MALT1 Protease Activation Triggers Acute Disruption of Endothelial Barrier Integrity via CYLD Cleavage.

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Cell Rep. Author manuscript; available in PMC 2016 October 31. Published in final edited form as: Cell Rep. 2016 September 27; 17(1): 221–232. doi:10.1016/j.celrep.2016.08.080.

MALT1 protease activation triggers acute disruption of endothelial barrier integrity via CYLD cleavage

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Linda R. Klei1,8, Dong Hu1,8, Robert Panek2,8, Danielle N. Alfano1, Rachel E. Bridwell1, Kelly M. Bailey3, Katherine I. Oravecz-Wilson2, Vincent J. Concel1, Emily M. Hess1, Matthew Van Beek2, Phillip C. Delekta2, Shufang Gu2, Simon C. Watkins4, Adrian T. Ting5, Peter J. Gough6, Kevin P. Foley6, John Bertin6, Linda M. McAllister-Lucas1,7,*, and Peter C. Lucas1,7,9,* 1Departments

of Pathology and Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15224, USA

2Department

of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109,

USA 3Department

of Pediatrics, University of Michigan Medical School, Ann Arbor, Michigan 48109,

USA 4Center

for Biologic Imaging, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA

5Immunology

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Institute & Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA 6Pattern

Recognition Receptor Discovery Performance Unit, Immuno-Inflammation Therapeutic Area, GlaxoSmithKline, Collegeville, Pennsylvania 19406, USA

7Vascular

Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA

SUMMARY Microvascular endothelial cells maintain a tight barrier to prevent passage of plasma and circulating immune cells into the extravascular tissue compartment. Yet endothelial cells respond rapidly to vasoactive substances, including thrombin, allowing transient paracellular permeability. This response is a cornerstone of acute inflammation but the mechanisms responsible are still

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*

Correspondence: [email protected] (P.C.L), [email protected] (L.M.M-L.). 8Co-first author 9Lead Contact

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. SUPPLEMENTAL INFORMATION Supplemental Information includes six figures and Supplemental Experimental Procedures. AUTHOR CONTRIBUTIONS Conceptualization, P.C.L., L.M.M-L., and R.P.; Methodology, L.R.K., D.H., D.L.A., R.P. and K.M.B.; Formal Analysis, V.J.C.; Investigation, L.R.K., R.P., D.H., D.L.A., R.E.B., K.M.B., K.I.O-W., E.M.H., M.V.B., P.C.D., S.G., and S.C.W.; Resources, S.C.W., A.T.T., P.J.G., K.P.F., and J.B.; Writing, P.C.L., L.M.M-L., L.R.K., D.H., and R.P.; Supervision, Project Administration, and Funding Acquisition, P.C.L., and L.M.M-L.

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incompletely understood. Here we demonstrate that thrombin triggers MALT1 to proteolytically cleave cylindromatosis (CYLD). Fragmentation of CYLD results in microtubule disruption and a cascade of events leading to endothelial cell retraction and an acute permeability response. This finding reveals an unexpected role for the MALT1 protease, which previously has been viewed mostly as a driver of pro-inflammatory NF-κB signaling in lymphocytes. Thus, MALT1 not only promotes immune cell activation but also acutely regulates endothelial cell biology, actions that together facilitate tissue inflammation. Pharmacologic inhibition of MALT1 may therefore have synergistic impact by targeting multiple disparate steps in the overall inflammatory response.

Graphical Abstract

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INTRODUCTION

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The CARMA1-Bcl10-MALT1 (CBM) signaling complex mediates NF-κB activation in Band T-lymphocytes in response to antigen presentation (Thome, 2004). As such, the complex plays a critical role in the adaptive immune system; deficiencies in any of the three components, in both humans and mice, lead to impaired lymphocyte activation in the face of antigenic challenge and to susceptibility to infection (Perez de Diego et al., 2015; Thome, 2004; Turvey et al., 2014). MALT1 is viewed as the effector protein of the CBM complex and operates through two distinct mechanisms (Afonina et al., 2015). First, MALT1 acts as a scaffold and recruits additional proteins, including the kinase TAK1 and the ubiquitin ligase TRAF6, which directly activate the IκB kinase (IKK) complex. IKK activation, in turn, induces the canonical NF-κB pathway. Second, MALT1 possesses a caspase-like protease activity which targets nine known cellular substrates for site-specific cleavage (Afonina et al., 2015; Juilland and Thome, 2016; McAllister-Lucas and Lucas, 2008). Several of these, including RelB, CYLD, and A20, are negative regulators of canonical NF-κB signaling, operating downstream from the IKK complex (Brummelkamp et al., 2003; Duwel et al., 2009; Hailfinger et al., 2011; Stilo et al., 2008; Sun, 2010), so that MALT1-dependent cleavage and destruction of these important regulators has the potential to undermine their Cell Rep. Author manuscript; available in PMC 2016 October 31.

