Formation Of Adherens Junctions Research Articles

Crucial Role of Rapgef2 and Rapgef6, a Family of Guanine Nucleotide Exchange Factors for Rap1 Small GTPase, in Formation of Apical Surface Adherens Junctions and Neural Progenitor Development in the Mouse Cerebral Cortex.

Cerebral neocortex development in mammals requires highly orchestrated events involving proliferation, differentiation, and migration of neural progenitors and neurons. Rapgef2 and Rapgef6 constitute a unique family of guanine nucleotide exchange factors for Rap1 small GTPase, which is known to play crucial roles in migration of postmitotic neurons. We previously reported that conditional knockout of Rapgef2 in dorsal telencephalon (Rapgef2-cKO) resulted in the formation of an ectopic cortical mass (ECM) resembling that of subcortical band heterotopia. Here we show that double knockout of Rapgef6 in Rapgef2-cKO mice (Rapgef2/6-dKO) results in marked enlargement of the ECM. While Rapgef2-cKO affects late-born neurons only, Rapgef2/6-dKO affects both early-born and late-born neurons. The Rapgef2-cKO cortex at embryonic day (E) 15.5, and the Rapgef2/6-dKO cortex at E13.5 and E15.5 show disruption of the adherens junctions (AJs) on the apical surface, detachment of radial glial cells (RGCs) from the apical surface and disorganization of the radial glial fiber system, which are accompanied by aberrant distribution of RGCs and intermediate progenitors, normally located in the ventricular zone and the subventricular zone, respectively, over the entire cerebral cortex. Moreover, intrauterine transduction of Cre recombinase into the Rapgef2flox/flox brains also results in the apical surface AJ disruption and the RGC detachment from the apical surface, both of which are effectively suppressed by cotransduction of the constitutively active Rap1 mutant Rap1G12V. These results demonstrate a cell-autonomous role of the Rapgef2/6-Rap1 pathway in maintaining the apical surface AJ structures, which is necessary for the proper development of neural progenitor cells.

Impaired endothelial barrier function in apolipoprotein M-deficient mice is dependent on sphingosine-1-phosphate receptor 1.

Apolipoprotein M (ApoM) transports sphingosine-1-phosphate (S1P) in plasma, and ApoM-deficient mice (Apom(-/-)) have ∼50% reduced plasma S1P levels. There are 5 known S1P receptors, and S1P induces adherens junction formation between endothelial cells through the S1P1 receptor, which in turn suppresses vascular leak. Increased vascular permeability is a hallmark of inflammation. The purpose of this study was to explore the relationships between vascular leakage in ApoM deficiency and S1P1 function in normal physiology and in inflammation. Vascular permeability in the lungs was assessed by accumulation of dextran molecules (70 kDa) and was increased ∼40% in Apom(-/-) mice compared to WT (C57Bl6/j) mice. Reconstitution of plasma ApoM/S1P or treatment with an S1P1 receptor agonist (SEW2871) rapidly reversed the vascular leakage to a level similar to that in WT mice, suggesting that it is caused by decreased plasma levels of S1P and reduced S1P1 stimulation. In a carrageenan-induced model of inflammation, Apom(-/-) mice had increased vascular leakage compared with that in WT mice. Adenoviral overexpression of ApoM in Apom(-/-) mice decreased the vascular leakage compared to adenoviral overexpression of green fluorescent protein. The study suggests that vascular leakage of albumin-sized particles in ApoM deficiency is S1P- and S1P1-dependent and this dependency exacerbates the response to inflammatory stimuli.-Christensen, P. M., Liu, C. H., Swendeman, S. L., Obinata, H., Qvortrup, K., Nielsen, L B., Hla, T., Di Lorenzo, A., Christoffersen, C. Impaired endothelial barrier function in apolipoprotein M-deficient mice is dependent on sphingosine-1-phosphate receptor 1.

Cortactin is a scaffolding platform for the E-cadherin adhesion complex and is regulated by protein kinase D1 phosphorylation.

