rac1. In contrast, Ang-1 can also enhance RhoA
activity near VEGFRs, reducing phosphorylation of VE-cadherin as well, which may stabilize cellcell junctions too.
18
Rho GEFs and GAPs and cell-cell junction regulation
As described in the previous chapter, each Rho GTPase can either initiate protection of the
endothelial barrier or can promote vascular permeability, which will depend on the level, timing,
and cellular location of their activation. Possibly GEFs and GAPs, functioning upstream of
RhoGTPases and activate and inhibit GTPases respectively, play an important role in the
spatiotemporal control of Rho GTPases. In this chapter we will focus on the GEFs and GAPs
known to control cell-cell junctions in all cell types in order to comprehend the regulation of
endothelial cell-cell junctions downstream of vascular cytokines.
GEFs and GAPs controlling Rac1 signaling involved in junction regulation.
The proposed roles of Rac1 in regulating AJs are conflicting. On one hand, Rac1 induces via its
effector PAK1 or via ROS formation phosphorylation of VE-cadherin, leading to VE-cadherin
internalization and loss of AJs. On the other hand, Rac1 is involved in barrier restoration after
Ang-1 stimulation and via the S1P pathway. Rac1 induces junction stabilization through
dislocation of IQGAP from cell junctions and activation p190RhoGAP, inhibiting RhoA. In both
situations, similar Rac1 GEFs are suggested to be involved as I will discuss here.
One of the GEFs that activates Rac1 in endothelial and epithelial cells is Tiam1 (Habets et al.,
1994; Sander et al., 1998). Tiam1 is recruited to the plasma membrane and associates with VEcadherin (Lampugnani et al., 2002) depending on the p110α subunit of PI3K downstream of e.g.
TNFα (Lee et al., 1999). Knockdown of this subunit results in decreased codistribution of the GEF
with VE-cadherin in HUVECs, suggesting that PI3K regulates Rac1 at AJs via recruitment of
Tiam1 (Cain et al., 2010). This leads to increase endothelial permeability (Cain et al., 2010). In
contrast, Tiam1 can be activated through increased cAMP concentrations, correlating with
increased barrier function Elevated cAMP levels, induced by prostaglandins, lead to activation of
PKA1, phosphorylating and activating Tiam1 in HUVECs (Kobayashi et al., 2013). Using lung
endothelial cells, similar results are reported: Tiam1 is involved in the recovery of cell-cell
junctions after thrombin exposure (Birukova et al., 2012). In support for a junction protection
function for Tiam1, in epithelial cells was found that Tiam1 is needed to maintain E-cadherin
mediated cell-cell contacts (Sander et al., 1998; Malliri et al., 2004). In addition, Tiam1 is
required for TJ formation in epidermal keratinocytes. Keratinocytes isolated from Tiam1
knockout mice display disturbed Rac1 activation and failure in TJ maturation (Mertens et al.,
2005). Thus, Tiam1 can either induce destabilization or stabilization of cell-cell junctions, but is
reported at least to be involved in junction regulation downstream of vascular cytokines.
The Rac-GEF Vav2 is responsive to VEGF (Garrett et al., 2007). Vav2-mediated activation of Rac1
leads to PAK1 activation, resulting in phosphorylation of VE-cadherin and subsequently in
internalization of VE-cadherin in mouse vascular endothelial cells (Gavard and Gutkind, 2006).
In human mammary epithelial cells is shown that Vav2 co-localizes with E-cadherin and that
activation of the Vav2/Rac1 pathway results in AJ disruption after stimulation with the growth
factor EGF (Duan et al., 2011). Overexpression of constitutively active Vav2 elevates Rac1
activity and remodels the actin cytoskeleton near cell-cell junctions. Vav2 knockdown
experiments significantly reduced the Rac1 activity and cells have less junctional F-actin,
resulting in destabilized junctions (Duan et al., 2011). Besides Rac1 activation, Vav2 may
activate Cdc42 as well (Liu et al., 2000). This signaling route is suggested to be involved in cell
migration, although, a role in cell-cell adhesion is not yet established. However, similar as
19
described for Tiam1, another study established that Vav2 is involved in the formation of
junctions and recovery of the barrier function after stimulation with thrombin (Birukova et al.,
2012). So, Vav2 can be involved in barrier function protection or destabilization.
