Adherens Junction E-cadherin Research Articles

HDAC-Mediated Glial Crosstalk Mediates Radiation Induced Memory Changes from Whole Brain Radiation in a Mouse Model

Prior work demonstrated radiation sensitivity can be regulated via Class III HDAC-mediated intracellular DNA damage response coordinated with Wnt-beta catenin signaling. We hypothesized that radiation induced functional alterations in HDAC mediated glial crosstalk in the CNS produce intercellular signaling alterations leading to phenotypic changes in cognition following whole brain radiation exposure. Primary human astrocytes, human astrocytoma (U251), and immortalized human microglia (HMC3) were examined in vitro in response to treatment with conditioned media isolated from irradiated glial cells. Prior to treatment with either single-dose or fractionated radiation schedules, cells were transfected with siRNA expression vector for Sirt2, a class III HDAC, versus scrambled controls. Quantitative PCR and immunoblots for neurotransmitters, metabolism, and inflammatory markers were obtained. Cells exposed to conditioned media were examined by immunofluorescence for cytoskeletal alterations and changes in junctional proteins. The findings were correlated with in vivo qPCR and immunohistochemical changes in brain tissue and functional memory changes in Sirt2 knockout versus wild type mice after whole brain radiation using novel object recognition testing at 2 weeks and 6 month post radiation timepoints. Statistical analysis was performed using paired Students T test between treated and control groups. Cells treated with conditioned media produced from irradiated U251, astrocytes and microglia produced increased expression of inflammatory cytokines IL-6, IL-8, GM-CSF and VEGF. However, immunofluorescence of cells treated with conditioned media from irradiated Sirt2 knockdown microglia and primary astrocytes demonstrated qualitative disruptions in glutamate neurotransmitter metabolism and actin cytoskeletal alterations with changes in pseudopodia and lamellipodia after staining with phalloidin and neurofilament L, suggesting altered intercellular communication. Connexin 43 in gap junctions was increased >2-fold (p<0.05) after exposure to conditioned media, whereas E-cadherin in adherens junctions was not significantly affected. Novel object recognition testing of Sirt2 knockout mice demonstrated resistance to radiation induced memory decline from whole brain radiation at pre radiation day 5 versus post radiation days 5 and 185 compared to controls (p<0.05). Glial crosstalk can be mediated via elimination of a Class III HDAC and appears to be a key mediator of radiation induced disruptions of intercellular communication in the CNS, connecting radiation to structural changes on the cell surface to synaptic activity and neurotransmitter metabolism, leading to functional disputations in recognition memory similar to what is experienced by patients receiving brain radiotherapy. These data suggest glial cross talk as a new therapeutic avenue to combat radiation induced cognitive dysfunction.

E-cadherin acts as a positive regulator of the JAK-STAT signaling pathway during Drosophila oogenesis.

The JAK-STAT pathway is evolutionary conserved. The simplicity of this signaling in Drosophila, due to the limited redundancy between pathway components, makes it an ideal model for investigation. In the Drosophila follicular epithelium, highly stereotyped functions of JAK-STAT signaling have been well characterized, but how signaling activity is regulated precisely to allow the different outcomes is not well understood. In this tissue, the ligand is secreted by the polar cells positioned at each follicle extremity, thus generating a gradient of JAK-STAT activity in adjacent cells. One way to control the delivered quantity of ligand is by regulating the number of polar cells, which is reduced by apoptosis to exactly two at each pole by mid-oogenesis. Hence, JAK-STAT activity is described as symmetrical between follicle anterior and posterior regions. Here, we show that JAK-STAT signaling activity is actually highly dynamic, resulting in asymmetry between poles by mid-oogenesis. Interestingly, we found similar temporal dynamics at follicle poles in the accumulation of the adherens junction E-cadherin protein. Remarkably, E-cadherin and JAK-STAT signaling not only display patterning overlaps but also share functions during oogenesis. In particular, we show that E-cadherin, like JAK-STAT signaling, regulates polar cell apoptosis non-cell-autonomously from follicle cells. Finally, our work reveals that E-cadherin is required for optimal JAK-STAT activity throughout oogenesis and that E-cadherin and Stat92E, the transcription factor of the pathway, form part of a physical complex in follicle cells. Taken together, our study establishes E-cadherin as a new positive regulator of JAK-STAT signaling during oogenesis.

