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Sample records for remodeling epithelial cell

  1. Epithelial Cell Proliferation Contributes to Airway Remodeling in Severe Asthma

    PubMed Central

    Cohen, Lance; E, Xueping; Tarsi, Jaime; Ramkumar, Thiruvamoor; Horiuchi, Todd K.; Cochran, Rebecca; DeMartino, Steve; Schechtman, Kenneth B.; Hussain, Iftikhar; Holtzman, Michael J.; Castro, Mario

    2007-01-01

    Rationale: Despite long-term therapy with corticosteroids, patients with severe asthma develop irreversible airway obstruction. Objectives: To evaluate if there are structural and functional differences in the airway epithelium in severe asthma associated with airway remodeling. Methods: In bronchial biopsies from 21 normal subjects, 11 subjects with chronic bronchitis, 9 subjects with mild asthma, and 31 subjects with severe asthma, we evaluated epithelial cell morphology: epithelial thickness, lamina reticularis (LR) thickness, and epithelial desquamation. Levels of retinoblastoma protein (Rb), Ki67, and Bcl-2 were measured, reflecting cellular proliferation and death. Terminal deoxynucleotidyl-mediated dUTP nick end labeling (TUNEL) was used to study cellular apoptosis. Measurements and Main Results: Airway epithelial and LR thickness was greater in subjects with severe asthma compared with those with mild asthma, normal subjects, and diseased control subjects (p = 0.009 and 0.033, respectively). There was no significant difference in epithelial desquamation between groups. Active, hypophosphorylated Rb expression was decreased (p = 0.002) and Ki67 was increased (p < 0.01) in the epithelium of subjects with severe asthma as compared with normal subjects, indicating increased cellular proliferation. Bcl-2 expression was decreased (p < 0.001), indicating decreased cell death suppression. There was a greater level of apoptotic activity in the airway biopsy in subjects with severe asthma as compared with the normal subjects using the TUNEL assay (p = 0.002), suggesting increased cell death. Conclusions: In subjects with severe asthma, as compared with subjects with mild asthma, normal subjects, and diseased control subjects, we found novel evidence of increased cellular proliferation in the airway contributing to a thickened epithelium and LR. These changes may contribute to the progressive decline in lung function and airway remodeling in patients with severe

  2. Heterogeneity and stochastic growth regulation of biliary epithelial cells dictate dynamic epithelial tissue remodeling.

    PubMed

    Kamimoto, Kenji; Kaneko, Kota; Kok, Cindy Yuet-Yin; Okada, Hajime; Miyajima, Atsushi; Itoh, Tohru

    2016-01-01

    Dynamic remodeling of the intrahepatic biliary epithelial tissue plays key roles in liver regeneration, yet the cellular basis for this process remains unclear. We took an unbiased approach based on in vivo clonal labeling and tracking of biliary epithelial cells in the three-dimensional landscape, in combination with mathematical simulation, to understand their mode of proliferation in a mouse liver injury model where the nascent biliary structure formed in a tissue-intrinsic manner. An apparent heterogeneity among biliary epithelial cells was observed: whereas most of the responders that entered the cell cycle upon injury exhibited a limited and tapering growth potential, a select population continued to proliferate, making a major contribution in sustaining the biliary expansion. Our study has highlighted a unique mode of epithelial tissue dynamics, which depends not on a hierarchical system driven by fixated stem cells, but rather, on a stochastically maintained progenitor population with persistent proliferative activity. PMID:27431614

  3. Heterogeneity and stochastic growth regulation of biliary epithelial cells dictate dynamic epithelial tissue remodeling

    PubMed Central

    Kamimoto, Kenji; Kaneko, Kota; Kok, Cindy Yuet-Yin; Okada, Hajime; Miyajima, Atsushi; Itoh, Tohru

    2016-01-01

    Dynamic remodeling of the intrahepatic biliary epithelial tissue plays key roles in liver regeneration, yet the cellular basis for this process remains unclear. We took an unbiased approach based on in vivo clonal labeling and tracking of biliary epithelial cells in the three-dimensional landscape, in combination with mathematical simulation, to understand their mode of proliferation in a mouse liver injury model where the nascent biliary structure formed in a tissue-intrinsic manner. An apparent heterogeneity among biliary epithelial cells was observed: whereas most of the responders that entered the cell cycle upon injury exhibited a limited and tapering growth potential, a select population continued to proliferate, making a major contribution in sustaining the biliary expansion. Our study has highlighted a unique mode of epithelial tissue dynamics, which depends not on a hierarchical system driven by fixated stem cells, but rather, on a stochastically maintained progenitor population with persistent proliferative activity. DOI: http://dx.doi.org/10.7554/eLife.15034.001 PMID:27431614

  4. SuperSILAC Quantitative Proteome Profiling of Murine Middle Ear Epithelial Cell Remodeling with NTHi

    PubMed Central

    Val, Stéphanie; Burgett, Katelyn; Brown, Kristy J.; Preciado, Diego

    2016-01-01

    Background Chronic Otitis Media with effusion (COME) develops after sustained inflammation and is characterized by secretory middle ear epithelial metaplasia and effusion, most frequently mucoid. Non-typeable Haemophilus influenzae (NTHi), the most common acute Otitis Media (OM) pathogen, is postulated to promote middle ear epithelial remodeling in the progression of OM from acute to chronic. The goals of this study were to examine histopathological and quantitative proteomic epithelial effects of NTHi challenge in a murine middle ear epithelial cell line. Methods NTHi lysates were generated and used to stimulate murine epithelial cells (mMEEC) cultured at air-liquid interface over 48 hours– 1 week. Conditional quantitative Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) of cell lysates was performed to interrogate the global protein production in the cells, using the SuperSILAC technique. Histology of the epithelium over time was done to measure bacterial dependent remodeling. Results Mass spectrometry analysis identified 2,565 proteins across samples, of which 74 exhibited differential enrichment or depletion in cell lysates (+/-2.0 fold-change; p value<0.05). The key molecular functions regulated by NTHi lysates exposure were related to cell proliferation, death, migration, adhesion and inflammation. Finally, chronic exposure induced significant epithelial thickening of cells grown at air liquid interface. Conclusions NTHi lysates drive pathways responsible of cell remodeling in murine middle ear epithelium which likely contributes to observed epithelial hyperplasia in vitro. Further elucidation of these mediators will be critical in understanding the progression of OM from acute to chronic at the molecular level. PMID:26859300

  5. Deciliation is associated with dramatic remodeling of epithelial cell junctions and surface domains.

    PubMed

    Overgaard, Christian E; Sanzone, Kaitlin M; Spiczka, Krystle S; Sheff, David R; Sandra, Alexander; Yeaman, Charles

    2009-01-01

    Stress-induced shedding of motile cilia (autotomy) has been documented in diverse organisms and likely represents a conserved cellular reaction. However, little is known about whether primary cilia are shed from mammalian epithelial cells and what impact deciliation has on polarized cellular organization. We show that several chemically distinct agents trigger autotomy in epithelial cells. Surprisingly, deciliation is associated with a significant, but reversible increase in transepithelial resistance. This reflects substantial reductions in tight junction proteins associated with "leaky" nephron segments (e.g., claudin-2). At the same time, apical trafficking of gp80/clusterin and gp114/CEACAM becomes randomized, basal-lateral delivery of Na,K-ATPase is reduced, and expression of the nonciliary apical protein gp135/podocalyxin is greatly decreased. However, ciliogenesis-impaired MDCK cells do not undergo continual junction remodeling, and mature cilia are not required for autotomy-associated remodeling events. Deciliation and epithelial remodeling may be mechanistically linked processes, because RNAi-mediated reduction of Exocyst subunit Sec6 inhibits ciliary shedding and specifically blocks deciliation-associated down-regulation of claudin-2 and gp135. We propose that ciliary autotomy represents a signaling pathway that impacts the organization and function of polarized epithelial cells. PMID:19005211

  6. Cytoskeleton remodelling of confluent epithelial cells cultured on porous substrates

    PubMed Central

    Rother, Jan; Büchsenschütz-Göbeler, Matthias; Nöding, Helen; Steltenkamp, Siegfried; Samwer, Konrad; Janshoff, Andreas

    2015-01-01

    The impact of substrate topography on the morphological and mechanical properties of confluent MDCK-II cells cultured on porous substrates was scrutinized by means of various imaging techniques as well as atomic force microscopy comprising force volume and microrheology measurements. Regardless of the pore size, ranging from 450 to 5500 nm in diameter, cells were able to span the pores. They did not crawl into the holes or grow around the pores. Generally, we found that cells cultured on non-porous surfaces are stiffer, i.e. cortical tension rises from 0.1 to 0.3 mN m−1, and less fluid than cells grown over pores. The mechanical data are corroborated by electron microscopy imaging showing more cytoskeletal filaments on flat samples in comparison to porous ones. By contrast, cellular compliance increases with pore size and cells display a more fluid-like behaviour on larger pores. Interestingly, cells on pores larger than 3500 nm produce thick actin bundles that bridge the pores and thereby strengthen the contact zone of the cells. PMID:25566882

  7. Airway basal stem cells: a perspective on their roles in epithelial homeostasis and remodeling.

    PubMed

    Rock, Jason R; Randell, Scott H; Hogan, Brigid L M

    2010-01-01

    The small airways of the human lung undergo pathological changes in pulmonary disorders, such as chronic obstructive pulmonary disease (COPD), asthma, bronchiolitis obliterans and cystic fibrosis. These clinical problems impose huge personal and societal healthcare burdens. The changes, termed 'pathological airway remodeling', affect the epithelium, the underlying mesenchyme and the reciprocal trophic interactions that occur between these tissues. Most of the normal human airway is lined by a pseudostratified epithelium of ciliated cells, secretory cells and 6-30% basal cells, the proportion of which varies along the proximal-distal axis. Epithelial abnormalities range from hypoplasia (failure to differentiate) to basal- and goblet-cell hyperplasia, squamous- and goblet-cell metaplasia, dysplasia and malignant transformation. Mesenchymal alterations include thickening of the basal lamina, smooth muscle hyperplasia, fibrosis and inflammatory cell accumulation. Paradoxically, given the prevalence and importance of airway remodeling in lung disease, its etiology is poorly understood. This is due, in part, to a lack of basic knowledge of the mechanisms that regulate the differentiation, maintenance and repair of the airway epithelium. Specifically, little is known about the proliferation and differentiation of basal cells, a multipotent stem cell population of the pseudostratified airway epithelium. This Perspective summarizes what we know, and what we need to know, about airway basal cells to evaluate their contributions to normal and abnormal airway remodeling. We contend that exploiting well-described model systems using both human airway epithelial cells and the pseudostratified epithelium of the genetically tractable mouse trachea will enable crucial discoveries regarding the pathogenesis of airway disease. PMID:20699479

  8. Aclidinium bromide combined with formoterol inhibits remodeling parameters in lung epithelial cells through cAMP.

    PubMed

    Lambers, Christopher; Costa, Luigi; Ying, Qi; Zhong, Jun; Lardinois, Didier; Dekan, Gerhard; Schuller, Elisabeth; Roth, Michael

    2015-12-01

    Combined muscarinic receptor antagonists and long acting β2-agonists improve symptom control in chronic obstructive pulmonary disease (COPD) significantly. In clinical studies aclidinium bromide achieved better beneficial effects than other bronchodilators; however, the underlying molecular mechanisms are unknown. This study assessed the effect of aclidinium bromide combined with formoterol on COPD lung (n=20) and non-COPD lung (n=10) derived epithelial cells stimulated with TGF-β1+carbachol on: (i) the generation of mesenchymal cells in relation to epithelial cells, (II) extracellular matrix (ECM) deposition, and (iii) the interaction of ECM on the generation of epithelial and mesenchymal cells. TGF-β1+carbachol enhanced the generation of mesenchymal cells, which was significantly reduced by aclidinium bromide or formoterol. The effect of combined drugs was additive. Inhibition of p38 MAP kinase and Smad by specific inhibitors or aclidinium bromide reduced the generation of mesenchymal cells. In mesenchymal cells, TGF-β1+carbachol induced the deposition of collagen-I and fibronectin which was prevented by both drugs dose-dependently. Formoterol alone reduced collagen-I deposition via cAMP, this however, was overruled by TGF-β1+carbachol and rescued by aclidinium bromide. Inhibition of fibronectin was cAMP independent, but involved p38 MAP kinase and Smad. Seeding epithelial cells on ECM collagen-I and fibronectin induced mesenchymal cell generation, which was reduced by aclidinium bromide and formoterol. Our results suggest that the beneficial effect of aclidinium bromide and formoterol involves cAMP affecting both, the accumulation of mesenchymal cells and ECM remodeling, which may explain the beneficial effect of the drugs on lung function in COPD. PMID:26546746

  9. Bioaerosols from a Food Waste Composting Plant Affect Human Airway Epithelial Cell Remodeling Genes

    PubMed Central

    Chang, Ming-Wei; Lee, Chung-Ru; Hung, Hsueh-Fen; Teng, Kuo-Sheng; Huang, Hsin; Chuang, Chun-Yu

    2013-01-01

    The composting procedure in food waste plants generates airborne bioaerosols that have the potential to damage human airway epithelial cells. Persistent inflammation and repair responses induce airway remodeling and damage to the respiratory system. This study elucidated the expression changes of airway remodeling genes in human lung mucoepidermoid NCI-H292 cells exposed to bioaerosols from a composting plant. Different types of microorganisms were detectable in the composting plant, using the agar culture method. Real-time polymerase chain reaction was used to quantify the level of Aspergillus fumigatus and the profile of remodeling genes. The real-time PCR results indicated that the amount of A. fumigatus in the composting hall was less than 102 conidia. The endotoxins in the field bioaerosols were determined using a limulus amebocyte lysate test. The endotoxin levels depended on the type of particulate matter (PM), with coarse particles (2.5–10 μm) having higher endotoxin levels than did fine particles (0.5–2.5 μm). After exposure to the conditioned medium of field bioaerosol samples, NCI-H292 cells showed increased pro-inflammatory interleukin (IL)-6 release and activated epidermal growth factor receptor (EGFR), transforming growth factor (TGF)-β1 and cyclin-dependent kinase inhibitor 1 (p21WAF1/CIP1) gene expression, but not of matrix metallopeptidase (MMP)-9. Airborne endotoxin levels were higher inside the composting hall than they were in other areas, and they were associated with PM. This suggested that airborne bioaerosols in the composting plant contained endotoxins and microorganisms besides A. fumigatus that cause the inflammatory cytokine secretion and augment the expression of remodeling genes in NCI-H292 cells. It is thus necessary to monitor potentially hazardous materials from bioaerosols in food composting plants, which could affect the health of workers. PMID:24368426

  10. The role of maintenance proteins in the preservation of epithelial cell identity during mammary gland remodeling and breast cancer initiation

    PubMed Central

    Coradini, Danila; Oriana, Saro

    2014-01-01

    During normal postnatal mammary gland development and adult remodeling related to the menstrual cycle, pregnancy, and lactation, ovarian hormones and peptide growth factors contribute to the delineation of a definite epithelial cell identity. This identity is maintained during cell replication in a heritable but DNA-independent manner. The preservation of cell identity is fundamental, especially when cells must undergo changes in response to intrinsic and extrinsic signals. The maintenance proteins, which are required for cell identity preservation, act epigenetically by regulating gene expression through DNA methylation, histone modification, and chromatin remodeling. Among the maintenance proteins, the Trithorax (TrxG) and Polycomb (PcG) group proteins are the best characterized. In this review, we summarize the structures and activities of the TrxG and PcG complexes and describe their pivotal roles in nuclear estrogen receptor activity. In addition, we provide evidence that perturbations in these epigenetic regulators are involved in disrupting epithelial cell identity, mammary gland remodeling, and breast cancer initiation. PMID:23845141

  11. KIF17 regulates RhoA-dependent actin remodeling at epithelial cell-cell adhesions.

    PubMed

    Acharya, Bipul R; Espenel, Cedric; Libanje, Fotine; Raingeaud, Joel; Morgan, Jessica; Jaulin, Fanny; Kreitzer, Geri

    2016-03-01

    The kinesin KIF17 localizes at microtubule plus-ends where it contributes to regulation of microtubule stabilization and epithelial polarization. We now show that KIF17 localizes at cell-cell adhesions and that KIF17 depletion inhibits accumulation of actin at the apical pole of cells grown in 3D organotypic cultures and alters the distribution of actin and E-cadherin in cells cultured in 2D on solid supports. Overexpression of full-length KIF17 constructs or truncation mutants containing the N-terminal motor domain resulted in accumulation of newly incorporated GFP-actin into junctional actin foci, cleared E-cadherin from cytoplasmic vesicles and stabilized cell-cell adhesions to challenge with calcium depletion. Expression of these KIF17 constructs also increased cellular levels of active RhoA, whereas active RhoA was diminished in KIF17-depleted cells. Inhibition of RhoA or its effector ROCK, or expression of LIMK1 kinase-dead or activated cofilin(S3A) inhibited KIF17-induced junctional actin accumulation. Interestingly, KIF17 activity toward actin depends on the motor domain but is independent of microtubule binding. Together, these data show that KIF17 can modify RhoA-GTPase signaling to influence junctional actin and the stability of the apical junctional complex of epithelial cells. PMID:26759174

  12. Linking progression of fibrotic lung remodeling and ultrastructural alterations of alveolar epithelial type II cells in the amiodarone mouse model.

    PubMed

    Birkelbach, Bastian; Lutz, Dennis; Ruppert, Clemens; Henneke, Ingrid; Lopez-Rodriguez, Elena; Günther, Andreas; Ochs, Matthias; Mahavadi, Poornima; Knudsen, Lars

    2015-07-01

    Chronic injury of alveolar epithelial type II cells (AE2 cells) represents a key event in the development of lung fibrosis in animal models and in humans, such as idiopathic pulmonary fibrosis (IPF). Intratracheal delivery of amiodarone to mice results in a profound injury and macroautophagy-dependent apoptosis of AE2 cells. Increased autophagy manifested in AE2 cells by disturbances of the intracellular surfactant. Hence, we hypothesized that ultrastructural alterations of the intracellular surfactant pool are signs of epithelial stress correlating with the severity of fibrotic remodeling. With the use of design-based stereology, the amiodarone model of pulmonary fibrosis in mice was characterized at the light and ultrastructural level during progression. Mean volume of AE2 cells, volume of lamellar bodies per AE2 cell, and mean size of lamellar bodies were correlated to structural parameters reflecting severity of fibrosis like collagen content. Within 2 wk amiodarone leads to an increase in septal wall thickness and a decrease in alveolar numbers due to irreversible alveolar collapse associated with alveolar surfactant dysfunction. Progressive hypertrophy of AE2 cells and increase in mean individual size and total volume of lamellar bodies per AE2 cell were observed. A high positive correlation of these AE2 cell-related ultrastructural changes and the deposition of collagen fibrils within septal walls were established. Qualitatively, similar alterations could be found in IPF samples with mild to moderate fibrosis. We conclude that ultrastructural alterations of AE2 cells including the surfactant system are tightly correlated with the progression of fibrotic remodeling. PMID:25957292

  13. Remodeling of Tight Junctions and Enhancement of Barrier Integrity of the CACO-2 Intestinal Epithelial Cell Layer by Micronutrients

    PubMed Central

    Valenzano, Mary Carmen; DiGuilio, Katherine; Mercado, Joanna; Teter, Mimi; To, Julie; Ferraro, Brendan; Mixson, Brittany; Manley, Isabel; Baker, Valerissa; Moore, Beverley A.; Wertheimer, Joshua; Mullin, James M.

    2015-01-01

    The micronutrients zinc, quercetin, butyrate, indole and berberine were evaluated for their ability to induce remodeling of epithelial tight junctions (TJs) and enhance barrier integrity in the CACO-2 gastrointestinal epithelial cell culture model. All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol. Increases of Rt as much as 200% of untreated controls were observed. Each of the five micronutrients also induced unique, signature-like changes in TJ protein composition, suggesting multiple pathways (and TJ arrangements) by which TJ barrier function can be enhanced. Decreases in abundance by as much as 90% were observed for claudin-2, and increases of over 300% could be seen for claudins -5 and -7. The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient. The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient. The implications of these findings for therapeutically decreasing morbidity in Inflammatory Bowel Disease are discussed. PMID:26226276

  14. Dynamics of adherens junctions in epithelial establishment, maintenance, and remodeling

    PubMed Central

    Baum, Buzz

    2011-01-01

    The epithelial cadherin (E-cadherin)–catenin complex binds to cytoskeletal components and regulatory and signaling molecules to form a mature adherens junction (AJ). This dynamic structure physically connects neighboring epithelial cells, couples intercellular adhesive contacts to the cytoskeleton, and helps define each cell’s apical–basal axis. Together these activities coordinate the form, polarity, and function of all cells in an epithelium. Several molecules regulate AJ formation and integrity, including Rho family GTPases and Par polarity proteins. However, only recently, with the development of live-cell imaging, has the extent to which E-cadherin is actively turned over at junctions begun to be appreciated. This turnover contributes to junction formation and to the maintenance of epithelial integrity during tissue homeostasis and remodeling. PMID:21422226

  15. Regulation of Lipid Droplet Size in Mammary Epithelial Cells by Remodeling of Membrane Lipid Composition—A Potential Mechanism

    PubMed Central

    Cohen, Bat-Chen; Shamay, Avi; Argov-Argaman, Nurit

    2015-01-01

    Milk fat globule size is determined by the size of its precursors—intracellular lipid droplets—and is tightly associated with its composition. We examined the relationship between phospholipid composition of mammary epithelial cells and the size of both intracellular and secreted milk fat globules. Primary culture of mammary epithelial cells was cultured in medium without free fatty acids (control) or with 0.1 mM free capric, palmitic or oleic acid for 24 h. The amount and composition of the cellular lipids and the size of the lipid droplets were determined in the cells and medium. Mitochondrial quantity and expression levels of genes associated with mitochondrial biogenesis and polar lipid composition were determined. Cells cultured with oleic and palmitic acids contained similar quantities of triglycerides, 3.1- and 3.8-fold higher than in controls, respectively (P < 0.0001). When cultured with oleic acid, 22% of the cells contained large lipid droplets (>3 μm) and phosphatidylethanolamine concentration was higher by 23 and 63% compared with that in the control and palmitic acid treatments, respectively (P < 0.0001). In the presence of palmitic acid, only 4% of the cells contained large lipid droplets and the membrane phosphatidylcholine concentration was 22% and 16% higher than that in the control and oleic acid treatments, respectively (P < 0.0001). In the oleic acid treatment, approximately 40% of the lipid droplets were larger than 5 μm whereas in that of the palmitic acid treatment, only 16% of the droplets were in this size range. Triglyceride secretion in the oleic acid treatment was 2- and 12-fold higher compared with that in the palmitic acid and control treatments, respectively. Results imply that membrane composition of bovine mammary epithelial cells plays a role in controlling intracellular and secreted lipid droplets size, and that this process is not associated with cellular triglyceride content. PMID:25756421

  16. Lacrimal Gland Inflammation Deregulates Extracellular Matrix Remodeling and Alters Molecular Signature of Epithelial Stem/Progenitor Cells

    PubMed Central

    Umazume, Takeshi; Thomas, William M.; Campbell, Sabrina; Aluri, Hema; Thotakura, Suharika; Zoukhri, Driss; Makarenkova, Helen P.

    2015-01-01

    Purpose The adult lacrimal gland (LG) is highly regenerative and is able to repair itself even after substantial damage; however, this ability to regenerate is lost with the development of dry eye conditions in chronically inflamed LGs.This study compares changes in the cell adhesion and cell matrix molecules and stem cell transcription factors in the LGs of healthy mice and of two mouse models of Sjögren's syndrome: nonobese diabetic (NOD) and MRL-lpr/lpr (MRL/lpr) mice during the early stage of inflammation. Methods The LGs from 12- to 13-week-old female MRL/lpr and male NOD mice along with their respective control strains were harvested and divided into three pieces and processed for quantitative (q) RT-PCR and qRT-PCR Arrays, histology, immunohistochemistry, and Western blotting. Results The extracellular matrix (ECM) and adhesion molecules RT2-PCR array combined with protein expression data revealed changes in the expression of integrins, matrix metalloproteinases, and other molecules, which are associated largely with invasion, attachment, and expansion of the lymphocytic cells, whereas changes in the stem cell transcription factors revealed substantial decrease in expression of transcription factors associated with epithelial stem/progenitor cell lineage. Conclusions We concluded that the expression of several important ECM components is significantly deregulated in the LG of two murine models of Sjögren's syndrome, suggesting an alteration of the epithelial stem/progenitor cell niche. This may result in profound effects on localization, activation, proliferation, and differentiation of the LG stem/progenitor cells and, therefore, LG regeneration. PMID:26747770

  17. Insights into the mechanical properties of epithelial cells: the effects of shear stress on the assembly and remodeling of keratin intermediate filaments

    PubMed Central

    Flitney, Eric W.; Kuczmarski, Edward R.; Adam, Stephen A.; Goldman, Robert D.

    2009-01-01

    The effects of shear stress on the keratin intermediate filament (KIF) cytoskeleton of cultured human alveolar epithelial (A549) cells have been investigated. Under normal culture conditions, immunofluorescence revealed a delicate network of fine tonofibrils containing KIFs, together with many nonfilamentous, keratin-containing “particles,” mostly containing either keratin 8 (K8) or 18 (K18), but not both. Triton X-100 extracted ∼10% of the cellular keratin, and this was accompanied by a loss of the particles but not the KIFs. Shear stress dramatically reduced the soluble keratin component and transformed the fine bundles of KIFs into thicker, “wavy” tonofibrils. Both effects were accompanied by the disappearance of most keratin particles and by increased phosphorylation of K8 and K18 on serine residues 73 and 33, respectively. The particles that remained after shearing were phosphorylated and were closely associated with KIFs. We suggest that keratin particles constitute a reservoir of protein that can be recruited into KIFs under flow, creating a more robust cytoskeleton able to withstand shear forces more effectively.—Flitney, E. W., Kuczmarski, E. R., Adam, S. A., Goldman, R. D. Insights into the mechanical properties of epithelial cells: the effects of shear stress on the assembly and remodeling of keratin intermediate filaments. PMID:19246484

  18. Apical Invasion of Intestinal Epithelial Cells by Salmonella typhimurium Requires Villin to Remodel the Brush Border Actin Cytoskeleton

    PubMed Central

    Lhocine, Nouara; Arena, Ellen T.; Bomme, Perrine; Ubelmann, Florent; Prévost, Marie-Christine; Robine, Sylvie; Sansonetti, Philippe J.

    2015-01-01

    Summary Salmonella invasion of intestinal epithelial cells requires extensive, though transient, actin modifications at the site of bacterial entry. The actin-modifying protein villin is present in the brush border where it participates in the constitution of microvilli and in epithelial restitution after damage through its actin-severing activity. We investigated a possible role for villin in Salmonella invasion. The absence of villin, which is normally located at the bacterial entry site, leads to a decrease in Salmonella invasion. Villin is necessary for early membrane-associated processes and for optimal ruffle assembly by balancing the steady-state level of actin. The severing activity of villin is important for Salmonella invasion in vivo. The bacterial phosphatase SptP tightly regulates villin phosphorylation, while the actin-binding effector SipA protects F-actin and counterbalances villin-severing activity. Thus, villin plays an important role in establishing the balance between actin polymerization and actin severing to facilitate the initial steps of Salmonella entry. PMID:25600187

  19. Cell Division Drives Epithelial Cell Rearrangements during Gastrulation in Chick.

    PubMed

    Firmino, Joao; Rocancourt, Didier; Saadaoui, Mehdi; Moreau, Chloe; Gros, Jerome

    2016-02-01

    During early embryonic development, cells are organized as cohesive epithelial sheets that are continuously growing and remodeled without losing their integrity, giving rise to a wide array of tissue shapes. Here, using live imaging in chick embryo, we investigate how epithelial cells rearrange during gastrulation. We find that cell division is a major rearrangement driver that powers dramatic epithelial cell intercalation events. We show that these cell division-mediated intercalations, which represent the majority of epithelial rearrangements within the early embryo, are absolutely necessary for the spatial patterning of gastrulation movements. Furthermore, we demonstrate that these intercalation events result from overall low cortical actomyosin accumulation within the epithelial cells of the embryo, which enables dividing cells to remodel junctions in their vicinity. These findings uncover a role for cell division as coordinator of epithelial growth and remodeling that might underlie various developmental, homeostatic, or pathological processes in amniotes. PMID:26859350

  20. EB1-recruited microtubule +TIP complexes coordinate protrusion dynamics during 3D epithelial remodeling

    PubMed Central

    Gierke, Sarah; Wittmann, Torsten

    2012-01-01

    SUMMARY Background Epithelial remodeling, in which apical-basal polarized cells switch to a migratory phenotype, plays a central role in development and disease of multicellular organisms. Although dynamic microtubules (MTs) are required for directed migration on flat surfaces, how MT dynamics are controlled or contribute to epithelial remodeling in a more physiological three-dimensional (3D) environment is not understood. We use confocal live cell imaging to analyze MT function and dynamics during 3D epithelial morphogenesis and remodeling of polarized Madin-Darby canine kidney (MDCK) epithelial cells that undergo partial epithelial-to-mesenchymal transition (EMT) in response to hepatocyte growth factor (HGF). Results We find that HGF treatment increases MT growth rate before morphological changes are evident, and that large numbers of MTs grow into HGF-induced cell extensions independent of centrosome reorientation. Using lentivirus-mediated shRNA, we demonstrate that EB1, an adaptor protein that mediates recruitment of numerous other +TIP proteins to growing MT plus ends, is required for this HGF-induced MT reorganization. We further show that protrusion and adhesion dynamics are disorganized, and that vesicular trafficking to the tip of HGF-induced cell extensions is disrupted in EB1-depleted cells. Conclusions We conclude that EB1-mediated interactions with growing MTs are important to coordinate cell shape changes and directed migration into the surrounding extracellular matrix during epithelial remodeling in a physiological 3D environment. In contrast, EB1 is not required for the establishment or maintenance of apical-basal cell polarity, suggesting different functions of +TIPs and MTs in different types of cell polarity. PMID:22483942

  1. Plant cell remodeling by autophagy

    PubMed Central

    Kim, Jimi; Lee, Han Nim; Chung, Taijoon

    2014-01-01

    Plant seedlings are not photoautotrophs until they are equipped with photosynthetic machinery. Some plant cells are remodeled after being exposed to light, and a group of peroxisomal proteins are degraded during the remodeling. Autophagy was proposed as one of the mechanisms for the degradation of peroxisomal proteins. We recently showed that ATG7-dependent autophagy is partially responsible for the degradation of obsolete peroxisomal proteins during Arabidopsis seedling growth. PMID:24492493

  2. Tubular endocytosis drives remodelling of the apical surface during epithelial morphogenesis in Drosophila.

    PubMed

    Fabrowski, Piotr; Necakov, Aleksandar S; Mumbauer, Simone; Loeser, Eva; Reversi, Alessandra; Streichan, Sebastian; Briggs, John A G; De Renzis, Stefano

    2013-01-01

    During morphogenesis, remodelling of cell shape requires the expansion or contraction of plasma membrane domains. Here we identify a mechanism underlying the restructuring of the apical surface during epithelial morphogenesis in Drosophila. We show that the retraction of villous protrusions and subsequent apical plasma membrane flattening is an endocytosis-driven morphogenetic process. Quantitation of endogenously tagged GFP::Rab5 dynamics reveals a massive increase in apical endocytosis that correlates with changes in apical morphology. This increase is accompanied by the formation of tubular plasma membrane invaginations that serve as platforms for the de novo generation of Rab5-positive endosomes. We identify the Rab5-effector Rabankyrin-5 as a regulator of this pathway and demonstrate that blocking dynamin activity results in the complete inhibition of tubular endocytosis, in the disappearance of Rab5 endosomes, and in the inhibition of surface flattening. These data collectively demonstrate a requirement for endocytosis in morphogenetic remodelling during epithelial development. PMID:23921440

  3. Regulation of epithelial function, differentiation, and remodeling in the epididymis

    PubMed Central

    Breton, Sylvie; Ruan, Ye Chun; Park, Yoo-Jin; Kim, Bongki

    2016-01-01

    The epididymis is a single convoluted tubule lined by a pseudostratified epithelium. Specialized epididymal epithelial cells, the so-called principal, basal, narrow, and clear cells, establish a unique luminal environment for the maturation and storage of spermatozoa. The epididymis is functionally and structurally divided into several segments and sub-segments that create regionally distinct luminal environments. This organ is immature at birth, and epithelial cells acquire their fully differentiated phenotype during an extended postnatal period, but the factors involved in this complex process remain incompletely characterized. In the adult epididymis, the establishment of an acidic luminal pH and low bicarbonate concentration in the epididymis contributes to preventing premature activation of spermatozoa during their maturation and storage. Clear cells are proton-secreting cells throughout the epididymis, but principal cells have distinct acid/base transport properties, depending on their localization within the epididymis. Basal cells are located in all epididymal segments, but they have a distinct morphology depending on the segment and species examined. How this structural plasticity of basal cells is regulated is discussed here. Also, the role of luminal factors and androgens in the regulation of epithelial cells is reviewed in relation to their respective localization in the proximal versus distal regions of the epididymis. Finally, we describe a novel role for CFTR in tubulogenesis and epithelial cell differentiation. PMID:26585699

  4. Smoking cessation and bronchial epithelial remodelling in COPD: a cross-sectional study

    PubMed Central

    Lapperre, Thérèse S; Sont, Jacob K; van Schadewijk, Annemarie; Gosman, Margot ME; Postma, Dirkje S; Bajema, Ingeborg M; Timens, Wim; Mauad, Thais; Hiemstra, Pieter S

    2007-01-01

    Background Chronic Obstructive Pulmonary Disease (COPD) is associated with bronchial epithelial changes, including squamous cell metaplasia and goblet cell hyperplasia. These features are partially attributed to activation of the epidermal growth factor receptor (EGFR). Whereas smoking cessation reduces respiratory symptoms and lung function decline in COPD, inflammation persists. We determined epithelial proliferation and composition in bronchial biopsies from current and ex-smokers with COPD, and its relation to duration of smoking cessation. Methods 114 COPD patients were studied cross-sectionally: 99 males/15 females, age 62 ± 8 years, median 42 pack-years, no corticosteroids, current (n = 72) or ex-smokers (n = 42, median cessation duration 3.5 years), postbronchodilator FEV1 63 ± 9% predicted. Squamous cell metaplasia (%), goblet cell (PAS/Alcian Blue+) area (%), proliferating (Ki-67+) cell numbers (/mm basement membrane), and EGFR expression (%) were measured in intact epithelium of bronchial biopsies. Results Ex-smokers with COPD had significantly less epithelial squamous cell metaplasia, proliferating cell numbers, and a trend towards reduced goblet cell area than current smokers with COPD (p = 0.025, p = 0.001, p = 0.081, respectively), but no significant difference in EGFR expression. Epithelial features were not different between short-term quitters (<3.5 years) and current smokers. Long-term quitters (≥3.5 years) had less goblet cell area than both current smokers and short-term quitters (medians: 7.9% vs. 14.4%, p = 0.005; 7.9% vs. 13.5%, p = 0.008; respectively), and less proliferating cell numbers than current smokers (2.8% vs. 18.6%, p < 0.001). Conclusion Ex-smokers with COPD had less bronchial epithelial remodelling than current smokers, which was only observed after long-term smoking cessation (>3.5 years). Trial registration NCT00158847 PMID:18039368

  5. Pak4 Is Required during Epithelial Polarity Remodeling through Regulating AJ Stability and Bazooka Retention at the ZA

    PubMed Central

    Walther, Rhian F.; Nunes de Almeida, Francisca; Vlassaks, Evi; Burden, Jemima J.; Pichaud, Franck

    2016-01-01

    Summary The ability of epithelial cells to assemble into sheets relies on their zonula adherens (ZA), a circumferential belt of adherens junction (AJ) material, which can be remodeled during development to shape organs. Here, we show that during ZA remodeling in a model neuroepithelial cell, the Cdc42 effector P21-activated kinase 4 (Pak4/Mbt) regulates AJ morphogenesis and stability through β-catenin (β-cat/Arm) phosphorylation. We find that β-catenin phosphorylation by Mbt, and associated AJ morphogenesis, is needed for the retention of the apical determinant Par3/Bazooka at the remodeling ZA. Importantly, this retention mechanism functions together with Par1-dependent lateral exclusion of Par3/Bazooka to regulate apical membrane differentiation. Our results reveal an important functional link between Pak4, AJ material morphogenesis, and polarity remodeling during organogenesis downstream of Par3. PMID:27052178

  6. Involvement of Toll-like receptor 2 and epidermal growth factor receptor signaling in epithelial expression of airway remodeling factors.

    PubMed

    Homma, Tetsuya; Kato, Atsushi; Sakashita, Masafumi; Norton, James E; Suh, Lydia A; Carter, Roderick G; Schleimer, Robert P

    2015-04-01

    Staphylococcus aureus (SA) colonization and infection is common, and may promote allergic or inflammatory airway diseases, such as asthma, cystic fibrosis, and chronic rhinosinusitis by interacting with airway epithelial cells. Airway epithelial cells not only comprise a physical barrier, but also play key roles in immune, inflammatory, repair, and remodeling responses upon encounters with pathogens. To elucidate the impact of SA on epithelial-mediated remodeling of allergic airways, we tested the hypothesis that SA can enhance the remodeling process. Normal human bronchial epithelial (NHBE) cells were stimulated with heat-killed SA (HKSA) or transforming growth factor (TGF) α. Cell extracts were collected to measure mRNA (real-time RT-PCR) and signaling molecules (Western blot); supernatants were collected to measure protein (ELISA) after 24 hours of stimulation. Epidermal growth factor receptor (EGFR) signaling inhibition experiments were performed using a specific EGFR kinase inhibitor (AG1478) and TGF-α was blocked with an anti-TGF-α antibody. HKSA induced both mRNA and protein for TGF-α and matrix metalloproteinase (MMP) 1 from NHBE cells by a Toll-like receptor 2-dependent mechanism. Recombinant human TGF-α also induced mRNA and protein for MMP-1 from NHBE cells; anti-TGF-α antibody inhibited HKSA-induced MMP-1, suggesting that endogenous TGF-α mediates the MMP-1 induction by HKSA. HKSA-induced MMP-1 expression was suppressed when a specific EGFR kinase inhibitor was added, suggesting that EGFR signaling was mediating the HKSA-induced MMP-1 release. Exposure or colonization by SA in the airway may enhance the remodeling of tissue through a TGF-α-dependent induction of MMP-1 expression, and may thereby promote remodeling in airway diseases in which SA is implicated, such as asthma and chronic rhinosinusitis. PMID:25180535

  7. Involvement of Toll-Like Receptor 2 and Epidermal Growth Factor Receptor Signaling in Epithelial Expression of Airway Remodeling Factors

    PubMed Central

    Kato, Atsushi; Sakashita, Masafumi; Norton, James E.; Suh, Lydia A.; Carter, Roderick G.; Schleimer, Robert P.

    2015-01-01

    Staphylococcus aureus (SA) colonization and infection is common, and may promote allergic or inflammatory airway diseases, such as asthma, cystic fibrosis, and chronic rhinosinusitis by interacting with airway epithelial cells. Airway epithelial cells not only comprise a physical barrier, but also play key roles in immune, inflammatory, repair, and remodeling responses upon encounters with pathogens. To elucidate the impact of SA on epithelial-mediated remodeling of allergic airways, we tested the hypothesis that SA can enhance the remodeling process. Normal human bronchial epithelial (NHBE) cells were stimulated with heat-killed SA (HKSA) or transforming growth factor (TGF) α. Cell extracts were collected to measure mRNA (real-time RT-PCR) and signaling molecules (Western blot); supernatants were collected to measure protein (ELISA) after 24 hours of stimulation. Epidermal growth factor receptor (EGFR) signaling inhibition experiments were performed using a specific EGFR kinase inhibitor (AG1478) and TGF-α was blocked with an anti–TGF-α antibody. HKSA induced both mRNA and protein for TGF-α and matrix metalloproteinase (MMP) 1 from NHBE cells by a Toll-like receptor 2–dependent mechanism. Recombinant human TGF-α also induced mRNA and protein for MMP-1 from NHBE cells; anti–TGF-α antibody inhibited HKSA-induced MMP-1, suggesting that endogenous TGF-α mediates the MMP-1 induction by HKSA. HKSA-induced MMP-1 expression was suppressed when a specific EGFR kinase inhibitor was added, suggesting that EGFR signaling was mediating the HKSA-induced MMP-1 release. Exposure or colonization by SA in the airway may enhance the remodeling of tissue through a TGF-α–dependent induction of MMP-1 expression, and may thereby promote remodeling in airway diseases in which SA is implicated, such as asthma and chronic rhinosinusitis. PMID:25180535

  8. Insights into the mechanical properties of epithelial cells: the effects of shear stress on the assembly and remodeling of keratin intermediate filaments.

    PubMed

    Flitney, Eric W; Kuczmarski, Edward R; Adam, Stephen A; Goldman, Robert D

    2009-07-01

    The effects of shear stress on the keratin intermediate filament (KIF) cytoskeleton of cultured human alveolar epithelial (A549) cells have been investigated. Under normal culture conditions, immunofluorescence revealed a delicate network of fine tonofibrils containing KIFs, together with many nonfilamentous, keratin-containing "particles," mostly containing either keratin 8 (K8) or 18 (K18), but not both. Triton X-100 extracted approximately 10% of the cellular keratin, and this was accompanied by a loss of the particles but not the KIFs. Shear stress dramatically reduced the soluble keratin component and transformed the fine bundles of KIFs into thicker, "wavy" tonofibrils. Both effects were accompanied by the disappearance of most keratin particles and by increased phosphorylation of K8 and K18 on serine residues 73 and 33, respectively. The particles that remained after shearing were phosphorylated and were closely associated with KIFs. We suggest that keratin particles constitute a reservoir of protein that can be recruited into KIFs under flow, creating a more robust cytoskeleton able to withstand shear forces more effectively. PMID:19246484

  9. Epithelial Cell Adhesion Molecule

    PubMed Central

    Trzpis, Monika; McLaughlin, Pamela M.J.; de Leij, Lou M.F.H.; Harmsen, Martin C.

    2007-01-01

    The epithelial cell adhesion molecule (EpCAM, CD326) is a glycoprotein of ∼40 kd that was originally identified as a marker for carcinoma, attributable to its high expression on rapidly proliferating tumors of epithelial origin. Normal epithelia express EpCAM at a variable but generally lower level than carcinoma cells. In early studies, EpCAM was proposed to be a cell-cell adhesion molecule. However, recent insights revealed a more versatile role for EpCAM that is not limited only to cell adhesion but includes diverse processes such as signaling, cell migration, proliferation, and differentiation. Cell surface expression of EpCAM may actually prevent cell-cell adhesion. Here, we provide a comprehensive review of the current knowledge on EpCAM biology in relation to other cell adhesion molecules. We discuss the implications of the newly identified functions of EpCAM in view of its prognostic relevance in carcinoma, inflammatory pathophysiology, and tissue development and regeneration as well as its role in normal epithelial homeostasis. PMID:17600130

  10. Inhalation exposure to ethylene induces eosinophilic rhinitis and nasal epithelial remodeling in Fischer 344 rats.

    PubMed

    Brandenberger, Christina; Hotchkiss, Jon A; Krieger, Shannon M; Pottenger, Lynn H; Harkema, Jack R

    2015-11-01

    This study investigated the time- and concentration-dependent effects of inhaled ethylene on eosinophilic rhinitis and nasal epithelial remodeling in Fisher 344 rats exposed to 0, 10, 50, 300, or 10,000 ppm ethylene, 6 h/day, 5 days/week for up to 4 weeks. Morphometric quantitation of eosinophilic inflammation and mucous cell metaplasia/hyperplasia (MCM) and nasal mucosal gene expression were evaluated at anatomic sites previously shown to undergo ethylene-induced epithelial remodeling. Serum levels of total IgE, IgG1 and IgG2a were measured to determine if ethylene exposure increased the expression of Th2-associated (IgE and IgG1) relative to Th1-associated (IgG2a) antibody isotypes. Rats exposed to 0 or 10,000 ppm for 1, 3, 5, 10, or 20 days were analyzed to assess the temporal pattern of ethylene-induced alterations in nasal epithelial cell proliferation, morphology and gene expression. Rats exposed to 0, 10, 50, 300, and 10,000 ppm ethylene for 20 days were analyzed to assess concentration-dependent effects on lesion development. Additional rats exposed 4 weeks to 0, 300, or 10,000 ppm ethylene were held for 13 weeks post-exposure to examine the persistence of ethylene-induced mucosal alterations. The data indicate that cell death and reparative cell proliferation were not a part of the pathogenesis of ethylene-induced nasal lesions. Enhanced gene expression of Th2 cytokines (e.g., IL-5, IL-13) and chitinase (YM1/2) in the nasal mucosa was much greater than that of Th1 cytokines (e.g., IFNγ) after ethylene exposure. A significant increase in MCM was measured after 5 days of exposure to 10,000 ppm ethylene and after 20 days of exposure 10 ppm ethylene. Ethylene-induced MCM was reversible after cessation of exposure. No increase in total serum IgE, IgG1 or IgG2a was measured in any ethylene-exposed group. These data do not support involvement of an immune-mediated allergic mechanism in the pathogenesis of ethylene-induced nasal lesions in rats. Repeated

  11. Epithelial stem cells.

    PubMed

    Draheim, Kyle M; Lyle, Stephen

    2011-01-01

    It is likely that adult epithelial stem cells will be useful in the treatment of diseases, such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa, and alopecias. Additionally, other skin problems such as burn wounds, chronic wounds, and ulcers will benefit from stem cell-related therapies. However, there are many questions that need to be answered before this goal can be realized. The most important of these questions is what regulates the adhesion of stem cells to the niche versus migration to the site of injury. We have started to identify the mechanisms involved in this decision-making process. PMID:21618097

  12. Thyroid hormone activates Wnt/β-catenin signaling involved in adult epithelial development during intestinal remodeling in Xenopus laevis.

    PubMed

    Hasebe, Takashi; Fujimoto, Kenta; Kajita, Mitsuko; Ishizuya-Oka, Atsuko

    2016-08-01

    During amphibian intestinal remodeling, thyroid hormone (TH) induces some larval epithelial cells to dedifferentiate into adult stem cells, which newly generate the absorptive epithelium analogous to the mammalian epithelium. To clarify molecular mechanisms underlying adult epithelial development, we here focus on TH response genes that are associated with the canonical Wnt pathway. Our quantitative reverse transcription plus polymerase chain reaction and immunohistochemical analyses indicate that all of the genes examined, including β-catenin, c-Myc and secreted frizzle-related protein 2 (SFRP2), are up-regulated in Xenopus laevis intestine during both natural and TH-induced metamorphosis. Moreover, immunoreactivity for nuclear β-catenin becomes detectable in adult stem cells from the start of their appearance and then increases in intensity in adult epithelial primordia derived from the stem cells, which actively proliferate and coexpress Wnt target genes c-Myc and LGR5. These expression profiles strongly suggest the involvement of the canonical Wnt pathway in the maintenance and/or proliferation of adult stem/progenitor cells. More importantly, by using organ cultures of the tadpole intestine, we have experimentally shown that the addition of exogenous SFRP2 protein to the culture medium promotes cell proliferation of the adult epithelial primordia, whereas inhibition of endogenous SFRP2 by its antibody suppresses their proliferation. The inhibition of SFRP2 suppresses larval epithelial changes in shape from simple columnar to stem-cell-like roundish cells, resulting in the failure of epithelial dedifferentiation. Thus, TH-up-regulated SFRP2 in the postembryonic intestine promotes adult stem cell development, possibly by acting as an agonist of both canonical and non-canonical Wnt signaling. PMID:27068920

  13. Left-right asymmetric cell intercalation drives directional collective cell movement in epithelial morphogenesis

    NASA Astrophysics Data System (ADS)

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Maekawa, Emi; Isomura, Ayako; Shibata, Tatsuo; Kuranaga, Erina

    2015-12-01

    Morphogenetic epithelial movement occurs during embryogenesis and drives complex tissue formation. However, how epithelial cells coordinate their unidirectional movement while maintaining epithelial integrity is unclear. Here we propose a novel mechanism for collective epithelial cell movement based on Drosophila genitalia rotation, in which epithelial tissue rotates clockwise around the genitalia. We found that this cell movement occurs autonomously and requires myosin II. The moving cells exhibit repeated left-right-biased junction remodelling, while maintaining adhesion with their neighbours, in association with a polarized myosin II distribution. Reducing myosinID, known to cause counter-clockwise epithelial-tissue movement, reverses the myosin II distribution. Numerical simulations revealed that a left-right asymmetry in cell intercalation is sufficient to induce unidirectional cellular movement. The cellular movement direction is also associated with planar cell-shape chirality. These findings support a model in which left-right asymmetric cell intercalation within an epithelial sheet drives collective cellular movement in the same direction.