A third GEF for Rac1 that is shown to be involved in junctional control is P-Rex1. P-Rex1 is
detected in vascular lung endothelial cells and can be activated via the TNFα/PI3K pathway,
resulting in Rac1 activation. P-Rex1 stimulates Rac1-mediated ROS production, inducing VEcadherin phosphorylation. Knockdown of P-Rex1 significantly reduces the level of Rac-GTP in
TNFα stimulated cells. The in vivo effects are studied as well in P-Rex1 knockout mice. The
knockout mice have reduced lung endothelial permeability and less lung edema after
intratracheal exposure to TNFα, compared to wild type mice. Moreover, P-Rex1-/- mice had less
transendothelial migration of leukocytes than wild type mice, suggesting that endothelial cellcell junctions had stabilized (Naikawadi et al., 2012). This clearly shows that TNFα-based
activation of the PI3K/P-Rex1/Rac1 pathway destabilizes endothelial cell-cell junctions. P-Rex2b
is also expressed in endothelial cells and is GEF for Rac1, however, a role for P-Rex2b in AJ
regulation is not yet reported (Li et al., 2005).
To sum up, at least three GEFs for Rac1 are expressed in endothelial cells. P-Rex1 functions
downstream of PI3K and disrupts junctions via Rac1 signaling. Both Vav2 and Tiam1 play a role
in junction disassembly as well. However, contradictory results are published. Tiam1 can also
become activate by cAMP, leading to enhancement of cell-cell contacts. Moreover, Tiam1
enhances TJs in epidermal keratinocytes. Intriguingly, the described regulation mechanisms for
these GEFs also do not explain how Ang-1 and S1P induced junction stabilization via Rac1.
Probably, it depends on the spatiotemporal control of the GEFs and their upstream activators,
but convincing data that deals with this dilemma is not published.
Cdc42 GEF and GAPS regulating cell-cell junctions
Cdc42 becomes active after thrombin stimulation and is shown to be essential in the restoration
of AJs, (Broman et al., 2007; Ramchandran et al., 2008). Mice expressing constitutively active
Cdc42 are more resistant to the induction of endothelial permeability by LPS (Ramchandran et
al., 2008). Moreover, Cdc42 is shown to assemble TJs, however, a role in destabilizing TJs is
reported as well (Wells et al., 2006; Otani et al., 2006).
Tuba is a Cdc42-specific GEF concentrated at the apical zone of epithelial cells, where it interacts
with the TJ-protein ZO-1 and contributes to TJ-stability (Otani et al., 2006). TJs and AJs are
disordered after silencing Tuba. However, the recruitment of ZO-1 to TJs on the plasma
membrane is not affected by Tuba depletion , in contrast, the organization of E-cadherin on the
membrane is strongly dependent on Tuba (Otani et al., 2006). Tuba is the first reported Cdc42
regulator that directly interacts with junctional proteins. It is not yet established whether Tuba
is expressed in endothelial cells.
Furthermore, the GAP Rich1 is found to regulate Cdc42. Rich1 localizes at TJs and AJs and
regulates the activity of Cdc42 at junctions in MDCK cells (Wells et al., 2006). A GAP-deficient
mutant of Rich1 induces improper localization of TJ proteins at the membrane, consequently
leading to instable TJs and increasing the permeability of the monolayer. However, Rich1 is not
necessary for the initiation of TJ formation. Moreover, silencing Rich1 by shRNA does not disturb
AJ organization. Additionally, the scaffold angiomotin (Amot) binds Rich1 and is recruited to TJs.
