Epithelial Junction Proteins Research Articles

Progesterone-based intrauterine device use is associated with a thinner apical layer of the human ectocervical epithelium and a lower ZO-1 mRNA expression.

Currently, whether hormonal contraceptives affect male to female human immunodeficiency virus (HIV) transmission is being debated. In this study, we investigated whether the use of progesterone-based intrauterine devices (pIUDs) is associated with a thinning effect on the ectocervical squamous epithelium, down-regulation of epithelial junction proteins, and/or alteration of HIV target cell distribution in the human ectocervix. Ectocervical tissue biopsies from healthy premenopausal volunteers using pIUDs were collected and compared to biopsies obtained from two control groups, namely women using combined oral contraceptives (COCs) or who do not use hormonal contraceptives. In situ staining and image analysis were used to measure epithelial thickness and the presence of HIV receptors in tissue biopsies. Messenger RNA levels of epithelial junction markers were measured by quantitative PCR. The epithelial thickness displayed by women in the pIUD group was similar to those in the COC group, but significantly thinner as compared to women in the no hormonal contraceptive group. The thinner epithelial layer of the pIUD group was specific to the apical layer of the ectocervix. Furthermore, the pIUD group expressed significantly lower levels of the tight junction marker ZO-1 within the epithelium as compared to the COC group. Similar expression levels of HIV receptors and coreceptors CD4, CCR5, DC-SIGN, and Langerin were observed in the three study groups. Thus, women using pIUD displayed a thinner apical layer of the ectocervical epithelium and reduced ZO-1 expression as compared to control groups. These data suggest that pIUD use may weaken the ectocervical epithelial barrier against invading pathogens, including HIV.

Antitumor progression potential of morusin suppressing STAT3 and NFκB in human hepatoma SK-Hep1 cells

Morusin is a prenylated flavonoid that has been isolated from the root bark of the mulberry tree (Morus species, Moraceae), a Chinese traditional medicine. It has been synthesized by our laboratory from commercially available phloroglucinol, and has demonstrated to possess antitumor effects of cell lines including A549, MCF-7, and MDA-MB-231. In this study, at non-cytotoxic concentrations, morusin altered invasive morphology and suppressed cell-matrix adhesion, cell motility and cell invasion in SK-Hep1 cells. Morusin also increased the expression of E-cadherin, an epithelial cell junction protein, decreased the expression of vimentin, a mesecnchymal marker, and α2-, α6-, β1- integrin, which regulated cancer attachment and migration. In addition, morusin reduced the activity of matrix metalloproteinase-2 and 9 (MMP-2 and MMP-9), which were involved in extracellular matrix (ECM) degradation and promoting cancer cell invasion. Furthermore, morusin suppressed the signal transducer and activator of transcription 3 (STAT3) and nuclear factor-κB (NFκB) signaling pathways, which modulate the protein expression involved in the invasion process. Finally, morusin decreased the lung colonization of the SK-Hep1 cells in the nude mice. These results indicate morusin possesses antitumor progression potential through suppressing STAT3 and NFκB.

P19 The Role Of Differential Tnfr Signalling In Maintenance Of Alveolar Epithelial Homeostasis

Alveolar epithelial activation and disruption of the alveolar epithelial barrier promote recruitment of neutrophils into the alveolar space and cause alveolar oedema respectively thereby playing key roles in the pathogenesis of Acute Respiratory Distress Syndrome (ARDS). Tumour necrosis factor alpha (TNF) is an early mediator of inflammation in ARDS. TNF signals through two cell surface receptors, TNFR1 and TNFR2 initiating distinct signalling pathways and cellular responses. Using a novel, highly selective TNFR1 domain antibody (dAb™), a dummy dAb and the dual TNFR antagonist Adalimumab™, we investigated the role of differential TNFR signalling on human pulmonary alveolar epithelial cell (human alveolar type 2 cells and A549 cell line) activation, permeability and repair. Human alveolar epithelial type 2 cells (haT2) expressed both TNFR, whilst A549 cells only expressed TNFR1. TNFR1 signalling mediated release of the neutrophil chemokines IL-8 and GMCSF as well as IL-6 in hAT2 (pin vitro scratch model of epithelial (haT2) wound repair. TNFR1 signalling induced pro-inflammatory cytokine expression from alveolar epithelial cells and mediated increased epithelial permeability. TNFR1 induced permeability did not appear to be due to disruption of epithelial junctional proteins; we speculate that this may alternatively be due to TNFR1 induced cell death.

