Arg mediates LPS-induced disruption of the pulmonary endothelial barrier.

Acute Respiratory Distress Syndrome (ARDS) is a devastating disease process that involves dysregulated inflammation and decreased alveolar-capillary barrier function. Despite increased understanding of the pathophysiology, no effective targeted therapies exist to treat ARDS. Recent preclinical studies suggest that the multi-tyrosine kinase inhibitor, imatinib, which targets the Abl kinases c-Abl and Arg, has the potential to restore endothelial dysfunction caused by inflammatory agonists. Prior work demonstrates that imatinib attenuates LPS (lipopolysaccharide)-induced vascular leak and inflammation; however, the mechanisms underlying these effects remain incompletely understood. In the current study, we demonstrate that imatinib inhibits LPS-induced increase in the phosphorylation of CrkL, a specific substrate of Abl kinases, in human pulmonary endothelial cells. Specific silencing of Arg, and not c-Abl, attenuated LPS-induced pulmonary vascular permeability as measured by electrical cellular impedance sensing (ECIS) and gap formation assays. In addition, direct activation of Abl family kinases with the small molecule activator DPH resulted in endothelial barrier disruption that was attenuated by Arg siRNA. In complementary studies to characterize the mechanisms by which Arg mediates endothelial barrier function, Arg silencing was found to inhibit LPS-induced disruption of adherens junctions and phosphorylation of myosin light chains (MLC). Overall, these results characterize the mechanisms by which imatinib protects against LPS-induced endothelial barrier disruption and suggest that Arg inhibition may represent a novel strategy to enhance endothelial barrier function.

Activation of Notch signaling by soluble Dll4 decreases vascular permeability via a cAMP/PKA-dependent pathway.

The Notch ligand delta-like ligand 4 (Dll4), upregulated by VEGF, is a key regulator of vessel morphogenesis and function, controlling tip and stalk cell selection during sprouting angiogenesis. Inhibition of Dll4 results in hypersprouting, nonfunctional, poorly perfused vessels, suggesting a role for Dll4 in the formation of mature, reactive, functional vessels, with low permeability and able to restrict fluid and solute exchange. We tested the hypothesis that Dll4 controls transvascular fluid exchange. A recombinant protein expressing only the extracellular portion of Dll4 [soluble Dll4 (sDll4)] induced Notch signaling in endothelial cells (ECs), resulting in increased expression of vascular-endothelial cadherin, but not the tight junctional protein zonula occludens 1, at intercellular junctions. sDll4 decreased the permeability of FITC-labeled albumin across EC monolayers, and this effect was abrogated by coculture with the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. One of the known molecular effectors responsible for strengthening EC-EC contacts is PKA, so we tested the effect of modulation of PKA on the sDll4-mediated reduction of permeability. Inhibition of PKA reversed the sDll4-mediated reduction in permeability and reduced expression of the Notch target gene Hey1. Knockdown of PKA reduced sDLL4-mediated vascular-endothelial cadherin junctional expression. sDll4 also caused a significant decrease in the hydraulic conductivity of rat mesenteric microvessels in vivo. This reduction was abolished upon coperfusion with the PKA inhibitor H89 dihydrochloride. These results indicate that Dll4 signaling through Notch activation acts through a cAMP/PKA pathway upon intercellular adherens junctions, but not tight junctions, to regulate endothelial barrier function. NEW & NOTEWORTHY Notch signaling reduces vascular permeability through stimulation of cAMP-dependent protein kinase A.

A non-canonical Notch complex regulates adherens junctions and vascular barrier function.