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capacity to inhibit NF-κB at distal points in the signaling pathway (Afonina et al., 2015). In this way, MALT1 proteolytic activity serves as a rheostat to optimize and sustain the overall NF-κB signal that is directly induced through MALT1 scaffolding activity.

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Over the last 15 years, our understanding of the CBM complex, and the functions of MALT1, have advanced primarily through the study of B- and T-cells. However, we and others have shown that a CARMA1 homologue, CARMA3 (CARD10/Bimp1), exists in a broad array of non-immune cells and directs NF-κB activation through an analogous CARMA3-containing CBM complex (Grabiner et al., 2007; McAllister-Lucas et al., 2001; McAllister-Lucas et al., 2007; Wang et al., 2001). In vascular endothelial cells, this CBM complex mediates pro-inflammatory NF-κB activation in response to at least three G protein-coupled receptor (GPCR) agonists, Angiotensin II, CXCL8/IL-8, and thrombin (Delekta et al., 2010; Martin et al., 2009; McAllister-Lucas et al., 2010; McAllister-Lucas et al., 2007). This in turn contributes to NF-κB dependent induction of adhesion molecules, facilitating the homing of immune cells to sites of tissue inflammation (Delekta et al., 2010). Yet, a fully effective inflammatory response also requires induced endothelial permeability to allow these cells to transmigrate from the vessel lumen into the subendothelial space. Here, we show that CBM activation in endothelial cells has an additional, unanticipated consequence in that the resulting MALT1 dependent cleavage of CYLD triggers disruption of the endothelial barrier and causes collapse of microvascular structures. These findings thereby provide direct demonstration of MALT1 proteolytic activity occurring in cells outside the immune system and in response to a GPCR agonist instead of an antigenic challenge. Further, the findings emphasize that MALT1 proteolytic activity can impact cell physiology in an NF-κB independent manner; MALT1-dependent CYLD cleavage in this setting appears to interfere with the role of CYLD in preserving microtubule integrity, which is critical for maintenance of barrier function and is distinct from the effects of CYLD on NF-κB signaling. Taken together, the findings described here suggest that pharmaceutical targeting of MALT1 protease activity may have broader implications than previously expected. Specifically, MALT1 inhibition may have applications that go beyond manipulation of immune cell function and could have utility in modulating acute vascular reactivity.

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RESULTS Thrombin induces endothelial permeability in a MALT1 protease dependent, but NF-κB independent, manner

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Previous studies have suggested that while MALT1 protease activity has a major role in finetuning the level of NF-κB activation achieved after lymphocytes are stimulated with antigen, there may be other roles for the protease. For example, MALT1-dependent cleavage of Bcl10 in lymphocytes has been linked to alterations in integrin function (Rebeaud et al., 2008). To further address this question, we asked if MALT1, and its proteolytic activity, impact any of the varied NF-κB independent effects of thrombin on endothelial cell biology. We had already shown that thrombin stimulation of protease activated receptor-1 (PAR1), the principal thrombin receptor on endothelial cells, induces NF-κB activation through the CBM pathway in a manner very much analogous to the mechanism by which antigen

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receptors induce NF-κB in lymphocytes (Delekta et al., 2010). However, PAR1 activation directs multiple other phenotypic alterations in these cells. Notably, PAR1 activation produces a rapid and reversible increase in paracellular endothelial permeability (Hirano, 2007; Vandenbroucke et al., 2008). This effect is thought to account for the ability of thrombin to induce microvascular leakage and edema, as well as facilitate the transendothelial migration of inflammatory cells at sites of vascular inflammation. Thus, we wondered if the CBM complex might also participate in this aspect of the thrombin response, despite the fact that rapid and transient permeability responses are not thought to depend on NF-κB activation (Kasa et al., 2015; Mehta and Malik, 2006; Sukriti et al., 2014).


