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Filling the gaps
The endothelium in regulating vascular leakage and leukocyte extravasation
Schimmel, L.
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2018
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Schimmel, L. (2018). Filling the gaps: The endothelium in regulating vascular leakage and
leukocyte extravasation.
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1Department of Plasma Proteins, Molecular Cell Biology Lab, Sanquin Research and Landsteiner
Labo-ratory, Academic Medical Center, University of Amsterdam, Plesmanlaan 125, Amsterdam 1066 CX, The Netherlands
Lilian Schimmel
1Summary and concluding
remarks
In this thesis, we focused on the active role of the endothelium during the process of leukocyte transendothelial migration (TEM). We showed that mul-tiple endothelial Rho GTPases have a role in maintaining vascular permea-bility and/or leukocyte TEM, with specific emphasis on signaling regulation by GEFs and GAPs. In this chapter we translate our findings into the general model of leukocyte TEM 1,2. Figure 1 shows an overview of the different
leu-kocyte TEM steps, as it is recognized nowadays. The involved molecules identified in the recent years are explained in detail in Chapter 1 and are in-dicated in grey writing. In bold, we indicate our findings, described in this the-sis, which we now incorporate in the model. We will discuss these findings in a broader context and propose research prospects and future directions.
Rolling
In response to inflammatory stimuli such as TNFα, ECs upregulate the ex-pression of E- and P-selectin, apical surface adhesion molecules that are es-sential for the first step of TEM: leukocyte rolling 3–6. In addition, ECs prepare
themselves for the next stage of TEM: leukocyte firm adhesion and crawling. Typically, small finger-like protrusions are found at the apical surface of the endothelium. Recently, it was shown that these structures are the result of TNFα-induced activation of the small GTPase Cdc42 7,8 and upregulation
of Myosin-X, which makes these structures being called filopodia 9. These
filopodia are rich in ICAM-1 and therefore function as adhesive structures 8.
Our work, presented in Chapter 5, builds on these findings and shows that the formation of ICAM-1-rich filopodia require the Cdc42-GEF FGD5 (Figure 1A). Whether FGD5 is directly activated by TNFα or if FGD5 activation requi-res adaptor proteins for recruitment towards the plasma membrane and to be able to exchange GDP for GTP on Cdc42 is not known and is currently under investigation. Interestingly, literature shows that the activation through tyro-sine phosphorylation of cortactin downstream of vascular endothelial growth factor receptor 2 (VEGFR2) depends on the presence of FGD5 10. Cortactin
is one of the adapter proteins that is involved in ICAM-1 clustering 11 and
might also be potentially involved in ICAM-1-rich filopodia formation.
Since it has been shown that activation of the TNFα-receptor 2 (TNFR2) by TNFα results not only in activation of the NFκB pathway, but also in the activation of VEGFR2, independent of its ligand VEGF 12, regulation of
both cortactin and Cdc42 by FGD5 as a downstream signaling pathway of TNFα-induced VEGFR2 activation might be a potential trigger in the forma-tion of ICAM-1-rich filopodia. This would include a novel role for endothelial FGD5, since so far, it has only been implicated in angiogenesis 13.
Once leukocytes strongly bind to the endothelium, they start crawling on the EC surface in a seemingly random fashion in order to find a proper place to cross the monolayer. However, studying in more detail the process of leukocyte TEM, it appears that certain “hot-spots” exist; predefined areas
on the endothelium that are preferred by the leukocyte to cross the endothe-lium. It has been suggested that such a spot may expose certain chemoki-nes or clusteres of specific adhesion molecules. Recently, the group of Von Andrian showed that the promiscuous chemokine receptor DARC is specifi-cally expressed on the endothelium of post-capillary vessels 14. This receptor
binds a variety of different chemokines, is non-functional in signaling and ser-ves primarily to immobilize the chemokine. Our preliminary data show that DARC localizes at cell-cell junctions throughout a HUVEC monolayer and is therefore not likely to be a candidate for apical surface hotspot recognition. However, DARC could still be a possible marker for junctional TEM hotspots and thereby direct leukocyte TEM towards a paracellular route, instead of a transcellular route. Depletion of endothelial DARC can be explored to see whether the route of neutrophil TEM switches from mainly paracellular to-wards more transcellular events.