Vasopressin regulation of epithelial colonic proliferation and permeability is mediated by pericryptal platelet-derived growth factor A.

Arginine vasopressin (AVP) has trophic effects on the rat distal colon, increasing the growth of pericryptal myofibroblasts and reducing the colonic crypt wall permeability. This study aimed to reproduce in vitro the effects of AVP observed in vivo using cultures of human CCD-18Co myofibroblasts and T84 colonic epithelial cells. Proliferation of myofibroblasts was quantified by bromodeoxyuridine incorporation; the expression of platelet-derived growth factorA (PDGFA), platelet-derived growth factorB, epidermal growth factor, transforming growth factor-β and vascular endothelial growth factor was measured by PCR and the expression of epithelial junction proteins by Western blot. Arginine vasopressin stimulated myofibroblast proliferation and the expression of PDGFA without affecting the expression of platelet-derived growth factorB, epidermal growth factor, transforming growth factor-β or vascular endothelial growth factor. These effects were prevented when AVP receptor inhibitors were present in the medium. Pre-incubation of CCD-18Co cells with anti-PDGF antibody or with an inhibitor of the PDGF receptor abolished the effects of AVP. When colonocytes were incubated with medium obtained from myofibroblasts incubated with AVP, both cell proliferation and the expression of epithelial junction proteins increased; however, direct incubation of colonocytes with AVP did not modify these variables. These results demonstrate that AVP stimulates myofibroblast proliferation and induces PDGFA secretion, implying that PDGFA mediates local myofibroblast proliferation by an autocrine feedback loop and regulates epithelial proliferation and permeability by a paracrine mechanism.

Immunohistological characterization of intercellular junction proteins in rhesus macaque intestine

Epithelial junctions play an important role in regulating paracellular permeability and intercellular adhesion. It has been reported that changes in the density of epithelial junctions and/or distribution pattern can contribute to various gastrointestinal (GI) disorders. In this study, we investigated the distribution of the tight junction (Claudins. 1, 3, 4, 5, 7, 10, Zonula Occludens (ZO-1), Occludin), adherens junction (E-cadherin), desmosome (Desmoglein 2, Desmocollin 2) and gap junction (Connexin 43) proteins in the jejunum, ileum and colonic epithelium of healthy rhesus macaques (RM) using immunofluorescence labeling. While proteins in these respective junctions were expressed throughout the jejunum, ileum and colon of RM, we observed differential labeling in epithelial cells from these sites. Claudins 1, 3, 4, 7, E-cadherin and Desmoglein 2 were distributed in the respective intercellular junctions with additional labeling in the lateral membrane of epithelial cells in both small and large intestine. However, claudin 5, claudin 10, ZO-1 and occludin showed uniform distribution in the intercellular junctions of crypt and surface epithelial cells of the intestine. Desmocollin 2 localized predominantly in the upper two thirds along the lateral membrane while desmoglein 2 was distributed along the entire lateral membrane of intestinal epithelial cells. In contrast, connexin 43 exhibited punctate lateral labeling in crypt epithelial cells of the small and large intestine. Our results show diverse localization of epithelial intercellular junction proteins along the intestinal tract of RM. These findings may correlate with differences in paracellular permeability and adhesion along the intestinal tract and could correlate with pathologic disease in these regions of the intestine.