This notion was motivated by the work of several laboratories which showed that, in endothelial and other cell types, CYLD has a critical role in binding to and stabilizing microtubules (Gao et al., 2008; Gao et al., 2010; Li et al., 2014; Trompouki et al., 2009; Wickstrom et al., 2010; Yang et al., 2014; Yang and Zhou, 2016). Upon CYLD depletion, microtubules are disassembled, an event that can release microtubule-bound factors including at least two Rho guanine nucleotide-exchange factors (GEFs), Trio and RhoGEFH1, as well as LIM kinase-1 (LIMK1); once released, these factors are able to initiate a cascade of signaling events that results in actin fiber reorganization and the conversion of cortical actin into actin stress fibers (Birukova et al., 2006; Gao et al., 2010; Gorovoy et al., 2005; Krendel et al., 2002; Mikelis et al., 2015). This, coupled with disruption of both adherens and tight junctions, leads to retraction of endothelial cells from one another and paracellular permeability. Since CYLD had been described as a MALT1 cleavage target in lymphocytes (Staal et al., 2011), we wondered if thrombin-induced, MALT1-dependent cleavage of CYLD would occur in endothelial cells and have an effect similar to the genetic deletion of CYLD. If so, rapid and dynamic CYLD cleavage would represent a novel physiologic mechanism governing induced endothelial permeability.

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To test this notion, we first evaluated two distinct endothelial cell lines, the human EA.hy926 line and the mouse SVEC line, for PAR1-dependent alterations in permeability. This was accomplished using Electrical Cell-substrate Impedance Sensing (ECIS), a technique whereby changes in electrical resistance are measured in real time, across confluent monolayers of endothelial cells grown on gold-plated electrodes (Stolwijk et al., 2015) (Figure 1A). We found that both cell lines express PAR1 and respond rapidly to thrombin with a drop in electrical resistance reflecting an increase in paracellular permeability (Figure S1A–S1C). Cells respond similarly when treated with Thrombin Receptor Activator Peptide-6 (TRAP-6), a six amino acid peptide that acts as a selective ligand for PAR1 and mimics thrombin activation of this receptor (Coughlin, 2000) (Figure S1D). We focused most subsequent evaluations on SVECs, since these cells are derived from microvascular endothelium, the subpopulation of endothelium most susceptible to physiologic, agonistinduced alterations in permeability (Mehta and Malik, 2006). Using siRNA transfection, we then knocked down MALT1 in SVECs and found that this significantly impaired the ability of TRAP-6 to induce an acute permeability response (Figures 1B and 1C). In parallel, we pre-treated SVECs with z-VRPR-fmk, a cell-permeable inhibitor of the MALT1 protease (Rebeaud et al., 2008). Upon subsequent addition of TRAP-6, cells showed a similarly blunted response in the permeability assay, suggesting that


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MALT1 protease inhibition is as effective as MALT1 knockdown with regard to blocking PAR1–responsive endothelial permeability (Figures 1D and 1E). Importantly, inhibiting canonical NF-κB activation with IKK-VI, a potent inhibitor of the IKK complex, had no effect (Figures 1F and 1G). This is consistent with the fact that the permeability response occurs very rapidly, within ~10 minutes, a time frame during which PAR1-dependent NF-κB activation is still at its early stages and before alterations in NF-κB target gene expression occur. As a control, we found that IKK-VI treatment completely blocked PAR1-dependent NF-κB activation in these cells, as evidenced by the lack of pIκB induction following TRAP-6 treatment (Figure S1E). In contrast, z-VRPR-fmk did not prevent canonical signaling to IκB (Figure S1E), consistent with what has been shown in lymphocytes following antigen receptor activation (Hailfinger et al., 2011). Taken together, these results indicate that the acute permeability response induced by PAR1 activation depends significantly on MALT1 and its proteolytic activity, but not on NF-κB activation.


To test for a potentially broader role of MALT1 in regulating endothelial permeability, we treated cells with two additional agonists, histamine and lysophosphatidic acid (LPA). Both induced rapid permeability responses, as evidenced by reductions in electrical resistance in the ECIS assay, mimicking the actions of thrombin (Figure S2A–S2D). Importantly, zVRPR-fmk was similarly effective at blocking the permeability response to either agonist (Figure S2A–S2D). As a control, the permeability response induced by nocodazole, a direct microtubule disruptor, could not be prevented by z-VRPR-fmk, indicating that only permeability responses that proceed through signaling pathways linked to the CBM complex are likely to be impacted by MALT1 inhibition (Figure S2E and S2F). Indeed, like thrombin, both histamine and LPA act through Gαq/11 / Gα12/13-linked GPCRs, which at least in the case of LPA receptors, have been shown to engage the CBM complex in other cellular contexts (Klemm et al., 2007; Wang et al., 2007). Thus, multiple GPCRs likely initiate permeability signals that converge on MALT1 in endothelial cells, so that MALT1 protease activation could act as a central switch controlling the response to a range of vasoactive substances. MALT1 protease activation disrupts endothelial capillary tubes in an NF-κB independent manner