Overexpression of Amot leads to disruption of TJs, (Wells et al., 2006). Since Rich1 can also
20
accelerates the intrinsic GTP hydrolysis of Rac1, Rich1 might be involved in Rac1 inactivation at
junctions as well (Richnau and Aspenström, 2001). Furthermore, in mice and zebrafish Amot
depletion leads to inactive Rac1 and vascular defects (Aase et al., 2007). Thus, Rich1 activation,
consequently inhibiting Cdc42 and Rac1, results in stable TJs and in decreased permeability of
cellular monolayers. It is reported that Rich1 is expressed in mouse embryonic microvascular
endothelial cells too (Aase et al., 2007)
A second Cdc42 GAP involved in cell-cell junction regulation is PX-RICS. In contrast to Rich1, this
GAP possibly regulates the activity of Cdc42 at the Golgi and not at the plasma membrane
(Nakamura et al., 2008). It is suggested that in HeLA cells, PX-RICS is involved in the transport of
the N-cadherin/β-catenin complex from the Golgi towards the plasma membrane (Nakamura et
al., 2008). This indicates that PX-RICS’s induced inactivation of Cdc42 supports repair of AJs.
Similar involvement of Cdc42 in the transport of E-cadherin from Golgi to membrane was
reported using MDCK cells (Wang et al., 2005).Thus far, PX-RICS expression is not reported to be
expressed in endothelial cells.
In conclusion, the Cdc42 GEF Tuba and the GAPs Rich1 and PX-RICS can all stabilize cell-cell
junctions. This is surprising, since the GEF activates Cdc42 and the GAPs inactivate Cdc42. This
again supports the notion that local regulation by GEFs or GAPs may differ at distinct junction
types and cell types. Tuba and Rich1 are involved in the regulation of TJs at the membrane,
whereas PX-RICS regulates the secretory pathway of N-cadherin towards the plasma membrane
near the Golgi. It will be interesting to study whether PX-RICS is also involved in the transport of
E- and VE-cadherin, although it is not yet established if PX-RICS is expressed in endothelial cells.
RhoA GEFs and GAPs controlling cell-cell junctions and the barrier function
The Rho GTPase RhoA acts downstream of several permeability inducing and protecting factors
(see previous chapter). The effectors of RhoA can either induce stabilization or disruption of
cellular junctions. Likely, the effect on the endothelial permeability will depend on the
spatiotemporal RhoA activation. RhoA specific GEFs and GAPs play an important role in this
tight regulation of RhoA. Some GEFs induce RhoA-mediated endothelial permeability, whereas
other GEFs stabilize junctions and have the ability to protect the barrier function. Also two GAPs
are known to regulate RhoA.
The first RhoA GEF we describe is GEF-H1, activated via the MAPK pathway downstream of
TNFα and required for TJ dissociation. Knockdown of GEF-H1 prevented activation of RhoA by
TNFα in renal epithelial cells (Kakiashvili et al., 2008). It is established that GEF-H1 is associated
with the TJ component ZO-1 in epithelial cells (Benais-Pont et al., 2003; Samarin et al., 2007) and
endothelial cells (Mckenzie, 2007). Overexpression of GEF-H1 does not induce changes in the
cytoskeleton and disruption of TJs in MDCKs, however, the intercellular permeability of the
epithelial monolayer is elevated (Benais-Pont et al., 2003). Another study found that GEF-H1 is
involved in the disassembly of the apical junctions in calcium depleted epithelial cells. RhoA and
ROCK increase the contractility of the actin cytoskeleton, providing force that disrupts junctions
(Samarin et al., 2007).
The RhoA GEF P115RhoGEF is activated via the G-protein subunit Gα12/13 downstream of
thrombin’s receptor PAR-1 (Birukova et al., 2004; Holinstat et al., 2003). In addition,
p115RhoGEF is phosphorylated within 1 min after thrombin through activated PKCα, an
intermediate downstream of TNFα and Gα11/q (Peng et al., 2011; Holinstat et al., 2003).
21
Knockdown of PKCα results in reduced phosphorylation of p115RhoGEF. As established in lung
endothelial cells, p115RhoGEF and RhoA both translocate towards the plasma membrane after
stimulation with thrombin. Subsequently, RhoA actives ROCK and induces actomyosin
contraction and phosphorylation of TJ proteins (Birukova et al., 2004; Yamamoto et al., 2008).