Dynamic regulation of cell-cell adhesion by the coordinated formation and dissolution of E-cadherin-based adherens junctions is crucial for tissue homeostasis. The actin-binding protein cortactin interacts with E-cadherin and enables F-actin accumulation at adherens junctions. Here, we were interested to study the broader functional interactions of cortactin in adhesion complexes. In line with literature, we demonstrate that cortactin binds to E-cadherin, and that a posttranslational modification of cortactin, RhoA-induced phosphorylation by protein kinase D1 (PKD1; also known as PRKD1) at S298, impairs adherens junction assembly and supports their dissolution. Two new S298-phosphorylation-dependent interactions were also identified, namely, that phosphorylation of cortactin decreases its interaction with β-catenin and the actin-binding protein vinculin. In addition, binding of vinculin to β-catenin, as well as linkage of vinculin to F-actin, are also significantly compromised upon phosphorylation of cortactin. Accordingly, we found that regulation of cell-cell adhesion by phosphorylation of cortactin downstream of RhoA and PKD1 is vitally dependent on vinculin-mediated protein interactions. Thus, cortactin, unexpectedly, is an important integration node for the dynamic regulation of protein complexes during breakdown and formation of adherens junctions.

Rab11a Mediates Vascular Endothelial-Cadherin Recycling and Controls Endothelial Barrier Function.

Vascular endothelial (VE)-cadherin is the predominant component of endothelial adherens junctions essential for cell-cell adhesion and formation of the vascular barrier. Endocytic recycling is an important mechanism for maintaining the expression of cell surface membrane proteins. However, little is known about the molecular mechanism of VE-cadherin recycling and its role in maintenance of vascular integrity. Using calcium-switch assay, confocal imaging, cell surface biotinylation, and flow cytometry, we showed that VE-cadherin recycling required Ras-related proteins in brain (Rab)11a and Rab11 family-interacting protein 2. Yeast 2-hybrid assay and coimmunoprecipitation demonstrated that direct interaction of VE-cadherin with family-interacting protein 2 (at aa 453-484) formed a ternary complex with Rab11a in human endothelial cells. Silencing of Rab11a or Rab11 family-interacting protein 2 in endothelial cells prevented VE-cadherin recycling and VE-cadherin expression at endothelial plasma membrane. Furthermore, inactivation of Rab11a signaling blocked junctional reannealing after vascular inflammation. Selective knockdown of Rab11a in pulmonary microvessels markedly increased vascular leakage in mice challenged with lipopolysaccharide or polymicrobial sepsis. Rab11a/Rab11 family-interacting protein 2-mediated VE-cadherin recycling is required for formation of adherens junctions and restoration of VE barrier integrity and hence a potential target for clinical intervention in inflammatory disease.

A local VE-cadherin and Trio-based signaling complex stabilizes endothelial junctions through Rac1

Endothelial cell-cell junctions maintain a restrictive barrier that is tightly regulated to allow dynamic responses to permeability-inducing angiogenic factors, as well as to inflammatory agents and adherent leukocytes. The ability of these stimuli to transiently remodel adherens junctions depends on Rho-GTPase-controlled cytoskeletal rearrangements. How the activity of Rho-GTPases is spatio-temporally controlled at endothelial adherens junctions by guanine-nucleotide exchange factors (GEFs) is incompletely understood. Here, we identify a crucial role for the Rho-GEF Trio in stabilizing junctions based around vascular endothelial (VE)-cadherin (also known as CDH5). Trio interacts with VE-cadherin and locally activates Rac1 at adherens junctions during the formation of nascent contacts, as assessed using a novel FRET-based Rac1 biosensor and biochemical assays. The Rac-GEF domain of Trio is responsible for the remodeling of junctional actin from radial into cortical actin bundles, a crucial step for junction stabilization. This promotes the formation of linear adherens junctions and increases endothelial monolayer resistance. Collectively, our data show the importance of spatio-temporal regulation of the actin cytoskeleton through Trio and Rac1 at VE-cadherin-based cell-cell junctions in the maintenance of the endothelial barrier.

The formation of ordered nanoclusters controls cadherin anchoring to actin and cell-cell contact fluidity.