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When plated in a 3D matrigel matrix, endothelial cells spontaneously organize into tube-like structures, recapitulating capillary networks (Sigurbjornsdottir et al., 2014). The integrity of these structures depends, in part, upon maintenance of continuous contact between the endothelial cells, so that the same stimuli that cause endothelial retraction and paracellular permeability can also induce tube collapse (Bayless and Davis, 2004). Consistent with this notion, we found that tubes formed from both SVEC and EA.hy926 cells are disrupted by treatment with either thrombin or TRAP-6 (Figure S3A–S3D). Importantly, tube disruption was dramatically mitigated when cells were first depleted of MALT1 through transfection with MALT1 siRNA (Figures 1H-1K). Similar to the permeability response, we found that treatment with z-VRPR-fmk was as effective as MALT1 knockdown at preventing PAR1induced tube disruption, while IKK-VI was unable to protect against the disruption (Figures 1L-1O). Together, the above studies demonstrate that PAR1 activation induces tube collapse through a mechanism that relies on MALT1 and its protease activity, but not on NF-κB


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activation. These findings parallel those from the permeability studies, underscoring how downstream signaling events diverge at the level of MALT1 in endothelial cells, with protease activity governing multiple related aspects of acute endothelial structural integrity through a mechanism that does not involve the canonical NF-κB pathway. Thrombin receptor activation induces MALT1-dependent CYLD cleavage in endothelial cells

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CYLD is known to bind microtubules through two tandem cytoskeleton-associated protein glycine-conserved (CAP-Gly) domains (Gao et al., 2008; Wickstrom et al., 2010), located in the N-terminus of the protein (Figure 2A). However, the CAP-Gly domains alone are unable to efficiently mediate microtubule stabilization (Trompouki et al., 2009). The C-terminus of CYLD, which contains a de-ubiquitination domain (DUB), is required for this functionality. Although not fully understood, the C-terminus appears to direct a complex array of binding and/or deubiquitination activities to inhibit microtubule-disruptors including dishelveled (dvl) and end-binding protein-1 (EB1) (Li et al., 2014; Yang et al., 2014; Yang and Zhou, 2016). Regardless of the precise mechanism, it appears that the N- and C-terminal domains function optimally only when together, with the N-terminal CAP-Gly domains mediating the recruitment of CYLD to microtubules, and the C-terminal domain mediating necessary enzymatic or other activities to fully stabilize microtubules.

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Interestingly, the MALT1-specific cleavage site, as determined through studies of activated T lymphocytes, is located just C-terminal to the CAP-Gly domains, following position R324 (Staal et al., 2011) (Figure 2A). As such, we reasoned that MALT1-dependent CYLD cleavage would separate the microtubule binding and stabilization domains present within CYLD, potentially rendering it incapable of maintaining microtubule integrity. To test this idea, we first looked for evidence of PAR1-dependent CYLD cleavage in SVEC and EA.hy926 cells, and found that 10 or more minutes of TRAP-6 treatment resulted in the generation of both the 70 and 40 kD CYLD fragments that would be predicted to result from cleavage at R324 (Figures 2B, 2C, S4A, and S4B). Treatment with the natural PAR1 agonist, thrombin, had a similar effect (Figure 2D). Importantly, we found that generation of the CYLD cleavage fragments is mediated by MALT1 proteolytic activity since it could be inhibited by pre-treatment with the MALT1-specific inhibitor, z-VRPR-fmk, but not by the IKKβ inhibitor, IKK-VI (Figures 2E, Figure S4C, and Figure S4D). To ensure that PAR1dependent CYLD cleavage is not a phenomenon restricted to immortalized endothelial cell lines, we confirmed that CYLD cleavage also occurs in primary human aortic endothelial cells (Figure S4E).

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MALT1-dependent CYLD cleavage drives microtubule disruption and associated ROCK activation We next testing whether blocking PAR1-induced MALT1 protease activity would correlate with a blockade in microtubule disruption. Since stable microtubules are characterized by high acetylated α-tubulin content, disruption of microtubules can be assessed by measuring a drop in cellular acetylated α-tubulin levels (Westermann and Weber, 2003). Indeed, TRAP-6 treated SVEC cells showed a dramatic reduction in acetylated α-tubulin content,


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but importantly, this reduction was completely prevented by pretreatment with the MALT1 protease inhibitor, z-VRPR-fmk (Figure 3A). We then asked whether the specific cleavage of CYLD, as opposed to cleavage of another unknown substrate of MALT1, is linked to α-tubulin deacetylation. To this end, we generated SVEC cells that stably express either wild-type CYLD or a non-cleavable mutant of CYLD, which contains a single amino acid substitution converting the critical P1 arginine at the cleavage site to alanine (R324A) (Staal et al., 2011) (Figure S5A). Cells expressing wild-type CYLD, or those with empty vector only, responded to thrombin with the expected drop in acetylated α-tubulin levels, while those expressing non-cleavable R324A CYLD maintained their acetylated α-tubulin levels in the face of thrombin treatment (Figure 3B).