Mast Cell Chymase/Mcpt4 Suppresses the Host Immune Response to Plasmodium yoelii, Limits Malaria-Associated Disruption of Intestinal Barrier Integrity and Reduces Parasite Transmission to Anopheles stephensi.

An increase in mast cells (MCs) and MCs mediators has been observed in malaria-associated bacteremia, however, the role of these granulocytes in malarial immunity is poorly understood. Herein, we studied the role of mouse MC protease (Mcpt) 4, an ortholog of human MC chymase, in malaria-induced bacteremia using Mcpt4 knockout (Mcpt4 -/-) mice and Mcpt4 +/+ C57BL/6J controls, and the non-lethal mouse parasite Plasmodium yoelii yoelii 17XNL. Significantly lower parasitemia was observed in Mcpt4 -/- mice compared with Mcpt4 +/+ controls by day 10 post infection (PI). Although bacterial 16S DNA levels in blood were not different between groups, increased intestinal permeability to FITC-dextran and altered ileal adherens junction E-cadherin were observed in Mcpt4 -/- mice. Relative to infected Mcpt4 +/+ mice, ileal MC accumulation in Mcpt4 -/- mice occurred two days earlier and IgE levels were higher by days 8-10 PI. Increased levels of circulating myeloperoxidase were observed at 6 and 10 days PI in Mcpt4 +/+ but not Mcpt4 -/- mice, affirming a role for neutrophil activation that was not predictive of parasitemia or bacterial 16S copies in blood. In contrast, early increased plasma levels of TNF-α, IL-12p40 and IL-3 were observed in Mcpt4 -/- mice, while levels of IL-2, IL-10 and MIP1β (CCL4) were increased over the same period in Mcpt4 +/+ mice, suggesting that the host response to infection was skewed toward a type-1 immune response in Mcpt4 -/- mice and type-2 response in Mcpt4 +/+ mice. Spearman analysis revealed an early (day 4 PI) correlation of Mcpt4 -/- parasitemia with TNF-α and IFN-γ, inflammatory cytokines known for their roles in pathogen clearance, a pattern that was observed in Mcpt4 +/+ mice much later (day 10 PI). Transmission success of P. y. yoelii 17XNL to Anopheles stephensi was significantly higher from infected Mcpt4 -/- mice compared with infected Mcpt4 +/+ mice, suggesting that Mcpt4 also impacts transmissibility of sexual stage parasites. Together, these results suggest that early MCs activation and release of Mcpt4 suppresses the host immune response to P. y. yoelii 17XNL, perhaps via degradation of TNF-α and promotion of a type-2 immune response that concordantly protects epithelial barrier integrity, while limiting the systemic response to bacteremia and parasite transmissibility.

Comparative Analysis of Cell-Cell Contact Abundance in Ovarian Carcinoma Cells Cultured in Two- and Three-Dimensional In Vitro Models.