Impaired epithelial integrity in the duodenal mucosa in early stages of celiac disease

The small-bowel mucosal damage characteristic of celiac disease (CD) develops from normal villus morphology to inflammation and finally to villus atrophy with crypt hyperplasia. Patients with early stage CD may already suffer from abdominal symptoms before the development of villus atrophy. Although epithelial junctional integrity is compromised in overt disease, the appearance of such changes in early phases of the disorder is not known. We investigated whether alterations in epithelial junction protein expression occur already in early stage CD with normal mucosal morphology, and whether this correlates with inflammation indicators and clinical symptoms. The study involved 10 patients with early stage and 10 patients with overt villus atrophy that were followed yearly according to the study protocol. As controls, 20 nonceliac subjects were included. The expression of junction proteins (occludin, claudin 3, zonula occludens 1, and E-cadherin) was studied in small-intestinal biopsies using immunohistochemistry and Western blot. The correlation between junctional proteins and mucosal morphology, autoantibodies, the number of intraepithelial lymphocytes (IELs), and gastrointestinal symptoms was assessed. The expression of all junction proteins was already decreased in early stage CD when compared with nonceliac controls (P<0.05). Junction protein expression correlated positively with mucosal villus morphology and negatively with the number of IELs, the intensity of small-intestinal autoantibody deposits, and serum autoantibodies. The expression of claudin 3 showed a negative correlation with diarrheal score (R=-0.314, P=0.04). These findings show that the mucosal epithelial integrity is disrupted already in early stage CD before the disorder progresses to full-blown enteropathy.

Transmural intestinal wall permeability in severe ischemia after enteral protease inhibition.

In intestinal ischemia, inflammatory mediators in the small intestine's lumen such as food byproducts, bacteria, and digestive enzymes leak into the peritoneal space, lymph, and circulation, but the mechanisms by which the intestinal wall permeability initially increases are not well defined. We hypothesize that wall protease activity (independent of luminal proteases) and apoptosis contribute to the increased transmural permeability of the intestine's wall in an acutely ischemic small intestine. To model intestinal ischemia, the proximal jejunum to the distal ileum in the rat was excised, the lumen was rapidly flushed with saline to remove luminal contents, sectioned into equal length segments, and filled with a tracer (fluorescein) in saline, glucose, or protease inhibitors. The transmural fluorescein transport was determined over 2 hours. Villi structure and epithelial junctional proteins were analyzed. After ischemia, there was increased transmural permeability, loss of villi structure, and destruction of epithelial proteins. Supplementation with luminal glucose preserved the epithelium and significantly attenuated permeability and villi damage. Matrix metalloproteinase (MMP) inhibitors (doxycycline, GM 6001), and serine protease inhibitor (tranexamic acid) in the lumen, significantly reduced the fluorescein transport compared to saline for 90 min of ischemia. Based on these results, we tested in an in-vivo model of hemorrhagic shock (90 min 30 mmHg, 3 hours observation) for intestinal lesion formation. Single enteral interventions (saline, glucose, tranexamic acid) did not prevent intestinal lesions, while the combination of enteral glucose and tranexamic acid prevented lesion formation after hemorrhagic shock. The results suggest that apoptotic and protease mediated breakdown cause increased permeability and damage to the intestinal wall. Metabolic support in the lumen of an ischemic intestine with glucose reduces the transport from the lumen across the wall and enteral proteolytic inhibition attenuates tissue breakdown. These combined interventions ameliorate lesion formation in the small intestine after hemorrhagic shock.

The Expression of Epithelial Intercellular Junctional Proteins in the Sinonasal Tissue of Subjects with Chronic Rhinosinusitis: A Histopathologic Study

Objective: To evaluate the expression of five epithelial intercellular junctional proteins in the sinonasal tissue of subjects with chronic rhinosinusitis (CRS). Methods: Forty-one samples of nasal polyp tissue of CRS patients with nasal polyps (wNP), 20 ethmoid sinus mucosa of CRS patients without nasal polyps (sNP) and 19 nasal mucosa of controls were collected and assessed for the expression of zonulae occludens (ZO-1), claudin-1, E-cadherin and desmoglein-1 and -2 (DSG1, DSG2) using immunohistochemical staining. Interleukin (IL)-5, IL-6 and IL-8 concentrations in the tissues were also measured using ELISA. Results: The expression of ZO-1, claudin-1, DSG1 and DSG2 in the CRSwNP patient group and the expression of claudin-1, DSG1 and DSG2 of the CRSsNP patient group was significantly lower compared to that of the control group. Furthermore, the expression of DSG1 in the CRSwNP patient group was also significantly lower than in the CRSsNP patient group. In contrast, the expression of E-cadherin in the CRSwNP and the CRSsNP patient groups was significantly greater compared to the controls. The assessment of associations between the expression of the intercellular junctional proteins and cytokines demonstrated negative correlations between IL-5 and claudin-1, IL-6 and claudin-1, IL-6 and DSG2, IL-8 and DSG1, and IL-8 and DSG2. In contrast, a positive correlation was found between IL-8 and E-cadherin. Conclusions: Differences in the expression of epithelial intercellular junctional proteins may play an important role in the pathogenesis of CRS.

