Localization Of N-cadherin Research Articles

Epithelial-To-Mesenchymal Transition and Its Correlation With Clinicopathologic Features in Patients With Urothelial Carcinoma of the Bladder

IntroductionEpithelial-to-mesenchymal transition (EMT) is a dynamic process in the pathogenesis of urinary bladder cancer. Despite significant advancements in its diagnosis and treatment, the outcomes have more or less remained the same. In the present study, the expression of EMT markers was investigated to evaluate its prognostic significance in patients with non–muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). Materials and MethodsThe present study was undertaken to examine the expression of EMT markers, including E-cadherin, N-cadherin, vimentin, Snail, Twist, Zeb, and Slug, on 28 bladder tumor tissues (15 cases of NMIBC and 13 of MIBC) using reverse transcription-polymerase chain reaction. Immunohistochemical (IHC) staining was performed to check the protein expression and localization of E-cadherin, N-cadherin, vimentin, Snail, and Slug. ResultsAt the message level, reduced expression of E-cadherin correlated with gender (P = .004), enhanced expression of N-cadherin correlated with stage and age (P = .02, for both), and increased expression of EMT transcription factors correlated significantly with stage, grade, or age. Inverse correlation of reduced levels of E-cadherin were observed with new expression of N-cadherin (P = .001; Mann-Whitney U test) and vimentin (P = .001; Mann-Whitney U test). On IHC, novel expression of vimentin and N-cadherin was associated with enhanced expression of Snail and Slug (P = .005; Wilcoxon signed rank test). ConclusionMolecular validation of the EMT marker profile proved to be a sensitive and an effective prognostic tool for objective and systematic investigation of EMT function in the pathogenesis of urinary bladder cancer. Nevertheless, further studies are required with a greater number of clinical samples.

Immunoexpression of Epithelial Mesenchymal Transition Proteins E-Cadherin, β-Catenin, and N-Cadherin in Oral Squamous Cell Carcinoma.

Aims Epithelial mesenchymal transition (EMT) is a crucial process for acquisition of malignant phenotype, aggressiveness, and metastatic capacity in neoplasms. It is characterized by loss of epithelial markers and gain of mesenchymal markers. Studies on EMT and its potential association with the histological grading are sparse in oral squamous cell carcinoma (OSCC). This study aims to evaluate the expression of EMT-associated proteins-E-cadherin, β-catenin, and N-cadherin-in different grades of OSCC. Methodology In all, 60 cases of OSCC further subdivided into 20 cases each of well-, moderately, and poorly differentiated OSCCs were stained immunohistochemically with E-cadherin, β-catenin, and N-cadherin antibodies. The differences in the expression were evaluated using χ2 and Fisher exact tests, whereas Spearman's correlation was used to analyze the correlation between the markers. Results A reduced E-cadherin expression noted in 40% of the OSCCs was associated with reduced β-catenin expression in 66.6% of the cases and increase in the expression of mesenchymal N-cadherin seen in 80% of cases. This expression pattern demonstrated a significant association with histological grades. A membrane to cytoplasmic shift of E-cadherin (73.3%) and β-catenin (78.3%) increased with histological grade. A negative correlation was observed with the E-cadherin and N-cadherin localization, though it did not reach statistical significance. Conclusion OSCC tissues had high levels of EMT phenotype as compared with the normal oral mucosa. This phenotype was characterized by reduced E-cadherin and β-catenin expression and overexpression of N-cadherin. Aberrant localization of the studied proteins was a hallmark for depicting EMT.

P120-Catenin Regulates Early Trafficking Stages of the N-Cadherin Precursor Complex.