The vascular barrier that separates blood from tissues is actively regulated by the endothelium and is essential for transport, inflammation, and haemostasis. Haemodynamic shear stress plays a critical role in maintaining endothelial barrier function, but how this occurs remains unknown. Here we use an engineered organotypic model of perfused microvessels to show that activation of the transmembrane receptor NOTCH1 directly regulates vascular barrier function through a non-canonical, transcription-independent signalling mechanism that drives assembly of adherens junctions, and confirm these findings in mouse models. Shear stress triggers DLL4-dependent proteolytic activation of NOTCH1 to expose the transmembrane domain of NOTCH1. This domain mediates establishment of the endothelial barrier; expression of the transmembrane domain of NOTCH1 is sufficient to rescue defects in barrier function induced by knockout of NOTCH1. The transmembrane domain restores barrier function by catalysing the formation of a receptor complex in the plasma membrane consisting of vascular endothelial cadherin, the transmembrane protein tyrosine phosphatase LAR, and the RAC1 guanidine-exchange factor TRIO. This complex activates RAC1 to drive assembly of adherens junctions and establish barrier function. Canonical transcriptional signalling via Notch is highly conserved in metazoans and is required for many processes in vascular development, including arterial-venous differentiation, angiogenesis and remodelling. We establish the existence of a non-canonical cortical NOTCH1 signalling pathway that regulates vascular barrier function, and thus provide a mechanism by which a single receptor might link transcriptional programs with adhesive and cytoskeletal remodelling.

Cdc42 regulates junctional actin but not cell polarization in the Caenorhabditis elegans epidermis.

During morphogenesis, adherens junctions (AJs) remodel to allow changes in cell shape and position while preserving adhesion. Here, we examine the function of Rho guanosine triphosphatase CDC-42 in AJ formation and regulation during Caenorhabditis elegans embryo elongation, a process driven by asymmetric epidermal cell shape changes. cdc-42 mutant embryos arrest during elongation with epidermal ruptures. Unexpectedly, we find using time-lapse fluorescence imaging that cdc-42 is not required for epidermal cell polarization or junction assembly, but rather is needed for proper junctional actin regulation during elongation. We show that the RhoGAP PAC-1/ARHGAP21 inhibits CDC-42 activity at AJs, and loss of PAC-1 or the interacting linker protein PICC-1/CCDC85A-C blocks elongation in embryos with compromised AJ function. pac-1 embryos exhibit dynamic accumulations of junctional F-actin and an increase in AJ protein levels. Our findings identify a previously unrecognized molecular mechanism for inhibiting junctional CDC-42 to control actin organization and AJ protein levels during epithelial morphogenesis.

ROCK1 but not ROCK2 contributes to RhoA signaling and NMIIA-mediated contractility at the epithelial zonula adherens.

Rho kinases (ROCK1 and ROCK2) function downstream of the small GTPase RhoA to drive actomyosin cytoskeletal remodeling. It has often been believed that ROCK1 and ROCK2 may be functionally redundant, as they share a highly conserved kinase domain. However, in this study, we report differential functional effects for these ROCKs at the epithelial zonula adherens (ZA). Using specific siRNA, we found that ROCK1 depletion disrupted cadherin organization at the ZA, accompanied by loss of F-actin and NMIIA, whereas ROCK2 knockdown had no significant effect. Further, ROCK1, but not ROCK2, was necessary to stabilize GTP-RhoA at the ZA, thereby sustaining junctional tension and inhibiting intraepithelial cell movement. We also found that nonmuscle myosin IIA is a major determinant of ROCK1 cortical stability. Thus, despite sharing the catalytic domain with ROCK2, ROCK1 appears to be the dominant kinase essential for junctional integrity and contractile tension at epithelial ZA.

Rac1 functions as a reversible tension modulator to stabilize VE-cadherin trans-interaction.

The role of the RhoGTPase Rac1 in stabilizing mature endothelial adherens junctions (AJs) is not well understood. In this paper, using a photoactivatable probe to control Rac1 activity at AJs, we addressed the relationship between Rac1 and the dynamics of vascular endothelial cadherin (VE-cadherin). We demonstrated that Rac1 activation reduced the rate of VE-cadherin dissociation, leading to increased density of VE-cadherin at AJs. This response was coupled to a reduction in actomyosin-dependent tension across VE-cadherin adhesion sites. We observed that inhibiting myosin II directly or through photo-release of the caged Rho kinase inhibitor also reduced the rate of VE-cadherin dissociation. Thus, Rac1 functions by stabilizing VE-cadherin trans-dimers in mature AJs by counteracting the actomyosin tension. The results suggest a new model of VE-cadherin adhesive interaction mediated by Rac1-induced reduction of mechanical tension at AJs, resulting in the stabilization of VE-cadherin adhesions.

Shockingly: the loss of Lyn leads to leakiness.