Oligomerization of cadherins could provide the stability to ensure tissue cohesion. Cadherins mediate cell-cell adhesion by forming trans-interactions. They form cis-interactions whose role could be essential to stabilize intercellular junctions by shifting cadherin clusters from a fluid to an ordered phase. However, no evidence has been provided so far for cadherin oligomerization in cellulo and for its impact on cell-cell contact stability. Visualizing single cadherins within cell membrane at a nanometric resolution, we show that E-cadherins arrange in ordered clusters, providing the first demonstration of the existence of oligomeric cadherins at cell-cell contacts. Studying the consequences of the disruption of the cis-interface, we show that it is not essential for adherens junction formation. Its disruption, however, increased the mobility of junctional E-cadherin. This destabilization strongly affected E-cadherin anchoring to actin and cell-cell rearrangement during collective cell migration, indicating that the formation of oligomeric clusters controls the anchoring of cadherin to actin and cell-cell contact fluidity.

SH2 Domain-Containing Protein Tyrosine Phosphatase 2 and Focal Adhesion Kinase Protein Interactions Regulate Pulmonary Endothelium Barrier Function

Enhanced protein tyrosine phosphorylation is associated with changes in vascular permeability through formation and dissolution of adherens junctions and regulation of stress fiber formation. Inhibition of the protein tyrosine phosphorylase SH2 domain-containing protein tyrosine phosphatase 2 (SHP2) increases tyrosine phosphorylation of vascular endothelial cadherin and β-catenin, resulting in disruption of the endothelial monolayer and edema formation in the pulmonary endothelium. Vascular permeability is a hallmark of acute lung injury (ALI); thus, enhanced SHP2 activity offers potential therapeutic value for the pulmonary vasculature in diseases such as ALI, but this has not been characterized. To assess whether SHP2 activity mediates protection against edema in the endothelium, we assessed the effect of molecular activation of SHP2 on lung endothelial barrier function in response to the edemagenic agents LPS and thrombin. Both LPS and thrombin reduced SHP2 activity, correlated with decreased focal adhesion kinase (FAK) phosphorylation (Y(397) and Y(925)) and diminished SHP2 protein-protein associations with FAK. Overexpression of constitutively active SHP2 (SHP2(D61A)) enhanced baseline endothelial monolayer resistance and completely blocked LPS- and thrombin-induced permeability in vitro and significantly blunted pulmonary edema formation induced by either endotoxin (LPS) or Pseudomonas aeruginosa exposure in vivo. Chemical inhibition of FAK decreased SHP2 protein-protein interactions with FAK concomitant with increased permeability; however, overexpression of SHP2(D61A) rescued the endothelium and maintained FAK activity and FAK-SHP2 protein interactions. Our data suggest that SHP2 activation offers the pulmonary endothelium protection against barrier permeability mediators downstream of the FAK signaling pathway. We postulate that further studies into the promotion of SHP2 activation in the pulmonary endothelium may offer a therapeutic approach for patients suffering from ALI.

An ELMO2-RhoG-ILK network modulates microtubule dynamics

ELMO2 belongs to a family of scaffold proteins involved in phagocytosis and cell motility. ELMO2 can simultaneously bind integrin-linked kinase (ILK) and RhoG, forming tripartite ERI complexes. These complexes are involved in promoting β1 integrin-dependent directional migration in undifferentiated epidermal keratinocytes. ELMO2 and ILK have also separately been implicated in microtubule regulation at integrin-containing focal adhesions. During differentiation, epidermal keratinocytes cease to express integrins, but ERI complexes persist. Here we show an integrin-independent role of ERI complexes in modulation of microtubule dynamics in differentiated keratinocytes. Depletion of ERI complexes by inactivating the Ilk gene in these cells reduces microtubule growth and increases the frequency of catastrophe. Reciprocally, exogenous expression of ELMO2 or RhoG stabilizes microtubules, but only if ILK is also present. Mechanistically, activation of Rac1 downstream from ERI complexes mediates their effects on microtubule stability. In this pathway, Rac1 serves as a hub to modulate microtubule dynamics through two different routes: 1) phosphorylation and inactivation of the microtubule-destabilizing protein stathmin and 2) phosphorylation and inactivation of GSK-3β, which leads to the activation of CRMP2, promoting microtubule growth. At the cellular level, the absence of ERI species impairs Ca(2+)-mediated formation of adherens junctions, critical to maintaining mechanical integrity in the epidermis. Our findings support a key role for ERI species in integrin-independent stabilization of the microtubule network in differentiated keratinocytes.