However, it has been shown that prostate cancer cells overexpressing PAR-1 still react on
thrombin after knockdown of p115RhoGEF (Wang et al., 2004), suggesting that also other GEFs
are able to activate RhoA. Moreover, in this cell type, depleting p115RhoGEF in mouse brain
microvascular endothelial cells inhibits RhoA activation and less intercellular gaps are formed
by thrombin (Peng et al., 2011). Another study in mouse brain endothelial cells showed that
knockdown of p115RhoGEF partly inhibits activation of RhoA, reducing the formation of stress
fibers and degradation of TJ proteins (Xiaolu et al., 2011). These data show that p115RhoGEFmediated RhoA activation is involved in breakdown of TJs in endothelial cells, contributing to
increased permeability.
RhoA activation through the above described GEFs disrupts junctions in endothelial and
epithelial cells. By contrast, Terry et al. suggest that the opposite occurs when RhoA is activated
via p114RhoGEF in epithelial cells. P114RhoGEF is found to be recruited towards TJs via
cingulin, a scaffold interacting with ZOs, JAM-A and actin (Bazzoni and Dejana, 2004; Shin et al.,
2006). Deletion of this GEF is associated with disassembly of TJs and activation of non-junctional
RhoA, resulting in MLC phosphorylation (Terry et al., 2011). However, another group found in a
different epithelial cell line that knockdown of P114RhoGEF did not result in disorganization of
the TJs, suggesting that the regulation of RhoA may vary between different cell types (Itoh et al.,
2012). Interestingly, p114RhoGEF is expressed in lung microvascular endothelial cells (Niu et al.,
2003). Thus, p114RhoGEF induces spatially restricted RhoA activation, supporting TJ formation
in some types of epithelial cells.
Additionally, the RhoA GEF PDZ-RhoGEF is shown to stabilize TJs. PDZ-RhoGEF binds directly to
ZO-1 and is expressed in both endothelium and epithelium (Basile et al., 2007; Itoh et al., 2012),
however, its function is only thoroughly studied in epithelial cells. PDZ-RhoGEF becomes
activated through Gα12/13, downstream of several GPCRs. Knockdown of PDZ-RhoGEF does not
result in diminished levels of ZO-1 at TJs. Although, depletion of PDZ-RhoGEF does affect the
cell-cell junction formation, resulting in attenuated barrier function. Moreover, MLC
phosphorylation is increased in the absence of PDZ-RhoGEF at apical TJs, leading to contraction
of the actomyosin cytoskeleton near cell-cell junctions. In epithelial cells, this actomyosin
contraction induces clustering of TJs and consequently polarizes the plasma membrane in an
apical and basolateral side (Itoh et al., 2012). Thus, this GEF also spatially stimulates RhoA to
regulate TJ formation in epithelium. Since the function of TJs in endothelium is not the same as
in epithelium, PDZ-RhoGEF might have other effects on the endothelial barrier, for which further
experimentation is necessary.
Another RhoA GEF supporting junctional integrity via junction-associated actomyosin is Ect2
(Ratheesh et al., 2012). During cell division the centralspindlin complex attracts this GEF
towards AJs at the cleavage furrow, where it activates RhoA. Centralspindlin is also involved in
inhibition of the p190RhoGAP function, thereby enhancing RhoA activity. As a result, active
RhoA initiates myosin II activation, leading to actomyosin contraction and stabilization of apical
AJs (Ratheesh et al., 2012). Interestingly, both PDZ-RhoGEF and Ect2 seem to underlie the
integrity of the zonula adherens in epithelial cells via RhoA mediated ROCK/myosin II activation.
Still, it is poorly understood how if these GEFs are regulated by inflammatory mediators. Both
22
GEFs are known to be expressed by endothelial cells, but no functions have been reported
(Basile et al., 2007; Nacak et al., 2007).
LARG, related to PDZ-RhoGEF and P115RhoGEF, can also be activated by Gα12/13, downstream of
thrombin and histamine signaling (Wang et al., 2004; Pfreimer et al., 2012). In podocytes,
epithelial cells in the glomerulus in the kidney, is shown that LARG can play a role in cell-cell
adhesion. It is suggested that a scaffolding protein recruits LARG appropriately at the AJs,
consequently activation RhoA at the junctions. RhoA on its turn can link actin to cadherin (Kim
et al., 2012). LARG expression is also detected in HUVECs (Geneprof, 2013a).