In this issue of Blood, Han et al demonstrate that endotoxin-induced mortality in a murine model of acute lung injury (ALI) was associated with increased vascular permeability attributable to loss of the Src family kinase (SFK) Lyn.

A critical role for Lyn kinase in strengthening endothelial integrity and barrier function.

The Src family kinases (SFKs) c-Src and Yes mediate vascular leakage in response to various stimuli including lipopolysaccharide (LPS) and vascular endothelial growth factor (VEGF). Here, we define an opposing function of another SFK, Lyn, which in contrast to other SFKs, strengthens endothelial junctions and thereby restrains the increase in vascular permeability. Mice lacking Lyn displayed increased mortality in LPS-induced endotoxemia and increased vascular permeability in response to LPS or VEGF challenge compared with wild-type littermates. Lyn knockout mice repopulated with wild-type bone marrow-derived cells have higher vascular permeability than wild-type mice, suggesting a role of endothelial Lyn in the maintenance of the vascular barrier. Small interfering RNA-mediated down-regulation of Lyn disrupted endothelial barrier integrity, whereas expression of a constitutively active mutant of Lyn enhanced the barrier. However, down-regulation of Lyn did not affect LPS-induced endothelial permeability. We demonstrate that Lyn association with focal adhesion kinase (FAK) and phosphorylation of FAK at tyrosine residues 576/577 and 925 were required for Lyn-dependent stabilization of endothelial adherens junctions. Thus, in contrast to c-Src and Yes, which increase vascular permeability in response to stimuli, Lyn stabilizes endothelial junctions through phosphorylation of FAK. Therefore, therapeutics activating Lyn kinase may strengthen the endothelial barrier junction and hence have anti-inflammatory potential.

Role of Janus kinase 3 in mucosal differentiation and predisposition to colitis.

Janus kinase 3 (Jak3) is a nonreceptor tyrosine kinase expressed in both hematopoietic and nonhematopoietic cells. Previously, we characterized the functions of Jak3 in cytoskeletal remodeling, epithelial wound healing, and mucosal homeostasis. However, the role of Jak3 in mucosal differentiation and inflammatory bowel disease was not known. In this report, we characterize the role of Jak3 in mucosal differentiation, basal colonic inflammation, and predisposition toward colitis. Using the Jak3 knock-out (KO) mouse model, we show that Jak3 is expressed in colonic mucosa of mice, and the loss of mucosal expression of Jak3 resulted in reduced expression of differentiation markers for the cells of both enterocytic and secretory lineages. Jak3 KO mice showed reduced expression of colonic villin, carbonic anhydrase, secretory mucin muc2, and increased basal colonic inflammation reflected by increased levels of pro-inflammatory cytokines IL-6 and IL-17A in colon along with increased colonic myeloperoxidase activity. The inflammations in KO mice were associated with shortening of colon length, reduced cecum length, decreased crypt heights, and increased severity toward dextran sulfate sodium-induced colitis. In differentiated human colonic epithelial cells, Jak3 redistributed to basolateral surfaces and interacted with adherens junction (AJ) protein ß-catenin. Jak3 expression in these cells was essential for AJ localization of ß-catenin and maintenance of epithelial barrier functions. Collectively, these results demonstrate the essential role of Jak3 in the colon where it facilitated mucosal differentiation by promoting the expression of differentiation markers and enhanced colonic barrier functions through AJ localization of ß-catenin.

Transcriptional corepressors HIPK1 and HIPK2 control angiogenesis via TGF-ß-TAK1-dependent mechanism.