Impairment of radial glial scaffold-dependent neuronal migration and formation of double cortex by genetic ablation of afadin

Studies of human brain malformations, such as lissencephaly and double cortex, have revealed the importance of neuronal migration during cortical development. Afadin, a membrane scaffolding protein, regulates the formation of adherens junctions (AJs) and cell migration to form and maintain tissue structures. Here, we report that mice with dorsal telencephalon-specific ablation of afadin gene exhibited defects similar to human double cortex, in which the heterotopic cortex was located underneath the normotopic cortex. The normotopic cortex of the mutant mice was arranged in the pattern similar to the cortex of the control mice, while the heterotopic cortex was disorganized. As seen in human patients, double cortex in the mutant mice was formed by impaired neuronal migration during cortical development. Genetic ablation of afadin in the embryonic cerebral cortex disrupted AJs of radial glial cells, likely resulting in the retraction of the apical endfeet from the ventricular surface and the dispersion of radial glial cells from the ventricular zone to the subventricular and intermediate zones. These results indicate that afadin is required for the maintenance of AJs of radial glial cells and that the disruption of AJs might cause an abnormal radial scaffold for neuronal migration. In contrast, the proliferation or differentiation of radial glial cells was not significantly affected. Taken together, these findings indicate that afadin is required for the maintenance of the radial glial scaffold for neuronal migration and that the genetic ablation of afadin leads to the formation of double cortex.

Junctional actin assembly is mediated by Formin-like 2 downstream of Rac1.

Epithelial integrity is vitally important, and its deregulation causes early stage cancer. De novo formation of an adherens junction (AJ) between single epithelial cells requires coordinated, spatial actin dynamics, but the mechanisms steering nascent actin polymerization for cell-cell adhesion initiation are not well understood. Here we investigated real-time actin assembly during daughter cell-cell adhesion formation in human breast epithelial cells in 3D environments. We identify formin-like 2 (FMNL2) as being specifically required for actin assembly and turnover at newly formed cell-cell contacts as well as for human epithelial lumen formation. FMNL2 associates with components of the AJ complex involving Rac1 activity and the FMNL2 C terminus. Optogenetic control of Rac1 in living cells rapidly drove FMNL2 to epithelial cell-cell contact zones. Furthermore, Rac1-induced actin assembly and subsequent AJ formation critically depends on FMNL2. These data uncover FMNL2 as a driver for human epithelial AJ formation downstream of Rac1.

Abstract 434: Formin Dependent Regulation of Adherens Junction Assembly

Adherens junctions are cadherin-dependent structures that mediate intercellular signaling and structural integrity. In endothelial cells, adherens junctions are the primary determiners of vascular permeability and endothelial barrier function. Adherens junctions connect directly to the actin cytoskeleton and this connection is essential for junction formation and maintenance. Formins are a highly conserved family of cytoskeletal remodeling proteins whose activity has been implicated in regulating junction formation in other cell-types. Therefore, we tested the hypothesis that formin activity is essential for adherens junction assembly in endothelial cells. A small-molecule formin inhibitor (smiFH2) was used to determine the effect of formin inhibition on junction formation in Telomerase-Immortalized Microvascular Endothelial (TIME) cells. Using an in vitro vascular permeability assay, we determined that smiFH2 treatment caused a dose-dependent inhibition of junction formation. We used siRNA to knockdown expression of seven of the formins expressed in TIME cells in order to determine which specific formins were required to maintain endothelial barrier function. We determined that individual knockdown of three formins, FHOD1, FHOD3 and Dia1, significantly increased the permeability of the endothelial monolayer. Interestingly, the knockdown of a fourth formin, FMNL2, had the opposite effect and actually potentiated barrier function. Knockdown of the remaining formins had little or no effect on junction formation. Knockdown of FHOD3 had the greatest inhibitory effect on junction assembly and VE-cadherin, β-catenin and α-catenin protein levels were decreased in FHOD3-depleted cells. The FHOD3 knockdown cells were also elongated in comparison to controls and formed thin linear adherens junctions and few focal adherens junctions. In contrast, the morphology of FMNL2-depleted cells did not appear obviously different from controls. In conclusion, our results suggest that multiple formins play diverse roles in endothelial cell adherens junction formation and maintenance.