Interleukin‐4 and interleukin‐13 compromise the sinonasal epithelial barrier and perturb intercellular junction protein expression

Altered expression of epithelial intercellular junction proteins has been observed in sinonasal biopsies from nasal polyps and epithelial layers cultured from nasal polyp patients. These alterations comprise a "leaky" epithelial barrier phenotype. We hypothesize that T helper 2 (Th2) cytokines interleukin (IL)-4 and IL-13 modulate epithelial junction proteins, thereby contributing to the leaky epithelial barrier. Differentiated primary sinonasal epithelial layers cultured at the air-liquid interface were exposed to IL-4, IL-13, and controls for 24 hours at 37°C. Epithelial resistance measurements were taken every 4 hours during cytokine exposure. Western blot and immunofluorescence staining/confocal microscopy were used to assess changes in a panel of tight and adherens junction proteins. Western blot densitometry was quantified with image analysis. IL-4 and IL-13 exposure resulted in a mean decrease in transepithelial resistance at 24 hours to 51.6% (n = 6) and 68.6% (n = 8) of baseline, respectively. Tight junction protein junctional adhesion molecule-A (JAM-A) expression decreased 42.2% with IL-4 exposure (n = 9) and 37.5% with IL-13 exposure (n = 9). Adherens junction protein E-cadherin expression decreased 35.3% with IL-4 exposure (n = 9) and 32.9% with IL-13 exposure (n = 9). Tight junction protein claudin-2 showed more variability but had a trend toward higher expression with Th2 cytokine exposure. There were no appreciable changes in claudin-1, occludin, or zonula occludens-1 (ZO-1) with IL-4 or IL-13 exposure. Sinonasal epithelial exposure to Th2 cytokines IL-4 and IL-13 results in alterations in intercellular junction proteins, reflecting increased epithelial permeability. Such changes may explain some of the phenotypic manifestations of Th2-mediated sinonasal disease, such as edema, nasal discharge, and environmental reactivity.

Penton-Dodecahedral Particles Trigger Opening of Intercellular Junctions and Facilitate Viral Spread during Adenovirus Serotype 3 Infection of Epithelial Cells

Human adenovirus serotypes Ad3, Ad7, Ad11, and Ad14 use the epithelial junction protein desmoglein 2 (DSG2) as a receptor for infection. During Ad infection, the fiber and penton base capsid proteins are produced in vast excess and form hetero-oligomers, called pentons. It has been shown for Ad3 that pentons self-assemble into penton-dodecahedra (PtDd). Our previous studies with recombinant purified Ad3 PtDd (produced in insect cells) showed that PtDd bind to DSG2 and trigger intracellular signaling resulting in the transient opening of junctions between epithelial cells. So far, a definitive proof for a function of Ad3 PtDd in the viral life cycle is elusive. Based on the recently published 3D structure of recombinant Ad3 PtDd, we generated a penton base mutant Ad3 vector (mu-Ad3GFP). mu-Ad3GFP is identical to its wild-type counterpart (wt-Ad3GFP) in the efficiency of progeny virus production; however, it is disabled in the production of PtDd. For infection studies we used polarized epithelial cancer cells or cell spheroids. We showed that in wt-Ad3GFP infected cultures, PtDd were released from cells before viral cytolysis and triggered the restructuring of epithelial junctions. This in turn facilitated lateral viral spread of de novo produced virions. These events were nearly absent in mu-Ad3GFP infected cultures. Our in vitro findings were consolidated in mice carrying xenograft tumors derived from human epithelial cancer cells. Furthermore, we provide first evidence that PtDd are also formed by another DSG2-interacting Ad serotype, the newly emerged, highly pathogenic Ad14 strain (Ad14p1). The central finding of this study is that a subgroup of Ads has evolved to generate PtDd as a strategy to achieve penetration into and dissemination in epithelial tissues. Our findings are relevant for basic and applied virology, specifically for cancer virotherapy.