Since acetylated α-tubulin levels are an indirect measure of microtubule stability, we sought to directly visualize and quantitate microtubule dynamics in cells that express either exogenous wild-type CYLD or the non-cleavable R324A CYLD mutant. Thus, we generated derivative lines that also express GFP-tagged EB1, which associates with the growing microtubule plus ends and can be used as a dynamic marker of microtubule polymerization and disruption via time-lapse confocal imaging (Akhmanova and Steinmetz, 2015; Applegate et al., 2011; Matov et al., 2010). Cells were imaged over a 2-minute period immediately before the addition of thrombin, and then again either immediately after addition (Figures 3C-3E), or following a 5-minute delay (Figure S5B and S5C). PlusTipTracker software was used to follow EB1 microtubule comets and categorize individual microtubules into fast or slow growth categories, reflecting the combined effects of microtubule catastrophe, pausing, and polymerization (Applegate et al., 2011). Consistent with previous work (Geyer et al., 2015), in the 2 minutes following thrombin stimulation, control SVEC cells responded with a significant reduction in overall microtubule growth rate, as did cells expressing wild-type CYLD (Figure 3C-3E). In contrast, cells expressing non-cleavable R324A CYLD showed no measurable changes in microtubule dynamics (Figures 3C-3E). At a point 5 minutes following thrombin stimulation, cells expressing the R324A CYLD mutant began to show a reduction in microtubule growth, but the overall growth rate remained elevated as compared to the growth rate in control cells at this time point (Figure S5B and S5C).

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Because microtubule disruption has been linked to release of at least two Rho GEFs, Trio and RhoGEF-H1 (Birukova et al., 2006; Krendel et al., 2002; Mikelis et al., 2015; van Haren et al., 2014), which subsequently activate ROCK, we sought to test whether the noncleavable R324A CYLD mutant can also suppress ROCK kinase activity following thrombin treatment. Remarkably, while control cells, and those expressing wild-type CYLD, responded to thrombin with rapid phosphorylation of the major ROCK substrate, myosin light chain (MLC), cells expressing R324A CYLD showed no detectable MLC phosphorylation (Figure 3F). Phosphorylation of another ROCK substrate, the regulatory subunit of MLC phosphatase (MYPT1) on S507 and T696 was partially blunted in cells expressing R324A CYLD. However, MYPT1 phosphorylation is known to be catalyzed not only by ROCK but also by several other kinases (Mikelis et al., 2015). Taken together, these results suggest that MALT1-dependent CYLD cleavage drives microtubule disruption and subsequent ROCK activation. ROCK, in turn, directly phosphorylates MLC to promote


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actin-myosin stress fiber formation, while it also contributes to the phosphorylation and inhibition of MLC phosphatase, thereby preserving levels of p-MLC. CYLD cleavage is mechanistically linked to endothelial barrier disruption

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Next, we asked if the observed PAR1-responsive CYLD cleavage is mechanistically linked to alterations in endothelial cell barrier integrity. Control cells, and those expressing exogenous wild-type CYLD, responded to TRAP-6 with the expected acute permeability, but strikingly, cells expressing the R324A CYLD mutant showed a blunted permeability response (Figures 4A and 4B). We then used the same approach to ask if PAR1-responsive CYLD cleavage is mechanistically linked to an overall destabilization of endothelial contacts, and the loss of structural integrity that underlies tube collapse. As with the permeability assay, cells expressing wild-type CYLD responded to TRAP-6 with the expected tube dissolution, but those expressing non-cleavable CYLD maintained nearly complete tube integrity in the face of TRAP-6 treatment (Figures 4C and 4D). Thus, this CYLD mutant, resistant to MALT1-dependent cleavage (Staal et al., 2011), acts in a dominant negative fashion, suggesting that CYLD cleavage is required for efficient PAR1induced endothelial permeability and tube disruption.

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The results support the overall hypothesis that cleavage-dependent separation of the CYLD N-terminal microtubule-binding and C-terminal microtubule-stabilization domains renders the protein unable to perform its functions with regard to maintaining microtubules. Viewed in this light, cleavage of CYLD would mechanistically represent a loss-of-function response. However, we also considered the possibility that CYLD cleavage might represent a gain-offunction response if either the newly-formed 40 kD fragment or 70 kD fragment gains new functionality, allowing it to actively promote microtubule disassembly. To rule out this possibility, we transfected cells with expression vectors encoding either wild-type CYLD or these CYLD fragments, in order to mimic their production in the setting of PAR1 stimulation (Figure S6A). In each case, cells established and maintained tube networks as efficiently as cells transfected with control GST-expressing vector (Figure S6B and S6C). Thus, it is unlikely that the generation of CYLD fragments unmasks new functionality that actively contributes to loss of endothelial structural integrity and tube disruption. MALT1PD/PD mice exhibit resistance to thrombin receptor-induced CYLD cleavage and endothelial cell permeability