It is well established that binding of p120 catenin to the cytoplasmic domain of surface cadherin prevents cadherin endocytosis and degradation, contributing to cell-cell adhesion. In the present work we show that p120 catenin bound to the N-cadherin precursor, contributes to its anterograde movement from the endoplasmic reticulum (ER) to the Golgi complex. In HeLa cells, depletion of p120 expression, or blocking its binding to N-cadherin, increased the accumulation of the precursor in the ER, while it decreased the localization of mature N-cadherin at intercellular junctions. Reconstitution experiments in p120-deficient SW48 cells with all three major isoforms of p120 (1, 3 and 4) had similar capacity to promote the processing of the N-cadherin precursor to the mature form, and its localization at cell-cell junctions. P120 catenin and protein tyrosine phosphatase PTP1B facilitated the recruitment of the N-ethylmaleimide sensitive factor (NSF), an ATPase involved in vesicular trafficking, to the N-cadherin precursor complex. Dominant negative NSF E329Q impaired N-cadherin trafficking, maturation and localization at cell-cell junctions. Our results uncover a new role for p120 catenin bound to the N-cadherin precursor ensuring its trafficking through the biosynthetic pathway towards the cell surface.

Reduced adhesive ligand density in engineered extracellular matrices induces an epithelial-mesenchymal-like transition

A synergistic effect of biochemical and mechanical cues emanating from the extracellular matrix (ECM) on inducing malignant transformation of epithelial cells has been observed recently. However, the effect of quantitative changes in biochemical stimuli on cell phenotype, without changes in ECM component and rigidity, remains unknown. To determine this effect, we grew Madin-Darby canine kidney epithelial cells (MDCK) on gold surfaces immobilized with varying densities of cyclic arginine-glycine-aspartate (cRGD) peptide and analyzed cell morphology, cell migration, cytoskeletal organization, and protein expression. Cells grown on a surface presenting a higher density of cRGD displayed an epithelial morphology and grew in clusters, while those grown on a diluted cRGD surface transformed into an elongated, fibroblast-like form with extensive scattering. Time-lapse imaging of cell clusters grown on the concentrated cRGD surface revealed collective migration with intact cell-cell contacts accompanied by the development of cortical actin. In contrast, cells migrated individually and formed stress fibers on the substrate with sparse cRGD. These data point towards transdifferentiation of epithelial cells to mesenchymal-like cells when plated on a diluted cRGD surface. Supporting this hypothesis, immunofluorescence microscopy and western blot analysis revealed increased membrane localization and total expression of N-cadherin and vimentin in cells undergoing mesenchymal-like transition. Taken together, these results suggest a possible role of decreased biochemical stimuli from the ECM in regulating epithelial phenotype switching. Statement of SignificanceEpithelial-mesenchymal transition (EMT) is a process where adherent epithelial cells convert into individual migratory mesenchymal phenotype. It plays an important role both in physiological and pathological processes. Recent studies demonstrate that the program is not only governed by soluble factors and gene expressions, but also modulated by biochemical and mechanical cues in ECMs. In this study, we developed chemically defined surfaces presenting controlled ECM ligand densities and studied their impact on the EMT progression. Morphological and biochemical analyses of epithelial cells cultured on the surfaces indicate the cells undergo an EMT-like transition on the diluted cRGD surface while retaining epithelial characteristics on the cRGD-rich substrate, suggesting an important role of the ECM ligand density in epithelial phenotype switching.

E-cadherin cytoplasmic domain inhibits cell surface localization of endogenous cadherins and fusion of C2C12 myoblasts.

ABSTRACTMyoblast fusion is a highly regulated process that is essential for skeletal muscle formation during muscle development and regeneration in mammals. Much remains to be elucidated about the molecular mechanism of myoblast fusion although cadherins, which are Ca2+-dependent cell–cell adhesion molecules, are thought to play a critical role in this process. Mouse myoblasts lacking either N-cadherin or M-cadherin can still fuse to form myotubes, indicating that they have no specific function in this process and may be functionally replaced by either M-cadherin or N-cadherin, respectively. In this study, we show that expressing the E-cadherin cytoplasmic domain ectopically in C2C12 myoblasts inhibits cell surface localization of endogenous M-cadherin and N-cadherin, as well as cell–cell fusion. This domain, however, does not inhibit myoblast differentiation according to microarray-based gene expression analysis. In contrast, expressing a dominant-negative β-catenin mutant ectopically, which suppresses Wnt/β-catenin signaling, did not inhibit cell–cell fusion. Therefore, the E-cadherin cytoplasmic domain inhibits cell–cell fusion by inhibiting cell surface localization of endogenous cadherins and not by inhibiting Wnt/β-catenin signaling.