In Chapter 7, we propose a novel model on how leukocytes find their ideal spot to cross the endothelium using inflammation-induced junctional membrane ruffles. We increased Rac1 activity by an active mutant of the Rac-GEF Tiam1 expressed in one EC, but not in the adjacent cell. This way, we induced an asymmetric junctional membrane ruffle: the junction displays a membrane ruffle from the active Rac1-positive cell but not from the other cell. Surprisingly, we found that leukocytes preferred these asymmetric junc-tions over juncjunc-tions formed by two Tiam1-positive cells or two control cells with similar Rac1 activity levels (Figure 1A). Interestingly, we could show that Rac1-induced junctional membrane ruffles are not specific for Tiam1, since asymmetric junctional membrane ruffles induced by the Rac-GEF TrioN also showed a clear preference for leukocytes to cross.
Because both Trio and Tiam1 localize at cell-cell junction regions, we hypothesize that local junctional Rac1 activity is required to induce the TEM hotspot membrane ruffles. Thus, from these findings we concluded that the morphology of the endothelium may determine preferred exit points for leukocytes, i.e. local “hot-spots”, rather than the local increase of adhesion molecules. Because both TrioN and Tiam1 increased active Rac1 levels, but TrioN did not alter adhesion molecules density in contrary to Tiam1, which lowered adhesion molecules expression levels.
Induction of Rac1-mediated junctional membrane ruffles has been shown before in ECs, but were only described in the context of facilitating EC migration during angiogenesis 15,16. Many cell types, such as fibroblasts
and smooth muscle cells, display a specific type of dorsal membrane ruffles, so-called circular dorsal ruffles (CDRs also referred to as waves or ring ruf-fles). Formation of these CDRs in smooth muscle cells is induced by the Rho-GEF Trio activating the small GTPase RhoG 17. Research from our group has
implicated both Trio and RhoG in leukocyte TEM by regulating the formation of the transmigratory cup or docking structure 18,19. These structures are
cally induced around an adherent leukocyte, presumably to prevent vascular leakage during leukocyte TEM 20–22. However, the activation of RhoG in both
processes is different: CDRs are formed upon growth factor stimulation 23,
whereas the endothelial docking structure is induced by leukocyte binding
24,25 and depends on signaling by the intracellular tail of ICAM-1 19,21, essential
for diapedesis but not for firm adhesion of leukocytes 26.
Interestingly, in vitro silencing of the RhoG-GEF SGEF resulted in
decreased leukocyte TEM 19, and in vivo depletion reduced the formation of
transmigratory cups 27. In addition, our group could show that upon ICAM-1
clustering, Rac1 was first activated followed by RhoG activation 18. Together
with the fact that SGEF specifically activates RhoG and not Rac1, we hypo-thesize that Rac1 is required for the initial induction of the membrane ruffles and RhoG is most likely required for the stabilization of the dorsal membra-ne ruffles, making RhoG essential for dorsal membramembra-ne ruffle formation. Ta-ken together, we can conclude that there are many similarities between the Rac1-driven junctional membrane ruffles, CDRs and transmigratory cups as they all clearly require GTPase activation 17,21,22.
However, the major difference is where and when formation of these membrane ruffles are induced. Junctional ruffles are induced by high Rac1 activity, prior to leukocyte adhesion, while the transmigratory cup structures also require Rac1 activity, but are induced after leukocyte binding. Therefore the transmigratory cups are excluded to predict the TEM hotspot, as they are only formed after adhesion of the leukocyte. Whether CDRs play a role in leukocyte TEM remains to be elucidated. We probably have never observed CDRs on ECs upon treatment with TNFα because they are mainly induced upon growth factor stimulation. Therefore we suggest that CDRs might be more similar to transmigratory cups, which are formed upon binding of the leukocyte, instead of marking the predicted TEM hotspot.