Furthermore, the GEF Synectin-binding RhoA exchange factor (Syx) is expressed in several cell
types, including endothelial cells and associates with AJ and TJ remodeling. Syx can be recruited
towards ZO-1 in HUVECs and MDCK cells (Garnaas et al., 2009; Ngok et al., 2012). The
localization of Syx can be regulated by VEGF and Ang-1 and is crucial for the regulation of
junction stability. VEGF promotes removal of Syx from TJs, preventing RhoA to become active
near TJs, whereas Ang-1 preserves Syx at the junctions and accordingly stabilizes the
intercellular interactions via RhoA effectors (Ngok et al., 2012). It is indicated that Dia1
functions downstream of Syx, since impaired ZO-1 localization at the plasma membrane in Syx
depleted cells can be restored through expression of constitutively active Dia1 (Ngok et al.,
2012). After Syx depletion, increased VE-cadherin internalized occurs, probably initiated by
increased Src-mediated VE-cadherin phosphorylation. Moreover, by controlling expression of
Syx in zebrafish, it was shown that this GEF is required for the sprouting at the intersomatic
vessels, showing that Syx is involved in angiogenesis which depends on endothelial cell-cell
junction remodeling. (Garnaas et al., 2009). Moreover, Syx KO mice showed malformed or absent
intercellular endothelial cell-cell junctions and vascular leakage compared to wild type mice. To
sum up, Syx is shown to induce RhoA-mediated activation of Dia and inhibition of Src near the
AJs and TJs, mediating reannealing of junctions, which potentially explains the crucial role of Syx
in angiogenesis.
Interestingly, not only Rac1 is suggested to initiate cadherin internalization. Also RhoA can play
a role the endocytosis of AJs, as established in drosophila (Levayer et al., 2011). The RhoA GEF
RhoGEF2 can initiate disruption of cell-cell adhesion during epithelial morphogenesis in
drosophila embryos. RhoGEF2 has three mammalian homologs: p115-RhoGEF, PDZ-RhoGEF and
LARG (Brumby et al., 2011). It localizes in epithelial cells near the plasma membrane, where it
activates Rho1 (drosophila ortholog of RhoA). It has been proposed that Rho1 activates both Dia
and ROCK near AJs, which in cooperation initiate the endocytosis of E-cadherin at the plasma
membrane. Dia and active myosin II are necessary for appropriate clathrin localization at the
junctions, since E-cadherin is internalized via clathrin-coated-endocytosis. Moreover, drosophila
embryos carrying a dominant negative RhoGEF2 have lower levels of clathrin around the cellcell junctions (Levayer et al., 2011). Reduction of RhoGEF2 activity also results in limited Ecadherin turnover during tracheal tube morphogenesis in drosophila (Warrington et al., 2013).
RhoGEF2 has been shown to be involved in the elongation of the epithelial tissue, a process that
requires remodeling of cell junctions (Warrington et al., 2013). Thus, it is established that
RhoGEF2 induces internalization of E-cadherin during early drosophila development, eventually
contributing to less cell-cell junctions.
In addition, also some GAPs are known to be involved in the RhoA-mediated cell-cell junction
regulation. One of them is GRAF2, a GAP colocalizing with α-catenin at AJs in several types of
23
epithelial cells. Without cadherin expression, GRAF2 is detected at the Golgi (Sousa et al., 2005).
Like RhoGEF2, this GAP is involved in E-cadherin internalization. The function of GRAF2 is
studied in the context of bacteria internalization via the cadherin-mediated endocytosis.
Overexpression of GRAF2 results in elevated α-catenin levels and disrupted actin fibers, whereas
knockdown via siRNA leads to impaired α-catenin recruitment towards AJs and weak junctions.
GRAF2 is suggested to coordinate AJ formation in epithelial cells (Sousa et al., 2005) and it is also
found to be expressed in HUVECs (Geneprof, 2013b).