Flavocoxid Protects Against Cadmium-Induced Disruption of the Blood–Testis Barrier and Improves Testicular Damage and Germ Cell Impairment in Mice [corrected

Cadmium (Cd) causes male infertility. There is the need to identify safe treatments counteracting this toxicity. Flavocoxid is a flavonoid that induces a balanced inhibition of cyclooxygenase (COX)-1 and COX-2 peroxidase moieties and of 5-lipoxygenase (LOX) and has efficacy in the male genitourinary system. We investigated flavocoxid effects on Cd-induced testicular toxicity in mice. Swiss mice were divided into 4 groups: 2 control groups received 0.9% NaCl (vehicle; 1 ml/kg/day) or flavocoxid (20 mg/kg/day ip); 2 groups were challenged with cadmium chloride (CdCl2; 2 mg/kg/day ip) and administered with vehicle or flavocoxid. The treatment lasted for 1 or 2 weeks. The testes were processed for biochemical and morphological studies. CdCl2 increased phosphorylated extracellular signal-regulated kinase (p-ERK) 1/2, tumor necrosis factor (TNF)-α, COX-2, 5-LOX, malondialdehyde (MDA), B-cell-lymphoma (Bcl)-2-associated X protein (Bax), follicle-stimulating hormone (FSH), luteinizing hormone (LH), transforming growth factor (TGF) -β3, decreased Bcl-2, testosterone, inhibin-B, occludin, N-Cadherin, induced structural damages in the testis and disrupted the blood-testis barrier. Many TUNEL-positive germ cells and changes in claudin-11, occludin, and N-cadherin localization were present. Flavocoxid administration reduced, in a time-dependent way, p-ERK 1/2, TNF-α, COX-2, 5-LOX, MDA, Bax, FSH, LH, TGF-β3, augmented Bcl-2, testosterone, inhibin B, occludin, N-Cadherin, and improved the structural organization of the testis and the blood-testis barrier. Few TUNEL-positive germ cells were present and a morphological retrieval of the intercellular junctions was observed. In conclusion, flavocoxid has a protective anti-inflammatory, antioxidant, and antiapoptotic function against Cd-induced toxicity in mice testis. We suggest that flavocoxid may play a relevant positive role against environmental levels of Cd, otherwise deleterious to gametogenesis and tubular integrity.

Myosin X regulates neuronal radial migration through interacting with N-cadherin.

Proper brain function depends on correct neuronal migration during development, which is known to be regulated by cytoskeletal dynamics and cell-cell adhesion. Myosin X (Myo10), an uncharacteristic member of the myosin family, is an important regulator of cytoskeleton that modulates cell motilities in many different cellular contexts. We previously reported that Myo10 was required for neuronal migration in the developing cerebral cortex, but the underlying mechanism was still largely unknown. Here, we found that knockdown of Myo10 expression disturbed the adherence of migrating neurons to radial glial fibers through abolishing surface Neuronal cadherin (N-cadherin) expression, thereby impaired neuronal migration in the developmental cortex. Next, we found Myo10 interacted with N-cadherin cellular domain through its FERM domain. Furthermore, we found knockdown of Myo10 disrupted N-cadherin subcellular distribution and led to localization of N-cadherin into Golgi apparatus and endosomal sorting vesicle. Taking together, these results reveal a novel mechanism of Myo10 interacting with N-cadherin and regulating its cell-surface expression, which is required for neuronal adhesion and migration.