Although the asymmetric junctional membrane ruffles are preferred by leukocytes to cross the endothelium, it requires further research to de-termine if the junctional membrane ruffles indeed can predict the site of leu-kocyte diapedesis and serve as a transmigration hotspot. If so, composition of such transmigration hotspots can be explored by using these ruffles as visualization tool to study the molecular mechanism how these structures can coordinate leukocyte exit. Moreover, components such as concentrated chemokines and adhesion molecules on these ruffles should be explored.
One possible candidate component of the hotspot could be platelets, since recently, platelets were described as a bridging factor between ECs and leukocytes to guide leukocytes to the appropriate TEM site 28,29.
Adhe-sion of platelets, specifically in venular microvessels at the endothelial cell-cell junctions, enhanced the capture of neutrophils and, in turn, monocytes. Crosstalk of PSGL-1 on the platelets with P-selectin on the neutrophils leads to activation of the leukocyte integrins, and thereby promotes local
extrava-sation of both neutrophils and monocytes 28–30. Because platelets adhered
to specific vascular beds, they could serve as possible markers of the TEM hotspot. Further research into the ability of Rac1-driven junctional membrane ruffles to bind platelets would be an exciting next step in confirming platelets as TEM hotspot component.
Firm adhesion and crawling
Upon firm adhesion of leukocytes on the ECs, clustering of ICAM-1 and VCAM-1 results in recruitment and activation of a plethora of proteins and protein complexes 31–35. We propose the term ICAM-1 adhesome for these
protein complexes on clustered ICAM-1 (Chapter 6). Among the recruited proteins, the Rho-GEF Trio is responsible for activation of the small GTPa-ses RhoG and Rac1 to induce actin branching and formation of endotheli-al apicendotheli-al membrane protrusions that facilitate firm adhesion and diapedesis
18,19,35. In addition, recruitment to ICAM-1 of actin binding proteins, such as
Filamin A, Filamin B, cortactin and α-actinin4 crosslink ICAM-1 to the actin cytoskeleton, and induce local actin remodeling to provide a suitable en-dothelial surface for leukocytes to spread on 11. In Chapter 6 we show that
DLC-1, independent of its Rho-GAP function, stabilizes the recruited protein complex downstream from ICAM-1 clustering and thereby regulates the for-mation of the ICAM-1 adhesome (Figure 1B). Interestingly, DLC-1 expres-sion is regulated by substrate stiffness, and our results show that DLC-1 is upregulated in stiffness-related diseases such atherosclerosis and pulmona-ry arterial hypertension (Chapter 6). Reducing the expression of endothelial DLC-1 resulted in leukocytes behaving like they are adhering on ECs cultu-red on a soft substrate; spreading was impaicultu-red, and leukocytes remained round. Moreover, we could rescue this round phenotype by overexpressing a shRNA-insensitive wildtype or GAP-dead DLC-1 mutant in either DLC-1 depleted ECs as well as in ECs cultures on soft substrate. As a result, leu-kocytes started spreading and crawling again. Taken together, DLC-1 pro-motes the transition of leukocytes from rolling to spreading in a relatively stiff microenvironment and the work in Chapter 6 proposes for the first time a molecular mechanism that is responsible for translating substrate stiffening by ECs to leukocytes via the upregulation of endothelial DLC-1.
Next to the novel molecular mechanism that we found for DLC-1, our results may also be of value in the field of mechano-signalling. We propose that by manipulating DLC-1 expression, one can mimic substrate stiffness
in vitro without culturing cells on potentially toxic substrates of variable
stiff-ness. This reasoning goes along the same lines as what was found for KLF2 with mechano-sensing shear stress. Previous studies on KLF2 showed that overexpression of this transcription factor mimics the response of EC align-ment to laminar flow 36,37.
This resulted in many studies using KLF2 overexpression as a tool
Rolling Leukocyte Endothelial cell Extracellular matrix P/E-selectin Cdc42 FGD5 Rac1/RhoA TNFa ICAM1 Rac1 Tiam1 TNFa-receptor Inflammation
Firm adhesion and crawling
Trio RhoGRac1 Actin binding proteins DLC1 Branched Actin ICAM1-adhesome ICAM1 clustering Actin polymerization Cup formation
Stiff extracellular matrix
Figure 1 (A)
Upon inflammatory stimuli like TNFα, ECs transmit the intracellular signal via the GEF FGD5 that activates Cdc42, re-sulting in the formation of ICAM-1-rich filopodia on the apical surface, that allow firm adhesion of leukocytes (Chapter 5). The stiffness and inflammatory sensitive GEF Tiam1 activates Rac1 in order to induce formation of dorsal endothelial junctional membrane ruffles that guide rolling and crawling leukocytes to the favored site of diapedesis and thereby marks the transmigration hotspot (Chap-ter 7).