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Finally, we took a genetic approach to testing the role of MALT1 proteolytic activity in promoting endothelial destabilization by generating knock-in mice, homozygous for a protease-dead MALT1 allele (MALT1PD/PD) (Yu et al., 2015). These mice were engineered to harbor a single codon missense mutation (TGT>GCC) in exon 12, resulting in a single amino acid substitution at the critical catalytic cysteine (C472A) which is known to cripple the protein’s enzymatic activity (Coornaert et al., 2008; Rebeaud et al., 2008). Primary pulmonary endothelial cells from 4 week-old mice were isolated and cultured in vitro, and compared with cells isolated from wild-type mice. As expected, MALT1PD/PD endothelial cells maintained responsiveness to TRAP-6 with respect to canonical NF-κB activation, demonstrating that the scaffolding role of MALT1 remains intact in the face of the C472A


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mutation, and activation of the IKK complex still occurs normally (Figure 5A). In contrast, TRAP-6-induced CYLD cleavage was abolished (Figures 5A and 5B). Further, we found that the loss of protease activity was linked to a loss in PAR1-responsive microtubule destabilization; while wild-type cells responded to TRAP-6 with a timedependent decrease in acetylated α-tubulin levels, reflecting microtubule disassembly, MALT1PD/PD cells showed no significant decrease (Figure 5C and 5D). In fact, there was a slight increase in acetylated α-tubulin content. Most remarkable, when evaluated by ECIS, we found that primary endothelial cells from MALT1PD/PD mice were also significantly protected from the TRAP-6 induced acute permeability response (Figures 5E and 5F).

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To test acute endothelial permeability responses in vivo, we utilized a pulmonary vascular leak model. Specifically, we perfused mice through the jugular vein with Evans blue dye along with either TRAP-6 or PBS vehicle, as described (Kaneider et al., 2007; Patterson et al., 1992). Mice were sacrificed and transendothelial extravasation of albumin-bound Evans blue dye was quantified in excised lungs. While lungs from wild-type mice showed visible and spectrophotometrically quantifiable increases in Evans blue dye extravasation (~40% increase), lungs from MALT1PD/PD mice showed a slight decrease in extravasation relative to PBS-treated controls (~30% decrease) (Figures 5G and 5H). Thus, MALT1PD/PD mice showed protection from thrombin-receptor induced acute pulmonary edema. Taken together, the in vitro and in vivo results indicate that altering the genome by introducing only a single codon mutation in the MALT1 gene to disable its protease domain is sufficient to effect a substantial change in endothelial cell reactivity.

DISCUSSION Author Manuscript

MALT1 has a well-known role as the effector protein of the CBM complex in immune cells, and mediates critical NF-κB-driven adaptive immune responses (Thome, 2004). The direct NF-κB induction mediated by MALT1 is thought to occur primarily through its scaffolding activity, whereby it recruits IKK activating enzymes to bring them into proximity with NEMO and the other components of the IKK complex (Afonina et al., 2015). The proteolytic activity of MALT1 in immune cells has been largely viewed as a mechanism for purging negative regulators of downstream NF-κB signaling, thereby maintaining optimal, sustained NF-κB dependent transcriptional activity.

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Beyond its role in immune cells, MALT1 promotes NF-κB activation in endothelial cells in response to several GPCR ligands (Delekta et al., 2010; Martin et al., 2009; McAllisterLucas et al., 2010). This results in expression of cell surface adhesion molecules such as ICAM and VCAM, which work in concert to recruit activated immune cells to sites of tissue inflammation. Studies described here now demonstrate MALT1 proteolytic activity occurring in cells outside the immune system, namely in endothelial cells. Furthermore, this MALT1 proteolytic activity has a surprising biologic effect unrelated to NF-κB signaling; specifically, MALT1-dependent CYLD cleavage acutely alters endothelial structural integrity, thereby impacting vascular permeability (Figure 6). Data presented here are consistent with a model whereby CYLD cleavage initiates microtubule disruption and release of Rho GEFs. As suggested by Mikelis et al., the redistribution of these Rho GEFs


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may allow them to interact directly with GPCR-proximal Gαq and subsequently activate RhoA and ROCK to promote MLC phosphorylation and the assembly of actin-myosin stress fibers (Figure 6). Although our data focus mostly on the role of this pathway in thrombin receptor (PAR1) mediated endothelial responses, findings presented in Figure S2 suggest that the pathway may also be relevant to histamine receptor (H1R) and LPA receptor (LPAR) mediated permeability responses. Thus, MALT1 appears to have a dual role in the vasculature, facilitating both leukocyte recruitment via induction of NF-κB-responsive adhesion molecules, and transmural leukocyte migration via an NF-κB-independent effect on the permeability barrier. Together, these concepts illustrate that MALT1 proteolytic activity has consequences that are substantially more far-reaching than previously appreciated, impacting cell biologic processes that are unrelated to NF-κB signaling or downstream transcriptional reprogramming.