Plakophilin-2 c.419C>T and risk of heart failure and arrhythmias in the general population

A rare genetic variant in the desmosomal gene plakophilin-2 (PKP2) c.419C>T(p.(S140F)) has repeatedly been identified in patients with dilated cardiomyopathy (DCM) and arrhythmogenic right ventricular cardiomyopathy (ARVC). Whether this is a disease-causing variant remains highly controversial. We tested this hypothesis using three approaches. Initially, in a prospective study of 10 407 individuals from the general population, including 2688 who developed heart failure or arrhythmias during >14 years of follow-up, PKP2 c.419C>T was identified in 98 individuals (0.94%). PKP2 genotype was not associated with electrocardiographic or echocardiographic changes, or with plasma levels of probrain natriuretic peptide (all P≥0.05). In c.419C>T carriers versus non-carriers, multifactorially adjusted hazard ratios were 1.26 (95% confidence interval: 0.77-2.07) for heart failure, 1.40 (0.90-2.17) for arrhythmias, 1.15 (0.78-1.71) for end points combined, and 1.33 (0.98-1.80) for all-cause mortality. The cumulative survival as a function of age and PKP2 genotype was similar among carriers and non-carriers (P=0.14). Second, comparing 517 patients referred for genetic testing with 1918 matched controls, odds ratios as a function of c.419C>T genotype were 2.11 (0.50-8.99) for ARVC, 0.72 (0.16-3.28) for hypertrophic cardiomyopathy (HCM)/DCM, and 1.28 (0.46-3.54) for end points combined. Third, in in vitro studies cellular localization of plakophilin-2, plakoglobin, connexin-43, or N-cadherin were similar in cells transfected with wild-type or mutant plakophilin-2. In conclusion, combining epidemiological data, with data on patients referred for genetic testing for ARVC or HCM/DCM, and data from in vitro studies, PKP2 c.419C>T did not associate with heart failure, arrhythmias, or premature death, with ARVC or HCM/DCM, or with effects in vitro, suggesting that this is not a disease-causing variant.

Cdon promotes neural crest migration by regulating N-cadherin localization

Neural crest cells (NCCs) are essential embryonic progenitor cells that are unique to vertebrates and form a remarkably complex and coordinated system of highly motile cells. Migration of NCCs occurs along specific pathways within the embryo in response to both environmental cues and cell–cell interactions within the neural crest population. Here, we demonstrate a novel role for the putative Sonic hedgehog (Shh) receptor and cell adhesion regulator, cdon, in zebrafish neural crest migration. cdon is expressed in developing premigratory NCCs but is downregulated once the cells become migratory. Knockdown of cdon results in aberrant migration of trunk NCCs: crestin positive cells can emigrate out of the neural tube but stall shortly after the initiation of migration. Live cell imaging analysis demonstrates reduced directedness of migration, increased velocity and mispositioned cell protrusions. In addition, transplantation analysis suggests that cdon is required cell-autonomously for directed NCC migration in the trunk. Interestingly, N-cadherin is mislocalized following cdon knockdown suggesting that the role of cdon in NCCs is to regulate N-cadherin localization. Our results reveal a novel role for cdon in zebrafish neural crest migration, and suggest a mechanism by which Cdon is required to localize N-cadherin to the cell membrane in migratory NCCs for directed migration.

Cardiac Development in Zebrafish and Human Embryonic Stem Cells Is Inhibited by Exposure to Tobacco Cigarettes and E-Cigarettes

BackgroundMaternal smoking is a risk factor for low birth weight and other adverse developmental outcomes.ObjectiveWe sought to determine the impact of standard tobacco cigarettes and e-cigarettes on heart development in vitro and in vivo.MethodsZebrafish (Danio rerio) were used to assess developmental effects in vivo and cardiac differentiation of human embryonic stem cells (hESCs) was used as a model for in vitro cardiac development.ResultsIn zebrafish, exposure to both types of cigarettes results in broad, dose-dependent developmental defects coupled with severe heart malformation, pericardial edema and reduced heart function. Tobacco cigarettes are more toxic than e-cigarettes at comparable nicotine concentrations. During cardiac differentiation of hESCs, tobacco smoke exposure results in a delayed transition through mesoderm. Both types of cigarettes decrease expression of cardiac transcription factors in cardiac progenitor cells, suggesting a persistent delay in differentiation. In definitive human cardiomyocytes, both e-cigarette- and tobacco cigarette-treated samples showed reduced expression of sarcomeric genes such as MLC2v and MYL6. Furthermore, tobacco cigarette-treated samples had delayed onset of beating and showed low levels and aberrant localization of N-cadherin, reduced myofilament content with significantly reduced sarcomere length, and increased expression of the immature cardiac marker smooth muscle alpha-actin.ConclusionThese data indicate a negative effect of both tobacco cigarettes and e-cigarettes on heart development in vitro and in vivo. Tobacco cigarettes are more toxic than E-cigarettes and exhibit a broader spectrum of cardiac developmental defects.