Figure 1 (B)
Interaction of the leukocyte integrins with endothelial ICAM-1 and VCAM-1 results in clustering of the adhesion mo-lecules and recruitment of actin binding proteins to the intracellular tail of ICAM-1 or VCAM-ICAM-1. The ICAM-ICAM-1 achesiome that consists of actin binding proteins and is crosslinked to the actin cytoske-leton is stabilized by DLC1 and allows leukocyte spreading. DLC1 is upregu-lated upon high stiffness substrates and thereby promotes leukocyte TEM in stiff environments by stimulating the transiti-on from rolling to firm adhesitransiti-on of leu-kocytes (Chapter 6).
to study EC alignment or using KLF2 expression levels as a marker for EC response to laminar shear stress 38–40. Based on our work, we suggest that
a similar application can be used for mechano-transduction studies in EC biology, and potentially also for other cell types.
Because of the effects of endothelial DLC-1 depletion on reduced leukocyte adhesion, it is reasonable to explore the possibilities of DLC-1-tar-geting drugs. In fact, tarDLC-1-tar-geting of DLC-1 for therapeutic purposes is already being explored. A TAT-RasGAP317-326 peptide that corresponds to amino acids 317-326 of p120RasGAP and is coupled to a cell permeable TAT-de-rived peptide, shows promising results as an inhibitor of cell migration and invasion of cancer cells 41. Interestingly, this peptide binds DLC-1 and inhibits
Diapedesis ICAM1 tension
LARG/Ect2
RhoA RhoA specific. RhoB and RhoC are not involved.
Actomyosin contraction Bundled Actin Closure Rac1 Tuba Cdc42 Actin polymerization Actin branching Figure 1 (C)
Pulling forces on ICAM-1 exerted by the moving leukocytes induce activation of RhoA via the GEFs LARG and Ect2. Myosin contractility downstream of RhoA signaling ensures the present actin ring that surrounds breaching leukocytes to seal off the transmigration pore du-ring passage of leukocytes (Chapter 3). Induction of the contractile F-actin ring is specific for RhoA activity, as endothe-lial RhoB and RhoC are dispensible for leukocyte diapedesis and for maintaing vascular integrity during diapedesis (Chapter 4).
Figure 1 (D)
Depletion of the Cdc42 specific GEF Tuba results in leukocyte induced vas-cular leakage. It is suggested that Cdc42 activity during the last stage of diape-desis is involved in inducing membra-ne protrusions that ensure pore closure next to Rac1 driven ventral lamellipodia (Chapter 5).
its GAP activity but stimulates a GAP-independent function of DLC-1 in order to inhibit cancer cell migration. It is suggested that the TAT-RasGAP317-326 peptide promotes DLC-1-PLCδ1 interaction, enhancing PIP2 hydrolysis and consequently preventing actin polymerization 42. Follow-up studies revealed
that the peptide not only blocked cell migration and invasion, but is also toxic since it resulted in cell death, independent of the caspase-, apoptosis and necroptosis- dependent pathways 43. Caution and careful testing both in vitro
and in vivo models for the possible application of TAT-RasGAP317-326 pep-tide is necessary. However, a comparable cell-permeable peppep-tide that would inhibit the GAP-independent function of endothelial DLC-1 in recruiting actin adaptor proteins upon ICAM-1 clustering, could possibly be used for explo-ring the ability of inhibiting leukocyte TEM. If such a peptide is indeed able to
inhibit DLC-1 mediated stabilization of the ICAM-1 adhesome and could the-reby decrease leukocyte TEM, clinical application could in the future possibly be explored. For instance treatment of stiffness-related vascular diseases that includes excessive leukocyte extravasation such as atherosclerosis and pulmonary arterial hypertension.