  14. Integrins and epithelial cell polarity

    PubMed Central

    Lee, Jessica L.; Streuli, Charles H.

    2014-01-01

    ABSTRACT Cell polarity is characterised by differences in structure, composition and function between at least two poles of a cell. In epithelial cells, these spatial differences allow for the formation of defined apical and basal membranes. It has been increasingly recognised that cell–matrix interactions and integrins play an essential role in creating epithelial cell polarity, although key gaps in our knowledge remain. This Commentary will discuss the mounting evidence for the role of integrins in polarising epithelial cells. We build a model in which both inside-out signals to polarise basement membrane assembly at the basal surface, and outside-in signals to control microtubule apical–basal orientation and vesicular trafficking are required for establishing and maintaining the orientation of epithelial cell polarity. Finally, we discuss the relevance of the basal integrin polarity axis to cancer. This article is part of a Minifocus on Establishing polarity. For further reading, please see related articles: ‘ERM proteins at a glance’ by Andrea McClatchey (J. Cell Sci. 127, 3199–3204). ‘Establishment of epithelial polarity – GEF who's minding the GAP?’ by Siu Ngok et al. (J. Cell Sci. 127, 3205–3215). PMID:24994933

  15. The SYT-SSX fusion protein and histological epithelial differentiation in synovial sarcoma: relationship with extracellular matrix remodeling.

    PubMed

    Saito, Tsuyoshi

    2013-01-01

    Synovial sarcoma (SS) tumor cells, which have the chromosomal translocation t(X;18)(p11.2;q11.2), have an inherently greater propensity for epithelial differentiation than other mesenchymal tumors, especially spindle cell sarcomas. This is caused by de-repression of the transcription of E-cadherin by SYT-SSX1 and SYT-SSX2, which dissociate Snail or Slug, respectively, from the E-cadherin promoter. However, a subset of SS with SYT-SSX1 loses E-cadherin expression despite adequate de-repression because of mutations in E-cadherin, resulting in monophasic histology. The ratio of the expression levels of SYT-SSX1 and Snail is also associated with E-cadherin expression: the lower the SYT-SSX1/Snail ratio, the lower the level of E-cadherin expression, and vice versa, thus affecting tumor histology. In addition, Wnt signal activation caused by mutation of β-catenin, APC, or Axin 1 and 2 is associated with monophasic histology. Remodeling of the extracellular matrix is also important. Only cells that survive all of these steps can finally exhibit biphasic histology. On the other hand, the SYT-SSX2 fusion has a weaker de-repression effect on the E-cadherin promoter than does SYT-SSX1, so it is difficult for SYT-SSX2-expressing tumors to achieve sufficient capacity for epithelial differentiation to form glandular structures. This review provides an interesting model for this epithelial differentiation that shows a possible mechanism for the aberrant mesenchymal to epithelial transition of SS and suggests that it might better be considered an epithelial to mesenchymal transition. PMID:24228088

  16. Alignment of cell division axes in directed epithelial cell migration

    NASA Astrophysics Data System (ADS)

    Marel, Anna-Kristina; Podewitz, Nils; Zorn, Matthias; Oskar Rädler, Joachim; Elgeti, Jens

    2014-11-01

    Cell division is an essential dynamic event in tissue remodeling during wound healing, cancer and embryogenesis. In collective migration, tensile stresses affect cell shape and polarity, hence, the orientation of the cell division axis is expected to depend on cellular flow patterns. Here, we study the degree of orientation of cell division axes in migrating and resting epithelial cell sheets. We use microstructured channels to create a defined scenario of directed cell invasion and compare this situation to resting but proliferating cell monolayers. In experiments, we find a strong alignment of the axis due to directed flow while resting sheets show very weak global order, but local flow gradients still correlate strongly with the cell division axis. We compare experimental results with a previously published mesoscopic particle based simulation model. Most of the observed effects are reproduced by the simulations.

  17. Remodeling of the Epithelial-Connective Tissue Interface in Oral Epithelial Dysplasia as Visualized by Noninvasive 3D Imaging.

    PubMed

    Pal, Rahul; Shilagard, Tuya; Yang, Jinping; Villarreal, Paula; Brown, Tyra; Qiu, Suimin; McCammon, Susan; Resto, Vicente; Vargas, Gracie

    2016-08-15

    Early neoplastic features in oral epithelial dysplasia are first evident at the basal epithelium positioned at the epithelial-connective tissue interface (ECTI), separating the basal epithelium from the underlying lamina propria. The ECTI undergoes significant deformation in early neoplasia due to focal epithelial expansion and proteolytic remodeling of the lamina propria, but few studies have examined these changes. In the present study, we quantitated alterations in ECTI topography in dysplasia using in vivo volumetric multiphoton autofluorescence microscopy and second harmonic generation microscopy. The label-free method allows direct noninvasive visualization of the ECTI surface without perturbing the epithelium. An image-based parameter, "ECTI contour," is described that indicates deformation of the ECTI surface. ECTI contour was higher in dysplasia than control or inflamed specimens, indicating transition from flat to a deformed surface. Cellular parameters of nuclear area, nuclear density, coefficient of variation in nuclear area in the basal epithelium and collagen density in areas adjacent to ECTI were measured. ECTI contour differentiated dysplasia from control/benign mucosa with higher sensitivity and specificity than basal nuclear density or basal nuclear area, comparable with coefficient of variation in nuclear area and collagen density. The presented method offers a unique opportunity to study ECTI in intact mucosa with simultaneous assessment of cellular and extracellular matrix features, expanding opportunities for studies of early neoplastic events near this critical interface and potentially leading to development of new approaches for detecting neoplasia in vivo Cancer Res; 76(16); 4637-47. ©2016 AACR. PMID:27302162

  18. Ion Channels in Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Palmer, Lawrence G.

    Ion channels in epithelial cells serve to move ions, and in some cases fluid, between compartments of the body. This function of the transfer of material is fundamentally different from that of the transfer of information, which is the main job of most channels in excitable cells. Nevertheless the basic construction of the channels is similar in many respects in the two tissue types. This chapter reviews the nature of channels in epithelia and discusses how their functions have evolved to accomplish the basic tasks for which they are responsible. I will focus on three channel types: epithelial Na+ channels, inward-rectifier K+ channels, and CFTR Cl- channels.

  19. Cell wall remodeling under abiotic stress

    PubMed Central

    Tenhaken, Raimund

    2015-01-01

    Plants exposed to abiotic stress respond to unfavorable conditions on multiple levels. One challenge under drought stress is to reduce shoot growth while maintaining root growth, a process requiring differential cell wall synthesis and remodeling. Key players in this process are the formation of reactive oxygen species (ROS) and peroxidases, which initially cross-link phenolic compounds and glycoproteins of the cell walls causing stiffening. The function of ROS shifts after having converted all the peroxidase substrates in the cell wall. If ROS-levels remain high during prolonged stress, OH°-radicals are formed which lead to polymer cleavage. In concert with xyloglucan modifying enzymes and expansins, the resulting cell wall loosening allows further growth of stressed organs. PMID:25709610

  20. Alveolar Epithelial Cells Undergo Epithelial-to-Mesenchymal Transition in Response to Endoplasmic Reticulum Stress*

    PubMed Central

    Tanjore, Harikrishna; Cheng, Dong-Sheng; Degryse, Amber L.; Zoz, Donald F.; Abdolrasulnia, Rasul; Lawson, William E.; Blackwell, Timothy S.

    2011-01-01

    Expression of mutant surfactant protein C (SFTPC) results in endoplasmic reticulum (ER) stress in type II alveolar epithelial cells (AECs). AECs have been implicated as a source of lung fibroblasts via epithelial-to-mesenchymal transition (EMT); therefore, we investigated whether ER stress contributes to EMT as a possible mechanism for fibrotic remodeling. ER stress was induced by tunicamyin administration or stable expression of mutant (L188Q) SFTPC in type II AEC lines. Both tunicamycin treatment and mutant SFTPC expression induced ER stress and the unfolded protein response. With tunicamycin or mutant SFTPC expression, phase contrast imaging revealed a change to a fibroblast-like appearance. During ER stress, expression of epithelial markers E-cadherin and Zonula occludens-1 decreased while expression of mesenchymal markers S100A4 and α-smooth muscle actin increased. Following induction of ER stress, we found activation of a number of pathways, including MAPK, Smad, β-catenin, and Src kinase. Using specific inhibitors, the combination of a Smad2/3 inhibitor (SB431542) and a Src kinase inhibitor (PP2) blocked EMT with maintenance of epithelial appearance and epithelial marker expression. Similar results were noted with siRNA targeting Smad2 and Src kinase. Together, these studies reveal that induction of ER stress leads to EMT in lung epithelial cells, suggesting possible cross-talk between Smad and Src kinase pathways. Dissecting pathways involved in ER stress-induced EMT may lead to new treatment strategies to limit fibrosis. PMID:21757695

  1. Osteocytes: The master cells in bone remodelling.

    PubMed

    Prideaux, Matthew; Findlay, David M; Atkins, Gerald J

    2016-06-01

    Bone remodelling is an essential process for shaping and maintaining bone mass in the mature skeleton. During our lifetime bone is constantly being removed by osteoclasts and new bone is formed by osteoblasts. The activities of osteoclasts and osteoblasts must be regulated under a strict balance to ensure that bone homeostasis is maintained. Osteocytes, which form an extensive, multi-functional syncytium throughout the bone, are increasingly considered to be the cells that maintain this balance. Current research is elucidating key signalling pathways by which the osteocyte exerts control over the other cell types in bone and over its own activities, and potential ways in which these pathways may be exploited therapeutically. PMID:26927500

  2. Microvesicles released from tumor cells disrupt epithelial cell morphology and contractility.

    PubMed

    Bordeleau, Francois; Chan, Bryan; Antonyak, Marc A; Lampi, Marsha C; Cerione, Richard A; Reinhart-King, Cynthia A

    2016-05-24

    During tumor progression, cancer cells interact and communicate with non-malignant cells within their local microenvironment. Microvesicles (MV) derived from human cancer cells play an important role in mediating this communication. Another critical aspect of cancer progression involves widespread ECM remodeling, which occur both at the primary and metastatic sites. ECM remodeling and reorganization within the tumor microenvironment is generally attributed to fibroblasts. Here, using MCF10a cells, a well-characterized breast epithelial cell line that exhibits a non-malignant epithelial phenotype, and MVs shed by aggressive MDA-MB-231 carcinoma cells, we show that non-malignant epithelial cells can participate in ECM reorganization of 3D collagen matrices following their treatment with cancer cell-derived MVs. In addition, MVs trigger several changes in epithelial cells under 3D culture conditions. Furthermore, we show that this ECM reorganization is associated with an increase in cellular traction force following MV treatment, higher acto-myosin contractility, and higher FAK activity. Overall, our findings suggest that MVs derived from tumor cells can contribute to ECM reorganization occurring within the tumor microenvironment by enhancing the contractility of non-malignant epithelial cells. PMID:26477404

  3. Lymphoid Tissue Mesenchymal Stromal Cells in Development and Tissue Remodeling

    PubMed Central

    2016-01-01

    Secondary lymphoid organs (SLOs) are sites that facilitate cell-cell interactions required for generating adaptive immune responses. Nonhematopoietic mesenchymal stromal cells have been shown to play a critical role in SLO function, organization, and tissue homeostasis. The stromal microenvironment undergoes profound remodeling to support immune responses. However, chronic inflammatory conditions can promote uncontrolled stromal cell activation and aberrant tissue remodeling including fibrosis, thus leading to tissue damage. Despite recent advancements, the origin and role of mesenchymal stromal cells involved in SLO development and remodeling remain unclear. PMID:27190524

  4. Lymphoid Tissue Mesenchymal Stromal Cells in Development and Tissue Remodeling.

    PubMed

    Genovese, Luca; Brendolan, Andrea

    2016-01-01

    Secondary lymphoid organs (SLOs) are sites that facilitate cell-cell interactions required for generating adaptive immune responses. Nonhematopoietic mesenchymal stromal cells have been shown to play a critical role in SLO function, organization, and tissue homeostasis. The stromal microenvironment undergoes profound remodeling to support immune responses. However, chronic inflammatory conditions can promote uncontrolled stromal cell activation and aberrant tissue remodeling including fibrosis, thus leading to tissue damage. Despite recent advancements, the origin and role of mesenchymal stromal cells involved in SLO development and remodeling remain unclear. PMID:27190524

  5. Epithelial Sodium Channels in Pulmonary Epithelial Progenitor and Stem Cells

    PubMed Central

    Liu, Yang; Jiang, Bi-Jie; Zhao, Run-Zhen; Ji, Hong-Long

    2016-01-01

    Regeneration of the epithelium of mammalian lungs is essential for restoring normal function following injury, and various cells and mechanisms contribute to this regeneration and repair. Club cells, bronchioalveolar stem cells (BASCs), and alveolar type II epithelial cells (ATII) are dominant stem/progenitor cells for maintaining epithelial turnover and repair. Epithelial Na+ channels (ENaC), a critical pathway for transapical salt and fluid transport, are expressed in lung epithelial progenitors, including club and ATII cells. Since ENaC activity and expression are development- and differentiation-dependent, apically located ENaC activity has therefore been used as a functional biomarker of lung injury repair. ENaC activity may be involved in the migration and differentiation of local and circulating stem/progenitor cells with diverse functions, eventually benefiting stem cells spreading to re-epithelialize injured lungs. This review summarizes the potential roles of ENaC expressed in native progenitor and stem cells in the development and regeneration of the respiratory epithelium. PMID:27570489

  6. Hyperoxia alters the mechanical properties of alveolar epithelial cells.

    PubMed

    Roan, Esra; Wilhelm, Kristina; Bada, Alex; Makena, Patrudu S; Gorantla, Vijay K; Sinclair, Scott E; Waters, Christopher M

    2012-06-15

    Patients with severe acute lung injury are frequently administered high concentrations of oxygen (>50%) during mechanical ventilation. Long-term exposure to high levels of oxygen can cause lung injury in the absence of mechanical ventilation, but the combination of the two accelerates and increases injury. Hyperoxia causes injury to cells through the generation of excessive reactive oxygen species. However, the precise mechanisms that lead to epithelial injury and the reasons for increased injury caused by mechanical ventilation are not well understood. We hypothesized that alveolar epithelial cells (AECs) may be more susceptible to injury caused by mechanical ventilation if hyperoxia alters the mechanical properties of the cells causing them to resist deformation. To test this hypothesis, we used atomic force microscopy in the indentation mode to measure the mechanical properties of cultured AECs. Exposure of AECs to hyperoxia for 24 to 48 h caused a significant increase in the elastic modulus (a measure of resistance to deformation) of both primary rat type II AECs and a cell line of mouse AECs (MLE-12). Hyperoxia also caused remodeling of both actin and microtubules. The increase in elastic modulus was blocked by treatment with cytochalasin D. Using finite element analysis, we showed that the increase in elastic modulus can lead to increased stress near the cell perimeter in the presence of stretch. We then demonstrated that cyclic stretch of hyperoxia-treated cells caused significant cell detachment. Our results suggest that exposure to hyperoxia causes structural remodeling of AECs that leads to decreased cell deformability. PMID:22467640

  7. Epithelial cells and Von Gierke's disease.

    PubMed

    Negishi, H; Benke, P J

    1977-08-01

    Epithelial cells and not fibroblasts from human liver and amniotic fluid contain inducible glucose-6-phosphatase (G-6-Pase) activity. The diagnosis of Von Gierke's disease has been made in a patient with hepatomegaly utilizing cultured epithelial cells grown from a liver biopsy. G-6-Pase activity in epithelial cells from this patient could not be induced by dibutyryl cyclic AMP and theophylline. This is the first use of epithelial cells for diagnosis of a metabolic disease. G-6-Pase activity in cloned epithelial cells from amniotic fluid increases 2- to 3-fold after 24-hr exposure to dibutyryl cyclic AMP and theophylline. The prenatal diagnosis of Von Gierke's disease may be possible in a laboratory experienced with these techniques if epithelial cell growth is obtained from amniotic fluid. PMID:196249

  8. Left-right asymmetric cell intercalation drives directional collective cell movement in epithelial morphogenesis.

    PubMed

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Maekawa, Emi; Isomura, Ayako; Shibata, Tatsuo; Kuranaga, Erina

    2015-01-01

    Morphogenetic epithelial movement occurs during embryogenesis and drives complex tissue formation. However, how epithelial cells coordinate their unidirectional movement while maintaining epithelial integrity is unclear. Here we propose a novel mechanism for collective epithelial cell movement based on Drosophila genitalia rotation, in which epithelial tissue rotates clockwise around the genitalia. We found that this cell movement occurs autonomously and requires myosin II. The moving cells exhibit repeated left-right-biased junction remodelling, while maintaining adhesion with their neighbours, in association with a polarized myosin II distribution. Reducing myosinID, known to cause counter-clockwise epithelial-tissue movement, reverses the myosin II distribution. Numerical simulations revealed that a left-right asymmetry in cell intercalation is sufficient to induce unidirectional cellular movement. The cellular movement direction is also associated with planar cell-shape chirality. These findings support a model in which left-right asymmetric cell intercalation within an epithelial sheet drives collective cellular movement in the same direction. PMID:26656655

  9. Left–right asymmetric cell intercalation drives directional collective cell movement in epithelial morphogenesis

    PubMed Central

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Maekawa, Emi; Isomura, Ayako; Shibata, Tatsuo; Kuranaga, Erina

    2015-01-01

    Morphogenetic epithelial movement occurs during embryogenesis and drives complex tissue formation. However, how epithelial cells coordinate their unidirectional movement while maintaining epithelial integrity is unclear. Here we propose a novel mechanism for collective epithelial cell movement based on Drosophila genitalia rotation, in which epithelial tissue rotates clockwise around the genitalia. We found that this cell movement occurs autonomously and requires myosin II. The moving cells exhibit repeated left–right-biased junction remodelling, while maintaining adhesion with their neighbours, in association with a polarized myosin II distribution. Reducing myosinID, known to cause counter-clockwise epithelial-tissue movement, reverses the myosin II distribution. Numerical simulations revealed that a left–right asymmetry in cell intercalation is sufficient to induce unidirectional cellular movement. The cellular movement direction is also associated with planar cell-shape chirality. These findings support a model in which left–right asymmetric cell intercalation within an epithelial sheet drives collective cellular movement in the same direction. PMID:26656655

  10. High mobility group box 1-induced epithelial mesenchymal transition in human airway epithelial cells.

    PubMed

    Chen, Yu-Ching; Statt, Sarah; Wu, Reen; Chang, Hao-Teng; Liao, Jiunn-Wang; Wang, Chien-Neng; Shyu, Woei-Cherng; Lee, Chen-Chen

    2016-01-01

    Epithelial-mesenchymal transition (EMT) is implicated in bronchial remodeling and loss of lung function in chronic inflammatory airway diseases. Previous studies showed the involvement of the high mobility group box 1 (HMGB1) protein in the pathology of chronic pulmonary inflammatory diseases. However, the role of HMGB1 in EMT of human airway epithelial cells is still unclear. In this study, we used RNA sequencing to show that HMGB1 treatment regulated EMT-related gene expression in human primary-airway epithelial cells. The top five upregulated genes were SNAI2, FGFBP1, VIM, SPARC (osteonectin), and SERPINE1, while the downregulated genes included OCLN, TJP1 (ZO-1), FZD7, CDH1 (E-cadherin), and LAMA5. We found that HMGB1 induced downregulation of E-cadherin and ZO-1, and upregulation of vimentin mRNA transcription and protein translation in a dose-dependent manner. Additionally, we observed that HMGB1 induced AKT phosphorylation, resulting in GSK3β inactivation, cytoplasmic accumulation, and nuclear translocation of β-catenin to induce EMT in human airway epithelial cells. Treatment with PI3K inhibitor (LY294006) and β-catenin shRNA reversed HMGB1-induced EMT. Moreover, HMGB1 induced expression of receptor for advanced glycation products (RAGE), but not that of Toll-like receptor (TLR) 2 or TLR4, and RAGE shRNA inhibited HMGB1-induced EMT in human airway epithelial cells. In conclusion, we found that HMGB1 induced EMT through RAGE and the PI3K/AKT/GSK3β/β-catenin signaling pathway. PMID:26739898

  11. LOXL2 in epithelial cell plasticity and tumor progression.

    PubMed

    Cano, Amparo; Santamaría, Patricia G; Moreno-Bueno, Gema

    2012-09-01

    Several members of the lysyl oxidase family have recently emerged as important regulators of tumor progression. Among them, LOXL2 has been shown to be involved in tumor progression and metastasis of several tumor types, including breast carcinomas. Secreted LOXL2 participates in the remodeling of the extracellular matrix of the tumor microenvironment, in a similar fashion to prototypical lysyl oxidase. In addition, new intracellular functions of LOXL2 have been described, such as its involvement in the regulation of the epithelial-to-mesenchymal transition, epithelial cell polarity and differentiation mediated by transcriptional repression mechanisms. Importantly, intracellular (perinuclear) expression of LOXL2 is associated with poor prognosis and distant metastasis of specific tumor types, such as larynx squamous cell carcinoma and basal breast carcinomas. These recent findings open new avenues for the therapeutic utility of LOXL2. PMID:23030485

  12. Epithelial organization, cell polarity and tumorigenesis.

    PubMed

    McCaffrey, Luke Martin; Macara, Ian G

    2011-12-01

    Epithelial cells comprise the foundation for the majority of organs in the mammalian body, and are the source of approximately 90% of all human cancers. Characteristically, epithelial cells form intercellular adhesions, exhibit apical/basal polarity, and orient their mitotic spindles in the plane of the epithelial sheet. Defects in these attributes result in the tissue disorganization associated with cancer. Epithelia undergo self-renewal from stem cells, which might in some cases be the cell of origin for cancers. The PAR polarity proteins are master regulators of epithelial organization, and are closely linked to signaling pathways such as Hippo, which orchestrate proliferation and apoptosis to control organ size. 3D ex vivo culture systems can now faithfully recapitulate epithelial organ morphogenesis, providing a powerful approach to study both normal development and the initiating events in carcinogenesis. PMID:21782440

  13. Epithelial TRPV1 signaling accelerates gingival epithelial cell proliferation.

    PubMed

    Takahashi, N; Matsuda, Y; Yamada, H; Tabeta, K; Nakajima, T; Murakami, S; Yamazaki, K

    2014-11-01

    Transient receptor potential cation channel subfamily V member 1 (TRPV1), a member of the calcium-permeable thermosensitive transient receptor potential superfamily, is a sensor of thermal and chemical stimuli. TRPV1 is activated by noxious heat (> 43°C), acidic conditions (pH < 6.6), capsaicin, and endovanilloids. This pain receptor was discovered on nociceptive fibers in the peripheral nervous system. TRPV1 was recently found to be expressed by non-neuronal cells, such as epithelial cells. The oral gingival epithelium is exposed to multiple noxious stimuli, including heat and acids derived from endogenous and exogenous substances; however, whether gingival epithelial cells (GECs) express TRPV1 is unknown. We show that both TRPV1 mRNA and protein are expressed by GECs. Capsaicin, a TRPV1 agonist, elevated intracellular Ca(2+) levels in the gingival epithelial cell line, epi 4. Moreover, TRPV1 activation in epi 4 cells accelerated proliferation. These responses to capsaicin were inhibited by a specific TRPV1 antagonist, SB-366791. We also observed GEC proliferation in capsaicin-treated mice in vivo. No effects were observed on GEC apoptosis by epithelial TRPV1 signaling. To examine the molecular mechanisms underlying this proliferative effect, we performed complementary (c)DNA microarray analysis of capsaicin-stimulated epi 4 cells. Compared with control conditions, 227 genes were up-regulated and 232 genes were down-regulated following capsaicin stimulation. Several proliferation-related genes were validated by independent experiments. Among them, fibroblast growth factor-17 and neuregulin 2 were significantly up-regulated in capsaicin-treated epi 4 cells. Our results suggest that functional TRPV1 is expressed by GECs and contributes to the regulation of cell proliferation. PMID:25266715

  14. Control of cell mechanics by RhoA and calcium fluxes during epithelial scattering.

    PubMed

    Haws, Hillary J; McNeil, Melissa A; Hansen, Marc D H

    2016-01-01

    Epithelial tissues use adherens junctions to maintain tight interactions and coordinate cellular activities. Adherens junctions are remodeled during epithelial morphogenesis, including instances of epithelial-mesenchymal transition, or EMT, wherein individual cells detach from the tissue and migrate as individual cells. EMT has been recapitulated by growth factor induction of epithelial scattering in cell culture. In culture systems, cells undergo a highly reproducible series of cell morphology changes, most notably cell spreading followed by cellular compaction and cell migration. These morphology changes are accompanied by striking actin rearrangements. The current evidence suggests that global changes in actomyosin-based cellular contractility, first a loss of contractility during spreading and its activation during cell compaction, are the main drivers of epithelial scattering. In this review, we focus on how spreading and contractility might be controlled during epithelial scattering. While we propose a central role for RhoA, which is well known to control cellular contractility in multiple systems and whose role in epithelial scattering is well accepted, we suggest potential roles for additional cellular systems whose role in epithelial cell biology has been less well documented. In particular, we propose critical roles for vesicle recycling, calcium channels, and calcium-dependent kinases. PMID:27583192

  15. Remodeling of Endogenous Mammary Epithelium by Breast Cancer Stem Cells

    PubMed Central

    Parashurama, Natesh; Lobo, Neethan A.; Ito, Ken; Mosley, Adriane R.; Habte, Frezghi G.; Zabala, Maider; Smith, Bryan R.; Lam, Jessica; Weissman, Irving L.; Clarke, Michael F.; Gambhir, Sanjiv S.

    2014-01-01

    Poorly regulated tissue remodeling results in increased breast cancer risk, yet how breast cancer stem cells (CSC) participate in remodeling is unknown. We performed in vivo imaging of changes in fluorescent, endogenous duct architecture as a metric for remodeling. First, we quantitatively imaged physiologic remodeling of primary branches of the developing and regenerating mammary tree. To assess CSC-specific remodeling events, we isolated CSC from MMTV-Wnt1 (mouse mammary tumor virus long-term repeat enhancer driving Wnt1 oncogene) breast tumors, a well studied model in which tissue remodeling affects tumorigenesis. We confirm that CSC drive tumorigenesis, suggesting a link between CSC and remodeling. We find that normal, regenerating, and developing gland maintain a specific branching pattern. In contrast, transplantation of CSC results in changes in the branching patterns of endogenous ducts while non-CSC do not. Specifically, in the presence of CSC, we identified an increased number of branches, branch points, ducts which have greater than 40 branches (5/33 for CSC and 0/39 for non-CSC), and histological evidence of increased branching. Moreover, we demonstrate that only CSC implants invade into surrounding stroma with structures similar to developing mammary ducts (nine for CSC and one for non-CSC). Overall, we demonstrate a novel approach for imaging physiologic and pathological remodeling. Furthermore, we identify unique, CSC-specific, remodeling events. Our data suggest that CSC interact with the microenvironment differently than non-CSC, and that this could eventually be a therapeutic approach for targeting CSC. PMID:22899386

  16. Remodeling of endogenous mammary epithelium by breast cancer stem cells.

    PubMed

    Parashurama, Natesh; Lobo, Neethan A; Ito, Ken; Mosley, Adriane R; Habte, Frezghi G; Zabala, Maider; Smith, Bryan R; Lam, Jessica; Weissman, Irving L; Clarke, Michael F; Gambhir, Sanjiv S

    2012-10-01

    Poorly regulated tissue remodeling results in increased breast cancer risk, yet how breast cancer stem cells (CSC) participate in remodeling is unknown. We performed in vivo imaging of changes in fluorescent, endogenous duct architecture as a metric for remodeling. First, we quantitatively imaged physiologic remodeling of primary branches of the developing and regenerating mammary tree. To assess CSC-specific remodeling events, we isolated CSC from MMTV-Wnt1 (mouse mammary tumor virus long-term repeat enhancer driving Wnt1 oncogene) breast tumors, a well studied model in which tissue remodeling affects tumorigenesis. We confirm that CSC drive tumorigenesis, suggesting a link between CSC and remodeling. We find that normal, regenerating, and developing gland maintain a specific branching pattern. In contrast, transplantation of CSC results in changes in the branching patterns of endogenous ducts while non-CSC do not. Specifically, in the presence of CSC, we identified an increased number of branches, branch points, ducts which have greater than 40 branches (5/33 for CSC and 0/39 for non-CSC), and histological evidence of increased branching. Moreover, we demonstrate that only CSC implants invade into surrounding stroma with structures similar to developing mammary ducts (nine for CSC and one for non-CSC). Overall, we demonstrate a novel approach for imaging physiologic and pathological remodeling. Furthermore, we identify unique, CSC-specific, remodeling events. Our data suggest that CSC interact with the microenvironment differently than non-CSC, and that this could eventually be a therapeutic approach for targeting CSC. PMID:22899386

  17. ATP-dependent chromatin remodeling in T cells

    PubMed Central

    Wurster, Andrea L.; Pazin, Michael J.

    2012-01-01

    One of the best studied systems for mammalian chromatin remodeling is transcriptional regulation during T cell development. The variety of these studies have led to important findings in T cell gene regulation and cell fate determination. Importantly, these findings have also advanced our knowledge of the function of remodeling enzymes in mammalian gene regulation. In this review, first we briefly present biochemical/cell-free analysis of 3 types of ATP dependent remodeling enzymes (SWI/SNF, Mi2, and ISWI), to construct an intellectual framework to understand how these enzymes might be working. Second, we compare and contrast the function of these enzymes, during early (thymic) and late (peripheral) T cell development. Finally, we examine some of the gaps in our present understanding. PMID:21999456

  18. High mobility group box 1-induced epithelial mesenchymal transition in human airway epithelial cells

    PubMed Central

    Chen, Yu-Ching; Statt, Sarah; Wu, Reen; Chang, Hao-Teng; Liao, Jiunn-Wang; Wang, Chien-Neng; Shyu, Woei-Cherng; Lee, Chen-Chen

    2016-01-01

    Epithelial–mesenchymal transition (EMT) is implicated in bronchial remodeling and loss of lung function in chronic inflammatory airway diseases. Previous studies showed the involvement of the high mobility group box 1 (HMGB1) protein in the pathology of chronic pulmonary inflammatory diseases. However, the role of HMGB1 in EMT of human airway epithelial cells is still unclear. In this study, we used RNA sequencing to show that HMGB1 treatment regulated EMT-related gene expression in human primary-airway epithelial cells. The top five upregulated genes were SNAI2, FGFBP1, VIM, SPARC (osteonectin), and SERPINE1, while the downregulated genes included OCLN, TJP1 (ZO-1), FZD7, CDH1 (E-cadherin), and LAMA5. We found that HMGB1 induced downregulation of E-cadherin and ZO-1, and upregulation of vimentin mRNA transcription and protein translation in a dose-dependent manner. Additionally, we observed that HMGB1 induced AKT phosphorylation, resulting in GSK3β inactivation, cytoplasmic accumulation, and nuclear translocation of β-catenin to induce EMT in human airway epithelial cells. Treatment with PI3K inhibitor (LY294006) and β-catenin shRNA reversed HMGB1-induced EMT. Moreover, HMGB1 induced expression of receptor for advanced glycation products (RAGE), but not that of Toll-like receptor (TLR) 2 or TLR4, and RAGE shRNA inhibited HMGB1-induced EMT in human airway epithelial cells. In conclusion, we found that HMGB1 induced EMT through RAGE and the PI3K/AKT/GSK3β/β-catenin signaling pathway. PMID:26739898

  19. Symmetry breaking mechanism for epithelial cell polarization

    NASA Astrophysics Data System (ADS)

    Veglio, A.; Gamba, A.; Nicodemi, M.; Bussolino, F.; Serini, G.

    2009-09-01

    In multicellular organisms, epithelial cells form layers separating compartments responsible for different physiological functions. At the early stage of epithelial layer formation, each cell of an aggregate defines an inner and an outer side by breaking the symmetry of its initial state, in a process known as epithelial polarization. By integrating recent biochemical and biophysical data with stochastic simulations of the relevant reaction-diffusion system, we provide evidence that epithelial cell polarization is a chemical phase-separation process induced by a local bistability in the signaling network at the level of the cell membrane. The early symmetry breaking event triggering phase separation is induced by adhesion-dependent mechanical forces localized in the point of convergence of cell surfaces when a threshold number of confluent cells is reached. The generality of the emerging phase-separation scenario is likely common to many processes of cell polarity formation.

  20. Airway epithelial cell responses to ozone injury

    SciTech Connect

    Leikauf, G.D.; Simpson, L.G.; Zhao, Qiyu

    1995-03-01

    The airway epithelial cell is an important target in ozone injury. Once activated, the airway epithelium responds in three phases. The initial, or immediate phase, involves activation of constitutive cells, often through direct covalent interactions including the formation of secondary ozonolysis products-hydroxyhydroperoxides, aldehydes, and hydrogen peroxide. Recently, we found hydroxyhydroperoxides to be potent agonists; of bioactive eicosanoid formation by human airway epithelial cells in culture. Other probable immediate events include activation and inactivation of enzymes present on the epithelial surface (e.g., neutral endopeptidase). During the next 2 to 24 hr, or early phase, epithelial cells respond by synthesis and release of chemotactic factors, including chemokines-macrophage inflammatory protein-2, RANTES, and interleukin-8. Infiltrating leukocytes during this period also release elastase, an important agonist of epithelial cell mucus secretion and additional chemokine formation. The third (late) phase of ozone injury is characterized by eosinophil or monocyte infiltration. Cytokine expression leads to alteration of structural protein synthesis, with increases in fibronectin evident by in situ hybridization. Synthesis of epithelial antiproteases, e.g., secretary leukocyte protease inhibitor, may also increase locally 24 to 48 hr after elastase concentrations become excessive. Thus, the epithelium is not merely a passive barrier to ozone injury but has a dynamic role in directing the migration, activating, and then counteracting inflammatory cells. Through these complex interactions, epithelial cells can be viewed as the initiators (alpha) and the receptors (omega) of ozone-induced airway disease. 51 refs., 2 figs., 3 tabs.

  1. Concise review: the relevance of human stem cell-derived organoid models for epithelial translational medicine.

    PubMed

    Hynds, Robert E; Giangreco, Adam

    2013-03-01

    Epithelial organ remodeling is a major contributing factor to worldwide death and disease, costing healthcare systems billions of dollars every year. Despite this, most fundamental epithelial organ research fails to produce new therapies and mortality rates for epithelial organ diseases remain unacceptably high. In large part, this failure in translating basic epithelial research into clinical therapy is due to a lack of relevance in existing preclinical models. To correct this, new models are required that improve preclinical target identification, pharmacological lead validation, and compound optimization. In this review, we discuss the relevance of human stem cell-derived, three-dimensional organoid models for addressing each of these challenges. We highlight the advantages of stem cell-derived organoid models over existing culture systems, discuss recent advances in epithelial tissue-specific organoids, and present a paradigm for using organoid models in human translational medicine. PMID:23203919

  2. Odontogenic epithelial stem cells: hidden sources.

    PubMed

    Padma Priya, Sivan; Higuchi, Akon; Abu Fanas, Salem; Pooi Ling, Mok; Kumari Neela, Vasantha; Sunil, P M; Saraswathi, T R; Murugan, Kadarkarai; Alarfaj, Abdullah A; Munusamy, Murugan A; Kumar, Suresh

    2015-12-01

    The ultimate goal of dental stem cell research is to construct a bioengineered tooth. Tooth formation occurs based on the well-organized reciprocal interaction of epithelial and mesenchymal cells. The dental mesenchymal stem cells are the best explored, but because the human odontogenic epithelium is lost after the completion of enamel formation, studies on these cells are scarce. The successful creation of a bioengineered tooth is achievable only when the odontogenic epithelium is reconstructed to produce a replica of natural enamel. This article discusses the untapped sources of odontogenic epithelial stem cells in humans, such as those present in the active dental lamina in postnatal life, in remnants of dental lamina (the gubernaculum cord), in the epithelial cell rests of Malassez, and in reduced enamel epithelium. The possible uses of these stem cells in regenerative medicine, not just for enamel formation, are discussed. PMID:26367485

  3. Pulmonary epithelial cancer cells and their exosomes metabolize myeloid cell-derived leukotriene C4 to leukotriene D4.

    PubMed

    Lukic, Ana; Ji, Jie; Idborg, Helena; Samuelsson, Bengt; Palmberg, Lena; Gabrielsson, Susanne; Rådmark, Olof

    2016-09-01

    Leukotrienes (LTs) play major roles in lung immune responses, and LTD4 is the most potent agonist for cysteinyl LT1, leading to bronchoconstriction and tissue remodeling. Here, we studied LT crosstalk between myeloid cells and pulmonary epithelial cells. Monocytic cells (Mono Mac 6 cell line, primary dendritic cells) and eosinophils produced primarily LTC4 In coincubations of these myeloid cells and epithelial cells, LTD4 became a prominent product. LTC4 released from the myeloid cells was further transformed by the epithelial cells in a transcellular manner. Formation of LTD4 was rapid when catalyzed by γ-glutamyl transpeptidase (GGT)1 in the A549 epithelial lung cancer cell line, but considerably slower when catalyzed by GGT5 in primary bronchial epithelial cells. When A549 cells were cultured in the presence of IL-1β, GGT1 expression increased about 2-fold. Also exosomes from A549 cells contained GGT1 and augmented LTD4 formation. Serine-borate complex (SBC), an inhibitor of GGT, inhibited conversion of LTC4 to LTD4 Unexpectedly, SBC also upregulated translocation of 5-lipoxygenase (LO) to the nucleus in Mono Mac 6 cells, and 5-LO activity. Our results demonstrate an active role for epithelial cells in biosynthesis of LTD4, which may be of particular relevance in the lung. PMID:27436590

  4. Physical principles of membrane remodelling during cell mechanoadaptation

    NASA Astrophysics Data System (ADS)

    Kosmalska, Anita Joanna; Casares, Laura; Elosegui-Artola, Alberto; Thottacherry, Joseph Jose; Moreno-Vicente, Roberto; González-Tarragó, Víctor; Del Pozo, Miguel Ángel; Mayor, Satyajit; Arroyo, Marino; Navajas, Daniel; Trepat, Xavier; Gauthier, Nils C.; Roca-Cusachs, Pere

    2015-06-01

    Biological processes in any physiological environment involve changes in cell shape, which must be accommodated by their physical envelope--the bilayer membrane. However, the fundamental biophysical principles by which the cell membrane allows for and responds to shape changes remain unclear. Here we show that the 3D remodelling of the membrane in response to a broad diversity of physiological perturbations can be explained by a purely mechanical process. This process is passive, local, almost instantaneous, before any active remodelling and generates different types of membrane invaginations that can repeatedly store and release large fractions of the cell membrane. We further demonstrate that the shape of those invaginations is determined by the minimum elastic and adhesive energy required to store both membrane area and liquid volume at the cell-substrate interface. Once formed, cells reabsorb the invaginations through an active process with duration of the order of minutes.

  5. Physical principles of membrane remodelling during cell mechanoadaptation.

    PubMed

    Kosmalska, Anita Joanna; Casares, Laura; Elosegui-Artola, Alberto; Thottacherry, Joseph Jose; Moreno-Vicente, Roberto; González-Tarragó, Víctor; del Pozo, Miguel Ángel; Mayor, Satyajit; Arroyo, Marino; Navajas, Daniel; Trepat, Xavier; Gauthier, Nils C; Roca-Cusachs, Pere

    2015-01-01

    Biological processes in any physiological environment involve changes in cell shape, which must be accommodated by their physical envelope--the bilayer membrane. However, the fundamental biophysical principles by which the cell membrane allows for and responds to shape changes remain unclear. Here we show that the 3D remodelling of the membrane in response to a broad diversity of physiological perturbations can be explained by a purely mechanical process. This process is passive, local, almost instantaneous, before any active remodelling and generates different types of membrane invaginations that can repeatedly store and release large fractions of the cell membrane. We further demonstrate that the shape of those invaginations is determined by the minimum elastic and adhesive energy required to store both membrane area and liquid volume at the cell-substrate interface. Once formed, cells reabsorb the invaginations through an active process with duration of the order of minutes. PMID:26073653

  6. Induced pluripotency of human prostatic epithelial cells.

    PubMed

    Zhao, Hongjuan; Sun, Ning; Young, Sarah R; Nolley, Rosalie; Santos, Jennifer; Wu, Joseph C; Peehl, Donna M

    2013-01-01

    Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen (PSA), a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified epigenetic changes in key pathways and genes involved in prostatic differentiation as E-PZ-iPS-like cells converted to differentiated AR- and PSA-expressing cells. Our results suggest that

  7. Differential remodeling of extracellular matrices by breast cancer initiating cells.

    PubMed

    Raja, Anju M; Xu, Shuoyu; Zhuo, Shuangmu; Tai, Dean C S; Sun, Wanxin; So, Peter T C; Welsch, Roy E; Chen, Chien-Shing; Yu, Hanry

    2015-10-01

    Cancer initiating cells (CICs) have been the focus of recent anti-cancer therapies, exhibiting strong invasion capability via potentially enhanced ability to remodel extracellular matrices (ECM). We have identified CICs in a human breast cancer cell line, MX-1, and developed a xenograft model in SCID mice. We investigated the CICs' matrix-remodeling effects using Second Harmonic Generation (SHG) microscopy to identify potential phenotypic signatures of the CIC-rich tumors. The isolated CICs exhibit higher proliferation, drug efflux and drug resistant properties in vitro; were more tumorigenic than non-CICs, resulting in more and larger tumors in the xenograft model. The CIC-rich tumors have less collagen in the tumor interior than in the CIC-poor tumors supporting the idea that the CICs can remodel the collagen more effectively. The collagen fibers were preferentially aligned perpendicular to the CIC-rich tumor boundary while parallel to the CIC-poor tumor boundary suggesting more invasive behavior of the CIC-rich tumors. These findings would provide potential translational values in quantifying and monitoring CIC-rich tumors in future anti-cancer therapies. CIC-rich tumors remodel the collagen matrix more than CIC-poor tumors. PMID:25597396

  8. Sustained Polymorphonuclear Leukocyte Transmigration Induces Apoptosis in T84 Intestinal Epithelial Cells

    PubMed Central

    Le'Negrate, Gaëlle; Selva, Eric; Auberger, Patrick; Rossi, Bernard; Hofman, Paul

    2000-01-01

    Acute colitis is characterized by a large number of polymorphonuclear leukocytes (PMNLs) migrating across the columnar epithelium in response to inflammatory stimuli. Several of these inflammatory factors have been characterized as proapoptotic inducers for intestinal epithelial cells. Our aim was to elucidate the role of PMNL transmigration in the onset of intestinal epithelial cell apoptosis. We found that PMNL migration, in response to N-formyl-methionyl-leucyl-phenylalanine across monolayers of intestinal epithelial cells (T84), was associated with activation of caspase-2, -3, and -9 and poly(ADP-ribose) polymerase cleavage within epithelial cells. Moreover, dihydrocytochalasin B treatment of T84 cells induced apoptosis with similar characteristics. Although Fas and Fas ligand were expressed on T84 cells and PMNLs, treatment of epithelial cells with an antagonistic anti-Fas antibody failed to prevent apoptosis induced by migrating PMNLs. Owing to the F-actin reorganization accompanying PMNL transmigration, these findings indicate a direct relationship between PMNL migration and induction of apoptosis in epithelial cells. This apoptotic process appears to involve remodeling of the actin cytoskeleton of enterocytes independent of the Fas/Fas ligand pathway. PMID:10995451

  9. Epithelial Cell Shedding and Barrier Function

    PubMed Central

    Williams, J. M.; Duckworth, C. A.; Burkitt, M. D.; Watson, A. J. M.; Campbell, B. J.

    2015-01-01

    The intestinal epithelium is a critical component of the gut barrier. Composed of a single layer of intestinal epithelial cells (IECs) held together by tight junctions, this delicate structure prevents the transfer of harmful microorganisms, antigens, and toxins from the gut lumen into the circulation. The equilibrium between the rate of apoptosis and shedding of senescent epithelial cells at the villus tip, and the generation of new cells in the crypt, is key to maintaining tissue homeostasis. However, in both localized and systemic inflammation, this balance may be disturbed as a result of pathological IEC shedding. Shedding of IECs from the epithelial monolayer may cause transient gaps or microerosions in the epithelial barrier, resulting in increased intestinal permeability. Although pathological IEC shedding has been observed in mouse models of inflammation and human intestinal conditions such as inflammatory bowel disease, understanding of the underlying mechanisms remains limited. This process may also be an important contributor to systemic and intestinal inflammatory diseases and gut barrier dysfunction in domestic animal species. This review aims to summarize current knowledge about intestinal epithelial cell shedding, its significance in gut barrier dysfunction and host-microbial interactions, and where research in this field is directed. PMID:25428410

  10. Interaction with Epithelial Cells Modifies Airway Macrophage Response to Ozone

    EPA Science Inventory

    The initial innate immune response to ozone (03) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived...

  11. Epithelial Cell Regulation of Allergic Diseases.

    PubMed

    Gour, Naina; Lajoie, Stephane

    2016-09-01

    Allergic diseases, which have escalated in prevalence in recent years, arise as a result of maladaptive immune responses to ubiquitous environmental stimuli. Why only certain individuals mount inappropriate type 2 immune responses to these otherwise harmless allergens has remained an unanswered question. Mounting evidence suggests that the epithelium, by sensing its environment, is the central regulator of allergic diseases. Once considered to be a passive barrier to allergens, epithelial cells at mucosal surfaces are now considered to be the cornerstone of the allergic diathesis. Beyond their function as maintaining barrier at mucosal surfaces, mucosal epithelial cells through the secretion of mediators like IL-25, IL-33, and TSLP control the fate of downstream allergic immune responses. In this review, we will discuss the advances in recent years regarding the process of allergen recognition and secretion of soluble mediators by epithelial cells that shape the development of the allergic response. PMID:27534656

  12. Innate lymphoid cells regulate intestinal epithelial cell glycosylation.

    PubMed

    Goto, Yoshiyuki; Obata, Takashi; Kunisawa, Jun; Sato, Shintaro; Ivanov, Ivaylo I; Lamichhane, Aayam; Takeyama, Natsumi; Kamioka, Mariko; Sakamoto, Mitsuo; Matsuki, Takahiro; Setoyama, Hiromi; Imaoka, Akemi; Uematsu, Satoshi; Akira, Shizuo; Domino, Steven E; Kulig, Paulina; Becher, Burkhard; Renauld, Jean-Christophe; Sasakawa, Chihiro; Umesaki, Yoshinori; Benno, Yoshimi; Kiyono, Hiroshi

    2014-09-12

    Fucosylation of intestinal epithelial cells, catalyzed by fucosyltransferase 2 (Fut2), is a major glycosylation mechanism of host-microbiota symbiosis. Commensal bacteria induce epithelial fucosylation, and epithelial fucose is used as a dietary carbohydrate by many of these bacteria. However, the molecular and cellular mechanisms that regulate the induction of epithelial fucosylation are unknown. Here, we show that type 3 innate lymphoid cells (ILC3) induced intestinal epithelial Fut2 expression and fucosylation in mice. This induction required the cytokines interleukin-22 and lymphotoxin in a commensal bacteria-dependent and -independent manner, respectively. Disruption of intestinal fucosylation led to increased susceptibility to infection by Salmonella typhimurium. Our data reveal a role for ILC3 in shaping the gut microenvironment through the regulation of epithelial glycosylation. PMID:25214634

  13. Expression of cyclin D{sub 1} during endotoxin-induced aleveolar type II cell hyperplasia in rat lung and the detection of apoptotic cells during the remodeling process

    SciTech Connect

    Tesfaigzi, J.; Wood, M.B.; Johnson, N.F.