Desmoplakin is involved in organization of an adhesion complex in peripheral nerve regeneration after injury

Peripheral nerves have the unique capability to regenerate after injury. Insights into regeneration of peripheral nerves after injury may have implications for neurodegenerative diseases of the nervous system. In this study, we analyzed the expression and function of desmoplakin in peripheral nerve regeneration. Desmoplakin was upregulated in spinal cord motoneurons after sciatic nerve injury. Conditional ablation of desmoplakin in motoneurons demonstrated that desmoplakin is necessary for normal motor regeneration. SiRNA and desmoplakin deletion-constructs revealed a role of desmoplakin in neurite extension in vitro. A complex of N-cadherin, plakoglobin, desmoplakin and vimentin was shown in motoneuronal cell cultures and peripheral nerves after injury in vivo. Motor nerve fiber regeneration and localization of N-cadherin and vimentin to axonal growth fronts were reduced in conditionally desmoplakin-ablated mice.These data indicate a function of desmoplakin in motor nerve regeneration by linking N-cadherin to intermediate filaments in regenerating motor axons.

Dlg5 regulates dendritic spine formation and synaptogenesis by controlling subcellular N-cadherin localization.

Most excitatory synapses in the mammalian brain are formed on dendritic spines, and spine density has a profound impact on synaptic transmission, integration, and plasticity. Membrane-associated guanylate kinase (MAGUK) proteins are intracellular scaffolding proteins with well established roles in synapse function. However, whether MAGUK proteins are required for the formation of dendritic spines in vivo is unclear. We isolated a novel disc large-5 (Dlg5) allele in mice, Dlg5(LP), which harbors a missense mutation in the DLG5 SH3 domain, greatly attenuating its ability to interact with the DLG5 GUK domain. We show here that DLG5 is a MAGUK protein that regulates spine formation, synaptogenesis, and synaptic transmission in cortical neurons. DLG5 regulates synaptogenesis by enhancing the cell surface localization of N-cadherin, revealing a key molecular mechanism for regulating the subcellular localization of this cell adhesion molecule during synaptogenesis.

Desmoglein-2 interaction is crucial for cardiomyocyte cohesion and function.

We determined the contribution of the desmosomal cadherin desmoglein-2 to cell-cell cohesion in cardiomyocytes. In the intercalated disc, providing mechanical strength and electrical communication between adjacent cardiomyocytes, desmoglein-2 is closely associated with N-cadherin and gap junctions. We studied intercalated discs of HL-1 cardiomyocytes by immunostaining of desmoglein-2 and N-cadherin. Cohesion was measured using a liberase-based dissociation-assay and compared with cell-free single-molecule atomic force microscopy measurements. L-tryptophan caused irregular desmoglein-2 condensation, weakened cell-cell cohesion and impaired both homophilic desmoglein-2 and N-cadherin trans-interaction, whereas l-phenylalanine had no effect. L-tryptophan did not affect N-cadherin localization and its inhibitory effect on cell-cohesion and desmoglein-2 binding, but not on N-cadherin interaction, was blocked by a desmoglein-specific tandem peptide. Moreover, Ca(2+)-depletion, desmoglein-2 knockdown, a desmoglein-specific single peptide and certain desmoglein-2 mutations associated with arrhythmogenic cardiomyopathy reduced cell-cell cohesion, whereas cell adhesion was strengthened by desmoglein-2 overexpression. Since single peptide did not interfere with N-cadherin trans-interaction, these data indicate that (i) desmoglein-2 binding is crucial for cardiomyocyte cohesion and (ii) L-tryptophan reduced both desmoglein-2 and N-cadherin binding, whereas single and tandem peptide can be used to specifically target desmoglein-2-mediated adhesion. L-tryptophan and single peptide also induced ultrastructural alterations of areae compositae. Functional analyses at the organ level revealed reduced cardiomyocyte function and inefficient response to adrenergic stimulation in both L-tryptophan- and single peptide-challenged murine Langendorff hearts paralleled by redistribution of connexin 43 in L-tryptophan-treated heart slices. Our data demonstrate that desmoglein-2 plays a critical role in cardiomyocyte cohesion and function.