Diapedesis
Molecular evidence for the uncoupling between leukocyte TEM and vascu-lar leakage was shown by phosphorylation of different tyrosine residues of VE-cadherin for both processes by the group of Vestweber 44. They showed
that leukocytes induced the dephosphorylation of Y731 through SHP2, whe-reas permeability factors VEGF and histamine induced the phosphorylation of Y685.
However, the exact mechanism how the endothelial monolayer main-tains its vascular barrier function when a leukocyte penetrates was unknown. Pulling forces on ICAM-1 induced the recruitment of the GEF LARG 45 and
together with the work in Chapter 3, we propose that ICAM-1 clustering by migrating PMNs induces the recruitment of the Rho-GEFs LARG and Ect2, which subsequently activate the small GTPase RhoA. Myosin-II contractility downstream of activated RhoA ensures the formation of an endothelial F-ac-tin-rich ring that surrounds breaching leukocytes to seal the transmigration pore and limit leakage of plasma proteins while the PMN cross the EC layer (Figure 1C) 20.
Our work suggests that clustering of ICAM-1 initiates the above-men-tioned signaling pathways. However, our results also showed that ICAM-1 is most likely not the only initiator for activating RhoA and the induction of the local contractile F-actin ring. As described in Chapter 2, other junctional pro-teins might be candidate Rho-activators as well. One of those is Junctional Adhesion Molecule A (JAM-A) which makes hemophilic interactions between ECs and leukocytes 46. JAM-A depletion in ECs decreases leukocyte
dia-pedesis 47 and upon induction of tension on the receptor, JAM-A activates
RhoA 48. Next to JAM-A, junctional CD99 might be a good candidate driving
RhoA activation. Because CD99 signaling through soluble adenylyl cyclase and PKA regulates diapedesis upon homotypic interactions with CD99 on monocytes, neutrophils and T-cells 49–51. We are currently investigating both
options.
Additionally, one should not neglect the many other (junctional) re-ceptors present on ECs. Due to the strong mechanical forces that leukocytes exert in order to breach the EC layer 52–54, receptors that respond to
mecha-nical cues are likely instrumental in TEM. Endothelial cells express a varie-ty of mechanosensors, which can be divided into three categories: apical, junctional, and basal. The apical mechanosensors mainly contribute to shear stress sensing and include ion channels, G-protein-coupled receptors, cilia,
the glycocalyx and caveolae 55. For leukocyte TEM, an increase of
intracel-lular endothelial calcium is essential to allow leukocytes to cross 56. One
particular Ca2+ channel, transient receptor potential canonical 6 (TRPC6), localizes with PECAM-1 around transmigrating leukocytes and is essential for mediating the required Ca2+ influx in ECs to allow leukocyte TEM. This occurs following adhesion to PECAM-1, through mobilization of the lateral recycling compartment 57.
The junctional mechanosensing complex, consists of PECAM-1, VE-cadherin and VEGFR2 and VEGFR3 55,58–60. This complex is of interest
since breaching leukocytes exert physical forces on EC junctions prior to diapedesis. However, our data showed that silencing of EC PECAM-1 or VE-cadherin had no effect on leukocyte-induced vascular leakage 20,
indica-ting that these components of the junctional mechanosensing complex are not involved in limiting vascular leakage during leukocyte TEM, which leaves the other junctional mechanosensors for future research. The mechanosen-sors at the basal side of ECs consist of integrins, which interact with the underlying extracellular matrix to measure substrate stiffness, and integrate this into a cellular response 55. A link between endothelial basal integrins and
leukocyte TEM remains unknown, but a role for the β1-subunit in maintai-ning basal vascular permeability by regulating VE-cadherin internalization has recently been described 61. This indicates that also physical forces such
as pushing, induced by membrane protrusion, or even disruption of EC inte-grins at the basal side, provoked by breaching leukocytes could activate in-tegrin signaling and thereby contribute to preservation of the vascular barrier function during leukocyte TEM. In conclusion, future research should reveal the upstream signaling receptors or mechanosensors that regulate formation and constriction of the F-actin-rich rings and limitation of leukocyte-induced vascular leakage.