    1995-12-01

    Our studies have shown that endotoxin intratracheally instilled into the rat lung induces proliferation of alveolar type II cells. In that study, the alveolar type II cells. In that study, the alveolar type II cell hyperplasia occurred 2 d after instillation of endotoxin and persisted for a further 2 d. After hyperplasia, the lung remodeled and returned to a normal state within 24-48 h. Understanding the mechanisms involved in the remodeling process of this transient hyperplasia may be useful to identify molecular changes that are altered in neoplasia. The purpose of the present study was to corroborate induction of epithelial cell hyperplasia by endotoxin and to delineate mechanisms involved in tissue remodeling after endotoxin-induced alveolar type II cell hyperplasia. In conclusion, immonostaining with cyclin D1 and cytokeratin shows that endotoxin induced epithelial cell proliferation and resulted in hyperplasia in the lung which persisted through 4 d post-instillation.

  14. Recruited alveolar macrophages, in response to airway epithelial-derived monocyte chemoattractant protein 1/CCl2, regulate airway inflammation and remodeling in allergic asthma.

    PubMed

    Lee, Yong Gyu; Jeong, Jong Jin; Nyenhuis, Sharmilee; Berdyshev, Evgeny; Chung, Sangwoon; Ranjan, Ravi; Karpurapu, Manjula; Deng, Jing; Qian, Feng; Kelly, Elizabeth A B; Jarjour, Nizar N; Ackerman, Steven J; Natarajan, Viswanathan; Christman, John W; Park, Gye Young

    2015-06-01

    Although alveolar macrophages (AMs) from patients with asthma are known to be functionally different from those of healthy individuals, the mechanism by which this transformation occurs has not been fully elucidated in asthma. The goal of this study was to define the mechanisms that control AM phenotypic and functional transformation in response to acute allergic airway inflammation. The phenotype and functional characteristics of AMs obtained from human subjects with asthma after subsegmental bronchoprovocation with allergen was studied. Using macrophage-depleted mice, the role and trafficking of AM populations was determined using an acute allergic lung inflammation model. We observed that depletion of AMs in a mouse allergic asthma model attenuates Th2-type allergic lung inflammation and its consequent airway remodeling. In both human and mouse, endobronchial challenge with allergen induced a marked increase in monocyte chemotactic proteins (MCPs) in bronchoalveolar fluid, concomitant with the rapid appearance of a monocyte-derived population of AMs. Furthermore, airway allergen challenge of allergic subjects with mild asthma skewed the pattern of AM gene expression toward high levels of the receptor for MCP1 (CCR2/MCP1R) and expression of M2 phenotypic proteins, whereas most proinflammatory genes were highly suppressed. CCL2/MCP-1 gene expression was prominent in bronchial epithelial cells in a mouse allergic asthma model, and in vitro studies indicate that bronchial epithelial cells produced abundant MCP-1 in response to house dust mite allergen. Thus, our study indicates that bronchial allergen challenge induces the recruitment of blood monocytes along a chemotactic gradient generated by allergen-exposed bronchial epithelial cells. PMID:25360868

  15. Protons Sensitize Epithelial Cells to Mesenchymal Transition

    PubMed Central

    Wang, Minli; Hada, Megumi; Saha, Janapriya; Sridharan, Deepa M.; Pluth, Janice M.; Cucinotta, Francis A.

    2012-01-01

    Proton radiotherapy has gained more favor among oncologists as a treatment option for localized and deep-seated tumors. In addition, protons are a major constituent of the space radiation astronauts receive during space flights. The potential for these exposures to lead to, or enhance cancer risk has not been well studied. Our objective is to study the biological effects of low energy protons on epithelial cells and its propensity to enhance transforming growth factor beta 1 (TGFβ1)-mediated epithelial-mesenchymal transition (EMT), a process occurring during tumor progression and critical for invasion and metastasis. Non-transformed mink lung epithelial cells (Mv1Lu) and hTERT- immortalized human esophageal epithelial cells (EPC) were used in this study. EMT was identified by alterations in cell morphology, EMT-related gene expression changes determined using real-time PCR, and EMT changes in specific cellular markers detected by immunostaining and western blotting. Although TGFβ1 treatment alone is able to induce EMT in both Mv1Lu and EPC cells, low energy protons (5 MeV) at doses as low as 0.1 Gy can enhance TGFβ1 induced EMT. Protons alone can also induce a mild induction of EMT. SD208, a potent TGFβ Receptor 1 (TGFβR1) kinase inhibitor, can efficiently block TGFβ1/Smad signaling and attenuate EMT induction. We suggest a model for EMT after proton irradiation in normal and cancerous tissue based on our results that showed that low and high doses of protons can sensitize normal human epithelial cells to mesenchymal transition, more prominently in the presence of TGFβ1, but also in the absence of TGFβ1. PMID:22844446

  16. Cell Wall Composition, Biosynthesis and Remodeling during Pollen Tube Growth

    PubMed Central

    Mollet, Jean-Claude; Leroux, Christelle; Dardelle, Flavien; Lehner, Arnaud

    2013-01-01

    The pollen tube is a fast tip-growing cell carrying the two sperm cells to the ovule allowing the double fertilization process and seed setting. To succeed in this process, the spatial and temporal controls of pollen tube growth within the female organ are critical. It requires a massive cell wall deposition to promote fast pollen tube elongation and a tight control of the cell wall remodeling to modify the mechanical properties. In addition, during its journey, the pollen tube interacts with the pistil, which plays key roles in pollen tube nutrition, guidance and in the rejection of the self-incompatible pollen. This review focuses on our current knowledge in the biochemistry and localization of the main cell wall polymers including pectin, hemicellulose, cellulose and callose from several pollen tube species. Moreover, based on transcriptomic data and functional genomic studies, the possible enzymes involved in the cell wall remodeling during pollen tube growth and their impact on the cell wall mechanics are also described. Finally, mutant analyses have permitted to gain insight in the function of several genes involved in the pollen tube cell wall biosynthesis and their roles in pollen tube growth are further discussed. PMID:27137369

  17. Respiratory epithelial cells orchestrate pulmonary innate immunity.

    PubMed

    Whitsett, Jeffrey A; Alenghat, Theresa

    2015-01-01

    The epithelial surfaces of the lungs are in direct contact with the environment and are subjected to dynamic physical forces as airway tubes and alveoli are stretched and compressed during ventilation. Mucociliary clearance in conducting airways, reduction of surface tension in the alveoli, and maintenance of near sterility have been accommodated by the evolution of a multi-tiered innate host-defense system. The biophysical nature of pulmonary host defenses are integrated with the ability of respiratory epithelial cells to respond to and 'instruct' the professional immune system to protect the lungs from infection and injury. PMID:25521682

  18. Thrombin-induced contraction in alveolar epithelial cells probed by traction microscopy.

    PubMed

    Gavara, Núria; Sunyer, Raimon; Roca-Cusachs, Pere; Farré, Ramon; Rotger, Mar; Navajas, Daniel

    2006-08-01

    Contractile tension of alveolar epithelial cells plays a major role in the force balance that regulates the structural integrity of the alveolar barrier. The aim of this work was to study thrombin-induced contractile forces of alveolar epithelial cells. A549 alveolar epithelial cells were challenged with thrombin, and time course of contractile forces was measured by traction microscopy. The cells exhibited basal contraction with total force magnitude 55.0 +/- 12.0 nN (mean +/- SE, n = 12). Traction forces were exerted predominantly at the cell periphery and pointed to the cell center. Thrombin (1 U/ml) induced a fast and sustained 2.5-fold increase in traction forces, which maintained peripheral and centripetal distribution. Actin fluorescent staining revealed F-actin polymerization and enhancement of peripheral actin rim. Disruption of actin cytoskeleton with cytochalasin D (5 microM, 30 min) and inhibition of myosin light chain kinase with ML-7 (10 microM, 30 min) and Rho kinase with Y-27632 (10 microM, 30 min) markedly depressed basal contractile tone and abolished thrombin-induced cell contraction. Therefore, the contractile response of alveolar epithelial cells to the inflammatory agonist thrombin was mediated by actin cytoskeleton remodeling and actomyosin activation through myosin light chain kinase and Rho kinase signaling pathways. Thrombin-induced contractile tension might further impair alveolar epithelial barrier integrity in the injured lung. PMID:16675616

  19. Transcriptional mechanisms link epithelial plasticity to adhesion and differentiation of epidermal progenitor cells

    PubMed Central

    Lee, Briana; Villarreal-Ponce, Alvaro; Fallahi, Magid; Ovadia, Jeremy; Sun, Peng; Yu, Qian-Chun; Ito, Seiji; Sinha, Satrajit; Nie, Qing; Dai, Xing

    2014-01-01

    During epithelial tissue morphogenesis, developmental progenitor cells undergo dynamic adhesive and cytoskeletal remodeling to trigger proliferation and migration. Transcriptional mechanisms that restrict such mild form of epithelial plasticity to maintain lineage-restricted differentiation in committed epithelial tissues are poorly understood. Here we report that simultaneous ablation of transcriptional repressor-encoding Ovol1 and Ovol2 results in expansion and blocked terminal differentiation of embryonic epidermal progenitor cells. Conversely, mice overexpressing Ovol2 in their skin epithelia exhibit precocious differentiation accompanied by smaller progenitor cell compartments. We show that Ovol1/2-deficient epidermal cells fail to undertake α-catenin–driven actin cytoskeletal reorganization and adhesive maturation, and exhibit changes that resemble epithelial-to-mesenchymal transition (EMT). Remarkably, these alterations as well as defective terminal differentiation are reversed upon depletion of EMT-promoting transcriptional factor Zeb1. Collectively, our findings reveal Ovol-Zeb1-α-catenin sequential repression and highlight functions of Ovol as gatekeepers of epithelial adhesion and differentiation by inhibiting progenitor-like traits and epithelial plasticity. PMID:24735878

  20. Ionizing radiation induces heritable disruption of epithelial cell interactions

    NASA Technical Reports Server (NTRS)

    Park, Catherine C.; Henshall-Powell, Rhonda L.; Erickson, Anna C.; Talhouk, Rabih; Parvin, Bahram; Bissell, Mina J.; Barcellos-Hoff, Mary Helen; Chatterjee, A. (Principal Investigator)

    2003-01-01

    Ionizing radiation (IR) is a known human breast carcinogen. Although the mutagenic capacity of IR is widely acknowledged as the basis for its action as a carcinogen, we and others have shown that IR can also induce growth factors and extracellular matrix remodeling. As a consequence, we have proposed that an additional factor contributing to IR carcinogenesis is the potential disruption of critical constraints that are imposed by normal cell interactions. To test this hypothesis, we asked whether IR affected the ability of nonmalignant human mammary epithelial cells (HMEC) to undergo tissue-specific morphogenesis in culture by using confocal microscopy and imaging bioinformatics. We found that irradiated single HMEC gave rise to colonies exhibiting decreased localization of E-cadherin, beta-catenin, and connexin-43, proteins necessary for the establishment of polarity and communication. Severely compromised acinar organization was manifested by the majority of irradiated HMEC progeny as quantified by image analysis. Disrupted cell-cell communication, aberrant cell-extracellular matrix interactions, and loss of tissue-specific architecture observed in the daughters of irradiated HMEC are characteristic of neoplastic progression. These data point to a heritable, nonmutational mechanism whereby IR compromises cell polarity and multicellular organization.

  1. Esophageal epithelial cells acquire functional characteristics of activated myofibroblasts after undergoing an epithelial to mesenchymal transition

    PubMed Central

    Muir, Amanda B.; Dods, Kara; Noah, Yuli; Toltzis, Sarit; Chandramouleeswaran, Prasanna Modayur; Lee, Anna; Benitez, Alain; Bedenbaugh, Adam; Falk, Gary W.; Wells, Rebecca G.; Nakagawa, Hiroshi; Wang, Mei-Lun

    2015-01-01

    Background and Aims Eosinophilic esophagitis (EoE) is an allergic inflammatory disease that leads to esophageal fibrosis and stricture. We have recently shown that in EoE, esophageal epithelial cells undergo an epithelial to mesenchymal transition (EMT), characterized by gain of mesenchymal markers and loss of epithelial gene expression. Whether epithelial cells exposed to profibrotic cytokines can also acquire the functional characteristics of activated myofibroblasts, including migration, contraction, and extracellular matrix deposition, is relevant to our understanding and treatment of EoE-associated fibrogenesis. In the current study, we characterize cell migration, contraction, and collagen production by esophageal epithelial cells that have undergone cytokine-induced EMT in vitro. Methods and Results Stimulation of human non-transformed immortalized esophageal epithelial cells (EPC2-hTERT) with profibrotic cytokines TNFα, TGFβ, and IL1β for three weeks led to acquisition of mesenchymal αSMA and vimentin, and loss of epithelial E-cadherin expression. Upon removal of the profibrotic stimulus, epithelial characteristics were partially rescued. TGFβ stimulation had a robust effect upon epithelial collagen production. Surprisingly, TNFα stimulation had the most potent effect upon cell migration and contraction, exceeding the effects of the prototypical profibrotic cytokine TGFβ. IL1β stimulation alone had minimal effect upon esophageal epithelial migration, contraction, and collagen production. Conclusions Esophageal epithelial cells that have undergone EMT acquire functional characteristics of activated myofibroblasts in vitro. Profibrotic cytokines exert differential effects upon esophageal epithelial cells, underscoring complexities of fibrogenesis in EoE, and implicating esophageal epithelial cells as effector cells in EoE-associated fibrogenesis. PMID:25183431

  2. EDAC: Epithelial defence against cancer-cell competition between normal and transformed epithelial cells in mammals.

    PubMed

    Kajita, Mihoko; Fujita, Yasuyuki

    2015-07-01

    During embryonic development or under certain pathological conditions, viable but suboptimal cells are often eliminated from the cellular society through a process termed cell competition. Cell competition was originally identified in Drosophila where cells with different properties compete for survival; 'loser' cells are eliminated from tissues and consequently 'winner' cells become dominant. Recent studies have shown that cell competition also occurs in mammals. While apoptotic cell death is the major fate for losers in Drosophila, outcompeted cells show more variable phenotypes in mammals, such as cell death-independent apical extrusion and cellular senescence. Molecular mechanisms underlying these processes have been recently revealed. Especially, in epithelial tissues, normal cells sense and actively eliminate the neighbouring transformed cells via cytoskeletal proteins by the process named epithelial defence against cancer (EDAC). Here, we introduce this newly emerging research field: cell competition in mammals. PMID:25991731

  3. Human Mammary Luminal Epithelial Cells Contain Progenitors to Myoepithelial Cells

    SciTech Connect

    Pechoux, Christine; Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Bissell, Mina J; Petersen, Ole

    1999-02-01

    The origin of the epithelial and myoepithelial cells in the human breast has not been delineated. In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other. We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture. The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity. The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis. Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and {alpha}-smooth muscle actin. We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor. The two different culture media supported each lineage for at least five passages without signs of interconversion. We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other. Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells. We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around.

  4. Tuft-cell-derived IL-25 regulates an intestinal ILC2-epithelial response circuit.

    PubMed

    von Moltke, Jakob; Ji, Ming; Liang, Hong-Erh; Locksley, Richard M

    2016-01-14

    Parasitic helminths and allergens induce a type 2 immune response leading to profound changes in tissue physiology, including hyperplasia of mucus-secreting goblet cells and smooth muscle hypercontractility. This response, known as 'weep and sweep', requires interleukin (IL)-13 production by tissue-resident group 2 innate lymphoid cells (ILC2s) and recruited type 2 helper T cells (TH2 cells). Experiments in mice and humans have demonstrated requirements for the epithelial cytokines IL-33, thymic stromal lymphopoietin (TSLP) and IL-25 in the activation of ILC2s, but the sources and regulation of these signals remain poorly defined. In the small intestine, the epithelium consists of at least five distinct cellular lineages, including the tuft cell, whose function is unclear. Here we show that tuft cells constitutively express IL-25 to sustain ILC2 homeostasis in the resting lamina propria in mice. After helminth infection, tuft-cell-derived IL-25 further activates ILC2s to secrete IL-13, which acts on epithelial crypt progenitors to promote differentiation of tuft and goblet cells, leading to increased frequencies of both. Tuft cells, ILC2s and epithelial progenitors therefore comprise a response circuit that mediates epithelial remodelling associated with type 2 immunity in the small intestine, and perhaps at other mucosal barriers populated by these cells. PMID:26675736

  5. Tuft-cell-derived IL-25 regulates an intestinal ILC2–epithelial response circuit

    PubMed Central

    von Moltke, Jakob; Ji, Ming; Liang, Hong-Erh; Locksley, Richard M.

    2016-01-01

    Parasitic helminths and allergens induce a type 2 immune response leading to profound changes in tissue physiology, including hyperplasia of mucus-secreting goblet cells1 and smooth muscle hypercontractility2. This response, known as ‘weep and sweep’, requires interleukin (IL)-13 production by tissue-resident group 2 innate lymphoid cells (ILC2s) and recruited type 2 helper T cells (TH2 cells)3. Experiments in mice and humans have demonstrated requirements for the epithelial cytokines IL-33, thymic stromal lymphopoietin (TSLP) and IL-25 in the activation of ILC2s4–11, but the sources and regulation of these signals remain poorly defined. In the small intestine, the epithelium consists of at least five distinct cellular lineages12, including the tuft cell, whose function is unclear. Here we show that tuft cells constitutively express IL-25 to sustain ILC2 homeostasis in the resting lamina propria in mice. After helminth infection, tuft-cell-derived IL-25 further activates ILC2s to secrete IL-13, which acts on epithelial crypt progenitors to promote differentiation of tuft and goblet cells, leading to increased frequencies of both. Tuft cells, ILC2s and epithelial progenitors therefore comprise a response circuit that mediates epithelial remodelling associated with type 2 immunity in the small intestine, and perhaps at other mucosal barriers populated by these cells. PMID:26675736

  6. Hox proteins drive cell segregation and non-autonomous apical remodelling during hindbrain segmentation

    PubMed Central

    Prin, Fabrice; Serpente, Patricia; Itasaki, Nobue; Gould, Alex P.

    2014-01-01

    Hox genes encode a conserved family of homeodomain transcription factors regulating development along the major body axis. During embryogenesis, Hox proteins are expressed in segment-specific patterns and control numerous different segment-specific cell fates. It has been unclear, however, whether Hox proteins drive the epithelial cell segregation mechanism that is thought to initiate the segmentation process. Here, we investigate the role of vertebrate Hox proteins during the partitioning of the developing hindbrain into lineage-restricted units called rhombomeres. Loss-of-function mutants and ectopic expression assays reveal that Hoxb4 and its paralogue Hoxd4 are necessary and sufficient for cell segregation, and for the most caudal rhombomere boundary (r6/r7). Hox4 proteins regulate Eph/ephrins and other cell-surface proteins, and can function in a non-cell-autonomous manner to induce apical cell enlargement on both sides of their expression border. Similarly, other Hox proteins expressed at more rostral rhombomere interfaces can also regulate Eph/ephrins, induce apical remodelling and drive cell segregation in ectopic expression assays. However, Krox20, a key segmentation factor expressed in odd rhombomeres (r3 and r5), can largely override Hox proteins at the level of regulation of a cell surface target, Epha4. This study suggests that most, if not all, Hox proteins share a common potential to induce cell segregation but in some contexts this is masked or modulated by other transcription factors. PMID:24574009

  7. Inhibition of airway epithelial-to-mesenchymal transition and fibrosis by kaempferol in endotoxin-induced epithelial cells and ovalbumin-sensitized mice.

    PubMed

    Gong, Ju-Hyun; Cho, In-Hee; Shin, Daekeun; Han, Seon-Young; Park, Sin-Hye; Kang, Young-Hee

    2014-03-01

    Chronic airway remodeling is characterized by structural changes within the airway wall, including smooth muscle hypertrophy, submucosal fibrosis and epithelial shedding. Epithelial-to-mesenchymal transition (EMT) is a fundamental mechanism of organ fibrosis, which can be induced by TGF-β. In the in vitro study, we investigated whether 1-20 μM kaempferol inhibited lipopolysaccharide (LPS)-induced bronchial EMT in BEAS-2B cells. The in vivo study explored demoting effects of 10-20 mg/kg kaempferol on airway fibrosis in BALB/c mice sensitized with ovalbumin (OVA). LPS induced airway epithelial TGF-β1 signaling that promoted EMT with concurrent loss of E-cadherin and induction of α-smooth muscle actin (α-SMA). Nontoxic kaempferol significantly inhibited TGF-β-induced EMT process through reversing E-cadherin expression and retarding the induction of N-cadherin and α-SMA. Consistently, OVA inhalation resulted in a striking loss of epithelial morphology by displaying myofibroblast appearance, which led to bronchial fibrosis with submucosal accumulation of collagen fibers. Oral administration of kaempferol suppressed collagen deposition, epithelial excrescency and goblet hyperplasia observed in the lung of OVA-challenged mice. The specific inhibition of TGF-β entailed epithelial protease-activated receptor-1 (PAR-1) as with 20 μM kaempferol. The epithelial PAR-1 inhibition by SCH-79797 restored E-cadherin induction and deterred α-SMA induction, indicating that epithelial PAR-1 localization was responsible for resulting in airway EMT. These results demonstrate that dietary kaempferol alleviated fibrotic airway remodeling via bronchial EMT by modulating PAR1 activation. Therefore, kaempferol may be a potential therapeutic agent targeting asthmatic airway constriction. PMID:24378645

  8. Perivascular mast cells regulate vein graft neointimal formation and remodeling

    PubMed Central

    Grassia, Gianluca; Cambrook, Helen; Ialenti, Armando; MacRitchie, Neil; Carberry, Jaclyn; Lawrence, Catherine

    2015-01-01

    Objective. Emerging evidence suggests an important role for mast cells in vein graft failure. This study addressed the hypothesis that perivascular mast cells regulate in situ vascular inflammatory and proliferative responses and subsequent vein graft neointimal lesion formation, using an optimized local mast cell reconstitution method. Methods and Results. Neointimal hyperplasia was induced by insertion of a vein graft into the right carotid artery in wild type and mast cell deficient KitW−sh/W−sh mice. In some experiments, mast cells were reconstituted systemically (tail vein injection of bone marrow-derived mast cells) or locally (directly into the right neck area) prior to vein grafting. Vein graft neointimal lesion formation was significantly (P < 0.05) reduced in KitW−sh/W−sh mice. Mast cell deficiency reduced the number of proliferating cells, and inhibited L-selectin, CCL2, M-CSF and MIP-3α expression in the vein grafts. Local but not systemic mast cell reconstitution restored a perivascular mast cell population that subsequently promoted neointimal formation in mast cell deficient mice. Conclusion. Our data demonstrate that perivascular mast cells play a key role in promoting neointima formation by inducing local acute inflammatory and proliferative responses. These results suggest that ex vivo intraoperative targeting of mast cells may have therapeutic potential for the prevention of pathological vein graft remodeling. PMID:26312183

  9. DNA typing of epithelial cells after strangulation.

    PubMed

    Wiegand, P; Kleiber, M

    1997-01-01

    DNA typing was carried out on epithelial cells which were transferred from the hands of the suspect onto the neck of the victim. In an experimental study 16 suspect-victim combinations were investigated for estimating the typing success. Alternatively to an attack against the neck, the upper arm was used for "strangulation". PCR typing was carried out using the short tandem repeat systems (STRs) HumCD4, HumVWF31A (VWA) and Hum-FIBRA (FGA) and the success rate was > 70% for all 3 systems. In most of the cases mixed patterns containing the phenotype of the suspect and the victim were obtained. In a case where strangulation was the cause of death, epithelial cells could be removed from the neck of the victim. The DNA pattern of the suspect could be successfully amplified using four STRs, demonstrating the applicability of this approach for practical casework. PMID:9274940

  10. Ionizing radiation induces heritable disruption of epithelial cell interactions

    PubMed Central

    Park, Catherine C.; Henshall-Powell, Rhonda L.; Erickson, Anna C.; Talhouk, Rabih; Parvin, Bahram; Bissell, Mina J.; Barcellos-Hoff, Mary Helen

    2003-01-01

    Ionizing radiation (IR) is a known human breast carcinogen. Although the mutagenic capacity of IR is widely acknowledged as the basis for its action as a carcinogen, we and others have shown that IR can also induce growth factors and extracellular matrix remodeling. As a consequence, we have proposed that an additional factor contributing to IR carcinogenesis is the potential disruption of critical constraints that are imposed by normal cell interactions. To test this hypothesis, we asked whether IR affected the ability of nonmalignant human mammary epithelial cells (HMEC) to undergo tissue-specific morphogenesis in culture by using confocal microscopy and imaging bioinformatics. We found that irradiated single HMEC gave rise to colonies exhibiting decreased localization of E-cadherin, β-catenin, and connexin-43, proteins necessary for the establishment of polarity and communication. Severely compromised acinar organization was manifested by the majority of irradiated HMEC progeny as quantified by image analysis. Disrupted cell–cell communication, aberrant cell–extracellular matrix interactions, and loss of tissue-specific architecture observed in the daughters of irradiated HMEC are characteristic of neoplastic progression. These data point to a heritable, nonmutational mechanism whereby IR compromises cell polarity and multicellular organization. PMID:12960393

  11. Annexin 2 Regulates Intestinal Epithelial Cell Spreading and Wound Closure through Rho-Related Signaling

    PubMed Central

    Babbin, Brian A.; Parkos, Charles A.; Mandell, Kenneth J.; Winfree, L. Matthew; Laur, Oskar; Ivanov, Andrei I.; Nusrat, Asma

    2007-01-01

    Epithelial cell migration is a critical event in gastrointestinal mucosal wound healing and is dependent on actin cytoskeletal reorganization. We observed increased expression of an actin regulatory protein, annexin 2, in migrating intestinal epithelial cells. Small interfering RNA (siRNA)-mediated knockdown of annexin 2 expression in Caco-2 epithelial cells resulted in significant reductions in cell spreading and wound closure associated with decreased formation of filamentous actin bundles along the base of migrating cells. Because annexin 2 has been shown to influences actin cytoskeletal remodeling through targeting signaling molecules to membrane domains, we examined the membrane association and activation status of Rho GTPases after annexin 2 knockdown. We observed Rho dissociation from membranes and decreased Rho activity following annexin 2 siRNA transfection. Inhibition of cell spreading and wound closure in annexin 2 siRNA-transfected cells was prevented by expression of constitutively active RhoA. Rho colocalized with annexin 2 in lamellipodia and along the cytoplasmic face of the plasma membrane. In addition, annexin 2 was observed to co-immunoprecipitate with endogenous Rho and constitutively active RhoA. These findings suggest that annexin 2 plays a role in targeting Rho to cellular membranes, thereby modulating Rho-related signaling events regulating cytoskeletal reorganization during epithelial cell migration. PMID:17322380

  12. Control of local immunity by airway epithelial cells.

    PubMed

    Weitnauer, M; Mijošek, V; Dalpke, A H

    2016-03-01

    The lung is ventilated by thousand liters of air per day. Inevitably, the respiratory system comes into contact with airborne microbial compounds, most of them harmless contaminants. Airway epithelial cells are known to have innate sensor functions, thus being able to detect microbial danger. To avoid chronic inflammation, the pulmonary system has developed specific means to control local immune responses. Even though airway epithelial cells can act as proinflammatory promoters, we propose that under homeostatic conditions airway epithelial cells are important modulators of immune responses in the lung. In this review, we discuss epithelial cell regulatory functions that control reactivity of professional immune cells within the microenvironment of the airways and how these mechanisms are altered in pulmonary diseases. Regulation by epithelial cells can be divided into two mechanisms: (1) mediators regulate epithelial cells' innate sensitivity in cis and (2) factors are produced that limit reactivity of immune cells in trans. PMID:26627458

  13. Generation of Mouse Lung Epithelial Cells

    PubMed Central

    Kasinski, Andrea L.; Slack, Frank J.

    2016-01-01

    Although in vivo models are excellent for assessing various facets of whole organism physiology, pathology, and overall response to treatments, evaluating basic cellular functions, and molecular events in mammalian model systems is challenging. It is therefore advantageous to perform these studies in a refined and less costly setting. One approach involves utilizing cells derived from the model under evaluation. The approach to generate such cells varies based on the cell of origin and often the genetics of the cell. Here we describe the steps involved in generating epithelial cells from the lungs of KrasLSL-G12D/+; p53LSL-R172/+ mice (Kasinski and Slack, 2012). These mice develop aggressive lung adenocarcinoma following cre-recombinase dependent removal of a stop cassette in the transgenes and subsequent expression of Kra-G12D and p53R172. While this protocol may be useful for the generation of epithelial lines from other genetic backgrounds, it should be noted that the Kras; p53 cell line generated here is capable of proliferating in culture without any additional genetic manipulation that is often needed for less aggressive backgrounds.

  14. Transcriptional Landscape of Glomerular Parietal Epithelial Cells

    PubMed Central

    Gharib, Sina A.; Pippin, Jeffrey W.; Ohse, Takamoto; Pickering, Scott G.; Krofft, Ronald D.; Shankland, Stuart J.

    2014-01-01

    Very little is known about the function of glomerular parietal epithelial cells (PECs). In this study, we performed genome-wide expression analysis on PEC-enriched capsulated vs. PEC-deprived decapsulated rat glomeruli to determine the transcriptional state of PECs under normal conditions. We identified hundreds of differentially expressed genes that mapped to distinct biologic modules including development, tight junction, ion transport, and metabolic processes. Since developmental programs were highly enriched in PECs, we characterized several of their candidate members at the protein level. Collectively, our findings confirm that PECs are multifaceted cells and help define their diverse functional repertoire. PMID:25127402

  15. Differential deposition of fibronectin by asthmatic bronchial epithelial cells.

    PubMed

    Ge, Qi; Zeng, Qingxiang; Tjin, Gavin; Lau, Edmund; Black, Judith L; Oliver, Brian G G; Burgess, Janette K

    2015-11-15

    Altered ECM protein deposition is a feature in asthmatic airways. Fibronectin (Fn), an ECM protein produced by human bronchial epithelial cells (HBECs), is increased in asthmatic airways. This study investigated the regulation of Fn production in asthmatic or nonasthmatic HBECs and whether Fn modulated HBEC proliferation and inflammatory mediator secretion. The signaling pathways underlying transforming growth factor (TGF)-β1-regulated Fn production were examined using specific inhibitors for ERK, JNK, p38 MAPK, phosphatidylinositol 3 kinase, and activin-like kinase 5 (ALK5). Asthmatic HBECs deposited higher levels of Fn in the ECM than nonasthmatic cells under basal conditions, whereas cells from the two groups had similar levels of Fn mRNA and soluble Fn. TGF-β1 increased mRNA levels and ECM and soluble forms of Fn but decreased cell proliferation in both cells. The rate of increase in Fn mRNA was higher in nonasthmatic cells. However, the excessive amounts of ECM Fn deposited by asthmatic cells after TGF-β1 stimulation persisted compared with nonasthmatic cells. Inhibition of ALK5 completely prevented TGF-β1-induced Fn deposition. Importantly, ECM Fn increased HBEC proliferation and IL-6 release, decreased PGE2 secretion, but had no effect on VEGF release. Soluble Fn had no effect on cell proliferation and inflammatory mediator release. Asthmatic HBECs are intrinsically primed to produce more ECM Fn, which when deposited into the ECM, is capable of driving remodeling and inflammation. The increased airway Fn may be one of the key driving factors in the persistence of asthma and represents a novel, therapeutic target. PMID:26342086

  16. Airway epithelial homeostasis and planar cell polarity signaling depend on multiciliated cell differentiation

    PubMed Central

    Vladar, Eszter K.; Nayak, Jayakar V.; Milla, Carlos E.; Axelrod, Jeffrey D.

    2016-01-01

    Motile airway cilia that propel contaminants out of the lung are oriented in a common direction by planar cell polarity (PCP) signaling, which localizes PCP protein complexes to opposite cell sides throughout the epithelium to orient cytoskeletal remodeling. In airway epithelia, PCP is determined in a 2-phase process. First, cell-cell communication via PCP complexes polarizes all cells with respect to the proximal-distal tissue axis. Second, during ciliogenesis, multiciliated cells (MCCs) undergo cytoskeletal remodeling to orient their cilia in the proximal direction. The second phase not only directs cilium polarization, but also consolidates polarization across the epithelium. Here, we demonstrate that in airway epithelia, PCP depends on MCC differentiation. PCP mutant epithelia have misaligned cilia, and also display defective barrier function and regeneration, indicating that PCP regulates multiple aspects of airway epithelial homeostasis. In humans, MCCs are often sparse in chronic inflammatory diseases, and these airways exhibit PCP dysfunction. The presence of insufficient MCCs impairs mucociliary clearance in part by disrupting PCP-driven polarization of the epithelium. Consistent with defective PCP, barrier function and regeneration are also disrupted. Pharmacological stimulation of MCC differentiation restores PCP and reverses these defects, suggesting its potential for broad therapeutic benefit in chronic inflammatory disease. PMID:27570836

  17. Cells involved in extracellular matrix remodeling after acute myocardial infarction

    PubMed Central

    Garcia, Larissa Ferraz; Mataveli, Fábio D’Aguiar; Mader, Ana Maria Amaral Antônio; Theodoro, Thérèse Rachell; Justo, Giselle Zenker; Pinhal, Maria Aparecida da Silva

    2015-01-01

    Objective Evaluate the effects of VEGF165 gene transfer in the process of remodeling of the extracellular matrix after an acute myocardial infarct. Methods Wistar rats were submitted to myocardial infarction, after the ligation of the left descending artery, and the left ventricle ejection fraction was used to classify the infarcts into large and small. The animals were divided into groups of ten, according to the size of infarcted area (large or small), and received or not VEGF165 treatment. Evaluation of different markers was performed using immunohistochemistry and digital quantification. The primary antibodies used in the analysis were anti-fibronectin, anti-vimentin, anti-CD44, anti-E-cadherin, anti-CD24, anti-alpha-1-actin, and anti-PCNA. The results were expressed as mean and standard error, and analyzed by ANOVA, considering statistically significant if p≤0.05. Results There was a significant increase in the expression of undifferentiated cell markers, such as fibronectin (protein present in the extracellular matrix) and CD44 (glycoprotein present in the endothelial cells). However, there was decreased expression of vimentin and PCNA, indicating a possible decrease in the process of cell proliferation after treatment with VEGF165. Markers of differentiated cells, E-cadherin (adhesion protein between myocardial cells), CD24 (protein present in the blood vessels), and alpha-1-actin (specific myocyte marker), showed higher expression in the groups submitted to gene therapy, compared to non-treated group. The value obtained by the relation between alpha-1-actin and vimentin was approximately three times higher in the groups treated with VEGF165, suggesting greater tissue differentiation. Conclusion The results demonstrated the important role of myocytes in the process of tissue remodeling, confirming that VEGF165 seems to provide a protective effect in the treatment of acute myocardial infarct. PMID:25993074

  18. Single-cell transcriptome analysis reveals coordinated ectopic gene expression patterns in medullary thymic epithelial cells

    PubMed Central

    Brennecke, Philip; Reyes, Alejandro; Pinto, Sheena; Rattay, Kristin; Nguyen, Michelle; Küchler, Rita; Huber, Wolfgang; Kyewski, Bruno; Steinmetz, Lars M.

    2015-01-01

    Expression of tissue-restricted self-antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for self-tolerance induction and prevents autoimmunity, with each TRA being expressed in only a few mTECs. How this process is regulated in single mTECs and coordinated at the population level, such that the varied single-cell patterns add up to faithfully represent TRAs, is poorly understood. Here we used single-cell RNA-sequencing and provide evidence for numerous recurring TRA co-expression patterns, each present in only a subset of mTECs. Co-expressed genes clustered in the genome and showed enhanced chromatin accessibility. Our findings characterize TRA expression in mTECs as a coordinated process, which might involve local re-modeling of chromatin and thus ensures a comprehensive representation of the immunological self. PMID:26237553

  19. Cytoskeletal changes induced by allosteric modulators of calcium-sensing receptor in esophageal epithelial cells

    PubMed Central

    Abdulnour-Nakhoul, Solange; Brown, Karen L; Rabon, Edd C; Al-Tawil, Youhanna; Islam, Mohammed T; Schmieg, John J; Nakhoul, Nazih L

    2015-01-01

    The calcium-sensing receptor (CaSR), a G-protein-coupled receptor, plays a role in glandular and fluid secretion in the gastrointestinal tract, and regulates differentiation and proliferation of epithelial cells. We examined the expression of CaSR in normal and pathological conditions of human esophagus and investigated the effect of a CaSR agonist, cinacalcet (CCT), and antagonist, calhex (CHX), on cell growth and cell–cell junctional proteins in primary cultures of porcine stratified squamous esophageal epithelium. We used immunohistochemistry and Western analysis to monitor expression of CaSR and cell–cell adhesion molecules, and MTT assay to monitor cell proliferation in cultured esophageal cells. CCT treatment significantly reduced proliferation, changed the cell shape from polygonal to spindle-like, and caused redistribution of E-cadherin and β-catenin from the cell membrane to the cytoplasm. Furthermore, it reduced expression of β-catenin by 35% (P < 0.02) and increased expression of a proteolysis cleavage fragment of E-cadherin, Ecad/CFT2, by 2.3 folds (P < 0.01). On the other hand, CHX treatment enhanced cell proliferation by 27% (P < 0.01), increased the expression of p120-catenin by 24% (P < 0.04), and of Rho, a GTPase involved in cytoskeleton remodeling, by 18% (P < 0.03). In conclusion, CaSR is expressed in normal esophagus as well as in Barrett’s, esophageal adenocarcinoma, squamous cell carcinoma, and eosinophilic esophagitis. Long-term activation of CaSR with CCT disrupted the cadherin–catenin complex, induced cytoskeletal remodeling, actin fiber formation, and redistribution of CaSR to the nuclear area. These changes indicate a significant and complex role of CaSR in epithelial remodeling and barrier function of esophageal cells. PMID:26603452

  20. The Dynamics in Epithelial Cell Intercalation in Drosophila Morphogenesis

    NASA Astrophysics Data System (ADS)

    Wolf, Fred; Reichl, Lars; Kong, Deqing; Zhang, Yujun; Eule, Stephan; Metzger, Jakob; Großhans, Jörg

    2015-03-01

    Epithelial cell rearrangement is important for many processes in morphogenesis. During germband extension in early gastrulation of Drosophila embryos, exchange of neighbors is achieved by junction remodeling that follows a topological T1 process. Its first step is the constriction of dorsal-ventral junctions and fusion of two 3x vertices into a 4x vertex a process believed to be junction autonomous. We established a high throughput imaging pipeline, by which we recorded, segmented and analysed more than 1000 neighbor exchanges in drosophila embryos. Characterizing the dynamics of junction lengths we find that the constriction of cell contacts follows intriguingly simple quantitative laws. (1) The mean contact length decreases approximately as a square root of time to collapse. (2) The time dependent variance of contact lengths is proportional to the square of the mean. (3) The time dependent probability density of the contact lengths remains close to Gaussian during the entire process. These observations are sufficient to derive a stochastic differential equation for contact length that captures the non-equilibrium statistical mechanics of contact collapse. Supported by the German Research Foundation.

  1. Cell Death and Tissue Remodeling in Planarian Regeneration

    PubMed Central

    Pellettieri, Jason; Fitzgerald, Patrick; Watanabe, Shigeki; Mancuso, Joel; Green, Douglas R.; Alvarado, Alejandro Sánchez

    2010-01-01

    Many long-lived organisms, including humans, can regenerate some adult tissues lost to physical injury or disease. Much of the previous research on mechanisms of regeneration has focused on adult stem cells, which give rise to new tissue necessary for the replacement of missing body parts. Here we report that apoptosis of differentiated cells complements stem cell division during regeneration in the planarian Schmidtea mediterranea. Specifically, we developed a whole-mount TUNEL assay that allowed us to document two dramatic increases in the rate of apoptosis following amputation – an intial localized response near the wound site and a subsequent systemic response that varies in magnitude depending on the type of fragment examined. The latter cell death response can be induced in uninjured organs, occurs in the absence of planarian stem cells, and can also be triggered by prolonged starvation. Taken together, our results implicate apoptosis in the restoration of proper anatomical scale and proportion through remodeling of existing tissues. We also report results from initial mechanistic studies of apoptosis in planarians, which revealed that a S. mediterranea homolog of the antiapoptotic gene BCL2 is required for cell survival in adult animals. We propose that apoptosis is a central mechanism working in concert with stem cell division to restore anatomical form and function during metazoan regeneration. PMID:19766622

  2. Remodeling of the thoracic aorta after bone marrow cell transplantation

    PubMed Central

    Felix, Alyne; Monteiro, Nemesis; Rocha, Vinícius Novaes; Oliveira, Genilza; Moraes, Alan Cesar; Andrade, Cherley; Nascimento, Ana Lucia; de Carvalho, Laís; Thole, Alessandra; Carvalho, Jorge

    2014-01-01

    Stem cells are characterized by their ability to differentiate into multiple cell lineages and display the paracrine effect. The aim of this work was to evaluate the effect of therapy with bone marrow cells (BMCs) on blood glucose, lipid metabolism and aortic wall remodeling in mice through the administration of a high fat diet and subsequent BMCs transplantation. C57BL/6 mice were fed a control diet (CO group) or an atherogenic diet (AT group). After 16 weeks, the AT group was divided into four groups: an AT 14 days group and AT 21 days group, that were given an injection of vehicle and sacrificed at 14 and 21 days after, respectively; AT-BMC 14 days group and AT-BMC 21 days group that was given an injection of BMCs and sacrificed at 14 and 21 days after. The CO group was sacrificed along with other groups. The BMCs transplant had reduced blood glucose, triglycerides and total cholesterol. The Qa (1/mm2) was quantitatively reduced in AT 14 days group, AT 21 days group and was high in AT-BMC 21 days group. The AT 21 days group exhibited increased tunica media and elastic system fibers. The immunolabeling for α-SMA and VEGF showed less immunolabeling in transplanted groups with BMCs. The immunostaining for PCNA seems to be more expressive in the group AT-BMC 21 days group. To conclude, our results support the concept that in mice, the injection of BMCs improve glucose levels, lipid metabolism and remodeling of the aortic wall in animals using atherogenic diet. PMID:25337194

  3. Epigenetics in Intestinal Epithelial Cell Renewal.

    PubMed

    Roostaee, Alireza; Benoit, Yannick D; Boudjadi, Salah; Beaulieu, Jean-François

    2016-11-01

    A controlled balance between cell proliferation and differentiation is essential to maintain normal intestinal tissue renewal and physiology. Such regulation is powered by several intracellular pathways that are translated into the establishment of specific transcription programs, which influence intestinal cell fate along the crypt-villus axis. One important check-point in this process occurs in the transit amplifying zone of the intestinal crypts where different signaling pathways and transcription factors cooperate to manage cellular proliferation and differentiation, before secretory or absorptive cell lineage terminal differentiation. However, the importance of epigenetic modifications such as histone methylation and acetylation in the regulation of these processes is still incompletely understood. There have been recent advances in identifying the impact of histone modifications and chromatin remodelers on the proliferation and differentiation of normal intestinal crypt cells. In this review we discuss recent discoveries on the role of the cellular epigenome in intestinal cell fate, development, and tissue renewal. J. Cell. Physiol. 231: 2361-2367, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. PMID:27061836

  4. Formation of a Neurosensory Organ by Epithelial Cell Slithering.

    PubMed

    Kuo, Christin S; Krasnow, Mark A

    2015-10-01

    Epithelial cells are normally stably anchored, maintaining their relative positions and association with the basement membrane. Developmental rearrangements occur through cell intercalation, and cells can delaminate during epithelial-mesenchymal transitions and metastasis. We mapped the formation of lung neuroepithelial bodies (NEBs), innervated clusters of neuroendocrine/neurosensory cells within the bronchial epithelium, revealing a targeted mode of cell migration that we named "slithering," in which cells transiently lose epithelial character but remain associated with the membrane while traversing neighboring epithelial cells to reach cluster sites. Immunostaining, lineage tracing, clonal analysis, and live imaging showed that NEB progenitors, initially distributed randomly, downregulate adhesion and polarity proteins, crawling over and between neighboring cells to converge at diametrically opposed positions at bronchial branchpoints, where they reestablish epithelial structure and express neuroendocrine genes. There is little accompanying progenitor proliferation or apoptosis. Activation of the slithering program may explain why lung cancers arising from neuroendocrine cells are highly metastatic. PMID:26435104

  5. Ouabain modulates ciliogenesis in epithelial cells

    PubMed Central

    Larre, Isabel; Castillo, Aida; Flores-Maldonado, Catalina; Contreras, Ruben G.; Galvan, Ivan; Muñoz-Estrada, Jesus; Cereijido, Marcelino

    2011-01-01

    The exchange of substances between higher organisms and the environment occurs across transporting epithelia whose basic features are tight junctions (TJs) that seal the intercellular space, and polarity, which enables cells to transport substances vectorially. In a previous study, we demonstrated that 10 nM ouabain modulates TJs, and we now show that it controls polarity as well. We gauge polarity through the development of a cilium at the apical domain of Madin-Darby canine kidney cells (MDCK, epithelial dog kidney). Ouabain accelerates ciliogenesis in an ERK1/2-dependent manner. Claudin-2, a molecule responsible for the Na+ and H2O permeability of the TJs, is also present at the cilium, as it colocalizes and coprecipitates with acetylated α-tubulin. Ouabain modulates claudin-2 localization at the cilium through ERK1/2. Comparing wild-type and ouabain-resistant MDCK cells, we show that ouabain acts through Na+,K+-ATPase. Taken together, our previous and present results support the possibility that ouabain constitutes a hormone that modulates the transporting epithelial phenotype, thereby playing a crucial role in metazoan life. PMID:22143774

  6. Is the inflammasome relevant for epithelial cell function?

    PubMed

    Santana, Patricia T; Martel, Jan; Lai, Hsin-Chih; Perfettini, Jean-Luc; Kanellopoulos, Jean M; Young, John D; Coutinho-Silva, Robson; Ojcius, David M

    2016-02-01

    Inflammasomes are intracellular protein complexes that sense microbial components and damage of infected cells. Following activation by molecules released by pathogens or injured cells, inflammasomes activate caspase-1, allowing secretion of the pro-inflammatory cytokines IL-1β and IL-18 from innate immune cells. Inflammasomes are also expressed in epithelial cells, where their function has attracted less attention. Nonetheless, depending on the tissue, epithelial inflammasomes can mediate inflammation, wound healing, and pain sensitivity. We review here recent findings on inflammasomes found in epithelial tissues, highlighting the importance of these protein complexes in the response of epithelial tissues to microbial infections. PMID:26546965

  7. Coevolution of neoplastic epithelial cells and multilineage stroma via polyploid giant cells during immortalization and transformation of mullerian epithelial cells

    PubMed Central

    Zhang, Shiwu; Mercado-Uribe, Imelda; Sood, Anil; Bast, Robert C.; Liu, Jinsong

    2016-01-01

    Stromal cells are generally considered to be derived primarily from the host's normal mesenchymal stromal cells or bone marrow. However, the origins of stromal cells have been quite controversial. To determine the role of polyploidy in tumor development, we examined the fate of normal mullerian epithelial cells during the immortalization and transformation process by tracing the expression of SV40 large T antigen. Here we show that immortalized or HRAS-transformed mullerian epithelial cells contain a subpopulation of polyploid giant cells that grow as multicellular spheroids expressing hematopoietic markers in response to treatment with CoCl2. The immortalized or transformed epithelial cells can transdifferentiate into stromal cells when transplanted into nude mice. Immunofluorescent staining revealed expression of stem cell factors OCT4, Nanog, and SOX-2 in spheroid, whereas expression of embryonic stem cell marker SSEA1 was increased in HRAS-transformed cells compared with their immortalized isogenic counterparts. These results suggest that normal mullerian epithelial cells are intrinsically highly plastic, via the formation of polyploid giant cells and activation of embryonic stem-like program, which work together to promote the coevolution of neoplastic epithelial cells and multiple lineage stromal cells. PMID:27382431

  8. Promotion of the induction of cell pluripotency through metabolic remodeling by thyroid hormone triiodothyronine-activated PI3K/AKT signal pathway

    PubMed Central

    Chen, Mengfei; Zhang, He; Wu, Jie; Xu, Liang; Xu, Di; Sun, Jinglan; He, Yixin; Zhou, Xin; Wang, Zhaojing; Wu, Lifang; Xu, Shaokun; Wang, Jinsong; Jiang, Shu; Zhou, Xiangjun; Hoffman, Andrew R.; Hu, Xiang; Hu, Jifan; Li, Tao

    2012-01-01

    Generation of induced pluripotent stem cells (iPSCs) from somatic cells by defined factors is a mechanism-unknown, yet extremely time-consuming process. Inefficient reprogramming leads to prolonged periods of in vitro iPSC selection, resulting in subtle genetic and epigenetic abnormalities. To facilitate pluripotent reprogramming, we have identified the thyroid hormone triiodothyronine (T3) as an endogenous factor that can enhance reprogramming of human dermal fibroblasts (HDF) and umbilical cord mesenchymal stem cells (UCMSC). This potentiation of iPSC induction is associated with metabolic remodeling activity, including up-regulation of key glycolytic genes, an increase in cell proliferation, and the induction of mesenchymal-epithelial transition (MET). We further identify the activation of the PI3K/AKT signal pathway by T3 as an underlying mechanism for the enhanced conversion to cell pluripotency in this model. These studies demonstrate that T3 enhances metabolic remodeling of donor cells in potentiating cell reprogramming. PMID:22575839

  9. Glycolysis is the primary bioenergetic pathway for cell motility and cytoskeletal remodeling in human prostate and breast cancer cells

    PubMed Central

    Shiraishi, Takumi; Verdone, James E.; Huang, Jessie; Kahlert, Ulf D.; Hernandez, James R.; Torga, Gonzalo; Zarif, Jelani C.; Epstein, Tamir; Gatenby, Robert; McCartney, Annemarie; Elisseeff, Jennifer H.; Mooney, Steven M.; An, Steven S.; Pienta, Kenneth J.