Digestion of epithelial tight junction proteins by the commensalClostridium perfringens

The enteric microbiota contributes to the pathogenesis of inflammatory bowel disease, but the pathways involved and bacterial participants may vary in different hosts. We previously reported that some components of the human commensal microbiota, particularly Clostridium perfringens (C. perfringens), have the proteolytic capacity for host matrix degradation and reduce transepithelial resistance. Here, we examined the C. perfringens-derived proteolytic activity against epithelial tight junction proteins using human intestinal epithelial cell lines. We showed that the protein levels of E-cadherin, occludin, and junctional adhesion molecule 1 decrease in colonic cells treated with C. perfringens culture supernatant. E-cadherin ectodomain shedding in C. perfringens-stimulated intestinal epithelial cells was detected with antibodies against the extracellular domain of E-cadherin, and we demonstrate that this process occurs in a time- and dose-dependent manner. In addition, we showed that the filtered sterile culture supernatant of C. perfringens has no cytotoxic activity on the human intestinal cells at the concentrations used in this study. The direct cleavage of E-cadherin by the proteases from the C. perfringens culture supernatant was confirmed by C. perfringens supernatant-induced in vitro degradation of the human recombinant E-cadherin. We conclude that C. perfringens culture supernatant mediates digestion of epithelial cell junctional proteins, which is likely to enable access to the extracellular matrix components by the paracellular pathway.

Transepithelial Riboflavin/Ultraviolet. A Corneal Cross-linking in Keratoconus: Morphologic Studies on Human Corneas

To evaluate histologic and molecular changes in human keratoconic corneas after the procedure of transepithelial collagen cross-linking (CXL), without the removal of corneal epithelium. Experimental laboratory investigation. Thirty corneal buttons were examined, 18 of which were from patients affected by severe keratoconus and submitted to penetrating keratoplasty (PK). Among these, 8 were analyzed without any treatment, 4 were treated with transepithelial CXL 2 hours before PK, and 6 were treated with transepithelial CXL 3 months before PK. Twelve normal corneal buttons from healthy donors were used as controls. The corneal buttons were then evaluated by hematoxylin-eosin staining and by immunostaining with markers of epithelial junction proteins (ß-catenin and connexin 43), of stromal keratocytes (CD34), of apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL] assay), and of collagen type I fibers. The analysis of epithelial markers showed a clear defective expression in keratoconic corneas before and soon after the transepithelial CXL treatment, returning to normal in corneas analyzed 3 months after transepithelial CXL. The analysis of stroma components indicated a loss of keratocytes in the upper stroma of keratoconic corneas and a trend toward a normal situation 3 months after transepithelial CXL; similarly, collagen fibers appeared disorganized in keratoconus, while their pattern appears to be close to normal 3 months after treatment. Histologic and immunohistochemical findings on human keratoconic corneas showed the presence of biochemical and morphologic alterations in the epithelium and the upper stroma that are significantly improved 3 months after transepithelial CXL. However, further studies are necessary to assess to what extent these results correlate with measurable biomechanical effects.

MiR-155-mediated loss of C/EBPβ shifts the TGF-β response from growth inhibition to epithelial-mesenchymal transition, invasion and metastasis in breast cancer