ADAM-10 could mediate cleavage of N-cadherin promoting apoptosis in human atherosclerotic lesions leading to vulnerable plaque: A morphological and immunohistochemical study

Atherosclerosis remains a major cause of mortality. Whereas the histopathological progression of atherosclerotic lesions is well documented, much less is known about the development of unstable or vulnerable plaque, which can rupture leading to thrombus, luminal occlusion and infarct. Apoptosis in the fibrous cap, which is rich in vascular smooth muscle cells (VSMCs) and macrophages, and its subsequent weakening or erosion seems to be an important regulator of plaque stability. The aim of our study was to improve our knowledge on the biological mechanisms that cause plaque instability in order to develop new therapies to maintain atherosclerotic plaque stability and avoid its rupture. In our study, we collected surgical specimens from atherosclerotic plaques in the right or left internal carotid artery of 62 patients with evident clinical symptoms. Histopathology and histochemistry were performed on wax-embedded sections. Immunohistochemical localization of caspase-3, N-cadherin and ADAM-10 was undertaken in order to highlight links between apoptosis, as expressed by caspase-3 immunostaining, and possible roles of N-cadherin, a cell-cell junction protein in VSMCs and macrophages that provides a pro-survival signal reducing apoptosis, and ADAM-10, a “disintegrin and metalloproteases” that is able to cleave N-cadherin in glioblastomas. Our results showed that when apoptosis, expressed by caspase-3 immunostaining, increased in the fibrous cap, rich in VSMCs and macrophages, the expression of N-cadherin decreased. The decreased N-cadherin expression, in turn, was linked to increased ADAM-10 expression. This study shows that apoptotic events are probably involved in the vulnerability of atherosclerotic plaque.

POPX2 phosphatase regulates cell polarity and centrosome placement

Proper centrosome positioning is critical for many cellular functions, such as cell migration and maintenance of polarity. During wound healing, fibroblasts orient their centrosomes such that they face the wound edge. The centrosome orientation determines the direction of cells’ migration so that they can close the wound effectively. In this study, we investigated the regulation of centrosome polarization and have identified the phosphatase POPX2 as an important regulator of centrosome orientation. We found that POPX2 inhibits centrosome centration, but not rearward nuclear movement, by regulating multiple proteins that function in centrosome positioning. High POPX2 levels result in reduced motility of the kinesin-2 motor, which, in turn, inhibits the transport of N-cadherin to the cell periphery and cell junctions. Loss of N-cadherin localization to the cell membrane affects the localization of focal adhesions and perturbs CDC42-Par6/PKCζ signaling. In addition, overexpression of POPX2 also results in a loss of Par3 localization to the cell periphery and reduced levels of LIC2 (dynein light intermediate chain 2), leading to defects in microtubule tethering and dynamics at cell-cell contacts. Therefore, POPX2 functions as a regulator of signaling pathways to modulate the positioning of centrosome in fibroblast during wound healing.