Role of Krev Interaction Trapped-1 in Prostacyclin-Induced Protection against Lung Vascular Permeability Induced by Excessive Mechanical Forces and Thrombin Receptor Activating Peptide 6.

Mechanisms of vascular endothelial cell (EC) barrier regulation during acute lung injury (ALI) or other pathologies associated with increased vascular leakiness are an active area of research. Adaptor protein krev interaction trapped-1 (KRIT1) participates in angiogenesis, lumen formation, and stabilization of EC adherens junctions (AJs) in mature vasculature. We tested a role of KRIT1 in the regulation of Rho-GTPase signaling induced by mechanical stimulation and barrier dysfunction relevant to ventilator-induced lung injury and investigated KRIT1 involvement in EC barrier protection by prostacyclin (PC). PC stimulated Ras-related protein 1 (Rap1)-dependent association of KRIT1 with vascular endothelial cadherin at AJs, with KRIT1-dependent cortical cytoskeletal remodeling leading to EC barrier enhancement. KRIT1 knockdown exacerbated Rho-GTPase activation and EC barrier disruption induced by pathologic 18% cyclic stretch and thrombin receptor activating peptide (TRAP) 6 and attenuated the protective effects of PC. In the two-hit model of ALI caused by high tidal volume (HTV) mechanical ventilation and TRAP6 injection, KRIT1 functional deficiency in KRIT1(+/-) mice increased basal lung vascular leak and augmented vascular leak and lung injury caused by exposure to HTV and TRAP6. Down-regulation of KRIT1 also diminished the protective effects of PC against TRAP6/HTV-induced lung injury. These results demonstrate a KRIT1-dependent mechanism of vascular EC barrier control in basal conditions and in the two-hit model of ALI caused by excessive mechanical forces and TRAP6 via negative regulation of Rho activity and enhancement of cell junctions. We also conclude that the stimulation of the Rap1-KRIT1 signaling module is a major mechanism of vascular endothelial barrier protection by PC in the injured lung.

RhoA and ROCK mediate histamine-induced vascular leakage and anaphylactic shock.

Histamine-induced vascular leakage is an integral component of many highly prevalent human diseases, including allergies, asthma, and anaphylaxis. Yet, how histamine induces the disruption of the endothelial barrier is not well defined. By using genetically modified animal models, pharmacologic inhibitors, and a synthetic biology approach, here we show that the small GTPase RhoA mediates histamine-induced vascular leakage. Histamine causes the rapid formation of focal adherens junctions, disrupting the endothelial barrier by acting on H1R Gαq-coupled receptors, which is blunted in endothelial Gαq/11 KO mice. Interfering with RhoA and ROCK function abolishes endothelial permeability, while phospholipase Cβ plays a limited role. Moreover, endothelial-specific RhoA gene deletion prevents vascular leakage and passive cutaneous anaphylaxis in vivo, and ROCK inhibitors protect from lethal systemic anaphylaxis. This study supports a key role for the RhoA signaling circuitry in vascular permeability, thereby identifying novel pharmacological targets for many human diseases characterized by aberrant vascular leakage.

Select Rab GTPases Mediate Pulmonary Endothelial Barrier

Vascular permeability, a major hallmark of acute lung Injury (ALI), is regulated in part by VE‐cadherin (VE‐cad) forming adherens junctions at the interendothelial junctions (IEJ). We have recently demonstrated that the endosomal protein, p18, binds to the early endosome to enhance recycling of VE‐cad to IEJ, improve endothelial (EC) barrier function and attenuate pulmonary edema formation. The endosome system is a dynamic process which sorts cargo and directs it either back to the plasma membrane, through Rab4‐mediated recycling or to the lysosome through Rab7 and Rab9. We thus hypothesize that Rab GTPases regulates EC barrier function through modulation of VE‐cad trafficking to and from the IEJ. Lung EC were transfected with wild type Rab4 (Rab4wt), GTP‐locked endosome‐anchored Rab4 (Rab4Q67L), GDP‐locked non‐endosome binding Rabs, Rab4S22N, Rab7T22N and Rab9S21N or GFP as control. The effects of Rab4, Rab7 and Rab9 on EC endocytosis of VE‐cad, in vitro EC barrier function and in vivo pulmonary edema formation were examined. We show that activation of pro‐recycling Rab4wt and Rab4Q67L or inhibition of pro‐degradative Rab9S21N protects EC barrier function in settings of ALI in vitro and in vivo in settings of LPS‐induced injury. In contrast, Rab4S22N or pro‐lysosome forming Rab7T22N play no protective role on EC barrier integrity. We further show enhanced levels of VE‐cad at the plasma membrane concomitant with improved EC barrier function. We conclude that Rab4 and Rab9 regulate pulmonary EC barrier function by enhancing recycling of VE‐cad+ endosomes to the IEJ. We propose that targeting Rab GTPase‐mediated endocytic trafficking represents a novel therapy in the treatment of ALI.