During breast cancer progression, transforming growth factor-beta (TGF-β) switches from acting as a growth inhibitor to become a major promoter of epithelial-mesenchymal transition (EMT), invasion and metastasis. However, the mechanisms involved in this switch are not clear. We found that loss of CCAAT-enhancer binding protein beta (C/EBPβ), a differentiation factor for the mammary epithelium, was associated with signs of EMT in triple-negative human breast cancer, and in invasive areas of mammary tumors in MMTV-PyMT mice. Using an established model of TGF-β-induced EMT in mouse mammary gland epithelial cells, we discovered that C/EBPβ was repressed during EMT by miR-155, an oncomiR in breast cancer. Depletion of C/EBPβ potentiated the TGF-β response towards EMT, and contributed to evasion of the growth inhibitory response to TGF-β. Furthermore, loss of C/EBPβ enhanced invasion and metastatic dissemination of the mouse mammary tumor cells to the lungs after subcutaneous injection into mice. The mechanism by which loss of C/EBPβ promoted the TGF-β response towards EMT, invasion and metastasis, was traced to a previously uncharacterized role of C/EBPβ as a transcriptional activator of genes encoding the epithelial junction proteins E-cadherin and coxsackie virus and adenovirus receptor. The results identify miR-155-mediated loss of C/EBPβ as a mechanism, which promotes breast cancer progression by shifting the TGF-β response from growth inhibition to EMT, invasion and metastasis.

Structural and Functional Studies on the Interaction of Adenovirus Fiber Knobs and Desmoglein 2

Human adenovirus (Ad) serotypes Ad3, Ad7, Ad11, and Ad14, as well as a recently emerged strain of Ad14 (Ad14p1), use the epithelial junction protein desmoglein 2 (DSG2) as a receptor for infection. Unlike Ad interaction with CAR and CD46, structural details for Ad binding to DSG2 are still elusive. Using an approach based on Escherichia coli expression libraries of random Ad3 and Ad14p1 fiber knob mutants, we identified amino acid residues that, when mutated individually, ablated or reduced Ad knob binding to DSG2. These residues formed three clusters inside one groove at the extreme distal end of the fiber knob. The Ad3 fiber knob mutant library was also used to identify variants with increased affinity to DSG2. We found a number of mutations within or near the EF loop of the Ad3 knob that resulted in affinities to DSG2 that were several orders of magnitude higher than those to the wild-type Ad3 knob. Crystal structure analysis of one of the mutants showed that the introduced mutations make the EF loop more flexible, which might facilitate the interaction with DSG2. Our findings have practical relevance for cancer therapy. We have recently reported that an Ad3 fiber knob-containing recombinant protein (JO-1) is able to trigger opening of junctions between epithelial cancer cells which, in turn, greatly improved the intratumoral penetration and efficacy of therapeutic agents (I. Beyer, et al., Clin. Cancer Res. 18:3340-3351, 2012; I. Beyer, et al., Cancer Res. 71:7080-7090, 2011). Here, we show that affinity-enhanced versions of JO-1 are therapeutically more potent than the parental protein in a series of cancer models.

The Transcription Factor Sterile Alpha Motif (SAM) Pointed Domain-containing ETS Transcription Factor (SPDEF) Is Required for E-cadherin Expression in Prostate Cancer Cells

Loss of E-cadherin is one of the key steps in tumor progression. Our previous studies demonstrate that SAM pointed domain-containing ETS transcription factor (SPDEF) inhibited prostate cancer metastasis in vitro and in vivo. In the present study, we evaluated the relationship between SPDEF and E-cadherin expression in an effort to better understand the mechanism of action of SPDEF in prostate tumor cell invasion and metastasis. The results presented here demonstrate a direct correlation between expression of E-cadherin and SPDEF in prostate cancer cells. Additional data demonstrate that modulation of E-cadherin and SPDEF had similar effects on cell migration/invasion. In addition, siRNA-mediated knockdown of E-cadherin was sufficient to block the effects of SPDEF on cell migration and invasion. We also show that stable forced expression of SPDEF results in increased expression of E-cadherin, whereas down-regulation of SPDEF decreased E-cadherin expression. In addition, we demonstrate that SPDEF expression is not regulated by E-cadherin. Moreover, our chromatin immunoprecipitation and luciferase reporter assay revealed that SPDEF occupies E-cadherin promoter site and acts as a direct transcriptional inducer of E-cadherin in prostate cancer cells. Taken together, to the best of our knowledge, these studies are the first demonstrating requirement of SPDEF for expression of E-cadherin, an essential epithelial cell junction protein. Given that loss of E-cadherin is a central tenant in tumor metastasis, the results of our studies, by providing a new mechanism for regulation of E-cadherin expression, could have far reaching impact.