P190RhoGAP is activated after stimulation of endothelial cells with inflammatory mediators
such as thrombin. Activated Rac1 induces to production of ROS, resulting in several responses
including translocation of p190RhoGAP towards AJs, where it interacts with p120-catenin
(Nimnual et al., 2003; Wildenberg et al., 2006). Also Ang-1 signals via Rac1 and p190RhoGAP to
inactivate RhoA. Knockdown of p190RhoGAP in endothelial cells eliminates the ability of Ang-1
to mitigate lung vascular permeability induced by inflammatory cytokines. However, depletion
of p190RhoGAP does not alter the permeability (in vivo and in vitro) in non-stimulated cells
(Mammoto et al., 2007).
To sum up, many GEFs and GAPs are suggested to regulate RhoA activity in epithelial and/or
endothelial cells. It is clear that the activation of RhoA by GEFs and GAPs is spatially restricted.
GEF-H1 and p115-RhoGEF induce the formation of stress fibers, providing tension on cell-cell
junctions. . The GEFs p114-RhoGEF and PDZ-RhoGEF are located near TJs in epithelial cells. In
this subcellular location, they activate RhoA, resulting in the stabilization of the TJs and so
mediating cell-cell adhesion. It has to be taken in account that there is a difference between
endothelial and epithelial cells. Both PDZ-RhoGEF and Ect2 enhance junctional stability via
induced actomyosin contractions near TJs. This results in clustering of the TJs and strengthening
of the cell-cell contacts. Since endothelial TJs are not organized similarly as in polarized
epithelial cells, it might be possible that these GEFs have different effects on endothelial cell-cell
junctions. Another important GEF stabilizing both AJs and TJs in endothelial cells is Syx, which
crucial function is also established in the vasculature of zebrafish and mice. Furthermore, in
drosophila embryos the p115-RhoGEF, PDZ-RhoGEF and LARG homologue RhoGEF2 induces
internalization of E-cadherin, which may contribute to regulation of cell-cell junctions in
mammalian cells. Lastly, two GAPs are associated with RhoA regulation and support cell-cell
contacts. GRAF2 inhibits RhoA near the plasma membrane and blocks internalization of Ecadherin. p190RhoGAP inhibits RhoA and prevents disassembly of junctions. P190RhoGAP lies
downstream of Rac1 signaling and once activated will restore cell-cell adhesion in endothelial
monolayers.
24
Discussion
Rho GEFs and GAPs involved in remodeling endothelial cell junctions, supporting endothelial
permeability
Rho GTPases are proteins that regulate the actin cytoskeleton and remodel cell junctions, such
as AJs and TJs. I focused on the role of Rho GTPases RhoA, Rac1 and Cdc42 in regulating the
endothelial permeability. Opening of the junctions between the endothelial cells in the vessel
wall allows leukocytes and molecules to pass the barrier and enter the surrounding tissue,
regulated by several inflammatory mediators. Rho GTPases play an important role downstream
of these factors. They can initiate the phosphorylation of AJ and TJ components, contraction of
the junctional-associated actomyosin cytoskeleton or regulate endocytosis of AJs. It becomes
clear that the activation/inactivation of RhoGTPases is complex: it depends on their cellular
localization and the available effectors whether a GTPase induces permeability of protects
against permeability. Each Rho GTPase is reported to induce both, depending on the biological
context. GEFs and GAPs are necessary in this complex regulation: their activation and subcellular
localization is crucial in the activating and inactivating of Rho GTPases respectively, but our
knowledge concerning the regulation of GEFs and GAPs is still in its infancy.
In this review, I paid special attention to the GEFs and GAPs known to regulate the junctions in
all cell types and animals to try to understand if they might be involved in regulation of cell-cell
junctions in vascular biology. It is shown that some GEFs/GAPs are involved in the regulation of
the endothelial barrier function in vivo. For example, Syx localizes near junctions after exposure
to Ang-1, resulting in RhoA/Dia activation. This stabilizes cell junctions and maintains of the
barrier function. Furthermore, P-Rex1 is activated via the TNFα/PI3K pathway, activating Rac1.
P-Rex1 knockout mice have less transendothelial leukocyte migration in the lungs after
exposure to TNFα, indicating that P-Rex1 reduces endothelial barrier function. The two Rac1
GEFs Vav2 and Tiam1 seem to be involved in the regulation of endothelial cell-cell junctions as
well. Their functions are widely studied in endothelial cells; however, it is still not clear whether
Vav2 and Tiam1 induce disruption or stabilization of junctions.