    2015-01-01

    The ability of a cancer cell to detach from the primary tumor and move to distant sites is fundamental to a lethal cancer phenotype. Metabolic transformations are associated with highly motile aggressive cellular phenotypes in tumor progression. Here, we report that cancer cell motility requires increased utilization of the glycolytic pathway. Mesenchymal cancer cells exhibited higher aerobic glycolysis compared to epithelial cancer cells while no significant change was observed in mitochondrial ATP production rate. Higher glycolysis was associated with increased rates of cytoskeletal remodeling, greater cell traction forces and faster cell migration, all of which were blocked by inhibition of glycolysis, but not by inhibition of mitochondrial ATP synthesis. Thus, our results demonstrate that cancer cell motility and cytoskeleton rearrangement is energetically dependent on aerobic glycolysis and not oxidative phosphorylation. Mitochondrial derived ATP is insufficient to compensate for inhibition of the glycolytic pathway with regard to cellular motility and CSK rearrangement, implying that localization of ATP derived from glycolytic enzymes near sites of active CSK rearrangement is more important for cell motility than total cellular ATP production rate. These results extend our understanding of cancer cell metabolism, potentially providing a target metabolic pathway associated with aggressive disease. PMID:25426557

  10. Extensive Translatome Remodeling during ER Stress Response in Mammalian Cells

    PubMed Central

    Ventoso, Iván; Kochetov, Alex; Montaner, David; Dopazo, Joaquín; Santoyo, Javier

    2012-01-01

    In this work we have described the translatome of two mammalian cell lines, NIH3T3 and Jurkat, by scoring the relative polysome association of ∼10,000 mRNA under normal and ER stress conditions. We have found that translation efficiencies of mRNA correlated poorly with transcript abundance, although a general tendency was observed so that the highest translation efficiencies were found in abundant mRNA. Despite the differences found between mouse (NIH3T3) and human (Jurkat) cells, both cell types share a common translatome composed by ∼800–900 mRNA that encode proteins involved in basic cellular functions. Upon stress, an extensive remodeling in translatomes was observed so that translation of ∼50% of mRNA was inhibited in both cell types, this effect being more dramatic for those mRNA that accounted for most of the cell translation. Interestingly, we found two subsets comprising 1000–1500 mRNA whose translation resisted or was induced by stress. Translation arrest resistant class includes many mRNA encoding aminoacyl tRNA synthetases, ATPases and enzymes involved in DNA replication and stress response such as BiP. This class of mRNA is characterized by high translation rates in both control and stress conditions. Translation inducible class includes mRNA whose translation was relieved after stress, showing a high enrichment in early response transcription factors of bZIP and zinc finger C2H2 classes. Unlike yeast, a general coordination between changes in translation and transcription upon stress (potentiation) was not observed in mammalian cells. Among the different features of mRNA analyzed, we found a relevant association of translation efficiency with the presence of upstream ATG in the 5′UTR and with the length of coding sequence of mRNA, and a looser association with other parameters such as the length and the G+C content of 5′UTR. A model for translatome remodeling during the acute phase of stress response in mammalian cells is proposed. PMID

  11. Establishment of Hertwig's epithelial root sheath/epithelial rests of Malassez cell line from human periodontium.

    PubMed

    Nam, Hyun; Kim, Ji-Hye; Kim, Jae-Won; Seo, Byoung-Moo; Park, Joo-Cheol; Kim, Jung-Wook; Lee, Gene

    2014-07-01

    Human Hertwig's epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cells are epithelial remnants of teeth residing in the periodontium. Although the functional roles of HERS/ERM cells have yet to be elucidated, they are a unique epithelial cell population in adult teeth and are reported to have stem cell characteristics. Therefore, HERS/ERM cells might play a role as an epithelial component for the repair or regeneration of dental hard tissues; however, they are very rare population in periodontium and the primary isolation of them is considered to be difficult. To overcome these problems, we immortalized primary HERS/ERM cells isolated from human periodontium using SV40 large T antigen (SV40 LT) and performed a characterization of the immortalized cell line. Primary HERS/ERM cells could not be maintained for more than 6 passages; however, immortalized HERS/ERM cells were maintained for more than 20 passages. There were no differences in the morphological and immunophenotypic characteristics of HERS/ERM cells and immortalized HERS/ERM cells. The expression of epithelial stem cell and embryonic stem cell markers was maintained in immortalized HERS/ERM cells. Moreover, immortalized HERS/ERM cells could acquire mesenchymal phenotypes through the epithelial-mesenchymal transition via TGF-β1. In conclusion, we established an immortalized human HERS/ERM cell line with SV40 LT and expect this cell line to contribute to the understanding of the functional roles of HERS/ERM cells and the tissue engineering of teeth. PMID:25081036

  12. Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

    SciTech Connect

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2004-07-14

    Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

  13. Stromal-epithelial interactions in aging and cancer: senescent fibroblasts alter epithelial cell differentiation

    PubMed Central

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2016-01-01

    Summary Cellular senescence suppresses cancer by arresting cells at risk of malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation and branching morphogenesis. Furthermore, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts – the ability to alter epithelial differentiation – that might also explain the loss of tissue function and organization that is a hallmark of aging. PMID:15657080

  14. Henipavirus Pathogenesis in Human Respiratory Epithelial Cells

    PubMed Central

    Escaffre, Olivier; Borisevich, Viktoriya; Carmical, J. Russ; Prusak, Deborah; Prescott, Joseph; Feldmann, Heinz

    2013-01-01

    Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection. PMID:23302882

  15. Henipavirus pathogenesis in human respiratory epithelial cells.

    PubMed

    Escaffre, Olivier; Borisevich, Viktoriya; Carmical, J Russ; Prusak, Deborah; Prescott, Joseph; Feldmann, Heinz; Rockx, Barry

    2013-03-01

    Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection. PMID:23302882

  16. Nuclear microscopy of rat colon epithelial cells

    NASA Astrophysics Data System (ADS)

    Ren, M.; Rajendran, Reshmi; Ng, Mary; Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank; Jenner, Andrew Michael

    2011-10-01

    Using Nuclear microscopy, we have investigated iron distributions in the colons of Sprague Dawley rats, in order to elucidate heme uptake. Four groups of five Sprague Dawley rats (mean weight 180 g) were fed different purified diets containing either heme diet (2.5% w/w hemoglobin), high fat diet (HFD) (18% w/w fat, 1% w/w cholesterol), 'western' diet (combination of hemoglobin 2.5% and 18% fat, 1% cholesterol) or control diet (7% w/w fat). After 4 weeks, animals were sacrificed by exsanguination after anaesthesia. Thin sections of frozen colon tissue were taken, freeze dried and scanned using nuclear microscopy utilising the techniques PIXE, RBS and STIM. The new data acquisition system (IonDaq) developed in CIBA was used to obtain high resolution images and line scans were used to map the iron distributions across the colon boundaries. The nuclear microscope results indicate that when HFD is given in addition to heme, the iron content of the epithelial cells that line the colon decreases, and the zinc in the smooth muscle wall increases. This implies that the level of heme and fat in diet has an important role in colon health, possibly by influencing epithelial cells directly or changing luminal composition such as bacterial flora or levels of metabolites and cytotoxins.

  17. Polarizing intestinal epithelial cells electrically through Ror2.

    PubMed

    Cao, Lin; McCaig, Colin D; Scott, Roderick H; Zhao, Siwei; Milne, Gillian; Clevers, Hans; Zhao, Min; Pu, Jin

    2014-08-01

    The apicobasal polarity of enterocytes determines where the brush border membrane (apical membrane) will form, but how this apical membrane faces the lumen is not well understood. The electrical signal across the epithelium could serve as a coordinating cue, orienting and polarizing enterocytes. Here, we show that applying a physiological electric field to intestinal epithelial cells, to mimic the natural electric field created by the transepithelial potential difference, polarized phosphorylation of the actin-binding protein ezrin, increased expression of intestinal alkaline phosphatase (ALPI, a differentiation marker) and remodeled the actin cytoskeleton selectively on the cathode side. In addition, an applied electric field also activated ERK1/2 and LKB1 (also known as STK11), key molecules in apical membrane formation. Disruption of the tyrosine protein kinase transmembrane receptor Ror2 suppressed activation of ERK1/2 and LKB1 significantly, and subsequently inhibited apical membrane formation in enterocytes. Our findings indicate that the endogenous electric field created by the transepithelial potential difference might act as an essential coordinating signal for apical membrane formation at a tissue level, through activation of LKB1 mediated by Ror2-ERK signaling. PMID:24928904

  18. Polarizing intestinal epithelial cells electrically through Ror2

    PubMed Central

    Cao, Lin; McCaig, Colin D.; Scott, Roderick H.; Zhao, Siwei; Milne, Gillian; Clevers, Hans; Zhao, Min; Pu, Jin

    2014-01-01

    ABSTRACT The apicobasal polarity of enterocytes determines where the brush border membrane (apical membrane) will form, but how this apical membrane faces the lumen is not well understood. The electrical signal across the epithelium could serve as a coordinating cue, orienting and polarizing enterocytes. Here, we show that applying a physiological electric field to intestinal epithelial cells, to mimic the natural electric field created by the transepithelial potential difference, polarized phosphorylation of the actin-binding protein ezrin, increased expression of intestinal alkaline phosphatase (ALPI, a differentiation marker) and remodeled the actin cytoskeleton selectively on the cathode side. In addition, an applied electric field also activated ERK1/2 and LKB1 (also known as STK11), key molecules in apical membrane formation. Disruption of the tyrosine protein kinase transmembrane receptor Ror2 suppressed activation of ERK1/2 and LKB1 significantly, and subsequently inhibited apical membrane formation in enterocytes. Our findings indicate that the endogenous electric field created by the transepithelial potential difference might act as an essential coordinating signal for apical membrane formation at a tissue level, through activation of LKB1 mediated by Ror2–ERK signaling. PMID:24928904

  19. Regulation of intestinal epithelial cells transcriptome by enteric glial cells: impact on intestinal epithelial barrier functions

    PubMed Central

    2009-01-01

    Background Emerging evidences suggest that enteric glial cells (EGC), a major constituent of the enteric nervous system (ENS), are key regulators of intestinal epithelial barrier (IEB) functions. Indeed EGC inhibit intestinal epithelial cells (IEC) proliferation and increase IEB paracellular permeability. However, the role of EGC on other important barrier functions and the signalling pathways involved in their effects are currently unknown. To achieve this goal, we aimed at identifying the impact of EGC upon IEC transcriptome by performing microarray studies. Results EGC induced significant changes in gene expression profiling of proliferating IEC after 24 hours of co-culture. 116 genes were identified as differentially expressed (70 up-regulated and 46 down-regulated) in IEC cultured with EGC compared to IEC cultured alone. By performing functional analysis of the 116 identified genes using Ingenuity Pathway Analysis, we showed that EGC induced a significant regulation of genes favoring both cell-to-cell and cell-to-matrix adhesion as well as cell differentiation. Consistently, functional studies showed that EGC induced a significant increase in cell adhesion. EGC also regulated genes involved in cell motility towards an enhancement of cell motility. In addition, EGC profoundly modulated expression of genes involved in cell proliferation and cell survival, although no clear functional trend could be identified. Finally, important genes involved in lipid and protein metabolism of epithelial cells were shown to be differentially regulated by EGC. Conclusion This study reinforces the emerging concept that EGC have major protective effects upon the IEB. EGC have a profound impact upon IEC transcriptome and induce a shift in IEC phenotype towards increased cell adhesion and cell differentiation. This concept needs to be further validated under both physiological and pathophysiological conditions. PMID:19883504

  20. Chromatin remodeling in somatic cells injected into mature pig oocytes.

    PubMed

    Bui, Hong-Thuy; Van Thuan, Nguyen; Wakayama, Teruhiko; Miyano, Takashi

    2006-06-01

    We examined the involvement of histone H3 modifications in the chromosome condensation and decondensation of somatic cell nuclei injected into mature pig oocytes. Nuclei of pig granulosa cells were transferred into in vitro matured intact pig oocytes, and histone H3 phosphorylation, acetylation, and methylation were examined by immunostaining with specific antibodies in relation to changes in chromosome morphology. In the condensed chromosomes of pig oocytes at metaphase II, histone H3 was phosphorylated at serine 10 (H3-S10) and serine 28 (H3-S28), and methylated at lysine 9 (H3-K9), but was not acetylated at lysine 9, 14 and 18 (H3-K9, H3-K14 and H3-K18). During the first 2 h after nuclear transfer, a series of events were observed in the somatic nuclei: nuclear membrane disassembly; chromosome condensation to form a metaphase-like configuration; an increase in histone H3 phosphorylation levels (H3-S10 and H3-S28). Next, pig oocytes injected with nuclei of somatic cells were electroactivated and the chromosome morphology of oocytes and somatic cells was examined along with histone modifications. Generally, chromosomes of the somatic cells showed a similar progression of cell cycle stage to that of oocytes, through anaphase II- and telophase II-like stages then formed pronucleus-like structures, although the morphology of the spindles differed from that of oocyte spindles. The chromosomes of somatic cells also showed changes in histone H3 dephosphorylation and reacetylation, similar to oocytes. In contrast, histone H3 methylation (H3-K9) of somatic cell nuclei did not show any significant change after injection and electroactivation of the oocytes. These results suggest that nuclear remodeling including histone H3 phosphorylation and acetylation of injected somatic nuclei took place in the oocytes under regulation by the oocyte cytoplasm. PMID:16735543

  1. Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells

    PubMed Central

    Celià-Terrassa, Toni; Meca-Cortés, Óscar; Mateo, Francesca; Martínez de Paz, Alexia; Rubio, Nuria; Arnal-Estapé, Anna; Ell, Brian J.; Bermudo, Raquel; Díaz, Alba; Guerra-Rebollo, Marta; Lozano, Juan José; Estarás, Conchi; Ulloa, Catalina; ρlvarez-Simón, Daniel; Milà, Jordi; Vilella, Ramón; Paciucci, Rosanna; Martínez-Balbás, Marian; García de Herreros, Antonio; Gomis, Roger R.; Kang, Yibin; Blanco, Jerónimo; Fernández, Pedro L.; Thomson, Timothy M.

    2012-01-01

    Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self renewal, survival under stress, and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here, we characterize 2 human cellular models derived from prostate and bladder cancer cell lines to better understand the relationship between TIC and EMT programs in local invasiveness and distant metastasis. The model tumor subpopulations that expressed a strong epithelial gene program were enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits was impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene program and suppressed their self renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions among epithelial, self-renewal, and mesenchymal gene programs determine the plasticity of epithelial TICs. PMID:22505459

  2. Documentation of angiotensin II receptors in glomerular epithelial cells

    NASA Technical Reports Server (NTRS)

    Sharma, M.; Sharma, R.; Greene, A. S.; McCarthy, E. T.; Savin, V. J.; Cowley, A. W. (Principal Investigator)

    1998-01-01

    Angiotensin II decreases glomerular filtration rate, renal plasma flow, and glomerular capillary hydraulic conductivity. Although angiotensin II receptors have been demonstrated in mesangial cells and proximal tubule cells, the presence of angiotensin II receptors in glomerular epithelial cells has not previously been shown. Previously, we have reported that angiotensin II caused an accumulation of cAMP and a reorganization of the actin cytoskeleton in cultured glomerular epithelial cells. Current studies were conducted to verify the presence of angiotensin II receptors by immunological and non-peptide receptor ligand binding techniques and to ascertain the activation of intracellular signal transduction in glomerular epithelial cells in response to angiotensin II. Confluent monolayer cultures of glomerular epithelial cells were incubated with angiotensin II, with or without losartan and/or PD-123,319 in the medium. Membrane vesicle preparations were obtained by homogenization of washed cells followed by centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins followed by multiscreen immunoblotting was used to determine the presence of angiotensin II receptor type 1 (AT1) or type 2 (AT2). Angiotensin II-mediated signal transduction in glomerular epithelial cells was studied by measuring the levels of cAMP, using radioimmunoassay. Results obtained in these experiments showed the presence of both AT1 and AT2 receptor types in glomerular epithelial cells. Angiotensin II was found to cause an accumulation of cAMP in glomerular epithelial cells, which could be prevented only by simultaneous use of losartan and PD-123,319, antagonists for AT1 and AT2, respectively. The presence of both AT1 and AT2 receptors and an increase in cAMP indicate that glomerular epithelial cells respond to angiotensin II in a manner distinct from that of mesangial cells or proximal tubular epithelial cells. Our results suggest that glomerular epithelial

  3. Intestinal epithelial tuft cells initiate type 2 mucosal immunity to helminth parasites.

    PubMed

    Gerbe, François; Sidot, Emmanuelle; Smyth, Danielle J; Ohmoto, Makoto; Matsumoto, Ichiro; Dardalhon, Valérie; Cesses, Pierre; Garnier, Laure; Pouzolles, Marie; Brulin, Bénédicte; Bruschi, Marco; Harcus, Yvonne; Zimmermann, Valérie S; Taylor, Naomi; Maizels, Rick M; Jay, Philippe

    2016-01-14

    Helminth parasitic infections are a major global health and social burden. The host defence against helminths such as Nippostrongylus brasiliensis is orchestrated by type 2 cell-mediated immunity. Induction of type 2 cytokines, including interleukins (IL) IL-4 and IL-13, induce goblet cell hyperplasia with mucus production, ultimately resulting in worm expulsion. However, the mechanisms underlying the initiation of type 2 responses remain incompletely understood. Here we show that tuft cells, a rare epithelial cell type in the steady-state intestinal epithelium, are responsible for initiating type 2 responses to parasites by a cytokine-mediated cellular relay. Tuft cells have a Th2-related gene expression signature and we demonstrate that they undergo a rapid and extensive IL-4Rα-dependent amplification following infection with helminth parasites, owing to direct differentiation of epithelial crypt progenitor cells. We find that the Pou2f3 gene is essential for tuft cell specification. Pou2f3(-/-) mice lack intestinal tuft cells and have defective mucosal type 2 responses to helminth infection; goblet cell hyperplasia is abrogated and worm expulsion is compromised. Notably, IL-4Rα signalling is sufficient to induce expansion of the tuft cell lineage, and ectopic stimulation of this signalling cascade obviates the need for tuft cells in the epithelial cell remodelling of the intestine. Moreover, tuft cells secrete IL-25, thereby regulating type 2 immune responses. Our data reveal a novel function of intestinal epithelial tuft cells and demonstrate a cellular relay required for initiating mucosal type 2 immunity to helminth infection. PMID:26762460

  4. Transcriptional Profiling of Coxiella burnetii Reveals Extensive Cell Wall Remodeling in the Small Cell Variant Developmental Form

    PubMed Central

    Sandoz, Kelsi M.; Popham, David L.; Beare, Paul A.; Sturdevant, Daniel E.; Hansen, Bryan; Nair, Vinod; Heinzen, Robert A.

    2016-01-01

    A hallmark of Coxiella burnetii, the bacterial cause of human Q fever, is a biphasic developmental cycle that generates biologically, ultrastructurally, and compositionally distinct large cell variant (LCV) and small cell variant (SCV) forms. LCVs are replicating, exponential phase forms while SCVs are non-replicating, stationary phase forms. The SCV has several properties, such as a condensed nucleoid and an unusual cell envelope, suspected of conferring enhanced environmental stability. To identify genetic determinants of the LCV to SCV transition, we profiled the C. burnetii transcriptome at 3 (early LCV), 5 (late LCV), 7 (intermediate forms), 14 (early SCV), and 21 days (late SCV) post-infection of Vero epithelial cells. Relative to early LCV, genes downregulated in the SCV were primarily involved in intermediary metabolism. Upregulated SCV genes included those involved in oxidative stress responses, arginine acquisition, and cell wall remodeling. A striking transcriptional signature of the SCV was induction (>7-fold) of five genes encoding predicted L,D transpeptidases that catalyze nonclassical 3–3 peptide cross-links in peptidoglycan (PG), a modification that can influence several biological traits in bacteria. Accordingly, of cross-links identified, muropeptide analysis showed PG of SCV with 46% 3–3 cross-links as opposed to 16% 3–3 cross-links for LCV. Moreover, electron microscopy revealed SCV with an unusually dense cell wall/outer membrane complex as compared to LCV with its clearly distinguishable periplasm and inner and outer membranes. Collectively, these results indicate the SCV produces a unique transcriptome with a major component directed towards remodeling a PG layer that likely contributes to Coxiella’s environmental resistance. PMID:26909555

  5. Transcriptional Profiling of Coxiella burnetii Reveals Extensive Cell Wall Remodeling in the Small Cell Variant Developmental Form.

    PubMed

    Sandoz, Kelsi M; Popham, David L; Beare, Paul A; Sturdevant, Daniel E; Hansen, Bryan; Nair, Vinod; Heinzen, Robert A

    2016-01-01

    A hallmark of Coxiella burnetii, the bacterial cause of human Q fever, is a biphasic developmental cycle that generates biologically, ultrastructurally, and compositionally distinct large cell variant (LCV) and small cell variant (SCV) forms. LCVs are replicating, exponential phase forms while SCVs are non-replicating, stationary phase forms. The SCV has several properties, such as a condensed nucleoid and an unusual cell envelope, suspected of conferring enhanced environmental stability. To identify genetic determinants of the LCV to SCV transition, we profiled the C. burnetii transcriptome at 3 (early LCV), 5 (late LCV), 7 (intermediate forms), 14 (early SCV), and 21 days (late SCV) post-infection of Vero epithelial cells. Relative to early LCV, genes downregulated in the SCV were primarily involved in intermediary metabolism. Upregulated SCV genes included those involved in oxidative stress responses, arginine acquisition, and cell wall remodeling. A striking transcriptional signature of the SCV was induction (>7-fold) of five genes encoding predicted L,D transpeptidases that catalyze nonclassical 3-3 peptide cross-links in peptidoglycan (PG), a modification that can influence several biological traits in bacteria. Accordingly, of cross-links identified, muropeptide analysis showed PG of SCV with 46% 3-3 cross-links as opposed to 16% 3-3 cross-links for LCV. Moreover, electron microscopy revealed SCV with an unusually dense cell wall/outer membrane complex as compared to LCV with its clearly distinguishable periplasm and inner and outer membranes. Collectively, these results indicate the SCV produces a unique transcriptome with a major component directed towards remodeling a PG layer that likely contributes to Coxiella's environmental resistance. PMID:26909555

  6. Applications of mouse airway epithelial cell culture for asthma research.

    PubMed

    Horani, Amjad; Dickinson, John D; Brody, Steven L

    2013-01-01

    Primary airway epithelial cell culture provides a valuable tool for studying cell differentiation, cell-cell interactions, and the role of immune system factors in asthma pathogenesis. In this chapter, we discuss the application of mouse tracheal epithelial cell cultures for the study of asthma biology. A major advantage of this system is the ability to use airway epithelial cells from mice with defined genetic backgrounds. The in vitro proliferation and differentiation of mouse airway epithelial cells uses the air-liquid interface condition to generate well-differentiated epithelia with characteristics of native airways. Protocols are provided for manipulation of differentiation, induction of mucous cell metaplasia, genetic modification, and cell and pathogen coculture. Assays for the assessment of gene expression, responses of cells, and analysis of specific cell subpopulations within the airway epithelium are included. PMID:23943446

  7. Klebsiella pneumoniae Is Able to Trigger Epithelial-Mesenchymal Transition Process in Cultured Airway Epithelial Cells

    PubMed Central

    Leone, Laura; Mazzetta, Francesca; Martinelli, Daniela; Valente, Sabatino; Alimandi, Maurizio; Raffa, Salvatore; Santino, Iolanda

    2016-01-01

    The ability of some bacterial pathogens to activate Epithelial-Mesenchymal Transition normally is a consequence of the persistence of a local chronic inflammatory response or depends on a direct interaction of the pathogens with the host epithelial cells. In this study we monitored the abilities of the K. pneumoniae to activate the expression of genes related to EMT-like processes and the occurrence of phenotypic changes in airway epithelial cells during the early steps of cell infection. We describe changes in the production of intracellular reactive oxygen species and increased HIF-1α mRNA expression in cells exposed to K. pneumoniae infection. We also describe the upregulation of a set of transcription factors implicated in the EMT processes, such as Twist, Snail and ZEB, indicating that the morphological changes of epithelial cells already appreciable after few hours from the K. pneumoniae infection are tightly regulated by the activation of transcriptional pathways, driving epithelial cells to EMT. These effects appear to be effectively counteracted by resveratrol, an antioxidant that is able to exert a sustained scavenging of the intracellular ROS. This is the first report indicating that strains of K. pneumoniae may promote EMT-like programs through direct interaction with epithelial cells without the involvement of inflammatory cells. PMID:26812644

  8. Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells

    PubMed Central

    de Dieuleveult, Maud; Yen, Kuangyu; Hmitou, Isabelle; Depaux, Arnaud; Boussouar, Fayçal; Dargham, Daria Bou; Jounier, Sylvie; Humbertclaude, Hélène; Ribierre, Florence; Baulard, Céline; Farrell, Nina P.; Park, Bongsoo; Keime, Céline; Carrière, Lucie; Berlivet, Soizick; Gut, Marta; Gut, Ivo; Werner, Michel; Deleuze, Jean-François; Olaso, Robert; Aude, Jean-Christophe; Chantalat, Sophie; Pugh, B. Franklin; Gérard, Matthieu

    2015-01-01

    Summary ATP-dependent chromatin remodellers allow access to DNA for transcription factors and the general transcription machinery, but whether mammalian chromatin remodellers1–3 target specific nucleosomes to regulate transcription is unclear. Here, we present genome-wide remodeller-nucleosome interaction profiles for Chd1, Chd2, Chd4, Chd6, Chd8, Chd9, Brg1 and Ep400 in mouse embryonic stem (ES) cells. These remodellers bind one or both full nucleosomes that flank MNase-defined nucleosome-free promoter regions (NFRs), where they separate divergent transcription. Surprisingly, large CpG-rich NFRs that extend downstream of annotated transcriptional start sites (TSSs) are nevertheless chromatinized with non-nucleosomal or subnucleosomal histone variants (H3.3 and H2A.Z) and modifications (H3K4me3 and H3K27ac). RNA polymerase (pol) II therefore navigates hundreds of bp of altered chromatin in the sense direction before encountering an MNase-resistant nucleosome at the 3′ end of the NFR. Transcriptome analysis upon remodeller depletion reveals reciprocal mechanisms of transcriptional regulation by remodellers. Whereas at active genes individual remodellers play either positive or negative roles via altering nucleosome stability, at polycomb-enriched bivalent genes the same remodellers act in an opposite manner. These findings indicate that remodellers target specific nucleosomes at the edge of NFRs, where they regulate ES cell transcriptional programs. PMID:26814966

  9. Persistent rhinitis and epithelial remodeling induced by cyclic ozone exposure in the nasal airways of infant monkeys

    PubMed Central

    Ballinger, Carol A.; Plopper, Charles G.; McDonald, Ruth J.; Bartolucci, Alfred A.; Postlethwait, Edward M.; Harkema, Jack R.

    2011-01-01

    Children chronically exposed to high levels of ozone (O3), the principal oxidant pollutant in photochemical smog, are more vulnerable to respiratory illness and infections. The specific factors underlying this differential susceptibility are unknown but may be related to air pollutant-induced nasal alterations during postnatal development that impair the normal physiological functions (e.g., filtration and mucociliary clearance) serving to protect the more distal airways from inhaled xenobiotics. In adult animal models, chronic ozone exposure is associated with adaptations leading to a decrease in airway injury. The purpose of our study was to determine whether cyclic ozone exposure induces persistent morphological and biochemical effects on the developing nasal airways of infant monkeys early in life. Infant (180-day-old) rhesus macaques were exposed to 5 consecutive days of O3 [0.5 parts per million (ppm), 8 h/day; “1-cycle”] or filtered air (FA) or 11 biweekly cycles of O3 (FA days 1–9; 0.5 ppm, 8 h/day on days 10–14; “11-cycle”). The left nasal passage was processed for light microscopy and morphometric analysis. Mucosal samples from the right nasal passage were processed for GSH, GSSG, ascorbate (AH2), and uric acid (UA) concentration. Eleven-cycle O3 induced persistent rhinitis, squamous metaplasia, and epithelial hyperplasia in the anterior nasal airways of infant monkeys, resulting in a 39% increase in the numeric density of epithelial cells. Eleven-cycle O3 also induced a 65% increase in GSH concentrations at this site. The persistence of epithelial hyperplasia was positively correlated with changes in GSH. These results indicate that early life ozone exposure causes persistent nasal epithelial alterations in infant monkeys and provide a potential mechanism for the increased susceptibility to respiratory illness exhibited by children in polluted environments. PMID:21131400

  10. Persistent rhinitis and epithelial remodeling induced by cyclic ozone exposure in the nasal airways of infant monkeys.

    PubMed

    Carey, Stephan A; Ballinger, Carol A; Plopper, Charles G; McDonald, Ruth J; Bartolucci, Alfred A; Postlethwait, Edward M; Harkema, Jack R

    2011-02-01

    Children chronically exposed to high levels of ozone (O(3)), the principal oxidant pollutant in photochemical smog, are more vulnerable to respiratory illness and infections. The specific factors underlying this differential susceptibility are unknown but may be related to air pollutant-induced nasal alterations during postnatal development that impair the normal physiological functions (e.g., filtration and mucociliary clearance) serving to protect the more distal airways from inhaled xenobiotics. In adult animal models, chronic ozone exposure is associated with adaptations leading to a decrease in airway injury. The purpose of our study was to determine whether cyclic ozone exposure induces persistent morphological and biochemical effects on the developing nasal airways of infant monkeys early in life. Infant (180-day-old) rhesus macaques were exposed to 5 consecutive days of O(3) [0.5 parts per million (ppm), 8 h/day; "1-cycle"] or filtered air (FA) or 11 biweekly cycles of O(3) (FA days 1-9; 0.5 ppm, 8 h/day on days 10-14; "11-cycle"). The left nasal passage was processed for light microscopy and morphometric analysis. Mucosal samples from the right nasal passage were processed for GSH, GSSG, ascorbate (AH(2)), and uric acid (UA) concentration. Eleven-cycle O(3) induced persistent rhinitis, squamous metaplasia, and epithelial hyperplasia in the anterior nasal airways of infant monkeys, resulting in a 39% increase in the numeric density of epithelial cells. Eleven-cycle O(3) also induced a 65% increase in GSH concentrations at this site. The persistence of epithelial hyperplasia was positively correlated with changes in GSH. These results indicate that early life ozone exposure causes persistent nasal epithelial alterations in infant monkeys and provide a potential mechanism for the increased susceptibility to respiratory illness exhibited by children in polluted environments. PMID:21131400

  11. Quantitative analysis of 3D extracellular matrix remodelling by pancreatic stellate cells

    PubMed Central

    Robinson, Benjamin K.; Cortes, Ernesto; Rice, Alistair J.; Sarper, Muge

    2016-01-01

    ABSTRACT Extracellular matrix (ECM) remodelling is integral to numerous physiological and pathological processes in biology, such as embryogenesis, wound healing, fibrosis and cancer. Until recently, most cellular studies have been conducted on 2D environments where mechanical cues significantly differ from physiologically relevant 3D environments, impacting cellular behaviour and masking the interpretation of cellular function in health and disease. We present an integrated methodology where cell-ECM interactions can be investigated in 3D environments via ECM remodelling. Monitoring and quantification of collagen-I structure in remodelled matrices, through designated algorithms, show that 3D matrices can be used to correlate remodelling with increased ECM stiffness observed in fibrosis. Pancreatic stellate cells (PSCs) are the key effectors of the stromal fibrosis associated to pancreatic cancer. We use PSCs to implement our methodology and demonstrate that PSC matrix remodelling capabilities depend on their contractile machinery and β1 integrin-mediated cell-ECM attachment. PMID:27170254

  12. Epithelial in vitro cell systems in carcinogenesis studies

    SciTech Connect

    Borek, C.

    1983-01-01

    The development of epithelial cells systems to study oncogenic transformation has presented a major challenge in the field of carcinogenesis. Because there exists in man a preponderance of carcinomas over sarcomas, the importance of studying oncogenic transformation in epithelial cells is of great relevance to human disease. The difficulty lies in the fact that different tissues contain epithelial cells with singular differentiated characteristics, which must be defined to assert the different nature of the cells being used. Liver cells in culture are a case in point. By careful maintenance and optimal culture conditions, one can maintain many of the differentiated characteristics of the cells for prolonged periods of time.

  13. Adipose Mesenchymal Stem Cell Secretome Modulated in Hypoxia for Remodeling of Radiation-Induced Salivary Gland Damage

    PubMed Central

    An, Hye-Young; Shin, Hyun-Soo; Choi, Jeong-Seok; Kim, Hun Jung

    2015-01-01

    Background and Purpose This study was conducted to determine whether a secretome from mesenchymal stem cells (MSC) modulated by hypoxic conditions to contain therapeutic factors contributes to salivary gland (SG) tissue remodeling and has the potential to improve irradiation (IR)-induced salivary hypofunction in a mouse model. Materials and Methods Human adipose mesenchymal stem cells (hAdMSC) were isolated, expanded, and exposed to hypoxic conditions (O2 < 5%). The hypoxia-conditioned medium was then filtered to a high molecular weight fraction and prepared as a hAdMSC secretome. The hAdMSC secretome was subsequently infused into the tail vein of C3H mice immediately after local IR once a day for seven consecutive days. The control group received equal volume (500 μL) of vehicle (PBS) only. SG function and structural tissue remodeling by the hAdMSC secretome were investigated. Human parotid epithelial cells (HPEC) were obtained, expanded in vitro, and then irradiated and treated with either the hypoxia-conditioned medium or a normoxic control medium. Cell proliferation and IR-induced cell death were examined to determine the mechanism by which the hAdMSC secretome exerted its effects. Results The conditioned hAdMSC secretome contained high levels of GM-CSF, VEGF, IL-6, and IGF-1. Repeated systemic infusion with the hAdMSC secretome resulted in improved salivation capacity and increased levels of salivary proteins, including amylase and EGF, relative to the PBS group. The microscopic structural integrity of SG was maintained and salivary epithelial (AQP-5), endothelial (CD31), myoepithelial (α-SMA) and SG progenitor cells (c-Kit) were successfully protected from radiation damage and remodeled. The hAdMSC secretome strongly induced proliferation of HPEC and led to a significant decrease in cell death in vivo and in vitro. Moreover, the anti-apoptotic effects of the hAdMSC secretome were found to be promoted after hypoxia-preconditioning relative to normoxia

  14. Isolation of Cancer Epithelial Cells from Mouse Mammary Tumors

    PubMed Central

    Johnson, Sara; Chen, Hexin; Lo, Pang-Kuo

    2016-01-01

    The isolation of cancer epithelial cells from mouse mammary tumor is accomplished by digestion of the solid tumor. Red blood cells and other contaminates are removed using several washing techniques such that primary epithelial cells can further enriched. This procedure yields primary tumor cells that can be used for in vitro tissue culture, fluorescence-activated cell sorting (FACS) and a wide variety of other experiments (Lo et al., 2012).

  15. Multi-functionality and plasticity characterize epithelial cells in Hydra

    PubMed Central

    Buzgariu, W; Al Haddad, S; Tomczyk, S; Wenger, Y; Galliot, B

    2015-01-01

    Epithelial sheets, a synapomorphy of all metazoans but porifers, are present as 2 layers in cnidarians, ectoderm and endoderm, joined at their basal side by an extra-cellular matrix named mesoglea. In the Hydra polyp, epithelial cells of the body column are unipotent stem cells that continuously self-renew and concomitantly express their epitheliomuscular features. These multifunctional contractile cells maintain homeostasis by providing a protective physical barrier, by digesting nutrients, by selecting a stable microbiota, and by rapidly closing wounds. In addition, epithelial cells are highly plastic, supporting the adaptation of Hydra to physiological and environmental changes, such as long starvation periods where survival relies on a highly dynamic autophagy flux. Epithelial cells also play key roles in developmental processes as evidenced by the organizer activity they develop to promote budding and regeneration. We propose here an integrative view of the homeostatic and developmental aspects of epithelial plasticity in Hydra. PMID:26716072

  16. Multi-functionality and plasticity characterize epithelial cells in Hydra.

    PubMed

    Buzgariu, W; Al Haddad, S; Tomczyk, S; Wenger, Y; Galliot, B

    2015-01-01

    Epithelial sheets, a synapomorphy of all metazoans but porifers, are present as 2 layers in cnidarians, ectoderm and endoderm, joined at their basal side by an extra-cellular matrix named mesoglea. In the Hydra polyp, epithelial cells of the body column are unipotent stem cells that continuously self-renew and concomitantly express their epitheliomuscular features. These multifunctional contractile cells maintain homeostasis by providing a protective physical barrier, by digesting nutrients, by selecting a stable microbiota, and by rapidly closing wounds. In addition, epithelial cells are highly plastic, supporting the adaptation of Hydra to physiological and environmental changes, such as long starvation periods where survival relies on a highly dynamic autophagy flux. Epithelial cells also play key roles in developmental processes as evidenced by the organizer activity they develop to promote budding and regeneration. We propose here an integrative view of the homeostatic and developmental aspects of epithelial plasticity in Hydra. PMID:26716072

  17. Sonic Hedgehog regulates thymic epithelial cell differentiation

    PubMed Central

    Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L.; Ono, Masahiro; Holländer, Georg; Crompton, Tessa

    2016-01-01

    Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus. PMID

  18. Sonic Hedgehog regulates thymic epithelial cell differentiation.

    PubMed

    Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L; Ono, Masahiro; Holländer, Georg; Crompton, Tessa

    2016-04-01

    Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus. PMID

  19. Collective Epithelial Migration and Cell Rearrangements Drive Mammary Branching Morphogenesis

    PubMed Central

    Ewald, Andrew J.; Brenot, Audrey; Duong, Myhanh; Chan, Bianca S.; Werb, Zena

    2009-01-01

    Summary Epithelial organs are built through the movement of groups of interconnected cells. We observed cells in elongating mammary ducts reorganize into a multilayered epithelium, migrate collectively, and rearrange dynamically, all without forming leading cellular extensions. Duct initiation required proliferation, Rac, and myosin light-chain kinase, whereas repolarization to a bilayer depended on Rho kinase. We observed that branching morphogenesis results from the active motility of both luminal and myoepithelial cells. Luminal epithelial cells advanced collectively, whereas myoepithelial cells appeared to restrain elongating ducts. Significantly, we observed that normal epithelium and neoplastic hyperplasias are organized similarly during morphogenesis, suggesting common mechanisms of epithelial growth. PMID:18410732

  20. Observing planar cell polarity in multiciliated mouse airway epithelial cells

    PubMed Central

    Vladar, Eszter K.; Lee, Yin Loon; Stearns, Tim; Axelrod, Jeffrey D.

    2015-01-01

    The concerted movement of cilia propels inhaled contaminants out of the lungs, safeguarding the respiratory system from toxins, pathogens, pollutants, and allergens. Motile cilia on the multiciliated cells (MCCs) of the airway epithelium are physically oriented along the tissue axis for directional motility, which depends on the planar cell polarity (PCP) signaling pathway. The MCCs of the mouse respiratory epithelium have emerged as an important model for the study of motile ciliogenesis and the PCP signaling mechanism. Unlike other motile ciliated or planar polarized tissues, airway epithelial cells are relatively easily accessible and primary cultures faithfully model many of the essential features of the in vivo tissue. There is growing interest in understanding how cells acquire and polarize motile cilia due to the impact of mucociliary clearance on respiratory health. Here, we present methods for observing and quantifying the planar polarized orientation of motile cilia both in vivo and in primary culture airway epithelial cells. We describe how to acquire and evaluate electron and light microscopy images of ciliary ultrastructural features that reveal planar polarized orientation. Furthermore, we describe the immunofluorescence localization of PCP pathway components as a simple readout for airway epithelial planar polarization and ciliary orientation. These methods can be adapted to observe ciliary orientation in other multi- and monociliated cells and to detect PCP pathway activity in any tissue or cell type. PMID:25837385

  1. Technical note: Isolation and characterization of porcine mammary epithelial cells.

    PubMed

    Dahanayaka, S; Rezaei, R; Porter, W W; Johnson, G A; Burghardt, R C; Bazer, F W; Hou, Y Q; Wu, Z L; Wu, G

    2015-11-01

    Within the mammary gland, functional synthesis of milk is performed by its epithelial (alveolar) cells. The availability of a stable mammary epithelial cell line is essential for biochemical studies to elucidate cellular and molecular mechanisms responsible for nutritional regulation of lactation. Therefore, porcine mammary epithelial cells (PMEC) were isolated from mammary glands of a 9-mo-old nonpregnant and nonlactating gilt and cultured to establish a nonimmortalized cell line. These cells were characterized by expression of cytokeratin-18 (an intermediate filament specific for epithelial cells), β-casein (a specific marker for mammary epithelial cells), and α-lactalbumin. In culture, the PMEC doubled in number every 24 h and maintained a cobblestone morphology, typical for cultured epithelial cells, for at least 15 passages. Addition of 0.2 to 2 μg/mL prolactin to culture medium for 3 d induced the production of β-casein and α-lactalbumin by PMEC in a dose-dependent manner. Thus, we have successfully developed a useful PMEC line for future studies of cellular and molecular regulation of milk synthesis by mammary epithelial cells of the sow. PMID:26641038

  2. Mesenchymal-epithelial interactions during digestive tract development and epithelial stem cell regeneration.

    PubMed

    Le Guen, Ludovic; Marchal, Stéphane; Faure, Sandrine; de Santa Barbara, Pascal

    2015-10-01

    The gastrointestinal tract develops from a simple and uniform tube into a complex organ with specific differentiation patterns along the anterior-posterior and dorso-ventral axes of asymmetry. It is derived from all three germ layers and their cross-talk is important for the regulated development of fetal and adult gastrointestinal structures and organs. Signals from the adjacent mesoderm are essential for the morphogenesis of the overlying epithelium. These mesenchymal-epithelial interactions govern the development and regionalization of the different gastrointestinal epithelia and involve most of the key morphogens and signaling pathways, such as the Hedgehog, BMPs, Notch, WNT, HOX, SOX and FOXF cascades. Moreover, the mechanisms underlying mesenchyme differentiation into smooth muscle cells influence the regionalization of the gastrointestinal epithelium through interactions with the enteric nervous system. In the neonatal and adult gastrointestinal tract, mesenchymal-epithelial interactions are essential for the maintenance of the epithelial regionalization and digestive epithelial homeostasis. Disruption of these interactions is also associated with bowel dysfunction potentially leading to epithelial tumor development. In this review, we will discuss various aspects of the mesenchymal-epithelial interactions observed during digestive epithelium development and differentiation and also during epithelial stem cell regeneration. PMID:26126787

  3. Human airway smooth muscle cells secrete amphiregulin via bradykinin/COX-2/PGE2, inducing COX-2, CXCL8, and VEGF expression in airway epithelial cells

    PubMed Central

    Knox, Alan J.

    2015-01-01

    Human airway smooth muscle cells (HASMC) contribute to asthma pathophysiology through an increased smooth muscle mass and elevated cytokine/chemokine output. Little is known about how HASMC and the airway epithelium interact to regulate chronic airway inflammation and remodeling. Amphiregulin is a member of the family of epidermal growth factor receptor (EGFR) agonists with cell growth and proinflammatory roles and increased expression in the lungs of asthma patients. Here we show that bradykinin (BK) stimulation of HASMC increases amphiregulin secretion in a mechanism dependent on BK-induced COX-2 expression, increased PGE2 output, and the stimulation of HASMC EP2 and EP4 receptors. Conditioned medium from BK treated HASMC induced CXCL8, VEGF, and COX-2 mRNA and protein accumulation in airway epithelial cells, which were blocked by anti-amphiregulin antibodies and amphiregulin siRNA, suggesting a paracrine effect of HASMC-derived amphiregulin on airway epithelial cells. Consistent with this, recombinant amphiregulin induced CXCL8, VEGF, and COX-2 in airway epithelial cells. Finally, we found that conditioned media from amphiregulin-stimulated airway epithelial cells induced amphiregulin expression in HASMC and that this was dependent on airway epithelial cell COX-2 activity. Our study provides evidence of a dynamic axis of interaction between HASMC and epithelial cells that amplifies CXCL8, VEGF, COX-2, and amphiregulin production. PMID:26047642

  4. Cell Chirality Induces Collective Cell Migration in Epithelial Sheets

    NASA Astrophysics Data System (ADS)

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Shibata, Tatsuo

    2015-10-01

    During early development, epithelial cells form a monolayer sheet and migrate in a uniform direction. Here, we address how this collective migration can occur without breaking the cell-to-cell attachments. Repeated contraction and expansion of the cell-to-cell interfaces enables the cells to rearrange their positions autonomously within the sheet. We show that when the interface tension is strengthened in a direction that is tilted from the body axis, cell rearrangements occur in such a way that unidirectional movement is induced. We use a vertex model to demonstrate that such anisotropic tension can generate the unidirectional motion of cell sheets. Our results suggest that cell chirality facilitates collective cell migration during tissue morphogenesis.