Several critical events dictate the successful establishment of nascent vasculature in yolk sac and in the developing embryos. These include aggregation of angioblasts to form the primitive vascular plexus, followed by the proliferation, differentiation, migration, and coalescence of endothelial cells. Although transforming growth factor-ß (TGF-ß) is known to regulate various aspects of vascular development, the signaling mechanism of TGF-ß remains unclear. Here we show that homeodomain interacting protein kinases, HIPK1 and HIPK2, are transcriptional corepressors that regulate TGF-ß-dependent angiogenesis during embryonic development. Loss of HIPK1 and HIPK2 leads to marked up-regulations of several potent angiogenic genes, including Mmp10 and Vegf, which result in excessive endothelial proliferation and poor adherens junction formation. This robust phenotype can be recapitulated by siRNA knockdown of Hipk1 and Hipk2 in human umbilical vein endothelial cells, as well as in endothelial cell-specific TGF-ß type II receptor (TßRII) conditional mutants. The effects of HIPK proteins are mediated through its interaction with MEF2C, and this interaction can be further enhanced by TGF-ß in a TAK1-dependent manner. Remarkably, TGF-ß-TAK1 signaling activates HIPK2 by phosphorylating a highly conserved tyrosine residue Y-361 within the kinase domain. Point mutation in this tyrosine completely eliminates the effect of HIPK2 as a transcriptional corepressor in luciferase assays. Our results reveal a previously unrecognized role of HIPK proteins in connecting TGF-ß signaling pathway with the transcriptional programs critical for angiogenesis in early embryonic development.

Prostaglandin D2-DP signaling promotes endothelial barrier function via the cAMP/PKA/Tiam1/Rac1 pathway.

OBJECTIVE: Prostaglandin D(2) (PGD(2)) is one of the prostanoids produced during inflammation. Although PGD(2) is known to decrease endothelial permeability through D prostanoid (DP) receptor stimulation, the detailed mechanism is unknown. METHODS AND RESULTS: Treatment with PGD(2) (0.1-3 µmol/L) or the DP receptor agonist, BW245C (0.1-3 µmol/L), dose-dependently increased transendothelial electrical resistance and decreased the FITC-dextran permeability of human umbilical vein endothelial cells. Both indicated decreased endothelial permeability. These phenomena were accompanied by Tiam1/Rac1-dependent cytoskeletal rearrangement. BW245C (0.3 µmol/L) increased the intracellular cAMP level and subsequent protein kinase A (PKA) activity. Pretreatment with PKA inhibitory peptide, but not gene depletion of exchange protein directly activated by cAMP 1 (Epac1), attenuated BW245C-induced Rac1 activation and transendothelial electric resistance increase. In vivo, application of 2.5% croton oil or histamine (100 µg) caused vascular leakage indexed by dye extravasation. Pretreatment with BW245C (1 mg/kg) attenuated the dye extravasation. Gene deficiency of DP abolished, or inhibition of PKA significantly reduced, the DP-mediated barrier enhancement. CONCLUSIONS: PGD(2)-DP signaling reduces vascular permeability both in vivo and in vitro. This phenomenon is mediated by cAMP/PKA/Tiam1-dependent Epac1-independent Rac1 activation and subsequent enhancement of adherens junction in endothelial cell.

Ve-ptp modulates vascular integrity by promoting adherens junction maturation.

BACKGROUND: Endothelial cell junctions control blood vessel permeability. Altered permeability can be associated with vascular fragility that leads to vessel weakness and haemorrhage formation. In vivo studies on the function of genes involved in the maintenance of vascular integrity are essential to better understand the molecular basis of diseases linked to permeability defects. Ve-ptp (Vascular Endothelial-Protein Tyrosine Phosphatase) is a transmembrane protein present at endothelial adherens junctions (AJs). METHODOLOGY/PRINCIPAL FINDINGS: We investigated the role of Ve-ptp in AJ maturation/stability and in the modulation of endothelial permeability using zebrafish (Danio rerio). Whole-mount in situ hybridizations revealed zve-ptp expression exclusively in the developing vascular system. Generation of altered zve-ptp transcripts, induced separately by two different splicing morpholinos, resulted in permeability defects closely linked to vascular wall fragility. The ultrastructural analysis revealed a statistically significant reduction of junction complexes and the presence of immature AJs in zve-ptp morphants but not in control embryos. CONCLUSIONS/SIGNIFICANCE: Here we show the first in vivo evidence of a potentially critical role played by Ve-ptp in AJ maturation, an important event for permeability modulation and for the development of a functional vascular system.

The tyrosine phosphatase SHP2 regulates recovery of endothelial adherens junctions through control of ß-catenin phosphorylation.