Simple SummaryTumor resistance to therapy is a crucial problem of today’s oncology. The emerging data indicate that tumor microenvironment is the key participant in the resistance development. One of the most basic aspect of tumor microenvironment is intercellular adhesion. Our data obtained using monolayer culture, matrix-free and matrix-based three-dimensional in vitro models indicate that the abundance of cell-cell contact proteins is varying depending on the microenvironment. These differences coincided with the degree of the resistance to therapeutics. The importance of adhesion proteins in tumor resistance may provide the fundamental basis for improving cancer treatment approaches and must be taken into account when screening candidate drugs. Tumor resistance to therapy is associated with the 3D organization and peculiarities of the tumor microenvironment, of which intercellular adhesion is a key participant. In this work, the abundance of contact proteins was compared in SKOV-3 and SKOV-3.ip human ovarian adenocarcinoma cell lines, cultivated in monolayers, tumor spheroids and collagen hydrogels. Three-dimensional models were characterized by extremely low expression of basic molecules of adherens junctions E-cadherin and demonstrated a simultaneous decrease in desmosomal protein desmoglein-2, gap junction protein connexin-43 and tight junction proteins occludin and ZO-1. The reduction in the level of contact proteins was most pronounced in collagen hydrogel, accompanied by significantly increased resistance to treatment with doxorubicin and targeted anticancer toxin DARPin-LoPE. Thus, we suggest that 3D models of ovarian cancer, especially matrix-based models, tend to recapitulate tumor microenvironment and treatment responsiveness to a greater extent than monolayer culture, so they can be used as a highly relevant platform for drug efficiency evaluation.

Longan pulp polysaccharides relieve intestinal injury in vivo and in vitro by promoting tight junction expression.

The integrity of the intestinal mucosal barrier is important for the health of the host. In this study, longan pulp polysaccharides (LP) prevented the intestinal mucosal injury by increasing the expression of mucin 2, tight junction proteins zonulae occludens-1 (ZO-1), claudin-1, claudin-4, and adherens junction E-cadherin in cyclophosphamide-treated mice. To further identify the principle bioactive component of LP, four acidic polysaccharides (LPIa, LPIIa, LPIIIa, and LPIVa) were purified, and their intestinal protection activity in vitro was compared. LPIa, LPIIa, and LPIIIa displayed an ability to increase mRNA expression of ZO-1, claudin-1, occludin, and E-cadherin in differentiated Caco-2 cells, especially LPIa. LPIa has specific structure characteristics: porous surface structure, a high molecular weight (1.47 × 105 Da), and two specific glycosidic linkages of α-Araf-(1→ and →5)-α-Araf-(1→. These structure characteristics might primarily contribute to greater intestinal barrier protective effect of LPIa.

E-cadherin expression pattern during zebrafish embryonic epidermis development

Background: E-cadherin is the major adhesion receptor in epithelial adherens junctions (AJs). On established epidermis, E-cadherin performs fine-tuned cell-cell contact remodeling to maintain tissue integrity, which is characterized by modulation of cell shape, size and packing density. In zebrafish, the organization and distribution of E-cadherin in AJs during embryonic epidermis development remain scarcely described. Methods: Combining classical immunofluorescence, deconvolution microscopy and 3D-segmentation of AJs in epithelial cells, a quantitative approach was implemented to assess the spatial and temporal distribution of E-cadherin across zebrafish epidermis between 24 and 72 hpf. Results: increasing levels of E-cadh protein parallel higher cell density and the appearance of hexagonal cells in the enveloping layer (EVL) as well as the establishments of new cell-cell contacts in the epidermal basal layer (EBL), being significantly between 31 and 48 hpf . Conclusions: Increasing levels of E-cadherin in AJs correlates with extensive changes in cell morphology towards hexagonal packing during the epidermis morphogenesis.

Deficiency of cathepsin C ameliorates severity of acute pancreatitis by reduction of neutrophil elastase activation and cleavage of E-cadherin