  5. Fungal glycan interactions with epithelial cells in allergic airway disease

    PubMed Central

    Roy, René M.; Klein, Bruce S.

    2014-01-01

    Human exposure to fungi results in a wide range of health outcomes, from invasive disease or allergy to immune tolerance. Inhaled fungi contact airway epithelial cells as an early event, and this host:fungal interaction can shape the eventual immunological outcome. Emerging evidence points to exposure to fungal cell wall carbohydrates in the development of allergic airway disease. Herein, we describe determinants of fungal allergenicity, and review the responses of airway epithelial cells to fungal carbohydrates. A greater understanding of the recognition of and response to fungal carbohydrates by airway epithelial cells may lead to the development of targeted therapies that ameliorate allergic airway disease. PMID:23602359

  6. Quantitative Assessment of Cytosolic Salmonella in Epithelial Cells

    PubMed Central

    Knodler, Leigh A.; Nair, Vinod; Steele-Mortimer, Olivia

    2014-01-01

    Within mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium) inhabits a membrane-bound vacuole known as the Salmonella-containing vacuole (SCV). We have recently shown that wild type S. Typhimurium also colonizes the cytosol of epithelial cells. Here we sought to quantify the contribution of cytosolic Salmonella to the total population over a time course of infection in different epithelial cell lines and under conditions of altered vacuolar escape. We found that the lysosomotropic agent, chloroquine, acts on vacuolar, but not cytosolic, Salmonella. After chloroquine treatment, vacuolar bacteria are not transcriptionally active or replicative and appear degraded. Using a chloroquine resistance assay, in addition to digitonin permeabilization, we found that S. Typhimurium lyses its nascent vacuole in numerous epithelial cell lines, albeit with different frequencies, and hyper-replication in the cytosol is also widespread. At later times post-infection, cytosolic bacteria account for half of the total population in some epithelial cell lines, namely HeLa and Caco-2 C2Bbe1. Both techniques accurately measured increased vacuole lysis in epithelial cells upon treatment with wortmannin. By chloroquine resistance assay, we also determined that Salmonella pathogenicity island-1 (SPI-1), but not SPI-2, the virulence plasmid nor the flagellar apparatus, was required for vacuolar escape and cytosolic replication in epithelial cells. Together, digitonin permeabilization and the chloroquine resistance assay will be useful, complementary tools for deciphering the mechanisms of SCV lysis and Salmonella replication in the epithelial cell cytosol. PMID:24400108

  7. Lung epithelial cells modulate the inflammatory response of alveolar macrophages.

    PubMed

    Rubovitch, Vardit; Gershnabel, Shoham; Kalina, Moshe

    2007-12-01

    The goal of this study was to examine the effect of alveolar epithelial cells on inflammatory responses in macrophages. Lung epithelial cells (either rat RLE-6TN or human A549 cells) reduced LPS-induced NO production in alveolar macrophages (AM) in a contact-independent mechanism. The inhibitory effect of the epithelial cells was present already at the transcriptional level: LPS-induced inducible NO synthase (iNOS) expression was significantly smaller. Surfactant protein A (SP-A)-induced NO production by alveolar macrophages was also reduced in the presence of A549 cells, though, by a different kinetics. LPS-induced interleukin-6 (IL-6) production (another inflammatory pathway) by alveolar macrophages was also reduced in the presence of RLE-6TN cells. These data suggest a role for lung epithelial cells in the complicated modulation of inflammatory processes, and provide an insight into the mechanism underlying. PMID:17851743

  8. Transforming Growth Factor β/activin signalling induces epithelial cell flattening during Drosophila oogenesis

    PubMed Central

    Brigaud, Isabelle; Duteyrat, Jean-Luc; Chlasta, Julien; Le Bail, Sandrine; Couderc, Jean-Louis; Grammont, Muriel

    2015-01-01

    ABSTRACT Although the regulation of epithelial morphogenesis is essential for the formation of tissues and organs in multicellular organisms, little is known about how signalling pathways control cell shape changes in space and time. In the Drosophila ovarian epithelium, the transition from a cuboidal to a squamous shape is accompanied by a wave of cell flattening and by the ordered remodelling of E-cadherin-based adherens junctions. We show that activation of the TGFβ pathway is crucial to determine the timing, the degree and the dynamic of cell flattening. Within these cells, TGFβ signalling controls cell-autonomously the formation of Actin filament and the localisation of activated Myosin II, indicating that internal forces are generated and used to remodel AJ and to promote cytoskeleton rearrangement. Our results also reveal that TGFβ signalling controls Notch activity and that its functions are partly executed through Notch. Thus, we demonstrate that the cells that undergo the cuboidal-to-squamous transition produce active cell-shaping mechanisms, rather than passively flattening in response to a global force generated by the growth of the underlying cells. Thus, our work on TGFβ signalling provides new insights into the mechanisms through which signal transduction cascades orchestrate cell shape changes to generate proper organ structure. PMID:25681395

  9. Mortality factor 4 like 1 protein mediates epithelial cell death in a mouse model of pneumonia.

    PubMed

    Zou, Chunbin; Li, Jin; Xiong, Sheng; Chen, Yan; Wu, Qin; Li, Xiuying; Weathington, Nathaniel M; Han, SeungHye; Snavely, Courtney; Chen, Bill B; Mallampalli, Rama K

    2015-10-28

    Unchecked epithelial cell death is fundamental to the pathogenesis of pneumonia. The recognition of unique signaling pathways that preserve epithelial cell viability may present new opportunities for interventional strategies. We describe that mortality factor 4 like 1 (Morf4l1), a protein involved in chromatin remodeling, is constitutively expressed at low levels in the lung because of its continuous degradation mediated by an orphan ubiquitin E3 ligase subunit, Fbxl18. Expression of Morf4l1 increases in humans with pneumonia and is up-regulated in lung epithelia after exposure to Pseudomonas aeruginosa or lipopolysaccharide. In a mouse model of pneumonia induced by P. aeruginosa, Morf4l1 is stabilized by acetylation that protects it from Fbxl18-mediated degradation. After P. aeruginosa infection of mice, overexpression of Morf4l1 resulted in lung epithelial cell death, whereas its depletion restored cell viability. Using in silico modeling and drug-target interaction studies, we identified that the U.S. Food and Drug Administration-approved thrombin inhibitor argatroban is a Morf4l1 antagonist. Argatroban inhibited Morf4l1-dependent histone acetylation, reduced its cytotoxicity, and improved survival of mice with experimental lung injury at doses that had no anticoagulant activity. These studies uncover a previously unrecognized biological mechanism whereby pathogens subvert cell viability by extending the life span of a cytotoxic host protein. Morf4l1 may be a potential molecular target for non-antibiotic pharmacotherapy during severe pulmonary infection. PMID:26511508

  10. Mortality factor 4 like 1 protein mediates epithelial cell death in a mouse model of pneumonia

    PubMed Central

    Zou, Chunbin; Li, Jin; Xiong, Sheng; Chen, Yan; Wu, Qin; Li, Xiuying; Weathington, Nathaniel M.; Han, Seung Hye; Snavely, Courtney; Chen, Bill B.; Mallampalli, Rama K.

    2016-01-01

    Unchecked epithelial cell death is fundamental to the pathogenesis of pneumonia. The recognition of unique signaling pathways that preserve epithelial cell viability may present new opportunities for interventional strategies. We describe that mortality factor 4 like 1 (Morf4l1), a protein involved in chromatin remodeling, is constitutively expressed at low levels in the lung because of its continuous degradation mediated by an orphan ubiquitin E3 ligase subunit, Fbxl18. Expression of Morf4l1 increases in humans with pneumonia and is up-regulated in lung epithelia after exposure to Pseudomonas aeruginosa or lipopolysaccharide. In a mouse model of pneumonia induced by P. aeruginosa, Morf4l1 is stabilized by acetylation that protects it from Fbxl18-mediated degradation. After P. aeruginosa infection of mice, overexpression of Morf4l1 resulted in lung epithelial cell death, whereas its depletion restored cell viability. Using in silico modeling and drug-target interaction studies, we identified that the U.S. Food and Drug Administration–approved thrombin inhibitor argatroban is a Morf4l1 antagonist. Argatroban inhibited Morf4l1-dependent histone acetylation, reduced its cytotoxicity, and improved survival of mice with experimental lung injury at doses that had no anticoagulant activity. These studies uncover a previously unrecognized biological mechanism whereby pathogens subvert cell viability by extending the life span of a cytotoxic host protein. Morf4l1 may be a potential molecular target for non-antibiotic pharmacotherapy during severe pulmonary infection. PMID:26511508

  11. Epithelial cells as alternative human biomatrices for comet assay

    PubMed Central

    Rojas, Emilio; Lorenzo, Yolanda; Haug, Kristiane; Nicolaissen, Bjørn; Valverde, Mahara

    2014-01-01

    The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases. PMID:25506353

  12. Intrinsic epithelial cells repair the kidney after injury.

    PubMed

    Humphreys, Benjamin D; Valerius, M Todd; Kobayashi, Akio; Mugford, Joshua W; Soeung, Savuth; Duffield, Jeremy S; McMahon, Andrew P; Bonventre, Joseph V

    2008-03-01

    Understanding the mechanisms of nephron repair is critical for the design of new therapeutic approaches to treat kidney disease. The kidney can repair after even a severe insult, but whether adult stem or progenitor cells contribute to epithelial renewal after injury and the cellular origin of regenerating cells remain controversial. Using genetic fate-mapping techniques, we generated transgenic mice in which 94%-95% of tubular epithelial cells, but no interstitial cells, were labeled with either beta-galactosidase (lacZ) or red fluorescent protein (RFP). Two days after ischemia-reperfusion injury (IRI), 50.5% of outer medullary epithelial cells coexpress Ki67 and RFP, indicating that differentiated epithelial cells that survived injury undergo proliferative expansion. After repair was complete, 66.9% of epithelial cells had incorporated BrdU, compared to only 3.5% of cells in the uninjured kidney. Despite this extensive cell proliferation, no dilution of either cell-fate marker was observed after repair. These results indicate that regeneration by surviving tubular epithelial cells is the predominant mechanism of repair after ischemic tubular injury in the adult mammalian kidney. PMID:18371453

  13. Clinical implications of epithelial cell plasticity in cancer progression.

    PubMed

    Aparicio, Luis A; Blanco, Moisés; Castosa, Raquel; Concha, Ángel; Valladares, Manuel; Calvo, Lourdes; Figueroa, Angélica

    2015-09-28

    In the last few years, the role of epithelial cell plasticity in cancer biology research has gained increasing attention. This concept refers to the ability of the epithelial cells to dynamically switch between different phenotypic cellular states. This programme is particularly relevant during the epithelial-to-mesenchymal transition (EMT) in cancer progression. During colonization, epithelial cells first activate the EMT programme to disseminate from a primary tumour to reach a distant tissue site. During this process, cells are transported into the circulation and are able to escape the immune system of the host. Then, a reverse process called mesenchymal-to-epithelial transition (MET) occurs on cells that settle in the distant organs. Although epithelial cell plasticity has an important impact on tumour biology, the clinical relevance of this concept remains to be recapitulated. In this review, we will update the current state of epithelial cell plasticity in cancer progression and its clinical implications for the design of therapeutic strategies, the acquisition of multidrug resistance, and future perspectives for the management of cancer patients. PMID:26099173

  14. Liver epithelial cells inhibit proliferation and invasiveness of hepatoma cells.

    PubMed

    Jeng, Kuo-Shyang; Jeng, Chi-Juei; Jeng, Wen-Juei; Sheen, I-Shyan; Li, Shih-Yun; Hung, Zih-Hang; Hsiau, Hsin-I; Yu, Ming-Che; Chang, Chiung-Fang

    2016-03-01

    Hepatocellular carcinoma (HCC) is a worldwide malignancy with poor prognosis. Liver progenitors or stem cells could be a potential therapy for HCC treatment since they migrate toward tumors. Rat liver epithelial (RLE) cells have both progenitor and stem cell-like properties. Therefore, our study elucidated the therapeutic effect of RLE cells in rat hepatoma cells. RLE cells were isolated from 10-day old rats and characterized for stem cell marker expression. RLE cells and rat hepatoma cells (H4-IIE-C3 cells) were co-cultured and divided into four groups with different ratios of RLE and hepatoma cells. Group A had only rat hepatoma cells as a control group. The ratios of rat hepatoma and RLE cells in group B, C and D were 5:1, 1:1 and 1:5, respectively. Effective inhibition of cell proliferation and migration was found in group D when compared to group A. There was a significant decrease in Bcl2 expression and increase in late apoptosis of rat hepatoma cells when adding more RLE cells. RLE cells reduced cell proliferation and migration of rat hepatoma cells. These results suggested that RLE cells could be used as a potential cell therapy. PMID:26647726

  15. Sodium selectivity of Reissner's membrane epithelial cells

    PubMed Central

    2011-01-01

    Background Sodium absorption by Reissner's membrane is thought to contribute to the homeostasis of the volume of cochlear endolymph. It was previously shown that the absorptive transepithelial current was blocked by amiloride and benzamil. The most commonly-observed target of these drugs is the epithelial sodium channel (ENaC), which is composed of the three subunits α-,β- and γ-ENaC. However, other less-selective cation channels have also been observed to be sensitive to benzamil and amiloride. The aim of this study was to determine whether Reissner's membrane epithelial cells could support parasensory K+ absorption via amiloride- and benzamil-sensitive electrogenic pathways. Results We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6196), RT-PCR, and whole-cell patch clamp. Transcript expression analysis of Reissner's membrane detected no amiloride-sensitive acid-sensing ion channels (ASIC1a, ASIC2a, ASIC2b) nor amiloride-sensitive cyclic-nucleotide gated channels (CNGA1, CNGA2, CNGA4, CNGB3). By contrast, α-,β- and γ-ENaC were all previously reported as present in Reissner's membrane. The selectivity of the benzamil-sensitive cation currents was observed in whole-cell patch clamp recordings under Cl--free conditions where cations were the only permeant species. The currents were carried by Na+ but not K+, and the permeability of Li+ was greater than that of Na+ in Reissner's membrane. Complete replacement of bath Na+ with the inpermeable cation NMDG+ led to the same inward current as with benzamil in a Na+ bath. Conclusions These results are consistent with the amiloride/benzamil-sensitive absorptive flux of Reissner's membrane mediated by a highly Na+-selective channel that has several key characteristics in common with αβγ-ENaC. The amiloride-sensitive pathway therefore absorbs only Na+ in this epithelium and does not provide a parasensory K+ efflux route from scala media. PMID:21284860

  16. Andrographolide suppresses epithelial mesenchymal transition by inhibition of MAPK signalling pathway in lens epithelial cells.

    PubMed

    Kayastha, Forum; Johar, Kaid; Gajjar, Devarshi; Arora, Anshul; Madhu, Hardik; Ganatra, Darshini; Vasavada, Abhay

    2015-06-01

    Epithelial mesenchymal transition (EMT) of lens epithelial cells (LECs) may contribute to the development of posterior capsular opacification (PCO), which leads to visual impairment. Andrographolide has been shown to have therapeutic potential against various cancers. However, its effect on human LECs is still unknown. The purpose of this study is to evaluate the effect of andrographolide on EMT induced by growth factors in the fetal human lens epithelial cell line (FHL 124). Initially the LECs were treated with growth factors (TGF-beta 2 and bFGF) to induce EMT. Subsequently these EMT-induced cells were treated with andrographolide at 100 and 500 nM concentrations for 24 h. Our results showed that FHL 124 cells treated with growth factors had a significant decrease in protein and m-RNA levels of epithelial markers pax6 and E-Cadherin. After administering andrographolide, these levels significantly increased. It was noticed that EMT markers alpha-SMA, fibronectin and collagen IV significantly decreased after treatment with andrographolide when compared to the other group. Treatment with andrographolide significantly inhibited phosphorylation of ERK and JNK. Cell cycle analysis showed that andrographolide did not arrest cells at G0/G1 or G2/M at tested concentrations. Our findings suggest that andrographolide helps sustain epithelial characteristics by modulating EMT markers and inhibiting the mitogen-activated protein kinase (MAPK) signalling pathway in LECs. Hence it can prove to be useful in curbing EMT-mediated PCO. PMID:25963259

  17. Serum-Induced Differentiation of Human Meibomian Gland Epithelial Cells

    PubMed Central

    Sullivan, David A.; Liu, Yang; Kam, Wendy R.; Ding, Juan; Green, Karin M.; Shaffer, Scott A.; Hatton, Mark P.; Liu, Shaohui

    2014-01-01

    Purpose. We hypothesize that culturing immortalized human meibomian gland epithelial cells in serum-containing medium will induce their differentiation. The purpose of this investigation was to begin to test our hypothesis, and explore the impact of serum on gene expression and lipid accumulation in human meibomian gland epithelial cells. Methods. Immortalized and primary human meibomian gland epithelial cells were cultured in the presence or absence of serum. Cells were evaluated for lysosome and lipid accumulation, polar and neutral lipid profiles, and gene expression. Results. Our results support our hypothesis that serum stimulates the differentiation of human meibomian gland epithelial cells. This serum-induced effect is associated with a significant increase in the expression of genes linked to cell differentiation, epithelium development, the endoplasmic reticulum, Golgi apparatus, vesicles, and lysosomes, and a significant decrease in gene activity related to the cell cycle, mitochondria, ribosomes, and translation. These cellular responses are accompanied by an accumulation of lipids within lysosomes, as well as alterations in the fatty acid content of polar and nonpolar lipids. Of particular importance, our results show that the molecular and biochemical changes of immortalized human meibomian gland epithelial cells during differentiation are analogous to those of primary cells. Conclusions. Overall, our findings indicate that immortalized human meibomian gland epithelial cells may serve as an ideal preclinical model to identify factors that control cellular differentiation in the meibomian gland. PMID:24867579

  18. Sepsis-associated AKI: epithelial cell dysfunction.

    PubMed

    Emlet, David R; Shaw, Andrew D; Kellum, John A

    2015-01-01

    Acute kidney injury (AKI) occurs frequently in critically ill patients with sepsis, in whom it doubles the mortality rate and half of the survivors suffer permanent kidney damage or chronic kidney disease. Failure in the development of viable therapies has prompted studies to better elucidate the cellular and molecular etiologies of AKI, which have generated novel theories and paradigms for the mechanisms of this disease. These studies have shown multifaceted origins and elements of AKI that, in addition to/in lieu of ischemia, include the generation of damage-associated molecular patterns and pathogen-associated molecular patterns, the inflammatory response, humoral and cellular immune activation, perturbation of microvascular flow and oxidative stress, bioenergetic alterations, cell-cycle alterations, and cellular de-differentiation/re-differentiation. It is becoming clear that a major etiologic effector of all these inputs is the renal tubule epithelial cell (RTEC). This review discusses these elements and their effects on RTECs, and reviews the current hypotheses of how these effects may determine the fate of RTECs during sepsis-induced AKI. PMID:25795502

  19. Sec3-containing exocyst complex is required for desmosome assembly in mammalian epithelial cells.

    PubMed

    Andersen, Nicholas J; Yeaman, Charles

    2010-01-01

    The Exocyst is a conserved multisubunit complex involved in the docking of post-Golgi transport vesicles to sites of membrane remodeling during cellular processes such as polarization, migration, and division. In mammalian epithelial cells, Exocyst complexes are recruited to nascent sites of cell-cell contact in response to E-cadherin-mediated adhesive interactions, and this event is an important early step in the assembly of intercellular junctions. Sec3 has been hypothesized to function as a spatial landmark for the development of polarity in budding yeast, but its role in epithelial cells has not been investigated. Here, we provide evidence in support of a function for a Sec3-containing Exocyst complex in the assembly or maintenance of desmosomes, adhesive junctions that link intermediate filament networks to sites of strong intercellular adhesion. We show that Sec3 associates with a subset of Exocyst complexes that are enriched at desmosomes. Moreover, we found that membrane recruitment of Sec3 is dependent on cadherin-mediated adhesion but occurs later than that of the known Exocyst components Sec6 and Sec8 that are recruited to adherens junctions. RNA interference-mediated suppression of Sec3 expression led to specific impairment of both the morphology and function of desmosomes, without noticeable effect on adherens junctions. These results suggest that two different exocyst complexes may function in basal-lateral membrane trafficking and will enable us to better understand how exocytosis is spatially organized during development of epithelial plasma membrane domains. PMID:19889837

  20. Genetics and epithelial cell dysfunction in cystic fibrosis

    SciTech Connect

    Riordan, J.R.; Buchwald, M.

    1987-01-01

    This book examines the advances being made in the study of the physiology, cell biology, and molecular genetics of cystic fibrosis. Emphasis is placed on various areas of research that involve epithelial cells (e.g., the CF-specific phenotypes exhibited by epithelial cells, abnormalities in epithelium ion transport, chloride channel regulation in CF epithelial.) Coverage is presented on the current status of CF, including data on the incidence of the disease, its mode of inheritance, chromosomal localization, genetic heterogeneity, and screening and management.

  1. Protein Kinase D Is Increased and Activated in Lung Epithelial Cells and Macrophages in Idiopathic Pulmonary Fibrosis

    PubMed Central

    Gan, Huachen; McKenzie, Raymond; Hao, Qin; Idell, Steven; Tang, Hua

    2014-01-01

    Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive and usually fatal lung disease of unknown etiology for which no effective treatments currently exist. Hence, there is a profound need for the identification of novel drugable targets to develop more specific and efficacious therapeutic intervention in IPF. In this study, we performed immunohistochemical analyses to assess the cell type-specific expression and activation of protein kinase D (PKD) family kinases in normal and IPF lung tissue sections. We also analyzed PKD activation and function in human lung epithelial cells. We found that PKD family kinases (PKD1, PKD2 and PKD3) were increased and activated in the hyperplastic and regenerative alveolar epithelial cells lining remodeled fibrotic alveolar septa and/or fibroblast foci in IPF lungs compared with normal controls. We also found that PKD family kinases were increased and activated in alveolar macrophages, bronchiolar epithelium, and honeycomb cysts in IPF lungs. Interestingly, PKD1 was highly expressed and activated in the cilia of IPF bronchiolar epithelial cells, while PKD2 and PKD3 were expressed in the cell cytoplasm and nuclei. In contrast, PKD family kinases were not apparently increased and activated in IPF fibroblasts or myofibroblasts. We lastly found that PKD was predominantly activated by poly-L-arginine, lysophosphatidic acid and thrombin in human lung epithelial cells and that PKD promoted epithelial barrier dysfunction. These findings suggest that PKD may participate in the pathogenesis of IPF and may be a novel target for therapeutic intervention in this disease. PMID:25000413

  2. Microfluidic approaches for epithelial cell layer culture and characterisation

    PubMed Central

    Thuenauer, Roland; Rodriguez-Boulan, Enrique; Römer, Winfried

    2014-01-01

    In higher eukaryotes, epithelial cell layers line most body cavities and form selective barriers that regulate the exchange of solutes between compartments. In order to fulfil these functions, the cells assume a polarised architecture and maintain two distinct plasma membrane domains, the apical domain facing the lumen and the basolateral domain facing other cells and the extracellular matrix. Microfluidic biochips offer the unique opportunity to establish novel in vitro models of epithelia in which the in vivo microenvironment of epithelial cells is precisely reconstituted. In addition, analytical tools to monitor biologically relevant parameters can be directly integrated on-chip. In this review we summarise recently developed biochip designs for culturing epithelial cell layers. Since endothelial cell layers, which line blood vessels, have similar barrier functions and polar organisation as epithelial cell layers, we also discuss biochips for culturing endothelial cell layers. Furthermore, we review approaches to integrate tools to analyse and manipulate epithelia and endothelia in microfluidic biochips, including methods to perform electrical impedance spectroscopy, methods to detect substances undergoing trans-epithelial transport via fluorescence, spectrophotometry, and mass spectrometry, techniques to mechanically stimulate cells via stretching and fluid flow-induced shear stress, and methods to carry out high-resolution imaging of vesicular trafficking with light microscopy. Taken together, this versatile microfluidic toolbox enables novel experimental approaches to characterise epithelial monolayers. PMID:24668405

  3. Microfluidic approaches for epithelial cell layer culture and characterisation.

    PubMed

    Thuenauer, Roland; Rodriguez-Boulan, Enrique; Römer, Winfried

    2014-07-01

    In higher eukaryotes, epithelial cell layers line most body cavities and form selective barriers that regulate the exchange of solutes between compartments. In order to fulfil these functions, the cells assume a polarised architecture and maintain two distinct plasma membrane domains, the apical domain facing the lumen and the basolateral domain facing other cells and the extracellular matrix. Microfluidic biochips offer the unique opportunity to establish novel in vitro models of epithelia in which the in vivo microenvironment of epithelial cells is precisely reconstituted. In addition, analytical tools to monitor biologically relevant parameters can be directly integrated on-chip. In this review we summarise recently developed biochip designs for culturing epithelial cell layers. Since endothelial cell layers, which line blood vessels, have similar barrier functions and polar organisation as epithelial cell layers, we also discuss biochips for culturing endothelial cell layers. Furthermore, we review approaches to integrate tools to analyse and manipulate epithelia and endothelia in microfluidic biochips; including methods to perform electrical impedance spectroscopy; methods to detect substances undergoing trans-epithelial transport via fluorescence, spectrophotometry, and mass spectrometry; techniques to mechanically stimulate cells via stretching and fluid flow-induced shear stress; and methods to carry out high-resolution imaging of vesicular trafficking using light microscopy. Taken together, this versatile microfluidic toolbox enables novel experimental approaches to characterise epithelial monolayers. PMID:24668405

  4. Molecular responses of rat tracheal epithelial cells to transmembrane pressure.

    PubMed

    Ressler, B; Lee, R T; Randell, S H; Drazen, J M; Kamm, R D

    2000-06-01

    Smooth muscle constriction in asthma causes the airway to buckle into a rosette pattern, folding the epithelium into deep crevasses. The epithelial cells in these folds are pushed up against each other and thereby experience compressive stresses. To study the epithelial cell response to compressive stress, we subjected primary cultures of rat tracheal epithelial cells to constant elevated pressures on their apical surface (i.e., a transmembrane pressure) and examined changes in the expression of genes that are important for extracellular matrix production and maintenance of smooth muscle activation. Northern blot analysis of RNA extracted from cells subjected to transmembrane pressure showed induction of early growth response-1 (Egr-1), endothelin-1, and transforming growth factor-beta1 in a pressure-dependent and time-dependent manner. Increases in Egr-1 protein were detected by immunohistochemistry. Our results demonstrate that airway epithelial cells respond rapidly to compressive stresses. Potential transduction mechanisms of transmembrane pressure were also investigated. PMID:10835333

  5. Cigarette Smoke Extract Induces a Phenotypic Shift in Epithelial Cells; Involvement of HIF1α in Mesenchymal Transition

    PubMed Central

    Eurlings, Irene M. J.; Reynaert, Niki L.; van den Beucken, Twan; Gosker, Harry R.; de Theije, C. C.; Verhamme, Fien M.; Bracke, Ken R.; Wouters, Emiel F. M.; Dentener, Mieke A.

    2014-01-01

    In COPD, matrix remodeling contributes to airflow limitation. Recent evidence suggests that next to fibroblasts, the process of epithelial-mesenchymal transition can contribute to matrix remodeling. CSE has been shown to induce EMT in lung epithelial cells, but the signaling mechanisms involved are largely unknown and subject of this study. EMT was assessed in A549 and BEAS2B cells stimulated with CSE by qPCR, Western blotting and immunofluorescence for epithelial and mesenchymal markers, as were collagen production, cell adhesion and barrier integrity as functional endpoints. Involvement of TGF-β and HIF1α signaling pathways were investigated. In addition, mouse models were used to examine the effects of CS on hypoxia signaling and of hypoxia per se on mesenchymal expression. CSE induced EMT characteristics in A549 and BEAS2B cells, evidenced by decreased expression of epithelial markers and a concomitant increase in mesenchymal marker expression after CSE exposure. Furthermore cells that underwent EMT showed increased production of collagen, decreased adhesion and disrupted barrier integrity. The induction of EMT was found to be independent of TGF-β signaling. On the contrary, CS was able to induce hypoxic signaling in A549 and BEAS2B cells as well as in mice lung tissue. Importantly, HIF1α knock-down prevented induction of mesenchymal markers, increased collagen production and decreased adhesion after CSE exposure, data that are in line with the observed induction of mesenchymal marker expression by hypoxia in vitro and in vivo. Together these data provide evidence that both bronchial and alveolar epithelial cells undergo a functional phenotypic shift in response to CSE exposure which can contribute to increased collagen deposition in COPD lungs. Moreover, HIF1α signaling appears to play an important role in this process. PMID:25329389

  6. Response of corneal epithelial cells to Staphylococcus aureus

    PubMed Central

    2010-01-01

    Staphylococcus aureus is a leading cause of invasive infection. It also infects wet mucosal tissues including the cornea and conjunctiva. Conflicting evidence exists on the expression of Toll-like receptors by human corneal epithelial cells. It was therefore of interest to determine how epithelial cells from this immune privileged tissue respond to S. aureus. Further, it was of interest to determine whether cytolytic toxins, with the potential to cause ion flux or potentially permit effector molecule movement across the target cell membrane, alter the response. Microarrays were used to globally assess the response of human corneal epithelial cells to S. aureus. A large increase in abundance of transcripts encoding the antimicrobial dendritic cell chemokine, CCL20, was observed. CCL20 release into the medium was detected, and this response was found to be largely TLR2 and NOD2 independent. Corneal epithelial cells also respond to S. aureus by increasing the intracellular abundance of mRNA for inflammatory mediators, transcription factors, and genes related to MAP kinase pathways, in ways similar to other cell types. The corneal epithelial cell response was surprisingly unaffected by toxin exposure. Toxin exposure did, however, induce a stress response. Although model toxigenic and non-toxigenic strains of S. aureus were employed in the present study, the results obtained were strikingly similar to those reported for stimulation of vaginal epithelial cells by clinical toxic shock toxin expressing isolates, demonstrating that the initial epithelial cellular responses to S. aureus are largely independent of strain as well as epithelial cell tissue source. PMID:21178447

  7. Flow Cytometry Analysis of Thymic Epithelial Cells and Their Subpopulations.

    PubMed

    Ohigashi, Izumi; Takahama, Yousuke

    2016-01-01

    The parenchyma of the thymus is compartmentalized into the cortex and the medulla, which are constructed by cortical thymic epithelial cells (cortical TECs, cTECs) and medullary thymic epithelial cells (mTECs), respectively. cTECs and mTECs essentially and differentially regulate the development and repertoire selection of T cells. Consequently, the biology of T cell development and selection includes the study of TECs in addition to the study of developing T cells and other hematopoietic cells including dendritic cells. In this chapter, we describe the methods for flow cytometric analysis and sorting of TECs and their subpopulations, including cTECs and mTECs. PMID:26294398

  8. Diversity of epithelial stem cell types in adult lung.

    PubMed

    Li, Feng; He, Jinxi; Wei, Jun; Cho, William C; Liu, Xiaoming

    2015-01-01

    Lung is a complex organ lined with epithelial cells. In order to maintain its homeostasis and normal functions following injuries caused by varied extraneous and intraneous insults, such as inhaled environmental pollutants and overwhelming inflammatory responses, the respiratory epithelium normally undergoes regenerations by the proliferation and differentiation of region-specific epithelial stem/progenitor cells that resided in distinct niches along the airway tree. The importance of local epithelial stem cell niches in the specification of lung stem/progenitor cells has been recently identified. Studies using cell differentiating and lineage tracing assays, in vitro and/or ex vivo models, and genetically engineered mice have suggested that these local epithelial stem/progenitor cells within spatially distinct regions along the pulmonary tree contribute to the injury repair of epithelium adjacent to their respective niches. This paper reviews recent findings in the identification and isolation of region-specific epithelial stem/progenitor cells and local niches along the airway tree and the potential link of epithelial stem cells for the development of lung cancer. PMID:25810726

  9. Diversity of Epithelial Stem Cell Types in Adult Lung

    PubMed Central

    Li, Feng; He, Jinxi; Wei, Jun; Cho, William C.; Liu, Xiaoming

    2015-01-01

    Lung is a complex organ lined with epithelial cells. In order to maintain its homeostasis and normal functions following injuries caused by varied extraneous and intraneous insults, such as inhaled environmental pollutants and overwhelming inflammatory responses, the respiratory epithelium normally undergoes regenerations by the proliferation and differentiation of region-specific epithelial stem/progenitor cells that resided in distinct niches along the airway tree. The importance of local epithelial stem cell niches in the specification of lung stem/progenitor cells has been recently identified. Studies using cell differentiating and lineage tracing assays, in vitro and/or ex vivo models, and genetically engineered mice have suggested that these local epithelial stem/progenitor cells within spatially distinct regions along the pulmonary tree contribute to the injury repair of epithelium adjacent to their respective niches. This paper reviews recent findings in the identification and isolation of region-specific epithelial stem/progenitor cells and local niches along the airway tree and the potential link of epithelial stem cells for the development of lung cancer. PMID:25810726

  10. Remodeling of ribosomal genes in somatic cells by Xenopus egg extract

    SciTech Connect

    Ostrup, Olga; Hyttel, Poul; Klaerke, Dan A.; Collas, Philippe

    2011-09-02

    Highlights: {yields} Xenopus egg extract remodels nuclei and alter cell growth characteristics. {yields} Ribosomal genes are reprogrammed within 6 h after extract exposure. {yields} rDNA reprogramming involves promoter targeting of SNF2H remodeling complex. {yields} Xenopus egg extract does not initiate stress-related response in somatic cells. {yields} Aza-cytidine elicits a stress-induced response in reprogrammed cells. -- Abstract: Extracts from Xenopus eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression. This occurs within hours of extract treatment and is distinct from a stress response. Egg extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by extract and elicits a stress-type nuclear response. Thus, an early event of Xenopus egg extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation of various reprogramming methods.

  11. ATRA mechanically reprograms pancreatic stellate cells to suppress matrix remodelling and inhibit cancer cell invasion.

    PubMed

    Chronopoulos, Antonios; Robinson, Benjamin; Sarper, Muge; Cortes, Ernesto; Auernheimer, Vera; Lachowski, Dariusz; Attwood, Simon; García, Rebeca; Ghassemi, Saba; Fabry, Ben; Del Río Hernández, Armando

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a dismal survival rate. Persistent activation of pancreatic stellate cells (PSCs) can perturb the biomechanical homoeostasis of the tumour microenvironment to favour cancer cell invasion. Here we report that ATRA, an active metabolite of vitamin A, restores mechanical quiescence in PSCs via a mechanism involving a retinoic acid receptor beta (RAR-β)-dependent downregulation of actomyosin (MLC-2) contractility. We show that ATRA reduces the ability of PSCs to generate high traction forces and adapt to extracellular mechanical cues (mechanosensing), as well as suppresses force-mediated extracellular matrix remodelling to inhibit local cancer cell invasion in 3D organotypic models. Our findings implicate a RAR-β/MLC-2 pathway in peritumoural stromal remodelling and mechanosensory-driven activation of PSCs, and further suggest that mechanical reprogramming of PSCs with retinoic acid derivatives might be a viable alternative to stromal ablation strategies for the treatment of PDAC. PMID:27600527

  12. HIV is inactivated after transepithelial migration via adult oral epithelial cells but not fetal epithelial cells

    PubMed Central

    Tugizov, Sharof M.; Herrera, Rossana; Veluppillai, Piri; Greenspan, Deborah; Soros, Vanessa; Greene, Warner C.; Levy, Jay A.; Palefsky, Joel M.

    2010-01-01

    Oral transmission of human immunodeficiency virus (HIV) in adult populations is rare. However, HIV spread across fetal/neonatal oropharyngeal epithelia could be important in mother-to-child transmission. Analysis of HIV transmission across polarized adult and fetal oral epithelial cells revealed that HIV transmigrates through both adult and fetal cells. However, only virions that passed through the fetal cells – and not those that passed through the adult cells – remained infectious. Analysis of expression of anti-HIV innate proteins beta-defensins 2 and 3, and secretory leukocyte protease inhibitor in adult, fetal, and infant oral epithelia showed that their expression is predominantly in the adult oral epithelium. Retention of HIV infectivity after transmigration correlated inversely with the expression of these innate proteins. Inactivation of innate proteins in adult oral keratinocytes restored HIV infectivity. These data suggest that high-level innate protein expression may contribute to the resistance of the adult oral epithelium to HIV transmission. PMID:21056450

  13. CUX1/Wnt signaling regulates Epithelial Mesenchymal Transition in EBV infected epithelial cells

    SciTech Connect

    Malizia, Andrea P.; Lacey, Noreen; Walls, Dermot; Egan, Jim J.; Doran, Peter P.

    2009-07-01

    Idiopathic pulmonary fibrosis (IPF) is a refractory and lethal interstitial lung disease characterized by alveolar epithelial cells apoptosis, fibroblast proliferation and extra-cellular matrix protein deposition. EBV, localised to alveolar epithelial cells of pulmonary fibrosis patients is associated with a poor prognosis. A strategy based on microarray-differential gene expression analysis to identify molecular drivers of EBV-associated lung fibrosis was utilized. Alveolar epithelial cells were infected with EBV to identify genes whose expression was altered following TGF{beta}1-mediated lytic phase. EBV lytic reactivation by TGF{beta}1 drives a selective alteration in CUX1 variant (a) (NCBI accession number NM{sub 1}81552) expression, inducing activation of non-canonical Wnt pathway mediators, implicating it in Epithelial Mesenchymal Transition (EMT), the molecular event underpinning scar production in tissue fibrosis. The role of EBV in EMT can be attenuated by antiviral strategies and inhibition of Wnt signaling by using All-Trans Retinoic Acids (ATRA). Activation of non-canonical Wnt signaling pathway by EBV in epithelial cells suggests a novel mechanism of EMT via CUX1 signaling. These data present a framework for further description of the link between infectious agents and fibrosis, a significant disease burden.

  14. Parvalbumin in cortical epithelial cells of the pigeon thymus

    PubMed Central

    ATOJI, YASURO; YAMAMOTO, YOSHIO; SUZUKI, YOSHITAKA

    2000-01-01

    We examined the distribution of parvalbumin in the pigeon thymus by light and electron microscopic immunohistochemistry. Tissues were also examined by conventional electron microscopy to determine the ultrastructure of immunoreactive cells. Parvalbumin immunoreaction was located in epithelial cells of the cortex, which formed dense mesh-like structures. Parvalbumin-positive epithelial cells were classified into 2 types. The first comprised elongated cells. In these, the nucleus was spindle-shaped, oval, or triangular, with a slightly irregular contour and contained rich heterochromatin peripherally. The cytoplasm was pale and processes extended laterally or ramified among the surrounding thymocytes. This type of cell formed the majority of immunoreactive cells. The other cell type consisted of polygonal epithelial cells. The nucleus was oval with deep indentations. Euchromatin occupied a large part of the nucleus. The cytoplasm contained numerous cell organelles compared with the elongated type, in particular, electron-dense vacuoles of various sizes and often bundles of tonofilaments. Both types of epithelial cell were interconnected by desmosomes. No secretory granules were found in the cytoplasm of elongated or polygonal cells. These results indicate the presence of heterogeneous group of parvalbumin-immunoreactive epithelial cells and suggest the likelihood of different functional roles for parvalbumin in the pigeon thymus. PMID:10853953

  15. Stochastic Terminal Dynamics in Epithelial Cell Intercalation

    NASA Astrophysics Data System (ADS)

    Eule, Stephan; Metzger, Jakob; Reichl, Lars; Kong, Deqing; Zhang, Yujun; Grosshans, Joerg; Wolf, Fred

    2015-03-01

    We found that the constriction of epithelial cell contacts during intercalation in germ band extension in Drosophila embryos follows intriguingly simple quantitative laws. The mean contact length < L > follows < L > (t) ~(T - t) α , where T is the finite collapse time; the time dependent variance of contact length is proportional to the square of the mean; finally the time dependent probability density of the contact lengths remains close to Gaussian during the entire process. These observations suggest that the dynamics of contact collapse can be captured by a stochastic differential equation analytically tractable in small noise approximation. Here, we present such a model, providing an effective description of the non-equilibrium statistical mechanics of contact collapse. All model parameters are fixed by measurements of time dependent mean and variance of contact lengths. The model predicts the contact length covariance function that we obtain in closed form. The contact length covariance function closely matches experimental observations suggesting that the model well captures the dynamics of contact collapse.

  16. Characteristics and EGFP expression of porcine mammary gland epithelial cells.

    PubMed

    Zheng, Yue-Mao; He, Xiao-Ying

    2010-12-01

    The aims of this study were to establish a porcine mammary gland epithelial (PMGE) cell line, and to determine if these PMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of PMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating pig. The passage sixteen PMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in PMGE cells was tested by immunofluorescence. Βeta-Casein gene mRNA was tested for PMGE cells by RT-PCR. The results showed that PMGE cells could form dome-like structure which looked like nipple, and the cells contained different cell types. The expression of Cell keratins demonstrated the property of epithelial cells, and the PMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the PMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected porcine mammary gland epithelial (ET-PMGE) cell line. PMID:20400167

  17. Desialylation of Spermatozoa and Epithelial Cell Glycocalyx Is a Consequence of Bacterial Infection of the Epididymis.

    PubMed

    Khosravi, Farhad; Michel, Vera; Galuska, Christina E; Bhushan, Sudhanshu; Christian, Philipp; Schuppe, Hans-Christian; Pilatz, Adrian; Galuska, Sebastian P; Meinhardt, Andreas

    2016-08-19

    Urinary tract infections caused by uropathogenic Escherichia coli (UPEC) pathovars belong to the most frequent infections in humans. In men, pathogens can also spread to the genital tract via the continuous ductal system, eliciting bacterial prostatitis and/or epididymo-orchitis. Antibiotic treatment usually clears pathogens in acute epididymitis; however, the fertility of patients can be permanently impaired. Because a premature acrosome reaction was observed in an UPEC epididymitis mouse model, and sialidases on the sperm surface are considered to be activated via proteases of the acrosome, we aimed to investigate whether alterations of the sialome of epididymal spermatozoa and surrounding epithelial cells occur during UPEC infection. In UPEC-elicited acute epididymitis in mice, a substantial loss of N-acetylneuraminic acid residues was detected in epididymal spermatozoa and epithelial cells using combined laser microdissection/HPLC-ESI-MS analysis. In support, a substantial reduction of sialic acid residues bound to the surface of spermatozoa was documented in men with a recent history of E. coli-associated epididymitis. In vitro, such an UPEC induced N-acetylneuraminic acid release from human spermatozoa was effectively counteracted by a sialidase inhibitor. These findings strongly suggest a substantial remodeling of the glycocalyx of spermatozoa and epididymal epithelial cells by endogenous sialidases after a premature acrosome reaction during acute epididymitis. PMID:27339898

  18. Bone marrow mesenchymal stem cells and TGF-β signaling in bone remodeling

    PubMed Central

    Crane, Janet L.; Cao, Xu

    2014-01-01

    During bone resorption, abundant factors previously buried in the bone matrix are released into the bone marrow microenvironment, which results in recruitment and differentiation of bone marrow mesenchymal stem cells (MSCs) for subsequent bone formation, temporally and spatially coupling bone remodeling. Parathyroid hormone (PTH) orchestrates the signaling of many pathways that direct MSC fate. The spatiotemporal release and activation of matrix TGF-β during osteoclast bone resorption recruits MSCs to bone-resorptive sites. Dysregulation of TGF-β alters MSC fate, uncoupling bone remodeling and causing skeletal disorders. Modulation of TGF-β or PTH signaling may reestablish coupled bone remodeling and be a potential therapy. PMID:24487640

  19. Cell volume regulation in epithelial physiology and cancer

    PubMed Central

    Pedersen, Stine F.; Hoffmann, Else K.; Novak, Ivana

    2013-01-01

    The physiological function of epithelia is transport of ions, nutrients, and fluid either in secretory or absorptive direction. All of these processes are closely related to cell volume changes, which are thus an integrated part of epithelial function. Transepithelial transport and cell volume regulation both rely on the spatially and temporally coordinated function of ion channels and transporters. In healthy epithelia, specific ion channels/transporters localize to the luminal and basolateral membranes, contributing to functional epithelial polarity. In pathophysiological processes such as cancer, transepithelial and cell volume regulatory ion transport are dys-regulated. Furthermore, epithelial architecture and coordinated ion transport function are lost, cell survival/death balance is altered, and new interactions with the stroma arise, all contributing to drug resistance. Since altered expression of ion transporters and channels is now recognized as one of the hallmarks of cancer, it is timely to consider this especially for epithelia. Epithelial cells are highly proliferative and epithelial cancers, carcinomas, account for about 90% of all cancers. In this review we will focus on ion transporters and channels with key physiological functions in epithelia and known roles in the development of cancer in these tissues. Their roles in cell survival, cell cycle progression, and development of drug resistance in epithelial cancers will be discussed. PMID:24009588

  20. MFGE8 regulates TGF-β-induced epithelial mesenchymal transition in endometrial epithelial cells in vitro.

    PubMed

    Yu, Liang; Hu, Rong; Sullivan, Claretta; Swanson, R James; Oehninger, Sergio; Sun, Ying-Pu; Bocca, Silvina

    2016-09-01

    This study investigated the role of milk fat globule-epidermal growth factor-factor 8 (MFGE8) in TGF-β-induced epithelial-mesenchymal transition (EMT) of endometrial epithelial cells. These were in vitro studies using human endometrial epithelial cells and mouse blastocysts. We investigated the ability of TGF-β to induce EMT in endometrial epithelial cells (HEC-1A) by assessment of cytological phenotype (by light and atomic force microscopy), changes in expression of the markers of cell adhesion/differentiation E- and N-cadherin, and of the transcription factor Snail (by immunofluorescence and immunoblotting), and competence to support embryo attachment in a mouse blastocyst outgrowth assay. We also studied the effects of E-cadherin expression in cells transfected by retroviral shRNA vectors specifically silencing MFGE8. Results demonstrated that TGF-β induced EMT as demonstrated by phenotypic cell changes, by a switch of cadherin expression as well as by upregulation of the expression of the mesenchymal markers Snail and Vimentin. Upon MFGE8 knockdown, these processes were interfered with, suggesting that MFGE8 and TGF-β together may participate in regulation of EMT. This study demonstrated for the first time that endometrial MFGE8 modulates TGF-β-induced EMT in human endometrium cells. PMID:27340235

  1. Morphological appearances of human lens epithelial cells in culture.

    PubMed

    Power, W; Neylan, D; Collum, L

    1993-01-01

    A system for culturing human lens epithelial cells in the laboratory was developed. The morphological appearances of the cells was studied using phase contrast, scanning and transmission electron microscopy. Cell marker studies using monoclonal antibodies to cytokeratin, vimentin and epithelial membrane antigen were also performed. There was a marked increase in cell size as a function of time in culture. After 3 to 4 weeks cells showed early signs of ageing. By 6 to 8 weeks the majority of the cells had become very irregular in shape and demonstrated irregularities of the plasma membrane and intra-cytoplasmic vacuole formation. The cells stained strongly for vimentin and epithelial membrane antigen. Staining with cytokeratin was somewhat weaker. This culture technique provides us with a suitable model for studying the growth behavior of these cells. PMID:7512459

  2. Regulated Mucin Secretion from Airway Epithelial Cells

    PubMed Central

    Adler, Kenneth B.; Tuvim, Michael J.; Dickey, Burton F.