Impaired endothelial barrier function results in a persistent increase in endothelial permeability and vascular leakage. Repair of a dysfunctional endothelial barrier requires controlled restoration of adherens junctions, comprising vascular endothelial (VE)-cadherin and associated ß-, γ-, α-, and p120-catenins. Little is known about the mechanisms by which recovery of VE-cadherin-mediated cell-cell junctions is regulated. Using the inflammatory mediator thrombin, we demonstrate an important role for the Src homology 2-domain containing tyrosine phosphatase (SHP2) in mediating recovery of the VE-cadherin-controlled endothelial barrier. Using SHP2 substrate-trapping mutants and an in vitro phosphatase activity assay, we validate ß-catenin as a bona fide SHP2 substrate. SHP2 silencing and SHP2 inhibition both result in delayed recovery of endothelial barrier function after thrombin stimulation. Moreover, on thrombin challenge, we find prolonged elevation in tyrosine phosphorylation levels of VE-cadherin-associated ß-catenin in SHP2-depleted cells. No disassembly of the VE-cadherin complex is observed throughout the thrombin response. Using fluorescence recovery after photobleaching, we show that loss of SHP2 reduces the mobility of VE-cadherin at recovered cell-cell junctions. In conclusion, our data show that the SHP2 phosphatase plays an important role in the recovery of disrupted endothelial cell-cell junctions by dephosphorylating VE-cadherin-associated ß-catenin and promoting the mobility of VE-cadherin at the plasma membrane.

Regulation of small GTPases at epithelial cell-cell junctions.

Small GTPases of the Rho family (RhoA, Rac1, and Cdc42) and the Ras family GTPase Rap1 are essential for the assembly and function of epithelial cell-cell junctions. Through their downstream effectors, small GTPases modulate junction formation and stability, primarily by orchestrating the polymerization and contractility of the actomyosin cytoskeleton. The major upstream regulators of small GTPases are guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Several GEFs and a few GAPs have been localized at epithelial junctions, and bind to specific junctional proteins. Thus, junctional proteins can regulate small GTPases at junctions, through their interactions with GEFs and GAPs. Here we review the current knowledge about the mechanisms of regulation of small GTPases by junctional proteins. Understanding these mechanisms will help to clarify at the molecular level how small GTPases control the morphogenesis and physiology of epithelial tissues, and how they are disregulated in disease.

Innate immune function of the adherens junction protein p120-catenin in endothelial response to endotoxin.

Sepsis-induced acute lung injury is a common clinical disorder in critically ill patients that is associated with high mortality. In this study, we investigated the role of p120-catenin (p120), a constituent of endothelial adherens junctions, in regulating the innate immune function of lungs. In mice in which acute lung injury was induced by i.p. administration of LPS, we observed a rapid decrease in the expression of p120 in lungs. The p120 protein expression was correlated inversely with severity of inflammation. Suppression of p120 expression in lung endothelial cells in mice using small interfering RNA resulted in high sensitivity to endotoxin and greatly increased the mortality compared with controls. Knockdown of p120 also increased the expression of ICAM-1, neutrophil recruitment, production of cytokines TNF-α and IL-6, pulmonary transvascular protein permeability, and lung water content in response to LPS. We demonstrated that endothelial p120 modulates lung innate immune function by interfering with the association of TLR4 with its adaptor MyD88 to block TLR4 signaling and NF-κB activation in endothelial cells. In conclusion, these studies have uncovered a novel innate immune function of endothelial p120 in downregulating the lung inflammatory response to endotoxin through the suppression of TLR4 signaling.

Adducins regulate remodeling of apical junctions in human epithelial cells.

Epithelial adherens junctions (AJs) and tight junctions (TJs) are dynamic structures that readily undergo disintegration and reassembly. Remodeling of the AJs and TJs depends on the orchestrated dynamics of the plasma membrane with its underlying F-actin cytoskeleton, and the membrane-cytoskeleton interface may play a key role in junctional regulation. Spectrin-adducin-ankyrin complexes link membranes to the actin cytoskeleton where adducins mediate specrtrin-actin interactions. This study elucidates roles of adducins in the remodeling of epithelial junctions in human SK-CO15 colonic and HPAF-II pancreatic epithelial cell monolayers. These cells expressed the α and γ isoforms of adducin that positively regulated each others protein level and colocalized with E-cadherin and ß-catenin at mature, internalized and newly assembled AJs. Small interfering RNA-mediated down-regulation of α- or γ-adducin expression significantly attenuated calcium-dependent AJ and TJ assembly and accelerated junctional disassembly triggered by activation of protein kinase C. Two mechanisms were found to mediate the impaired AJ and TJ assembly in adducin-depleted cells. One mechanism involved diminished expression and junctional recruitment of ßII-spectrin, and the other mechanism involved the decrease in the amount of cellular F-actin and impaired assembly of perijunctional actin bundles. These findings suggest novel roles for adducins in stabilization of epithelial junctions and regulation of junctional remodeling.