Where Chapter 3 showed specific and local activation of RhoA during mid and late diapedesis, it is very likely that the other TEM steps, i.e. the ope-ning and closure of the pore after leukocyte passage, require activation of two GTPases that are highly similar to RhoA; RhoB and RhoC. The results in Chapter 4 showed that, in contrast to RhoA, no additional function for RhoB or RhoC was found in limiting vascular leakage during leukocyte TEM or in regulating leukocyte TEM itself (Figure 1C). It is remarkable that such clo-sely related GTPases do not show any redundancy, as this has been shown before in ECs during barrier restoration after acute cell contraction 62–64.
Ho-wever, it needs to be stated that it is difficult to completely exclude any com-pensation by RhoA after RhoB or RhoC depletion, due to the indispensable function of RhoA in maintaining the endothelial barrier during leukocyte TEM
20. Knockdown of all three GTPases, RhoA, RhoB and RhoC in HUVECs did
not impair T-cell transmigration efficiency compared to single knockdowns 62.
But this does not directly exclude a difference in maintaining
lar barrier function upon depletion of the three GTPases compared to sin-gle knockdowns because leukocyte TEM and vascular permeability are two uncoupled events. A recent study to the control of endothelial barrier function by RhoA, RhoB and RhoC used single, double and triple knockdowns to show differential control of the three homologous RhoGTPases 65. RhoB, in
addition to RhoA, is important loss of basal EC barrier function upon throm-bin. This is in contrast to RhoC, which appeard to be required for recovery after thrombin in combination with Rac1 and Cdc42 65. Future studies with
tri-ple knockdowns will reveal if similar regulation mechanisms apply for RhoA, RhoB and RhoC in leukocyte-induced vascular leakage as in basal barrier recovery upon thromin.
Gap closure
After crossing of leukocytes through the EC layer, it is essential to rapidly close the induced gap or pore. Using mechanical-induced micro-wounds to mimic leukocyte-induced EC gap formation, the group of Carman showed that Rac1-driven ventral lamellipodia rapidly restored the endothelial barrier layer 66. In addition, local activation of Rac1 not only induces ventral
lamel-lipodia to close the endothelial gaps, it is also involved in regulating restorati-on of VE-cadherin-based cell-cell junctirestorati-ons through the Rac1-regulated actin nucleator ARP2/3 67–69.
Next to Rac1, our work presented in Chapter 5 suggests a role for Cdc42 in regulating leukocyte-induced vascular leakage. Depletion of the Cdc42-specific GEF Tuba results in vascular leakage upon leukocyte TEM, making this an interesting target for future studies on the control of vascular leakage during leukocyte extravasation (Figure 1D. The exact mechanism and localization of endothelial Tuba in relation to the transmigration pore is currently being investigated by our group.
Since Tuba lacks the classical plekstrin-homology (PH) domain, but instead contains a Bin/amphiphysin/Rys (BAR) domain for membrane loca-lization, it is only recruited towards sites of curved membranes 70,71. Recent
literature described recruitment of another BAR domain containing protein, Pacsin2, to VE-cadherin upon induction of membrane curvature due to pul-ling forces between ECs. Based on this, we speculate that Tuba is recruited towards the leukocyte docking structure and/or transmigration pore, structu-res that show curved membranes.
In addition, previous research using epithelial cells showed a role for Tuba in regulating Cdc42 specifically at the cell junctions to reseal the cell-cell junctions 72. Future research should reveal the existence of such
me-chanism in ECs, where Tuba regulates resealing of the pore after leukocyte diapedesis by reformation of Cdc42-induced cell-cell junctions.
Regulating the regulators
The work described in this thesis focusses primarily on the spatiotempo-ral regulation of small Rho-GTPases by their regulators, the GEFs and the GAPs. However, regulation of the involved GEFs and GAPs remains a rela-tively unstudied field. It is assumed that activation of Rho-GTPases is mainly induced by GEF-mediated nucleotide exchange instead of by inhibition of the corresponding GAP. As a result, more studies focused on GEF-mediated activition rather than GAP-mediated inhibition of small GTPases.