Sphingomyelin metabolism is involved in the differentiation of MDCK cells induced by environmental hypertonicity

Sphingolipids (SLs) are relevant lipid components of eukaryotic cells. Besides regulating various cellular processes, SLs provide the structural framework for plasma membrane organization. Particularly, SM is associated with detergent-resistant microdomains. We have previously shown that the adherens junction (AJ) complex, the relevant cell-cell adhesion structure involved in cell differentiation and tissue organization, is located in an SM-rich membrane lipid domain. We have also demonstrated that under hypertonic conditions, Madin-Darby canine kidney (MDCK) cells acquire a differentiated phenotype with changes in SL metabolism. For these reasons, we decided to evaluate whether SM metabolism is involved in the acquisition of the differentiated phenotype of MDCK cells. We found that SM synthesis mediated by SM synthase 1 is involved in hypertonicity-induced formation of mature AJs, necessary for correct epithelial cell differentiation. Inhibition of SM synthesis impaired the acquisition of mature AJs, evoking a disintegration-like process reflected by the dissipation of E-cadherin and β- and α-catenins from the AJ complex. As a consequence, MDCK cells did not develop the hypertonicity-induced differentiated epithelial cell phenotype.

Sphingosine 1 Phosphate Synthesis Controls the Preservation of Epithelial Barrier During MDCK Cell Differentiation

Sphingosine 1-Phophate (S1P) is a sphingolipid mediator of cellular fate. We have demonstrated that hypertonicity induces epithelial tissue organization by the establishment of mature adherent junctions. Organization and preservation of epithelial tissue requires an efficient cell extrusion followed by a closing of cellular gap to preserve tissue permeability. We study the involvement of sphingosine kinase (SK) activity in the transition from polarized to differentiated MDCK cells. Confluent MDCK cells were subjected to hypertonic medium with the concomitant knock down of SK or not (control), or pharmacological inhibition of SK. After 48 h, cell phenotype was visualized by fluorescence microscopy, evaluating actin cytoskeleton and Adherens Junction (AJ) formation. By performing a confocal z-plane reconstruction we visualized extruding cells and found that the inhibition of SK evoked an alteration of efficient cell extrusion. SK knock-down induced AJ disassembling, and actin cytoskeleton reorganization. SK inhibition produced an increase in de novo sphingolipid synthesis with Ceramide (Cer) accumulation. In order to evaluate whether AJ disassembly is due to Cer accumulation, Fumonicin B1 (FB1) was used, as inhibitor of Ceramide Synthase. FB1 treatment recovers MDCK phenotype, suggesting that the disassembly of AJ due to inhibition of SK activity is an indirect effect produced by Cer accumulation. Supported by grants from Conicet and University of Buenos Aires-Argentina

α-Catenin phosphorylation promotes intercellular adhesion through a dual-kinase mechanism

The cadherin-catenin adhesion complex is a key contributor to epithelial tissue stability and dynamic cell movements during development and tissue renewal. How this complex is regulated to accomplish these functions is not fully understood. We identified several phosphorylation sites in mammalian αE-catenin (also known as catenin α-1) and Drosophila α-Catenin within a flexible linker located between the middle (M)-region and the carboxy-terminal actin-binding domain. We show that this phospho-linker (P-linker) is the main phosphorylated region of α-catenin in cells and is sequentially modified at casein kinase 2 and 1 consensus sites. In Drosophila, the P-linker is required for normal α-catenin function during development and collective cell migration, although no obvious defects were found in cadherin-catenin complex assembly or adherens junction formation. In mammalian cells, non-phosphorylatable forms of α-catenin showed defects in intercellular adhesion using a mechanical dispersion assay. Epithelial sheets expressing phosphomimetic forms of α-catenin showed faster and more coordinated migrations after scratch wounding. These findings suggest that phosphorylation and dephosphorylation of the α-catenin P-linker are required for normal cadherin-catenin complex function in Drosophila and mammalian cells.