Acute pancreatitis is characterized by premature intracellular protease activation and infiltration of inflammatory cells, mainly neutrophil granulocytes and macrophages, into the organ. The lysosomal proteases cathepsin B, D, and L have been identified as regulators of early zymogen activation and thus modulators of the severity of pancreatitis. Cathepsin C (CTSC, syn. dipeptidly-peptidase I) is a widely expressed, exo-cystein-protease involved in the proteolytic processing of various other lysosomal enzymes. We have studied its role in pancreatitis. We used CTSC-deleted mice and their WT littermates in two experimental models of pancreatitis. The mild model involved eight hourly caerulein injections and the severe model partial duct ligation. Isolated pancreatic acini and spleen-derived leukocytes were used for ex vivo experiments. CTSC is expressed in the pancreas and in inflammatory cells. CTSC deletion reduced the severity of pancreatitis (more prominently in the milder model) without directly affecting intra-acinar cell trypsin activation in vitro The absence of CTSC reduced infiltration of neutrophil granulocytes impaired their capacity for cleaving E-cadherin in adherens junctions between acinar cells and reduced the activity of neutrophil serine proteases polymorphonuclear (neutrophil) elastase, cathepsin G, and proteinase 3, but not neutrophil motility. Macrophage invasion was not dependent on the presence of CTSC. CTSC is a regulator and activator of various lysosomal enzymes such as cathepsin B, D, and L. Its loss mitigates the severity of pancreatitis not by reducing intra-acinar cell zymogen activation but by reducing infiltration of neutrophil granulocytes into the pancreas. In this context one of its key roles is that of an activator of neutrophil elastase.

E-cadherin expression pattern during zebrafish embryonic epidermis development

Background: E-cadherin is the major adhesion receptor in epithelial adherens junctions (AJs). On established epidermis, E-cadherin performs fine-tuned cell-cell contact remodeling to maintain tissue integrity, which is characterized by modulation of cell shape, size and packing density. In zebrafish, the organization and distribution of E-cadherin in AJs during embryonic epidermis development remain scarcely described.Methods:Combining classical immunofluorescence, deconvolution microscopy and 3D-segmentation of AJs in epithelial cells, a quantitative approach was implemented to assess the spatial and temporal distribution of E-cadherin across zebrafish epidermis between 24 and 72 hpf.Results:increasing levels of E-cadh protein parallel higher cell density and the appearance of hexagonal cells in the enveloping layer (EVL) as well as the establishments of new cell-cell contacts in the epidermal basal layer (EBL), being significantly between 31 and 48 hpf.Conclusions:Increasing levels of E-cadherin in AJs correlates with extensive changes in cell morphology towards hexagonal packing during the epidermis morphogenesis.

E-cadherin expression pattern during zebrafish embryonic epidermis development

Background: E-cadherin is the major adhesion receptor in epithelial adherens junctions (AJs). On established epidermis, E-cadherin performs fine-tuned cell-cell contact remodeling to maintain tissue integrity, which is characterized by modulation of cell shape, size and packing density. In zebrafish, the organization and distribution of E-cadherin in AJs during embryonic epidermis development remain scarcely described. Methods: Combining classical immunofluorescence, deconvolution microscopy and 3D-segmentation of AJs in epithelial cells, a quantitative approach was implemented to assess the spatial and temporal distribution of E-cadherin across zebrafish epidermis between 24 and 72 hpf. Results: increasing levels of E-cadh protein parallel higher cell density and the appearance of hexagonal cells in the enveloping layer (EVL) as well as the establishments of new cell-cell contacts in the epidermal basal layer (EBL), being significantly between 31 and 48 hpf . Conclusions: Increasing levels of E-cadherin in AJs correlates with extensive changes in cell morphology towards hexagonal packing during the epidermis morphogenesis.

E-cadherin expression pattern during zebrafish embryonic epidermis development.

Background: E-cadherin is the major adhesion receptor in epithelial adherens junctions (AJs). On established epidermis, E-cadherin performs fine-tuned cell-cell contact remodeling to maintain tissue integrity, which is characterized by modulation of cell shape, size and packing density. In zebrafish, the organization and distribution of E-cadherin in AJs during embryonic epidermis development remain scarcely described. Methods: Combining classical immunofluorescence, deconvolution microscopy and 3D-segmentation of AJs in epithelial cells, a quantitative approach was implemented to assess the spatial and temporal distribution of E-cadherin across zebrafish epidermis between 24 and 72 hpf. Results: increasing levels of E-cadh protein parallel higher cell density and the appearance of hexagonal cells in the enveloping layer (EVL) as well as the establishments of new cell-cell contacts in the epidermal basal layer (EBL), being significantly between 31 and 48 hpf .Conclusions: Increasing levels of E-cadherin in AJs correlates with extensive changes in cell morphology towards hexagonal packing during the epidermis morphogenesis.