These findings also suggest that pharmaceutical targeting of the MALT1 protease may have impact on aspects of inflammation that relate not only to lymphocytes and other immune cells, but also to the cells of the microenvironment in which inflammation takes place. Specifically, MALT1 inhibition may abrogate the transmigration of activated immune cells through the endothelial barrier, dampening localized tissue inflammation. There are multiple settings in which this could have therapeutic benefit. For example, widespread pulmonary endothelial permeability, with concomitant pulmonary edema and airspace inflammation, is a major pathologic feature of sepsis-related acute lung injury (ALI) and associated acute respiratory distress syndrome (ARDS) (Matthay et al., 2012). Vasoactive substances, including thrombin, histamine, and LPA, mediate this dramatic endothelial response, yet most current therapies are generally supportive in nature, rather than based on a mechanistic understanding of disease pathogenesis (Matthay et al., 2012). We propose that in the acute setting, MALT1 protease inhibitors might have efficacy in reducing this underlying pathogenic feature of ARDS by specifically targeting a mechanism driving endothelial permeability. In the chronic setting, MALT1 inhibitors might be effective in reducing the intimal inflammation of atherosclerotic disease. Since active inflammation in the intimal/ sub-endothelial space is a critical feature that distinguishes unstable atherosclerotic plaque, susceptible to rupture, from stable plaque (Hansson et al., 2015; Weber and Noels, 2011), one could speculate that MALT1 inhibition might reduce transmigration of activated leukocytes through the arterial endothelial barrier and into this space, thereby reducing the risk of acute coronary events in the setting of advanced atherosclerosis.

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Recently, several pharmaceuticals have been identified that act as selective MALT1 protease inhibitors (Fontan et al., 2012; Nagel et al., 2012; Schlauderer et al., 2013; Xin et al., 2016). Since some of these are well-known phenothiazines, a class of pharmaceuticals that have been used for treatment of psychiatric disorders, the potential exists for their rapid redeployment to these novel therapeutic arenas. Thus, the mechanistic insights described here lay the foundation for an exciting and unexpected opportunity to explore a specific concept for drug repurposing, with application to the field of vascular biology.

EXPERIMENTAL PROCEDURES An extended version can be found in Supplemental Experimental Procedures.


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Mice

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MALT1PD/PD mice were generated with the assistance of genOway (Lyon, France) and have been recently described (Yu et al., 2015). See Supplemental Experimental Procedures for a detailed description. Cell lines and reagents EA.hy926 cells were a gift from Cora-Jean Edgell (University of North Carolina). SVEC (SVEC4–10) cells were obtained from ATCC. Primary human coronary artery endothelial cells (HcAECs) were purchased from Lonza, cultured in EGM-2MV, and used for no more than five passages. See Supplemental Experimental Procedures for details on these and all other reagents. Endothelial cell isolation

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Mouse primary pulmonary endothelial cells were isolated using a two-step selection procedure, based on sequential purification with anti-mouse CD31 and anti-mouse CD102. See Supplemental Experimental Procedures for details. Tube formation and Electric Cell-substrate Impedance Sensing (ECIS)

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Tube formation assays were performed on growth factor-reduced (GFR) Matrigel™ Matrix (BD Biosciences) and visualized using either phase contrast microscopy or fluorescence microscopy after staining with ActinRed™ 555 ReadyProbes® reagent (Life Technologies). Quantification was performed with either Image-ProPlus optical imaging software (Media Cybernetics) or Nikon Elements software. Endothelial cell permeability was measured using an ECIS® ZΘ instrument (Applied Biophysics), with a 16-well array station and 8-well chamber slides (8W10E+). See Supplemental Experimental Procedures for details on both these procedures. Live-cell Imaging of Microtubule Dynamics Imaging was carried out using a Nikon A1plus confocal galvano scanner mounted on a Nikon Eclipse Ti microscope. Images were acquired at 1–2 sec intervals for 2 min and microtubules were automatically tracked using PlusTipTracker software. See Supplemental Experimental Procedures for a detailed description. Assessment of pulmonary vascular leakage