The X-ray structure of human P-cadherin EC1-EC2 in a closed conformation provides insight into the type I cadherin dimerization pathway.

Cadherins are a large family of calcium-dependent proteins that mediate cellular adherens junction formation and tissue morphogenesis. To date, the most studied cadherins are those classified as classical, which are further divided into type I or type II depending on selected sequence features. Unlike other members of the classical cadherin family, a detailed structural characterization of P-cadherin has not yet been fully obtained. Here, the high-resolution crystal structure determination of the closed form of human P-cadherin EC1-EC2 is reported. The structure shows a novel, monomeric packing arrangement that provides a further snapshot in the yet-to-be-achieved complete description of the highly dynamic cadherin dimerization pathway. Moreover, this is the first multidomain cadherin fragment to be crystallized and structurally characterized in its closed conformation that does not carry any extra N-terminal residues before the naturally occurring aspartic acid at position 1. Finally, two clear alternate conformations are observed for the critical Trp2 residue, suggestive of a transient, metastable state. The P-cadherin structure and packing arrangement shown here provide new and valuable information towards the complete structural characterization of the still largely elusive cadherin dimerization pathway.

P120-Catenin Expressed in Alveolar Type II Cells Is Essential for the Regulation of Lung Innate Immune Response

The integrity of the lung alveolar epithelial barrier is required for the gas exchange and is important for immune regulation. Alveolar epithelial barrier is composed of flat type I cells, which make up approximately 95% of the gas-exchange surface, and cuboidal type II cells, which secrete surfactants and modulate lung immunity. p120-catenin (p120; gene symbol CTNND1) is an important component of adherens junctions of epithelial cells; however, its function in lung alveolar epithelial barrier has not been addressed in genetic models. Here, we created an inducible type II cell-specific p120-knockout mouse (p120EKO). The mutant lungs showed chronic inflammation, and the alveolar epithelial barrier was leaky to (125)I-albumin tracer compared to wild type. The mutant lungs also demonstrated marked infiltration of inflammatory cells and activation of NF-κB. Intracellular adhesion molecule 1, Toll-like receptor 4, and macrophage inflammatory protein 2 were all up-regulated. p120EKO lungs showed increased expression of the surfactant proteins Sp-B, Sp-C, and Sp-D, and displayed severe inflammation after pneumonia caused by Pseudomonas aeruginosa compared with wild type. In p120-deficient type II cell monolayers, we observed reduced transepithelial resistance compared to control, consistent with formation of defective adherens junctions. Thus, although type II cells constitute only 5% of the alveolar surface area, p120 expressed in these cells plays a critical role in regulating the innate immunity of the entire lung.

Nectin expression in pancreatic adenocarcinoma: nectin-3 is associated with a poor prognosis.

PurposeNectins are cell adhesion molecules that regulate the formation of adherens junctions and are linked with E-cadherin-based cell–cell adherens junctions. In pancreatic cancer, the expression of E-cadherin and nectins is considered to be related to metastasis, invasion and prognosis.MethodsWe evaluated the distribution of cells that were positive for nectin subtypes and E-cadherin using immunohistochemistry in specimens of human pancreatic adenocarcinoma, and correlated these results with the clinicopathological features and patient outcomes.ResultsThe immunohistochemical distribution of nectin-1 and E-cadherin showed a good correlation (r = 0.523, p < 0.01). Tumors over 4 cm in diameter had more intense staining for nectin-4 than smaller tumors (p = 0.035). Nectin-2 expression correlated with a poorer histological grade (p = 0.04). The cases that showed diffuse nectin-3 expression had a better prognosis than those with negative expression (p = 0.018).ConclusionOur results showed that the expression of nectin-3 in pancreatic cancer can be a prognostic factor.