Nevertheless, each member of the GEF family contains its specific regulatory mechanism, based on 4 main principles: 1. Intramolecular inhibition, 2. Pro-tein-protein interaction, 3. Re-localization and 4. Deactivation/degradation 73.
1. Intramolecular inhibition
Many GEFs are regulated via an intramolecular inhibitory sequence that blocks activation. Removal of the inhibitory N-terminal domain results in con-stitutively active mutants which has been shown for several GEFs of which Vav is the most well described, and Tiam1 we used for our work in Chapter 7 74–76. Activation of the full-length GEF is achieved through release of the
auto-inhibitory interaction either through phosphorylation or interaction with other proteins or lipids 73,77,78.
2. Protein-protein interaction
Where protein interaction could result in release of the auto-inhibitory do-main, there are also interactions that result in the activation of GEFs that do not contain an auto-inhibitory domain. Binding to activated subunits of G protein-coupled receptors result in the activation of PDZ-GEF and LARG
79,80. Activation of LARG by G protein-couples receptors might be of specific
interest regarding our work in Chapter 3 about RhoA-driven F-actin ring that surrounds transmigrating leukocytes 20. Since we are still in search of the
initial receptor, besides ICAM-1, that induces LARG and Ect2 activation, G protein-coupled receptors might be potential candidates since they can also act as mechanosensors.
Next to interaction with other proteins, dimerization of Dbl enhances its activity and potency of activating its downstream targets Cdc42 and Rho
in vivo 73.
3. Relocalization
The main mechanism of regulating GEF activity is by localization. For many GEFs, including Tiam1, membrane localization via its PH domain is asso-ciated with their specific activation 81. Relocalization of GEFs towards the
plasma membrane can occur upon responses to cellular activation of surface receptors that induce calcium influx, but also by direct recruitment towards activated cell-surface receptors. Interestingly, a rise in intracellular calcium in
ECs is one of the first signals induced upon leukocyte binding 56. For a
sub-set of GEFs, there is a specific regulation of their activity by nuclear retenti-on, which sequesters the GEFs away from the cytoplasmic substrates. For example, by phosphorylation or due to nuclear envelope breakdown during cell cycle progression, the RhoA-GEF Ect2, which is nuclear in resting cells, is released into the cytoplasm where it activates Rho at the cleavage furrow
82.
4. Deactivation/degradation
Active GEFs can be inactivated, although very little is known about the rele-vant mechanisms. Binding to other proteins can inhibit the GEF activity, e.g. binding of nm23H1 to the N-terminus of Tiam1 inhibits its GEF activity 83,
alt-hough the exact mechanism remains unclear. For the Rac1-GEF Vav it has been shown that interaction with suppressor of cytokine signaling 1 (SOCS1) results in poly-ubiquitination and subsequently degradation 84.
Ubiquitinati-on of junctiUbiquitinati-onal Tiam1 Ubiquitinati-on lysine 595 in lung cancer cells by the E3 ligase HUWE1 results in proteasomal degradation and consequently disassembly of cadherin-based cell junctions, to allow cancer cell migration and invasion
85,86. Besides ubiquitination of junctional Tiam1 by HUWE1, also SCFβ-TRCP
is able to target Tiam1 for degradation via ubiquitination to terminate mTOR-S6K signaling after stimulation with mitogens 87,88. Because we observed
de-creased levels of Tiam1 when ECs are cultured on soft substrates (Chapter 7), elucidating substrate stiffness-induced ubiquitin targeted degradation of Tiam1, and following consequences for EC junction stability could be of inte-rest for future research.
Concluding remarks
The work presented in this thesis shows the involvement of multiple GEFs in regulating different stages of leukocyte TEM and/or vascular permeability. Our work therefore significantly contributes to understanding the different steps of the leukocyte transendothelial migration cascade from the endot-helial cell point of view. However, future studies into the upstream activation and regulation of the involved GEFs is essential to completely understand the exact signaling pathways and mechanisms. Once the signaling pathways are elucidated, we can use this knowledge to interfere with these processes to develop potential therapies to target vascular diseases in which leukocyte extravasation is the underlying cause.
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