TSLP signaling blocking alleviates E-cadherin dysfunction of airway epithelium in a HDM-induced asthma model

Recent studies have indicated that Thymic stromal lymphopoietin (TSLP) plays an important role in the prevention and treatment of asthma. However the role of TSLP in dysfunction of airway epithelial adherens junctions E-cadherin in house dust mite (HDM)-induced asthma has not been addressed. We hypothesized that TSLP contributed to HDM-induced E-cadherin dysfunction in asthmatic BALB/c mice and 16HBE cells. In vivo, a HDM-induced asthma mouse model was set up for 8weeks. Mice inhaled an anti-TSLP monoclonal antibody (mAb) before HDM. The mice treated with the anti-TSLP mAb ameliorated airway inflammation, the decreasing and aberrant distribution of E-cadherin and β-catenin as well as phosphorylation(p)-AKT induced by HDM. In vitro, HDM increased the expression of TSLP and E-cadherin dysfunction by PI3K/Akt signaling pathway. The exposure of 16HBE to TSLP resulted in redistribution of E-cadherin. These results indicate that TSLP may be an important contributor in E-cadherin dysfunction of HDM-induced asthma. TSLP signaling blocking shows a protective effect in mice and that the PI3K/Akt pathway may play a role in this process.

H19 Long Noncoding RNA Regulates Intestinal Epithelial Barrier Function via MicroRNA 675 by Interacting with RNA-Binding Protein HuR.

The disruption of the intestinal epithelial barrier function occurs commonly in various pathologies, but the exact mechanisms responsible are unclear. The H19 long noncoding RNA (lncRNA) regulates the expression of different genes and has been implicated in human genetic disorders and cancer. Here, we report that H19 plays an important role in controlling the intestinal epithelial barrier function by serving as a precursor for microRNA 675 (miR-675). H19 overexpression increased the cellular abundance of miR-675, which in turn destabilized and repressed the translation of mRNAs encoding tight junction protein ZO-1 and adherens junction E-cadherin, resulting in the dysfunction of the epithelial barrier. Increasing the level of the RNA-binding protein HuR in cells overexpressing H19 prevented the stimulation of miR-675 processing from H19, promoted ZO-1 and E-cadherin expression, and restored the epithelial barrier function to a nearly normal level. In contrast, the targeted deletion of HuR in intestinal epithelial cells enhanced miR-675 production in the mucosa and delayed the recovery of the gut barrier function after exposure to mesenteric ischemia/reperfusion. These results indicate that H19 interacts with HuR and regulates the intestinal epithelial barrier function via the H19-encoded miR-675 by altering ZO-1 and E-cadherin expression posttranscriptionally.

‘Farm dust extract increases the epithelial barrier function of human bronchial epithelial cells partly via MyD88 signalling’