Label-free mass spectrometry proteome quantification of human embryonic kidney cells following 24 hours of sialic acid overproduction

BackgroundCell surface glycoprotein sialylation is one of the most ubiquitous glycan modifications found on higher eukaryotes. The surface sialylation pattern of cells is influenced by the cellular environment but also by the Golgi sialyltransferase activity and flux of metabolites through sialic acid producing pathways. Altered cell surface sialic acid patterns have been observed in several cancers and other pathological conditions. In this experiment we examined the cellular proteomic changes that occur in human embryonic kidney cells after 24 hours of sialic acid overproduction using N-Acetylmannosamine. We utilized high resolution mass spectrometry and label free protein quantification to characterize the relative changes in protein abundance as well as multiple reaction monitoring to quantify the cellular sialic acid levels.ResultsUsing N-Acetylmannosamine we were able to induce sialic acid production to almost 70-fold compared to non-induced control cells. Mass spectrometric analysis of cellular proteome of control and induced cells identified 1802 proteins of which 105 displayed significant changes in abundance. Functional analysis of the resulting relative changes in protein abundance revealed regulation of several cellular pathways including protein transport, metabolic and signaling pathways and remodeling of epithelial adherens junctions. We also identified several physically interacting co-regulated proteins in the set of changed proteins.ConclusionsIn this experiment we show that increased metabolic flux through sialic acid producing pathway affects the abundance of several protein transport, epithelial adherens junction, signaling and metabolic pathway related proteins.

Epithelial permeability alterations in an in vitro air–liquid interface model of allergic fungal rhinosinusitis

Chronic rhinosinusitis (CRS) is an inflammatory upper-airway disease with numerous etiologies. Patients with a characteristic subtype of CRS, allergic fungal rhinosinusitis (AFRS), display increased expression of T helper 2 (Th2) cytokines and antigen-specific immunoglobulin E (IgE). Various sinonasal inflammatory conditions are associated with alterations in epithelial barrier function. The aim of this study was to compare epithelial permeability and intercellular junctional protein expression among cultured primary sinonasal cells from AFRS patients vs noninflammatory controls. Epithelial cells isolated from paranasal sinus mucosa of AFRS and noninflammatory control patients were grown to confluence on permeable supports and transitioned to air-liquid interface (ALI). Transepithelial resistance (TER) was measured with a horizontal Ussing chamber to characterize the functional permeability of each cell type. After TER recordings were complete, a panel of intercellular junctional proteins was assessed by Western blot and immunofluorescence labeling followed by confocal microscopy. After 12 samples were measured from each group, we observed a 41% mean decrease in TER in AFRS cells (296 ± 89 ohms × cm(2) ) compared to control (503 ± 134 ohms × cm(2) , p = 0.006). TER deficits observed in AFRS were associated with decreased expression of the tight junction proteins occludin and junctional adhesion molecule-A (JAM-A), and increased expression of a leaky tight junction protein claudin-2. Cultured sinonasal epithelium from AFRS patients displayed increased epithelial permeability and altered expression of intercellular junctional proteins. Given that these cells were not incubated with inflammatory cytokines in vitro, the cultured AFRS epithelial alterations may represent a retained modification in protein expression from the in vivo phenotype.

E-cadherin is required for intestinal morphogenesis in the mouse

E-cadherin, the primary epithelial adherens junction protein, has been implicated as playing a critical role in nucleating formation of adherens junctions, tight junctions, and desmosomes. In addition to its role in maintaining structural tissue integrity, E-cadherin has also been suggested as an important modulator of cell signaling via interactions with its cytoplasmic binding partners, catenins, as well as with growth factor receptors. Therefore, we proposed that loss of E-cadherin from the developing mouse intestinal epithelium would disrupt intestinal epithelial morphogenesis and function. To test this hypothesis, we used a conditional knockout approach to eliminate E-cadherin specifically in the intestinal epithelium during embryonic development. We found that E-cadherin conditional knockout mice failed to survive, dying within the first 24 hours of birth. Examination of intestinal architecture at E18.5 demonstrated severe disruption to intestinal morphogenesis in animals lacking E-cadherin in the epithelium of the small intestine. We observed changes in epithelial cell shape as well as in the morphology of villi. Although junctional complexes were evident, junctions were abnormal, and barrier function was compromised in E-cadherin mutant intestine. We also identified changes in the epithelial cell populations present in E-cadherin conditional knockout animals. The number of proliferating cells was increased, whereas the number of enterocytes was decreased. Although Wnt/β-catenin target mRNAs were more abundant in mutants compared with controls, the amount of nuclear activated β-catenin protein was dramatically lower in mutants compared with controls. In summary, our data demonstrate that E-cadherin is essential for intestinal epithelial morphogenesis and homeostasis during embryonic development.