Two RhoA GEFs and one RhoA GAP are established to regulate endothelial junctions in vitro via
RhoA activation. GEF-H1 and p115-RhoGEF induce the formation of stress fibers, creating force
on junctions. Both effects cause an improved barrier function. Moreover, p115-RhoGEF
stimulates breakdown of TJs. p190RhoGAP is necessary for barrier protection, indicating that
p190RhoGAP plays an important role in supporting endothelial permeability. Mouse models
may confirm whether these regulatory proteins indeed regulate the endothelial barrier function
via RhoA activation or inactivation in vivo.
Additionally, the GEFs and GAPs that may be be involved in junction regulation are PDZ-RhoGEF,
p114RhoGEF, LARG, GRAF1, Rich1 and PX-RICs. They are detected in endothelial cells; however,
their function in the regulation of cell-cell junction is only established in epithelial cells. PDZRhoGEF, p114RhoGEF and LARG activate RhoA near junctions and it has been shown that this
signaling stabilizes AJs and TJs. Since TJs are more organized in epithelium than in endothelium
(Itoh et al., 2012), spatial activation of RhoA by PDZ-RhoGEF and LARG could have different
effects on endothelial cell-cell adhesions. Another RhoA regulation protein is the GAP GRAF2,
stabilizing cell-cell contacts via inhibition of RhoA. Furthermore, one GEF and two GAPs are
25
suggested to regulate Cdc42; however, they have contrasting effects. The GEF Tuba activates
Cdc42, whereas the GAPs Rich1 and PX-RICS inhibit Cdc42 at TJs and AJs.
As illustrated in this thesis, all three Rho GTPases can have various functions on the endothelial
barrier function, depending on their spatiotemporal activation. For example, local Rac1
activation seems critical, since the same upstream pathway (PI3K) can induce either
Rac1/Tiam1-mediated barrier weakening as barrier strengthening. Also for RhoA is shown that
different GEFs can control RhoA at different subcellular locations, leading to distinct effects on
the cell-cell junctions. RhoA can either regulate junctional actomyosin, non-junctional
actomyosin, the phosphorylation of junctional proteins and the internalization of cell junction
complexes, making the interpretation of biochemical experiments on MLCK and MLC even more
complex. Also the regulation and localization of the upstream Rho GEFs and GAPs is crucial. Not
for all GEFs/GAPs is yet established how their activation is induced by vascular cytokines (Table
1). Exploring the upstream regulation of GEFs/GAPs will uncover whether a GEF/GAP might be
biological relevant. Can it be activated by inflammatory mediators?
To gain more insight in the regulation of the Rho GTPases in endothelial cells, it is necessary to
perform experiments that can clarify where and by which GEF/GAP a GTPase is (in)activated. To
establish their regulation in space and time, bimolecular fluorescence resonance energy transfer
(FRET)-probes that visualize local Rho GTPase activities would be useful. The role of the
GEFs/GAPs can be studied by using knockdown approaches, in combination with vascular
cytokine stimuli. Of note, cultured in vitro cells can have other characteristics than the same cell
type in vivo. In addition, HUVECs are often used for studying the endothelial permeability;
however, they may not represent the exact responses to inflammatory mediators of other
endothelial cells in the vascular tree. It will be interesting to determine whether the reaction of
GEFs/GAPs is different between specific subtypes of endothelial cells (microvascular, arterial,
venal) and organs (brain, lung, kidney, etc.). Furthermore, using also an in vivo model can give
more understanding of the signaling networks. Knockout mice can be used for investigating the
effect of GEFs and GAPs on e.g. vascular leakage. When embryonic lethal, a conditional knockout
system can be the solution. With these techniques, new GEFs/GAPs involved in angiogenesis
and/or inflammation can be explored. These GEFs/GAPs can be interesting therapeutic targets
to reduce vascular leakage in patients with e.g. atherosclerosis or to prevent cancer metastasis
via angiogenesis.