Cdc42 regulates apical junction formation in human bronchial epithelial cells through PAK4 and Par6B.

Cdc42 has been implicated in numerous biochemical pathways during epithelial morphogenesis, including the control of spindle orientation during mitosis, the establishment of apical-basal polarity, the formation of apical cell-cell junctions, and polarized secretion. To investigate the signaling pathways through which Cdc42 mediates these diverse effects, we have screened an siRNA library corresponding to the 36 known Cdc42 target proteins, in a human bronchial epithelial cell line. Two targets, PAK4 and Par6B, were identified as necessary for the formation of apical junctions. PAK4 is recruited to nascent cell-cell contacts in a Cdc42-dependent manner, where it is required for the maturation of primordial junctions into apical junctions. PAK4 kinase activity is essential for junction maturation, but overexpression of an activated PAK4 mutant disrupts this process. Par6B, together with its binding partner aPKC, is necessary both for junction maturation and for the retention of PAK4 at sites of cell-cell contact. This study demonstrates that controlled regulation of PAK4 is required for apical junction formation in lung epithelial cells and highlights potential cross-talk between two Cdc42 targets, PAK4 and Par6B.

PTP-PEST controls motility, adherens junction assembly, and Rho GTPase activity in colon cancer cells.

An important step in carcinoma progression is loss of cell-cell adhesion leading to increased invasion and metastasis. We show here that the protein tyrosine phosphatase, PTP-PEST, is a critical regulator of cell-cell junction integrity and epithelial cell motility. Using colon carcinoma cells, we show that the expression level of PTP-PEST regulates cell motility. Either transient small interfering RNA or stable short hairpin RNA knockdown of PTP-PEST enhances haptotactic and chemotactic migration of KM12C colon carcinoma cells. Furthermore, KM12C cells with stably knocked down PTP-PEST exhibit a mesenchymal-like phenotype with prominent membrane ruffles and lamellae. In contrast, ectopic expression of PTP-PEST in KM20 or DLD-1 cells, which lack detectable endogenous PTP-PEST expression, suppresses haptotactic migration. Importantly, we find that PTP-PEST localizes in adherens junctions. Concomitant with enhanced motility, stable knockdown of PTP-PEST causes a disruption of cell-cell junctions. These effects are due to a defect in junctional assembly and not to a loss of E-cadherin expression. Adherens junction assembly is impaired following calcium switch in KM12C cells with stably knocked down PTP-PEST and is accompanied by an increase in the activity of Rac1 and a suppression of RhoA activity in response to cadherin engagement. Taken together, these results suggest that PTP-PEST functions as a suppressor of epithelial cell motility by controlling Rho GTPase activity and the assembly of adherens junctions.

Remodeling of the adherens junctions during morphogenesis.

Morphogenesis of epithelial tissues involves various forms of reshaping of cell layers, such as invagination or bending, convergent extension, and epithelial-mesenchymal transition. At the cellular level, these processes include changes in the shape, position, and assembly pattern of cells. During such morphogenetic processes, epithelial sheets in general maintain their multicellular architecture, implying that they must engage the mechanisms to change the spatial relationship with their neighbors without disrupting the junctions. A major junctional structure in epithelial tissues is the "adherens junction," which is composed of cadherin adhesion receptors and associated proteins including F-actin. The adherens junctions are required for the firm associations between cells, as disruption of them causes disorganization of the epithelial architecture. The adherens junctions, however, appear to be a dynamic entity, allowing the rearrangement of cells within cell sheets. This dynamic nature of the adherens junctions seems to be supported by various mechanisms, such as the interactions of cadherins with actin cytoskeleton, endocytosis and recycling of cadherins, and the cooperation of cadherins with other adhesion receptors. In this chapter, we provide an overview of these mechanisms analyzed in vitro and in vivo.