    2013-01-01

    Secretory epithelial cells of the proximal airways synthesize and secrete gel-forming polymeric mucins. The secreted mucins adsorb water to form mucus that is propelled by neighboring ciliated cells, providing a mobile barrier which removes inhaled particles and pathogens from the lungs. Several features of the intracellular trafficking of mucins make the airway secretory cell an interesting comparator for the cell biology of regulated exocytosis. Polymeric mucins are exceedingly large molecules (up to 3 × 106 Da per monomer) whose folding and initial polymerization in the ER requires the protein disulfide isomerase Agr2. In the Golgi, mucins further polymerize to form chains and possibly branched networks comprising more than 20 monomers. The large size of mucin polymers imposes constraints on their packaging into transport vesicles along the secretory pathway. Sugar side chains account for >70% of the mass of mucins, and their attachment to the protein core by O-glycosylation occurs in the Golgi. Mature polymeric mucins are stored in large secretory granules ∼1 μm in diameter. These are translocated to the apical membrane to be positioned for exocytosis by cooperative interactions among myristoylated alanine-rich C kinase substrate, cysteine string protein, heat shock protein 70, and the cytoskeleton. Mucin granules undergo exocytic fusion with the plasma membrane at a low basal rate and a high stimulated rate. Both rates are mediated by a regulated exocytic mechanism as indicated by phenotypes in both basal and stimulated secretion in mice lacking Munc13-2, a sensor of the second messengers calcium and diacylglycerol (DAG). Basal secretion is induced by low levels of activation of P2Y2 purinergic and A3 adenosine receptors by extracellular ATP released in paracrine fashion and its metabolite adenosine. Stimulated secretion is induced by high levels of the same ligands, and possibly by inflammatory mediators as well. Activated receptors are coupled to

  3. Effect of freezing on lens epithelial cell growth.

    PubMed

    Fukaya, Y; Hara, T; Hara, T; Iwata, S

    1988-05-01

    The effect of freezing on the growth of rat lens epithelial cells was studied in vitro. We found that 80% of the lens epithelial cells died after freezing at -45 degrees C for two hours and that the surviving cells could grow with the addition of growth factors or when placed on a sheet of type 4 collagen, but not when placed on a plain plastic culture dish. These results suggest that the surviving cells are at the Go phase of the cell cycle and that type 4 collagen or growth factors can initiate cell division. PMID:3294380

  4. Disruption of TGF-β signaling in smooth muscle cell prevents flow-induced vascular remodeling

    SciTech Connect

    Gao, Fu; Chambon, Pierre; Tellides, George; Kong, Wei; Zhang, Xiaoming; Li, Wei

    2014-11-07

    Highlights: • TGF-β signaling in SMC contributes to the flow-induced vascular remodeling. • Disruption of TGF-β signaling in SMC can prevent this process. • Targeting SM-specific Tgfbr2 could be a novel therapeutic strategy for vascular remodeling. - Abstract: Transforming growth factor-β (TGF-β) signaling has been prominently implicated in the pathogenesis of vascular remodeling, especially the initiation and progression of flow-induced vascular remodeling. Smooth muscle cells (SMCs) are the principal resident cells in arterial wall and are critical for arterial remodeling. However, the role of TGF-β signaling in SMC for flow-induced vascular remodeling remains unknown. Therefore, the goal of our study was to determine the effect of TGF-β pathway in SMC for vascular remodeling, by using a genetical smooth muscle-specific (SM-specific) TGF-β type II receptor (Tgfbr2) deletion mice model. Mice deficient in the expression of Tgfbr2 (MyhCre.Tgfbr2{sup f/f}) and their corresponding wild-type background mice (MyhCre.Tgfbr2{sup WT/WT}) underwent partial ligation of left common carotid artery for 1, 2, or 4 weeks. Then the carotid arteries were harvested and indicated that the disruption of Tgfbr2 in SMC provided prominent inhibition of vascular remodeling. And the thickening of carotid media, proliferation of SMC, infiltration of macrophage, and expression of matrix metalloproteinase (MMP) were all significantly attenuated in Tgfbr2 disruption mice. Our study demonstrated, for the first time, that the TGF-β signaling in SMC plays an essential role in flow-induced vascular remodeling and disruption can prevent this process.

  5. ONCOGENE ALTERNATIONS IN IN VITRO TRANSFORMED RAT TRACHEAL EPITHELIAL CELLS

    EPA Science Inventory

    Ten derivations of rat tracheal epithelial (RTE) cells, including normal cells, normal primary cultures, 7 tumorigenic cell lines and 1 non-tumorigenic cell line transformed by treatment with 7,12-dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene (BP) and/or 12-0-tetradecanoylphor...

  6. How Shigella Utilizes Ca2+ Jagged Edge Signals during Invasion of Epithelial Cells

    PubMed Central

    Bonnet, Mariette; Tran Van Nhieu, Guy

    2016-01-01

    Shigella, the causative agent of bacillary dysentery invades intestinal epithelial cells using a type III secretion system (T3SS). Through the injection of type III effectors, Shigella manipulates the actin cytoskeleton to induce its internalization in epithelial cells. At early invasion stages, Shigella induces atypical Ca2+ responses confined at entry sites allowing local cytoskeletal remodeling for bacteria engulfment. Global Ca2+ increase in the cell triggers the opening of connexin hemichannels at the plasma membrane that releases ATP in the extracellular milieu, favoring Shigella invasion and spreading through purinergic receptor signaling. During intracellular replication, Shigella regulates inflammatory and death pathways to disseminate within the epithelium. At later stages of infection, Shigella downregulates hemichannel opening and the release of extracellular ATP to dampen inflammatory signals. To avoid premature cell death, Shigella activates cell survival by upregulating the PI3K/Akt pathway and downregulating the levels of p53. Furthermore, Shigella interferes with pro-apoptotic caspases, and orients infected cells toward a slow necrotic cell death linked to mitochondrial Ca2+ overload. In this review, we will focus on the role of Ca2+ responses and their regulation by Shigella during the different stages of bacterial infection. PMID:26904514

  7. How Shigella Utilizes Ca(2+) Jagged Edge Signals during Invasion of Epithelial Cells.

    PubMed

    Bonnet, Mariette; Tran Van Nhieu, Guy

    2016-01-01

    Shigella, the causative agent of bacillary dysentery invades intestinal epithelial cells using a type III secretion system (T3SS). Through the injection of type III effectors, Shigella manipulates the actin cytoskeleton to induce its internalization in epithelial cells. At early invasion stages, Shigella induces atypical Ca(2+) responses confined at entry sites allowing local cytoskeletal remodeling for bacteria engulfment. Global Ca(2+) increase in the cell triggers the opening of connexin hemichannels at the plasma membrane that releases ATP in the extracellular milieu, favoring Shigella invasion and spreading through purinergic receptor signaling. During intracellular replication, Shigella regulates inflammatory and death pathways to disseminate within the epithelium. At later stages of infection, Shigella downregulates hemichannel opening and the release of extracellular ATP to dampen inflammatory signals. To avoid premature cell death, Shigella activates cell survival by upregulating the PI3K/Akt pathway and downregulating the levels of p53. Furthermore, Shigella interferes with pro-apoptotic caspases, and orients infected cells toward a slow necrotic cell death linked to mitochondrial Ca(2+) overload. In this review, we will focus on the role of Ca(2+) responses and their regulation by Shigella during the different stages of bacterial infection. PMID:26904514

  8. Control of Francisella tularensis Intracellular Growth by Pulmonary Epithelial Cells

    PubMed Central

    Maggio, Savannah; Takeda, Kazuyo; Stark, Felicity; Meierovics, Anda I.; Yabe, Idalia; Cowley, Siobhan C.

    2015-01-01

    The virulence of F. tularensis is often associated with its ability to grow in macrophages, although recent studies show that Francisella proliferates in multiple host cell types, including pulmonary epithelial cells. Thus far little is known about the requirements for killing of F. tularensis in the non-macrophage host cell types that support replication of this organism. Here we sought to address this question through the use of a murine lung epithelial cell line (TC-1 cells). Our data show that combinations of the cytokines IFN-γ, TNF, and IL-17A activated murine pulmonary epithelial cells to inhibit the intracellular growth of the F. tularensis Live Vaccine Strain (LVS) and the highly virulent F. tularensis Schu S4 strain. Although paired combinations of IFN-γ, TNF, and IL-17A all significantly controlled LVS growth, simultaneous treatment with all three cytokines had the greatest effect on LVS growth inhibition. In contrast, Schu S4 was more resistant to cytokine-induced growth effects, exhibiting significant growth inhibition only in response to all three cytokines. Since one of the main antimicrobial mechanisms of activated macrophages is the release of reactive nitrogen intermediates (RNI) via the activity of iNOS, we investigated the role of RNI and iNOS in Francisella growth control by pulmonary epithelial cells. NOS2 gene expression was significantly up-regulated in infected, cytokine-treated pulmonary epithelial cells in a manner that correlated with LVS and Schu S4 growth control. Treatment of LVS-infected cells with an iNOS inhibitor significantly reversed LVS killing in cytokine-treated cultures. Further, we found that mouse pulmonary epithelial cells produced iNOS during in vivo respiratory LVS infection. Overall, these data demonstrate that lung epithelial cells produce iNOS both in vitro and in vivo, and can inhibit Francisella intracellular growth via reactive nitrogen intermediates. PMID:26379269

  9. Epimorphin Functions as a Key Morphoregulator for Mammary Epithelial Cells

    SciTech Connect

    Hirai, H.; Lochter, A.; Galosy, S.; Koshida, S.; Niwa, S.; Bissell, M.J.

    1997-10-13

    Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.

  10. Interferons Mediate Terminal Differentiation of Human Cortical Thymic Epithelial Cells

    PubMed Central

    Vidalain, Pierre-Olivier; Laine, David; Zaffran, Yona; Azocar, Olga; Servet-Delprat, Christine; Wild, T. Fabian; Rabourdin-Combe, Chantal; Valentin, Hélène

    2002-01-01

    In the thymus, epithelial cells comprise a heterogeneous population required for the generation of functional T lymphocytes, suggesting that thymic epithelium disruption by viruses may compromise T-cell lymphopoiesis in this organ. In a previous report, we demonstrated that in vitro, measles virus induced differentiation of cortical thymic epithelial cells as characterized by (i) cell growth arrest, (ii) morphological and phenotypic changes, and (iii) apoptotis as a final step of this process. In the present report, we have analyzed the mechanisms involved. First, measles virus-induced differentiation of thymic epithelial cells is shown to be strictly dependent on beta interferon (IFN-β) secretion. In addition, transfection with double-stranded RNA, a common intermediate of replication for a broad spectrum of viruses, is reported to similarly mediate thymic epithelial cell differentiation through IFN-β induction. Finally, we demonstrated that recombinant IFN-α, IFN-β, or IFN-γ was sufficient to induce differentiation and apoptosis of uninfected thymic epithelial cells. These observations suggested that interferon secretion by either infected cells or activated leukocytes, such as plasmacytoid dendritic cells or lymphocytes, may induce thymic epithelium disruption in a pathological context. Thus, we have identified a new mechanism that may contribute to thymic atrophy and altered T-cell lymphopoiesis associated with many infections. PMID:12050353

  11. Inhibition of corneal epithelial cell migration by cadmium and mercury

    SciTech Connect

    Ubels, J.L.; Osgood, T.B. Medical Coll. of Wisconsin, Milwaukee )

    1991-02-01

    In a previous comparative study of corneal healing in fish, the authors observed that corneal epithelial healing occurs very rapidly in vivo in the marine teleost Myoxocephalus octodecimspinosus (longhorn sculpin) with a 6-mm diameter wound on the mammalian cornea. This rapid healing which permits prompt restoration of the epithelial barrier is apparently an adaptation to the large ionic and osmotic gradients between the environment and the intraocular fluids of the fish. These observations suggested that epithelial healing in the sculpin cornea might be useful model in aquatic biomedical toxicology if an in vitro method for measurement of healing rates could be developed. In this report the authors demonstrate that sculpin eyes maintained in short-term organ culture have a rapid corneal epithelial healing response and that this model can be used to demonstrate the toxic effects of heavy metals on epithelial cell migration.

  12. Role of autophagy in the regulation of epithelial cell junctions.

    PubMed

    Nighot, Prashant; Ma, Thomas

    2016-01-01

    Autophagy is a cell survival mechanism by which bulk cytoplasmic material, including soluble macromolecules and organelles, is targeted for lysosomal degradation. The role of autophagy in diverse cellular processes such as metabolic stress, neurodegeneration, cancer, aging, immunity, and inflammatory diseases is being increasingly recognized. Epithelial cell junctions play an integral role in the cell homeostasis via physical binding, regulating paracellular pathways, integrating extracellular cues into intracellular signaling, and cell-cell communication. Recent data indicates that cell junction composition is very dynamic. The junctional protein complexes are actively regulated in response to various intra- and extra-cellular clues by intracellular trafficking and degradation pathways. This review discusses the recent and emerging information on how autophagy regulates various epithelial cell junctions. The knowledge of autophagy regulation of epithelial junctions will provide further rationale for targeting autophagy in a wide variety of human disease conditions. PMID:27583189

  13. Characteristics and EGFP expression of goat mammary gland epithelial cells.

    PubMed

    Zheng, Y-M; He, X-Y; Zhang, Y

    2010-12-01

    The aims of this study were (i) to establish a goat mammary gland epithelial (GMGE) cell line, and (ii) to determine if these GMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of GMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating goat. The passage 16 GMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in GMGE cells was test by immunofluorescence. Βeta-Casein gene mRNA was test for GMGE cells by RT-PCR. The results showed that when grown at low density on a plastic substratum, the GMGE cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobble-stone morphology of epithelial cells. GMGE cells could form dome-like structure which looked like nipple, and the lumen-like structures formed among the cells. Several blister-like structures appeared in the appearance of the cells. The GMGE cells contained different cell types, majority of the cells were short shuttle-like or polygon which were beehive-like. A part of cells were round and flat, a small number of cells were elongated. Some of the GMGE cells contained milk drops. The cell nuclei were round which had 2-4 obvious cores. The expression of Cell keratins demonstrated the property of epithelial cells in GMGE cells by immunofluorescence. The GMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the GMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected GMGE (ET-GMGE) cell line and maintained it long-term in culture by continuous subculturing. PMID:20113446

  14. The Epithelial Cell in Lung Health and Emphysema Pathogenesis

    PubMed Central

    Mercer, Becky A.; Lemaître, Vincent; Powell, Charles A.; D’Armiento, Jeanine

    2009-01-01

    Cigarette smoking is the primary cause of the irreversible lung disease emphysema. Historically, inflammatory cells such as macrophages and neutrophils have been studied for their role in emphysema pathology. However, recent studies indicate that the lung epithelium is an active participant in emphysema pathogenesis and plays a critical role in the lung’s response to cigarette smoke. Tobacco smoke increases protease production and alters cytokine expression in isolated epithelial cells, suggesting that these cells respond potently even in the absence of a complete inflammatory program. Tobacco smoke also acts as an immunosuppressant, reducing the defense function of airway epithelial cells and enhancing colonization of the lower airways. Thus, the paradigm that emphysema is strictly an inflammatory-cell based disease is shifting to consider the involvement of resident epithelial cells. Here we review the role of epithelial cells in lung development and emphysema. To better understand tobacco-epithelial interactions we performed microarray analyses of RNA from human airway epithelial cells exposed to smoke extract for 24 hours. These studies identified differential regulation of 425 genes involved in diverse biological processes, such as apoptosis, immune function, cell cycle, signal transduction, proliferation, and antioxidants. Some of these genes, including VEGF, glutathione peroxidase, IL-13 receptor, and cytochrome P450, have been previously reported to be altered in the lungs of smokers. Others, such as pirin, cathepsin L, STAT1, and BMP2, are shown here for the first time to have a potential role in smoke-associated injury. These data broaden our understanding of the importance of epithelial cells in lung health and cigarette smoke-induced emphysema. PMID:19662102

  15. Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties

    PubMed Central

    Gudjonsson, Thorarinn; Villadsen, René; Nielsen, Helga Lind; Rønnov-Jessen, Lone; Bissell, Mina J.; Petersen, Ole William

    2002-01-01

    The epithelial compartment of the human breast comprises two distinct lineages: the luminal epithelial and the myoepithelial lineage. We have shown previously that a subset of the luminal epithelial cells could convert to myoepithelial cells in culture signifying the possible existence of a progenitor cell. We therefore set out to identify and isolate the putative precursor in the luminal epithelial compartment. Using cell surface markers and immunomagnetic sorting, we isolated two luminal epithelial cell populations from primary cultures of reduction mammoplasties. The major population coexpresses sialomucin (MUC+) and epithelial-specific antigen (ESA+) whereas the minor population has a suprabasal position and expresses epithelial specific antigen but no sialomucin (MUC−/ESA+). Two cell lines were further established by transduction of the E6/E7 genes from human papilloma virus type 16. Both cell lines maintained a luminal epithelial phenotype as evidenced by expression of the tight junction proteins, claudin-1 and occludin, and by generation of a high transepithelial electrical resistance on semipermeable filters. Whereas in clonal cultures, the MUC+/ESA+ epithelial cell line was luminal epithelial restricted in its differentiation repertoire, the suprabasal-derived MUC−/ESA+ epithelial cell line was able to generate itself as well as MUC+/ESA+ epithelial cells and Thy-1+/α-smooth muscle actin+ (ASMA+) myoepithelial cells. The MUC−/ESA+ epithelial cell line further differed from the MUC+/ESA+ epithelial cell line by the expression of keratin K19, a feature of a subpopulation of epithelial cells in terminal duct lobular units in vivo. Within a reconstituted basement membrane, the MUC+/ESA+ epithelial cell line formed acinus-like spheres. In contrast, the MUC−/ESA+ epithelial cell line formed elaborate branching structures resembling uncultured terminal duct lobular units both by morphology and marker expression. Similar structures were obtained by

  16. Phototoxic aptamers selectively enter and kill epithelial cancer cells

    PubMed Central

    Ferreira, Cátia S. M.; Cheung, Melissa C.; Missailidis, Sotiris; Bisland, Stuart; Gariépy, Jean

    2009-01-01

    The majority of cancers arise from malignant epithelial cells. We report the design of synthetic oligonucleotides (aptamers) that are only internalized by epithelial cancer cells and can be precisely activated by light to kill such cells. Specifically, phototoxic DNA aptamers were selected to bind to unique short O-glycan-peptide signatures on the surface of breast, colon, lung, ovarian and pancreatic cancer cells. These surface antigens are not present on normal epithelial cells but are internalized and routed through endosomal and Golgi compartments by cancer cells, thus providing a focused mechanism for their intracellular delivery. When modified at their 5′ end with the photodynamic therapy agent chlorin e6 and delivered to epithelial cancer cells, these aptamers exhibited a remarkable enhancement (>500-fold increase) in toxicity upon light activation, compared to the drug alone and were not cytotoxic towards cell types lacking such O-glycan-peptide markers. Our findings suggest that these synthetic oligonucleotide aptamers can serve as delivery vehicles in precisely routing cytotoxic cargoes to and into epithelial cancer cells. PMID:19103663

  17. Probiotics promote endocytic allergen degradation in gut epithelial cells

    SciTech Connect

    Song, Chun-Hua; Liu, Zhi-Qiang; Huang, Shelly; Zheng, Peng-Yuan; Yang, Ping-Chang

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Knockdown of A20 compromised the epithelial barrier function. Black-Right-Pointing-Pointer The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Black-Right-Pointing-Pointer Antigens transported across A20-deficient HT-29 monolayers conserved antigenicity. Black-Right-Pointing-Pointer Probiotic proteins increased the expression of A20 in HT-29 cells. -- Abstract: Background and aims: Epithelial barrier dysfunction plays a critical role in the pathogenesis of allergic diseases; the mechanism is to be further understood. The ubiquitin E3 ligase A20 (A20) plays a role in the endocytic protein degradation in the cells. This study aims to elucidate the role of A20 in the maintenance of gut epithelial barrier function. Methods: Gut epithelial cell line, HT-29 cell, was cultured into monolayers to evaluate the barrier function in transwells. RNA interference was employed to knock down the A20 gene in HT-29 cells to test the role of A20 in the maintenance of epithelial barrier function. Probiotic derived proteins were extracted from the culture supernatants using to enhance the expression of A20 in HT-29 cells. Results: The results showed that the knockdown of A20 compromised the epithelial barrier function in HT-29 monolayers, mainly increased the intracellular permeability. The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Allergens collected from the transwell basal chambers of A20-deficient HT-29 monolayers still conserved functional antigenicity. Treating with probiotic derived proteins increased the expression of A20 in HT-29 cells and promote the barrier function. Conclusion: A20 plays an important role in the maintenance of epithelial barrier function as shown by HT-29 monolayer. Probiotic derived protein increases the expression of A20 and promote the HT-29 monolayer barrier function.

  18. Interplay of cell dynamics and epithelial tension during morphogenesis of the Drosophila pupal wing

    PubMed Central

    Etournay, Raphaël; Popović, Marko; Merkel, Matthias; Nandi, Amitabha; Blasse, Corinna; Aigouy, Benoît; Brandl, Holger; Myers, Gene; Salbreux, Guillaume; Jülicher, Frank; Eaton, Suzanne

    2015-01-01

    How tissue shape emerges from the collective mechanical properties and behavior of individual cells is not understood. We combine experiment and theory to study this problem in the developing wing epithelium of Drosophila. At pupal stages, the wing-hinge contraction contributes to anisotropic tissue flows that reshape the wing blade. Here, we quantitatively account for this wing-blade shape change on the basis of cell divisions, cell rearrangements and cell shape changes. We show that cells both generate and respond to epithelial stresses during this process, and that the nature of this interplay specifies the pattern of junctional network remodeling that changes wing shape. We show that patterned constraints exerted on the tissue by the extracellular matrix are key to force the tissue into the right shape. We present a continuum mechanical model that quantitatively describes the relationship between epithelial stresses and cell dynamics, and how their interplay reshapes the wing. DOI: http://dx.doi.org/10.7554/eLife.07090.001 PMID:26102528

  19. Transdifferentiation of endothelial cells to smooth muscle cells play an important role in vascular remodelling.

    PubMed

    Coll-Bonfill, Núria; Musri, Melina Mara; Ivo, Victor; Barberà, Joan Albert; Tura-Ceide, Olga

    2015-01-01

    Pulmonary artery remodelling it is a major feature of pulmonary hypertension (PH). It is characterised by cellular and structural changes of the pulmonary arteries causing higher pulmonar vascular resistance and right ventricular failure. Abnormal deposition of smooth muscle-like (SM-like) cells in normally non-muscular, small diameter vessels and a deregulated control of endothelial cells are considered pathological features of PH. The origin of the SM-like cells and the mechanisms underlying the development and progression of this remodelling process are not understood. Endothelial cells within the intima may migrate from their organised layer of cells and transition to mesenchymal or SM-like phenotype in a process called endothelial-mesenchymal transition (EnMT). Traditionally, Waddington's epigenetic landscape illustrates that fates of somatic cells are progressively determined to compulsorily follow a downhill differentiation pathway. EnMT induces the transformation of cells with stem cell traits, therefore contrasting Waddington's theory and confirming that cell fate seems to be far more flexible than previously thought. The prospect of therapeutic inhibition of EnMT to delay or prevent PH may represent a promising new treatment modality. PMID:25973327

  20. EMMPRIN Is Secreted by Human Uterine Epithelial Cells in Microvesicles and Stimulates Metalloproteinase Production by Human Uterine Fibroblast Cells

    PubMed Central

    Dayger, C. A.; Mehrotra, P.; Belton, R. J.; Nowak, R. A.

    2012-01-01

    Endometrial remodeling is a physiological process involved in the gynecological disease, endometriosis. Tissue remodeling is directed by uterine fibroblast production of matrix metalloproteinases (MMPs). Several MMPs are regulated directly by the protein extracellular matrix metalloproteinase inducer (EMMPRIN) and also by proinflammatory cytokines such as interleukin (IL)1-α/β. We hypothesized that human uterine epithelial cells (HESs) secrete intact EMMPRIN to stimulate MMPs. Microvesicles from HES cell-conditioned medium (CM) expressed intact EMMPRIN protein. Treatment of HES cells with estradiol or phorbyl 12-myristate-13-acetate increased the release of EMMPRIN-containing microvesicles. The HES CM stimulated MMP-1, -2, and -3 messenger RNA levels in human uterine fibroblasts (HUFs) and EMMPRIN immunodepletion from HES-cell concentrated CM reduced MMP stimulation (P < .05). Treatment of HUF cells with low concentrations of IL-1β/α stimulated MMP production (P < .05). These results indicate that HES cells regulate MMP production by HUF cells by secretion of EMMPRIN, in response to ovarian hormones, proinflammatory cytokines as well as activation of protein kinase C. PMID:22729071

  1. Lateral adhesion drives reintegration of misplaced cells into epithelial monolayers.

    PubMed

    Bergstralh, Dan T; Lovegrove, Holly E; St Johnston, Daniel

    2015-11-01

    Cells in simple epithelia orient their mitotic spindles in the plane of the epithelium so that both daughter cells are born within the epithelial sheet. This is assumed to be important to maintain epithelial integrity and prevent hyperplasia, because misaligned divisions give rise to cells outside the epithelium. Here we test this assumption in three types of Drosophila epithelium; the cuboidal follicle epithelium, the columnar early embryonic ectoderm, and the pseudostratified neuroepithelium. Ectopic expression of Inscuteable in these tissues reorients mitotic spindles, resulting in one daughter cell being born outside the epithelial layer. Live imaging reveals that these misplaced cells reintegrate into the tissue. Reducing the levels of the lateral homophilic adhesion molecules Neuroglian or Fasciclin 2 disrupts reintegration, giving rise to extra-epithelial cells, whereas disruption of adherens junctions has no effect. Thus, the reinsertion of misplaced cells seems to be driven by lateral adhesion, which pulls cells born outside the epithelial layer back into it. Our findings reveal a robust mechanism that protects epithelia against the consequences of misoriented divisions. PMID:26414404

  2. Lingual Epithelial Stem Cells and Organoid Culture of Them

    PubMed Central

    Hisha, Hiroko; Tanaka, Toshihiro; Ueno, Hiroo

    2016-01-01

    As tongue cancer is one of the major malignant cancers in the world, understanding the mechanism of maintenance of lingual epithelial tissue, which is known to be the origin of tongue cancer, is unquestionably important. However, the actual stem cells that are responsible for the long-term maintenance of the lingual epithelium have not been identified. Moreover, a simple and convenient culture method for lingual epithelial stem cells has not yet been established. Recently, we have shown that Bmi1-positive cells, residing at the second or third layer of the epithelial cell layer at the base of the interpapillary pit (IPP), were slow-cycling and could supply keratinized epithelial cells for over one year, indicating that Bmi1-positive cells are long-term lingual epithelial stem cells. In addition, we have developed a novel lingual epithelium organoid culture system using a three-dimensional matrix and growth factors. Here, we discuss current progress in the identification of lingual stem cells and future applications of the lingual culture system for studying the regulatory mechanisms of the lingual epithelium and for regenerative medicine. PMID:26828484

  3. Effects of ethanol on an intestinal epithelial cell line

    SciTech Connect

    Nano, J.L.; Cefai, D.; Rampal, P. )

    1990-02-01

    The effect of exposure of an intestinal epithelial cell line to various concentrations of ethanol (217 mM (1%) to 652 mM (3%)) during 24, 48, and 72 hr was investigated in vitro using a rat intestinal epithelial cell line (IRD 98). Incubation of these cells in the presence of ethanol significantly decreased cell growth. This inhibition was accompanied by a strong increase in cellular protein. Stimulation of specific disaccharidases, gamma-glutamyl transferase, and aminopeptidase activities by ethanol was dose- and time-dependent. Ethanol induces a change in the relative proportions of the different lipid classes synthesized; triglycerides, fatty acids, and cholesterol esters were preferentially synthethysed. Our findings show that cell lines are good models for investigation of the effects of ethanol, and that alcohol considerably modifies the functions of intestinal epithelial cells.

  4. Development of human epithelial cell systems for radiation risk assessment

    NASA Technical Reports Server (NTRS)

    Yang, C. H.; Craise, L. M.

    1994-01-01

    The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-Linear Energy Transfer (LET) radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic tranformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.

  5. Medullary thymic epithelial stem cells: role in thymic epithelial cell maintenance and thymic involution.

    PubMed

    Hamazaki, Yoko; Sekai, Miho; Minato, Nagahiro

    2016-05-01

    The thymus consists of two distinct anatomical regions, the cortex and the medulla; medullary thymic epithelial cells (mTECs) play a crucial role in establishing central T-cell tolerance for self-antigens. Although the understanding of mTEC development in thymic organogenesis as well as the regulation of their differentiation and maturation has improved, the mechanisms of postnatal maintenance remain poorly understood. This issue has a central importance in immune homeostasis and physiological thymic involution as well as autoimmune disorders in various clinicopathological settings. Recently, several reports have demonstrated the existence of TEC stem or progenitor cells in the postnatal thymus, which are either bipotent or unipotent. We identified stem cells specified for mTEC-lineage that are generated in the thymic ontogeny and may sustain mTEC regeneration and lifelong central T-cell self-tolerance. This finding suggested that the thymic medulla is maintained autonomously by its own stem cells. Although several issues, including the relationship with other putative TEC stem/progenitors, remain unclear, further examination of mTEC stem cells (mTECSCs) and their regulatory mechanisms may contribute to the understanding of postnatal immune homeostasis. Possible relationships between decline of mTECSC activity and early thymic involution as well as various autoimmune disorders are discussed. PMID:27088906

  6. Induction of apoptosis in oral epithelial cells by Candida albicans.

    PubMed

    Villar, C Cunha; Chukwuedum Aniemeke, J; Zhao, X-R; Huynh-Ba, G

    2012-12-01

    During infection, interactions between Candida albicans and oral epithelial cells result in oral epithelial cell death. This is clinically manifested by the development of oral mucosal ulcerations generally associated with discomfort. In vitro studies have shown that C. albicans induces early apoptotic alterations in oral epithelial cells; however, these studies have also shown that treatment of infected cells with caspase inhibitors does not prevent their death. The reasons for these contradictory results are unknown and it is still not clear if C. albicans stimulates oral epithelial signaling pathways that promote apoptotic cell death. Activation of specific death pathways in response to microbial organisms plays an essential role in modulating the pathogenesis of a variety of infectious diseases. The aim of this study was to (i) characterize C. albicans-induced apoptotic morphological alterations in oral epithelial cells, and (ii) investigate the activation of apoptotic signaling pathways and expression of apoptotic genes during infection. Candida albicans induced early apoptotic changes in over 50% of oral epithelial cells. However, only 15% of those showed mid-late apoptotic alterations. At the molecular level, C. albicans caused a loss of the mitochondrial transmembrane potential and translocation of mitochondrial cytochrome c. Caspase-3/9 activities increased only during the first hours of infection. Moreover, poly[ADP ribose] polymerase 1 was cleaved into apoptotic and necrotic-like fragments. Finally, five anti-apoptotic genes were significantly upregulated and two pro-apoptotic genes were downregulated during infection. Altogether, these findings indicate that epithelial apoptotic pathways are activated in response to C. albicans, but fail to progress and promote apoptotic cell death. PMID:23134609

  7. Quantification of Three-Dimensional Cell-Mediated Collagen Remodeling Using Graph Theory

    PubMed Central

    Bilgin, Cemal Cagatay; Lund, Amanda W.; Can, Ali; Plopper, George E.; Yener, Bülent

    2010-01-01

    Background Cell cooperation is a critical event during tissue development. We present the first precise metrics to quantify the interaction between mesenchymal stem cells (MSCs) and extra cellular matrix (ECM). In particular, we describe cooperative collagen alignment process with respect to the spatio-temporal organization and function of mesenchymal stem cells in three dimensions. Methodology/Principal Findings We defined two precise metrics: Collagen Alignment Index and Cell Dissatisfaction Level, for quantitatively tracking type I collagen and fibrillogenesis remodeling by mesenchymal stem cells over time. Computation of these metrics was based on graph theory and vector calculus. The cells and their three dimensional type I collagen microenvironment were modeled by three dimensional cell-graphs and collagen fiber organization was calculated from gradient vectors. With the enhancement of mesenchymal stem cell differentiation, acceleration through different phases was quantitatively demonstrated. The phases were clustered in a statistically significant manner based on collagen organization, with late phases of remodeling by untreated cells clustering strongly with early phases of remodeling by differentiating cells. The experiments were repeated three times to conclude that the metrics could successfully identify critical phases of collagen remodeling that were dependent upon cooperativity within the cell population. Conclusions/Significance Definition of early metrics that are able to predict long-term functionality by linking engineered tissue structure to function is an important step toward optimizing biomaterials for the purposes of regenerative medicine. PMID:20927339

  8. Fabrication of transplantable corneal epithelial and oral mucosal epithelial cell sheets using a novel temperature-responsive closed culture device.

    PubMed

    Nakajima, Ryota; Kobayashi, Toyoshige; Kikuchi, Tetsutaro; Kitano, Yuriko; Watanabe, Hiroya; Mizutani, Manabu; Nozaki, Takayuki; Senda, Naoko; Saitoh, Kazuo; Takagi, Ryo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

    2015-05-01

    Temperature-responsive culture surfaces make it possible to harvest transplantable carrier-free cell sheets. Here, we applied temperature-responsive polymer for polycarbonate surfaces with previously developed closed culture devices for an automated culture system in order to fabricate transplantable stratified epithelial cell sheets. Histological and immunohistochemical analyses and colony-forming assays revealed that corneal epithelial and oral mucosal epithelial cell sheets could be harvested with the temperature-responsive closed culture devices. The results were similar to those obtained using temperature-responsive culture inserts. These results indicate that the novel temperature-responsive closed culture device is useful for fabricating transplantable stratified epithelial cell sheets. PMID:23475606

  9. CHARACTERIZATION OF ALVEOLAR EPITHELIAL CELLS CULTURED IN SEMIPERMEABLE HOLLOW FIBERS

    PubMed Central

    Grek, Christina L.; Newton, Danforth A.; Qiu, Yonhzhi; Wen, Xuejun; Spyropoulos, Demetri D.; Baatz, John E.

    2012-01-01

    Cell culture methods commonly used to represent alveolar epithelial cells in vivo have lacked airflow, a 3-dimensional air-liquid interface, and dynamic stretching characteristics of native lung tissue—physiological parameters critical for normal phenotypic gene expression and cellular function. Here the authors report the development of a selectively semipermeable hollow fiber culture system that more accurately mimics the in vivo microenvironment experienced by mammalian distal airway cells than in conventional or standard air-liquid interface culture. Murine lung epithelial cells (MLE-15) were cultured within semipermeable polyurethane hollow fibers and introduced to controlled airflow through the microfiber interior. Under these conditions, MLE-15 cells formed confluent monolayers, demonstrated a cuboidal morphology, formed tight junctions, and produced and secreted surfactant proteins. Numerous lamellar bodies and microvilli were present in MLE-15 cells grown in hollow fiber culture. Conversely, these alveolar type II cell characteristics were reduced in MLE-15 cells cultured in conventional 2D static culture systems. These data support the hypothesis that MLE-15 cells grown within our microfiber culture system in the presence of airflow maintain the phenotypic characteristics of type II cells to a higher degree than those grown in standard in vitro cell culture models. Application of our novel model system may prove advantageous for future studies of specific gene and protein expression involving alveolar epithelial or bronchiolar epithelial cells. PMID:19263283

  10. Apoptotic epithelial cells control the abundance of Treg cells at barrier surfaces.

    PubMed

    Nakahashi-Oda, Chigusa; Udayanga, Kankanam Gamage Sanath; Nakamura, Yoshiyuki; Nakazawa, Yuta; Totsuka, Naoya; Miki, Haruka; Iino, Shuichi; Tahara-Hanaoka, Satoko; Honda, Shin-ichiro; Shibuya, Kazuko; Shibuya, Akira

    2016-04-01

    Epithelial tissues continually undergo apoptosis. Commensal organisms that inhabit the epithelium influence tissue homeostasis, in which regulatory T cells (Treg cells) have a central role. However, the physiological importance of epithelial cell apoptosis and how the number of Treg cells is regulated are both incompletely understood. Here we found that apoptotic epithelial cells negatively regulated the commensal-stimulated proliferation of Treg cells. Gut commensals stimulated CX3CR1(+)CD103(-)CD11b(+) dendritic cells (DCs) to produce interferon-β (IFN-β), which augmented the proliferation of Treg cells in the intestine. Conversely, phosphatidylserine exposed on apoptotic epithelial cells suppressed IFN-β production by the DCs via inhibitory signaling mediated by the cell-surface glycoprotein CD300a and thus suppressed Treg cell proliferation. Our findings reveal a regulatory role for apoptotic epithelial cells in maintaining the number of Treg cell and tissue homeostasis. PMID:26855029

  11. Mechanobiology in Lung Epithelial Cells: Measurements, Perturbations, and Responses

    PubMed Central

    Waters, Christopher M.; Roan, Esra; Navajas, Daniel

    2015-01-01

    Epithelial cells of the lung are located at the interface between the environment and the organism and serve many important functions including barrier protection, fluid balance, clearance of particulate, initiation of immune responses, mucus and surfactant production, and repair following injury. Because of the complex structure of the lung and its cyclic deformation during the respiratory cycle, epithelial cells are exposed to continuously varying levels of mechanical stresses. While normal lung function is maintained under these conditions, changes in mechanical stresses can have profound effects on the function of epithelial cells and therefore the function of the organ. In this review, we will describe the types of stresses and strains in the lungs, how these are transmitted, and how these may vary in human disease or animal models. Many approaches have been developed to better understand how cells sense and respond to mechanical stresses, and we will discuss these approaches and how they have been used to study lung epithelial cells in culture. Understanding how cells sense and respond to changes in mechanical stresses will contribute to our understanding of the role of lung epithelial cells during normal function and development and how their function may change in diseases such as acute lung injury, asthma, emphysema, and fibrosis. PMID:23728969

  12. RNA-seq analysis of impact of PNN on gene expression and alternative splicing in corneal epithelial cells

    PubMed Central

    Akin, Debra; Newman, Jeremy R.B.; McIntyre, Lauren M.

    2016-01-01

    Purpose The specialized corneal epithelium requires differentiated properties, specific for its role at the anterior surface of the eye. Thus, tight maintenance of the differentiated qualities of the corneal epithelial is essential. Pinin (PNN) is an exon junction component (EJC) that has dramatic implications for corneal epithelial cell differentiation and may act as a stabilizer of the corneal epithelial cell phenotype. Our studies revealed that PNN is involved in transcriptional repression complexes and spliceosomal complexes, placing PNN at the fulcrum between chromatin and mRNA splicing. Transcriptome analysis of PNN-knockdown cells revealed clear and reproducible alterations in transcript profiles and splicing patterns of a subset of genes that would significantly impact the epithelial cell phenotype. We further investigated PNN’s role in the regulation of gene expression and alternative splicing (AS) in a corneal epithelial context. Methods Human corneal epithelial (HCET) cells that carry the doxycycline-inducible PNN-knockdown shRNA vector were used to perform RNA-seq to determine differential gene expression and differential AS events. Results Multiple genes and AS events were identified as differentially expressed between PNN-knockdown and control cells. Genes upregulated by PNN knockdown included a large proportion of genes that are associated with enhanced cell migration and ECM remodeling processes, such as MMPs, ADAMs, HAS2, LAMA3, CXCRs, and UNC5C. Genes downregulated in response to PNN depletion included IGFBP5, FGD3, FGFR2, PAX6, RARG, and SOX10. AS events in PNN-knockdown cells compared to control cells were also more likely to be detected, and upregulated. In particular, 60% of exon-skipping events, detected in only one condition, were detected in PNN-knockdown cells and of the shared exon-skipping events, 92% of those differentially expressed were more frequent in the PNN knockdown. Conclusions These data suggest that lowering of PNN levels in

  13. Herpes simplex virus type 1 entry into epithelial MDCKII cells: role of VASP activities.

    PubMed

    Jaeger, Verena; Hoppe, Sven; Petermann, Philipp; Liebig, Timo; Jansen, Matthias K; Renné, Thomas; Knebel-Mörsdorf, Dagmar

    2010-09-01

    VASP is an actin-regulatory protein that links signalling to remodelling of the cytoskeleton. We investigated the role of VASP during entry of herpes simplex viruses into epithelial MDCKII cells. As VASP functions are regulated by phosphorylations, the phosphorylation pattern was determined upon infection. Phosphorylated VASP decreased temporarily at 15 and 30 min after infection. The impact of phosphorylated VASP was addressed by overexpression of phosphomimetic VASP mutants. Our results revealed that phosphorylated VASP slightly reduced the number of infected cells. Expression studies with deletion mutants further indicated minor effects of VASP on infection efficiency, whereas RNA interference studies demonstrated that reduced VASP expression did not suppress infection. We conclude that VASP activities alone may contribute to herpes simplex virus infection to only a minor extent. PMID:20463151

  14. Lymphotoxin beta receptor signaling limits mucosal damage through driving IL-23 production by epithelial cells.

    PubMed

    Macho-Fernandez, E; Koroleva, E P; Spencer, C M; Tighe, M; Torrado, E; Cooper, A M; Fu, Y-X; Tumanov, A V

    2015-03-01

    The immune mechanisms regulating epithelial cell repair after injury remain poorly defined. We demonstrate here that lymphotoxin beta receptor (LTβR) signaling in intestinal epithelial cells promotes self-repair after mucosal damage. Using a conditional gene-targeted approach, we demonstrate that LTβR signaling in intestinal epithelial cells is essential for epithelial interleukin-23 (IL-23) production and protection against epithelial injury. We further show that epithelial-derived IL-23 promotes mucosal wound healing by inducing the IL-22-mediated proliferation and survival of epithelial cells and mucus production. Additionally, we identified CD4(-)CCR6(+)T-bet(-) RAR-related orphan receptor gamma t (RORγt)(+) lymphoid tissue inducer cells as the main producers of protective IL-22 after epithelial damage. Thus, our results reveal a novel role for LTβR signaling in epithelial cells in the regulation of intestinal epithelial cell homeostasis to limit mucosal damage. PMID:25183367

  15. D prostanoid receptor 2 (chemoattractant receptor–homologous molecule expressed on TH2 cells) protein expression in asthmatic patients and its effects on bronchial epithelial cells

    PubMed Central

    Stinson, Sally E.; Amrani, Yassine; Brightling, Christopher E.

    2015-01-01

    Background The D prostanoid receptor 2 (DP2; also known as chemoattractant receptor–homologous molecule expressed on TH2 cells) is implicated in the pathogenesis of asthma, but its expression within bronchial biopsy specimens is unknown. Objectives We sought to investigate the bronchial submucosal DP2 expression in asthmatic patients and healthy control subjects and to explore its functional role in epithelial cells. Methods DP2 protein expression was assessed in bronchial biopsy specimens from asthmatic patients (n = 22) and healthy control subjects (n = 10) by using immunohistochemistry and in primary epithelial cells by using flow cytometry, immunofluorescence, and quantitative RT-PCR. The effects of the selective DP2 agonist 13, 14-dihydro-15-keto prostaglandin D2 on epithelial cell migration and differentiation were determined. Results Numbers of submucosal DP2+ cells were increased in asthmatic patients compared with those in healthy control subjects (mean [SEM]: 78 [5] vs 22 [3]/mm2 submucosa, P < .001). The bronchial epithelium expressed DP2, but its expression was decreased in asthmatic patients compared with that seen in healthy control subjects (mean [SEM]: 21 [3] vs 72 [11]/10 mm2 epithelial area, P = .001), with similar differences observed in vitro by primary epithelial cells. Squamous metaplasia of the bronchial epithelium was increased in asthmatic patients and related to decreased DP2 expression (rs = 0.69, P < .001). 13, 14-Dihydro-15-keto prostaglandin D2 promoted epithelial cell migration and at air-liquid interface cultures increased the number of MUC5AC+ and involucrin-positive cells, which were blocked with the DP2-selective antagonist AZD6430. Conclusions DP2 is expressed by the bronchial epithelium, and its activation drives epithelial differentiation, suggesting that in addition to its well-characterized role in inflammatory cell migration, DP2 might contribute to airway remodeling in asthmatic patients. PMID:25312757

  16. Change in cell shape is required for matrix metalloproteinase-induced epithelial-mesenchymal transition of mammary epithelial cells

    SciTech Connect

    Nelson, Celeste M.; Khauv, Davitte; Bissell, Mina J.; Radisky, Derek C.

    2008-06-26

    Cell morphology dictates response to a wide variety of stimuli, controlling cell metabolism, differentiation, proliferation, and death. Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells acquire migratory characteristics, and in the process convert from a 'cuboidal' epithelial structure into an elongated mesenchymal shape. We had shown previously that matrix metalloproteinase-3 (MMP3) can stimulate EMT of cultured mouse mammary epithelial cells through a process that involves increased expression of Rac1b, a protein that stimulates alterations in cytoskeletal structure. We show here that cells treated with MMP-3 or induced to express Rac1b spread to cover a larger surface, and that this induction of cell spreading is a requirement of MMP-3/Rac1b-induced EMT. We find that limiting cell spreading, either by increasing cell density or by culturing cells on precisely defined micropatterned substrata, blocks expression of characteristic markers of EMT in cells treated with MMP-3. These effects are not caused by general disruptions in cell signaling pathways, as TGF-{beta}-induced EMT is not affected by similar limitations on cell spreading. Our data reveal a previously unanticipated cell shape-dependent mechanism that controls this key phenotypic alteration and provide insight into the distinct mechanisms activated by different EMT-inducing agents.

  17. Porphyromonas gingivalis invades oral epithelial cells in vitro.

    PubMed

    Sandros, J; Papapanou, P; Dahlén, G

    1993-05-01

    The aim of the present study was to analyze the adhesive and invasive potential of a number of P. gingivalis strains, in an in vitro system utilizing cultures of human oral epithelial cells (KB cell line, ATCC CCL 17). P. gingivalis strains W50 and FDC 381 (laboratory strains) and OMGS 1738, 1743 and 1439 (clinical isolates) as well as E. coli strain HB 101 (non-adhering, non-invasive control) were used. Adherence was assessed by means of scintillation counting and light microscopy, after incubation of radiolabelled bacteria with epithelial cells. In the invasion assay, monolayers were infected with the P. gingivalis and E. coli strains and further incubated with an antibiotic mixture (metronidazole 0.1 mg/ml and gentamicin 0.5 mg/ml). Invasion was evaluated by (i) assessing presence of bacteria surviving the antibiotic treatment, and (ii) electron microscopy. All P. gingivalis strains adhered to and entered into the oral epithelial cells. After 3 hours of incubation, bacteria were frequently identified intracellularly by means of electron microscopy. The cellular membranes, encapsulating the microorganisms in early stages of the invasive process, appeared later to disintegrate. The presence of coated pits on the epithelial cell surfaces suggested that internalization of P. gingivalis was associated with receptor-mediated endocytosis (RME). Formation of outer membrane vesicles (blebs) by intracellular bacteria indicated that internalized P. gingivalis was able to retain its viability. E. coli strain HB 101 neither adhered to nor invaded epithelial cells. PMID:8388449

  18. Porphyromonas gingivalis Fimbriae Bind to Cytokeratin of Epithelial Cells

    PubMed Central

    Sojar, Hakimuddin T.; Sharma, Ashu; Genco, Robert J.