Abstract Epidemiological studies showed that farmer’s children have a lower incidence of childhood asthma, which was partly attributed to a higher microbial exposure. Recently, distorted airway epithelium is considered an active player in pathogenesis of asthma. Interestingly, Toll-like receptor (TLR) ligation in the gut was found to enhance the gut epithelial barrier function. We hypothesize that exposure of the airways to microbial-rich farm dust may have barrier-enhancing effects on the airway epithelium and thereby may contribute to the protection against asthma in farmer’s children. In this study, the effect of farm dust extracts (FDE) collected from traditional German farms was investigated on barrier function and -repair in primary bronchial epithelial cells (PBECs). The expression of tight (TJs) and adherens junctions (AJs)-important to maintain the barrier function- and the involvement of TLR-induced MyD88 signalling were investigated. FDE exposure induced a dose-dependent two-fold increase in resistance measured by ECIS and up to 70% reduction in permeability in undifferentiated PBECs. Furthermore, restoration of barrier function of fully differentiated PBECs after cigarette smoke exposure was accelerated in the presence of FDE. The increase in barrier function was accompanied by an 1.5 to 2-fold enhanced mRNA expression of TJs occludin-1, claudin-1 and −4 and AJ E-cadherin. In search for the underlying mechanisms, we found that the increased barrier resistance could be significantly attenuated by blocking MyD88 signalling. These data altogether suggest that FDE exposure strengthen the epithelial barrier function partly via MyD88 signalling and, thereby, possibly reduce the risk of development of asthma and allergies.

Mechanical Induction of the Tumorigenic β-Catenin Pathway by Tumour Growth Pressure in vivo

The tumour microenvironment may contribute to tumorigenesis due to mechanical forces such as fibrotic stiffness or mechanical pressure caused by the expansion of hyper-proliferative cells. Here we explore the contribution of the mechanical pressure exerted by tumour growth onto non-tumorous adjacent epithelium. In the early stage of mouse colon tumour development in the Notch+Apc+/1638N mouse model, we observed mechanistic pressure stress in the non-tumorous epithelial cells caused by hyper-proliferative adjacent crypts overexpressing active Notch, associated with increased Ret and beta-catenin signalling. We thus developed a method that allows the delivery of a defined mechanical pressure in vivo, by subcutaneously inserting a magnet close to the mouse colon. The implanted magnet generated a magnetic force on ultra-magnetic liposomes, stabilized in the mesenchymal cells of the connective tissue surrounding colonic crypts after intravenous injection. The magnetically induced pressure quantitatively mimicked the endogenous early tumour growth stress in the order of 1,200 Pa, without affecting tissue stiffness, as monitored by acoustic strain imaging and shear wave elastography. Exertion of pressure mimicking that of tumour growth led to rapid Ret activation and downstream phosphorylation of beta-catenin on Tyr654, which impairs its interaction with the E-cadherin in adherens junctions, and which was followed by beta-catenin nuclear translocation after 15 days. As a consequence, elevated expression of beta-catenin-target genes was observed at 1 month, together with crypt enlargement accompanying the formation of early tumorous aberrant crypt foci. Mechanical activation of the tumorigenic beta-catenin pathway, conserved from early embryos beta-catenin dependent mechanical induction of patterning-genes expression, suggests unexplored modes of tumour propagation based on mechanical signalling pathways in healthy epithelial cells surrounding the tumour, which may contribute to tumour heterogeneity.Fernandez-Sanchez, Barbier et al, Nature 2015, Jul 2;523(7558):92-5. http://dx.doi.org/10.1038/nature14329. Epub 2015 May 11.

Mechanical induction of the tumorigenic β-catenin pathway by tumour growth pressure.