ICAM-2 regulates vascular permeability and N-cadherin localization through ezrin-radixin-moesin (ERM) proteins and Rac-1 signalling

BackgroundEndothelial junctions control functions such as permeability, angiogenesis and contact inhibition. VE-Cadherin (VECad) is essential for the maintenance of intercellular contacts. In confluent endothelial monolayers, N-Cadherin (NCad) is mostly expressed on the apical and basal membrane, but in the absence of VECad it localizes at junctions. Both cadherins are required for vascular development. The intercellular adhesion molecule (ICAM)-2, also localized at endothelial junctions, is involved in leukocyte recruitment and angiogenesis.ResultsIn human umbilical vein endothelial cells (HUVEC), both VECad and NCad were found at nascent cell contacts of sub-confluent monolayers, but only VECad localized at the mature junctions of confluent monolayers. Inhibition of ICAM-2 expression by siRNA caused the appearance of small gaps at the junctions and a decrease in NCad junctional staining in sub-confluent monolayers. Endothelioma lines derived from WT or ICAM-2-deficient mice (IC2neg) lacked VECad and failed to form junctions, with loss of contact inhibition. Re-expression of full-length ICAM-2 (IC2 FL) in IC2neg cells restored contact inhibition through recruitment of NCad at the junctions. Mutant ICAM-2 lacking the binding site for ERM proteins (IC2 ΔERM) or the cytoplasmic tail (IC2 ΔTAIL) failed to restore junctions. ICAM-2-dependent Rac-1 activation was also decreased in these mutant cell lines. Barrier function, measured in vitro via transendothelial electrical resistance, was decreased in IC2neg cells, both in resting conditions and after thrombin stimulation. This was dependent on ICAM-2 signalling to the small GTPase Rac-1, since transendothelial electrical resistance of IC2neg cells was restored by constitutively active Rac-1. In vivo, thrombin-induced extravasation of FITC-labeled albumin measured by intravital fluorescence microscopy in the mouse cremaster muscle showed that permeability was increased in ICAM-2-deficient mice compared to controls.ConclusionsThese results indicate that ICAM-2 regulates endothelial barrier function and permeability through a pathway involving N-Cadherin, ERMs and Rac-1.

Critical function of RA-GEF-2/Rapgef6, a guanine nucleotide exchange factor for Rap1, in mouse spermatogenesis

Small GTPase Rap1 has been implicated in the proper differentiation of testicular germ cells. In the present study, we investigated the functional significance of RA-GEF-2/Rapgef6, a guanine nucleotide exchange factor for Rap1, in testicular differentiation using mice lacking RA-GEF-2. RA-GEF-2 was expressed predominantly on the luminal side of the seminiferous tubules in wild-type mice. No significant differences were observed in the body weights or hormonal parameters of RA-GEF-2−/− and wild-type mice. However, the testes of RA-GEF-2−/− male mice were significantly smaller than those of wild-type mice and were markedly atrophied as well as hypospermatogenic. The concentration and motility of epididymal sperm were also markedly reduced and frequently had an abnormal shape. The pregnancy rate and number of fetuses were markedly lower in wild-type females after they mated with RA-GEF-2−/− males than with wild-type males, which demonstrated the male infertility phenotype of RA-GEF-2−/− mice. Furthermore, a significant reduction and alteration were observed in the expression level and cell junctional localization of N-cadherin, respectively, in RA-GEF-2−/− testes, which may, at least in part, account for the defects in testicular differentiation and spermatogenesis in these mice.

N-Cadherin Relocalizes from the Periphery to the Center of the Synapse after Transient Synaptic Stimulation in Hippocampal Neurons

N-cadherin is a cell adhesion molecule which is enriched at synapses. Binding of N-cadherin molecules to each other across the synaptic cleft has been postulated to stabilize adhesion between the presynaptic bouton and the postsynaptic terminal. N-cadherin is also required for activity-induced changes at synapses, including hippocampal long term potentiation and activity-induced spine expansion and stabilization. We hypothesized that these activity-dependent changes might involve changes in N-cadherin localization within synapses. To determine whether synaptic activity changes the localization of N-cadherin, we used structured illumination microscopy, a super-resolution approach which overcomes the conventional resolution limits of light microscopy, to visualize the localization of N-cadherin within synapses of hippocampal neurons. We found that synaptic N-cadherin exhibits a spectrum of localization patterns, ranging from puncta at the periphery of the synapse adjacent to the active zone to an even distribution along the synaptic cleft. Furthermore, the N-cadherin localization pattern within synapses changes during KCl depolarization and after transient synaptic stimulation. During KCl depolarization, N-cadherin relocalizes away from the central region of the synaptic cleft to the periphery of the synapse. In contrast, after transient synaptic stimulation with KCl followed by a period of rest in normal media, fewer synapses have N-cadherin present as puncta at the periphery and more synapses have N-cadherin present more centrally and uniformly along the synapse compared to unstimulated cells. This indicates that transient synaptic stimulation modulates N-cadherin localization within the synapse. These results bring new information to the structural organization and activity-induced changes occurring at synapses, and suggest that N-cadherin relocalization may contribute to activity dependent changes at synapses.