    2002-01-01

    The adherence of Porphyromonas gingivalis to host cells is likely a prerequisite step in the pathogenesis of P. gingivalis-induced periodontal disease. P. gingivalis binds to and invades epithelial cells, and fimbriae are shown to be involved in this process. Little is known regarding epithelial receptor(s) involved in binding of P. gingivalis fimbriae. Using an overlay assay with purified P. gingivalis fimbriae as a probe, two major epithelial cell proteins with masses of 50 and 40 kDa were identified by immunoblotting with fimbria-specific antibodies. Iodinated purified fimbriae also bound to the same two epithelial cell proteins. An affinity chromatography technique was utilized to isolate and purify the epithelial components to which P. gingivalis fimbriae bind. Purified fimbriae were coupled to CNBr-activated Sepharose-4B, and the solubilized epithelial cell extract proteins bound to the immobilized fimbriae were isolated from the column. A major 50-kDa component and a minor 40-kDa component were purified and could be digested with trypsin, suggesting that they were proteins. These affinity-eluted 50- and 40-kDa proteins were then subjected to amino-terminal sequencing, and no sequence could be determined, suggesting that these proteins have blocked amino-terminal residues. CNBr digestion of the 50-kDa component resulted in an internal sequence homologous to that of Keratin I molecules. Further evidence that P. gingivalis fimbriae bind to cytokeratin molecule(s) comes from studies showing that multicytokeratin rabbit polyclonal antibodies cross-react with the affinity-purified 50-kDa epithelial cell surface component. Also, binding of purified P. gingivalis fimbriae to epithelial components can be inhibited in an overlay assay by multicytokeratin rabbit polyclonal antibodies. Furthermore, we showed that biotinylated purified fimbriae bind to purified human epidermal keratin in an overlay assay. These studies suggest that the surface-accessible epithelial

  19. Nivalenol and Deoxynivalenol Affect Rat Intestinal Epithelial Cells: A Concentration Related Study

    PubMed Central

    Bianco, Giuseppe; Fontanella, Bianca; Severino, Lorella; Quaroni, Andrea; Autore, Giuseppina; Marzocco, Stefania

    2012-01-01

    The integrity of the gastrointestinal tract represents a crucial first level defence against ingested toxins. Among them, Nivalenol is a trichotecenes mycotoxin frequently found on cereals and processed grains; when it contaminates human food and animal feed it is often associated with another widespread contaminant, Deoxynivalenol. Following their ingestion, intestinal epithelial cells are exposed to concentrations of these trichothecenes high enough to cause mycotoxicosis. In this study we have investigated the effects of Nivalenol and Deoxynivalenol on intestinal cells in an in vitro model system utilizing the non-tumorigenic rat intestinal epithelial cell line IEC-6. Both Nivalenol and Deoxynivalenol (5–80 µM) significantly affected IEC-6 viability through a pro-apoptotic process which mainly involved the following steps: (i) Bax induction; (ii) Bcl-2 inhibition, and (iii) caspase-3 activation. Moreover, treatment with Nivalenol produced a significant cell cycle arrest of IEC-6 cells, primarily at the G0/G1 interphase and in the S phase, with a concomitant reduction in the fraction of cells in G2. Interestingly, when administered at lower concentrations (0.1–2.5 µM), both Nivalenol and Deoxynivalenol affected epithelial cell migration (restitution), representing the initial step in gastrointestinal wound healing in the gut. This reduced motility was associated with significant remodelling of the actin cytoskeleton, and changes in expression of connexin-43 and focal adhesion kinase. The concentration range of Nivalenol or Deoxynivalenol we have tested is comparable with the mean estimated daily intake of consumers eating contaminated food. Thus, our results further highlight the risks associated with intake of even low levels of these toxins. PMID:23251682

  20. Nivalenol and deoxynivalenol affect rat intestinal epithelial cells: a concentration related study.

    PubMed

    Bianco, Giuseppe; Fontanella, Bianca; Severino, Lorella; Quaroni, Andrea; Autore, Giuseppina; Marzocco, Stefania

    2012-01-01

    The integrity of the gastrointestinal tract represents a crucial first level defence against ingested toxins. Among them, Nivalenol is a trichotecenes mycotoxin frequently found on cereals and processed grains; when it contaminates human food and animal feed it is often associated with another widespread contaminant, Deoxynivalenol. Following their ingestion, intestinal epithelial cells are exposed to concentrations of these trichothecenes high enough to cause mycotoxicosis. In this study we have investigated the effects of Nivalenol and Deoxynivalenol on intestinal cells in an in vitro model system utilizing the non-tumorigenic rat intestinal epithelial cell line IEC-6. Both Nivalenol and Deoxynivalenol (5-80 µM) significantly affected IEC-6 viability through a pro-apoptotic process which mainly involved the following steps: (i) Bax induction; (ii) Bcl-2 inhibition, and (iii) caspase-3 activation. Moreover, treatment with Nivalenol produced a significant cell cycle arrest of IEC-6 cells, primarily at the G(0)/G(1) interphase and in the S phase, with a concomitant reduction in the fraction of cells in G(2). Interestingly, when administered at lower concentrations (0.1-2.5 µM), both Nivalenol and Deoxynivalenol affected epithelial cell migration (restitution), representing the initial step in gastrointestinal wound healing in the gut. This reduced motility was associated with significant remodelling of the actin cytoskeleton, and changes in expression of connexin-43 and focal adhesion kinase. The concentration range of Nivalenol or Deoxynivalenol we have tested is comparable with the mean estimated daily intake of consumers eating contaminated food. Thus, our results further highlight the risks associated with intake of even low levels of these toxins. PMID:23251682

  1. Differentiation of porcine mesenchymal stem cells into epithelial cells as a potential therapeutic application to facilitate epithelial regeneration.

    PubMed

    Kokubun, Kelsey; Pankajakshan, Divya; Kim, Min-Jung; Agrawal, Devendra K

    2016-02-01

    Epithelial denudation is one of the characteristics of chronic asthma. To restore its functions, the airway epithelium has to rapidly repair the injuries and regenerate its structure and integrity. Mesenchymal stem cells (MSCs) have the ability to differentiate into many cell lineages. However, the differentiation of MSCs into epithelial cells has not been fully studied. Here, we examined the differentiation of MSCs into epithelial cells using three different media compositions with various growth supplementations. The MSCs were isolated from porcine bone marrow by density gradient centrifugation. The isolated MSCs were CD11(-) CD34(-) CD45(-) CD44(+) CD90(+) and CD105(+) by immunostaining and flow cytometry. MSCs were stimulated with EpiGRO (Millipore), BEpiCM (ScienCell) and AECGM (PromoCell) media for 5 and 10 days, and epithelial differentiation was assessed by qPCR (keratin 14, 18 and EpCAM), fluorometry (cytokeratin 7-8, cytokeratin 14-15-16-19 and EpCAM), western blot analysis (pancytokeratin, EpCAM) and flow cytometry (cytokeratin 7-8, cytokeratin 14-15-16-19 and EpCAM). The functional marker MUC1 was also assessed after 10 days of air-liquid interface (ALI) culture in optimized media. Cells cultured in BEpiCM containing fibroblast growth factor and prostaglandin E2 showed the highest expression of the epithelial markers: CK7-8 (85.90%); CK-14-15-16-19 (10.14%); and EpCAM (64.61%). The cells also expressed functional marker MUC1 after ALI culture. The differentiated MSCs when cultured in BEpiCM medium ex vivo in a bioreactor on a decellularized trachea for 10 days retained the epithelial-like phenotype. In conclusion, porcine bone marrow-derived MSCs demonstrate commitment to the epithelial lineage and might be a potential therapy for facilitating the repair of denuded airway epithelium. PMID:23696537

  2. Slug Is Increased in Vascular Remodeling and Induces a Smooth Muscle Cell Proliferative Phenotype

    PubMed Central

    Coll-Bonfill, Núria; Peinado, Victor I.; Pisano, María V.; Párrizas, Marcelina; Blanco, Isabel; Evers, Maurits; Engelmann, Julia C.; García-Lucio, Jessica; Tura-Ceide, Olga; Meister, Gunter

    2016-01-01

    Objective Previous studies have confirmed Slug as a key player in regulating phenotypic changes in several cell models, however, its role in smooth muscle cells (SMC) has never been assessed. The purpose of this study was to evaluate the expression of Slug during the phenotypic switch of SMC in vitro and throughout the development of vascular remodeling. Methods and Results Slug expression was decreased during both cell-to-cell contact and TGFβ1 induced SMC differentiation. Tumor necrosis factor-α (TNFα), a known inductor of a proliferative/dedifferentiated SMC phenotype, induces the expression of Slug in SMC. Slug knockdown blocked TNFα-induced SMC phenotypic change and significantly reduced both SMC proliferation and migration, while its overexpression blocked the TGFβ1-induced SMC differentiation and induced proliferation and migration. Genome-wide transcriptomic analysis showed that in SMC, Slug knockdown induced changes mainly in genes related to proliferation and migration, indicating that Slug controls these processes in SMC. Notably, Slug expression was significantly up-regulated in lungs of mice using a model of pulmonary hypertension-related vascular remodeling. Highly remodeled human pulmonary arteries also showed an increase of Slug expression compared to less remodeled arteries. Conclusions Slug emerges as a key transcription factor driving SMC towards a proliferative phenotype. The increased Slug expression observed in vivo in highly remodeled arteries of mice and human suggests a role of Slug in the pathogenesis of pulmonary vascular diseases. PMID:27441378

  3. The Effects of Matrix Metalloproteinase-9 on Dairy Goat Mastitis and Cell Survival of Goat Mammary Epithelial Cells

    PubMed Central

    Li, Hui; Zheng, Huiling; Li, Lihui; Shen, Xingai; Zang, Wenjuan; Sun, Yongsen

    2016-01-01

    Matrix metalloproteinase-9 (MMP-9) is a zinc-dependent enzyme, and plays a crucial role in extracellular matrix degeneration, inflammation and tissue remodeling. However, the relationship between MMP-9 and somatic cell count (SCC) in goat milk and the role of MMP-9 in the regulation of mastitis are still unknown. In this study, we found MMP-9 was predominantly expressed in the spleen, intestine and mammary gland. The SCC in goat milk was positively correlated with MMP-9 expression, and staphylococcus aureus could markedly increase MMP-9 expression in goat mammary epithelial cells (GMEC) in dosage and time dependent manner. We also demonstrated that SB-3CT, an inhibitor of MMP-9, promoted apoptosis and inhibited proliferation in GMEC. Thus, MMP-9 may emerge as an easily measurable and sensitive parameter that reflects the number of somatic cells present in milk and a regulatory factor of apoptosis in GMEC. PMID:27518717

  4. The Effects of Matrix Metalloproteinase-9 on Dairy Goat Mastitis and Cell Survival of Goat Mammary Epithelial Cells.

    PubMed

    Li, Hui; Zheng, Huiling; Li, Lihui; Shen, Xingai; Zang, Wenjuan; Sun, Yongsen

    2016-01-01

    Matrix metalloproteinase-9 (MMP-9) is a zinc-dependent enzyme, and plays a crucial role in extracellular matrix degeneration, inflammation and tissue remodeling. However, the relationship between MMP-9 and somatic cell count (SCC) in goat milk and the role of MMP-9 in the regulation of mastitis are still unknown. In this study, we found MMP-9 was predominantly expressed in the spleen, intestine and mammary gland. The SCC in goat milk was positively correlated with MMP-9 expression, and staphylococcus aureus could markedly increase MMP-9 expression in goat mammary epithelial cells (GMEC) in dosage and time dependent manner. We also demonstrated that SB-3CT, an inhibitor of MMP-9, promoted apoptosis and inhibited proliferation in GMEC. Thus, MMP-9 may emerge as an easily measurable and sensitive parameter that reflects the number of somatic cells present in milk and a regulatory factor of apoptosis in GMEC. PMID:27518717

  5. Amniotic epithelial cells promote wound healing in mice through high epithelialization and engraftment.

    PubMed

    Jin, Enze; Kim, Tae-Hee; Han, Seongho; Kim, Sung-Whan

    2016-07-01

    Although human amniotic epithelial cells (AMEs) are an attractive source of stem cells, their therapeutic potential in wound healing has not been fully investigated. We evaluated the therapeutic potential of AMEs for wound healing. Real-time PCR showed that the epithelialization growth factors epidermal growth factor (EGF), platelet-derived growth factor (PDGF)-B and chemotactic factors interleukin-8 (IL-8 or CXCL8) and neutrophil-activating protein-2 (NAP-2 or CXCL7) were upregulated in AMEs compared with adipose-derived mesenchymal stem cells (ADMs). In vitro scratch wound assays revealed that AME-derived conditioned medium substantially accelerated wound closure. Wounds in NOD/SCID mice were created by skin excision, followed by AME transplantation. AMEs implantation significantly accelerated wound healing and increased cellularity and re-epithelialization. Transplanted AMEs exhibited high engraftment rates and expressed keratinocyte-specific proteins and cytokeratin in the wound area, suggesting direct benefits for cutaneous closure. Taken together, these data indicate that AMEs possess therapeutic capability for wound healing through the secretion of epithelialization growth factors and enhanced engraftment properties. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26174407

  6. The TNF Family Molecules LIGHT and Lymphotoxin αβ Induce a Distinct Steroid-Resistant Inflammatory Phenotype in Human Lung Epithelial Cells.

    PubMed

    da Silva Antunes, Ricardo; Madge, Lisa; Soroosh, Pejman; Tocker, Joel; Croft, Michael

    2015-09-01

    Lung epithelial cells are considered important sources of inflammatory molecules and extracellular matrix proteins that contribute to diseases such as asthma. Understanding the factors that stimulate epithelial cells may lead to new insights into controlling lung inflammation. This study sought to investigate the responsiveness of human lung epithelial cells to the TNF family molecules LIGHT and lymphotoxin αβ (LTαβ). Bronchial and alveolar epithelial cell lines, and primary human bronchial epithelial cells, were stimulated with LIGHT and LTαβ, and expression of inflammatory cytokines and chemokines and markers of epithelial-mesenchymal transition and fibrosis/remodeling was measured. LTβ receptor, the receptor shared by LIGHT and LTαβ, was constitutively expressed on all epithelial cells. Correspondingly, LIGHT and LTαβ strongly induced a limited but highly distinct set of inflammatory genes in all epithelial cells tested, namely the adhesion molecules ICAM-1 and VCAM-1; the chemokines CCL5, CCL20, CXCL1, CXCL3, CXCL5, and CXCL11; the cytokines IL-6, activin A and GM-CSF; and metalloproteinases matrix metalloproteinase-9 and a disintegrin and metalloproteinase domain-8. Importantly, induction of the majority of these inflammatory molecules was insensitive to the suppressive effects of the corticosteroid budesonide. LIGHT and LTαβ also moderately downregulated E-cadherin, a protein associated with maintaining epithelial integrity, but did not significantly drive production of extracellular matrix proteins or α-smooth muscle actin. Thus, LIGHT and LTαβ induce a distinct steroid-resistant inflammatory signature in airway epithelial cells via constitutively expressed LTβ receptor. These findings support our prior murine studies that suggested the receptors for LIGHT and LTαβ contribute to development of lung inflammation characteristic of asthma and idiopathic pulmonary fibrosis. PMID:26209626

  7. Salivary epithelial cells: an unassuming target site for gene therapeutics

    PubMed Central

    Perez, Paola; Rowzee, Anne M.; Zheng, Changyu; Adriaansen, Janik; Baum, Bruce J.

    2010-01-01

    Salivary glands are classical exocrine glands whose external secretions result in the production of saliva. However, in addition to the secretion of exocrine proteins, salivary epithelial cells are also capable of secreting proteins internally, into the bloodstream. This brief review examines the potential for using salivary epithelial cells as a target site for in situ gene transfer, with an ultimate goal of producing therapeutic proteins for treating both systemic and upper gastrointestinal tract disorders. The review discusses the protein secretory pathways reported to be present in salivary epithelial cells, the viral gene transfer vectors shown useful for transducing these cells, model transgenic secretory proteins examined, and some clinical conditions that might benefit from such salivary gland gene transfer. PMID:20219693

  8. Spleen tyrosine kinase regulates mammary epithelial cell proliferation in mammary glands of dairy cows.

    PubMed

    Hou, Xiaoming; Lin, Lin; Xing, Weinan; Yang, Yang; Duan, Xiaoyu; Li, Qingzhang; Gao, Xuejun; Lin, Ye

    2016-05-01

    Spleen tyrosine kinase (SYK) is a nonreceptor tyrosine kinase that has been considered a hematopoietic cell-specific signal transducer involved in cell proliferation and differentiation. However, the role of SYK in normal mammary gland is still poorly understood. Here we show that SYK is expressed in mammary glands of dairy cows. Expression of SYK was higher in dry period mammary tissues than in lactating mammary tissues. Knockdown and overexpression of SYK affected dairy cow mammary epithelial cell proliferation as well as the expression of signal molecules involved in proliferation, including protein kinase B (PKB, also known as AKT1), p42/44 mitogen-activated protein kinase (MAPK), and signal transducer and activator of transcription 5 (STAT5). Dual-luciferase reporter assay showed that SYK increased the transcriptional activity of the AKT1 promoter, and cis-elements within the AKT1 promoter region from -439 to -84 bp mediated this regulation. These results suggest that SYK affects mammary epithelial cell proliferation by activating AKT1 at the transcriptional level in mammary glands of dairy cows, which is important for the mammary remodeling process in dry cows as well as for increasing persistency of lactation in lactating cows. PMID:26947307

  9. Establishment and Characterization of Immortalized Human Amniotic Epithelial Cells

    PubMed Central

    Zhou, Kaixuan; Koike, Chika; Yoshida, Toshiko; Okabe, Motonori; Fathy, Moustafa; Kyo, Satoru; Kiyono, Tohru; Saito, Shigeru

    2013-01-01

    Abstract Human amniotic epithelial cells (HAEs) have a low immunogenic profile and possess potent immunosuppressive properties. HAEs also have several characteristics similar to stem cells, and they are discarded after parturition. Thus, they could potentially be used in cell therapy with fewer ethical problems. HAEs have a short life, so our aim is to establish and characterize immortalized human amniotic epithelial cells (iHAEs). HAEs were introduced with viral oncogenes E6/E7 and with human telomerase reverse transcriptase (hTERT) to create iHAEs. These iHAEs have proliferated around 200 population doublings (PDs) for at least 12 months. High expression of stem cell markers (Oct 3/4, Nanog, Sox2, Klf4) and epithelial markers (CK5, CK18) were detected by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). These iHAEs were expanded in ultra-low-attachment dishes to form spheroids similarly to epithelial stem/precursor cells. High expression of mesenchymal (CD44, CD73, CD90, CD105) and somatic (CD24, CD29, CD271, Nestin) stem cell markers was detected by flow cytometry. The iHAEs showed adipogenic, osteogenic, neuronal, and cardiac differentiation abilities. In conclusion, the immortalization of HAEs with the characteristics of stem cells has been established, allowing these iHAEs to become useful for cell therapy and regenerative medicine. PMID:23298399

  10. Cell Surface Glycan Changes in the Spontaneous Epithelial-Mesenchymal Transition of Equine Amniotic Multipotent Progenitor Cells.

    PubMed

    Lange-Consiglio, Anna; Accogli, Gianluca; Cremonesi, Fausto; Desantis, Salvatore

    2014-01-01

    Amniotic epithelial cells (AECs) spontaneously transform into amniotic mesenchymal cells (AMCs) in vitro during cell culture. Glycocalyx was analyzed to identify the glycan pattern in AECs, AMCs and epithelial-mesenchymal transdifferentiated cells (EMTCs). Pure cell cultures were derived using cloned AEC and AMC cell lines obtained by the dilution technique from amniotic membranes. Mesenchymal cells generated by differentiation of clonal epithelial cells were considered transdifferentiated. Immunocytoscreen, in vitro multipotent differentiation and molecular characterization of EMTCs were performed. In combination with saponification and sialidase digestion, a panel of 12 lectins was used to analyze the glycan pattern of AEC, AMC and EMTC glycocalyx. Cytokeratin cell markers were lost in EMTCs and typical mesenchymal markers, such as vimentin, appeared. These cells retained their differentiation potential. Lectin histochemistry revealed a cell-specific glycan profile. Galactose (Gal)β1,4GlcNAc, Neu5Acα2,6Gal/GalNAc and N-acetyl neuraminic (sialic) acid (NeuNAc)α2,3Galβ1,3(±NeuNAcα2,6)GalNAc were highly expressed on the surface of all the amniotic cell cultures. AECs expressed asialoglycans with terminal GalNAc and GlcNAc. More highly mannosylated N-linked glycans and NeuNAcα2,3Galβ1,3GalNAc in O-linked glycans were expressed by EMTCs, but these cells had fewer glycans ending with fucose (Fuc), Gal, GlcNAc and GalNAc than AECs. GlcNAc- and GalNAc-terminating glycans were similarly expressed on the glycocalyx of the mesenchymal cell populations (EMTCs and AMCs). These results demonstrate for the first time that the spontaneous epithelial-mesenchymal transition (EMT) of equine amnion cells is characterized by cell surface glycan remodeling and that glycosylation changes result in a cell type-specific glycan profile. The glycopattern of equine amnion spontaneous EMTCs differs from EMT of tumoral cells. PMID:26337136

  11. [Disodium cromoglycate--mast cell degranulation blocker in the process of tissue remodelation].

    PubMed

    Maxová, H; Vasilková, M; Tkaczyk, J; Vízek, M

    2010-01-01

    Disodium cromoglycate (DSCG) is a compound commonly used in the treatment of allergic diseases. The effect of DSCG is due to its ability to stabilize the mast cell membrane and to prevent release of histamine and inflammatory mediators. Mast cells are also an abundant source of tissue metalloproteinases, serine proteases and growth factors, which play an important role in the processes of the tissue remodeling. In this view the DSCG is a substance which allows us to study the mechanisms of the pulmonary vascular bed remodeling in the experimental animals exposed to chronic hypoxia and in a phase of the recovery from hypoxia. PMID:21254664

  12. Transcriptional PROFILING OF MUCOCILIARY DIFFERENTIATION IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    When cultured at an air-liquid interface (ALI) in the appropriate medium, primary human airway epithelial cells form a polarized, pseudostratified epithelium composed of ciliated and mucus-secreting cells. This culture system provides a useful tool for the in vitro study of...

  13. Lateral adhesion drives reintegration of misplaced cells into epithelial monolayers

    PubMed Central

    St Johnston, Daniel

    2016-01-01

    Cells in simple epithelia orient their mitotic spindles in the plane of the epithelium so that both daughter cells are born within the epithelial sheet. This is assumed to be important to maintain epithelial integrity and prevent hyperplasia, because misaligned divisions give rise to cells outside the epithelium1,2. Here we test this assumption in three types of Drosophila epithelia; the cuboidal follicle epithelium, the columnar early embryonic ectoderm, and the pseudostratified neuroepithelium. Ectopic expression of Inscuteable in these tissues reorients mitotic spindles, resulting in one daughter cell being born outside of the epithelial layer. Live imaging reveals that these misplaced cells reintegrate into the tissue. Reducing the levels of the lateral homophilic adhesion molecules Neuroglian or Fasciclin 2 disrupts reintegration, giving rise to extra-epithelial cells, whereas disruption of adherens junctions has no effect. Thus, the reinsertion of misplaced cells appears to be driven by lateral adhesion, which pulls cells born outside the epithelia layer back into it. Our findings reveal a robust mechanism that protects epithelia against the consequences of misoriented divisions. PMID:26414404

  14. Epithelial stem cells and implications for wound repair.

    PubMed

    Plikus, Maksim V; Gay, Denise L; Treffeisen, Elsa; Wang, Anne; Supapannachart, Rarinthip June; Cotsarelis, George

    2012-12-01

    Activation of epithelial stem cells and efficient recruitment of their proliferating progeny plays a critical role in cutaneous wound healing. The reepithelialized wound epidermis has a mosaic composition consisting of progeny that can be traced back both to epidermal and several types of hair follicle stem cells. The contribution of hair follicle stem cells to wound epidermis is particularly intriguing as it involves lineage identity change from follicular to epidermal. Studies from our laboratory show that hair follicle-fated bulge stem cells commit only transient amplifying epidermal progeny that participate in the initial wound re-epithelialization, but eventually are outcompeted by other epidermal clones and largely disappear after a few months. Conversely, recently described stem cell populations residing in the isthmus portion of hair follicle contribute long-lasting progeny toward wound epidermis and, arguably, give rise to new interfollicular epidermal stem cells. The role of epithelial stem cells during wound healing is not limited to regenerating stratified epidermis. By studying regenerative response in large cutaneous wounds, our laboratory uncovered that epithelial cells in the center of the wound can acquire greater morphogenetic plasticity and, together with the underlying wound dermis, can engage in an embryonic-like process of hair follicle neogenesis. Future studies should uncover the cellular and signaling basis of this remarkable adult wound regeneration phenomenon. PMID:23085626

  15. Epithelial Stem Cells and Implications for Wound Repair

    PubMed Central

    Plikus, Maksim V.; Gay, Denise L.; Treffeisen, Elsa; Wang, Anne; Supapannachart, Rarinthip June; Cotsarelis, George

    2012-01-01

    Activation of epithelial stem cells and efficient recruitment of their proliferating progeny plays a critical role in cutaneous wound healing. The reepithelialized wound epidermis hasa mosaic composition consisting of progeny that can be traced back both to epidermal and several types of hair follicle stem cells. The contribution of hair follicle stem cells to wound epidermis is particularly intriguing as it involves lineage identity change from follicular to epidermal. Studies from our laboratory show that hair follicle-fated bulge stem cells commit only transient amplifying epidermal progeny that participate in the initial wound re-epithelialization, but eventually are outcompeted by other epidermal clones and largely disappear after a few months. Conversely, recently described stem cell populations residing in the isthmus portion of hair follicle contribute long-lasting progeny toward wound epidermis and, arguably, give rise to new inter-follicular epidermal stem cells. The role of epithelial stem cells during wound healing is not limited to regenerating stratified epidermis. By studying regenerative response in large cutaneous wounds, our laboratory uncovered that epithelial cells in the center of the wound can acquire greater morphogenetic plasticity and, together with the underlying wound dermis, can engage in an embryonic-like process of hair follicle neogenesis. Future studies should uncover cellular and signaling basis of this remarkable adult wound regeneration phenomenon. PMID:23085626

  16. AN IN VITRO MODEL FOR MURINE URETERIC EPITHELIAL CELLS

    EPA Science Inventory

    This report presents a model developed to study growth and differentiation of primary cultures of ureteric epithelial cells from embryonic C57BL/6N mouse urinary tracts. Single cells were resuspended in medium and plated onto transwells coated with collagen IV and laminin. Basa...

  17. [Isolation, purification and identification of epithelial cells derived from fetal islet-like cell clusters].

    PubMed

    Qiao, Hai; Zhao, Ting; Wang, Yun; Yang, Chun-Rong; Xiao, Mei; Dou, Zhong-Ying

    2007-03-01

    The aim of this article is to provide methods for the isolation and identification of pancreatic stem cells and cell source for research and therapy of diabetes. ICCs were isolated by collagenase IV digesting and then cultured; epithelial cells were purified from monolayer cultured ICCs. The growth curve of the epithelial cells was measured by MTT. The expression of molecular markers in the cells was identified by immunohistochemical staining. The surface markers in the epithelial cells were analyzed by FACS. Epithelial cells were purified from isolated human fetal ICCs and passaged 40 times, and 10(6) - 10(8) cells were cryopreservated per passage. The growth curve demonstrated that the epithelial cells proliferated rapidly. The epithelial cells expressed PDX-1, PCNA, CK-7, CK-19, Nestin, Glut2, and Vimentin, but Insulin was undetected. The cells expressed CD29, CD44, and CD166, but did not express CD11a, CD14, CD34, CD45, CD90, CD105, and CD117. Taken together, these results indicate that self-renewable epithelial cells can be isolated and purified from human fetal pancreas. These also show that the epithelial cells originate from ducts and have the characteristics of pancreatic stem cells. PMID:17460896

  18. The syncytial nature of epithelial cells in the thymic cortex.

    PubMed Central

    Kendall, M D

    1986-01-01

    The epithelial cells of the cortex of human and rodent thymus glands were examined by light and electron microscopy, and the intracellular membrane potentials measured from the subcapsular, cortical and medullary regions. In the human thymus cortex, there is a highly correlated age-independent relationship (r = 0.78) between the distance in micron from one adjacent Type 2/3 epithelial nucleus to another, and the number of thymocytes between them. In rodent glands that had undergone some degree of involution due to hypoxia simulating an altitude of 17 000 feet or following the injection of phenylhydrazine, Type 2/3 epithelial cells were often found to be bi- or multinucleated. Electrophysiological studies of 10 mouse thymus lobes using 0.2 micron tipped electrodes showed that there were highly significant differences (P less than 0.0001) between the intracellular membrane potentials of the subcapsular zone, the cortex and the medulla. When dyes were injected intracellularly (through 0.5 micron tipped electrodes) into individual epithelial cells, methylene blue remained within the cytoplasm, but procion yellow passed in 30 minutes into the nuclei of all the epithelial cells of the cortex but not those of the subcapsular zone, nor the medulla. This indicates that the cortex must be a functional syncytium and it differs in this respect from the rest of the gland. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 PMID:3319999

  19. Switched-memory B cells remodel B cell receptors within secondary germinal centers

    PubMed Central

    Okitsu, Shinji L.; McHeyzer-Williams, Michael G.

    2015-01-01

    Effective vaccines induce high-affinity memory B cells and durable antibody responses through accelerated mechanisms of natural selection. Secondary changes in antibody repertoires after vaccine boosts suggest progressive B cell receptor (BCR) re-diversification, but underlying mechanisms remain unresolved. Here integrated specificity and function of individual memory B cell progeny reveal ongoing evolution of polyclonal antibody specificities through germinal center (GC) specific transcriptional activity. At the clonal and sub-clonal levels, single cell expression of Cd83 and Pol□ segregates the secondary GC transcriptional program into 4 stages that regulate divergent mechanisms of memory BCR evolution. These studies demonstrate that vaccine boosts re-activate a cyclic program of GC function in switched-memory B cells to remodel existing antibody specificities and enhance durable immune protection. PMID:25642821

  20. The potential role of polyamines in gill epithelial remodeling during extreme hypoosmotic challenges in the Gulf killifish, Fundulus grandis.

    PubMed

    Guan, Ying; Zhang, Guo-Xia; Zhang, Shujun; Domangue, Beau; Galvez, Fernando

    2016-01-01

    Polyamines are a family of low molecular weight organic cations produced in part by the coordinated actions of arginase II (Arg II) and ornithine decarboxylase (Odc). Although gill polyamine homeostasis is affected by acute transfer to fresh water, little is known of its function in fish osmoregulation. The current study investigated the role of polyamines in the compensatory response of hypoosmotic challenge in the euryhaline fish, Fundulus grandis. Adult F. grandis were acclimated to 5 ppt water, transferred abruptly to 5, 2, 1, 0.5 and 0.1 ppt water, and assessed for osmoregulatory function, gill morphology, and polyamine homeostasis. The plasma osmolality, Na(+) concentration, and Cl(-) concentration were only significantly reduced during exposure to salinities at or below 0.5 ppt, although these effects were transient except in the 0.1 ppt treatment. The phenotype of mitochondrion-rich cells (MRCs) shifted from a seawater-type to a freshwater-type only at salinities that also produced a plasma osmotic disturbance. Hypoosmotic exposure increased the concentrations of putrescine, spermidine, and spermine in the gill over the entire 7 day period. Exposure to 0.1 ppt water also transiently increased gill caspase-3 activity and gill mRNA levels of the immediate-early response genes, c-fos and c-myc, thus tightly associating polyamines with gill remodeling during freshwater acclimation. Furthermore, arginase II and ornithine decarboxylase mRNA levels were most highly expressed in MRCs, and these levels were further increased only in the 0.1 ppt treatment. Reduction of gill polyamine levels following administration of the Odc inhibitor, alpha-dl-difluoromethylornithine (DFMO), inhibited gill caspase-3 activity, but surprisingly reduced the magnitude of the plasma osmotic imbalance elicited by exposure to 0.1 ppt water. We used isolated opercular epithelia mounted on Ussing chambers to assess the influence of polyamines on the attenuating response of hypotonic shock

  1. Protrusive Activity Guides Changes in Cell-Cell Tension during Epithelial Cell Scattering

    PubMed Central

    Maruthamuthu, Venkat; Gardel, Margaret L.

    2014-01-01

    Knowing how epithelial cells regulate cell-matrix and cell-cell adhesions is essential to understand key events in morphogenesis as well as pathological events such as metastasis. During epithelial cell scattering, epithelial cell islands rupture their cell-cell contacts and migrate away as single cells on the extracellular matrix (ECM) within hours of growth factor stimulation, even as adhesion molecules such as E-cadherin are present at the cell-cell contact. How the stability of cell-cell contacts is modulated to effect such morphological transitions is still unclear. Here, we report that in the absence of ECM, E-cadherin adhesions continue to sustain substantial cell-generated forces upon hepatocyte growth factor (HGF) stimulation, consistent with undiminished adhesion strength. In the presence of focal adhesions, constraints that preclude the spreading and movement of cells at free island edges also prevent HGF-mediated contact rupture. To explore the role of cell motion and cell-cell contact rupture, we examine the biophysical changes that occur during the scattering of cell pairs. We show that the direction of cell movement with respect to the cell-cell contact is correlated with changes in the average intercellular force as well as the initial direction of cell-cell contact rupture. Our results suggest an important role for protrusive activity resulting in cell displacement and force redistribution in guiding cell-cell contact rupture during scattering. PMID:25099795

  2. Evaluating alternative stem cell hypotheses for adult corneal epithelial maintenance

    PubMed Central

    West, John D; Dorà, Natalie J; Collinson, J Martin

    2015-01-01

    In this review we evaluate evidence for three different hypotheses that explain how the corneal epithelium is maintained. The limbal epithelial stem cell (LESC) hypothesis is most widely accepted. This proposes that stem cells in the basal layer of the limbal epithelium, at the periphery of the cornea, maintain themselves and also produce transient (or transit) amplifying cells (TACs). TACs then move centripetally to the centre of the cornea in the basal layer of the corneal epithelium and also replenish cells in the overlying suprabasal layers. The LESCs maintain the corneal epithelium during normal homeostasis and become more active to repair significant wounds. Second, the corneal epithelial stem cell (CESC) hypothesis postulates that, during normal homeostasis, stem cells distributed throughout the basal corneal epithelium, maintain the tissue. According to this hypothesis, LESCs are present in the limbus but are only active during wound healing. We also consider a third possibility, that the corneal epithelium is maintained during normal homeostasis by proliferation of basal corneal epithelial cells without any input from stem cells. After reviewing the published evidence, we conclude that the LESC and CESC hypotheses are consistent with more of the evidence than the third hypothesis, so we do not consider this further. The LESC and CESC hypotheses each have difficulty accounting for one main type of evidence so we evaluate the two key lines of evidence that discriminate between them. Finally, we discuss how lineage-tracing experiments have begun to resolve the debate in favour of the LESC hypothesis. Nevertheless, it also seems likely that some basal corneal epithelial cells can act as long-term progenitors if limbal stem cell function is compromised. Thus, this aspect of the CESC hypothesis may have a lasting impact on our understanding of corneal epithelial maintenance, even if it is eventually shown that stem cells are restricted to the limbus as proposed

  3. [Epithelial cell in intestinal homeostasis and inflammatory bowel diseases].

    PubMed

    Zouiten-Mekki, Lilia; Serghini, Meriem; Fekih, Monia; Kallel, Lamia; Matri, Samira; Ben Mustapha, Nadia; Boubaker, Jalel; Filali, Azza

    2013-12-01

    Crohn's disease (CD) and ulcerative colitis (UC) are the principal inflammatory bowel diseases (IBD) which physiopathology is currently poorly elucidated. During these diseases, the participation of the epithelial cell in the installation and the perpetuation of the intestinal inflammation is now clearly implicated. In fact, the intestinal epithelium located at the interface between the internal environment and the intestinal luminal, is key to the homeostatic regulation of the intestinal barrier. This barrier can schematically be regarded as being three barriers in one: a physical, chemical and immune barrier. The barrier function of epithelial cell can be altered by various mechanisms as occurs in IBD. The goal of this article is to review the literature on the role of the epithelial cell in intestinal homeostasis and its implication in the IBD. PMID:24356146

  4. Cell Wall Remodeling Enzymes Modulate Fungal Cell Wall Elasticity and Osmotic Stress Resistance

    PubMed Central

    Ene, Iuliana V.; Walker, Louise A.; Schiavone, Marion; Lee, Keunsook K.; Martin-Yken, Hélène; Dague, Etienne; Gow, Neil A. R.; Munro, Carol A.

    2015-01-01

    ABSTRACT The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock. PMID:26220968

  5. Oxidized alginate hydrogels as niche environments for corneal epithelial cells

    PubMed Central

    Wright, Bernice; De Bank, Paul A; Luetchford, Kim A; Acosta, Fernando R; Connon, Che J

    2014-01-01

    Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P ≤ 0.05) and this improved further with addition of collagen IV (P ≤ 0.01). Oxidised gels presented larger internal pores (diameter: 0.2–0.8 µm) than unmodified gels (pore diameter: 0.05–0.1 µm) and were significantly less stiff (P ≤ 0.001), indicating that an increase in pore size and a decrease in stiffness contributed to improved cell viability. The diffusion of collagen IV from oxidised alginate gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy. © 2013 The Authors. Journal of Biomedical Materials Research Part A Published byWiley Periodicals, Inc. Part A: 102A: 3393–3400, 2014. PMID:24142706

  6. Twist1 and Slug mediate H2AX-regulated epithelial-mesenchymal transition in breast cells.

    PubMed

    Weyemi, Urbain; Redon, Christophe E; Sethi, Taresh K; Burrell, Allison S; Jailwala, Parthav; Kasoji, Manjula; Abrams, Natalie; Merchant, Anand; Bonner, William M

    2016-09-16

    The epithelial-mesenchymal transition (EMT) is thought to be essential for cancer metastasis. While chromatin remodeling is involved in EMT, which processes contribute to this remodeling remain poorly investigated. Recently, we showed that silencing or removal of the histone variant H2A.X induced mesenchymal-like characteristics, including activation of the EMT transcription factors, Slug and Zeb1 in human colon cancer cells. Here, we provide the evidence that H2A.X loss in human non-tumorigenic breast cell line MCF10A results in a robust EMT activation, as substantiated by a genome-wide expression analysis. Cells deficient for H2A.X exhibit enhanced migration and invasion, along with an activation of a set of mesenchymal genes and a concomitant repression of epithelial genes. In the breast model, the EMT-related transcription factor Twist1 cooperates with Slug to regulate EMT upon H2A.X Loss. Of interest, H2A.X expression level tightly correlates with Twist1, and to a lesser extent with Slug in the panel of human breast cancer cell lines of the NCI-60 datasets. These new findings indicate that H2A.X is involved in the EMT processes in cells of different origins but pairing with transcription factors for EMT may be tissue specific. PMID:27315462

  7. Dysfunctional resident lung mesenchymal stem cells contribute to pulmonary microvascular remodeling

    PubMed Central

    Chow, Kelsey; Fessel, Joshua P.; KaoriIhida-Stansbury; Schmidt, Eric P.; Gaskill, Christa; Alvarez, Diego; Graham, Brian; Harrison, David G.; Wagner, David H.; Nozik-Grayck, Eva; West, James D.; Klemm, Dwight J.; Majka, Susan M.

    2013-01-01

    Pulmonary vascular remodeling and oxidative stress are common to many adult lung diseases. However, little is known about the relevance of lung mesenchymal stem cells (MSCs) in these processes. We tested the hypothesis that dysfunctional lung MSCs directly participate in remodeling of the microcirculation. We employed a genetic model to deplete extracellular superoxide dismutase (EC-SOD) in lung MSCs coupled with lineage tracing analysis. We crossed floxpsod3 and mT/mG reporter mice to a strain expressing Cre recombinase under the control of the ABCG2 promoter. We demonstrated In vivo that depletion of EC-SOD in lung MSCs resulted in their contribution to microvascular remodeling in the smooth muscle actin positive layer. We further characterized lung MSCs to be multipotent vascular precursors, capable of myofibroblast, endothelial and pericyte differentiation in vitro. EC-SOD deficiency in cultured lung MSCs accelerated proliferation and apoptosis, restricted colony-forming ability, multilineage differentiation potential and promoted the transition to a contractile phenotype. Further studies correlated cell dysfunction to alterations in canonical Wnt/β-catenin signaling, which were more evident under conditions of oxidative stress. Our data establish that lung MSCs are a multipotent vascular precursor population, a population which has the capacity to participate in vascular remodeling and their function is likely regulated in part by the Wnt/β-catenin signaling pathway. These studies highlight an important role for microenviromental regulation of multipotent MSC function as well as their potential to contribute to tissue remodeling. PMID:23662173

  8. The innate immune function of airway epithelial cells in inflammatory lung disease.

    PubMed

    Hiemstra, Pieter S; McCray, Paul B; Bals, Robert

    2015-04-01

    The airway epithelium is now considered to be central to the orchestration of pulmonary inflammatory and immune responses, and is also key to tissue remodelling. It acts as the first barrier in the defence against a wide range of inhaled challenges, and is critically involved in the regulation of both innate and adaptive immune responses to these challenges. Recent progress in our understanding of the developmental regulation of this tissue, the differentiation pathways, recognition of pathogens and antimicrobial responses is now exploited to help understand how epithelial cell function and dysfunction contributes to the pathogenesis of a variety of inflammatory lung diseases. Herein, advances in our knowledge of the biology of airway epithelium, as well as its role and (dys)function in asthma, chronic obstructive pulmonary fibrosis and cystic fibrosis will be discussed. PMID:25700381

  9. The innate immune function of airway epithelial cells in inflammatory lung disease

    PubMed Central

    Hiemstra, Pieter S.; McCray, Paul B.; Bals, Robert

    2016-01-01

    The airway epithelium is now considered central to the orchestration of pulmonary inflammatory and immune responses, and is also key to tissue remodelling. It acts as a first barrier in the defence against a wide range of inhaled challenges, and is critically involved in the regulation of both innate and adaptive immune responses to these challenges. Recent progress in our understanding of the developmental regulation of this tissue, the differentiation pathways, recognition of pathogens and antimicrobial responses is now exploited to help understand how epithelial cell function and dysfunction contributes to the pathogenesis of a variety of inflammatory lung diseases. In the review, advances in our knowledge of the biology of airway epithelium, as well as its role and (dys)function in asthma, COPD and cystic fibrosis, are discussed. PMID:25700381

  10. Osteogenic Differentiation of Human Amniotic Epithelial Cells and Its Application in Alveolar Defect Restoration

    PubMed Central

    Jiawen, Si; Jianjun, Zhang; Jiewen, Dai; Dedong, Yu; Hongbo, Yu; Jun, Shi; Xudong, Wang; Shen, Steve G.F.

    2014-01-01

    The present study investigated the detailed in vitro osteogenic differentiation process and in vivo bone regenerative property of human amniotic epithelial cells (hAECs). The in vitro osteogenic differentiation process of hAECs was evaluated by biochemical staining, real-time polymerase chain reaction, and immunofluorescence. Next, β-tricalcium phosphate (β-TCP) scaffolds alone or loaded with hAECs were implanted into the alveolar defects of rats. Micro-computed tomography evaluation and histologic studies were conducted. Our results validated the in vitro osteogenic capacity of hAECs by upregulation of Runx2, osterix, alkaline phosphatase, collagen I, and osteopontin, with positive biochemical staining for osteoblasts. An epithelial-mesenchymal transformation process might be involved in the osteogenic differentiation of hAECs by increased expression of transforming growth factor-β1. Our data also demonstrated that in vivo implantation of hAECs loaded on β-TCP scaffolds, not only improved bone regeneration by direct participation, but also reduced the early host immune response to the scaffolds. The presented data indicate that hAECs possess proper osteogenic differentiation potential and a modulatory influence on the early tissue remodeling process, making these cells a potential source of progenitor cells for clinical restoration of the alveolar defect. PMID:25368378

  11. Epithelial cell cultures from normal and cancerous human tissues.

    PubMed

    Owens, R B; Smith, H S; Nelson-Rees, W A; Springer, E L

    1976-04-01

    Thirty epithelial cell strains were isolated from human carcinomas and normal epithelial tissues by collagenase digestion and selective removal of fibroblasts with trypsin-Versene. Most strains were obtained from metastatic carcinomas or epithelia of the urinary and intestinal tracts. The success rate for growth of both neoplastic and normal tissues (excluding skin) was 38%. Six of these strains showed gross morphologic and chromosome changes typical of malignant cells. Nine resembled normal epithelium. The other 15 exhibited some degree of morphologic change from normal. PMID:176412

  12. Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro.

    PubMed

    Hu, Han; Zheng, Nan; Gao, Haina; Dai, Wenting; Zhang, Yangdong; Li, Songli; Wang, Jiaqi

    2016-08-01

    The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, β-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland. PMID:27189858

  13. CHANGES IN CARBOHYDRATE COORDINATED PARTITIONING AND CELL WALL REMODELING WITH STRESS-INDUCED PATHOGENESIS IN WHEAT SHEATHS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant cell walls are dynamic structures that undergo specific remodeling events during plant defense responses. Changes in the coordinated partitioning of carbohydrates between the cytosol and the extracellular milieu may direct sheath cell wall remodeling that occurs in a wheat-endophytic interacti...

  14. Expression of the FGFR2 mesenchymal splicing variant in epithelial cells drives epithelial-mesenchymal transition

    PubMed Central

    Ranieri, Danilo; Rosato, Benedetta; Nanni, Monica; Magenta, Alessandra

    2016-01-01

    The FGFRs are receptor tyrosine kinases expressed by tissue-specific alternative splicing in epithelial IIIb or mesenchymal IIIc isoforms. Deregulation of FGF/FGFR signaling unbalances the epithelial-stromal homeostasis and may lead to cancer development. In the epithelial-context, while FGFR2b/KGFR acts as tumor suppressor, FGFR2c appears to play an oncogenic role. Based on our recent observation that the switching of FGFR2b versus FGFR2c induces EMT, here we investigated the biological outcome of the ectopic expression of FGFR2c in normal human keratinocytes. Morphological analysis showed that, differently from FGFR2b overexpression, the forced expression and activation of FGFR2c drive the epithelial cells to acquire a mesenchymal-like shape and actin reorganization. Moreover, the appearance of invasiveness and anchorage-independent growth ability in FGFR2c transfected keratinocytes was consistent with the potential tumorigenic role proposed for this receptor variant. Biochemical and molecular approaches revealed that the observed phenotypic changes were accompanied by modulation of EMT biomarkers and indicated the involvement of EMT transcription factors and miRs. Finally, the analysis of the expression pattern of discriminating markers strongly suggested that activation of FGFR2c triggers a process corresponding to the initiation of the pathological type III EMT, but not to the more physiological type II EMT occurring during FGFR2b-mediated wound healing. PMID:26713601

  15. Sp3 regulates fas expression in lung epithelial cells.

    PubMed Central

    Pang, H; Miranda, K; Fine, A

    1998-01-01

    By transducing an apoptotic signal in immune effector cells, Fas has been directly implicated in the control of immunological activity. Expression and functional results, however, have also suggested a role for Fas in regulating cell turnover in specific epithelial populations. To characterize factors responsible for Fas expression in epithelial cells, approximately 3 kb of the 5' flanking region of the mouse Fas gene was isolated. By rapid amplification of cDNA ends and primer extension, transcriptional start sites were identified within 50 bp upstream of the translation start site. Transient transfection of promoter-luciferase constructs in a mouse lung epithelial cell line, MLE-15, localized promoter activity to the first 77 bp of upstream sequence. By using a 60 bp DNA probe (-18 to -77) in electrophoretic mobility-shift assays, three shifted complexes were found. Incubation with excess cold Sp1 oligonucleotide or an anti-Sp3 antibody inhibited complex formation. Site-directed mutagenesis of the Sp1 site resulted in 60-70% loss of promoter activity. In Drosophila SL-2 cells, promoter activity was markedly increased by co-transfection of an Sp3 expression construct. These results show that the Sp3 protein is involved in regulating Fas gene expression in lung epithelial cells. PMID:9639581

  16. Mechanical strain induces involution-associated events in mammary epithelial cells

    PubMed Central

    Quaglino, Ana; Salierno, Marcelo; Pellegrotti, Jesica; Rubinstein, Natalia; Kordon, Edith C

    2009-01-01

    Background Shortly after weaning, a complex multi-step process that leads to massive epithelial apoptosis is triggered by tissue local factors in the mouse mammary gland. Several reports have demonstrated the relevance of mechanical stress to induce adaptive responses in different cell types. Interestingly, these signaling pathways also participate in mammary gland involution. Then, it has been suggested that cell stretching caused by milk accumulation after weaning might be the first stimulus that initiates the complete remodeling of the mammary gland. However, no previous report has demonstrated the impact of mechanical stress on mammary cell physiology. To address this issue, we have designed a new practical device that allowed us to evaluate the effects of radial stretching on mammary epithelial cells in culture. Results We have designed and built a new device to analyze the biological consequences of applying mechanical stress to cells cultured on flexible silicone membranes. Subsequently, a geometrical model that predicted the percentage of radial strain applied to the elastic substrate was developed. By microscopic image analysis, the adjustment of these calculations to the actual strain exerted on the attached cells was verified. The studies described herein were all performed in the HC11 non-tumorigenic mammary epithelial cell line, which was originated from a pregnant BALB/c mouse. In these cells, as previously observed in other tissue types, mechanical stress induced ERK1/2 phosphorylation and c-Fos mRNA and protein expression. In addition, we found that mammary cell stretching triggered involution associated cellular events as Leukemia Inhibitory Factor (LIF) expression induction, STAT3 activation and AKT phosphorylation inhibition. Conclusion Here, we show for the first time, that mechanical strain is able to induce weaning-associated events in cultured mammary epithelial cells. These results were obtained using a new practical and affordable device

  17. A conformational switch controls cell wall remodeling enzymes required for bacterial cell division

    PubMed Central

    Yang, Desirée C.; Tan, Kemin; Joachimiak, Andrzej; Bernhardt, Thomas G.