The tumour microenvironment may contribute to tumorigenesis owing to mechanical forces such as fibrotic stiffness or mechanical pressure caused by the expansion of hyper-proliferative cells. Here we explore the contribution of the mechanical pressure exerted by tumour growth onto non-tumorous adjacent epithelium. In the early stage of mouse colon tumour development in the Notch(+)Apc(+/1638N) mouse model, we observed mechanistic pressure stress in the non-tumorous epithelial cells caused by hyper-proliferative adjacent crypts overexpressing active Notch, which is associated with increased Ret and β-catenin signalling. We thus developed a method that allows the delivery of a defined mechanical pressure in vivo, by subcutaneously inserting a magnet close to the mouse colon. The implanted magnet generated a magnetic force on ultra-magnetic liposomes, stabilized in the mesenchymal cells of the connective tissue surrounding colonic crypts after intravenous injection. The magnetically induced pressure quantitatively mimicked the endogenous early tumour growth stress in the order of 1,200 Pa, without affecting tissue stiffness, as monitored by ultrasound strain imaging and shear wave elastography. The exertion of pressure mimicking that of tumour growth led to rapid Ret activation and downstream phosphorylation of β-catenin on Tyr654, imparing its interaction with the E-cadherin in adherens junctions, and which was followed by β-catenin nuclear translocation after 15 days. As a consequence, increased expression of β-catenin-target genes was observed at 1 month, together with crypt enlargement accompanying the formation of early tumorous aberrant crypt foci. Mechanical activation of the tumorigenic β-catenin pathway suggests unexplored modes of tumour propagation based on mechanical signalling pathways in healthy epithelial cells surrounding the tumour, which may contribute to tumour heterogeneity.

Roles of lncRNA in breast cancer

Epithelial-to-mesenchimal transition (EMT) is a process involved in invasion and metastasis of tumors. The occurrence of EMT during tumor progression resembles the developmental scenario and sheds light on important mechanisms for the initial step of metastasis - invasion where noninvasive tumor cells acquire motility and ultimately disseminate to distant organs. The hallmark of EMT is the loss of expression of the cell-cell adhesion molecule E-cadherin. The numerous reports by many authors as well as our own results indicate that E-cadherin plays a role in CNS tumors - meningiomas. Our studies showed that 73 % of meningiomas had downregulation of E-cadherin. Moreover, loss of heterozygosity of E-cadherin was observed in 32 % of meningiomas. Bound to E-cadherin in adherens junctions is beta-catenin, whose translocation to the nucleus is yet another molecular event involved in EMT. In our study beta-catenin was progressively upregulated from meningothelial to atypical, while 60 % of anaplastic meningiomas showed upregulation and nuclear localization of the protein. The elucidation of molecular mechanisms that govern EMT will offer new approaches and targets to restrain metastasis.

An innovative three-dimensional model of normal human skin to study the proinflammatory psoriatic effects of tumor necrosis factor-alpha and interleukin-17

BackgroundAmong all cytokines involved in the pathogenesis and in the progression of psoriasis, Tumor Necrosis Factor (TNF)-alpha and interleukin (IL)-17 play a pivotal role. ObjectiveThe aim of the present study was to mimic a psoriatic microenvironment and to investigate the early effects of TNF-alpha and IL-17 in a three-dimensional model of organotypic normal human skin. MethodsHuman skin explants were obtained from plastic aesthetic surgery of healthy young women 20–40years old (n=7). The study was approved by the Institutional Review Board and written informed consent was obtained from all subjects. Bioptic fragments were cultured at the air–liquid interface overnight in a Transwell system and further divided before adding either 50ng/ml IL-17 or 100ng/ml TNF-alpha or a combination of both cytokines. For each subject, a control sample was cultured without any cytokine. Samples were harvested 24 or 48h after cytokine incubation. At both time points and for all cytokine treatments a bioptic fragment obtained from each patient was processed. Epidermal proliferation, expressions of terminal differentiation (keratin 10, K10, and 14, K14) and of intercellular adhesion (occludin for tight junctions and E-cadherin for adherens junctions) biomarkers were investigated by indirect immunofluorescence. ResultsIL-17 and TNF-alpha induced an early and statistically significant inhibition of keratinocyte proliferation (more than 80% compared with their respective controls). At 24h, the combination of both cytokines did not further reduce cell proliferation. Starting from 24h of incubation, a non-continuous occludin expression in the granular layer was observed after both IL-17 and TNF-alpha exposure. Immunolabelling for E-cadherin in adherens junctions, for K10 in the suprabasal layers, and for K14 in the basal layer was similar in all experimental groups and unaffected after cytokine treatment. ConclusionsThese results suggest that in this experimental model IL-17 and TNF-alpha induced an early alteration of the homeostasis of the inner proliferative layer and of the upper granular layer, as shown by cell proliferation inhibition and occludin expression.