Correlation of epithelial-mesenchymal transition markers with clinicopathologic parameters in adenocarcinomas and squamous cell carcinoma of the lung.

Epithelial-mesenchymal transition (EMT) is characterized by the loss of epithelial cell junction proteins and the gain of mesenchymal markers. The aim of this study was to analyze the associations between the EMT-related markers vimentin, E-cadherin, β-catenin, slug, snail, and twist1 and clinicopathologic parameters as well as epidermal growth factor receptor (EGFR) gene copy number and protein expression in non-small cell lung carcinoma (NSCLC). Fifty-nine squamous cell carcinomas (SCC) and 43 adenocarcinomas (AD) were immunohistochemically examined for respective EMT markers and for EGFR, using the EGFR PharmDx kit (Dako) for protein expression and automated silver enhanced in situ hybridization (SISH) for copy number. Vimentin expression in tumor epithelia was significantly higher in AD samples than in SCC samples (P=0.015). Among AD samples, vimentin expression was positively correlated with histologic grade (2 vs. 3; P=0.021) and exhibited a tendency toward a positive correlation with pTNM stage (I vs. II-IV; P=0.052). EGFR gene copy number was positively correlated with EGFR protein expression among both AD samples (P=0.008) and SCC samples (P=0.042), with EGFR protein expression being significantly higher in SCC samples compared with AD (P=0.038). Among AD samples, EGFR protein expression was associated with higher cytoplasmic expression of β-catenin (P=0.031). Among SCC samples, EGFR protein expression was negatively correlated with nuclear expression of β-catenin (P=0.033) but positively with nuclear slug (P=0.021). The expression pattern of EMT markers in AD suggests that vimentin is a possible immunohistochemical predictor of tumor progression.

Tu1848 Digestion of Epithelial Tight Junction Proteins by the Commensal Clostridium Perfringens

Pruteanu M, Shanahan F. Digestion of epithelial tight junction proteins by the commensal Clostridium perfringens. Am J Physiol Gastrointest Liver Physiol 305: G740–G748, 2013. First published September 26, 2013; doi:10.1152/ajpgi.00316.2012.—The enteric microbiota contributes to the pathogenesis of inflammatory bowel disease, but the pathways involved and bacterial participants may vary in different hosts. We previously reported that some components of the human commensal microbiota, particularly Clostridium perfringens (C. perfringens), have the proteolytic capacity for host matrix degradation and reduce transepithelial resistance. Here, we examined the C. perfringens-derived proteolytic activity against epithelial tight junction proteins using human intestinal epithelial cell lines. We showed that the protein levels of E-cadherin, occludin, and junctional adhesion molecule 1 decrease in colonic cells treated with C. perfringens culture supernatant. E-cadherin ectodomain shedding in C. perfringens-stimulated intestinal epithelial cells was detected with antibodies against the extracellular domain of E-cadherin, and we demonstrate that this process occurs in a timeand dose-dependent manner. In addition, we showed that the filtered sterile culture supernatant of C. perfringens has no cytotoxic activity on the human intestinal cells at the concentrations used in this study. The direct cleavage of Ecadherin by the proteases from the C. perfringens culture supernatant was confirmed by C. perfringens supernatant-induced in vitro degradation of the human recombinant E-cadherin. We conclude that C. perfringens culture supernatant mediates digestion of epithelial cell junctional proteins, which is likely to enable access to the extracellular matrix components by the paracellular pathway.