26
GEF
Rho
GTPase
Upstream
regulator
Junction
type
(AJ/TJ)
Tiam1
Rac1
TNFα, S1P,
AJ, TJ
Vav2
Rac1,
Cdc42
VEGF, EGF
AJ
P-Rex1
Rac1
TNFα
AJ
RhoGEF2
RhoA
?
AJ
Ect2
Syx
RhoA
RhoA
P115RhoGEF/
ARHGEF1
RhoA
?
VEGF,
Ang-1
TNFα,
Thrombin
GEF-H1/
ARHGEF2/ Lfc
RhoA
PDZ-RhoGEF/
ARHGEF11
LARG/
ARHGEF12
P114RhoGEF/
ARHGEF18
Tuba
RhoA
RhoA
TNFα
Contractility
actin
cytoskeleton
TJ
TJ
x
TJ
x
TJ
x
Cell
type(s)
used
for
experiments
In
vivo
support
Expressed in
endothelium
Epithelium,
HUVECs
Lung
endothelium,
HUVECs,
epithelium
Lung
endothelium
Epithelium
(epidermis)
Epithelium
Endothelium
mice
yes
yes
mice
yes
drosophila
?
mice,
zebrafish
?
yes
Lung and brain
endothelium,
HUVECs
Epithelium
(MDCK
and
renal), HUVECs
Epithelium
yes
Epithelium
yes
yes
yes
?
RhoA
Histamine,
Thrombin
?
TJ
Epithelium
yes
Cdc42
?
TJ
epithelium
?
GAP
Rho
GTPase
Upstream
regulator
Junction
type
(AJ/TJ)
GRAF2/
ARHGAP10
P190RhoGAP
RhoA
?
AJ
Epithelium
RhoA
Thrombin,
Ang-1
AJ
RICH1/
ARHGAP17
PX-RICS/
p250RhoGAP/
p200RhoGAP/
Grit
Cdc42,
Rac1
Cdc42
?
TJ
?
AJ
Epithelium,
endothelium,
fibroblasts
Epithelium
(MDCK)
HeLa cells
Contractility
actin
cytoskeleton
Cell
type(s)
used
for
experiments
In
vivo
support
Expressed in
endothelium
yes
mice
yes
yes
?
Table 1. Rho GEFs and GAPs regulating cell junctions. The GEFS and GAPs mentioned are suggested to
play a role in the regulation of cell-cell junctions. For each GEF/GAP is depicted which Rho GTPase(s) it can
activate or inactivate respectively. If known, the upstream regulators are shown. In the third and fourth
columns is described which type of junction the GEF/GAP can regulate and whether they induce contractility
of the actin cytoskeleton. Next is shown which cell types are used to study the function of the GEF/GAP and
whether the function is also studied in an animal model. In the last column is mentioned whether the
GEF/GAP is expressed in endothelial cells.
27
Abbreviations
AJ
Ang-1
DH domain
DRFs
E-cadherin
GAP
GDI
GEF
GPCR
GTPase
HUVEC
JAM
LIMK
MLC
MLCK
N-cadherin
PH domain
PLCγ
Rho GTPase
ROCK
ROS
S1P
SPHK
TER
TJ
TNFα
TRK
VE-cadherin
VEGF
VEGFR
ZO
Adherens junction
Angiopoietin-1
Dbl homology domain
Diaphanous related formins
Epithelial cadherin
GTPase activating protein
Guanine nucleotide dissociation inhibitor
Guanine nucleotide exchange factor
G-protein-coupled receptor
Guanine triphosphate phosphohydrolase
Human umbilical vein endothelial cell
Junctional adhesion molecule
LIM kinase
Myosin light chain
MLC kinase
Neuronal cadherin
Pleckstrin homology domain
Phospholipase Cγ
Ras homologous GTPase
Rho-associated kinase
Reactive oxygen species
Sphingosine 1-phosphate
Sphingosine kinase
Transendothelial/-epithelial electrical resistance
Tight junction
Tumor necrosis factor α
Tyrosine kinase receptor
Vascular endothelial cadherin
Vascular endothelial growth factor
VEGF receptor
Zona occludens
Acknowledgements
I thank Stephan Huveneers for the opportunity to write my thesis under his supervision and for
his feedback on my literature study and ideas.
28
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