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Mice were perfused with Evans blue dye, administered through the external jugular vein, and treated ± TRAP-6. Extravascular Evans blue dye was quantified from perfused, excised lungs using formamide extraction and spectrophotometric quantification at 620 and 740 nm. See Supplemental Experimental Procedures for a detailed description. Statistical analysis When experiments involved only two conditions, differences in means were evaluated for statistical significance using one- or two-tailed, unpaired Student’s t-test, as appropriate. For data sets involving multiple treatments and a control, data were analyzed using one-way analysis of variance (ANOVA). In the latter case, significant differences between treatments


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were assessed using Tukey’s test, while Dunnet’s test was used to compare each individual treatment to controls. Sidak’s correction was used to account for multiple testing. All statistical analyses were performed using GraphPad Prism, version 6.0 (GraphPad Software). Data are presented as means ± SEM or as percent of control, and P values are shown in either the figures or the figure legends.

Supplementary Material Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

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This work was supported by NIH/NHLBI grant R01-HL082914. Resources for microscopic imaging were provided by the University of Pittsburgh Center for Biologic Imaging (CBI); Simon Watkins, Director. The authors wish to thank all members of the Lucas/McAllister lab for their insights and support. We also thank David Antonetti (University of Michigan) for guidance and use of ECIS equipment during the early phase of this project, Yulia Komarova (University of Illinois College of Medicine, Chicago) for advice on microtubule imaging and quantification, and Cora-Jean Edgell (University of North Carolina) for the gift of EA.hy926 cells.

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Figure 1. Thrombin receptor (PAR1) induces acute endothelial permeability and tube disruption in a MALT1 protease dependent, but NF-κB independent manner

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(A) A schematic of the ECIS system is shown. Paracellular permeability is indirectly proportional to the electrical resistance measured across the endothelial monolayer (B,C) MALT1 knockdown abrogates TRAP-6 induced acute endothelial permeability. SVECs were transfected with either control or MALT1-specific siRNAs prior to measuring the permeability response to TRAP-6. (B) Representative tracings, (C) percent maximal decrease in resistance (mean ± SEM, n=3, *P

Two Antagonistic MALT1 Auto-Cleavage Mechanisms Reveal a Role for TRAF6 to Unleash MALT1 Activation.

Hyperglycemia via activation of thromboxane A2 receptor impairs the integrity and function of blood-brain barrier in microvascular endothelial cells.

Hepatocyte growth factor triggers distinct mechanisms of Asef and Tiam1 activation to induce endothelial barrier enhancement.

MALT1 protease: equilibrating immunity versus tolerance.

Lung endothelial barrier disruption in Lyl1-deficient mice.

Hyperhomocysteinemia Alters Retinal Endothelial Cells Barrier Function and Angiogenic Potential via Activation of Oxidative Stress.

cAMP controls the restoration of endothelial barrier function after thrombin-induced hyperpermeability via Rac1 activation.

Activation of AMPK attenuates lipopolysaccharide-impaired integrity and function of blood-brain barrier in human brain microvascular endothelial cells.

Resolvin D1 via prevention of ROS-mediated SHP2 inactivation protects endothelial adherens junction integrity and barrier function.

Pharmacological activation of Rap1 antagonizes the endothelial barrier disruption induced by exotoxins ExoS and ExoT of Pseudomonas aeruginosa.

Exendin-4 promotes endothelial barrier enhancement via PKA- and Epac1-dependent Rac1 activation.

Pericytes contribute to the disruption of the cerebral endothelial barrier via increasing VEGF expression: implications for stroke.

Heat Shock Protein 70 Prevents Hyperoxia-Induced Disruption of Lung Endothelial Barrier via Caspase-Dependent and AIF-Dependent Pathways.

Soluble VCAM-1 impairs human brain endothelial barrier integrity via integrin α-4-transduced outside-in signalling.

SARM1 activation triggers axon degeneration locally via NAD⁺ destruction.

Disruption of endothelial barrier function by lipolytic remnants of triglyceride-rich lipoproteins.

Infection of pericytes in vitro by Japanese encephalitis virus disrupts the integrity of the endothelial barrier.

Rac1 pathway after acute spinal cord injury.

Tumor necrosis factor-α disruption of brain endothelial cell barrier is mediated through matrix metalloproteinase-9.

Regulation of Thrombin-Induced Lung Endothelial Cell Barrier Disruption by Protein Kinase C Delta.

Nrf1 by the aspartic protease DDI-1.

Lipopolysaccharide-induced pulmonary endothelial barrier disruption and lung edema: critical role for bicarbonate stimulation of AC10.

Hantavirus-induced disruption of the endothelial barrier: neutrophils are on the payroll.

Blood-tumor barrier disruption controversies.