Protein Ser/Thr phosphatase-6 is required for maintenance of E-cadherin at adherens junctions

BackgroundEpithelial tissues depend on intercellular homodimerization of E-cadherin and loss of E-cadherin is central to the epithelial to mesenchymal transition seen in multiple human diseases. Signaling pathways regulate E-cadherin function and cellular distribution via phosphorylation of the cytoplasmic region by kinases such as casein kinases but the protein phosphatases involved have not been identified.ResultsThis study shows protein Ser/Thr phosphatase-6 catalytic subunit (PP6c) is expressed in epithelial tissue and its mRNA and protein are robustly up-regulated in epithelial cell lines at high vs. low density. PP6c accumulates at adherens junctions, not tight junctions, co-immunoprecipitates with E-cadherin-catenin complexes without a canonical SAPS subunit, and associates directly with the E-cadherin cytoplasmic tail. Inducible shRNA knockdown of PP6c dispersed E-cadherin from the cell surface and this response was reversed by chemical inhibition of casein kinase-1 and prevented by alanine substitution of Ser846 in murine E-cadherin.ConclusionsPP6c associates with E-cadherin in adherens junctions and is required to oppose casein kinase-1 to maintain cell surface localization of E-cadherin. There is feedback signaling to enhance PP6c transcription and boost protein levels in high density epithelial cells.

Epithelial-to-mesenchymal transition possible role in meningiomas

Epithelial-to-mesenchimal transition (EMT) is a process involved in invasion and metastasis of tumors. The occurrence of EMT during tumor progression resembles the developmental scenario and sheds light on important mechanisms for the initial step of metastasis - invasion where noninvasive tumor cells acquire motility and ultimately disseminate to distant organs. The hallmark of EMT is the loss of expression of the cell-cell adhesion molecule E-cadherin. The numerous reports by many authors as well as our own results indicate that E-cadherin plays a role in CNS tumors - meningiomas. Our studies showed that 73 % of meningiomas had downregulation of E-cadherin. Moreover, loss of heterozygosity of E-cadherin was observed in 32 % of meningiomas. Bound to E-cadherin in adherens junctions is beta-catenin, whose translocation to the nucleus is yet another molecular event involved in EMT. In our study beta-catenin was progressively upregulated from meningothelial to atypical, while 60 % of anaplastic meningiomas showed upregulation and nuclear localization of the protein. The elucidation of molecular mechanisms that govern EMT will offer new approaches and targets to restrain metastasis.

Integrin α3β1–dependent β-catenin phosphorylation links epithelial Smad signaling to cell contacts

Injury-initiated epithelial to mesenchymal transition (EMT) depends on contextual signals from the extracellular matrix, suggesting a role for integrin signaling. Primary epithelial cells deficient in their prominent laminin receptor, α3β1, were found to have a markedly blunted EMT response to TGF-β1. A mechanism for this defect was explored in α3-null cells reconstituted with wild-type (wt) α3 or point mutants unable to engage laminin 5 (G163A) or epithelial cadherin (E-cadherin; H245A). After TGF-β1 stimulation, wt epithelial cells but not cells expressing the H245A mutant internalize complexes of E-cadherin and TGF-β1 receptors, generate phospho-Smad2 (p-Smad2)–pY654–β-catenin complexes, and up-regulate mesenchymal target genes. Although Smad2 phosphorylation is normal, p-Smad2–pY654–β-catenin complexes do not form in the absence of α3 or when α3β1 is mainly engaged on laminin 5 or E-cadherin in adherens junctions, leading to attenuated EMT. These findings demonstrate that α3β1 coordinates cross talk between β-catenin and Smad signaling pathways as a function of extracellular contact cues and thereby regulates responses to TGF-β1 activation.