Effects of TGF-β2 on Cadherins and β-Catenin in Human Trabecular Meshwork Cells

The effects of TGF-β2 on cadherin-mediated cell-cell adhesion in human trabecular meshwork (HTM) cells were characterized, since TGF-β-induced changes in the cytoskeleton, cell-cell, and cell-matrix interactions of HTM cells are suggested to have a significant role in primary open angle glaucoma. The HTM cells were derived from donor cornea rings and treated with TGF-β2 or vehicle, and protein expression was studied by Western Blot, while protein localization was studied by fractionation of lysates and by confocal immunofluorescence microscopy. Cell-cell adhesion was assessed functionally in dissociation experiments and N-cadherin-mediated cell contact formation in cell spreading experiments on cadherin-coated substrates. A rho-associated protein kinase (ROCK) inhibitor was used to evaluate the contribution of cytoskeletal tension to TGF-β2-induced changes in protein expression and localization. TGF-β2 activated Smad-2/3, serine-threonine kinase (AKT), and extracellular signal-regulated kinase (ERK) signaling, and enhanced expression of β-catenin as well as N- and OB-cadherin. The nuclear fraction of β-catenin was not enhanced by TGF-β2. Immunofluorescence microscopy revealed an increased localization of N-cadherin and β-catenin to cell-cell adhesions, and an increase in F-actin. The TGF-β2 increased cell-cell adhesion strength and enhanced N-cadherin-mediated cell contact formation. This effect was blocked by inhibition of mitogen-activated protein kinase kinase (MEK) or AKT. Cytoskeletal relaxation by a ROCK inhibitor did not prevent a TGF-β2-induced increase in cadherin and β-catenin expression. The cytokine TGF-β2 enhances cadherin-mediated cell-cell adhesion and β-catenin expression in HTM cells. Increased cell-cell adhesion may contribute to biomechanical alterations in glaucomatous trabecular meshwork (TM), and changes in β-catenin levels and its possible sequestration to cell adhesion sites may affect Wnt signaling. Thus, the crosstalk of TGF-β2 and Wnt signaling in TM may deserve further investigation.

Cardiomyocyte FGF signaling is required for Cx43 phosphorylation and cardiac gap junction maintenance

Cardiac remodeling resulting from impairment of myocardial integrity leads to heart failure, through still incompletely understood mechanisms. The fibroblast growth factor (FGF) system has been implicated in tissue maintenance, but its role in the adult heart is not well defined. We hypothesized that the FGF system plays a role in the maintenance of cardiac homeostasis, and the impairment of cardiomyocyte FGF signaling leads to pathological cardiac remodeling.We showed that FGF signaling is required for connexin 43 (Cx43) localization at cell–cell contacts in isolated cardiomyocytes and COS7 cells. Lack of FGF signaling led to decreased Cx43 phosphorylation at serines 325/328/330 (S325/328/330), sites known to be important for assembly of gap junctions. Cx43 instability induced by FGF inhibition was restored by the Cx43 S325/328/330 phospho-mimetic mutant, suggesting FGF-dependent phosphorylation of these sites. Consistent with these in vitro findings, cardiomyocyte-specific inhibition of FGF signaling in adult mice demonstrated mislocalization of Cx43 at intercalated discs, whereas localization of N-cadherin and desmoplakin was not affected. This led to premature death resulting from impaired cardiac remodeling.We conclude that cardiomyocyte FGF signaling is essential for cardiomyocyte homeostasis through phosphorylation of Cx43 at S325/328/330 residues which are important for the maintenance of gap junction.