    2012-01-01

    Summary Remodeling of the peptidoglycan (PG) exoskeleton is intimately tied to the growth and division of bacteria. Enzymes that hydrolyze PG are critical for these processes, but their activities must be tightly regulated to prevent the generation of lethal breaches in the PG matrix. Despite their importance, the mechanisms regulating PG hydrolase activity have remained elusive. Here we investigate the control of cell division hydrolases called amidases (AmiA, AmiB, and AmiC) required for Escherichia coli cell division. Poorly regulated amiB mutants were isolated encoding lytic AmiB variants with elevated basal PG hydrolase activities in vitro. The structure of an AmiB ortholog was also solved, revealing that the active site of AmiB is occluded by a conserved alpha-helix. Strikingly, most of the amino acid substitutions in the lytic AmiB variants mapped to this domain and are predicted to disrupt its interaction with the active site. Our results therefore support a model in which cell separation is stimulated by the reversible relief of amidase auto-inhibition governed by conserved sub-complexes within the cytokinetic ring. Analogous conformational control mechanisms are likely to be part of a general strategy used to control PG hydrolases present within multi-enzyme PG remodeling machines. PMID:22715947

  18. Myeloid-Epithelial-Reproductive Receptor Tyrosine Kinase and Milk Fat Globule Epidermal Growth Factor 8 Coordinately Improve Remodeling After Myocardial Infarction via Local Delivery of Vascular Endothelial Growth Factor

    PubMed Central

    Howangyin, Kiave-Yune; Zlatanova, Ivana; Pinto, Cristina; Ngkelo, Anta; Cochain, Clément; Rouanet, Marie; Vilar, José; Lemitre, Mathilde; Stockmann, Christian; Fleischmann, Bernd K.; Mallat, Ziad

    2016-01-01

    Background— In infarcted heart, improper clearance of dying cells by activated neighboring phagocytes may precipitate the transition to heart failure. We analyzed the coordinated role of 2 major mediators of efferocytosis, the myeloid-epithelial-reproductive protein tyrosine kinase (Mertk) and the milk fat globule epidermal growth factor (Mfge8), in directing cardiac remodeling by skewing the inflammatory response after myocardial infarction. Methods and Results— We generated double-deficient mice for Mertk and Mfge8 (Mertk−/−/Mfge8−/−) and challenged them with acute coronary ligature. Compared with wild-type, Mertk-deficient (Mertk−/−), or Mfge8-deficient (Mfge8−/−) animals, Mertk−/−/Mfge8−/− mice displayed greater alteration in cardiac function and remodeling. Mertk and Mfge8 were expressed mainly by cardiac Ly6CHigh and Low monocytes and macrophages. In parallel, Mertk−/−/Mfge8−/− bone marrow chimeras manifested increased accumulation of apoptotic cells, enhanced fibrotic area, and larger infarct size, as well as reduced angiogenesis. We found that the abrogation of efferocytosis affected neither the ability of circulating monocytes to infiltrate cardiac tissue nor the number of resident Ly6CHigh and Ly6CHow monocytes/macrophages populating the infarcted milieu. In contrast, combined Mertk and Mfge8 deficiency in Ly6CHigh/Ly6CLow monocytes/macrophages either obtained from in vitro differentiation of bone marrow cells or isolated from infarcted hearts altered their capacity of efferocytosis and subsequently blunted vascular endothelial growth factor A (VEGFA) release. Using LysMCre+/VEGFAfl/fl mice, we further identified an important role for myeloid-derived VEGFA in improving cardiac function and angiogenesis. Conclusions— After myocardial infarction, Mertk- and Mfge8-expressing monocyte/macrophages synergistically engage the clearance of injured cardiomyocytes, favoring the secretion of VEGFA to locally repair the

  19. Airway epithelial cell response to human metapneumovirus infection

    SciTech Connect

    Bao, X.; Liu, T.; Spetch, L.; Kolli, D.; Garofalo, R.P.; Casola, A.

    2007-11-10

    Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections (LRTIs) in infants, elderly and immunocompromised patients. In this study, we show that hMPV can infect in a similar manner epithelial cells representative of different tracts of the airways. hMPV-induced expression of chemokines IL-8 and RANTES in primary small alveolar epithelial cells (SAE) and in a human alveolar type II-like epithelial cell line (A549) was similar, suggesting that A549 cells can be used as a model to study lower airway epithelial cell responses to hMPV infection. A549 secreted a variety of CXC and CC chemokines, cytokines and type I interferons, following hMPV infection. hMPV was also a strong inducer of transcription factors belonging to nuclear factor (NF)-{kappa}B, interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) families, which are known to orchestrate the expression of inflammatory and immunomodulatory mediators.

  20. Cigarette smoke extract affects mitochondrial function in alveolar epithelial cells.

    PubMed

    Ballweg, Korbinian; Mutze, Kathrin; Königshoff, Melanie; Eickelberg, Oliver; Meiners, Silke

    2014-12-01

    Cigarette smoke is the main risk factor for chronic obstructive pulmonary disease (COPD). Exposure of cells to cigarette smoke induces an initial adaptive cellular stress response involving increased oxidative stress and induction of inflammatory signaling pathways. Exposure of mitochondria to cellular stress alters their fusion/fission dynamics. Whereas mild stress induces a prosurvival response termed stress-induced mitochondrial hyperfusion, severe stress results in mitochondrial fragmentation and mitophagy. In the present study, we analyzed the mitochondrial response to mild and nontoxic doses of cigarette smoke extract (CSE) in alveolar epithelial cells. We characterized mitochondrial morphology, expression of mitochondrial fusion and fission genes, markers of mitochondrial proteostasis, as well as mitochondrial functions such as membrane potential and oxygen consumption. Murine lung epithelial (MLE)12 and primary mouse alveolar epithelial cells revealed pronounced mitochondrial hyperfusion upon treatment with CSE, accompanied by increased expression of the mitochondrial fusion protein mitofusin 2 and increased metabolic activity. We did not observe any alterations in mitochondrial proteostasis, i.e., induction of the mitochondrial unfolded protein response or mitophagy. Therefore, our data indicate an adaptive prosurvival response of mitochondria of alveolar epithelial cells to nontoxic concentrations of CSE. A hyperfused mitochondrial network, however, renders the cell more vulnerable to additional stress, such as sustained cigarette smoke exposure. As such, cigarette smoke-induced mitochondrial hyperfusion, although part of a beneficial adaptive stress response in the first place, may contribute to the pathogenesis of COPD. PMID:25326581

  1. AIRWAY EPITHELIAL CELL RESPONSE TO HUMAN METAPNEUMOVIRUS INFECTION

    PubMed Central

    X, Bao; T, Liu; L, Spetch; D, Kolli; R.P, Garofalo; A, Casola

    2007-01-01

    Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections (LRTIs) in infants, elderly and immunocompromised patients. In this study, we show that hMPV can infect in a similar manner epithelial cells representative of different tracts of the airways. hMPV-induced expression of chemokines IL-8 and RANTES in primary small alveolar epithelial cells (SAE) and in a human alveolar type II-like epithelial cell line (A549) was similar, suggesting that A549 cells can be used as a model to study lower airway epithelial cell responses to hMPV infection. A549 secreted a variety of CXC and CC chemokines, cytokines and type I interferons, following hMPV infection. hMPV was also a strong inducer of transcription factors belonging to nuclear factor (NF)-κB, interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) families, which are known to orchestrate the expression of inflammatory and immuno-modulatory mediators. PMID:17655903

  2. Apicobasal polarity controls lymphocyte adhesion to hepatic epithelial cells.

    PubMed

    Reglero-Real, Natalia; Alvarez-Varela, Adrián; Cernuda-Morollón, Eva; Feito, Jorge; Marcos-Ramiro, Beatriz; Fernández-Martín, Laura; Gómez-Lechón, Maria José; Muntané, Jordi; Sandoval, Pilar; Majano, Pedro L; Correas, Isabel; Alonso, Miguel A; Millán, Jaime

    2014-09-25

    Loss of apicobasal polarity is a hallmark of epithelial pathologies. Leukocyte infiltration and crosstalk with dysfunctional epithelial barriers are crucial for the inflammatory response. Here, we show that apicobasal architecture regulates the adhesion between hepatic epithelial cells and lymphocytes. Polarized hepatocytes and epithelium from bile ducts segregate the intercellular adhesion molecule 1 (ICAM-1) adhesion receptor onto their apical, microvilli-rich membranes, which are less accessible by circulating immune cells. Upon cell depolarization, hepatic ICAM-1 becomes exposed and increases lymphocyte binding. Polarized hepatic cells prevent ICAM-1 exposure to lymphocytes by redirecting basolateral ICAM-1 to apical domains. Loss of ICAM-1 polarity occurs in human inflammatory liver diseases and can be induced by the inflammatory cytokine tumor necrosis factor alpha (TNF-α). We propose that adhesion receptor polarization is a parenchymal immune checkpoint that allows functional epithelium to hamper leukocyte binding. This contributes to the haptotactic guidance of leukocytes toward neighboring damaged or chronically inflamed epithelial cells that expose their adhesion machinery. PMID:25242329

  3. SIGNALING HIERARCHY THAT REGULATES ENDOTHELIAL CELL PROLIFERATION AND VASCULAR REMODELING DURING VASCULOGENESIS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We previously demonstrated that during vascular morphogenesis, retinoic acid (RA) is required for the control of endothelial cell proliferation and capillary plexus remodeling. In the present studies, we define the signaling hierarchy downstream of RA that independently regulates these cellular eve...

  4. Stem cell mechanisms during left ventricular remodeling post-myocardial infarction: Repair and regeneration

    PubMed Central

    Zamilpa, Rogelio; Navarro, Mary M; Flores, Iris; Griffey, Sy

    2014-01-01

    Post-myocardial infarction (MI), the left ventricle (LV) undergoes a series of events collectively referred to as remodeling. As a result, damaged myocardium is replaced with fibrotic tissue consequently leading to contractile dysfunction and ultimately heart failure. LV remodeling post-MI includes inflammatory, fibrotic, and neovascularization responses that involve regulated cell recruitment and function. Stem cells (SCs) have been transplanted post-MI for treatment of LV remodeling and shown to improve LV function by reduction in scar tissue formation in humans and animal models of MI. The promising results obtained from the application of SCs post-MI have sparked a massive effort to identify the optimal SC for regeneration of cardiomyocytes and the paradigm for clinical applications. Although SC transplantations are generally associated with new tissue formation, SCs also secrete cytokines, chemokines and growth factors that robustly regulate cell behavior in a paracrine fashion during the remodeling process. In this review, the different types of SCs used for cardiomyogenesis, markers of differentiation, paracrine factor secretion, and strategies for cell recruitment and delivery are addressed. PMID:25068021

  5. Epithelial cell adhesion and gastrointestinal colonization of Lactobacillus in poultry.

    PubMed

    Spivey, Megan A; Dunn-Horrocks, Sadie L; Duong, Tri

    2014-11-01

    Administration of probiotic Lactobacillus cultures is an important alternative to the use of antibiotic growth promoters and has been demonstrated to improve animal health, growth performance, and preharvest food safety in poultry production. Whereas gastrointestinal colonization is thought to be critical to their probiotic functionality, factors important to Lactobacillus colonization in chickens are not well understood. In this study we investigate epithelial cell adhesion in vitro and colonization of Lactobacillusin vivo in broiler chickens. Adhesion of Lactobacillus cultures to epithelial cells was evaluated using the chicken LMH cell line. Lactobacillus cultures were able adhere effectively to LMH cells relative to Bacillus subtilis and Salmonella Typhimurium. Epithelial cell adhesion was similar for Lactobacillus crispatus TDCC 75, L. cristpatus TDCC 76, and Lactobacillus gallinarum TDCC 77, and all 3 were more adherent than L. gallinarum TDCC 78. However, when colonization was evaluated in the ileum and cecum of broiler chicks, L. crispatus TDCC 75 and L. gallinarum TDCC 77 were more persistent than L. crispatus TDCC 76 and L. gallinarum TDCC 78. The reduction of growth in medium supplemented with oxgal was greater for L. gallinarum TDCC 78 than L. gallinarum TDCC 77, suggesting that whereas adhesion was similar for the 2 strains, the difference in colonization between L. gallinarum strains may be due in part to their bile sensitivity. This study demonstrates that whereas adhesion to epithelial cells may be important in predicting gastrointestinal colonization, other factors including bile tolerance may also contribute to the colonization of Lactobacillus in poultry. Additionally, the chicken LMH cell line is expected to provide a platform for investigating mechanisms of Lactobacillus adhesion to epithelial tissue and evaluating the probiotic potential Lactobacillus in poultry. PMID:25239531

  6. Mitochondrial Biogenesis and Proteome Remodeling Promote One-Carbon Metabolism for T Cell Activation.

    PubMed

    Ron-Harel, Noga; Santos, Daniel; Ghergurovich, Jonathan M; Sage, Peter T; Reddy, Anita; Lovitch, Scott B; Dephoure, Noah; Satterstrom, F Kyle; Sheffer, Michal; Spinelli, Jessica B; Gygi, Steven; Rabinowitz, Joshua D; Sharpe, Arlene H; Haigis, Marcia C

    2016-07-12

    Naive T cell stimulation activates anabolic metabolism to fuel the transition from quiescence to growth and proliferation. Here we show that naive CD4(+) T cell activation induces a unique program of mitochondrial biogenesis and remodeling. Using mass spectrometry, we quantified protein dynamics during T cell activation. We identified substantial remodeling of the mitochondrial proteome over the first 24 hr of T cell activation to generate mitochondria with a distinct metabolic signature, with one-carbon metabolism as the most induced pathway. Salvage pathways and mitochondrial one-carbon metabolism, fed by serine, contribute to purine and thymidine synthesis to enable T cell proliferation and survival. Genetic inhibition of the mitochondrial serine catabolic enzyme SHMT2 impaired T cell survival in culture and antigen-specific T cell abundance in vivo. Thus, during T cell activation, mitochondrial proteome remodeling generates specialized mitochondria with enhanced one-carbon metabolism that is critical for T cell activation and survival. PMID:27411012

  7. Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm

    SciTech Connect

    Alberio, Ramiro; Johnson, Andrew D.; Stick, Reimer; Campbell, Keith H.S. . E-mail: keith.campbell@nottingham.ac.uk

    2005-07-01

    The mechanisms governing nuclear reprogramming have not been fully elucidated yet; however, recent studies show a universally conserved ability of both oocyte and egg components to reprogram gene expression in somatic cells. The activation of genes associated with pluripotency by oocyte/egg components may require the remodeling of nuclear structures, such that they can acquire the features of early embryos and pluripotent cells. Here, we report on the remodeling of the nuclear lamina of mammalian cells by Xenopus oocyte and egg extracts. Lamin A/C is removed from somatic cells incubated in oocyte and egg extracts in an active process that requires permeable nuclear pores. Removal of lamin A/C is specific, since B-type lamins are not changed, and it is not dependent on the incorporation Xenopus egg specific lamin III. Moreover, transcriptional activity is differentially regulated in somatic cells incubated in the extracts. Pol I and II transcriptions are maintained in cells in oocyte extracts; however, both activities are abolished in egg extracts. Our study shows that components of oocyte and egg extracts can modify the nuclear lamina of somatic cells and that this nuclear remodeling induces a structural change in the nucleus which may have implications for transcriptional activity. These experiments suggest that modifications in the nuclear lamina structure by the removal of somatic proteins and the incorporation of oocyte/egg components may contribute to the reprogramming of somatic cell nuclei and may define a characteristic configuration of pluripotent cells.

  8. Modulation of Candida albicans attachment to human epithelial cells by bacteria and carbohydrates.

    PubMed Central

    Centeno, A; Davis, C P; Cohen, M S; Warren, M M

    1983-01-01

    The effects of carbohydrates (mannose and dextrose). Escherichia coli 07KL. and Klebsiella pneumoniae on Candida albicans attachment to epithelial cells was studied. Dextrose had no effect on yeast attachment to epithelial cells. Conversely, mannose significantly decreased both yeast and piliated bacterial attachment (E. coli 07KL, heavily piliated K. pneumoniae) whereas having no effect on nonpiliated K. pneumoniae attachment to epithelial cells. The number of yeasts attaching to epithelial cells was enhanced by preincubation of epithelial cells with piliated strains of bacteria, whereas preincubation with nonpiliated strains of bacteria had no effect on yeast attachment. Scanning electron microscopy showed that piliated bacteria and yeasts were juxtaposed on the epithelial cell surface. These data suggest that certain piliated strains of bacteria can enhance C. albicans attachment to epithelial cells and that type 1 pili of bacteria can be a factor in the enhanced attachment of C. albicans to epithelial cells. Images PMID:6132878

  9. Renal epithelial cells can release ATP by vesicular fusion

    PubMed Central

    Bjaelde, Randi G.; Arnadottir, Sigrid S.; Overgaard, Morten T.; Leipziger, Jens; Praetorius, Helle A.

    2013-01-01

    Renal epithelial cells have the ability to release nucleotides as paracrine factors. In the intercalated cells of the collecting duct, ATP is released by connexin30 (cx30), which is selectively expressed in this cell type. However, ATP is released by virtually all renal epithelia and the aim of the present study was to identify possible alternative nucleotide release pathways in a renal epithelial cell model. We used MDCK (type1) cells to screen for various potential ATP release pathways. In these cells, inhibition of the vesicular H+-ATPases (bafilomycin) reduced both the spontaneous and hypotonically (80%)-induced nucleotide release. Interference with vesicular fusion using N-ethylamide markedly reduced the spontaneous nucleotide release, as did interference with trafficking from the endoplasmic reticulum to the Golgi apparatus (brefeldin A1) and vesicular transport (nocodazole). These findings were substantiated using a siRNA directed against SNAP-23, which significantly reduced spontaneous ATP release. Inhibition of pannexin and connexins did not affect the spontaneous ATP release in this cell type, which consists of ~90% principal cells. TIRF-microscopy of either fluorescently-labeled ATP (MANT-ATP) or quinacrine-loaded vesicles, revealed that spontaneous release of single vesicles could be promoted by either hypoosmolality (50%) or ionomycin. This vesicular release decreased the overall cellular fluorescence by 5.8 and 7.6% respectively. In summary, this study supports the notion that spontaneous and induced ATP release can occur via exocytosis in renal epithelial cells. PMID:24065923

  10. Lung epithelial cell death induced by oil-dispersant mixtures.

    PubMed

    Wang, He; Shi, Yongli; Major, Danielle; Yang, Zhanjun

    2012-08-01

    The dispersants used in oil spill disasters are claimed to be safe, but increased solubility of high-molecular-weight components in crude oil is of public health concern. The water-accommodated fractions (WAF) of crude oil mixed with dispersants may become airborne and cause lung epithelial damage when inhaled. This study was designed to examine the cell death and related death pathways of lung epithelial cells in response to WAF. Cultured A549 cells were treated for 2 or 24h with different concentrations of WAF. The WAF was prepared by mixing each of the dispersants (Corexit EC9527A, Corexit EC9500A and Corexit EC9580A) with crude oil for extraction with PBS. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay, lactate dehydrogenase assay, morphology and cleaved caspase 9 protein, and microtubule-associated protein 1 light chain 3 were all used to measure cell viability, necrosis, apoptosis and autophagy quantitation, respectively. Results showed that the WAF of oil-dispersant mixtures caused cell death in the lung epithelial cells, in a dose-dependent manner, with the major cellular pathways of necrosis and apoptosis involved. Autophagy also occurred in cells exposed to WAF mixtures at lower concentrations before any detectable cell death, indicating greater sensitivity to WAF exposure. The three types of cell behavior, namely necrosis, apoptosis and autophagy, may play different roles in oil spill-related respiratory disorders. PMID:22504303

  11. Chronic stress in mice remodels lymph vasculature to promote tumour cell dissemination

    PubMed Central

    Le, Caroline P.; Nowell, Cameron J.; Kim-Fuchs, Corina; Botteri, Edoardo; Hiller, Jonathan G.; Ismail, Hilmy; Pimentel, Matthew A.; Chai, Ming G.; Karnezis, Tara; Rotmensz, Nicole; Renne, Giuseppe; Gandini, Sara; Pouton, Colin W.; Ferrari, Davide; Möller, Andreas; Stacker, Steven A.; Sloan, Erica K.

    2016-01-01

    Chronic stress induces signalling from the sympathetic nervous system (SNS) and drives cancer progression, although the pathways of tumour cell dissemination are unclear. Here we show that chronic stress restructures lymphatic networks within and around tumours to provide pathways for tumour cell escape. We show that VEGFC derived from tumour cells is required for stress to induce lymphatic remodelling and that this depends on COX2 inflammatory signalling from macrophages. Pharmacological inhibition of SNS signalling blocks the effect of chronic stress on lymphatic remodelling in vivo and reduces lymphatic metastasis in preclinical cancer models and in patients with breast cancer. These findings reveal unanticipated communication between stress-induced neural signalling and inflammation, which regulates tumour lymphatic architecture and lymphogenous tumour cell dissemination. These findings suggest that limiting the effects of SNS signalling to prevent tumour cell dissemination through lymphatic routes may provide a strategy to improve cancer outcomes. PMID:26925549

  12. Chronic stress in mice remodels lymph vasculature to promote tumour cell dissemination.

    PubMed

    Le, Caroline P; Nowell, Cameron J; Kim-Fuchs, Corina; Botteri, Edoardo; Hiller, Jonathan G; Ismail, Hilmy; Pimentel, Matthew A; Chai, Ming G; Karnezis, Tara; Rotmensz, Nicole; Renne, Giuseppe; Gandini, Sara; Pouton, Colin W; Ferrari, Davide; Möller, Andreas; Stacker, Steven A; Sloan, Erica K

    2016-01-01

    Chronic stress induces signalling from the sympathetic nervous system (SNS) and drives cancer progression, although the pathways of tumour cell dissemination are unclear. Here we show that chronic stress restructures lymphatic networks within and around tumours to provide pathways for tumour cell escape. We show that VEGFC derived from tumour cells is required for stress to induce lymphatic remodelling and that this depends on COX2 inflammatory signalling from macrophages. Pharmacological inhibition of SNS signalling blocks the effect of chronic stress on lymphatic remodelling in vivo and reduces lymphatic metastasis in preclinical cancer models and in patients with breast cancer. These findings reveal unanticipated communication between stress-induced neural signalling and inflammation, which regulates tumour lymphatic architecture and lymphogenous tumour cell dissemination. These findings suggest that limiting the effects of SNS signalling to prevent tumour cell dissemination through lymphatic routes may provide a strategy to improve cancer outcomes. PMID:26925549

  13. Metabolic cooperativity between epithelial cells and adipocytes of mice

    SciTech Connect

    Bartley, J.C.; Emerman, J.T.; Bissell, M.J.

    1981-01-01

    We have demonstrated that glycogen and lipid synthesis in adipocytes is modulated by the lactational state and that this modulation in mammary adipocytes requires the presence of the adjacent epithelial cells. Glycogen and lipid synthesis from (/sup 14/C)glucose was measured in mammary fat pads cleared of epithelium, in abdominal fat pads, and in adipocytes from both sources and from intact mammary gland of mature virgin, pregnant, and lactating mice. Accumulation of glycogen, the activity of glycogen synthase, and the lipogenic rate in abdominal and mammary adipocytes remained high during pregnancy but decreased to insignificant levels by early lactation. The depressant effects of lactation were observed solely in those mammary adipocytes isolated from intact glands. The presence of mammary epithelial cells was also required to effect the stimulated lipogenesis in mammary adipocytes during pregnancy. We conclude that the metabolic activity of adipocytes is modulated both during pregnancy and lactation to channel nutrients to the mammary epithelial cell. The fact that the changes occur in mammary adipocytes only when epithelial cells are present indicates that local as well as systemic factors are operating in these modulations.

  14. NITROTYROSINE ATTENUATES RSV-INDUCED INFLAMMATION IN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    Nitrotyrosine attenuates RSV-induced inflammation in airway epithelial cells. Joleen Soukup, Zuowei Li, Susanne Becker and Yuh-Chin Huang. NHEERL, ORD, USEPA, RTP, North Carolina, CEMALB, University of North Carolina, Chapel Hill, North Carolina

    Nitrotyrosine (NO2Tyr) is a...

  15. Interactions between Periodontal Bacteria and Human Oral Epithelial Cells: Fusobacterium nucleatum Adheres to and Invades Epithelial Cells

    PubMed Central

    Han, Yiping W.; Shi, Wenyuan; Huang, George T.-J.; Kinder Haake, Susan; Park, No-Hee; Kuramitsu, Howard; Genco, Robert J.

    2000-01-01

    Bacteria are causative agents of periodontal diseases. Interactions between oral bacteria and gingival epithelial cells are essential aspects of periodontal infections. Using an in vitro tissue culture model, a selected group of gram-negative anaerobic bacteria frequently associated with periodontal diseases, including Bacteroides forsythus, Campylobacter curvus, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia, were examined for their ability to adhere to and invade primary cultures of human gingival epithelial cells (HGEC). The effects of these bacteria on the production of interleukin-8 (IL-8), a proinflammatory chemokine, were also measured. These studies provided an initial demonstration that F. nucleatum adhered to and invaded HGEC and that this was accompanied by high levels of IL-8 secretion from the epithelial cells. The attachment and invasion characteristics of F. nucleatum were also tested using KB cells, an oral epithelial cell line. The invasion was verified by transmission electron microscopy and with metabolic inhibitors. Invasion appeared to occur via a “zipping” mechanism and required the involvement of actins, microtubules, signal transduction, protein synthesis, and energy metabolism of the epithelial cell, as well as protein synthesis by F. nucleatum. A spontaneous mutant, lam, of F. nucleatum, isolated as defective in autoagglutination, was unable to attach to or invade HGEC or KB cells, further indicating the requirement of bacterial components in these processes. Sugar inhibition assays indicated that lectin-like interactions were involved in the attachment of F. nucleatum to KB cells. Investigation of these new virulence phenotypes should improve our understanding of the role of F. nucleatum in periodontal infections. PMID:10816455

  16. Culture and characterization of human junctional epithelial cells.

    PubMed

    Matsuyama, T; Izumi, Y; Sueda, T

    1997-03-01

    This study was undertaken to establish a culture of junctional epithelial cells derived from gingival tissue attached to the tooth surface and to characterize these cells immunocytochemically and ultrastructurally. Primary cultures of cells were obtained from the junctional tissue explanted on type I collagen-coated dishes and immersed in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS). Cells were subcultured with conditioned serum-free keratinocyte medium (keratinocyte-SFM + 5% FBS) on dishes coated with solubilized extract of the basement membrane. After 24 hours, the medium was changed to keratinocyte-SFM (0.09 mM Ca2+). The cell-doubling time was 40.5 hours. As a control, cells from gingival tissue were cultured by the same method. Cells from junctional tissue and gingival tissue were compared immunocytochemically using monoclonal antibodies to keratin, vimentin, and desmoplakins I and II and using Dolichos biflorus agglutinin (DBA). The keratin AE1 and AE3 was expressed by all of culture cells. The vimentin (specific for the intermediate filament of mesenchymal cells) was also expressed by all cells. The expression pattern of keratin 19 was observed not only by cells from junctional tissue but also by cells from gingival tissue. All keratin peptides were expressed in both cells. However, DBA reacted only with cells from the junctional tissue. Anti-desmoplakin I and II reacted with both cells, however, the staining patterns differed. DBA-positive cultured epithelial cells from the junctional tissue showed poor tonofilament bundles and were rich in cytoplasmic organelles. These findings suggest that junctional epithelial cells can be isolated from junctional tissue and cultured under improved conditions. PMID:9100198

  17. Lactobacillus equigenerosi Strain Le1 Invades Equine Epithelial Cells

    PubMed Central

    Botha, Marlie; Botes, Marelize; Loos, Ben; Smith, Carine

    2012-01-01

    Lactobacillus equigenerosi strain Le1, a natural inhabitant of the equine gastrointestinal tract, survived pH 3.0 and incubation in the presence of 1.5% (wt/vol) bile salts for at least 2 h. Strain Le1 showed 8% cell surface hydrophobicity, 60% auto-aggregation, and 47% coaggregation with Clostridium difficile C6. Only 1% of the cells adhered to viable buccal epithelial cells and invaded the cells within 20 min after contact. Preincubation of strain Le1 in a buffer containing pronase prevented adhesion to viable epithelial cells. Preincubation in a pepsin buffer delayed invasion from 20 min to 1 h. Strain Le1 did not adhere to nonviable epithelial cells. Administration of L. equigenerosi Le1 (1 × 109 CFU per 50 kg body weight) to healthy horses did not increase white blood cell numbers. Differential white blood cell counts and aspartate aminotransferase levels remained constant. Glucose, lactate, cholesterol, and urea levels remained constant during administration with L. equigenerosi Le1 but decreased during the week after administration. PMID:22504808

  18. Differentiation of cultured epithelial cells: Response to toxic agents

    SciTech Connect

    Rice, R.H.; LaMontagne, A.D.; Petito, C.T.; Rong, Xianhui )

    1989-03-01

    Cell culture systems are instrumental in elucidating regulation of normal function and mechanisms of its perturbation by toxic substances. To this end, three applications of epithelial cells cultured with 3T3 feeder layer support are described. First, treatment of the premalignant human epidermal keratinocyte line SCC-12F2 with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate suppressed cell growth and differentiation. This agent produced a biphasic growth response greatly inhibiting cell growth at 1 to 10 nM, but much less above 100 nM. Expression of the differentiated functions involucrin and transglutaminase was found to be inhibited markedly at concentrations above 10 nM. Second, 3-methylcholanthrene toxicity was surveyed in a variety of rat epithelial cell types. The two most sensitive to growth inhibition were epidermal and mammary epithelial cells, while those from bladder, prostate, thyroid, and endometrium were insensitive to growth inhibition. Finally, expression of estrogen receptors in rat endometrial cells was shown to be stimulated by the cAmP-elevating agent forskolin. Maximal stimulation of 3- to 6-fold occurred in 6 hr, compatible with a requirement for protein synthesis. Pursuit of such results will aid in understanding differences in response among cell types and species, in elucidating mechanisms of action of known toxic substances and, ultimately, in predicting toxicity of less well understood agents.

  19. Collective Epithelial and Mesenchymal Cell Migration During Gastrulation

    PubMed Central

    Chuai, Manli; Hughes, David; Weijer, Cornelis J

    2012-01-01

    Gastrulation, the process that puts the three major germlayers, the ectoderm, mesoderm and endoderm in their correct topological position in the developing embryo, is characterised by extensive highly organised collective cell migration of epithelial and mesenchymal cells. We discuss current knowledge and insights in the mechanisms controlling these cell behaviours during gastrulation in the chick embryo. We discuss several ideas that have been proposed to explain the observed large scale vortex movements of epithelial cells in the epiblast during formation of the primitive streak. We review current insights in the control and execution of the epithelial to mesenchymal transition (EMT) underlying the formation of the hypoblast and the ingression of the mesendoderm cells through the streak. We discuss the mechanisms by which the mesendoderm cells move, the nature and dynamics of the signals that guide these movements, as well as the interplay between signalling and movement that result in tissue patterning and morphogenesis. We argue that instructive cell-cell signaling and directed chemotactic movement responses to these signals are instrumental in the execution of all phases of gastrulation. PMID:23204916

  20. Ciliary neurotrophic factor promotes the activation of corneal epithelial stem/progenitor cells and accelerates corneal epithelial wound healing.

    PubMed

    Zhou, Qingjun; Chen, Peng; Di, Guohu; Zhang, Yangyang; Wang, Yao; Qi, Xia; Duan, Haoyun; Xie, Lixin

    2015-05-01

    Ciliary neurotrophic factor (CNTF), a well-known neuroprotective cytokine, has been found to play an important role in neurogenesis and functional regulations of neural stem cells. As one of the most innervated tissue, however, the role of CNTF in cornea epithelium remains unclear. This study was to explore the roles and mechanisms of CNTF in the activation of corneal epithelial stem/progenitor cells and wound healing of both normal and diabetic mouse corneal epithelium. In mice subjecting to mechanical removal of corneal epithelium, the corneal epithelial stem/progenitor cell activation and wound healing were promoted by exogenous CNTF application, while delayed by CNTF neutralizing antibody. In cultured corneal epithelial stem/progenitor cells, CNTF enhanced the colony-forming efficiency, stimulated the mitogenic proliferation, and upregulated the expression levels of corneal epithelial stem/progenitor cell-associated transcription factors. Furthermore, the promotion of CNTF on the corneal epithelial stem/progenitor cell activation and wound healing was mediated by the activation of STAT3. Moreover, in diabetic mice, the content of CNTF in corneal epithelium decreased significantly when compared with that of normal mice, and the supplement of CNTF promoted the diabetic corneal epithelial wound healing, accompanied with the advanced activation of corneal epithelial stem/progenitor cells and the regeneration of corneal nerve fibers. Thus, the capability of expanding corneal epithelial stem/progenitor cells and promoting corneal epithelial wound healing and nerve regeneration indicates the potential application of CNTF in ameliorating limbal stem cell deficiency and treating diabetic keratopathy. PMID:25546438

  1. Radical-containing ultrafine particulate matter initiates epithelial-to-mesenchymal transitions in airway epithelial cells.

    PubMed

    Thevenot, Paul T; Saravia, Jordy; Jin, Nili; Giaimo, Joseph D; Chustz, Regina E; Mahne, Sarah; Kelley, Matthew A; Hebert, Valeria Y; Dellinger, Barry; Dugas, Tammy R; Demayo, Francesco J; Cormier, Stephania A

    2013-02-01

    Environmentally persistent free radicals (EPFRs) in combustion-generated particulate matter (PM) are capable of inducing pulmonary pathologies and contributing to the development of environmental asthma. In vivo exposure of infant rats to EPFRs demonstrates their ability to induce airway hyperresponsiveness to methacholine, a hallmark of asthma. However, the mechanisms by which combustion-derived EPFRs elicit in vivo responses remain elusive. In this study, we used a chemically defined EPFR consisting of approximately 0.2 μm amorphrous silica containing 3% cupric oxide with the organic pollutant 1,2-dichlorobenzene (DCB-230). DCB-230 possesses similar radical content to urban-collected EPFRs but offers several advantages, including lack of contaminants and chemical uniformity. DCB-230 was readily taken up by BEAS-2B and at high doses (200 μg/cm(2)) caused substantial necrosis. At low doses (20 μg/cm(2)), DCB-230 particles caused lysosomal membrane permeabilization, oxidative stress, and lipid peroxidation within 24 hours of exposure. During this period, BEAS-2B underwent epithelial-to-mesenchymal transition (EMT), including loss of epithelial cell morphology, decreased E-cadherin expression, and increased α-smooth muscle actin (α-SMA) and collagen I production. Similar results were observed in neonatal air-liquid interface culture (i.e., disruption of epithelial integrity and EMT). Acute exposure of infant mice to DCB-230 resulted in EMT, as confirmed by lineage tracing studies and evidenced by coexpression of epithelial E-cadherin and mesenchymal α-SMA proteins in airway cells and increased SNAI1 expression in the lungs. EMT in neonatal mouse lungs after EPFR exposure may provide an explanation for epidemiological evidence supporting PM exposure and increased risk of asthma. PMID:23087054

  2. Modeling Alveolar Epithelial Cell Behavior In Spatially Designed Hydrogel Microenvironments

    NASA Astrophysics Data System (ADS)

    Lewis, Katherine Jean Reeder

    The alveolar epithelium consists of two cell phenotypes, elongated alveolar type I cells (AT1) and rounded alveolar type II cells (ATII), and exists in a complex three-dimensional environment as a polarized cell layer attached to a thin basement membrane and enclosing a roughly spherical lumen. Closely surrounding the alveolar cysts are capillary endothelial cells as well as interstitial pulmonary fibroblasts. Many factors are thought to influence alveolar epithelial cell differentiation during lung development and wound repair, including physical and biochemical signals from the extracellular matrix (ECM), and paracrine signals from the surrounding mesenchyme. In particular, disrupted signaling between the alveolar epithelium and local fibroblasts has been implicated in the progression of several pulmonary diseases. However, given the complexity of alveolar tissue architecture and the multitude of signaling pathways involved, designing appropriate experimental platforms for this biological system has been difficult. In order to isolate key factors regulating cellular behavior, the researcher ideally should have control over biophysical properties of the ECM, as well as the ability to organize multiple cell types within the scaffold. This thesis aimed to develop a 3D synthetic hydrogel platform to control alveolar epithelial cyst formation, which could then be used to explore how extracellular cues influence cell behavior in a tissue-relevant cellular arrangement. To accomplish this, a poly(ethylene glycol) (PEG) hydrogel network containing enzymatically-degradable crosslinks and bioadhesive pendant peptides was employed as a base material for encapsulating primary alveolar epithelial cells. First, an array of microwells of various cross-sectional shapes was photopatterned into a PEG gel containing photo-labile crosslinks, and primary ATII cells were seeded into the wells to examine the role of geometric confinement on differentiation and multicellular arrangement

  3. Single cell mass cytometry reveals remodeling of human T cell phenotypes by varicella zoster virus.

    PubMed

    Sen, Nandini; Mukherjee, Gourab; Arvin, Ann M

    2015-11-15

    The recent application of mass cytometry (CyTOF) to biology provides a 'systems' approach to monitor concurrent changes in multiple host cell factors at the single cell level. We used CyTOF to evaluate T cells infected with varicella zoster virus (VZV) infection, documenting virus-mediated phenotypic and functional changes caused by this T cell tropic human herpesvirus. Here we summarize our findings using two complementary panels of antibodies against surface and intracellular signaling proteins to elucidate the consequences of VZV-mediated perturbations on the surface and in signaling networks of infected T cells. CyTOF data was analyzed by several statistical, analytical and visualization tools including hierarchical clustering, orthogonal scaling, SPADE, viSNE, and SLIDE. Data from the mass cytometry studies demonstrated that VZV infection led to 'remodeling' of the surface architecture of T cells, promoting skin trafficking phenotypes and associated with concomitant activation of T-cell receptor and PI3-kinase pathways. This method offers a novel approach for understanding viral interactions with differentiated host cells important for pathogenesis. PMID:26213183

  4. Nesfatin-1 inhibits ovarian epithelial carcinoma cell proliferation in vitro

    SciTech Connect

    Xu, Yang; Pang, Xiaoyan; Dong, Mei; Wen, Fang Zhang, Yi

    2013-11-01

    Highlights: •Nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest. •Nesfatin-1 enhances HO-8910 cell apoptosis. •Nesfatin-1 inhibits HO-8910 cell proliferation via mTOR and RhoA/ROCK signaling pathway. •The first report of nesfatin-1-mediated proliferation in ovarian epithelial carcinoma. -- Abstract: Nesfatin-1, an 82-amino-acid peptide derived from a 396-amino-acid precursor protein nucleobindin 2 (NUCB2), was originally identified in hypothalamic nuclei involved in the regulation of food intake. It was recently reported that nesfatin-1 is a novel depot specific adipokine preferentially produced by subcutaneous tissue, with obesity- and food deprivation-regulated expression. Although a relation between ovarian cancer mortality and obesity has been previously established, a role of nesfatin-1 in ovarian epithelial carcinoma remains unknown. The aim of the present study is to examine the effect of nesfatin-1 on ovary carcinoma cells proliferation. We found that nesfatin-1 inhibits the proliferation and growth of HO-8910 cells by G1 phase arrest, this inhibition could be abolished by nesfatin-1 neutralizing antibody. Nesfatin-1 enhances HO-8910 cell apoptosis, activation of mammalian target of rapamycin (mTOR) and RhoA/ROCK signaling pathway block the effects of nesfatin-1-induced apoptosis, therefore reverses the inhibition of HO-8910 cell proliferation by nesfatin-1. In conclusion, the present study demonstrated that nesfatin-1 can inhibit the proliferation in human ovarian epithelial carcinoma cell line HO-8910 cells through inducing apoptosis via mTOR and RhoA/ROCK signaling pathway. This study provides a novel regulatory signaling pathway of nesfatin-1-regulated ovarian epithelial carcinoma growth and may contribute to ovarian cancer prevention and therapy, especially in obese patients.

  5. Cytotoxic Effect of Lipophilic Bismuth Dimercaptopropanol Nanoparticles on Epithelial Cells.

    PubMed

    Rene, Hernandez-Delgadillo; Badireddy, Appala Raju; José, Martínez-Sanmiguel Juan; Francisco, Contreras-Cordero Juan; Israel, Martinez-Gonzalez Gustavo; Isela, Sánchez-Nájera Rosa; Chellam, Shankararaman; Claudio, Cabral-Romero

    2016-01-01

    Bismuth nanoparticles have many interesting properties to be applied in biomedical and medicinal sectors, however their safety in humans have not been comprehensively investigated. The objective of this research was to determine the cytotoxic effect of bismuth dimercaptopropanol nanoparticles (BisBAL NPs) on epithelial cells. The nanoparticles are composed of 18.7 nm crystallites on average and have a rhombohedral structure, agglomerating into chains-like or clusters of small nanoparticles. Based on MTT viability assay and fluorescence microscopy, cytotoxicity was not observed on monkey kidney cells after growing with 5 µM of BisBAL NPs for 24 h. Employing same techniques, identical results were obtained with human epithelial cells (HeLa), showing a not strain-dependent phenomenon. The absence of toxic effects on epithelial cells growing with BisBAL NPs was corroborated with long-time experiments (24-72 hrs.), showing no difference in comparison with growing control (cells without nanoparticles). Further, genotoxicity assays, comet assay and fluorescent microscopy and electrophoresis in bromide-stained agarose gel revealed no damage to genomic DNA of MA104 cells after 24 h. of exposition to BisBAL NPs. Finally, the effect of bismuth nanoparticles on protein synthesis was studied in cells growing with BisBAL NPs for 24 h. SDS-PAGE assays showed no difference between treated and untreated cells, suggesting that BisBAL NPs did not interfere with protein synthesis. Hence BisBAL NPs do not appear to exert cytotoxic effects suggesting their biological compatibility with epithelial cells. PMID:27398446

  6. Epigenetic Regulation of Stem Cell Maintenance in the Drosophila Testis via the Nucleosome Remodeling Factor NURF

    PubMed Central

    Cherry, Christopher M.; Matunis, Erika L.

    2010-01-01

    SUMMARY Regulation of stem cells depends on both tissue-specific transcriptional regulators and changes in chromatin organization, yet the coordination of these events in endogenous niches is poorly understood. In the Drosophila testis, local JAK-STAT signaling maintains germline and somatic stem cells (GSCs and cyst progenitor cells, or CPCs) in a single niche. Here we show that epigenetic regulation via the nucleosome remodeling factor (NURF) complex ensures GSC and CPC maintenance by positively regulating JAK-STAT signaling, thereby preventing premature differentiation. Conversely, NURF is not required in early differentiating daughter cells of either lineage. Since three additional ATP-dependent chromatin remodelers (ACF, CHRAC, and dMi-2/NuRD) are dispensable for stem cell maintenance in the testis, epigenetic regulation of stem cells within this niche may rely primarily on NURF. Thus, local signals cooperate with specific chromatin remodeling complexes in intact niches to coordinately regulate a common set of target genes to prevent premature stem cell differentiation. PMID:20569693

  7. Differentiated kidney epithelial cells repair injured proximal tubule.

    PubMed

    Kusaba, Tetsuro; Lalli, Matthew; Kramann, Rafael; Kobayashi, Akio; Humphreys, Benjamin D

    2014-01-28

    Whether kidney proximal tubule harbors a scattered population of epithelial stem cells is a major unsolved question. Lineage-tracing studies, histologic characterization, and ex vivo functional analysis results conflict. To address this controversy, we analyzed the lineage and clonal behavior of fully differentiated proximal tubule epithelial cells after injury. A CreER(T2) cassette was knocked into the sodium-dependent inorganic phosphate transporter SLC34a1 locus, which is expressed only in differentiated proximal tubule. Tamoxifen-dependent recombination was absolutely specific to proximal tubule. Clonal analysis after injury and repair showed that the bulk of labeled cells proliferate after injury with increased clone size after severe compared with mild injury. Injury to labeled proximal tubule epithelia induced expression of CD24, CD133, vimentin, and kidney-injury molecule-1, markers of putative epithelial stem cells in the human kidney. Similar results were observed in cultured proximal tubules, in which labeled clones proliferated and expressed dedifferentiation and injury markers. When mice with completely labeled kidneys were subject to injury and repair there was no dilution of fate marker despite substantial proliferation, indicating that unlabeled progenitors do not contribute to kidney repair. During nephrogenesis and early kidney growth, single proximal tubule clones expanded, suggesting that differentiated cells also contribute to tubule elongation. These findings provide no evidence for an intratubular stem-cell population, but rather indicate that terminally differentiated epithelia reexpress apparent stem-cell markers during injury-induced dedifferentiation and repair. PMID:24127583

  8. Interleukin-22 Promotes Intestinal Stem Cell-Mediated Epithelial Regeneration

    PubMed Central

    Dudakov, Jarrod A.; Jenq, Robert R.; Velardi, Enrico; Young, Lauren F.; Smith, Odette M.; Lawrence, Gillian; Ivanov, Juliet A.; Fu, Ya-Yuan; Takashima, Shuichiro; Hua, Guoqiang; Martin, Maria L.; O'Rourke, Kevin P.; Lo, Yuan-Hung; Mokry, Michal; Romera-Hernandez, Monica; Cupedo, Tom; Dow, Lukas; Nieuwenhuis, Edward E.; Shroyer, Noah F.; Liu, Chen; Kolesnick, Richard

    2015-01-01

    Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch, and epidermal growth factor (EGF) signals supporting Lgr5+ crypt base columnar ISCs for normal epithelial maintenance1,2. However, little is known about the regulation of the ISC compartment after tissue damage. Utilizing ex vivo organoid cultures, we provide evidence that innate lymphoid cells (ILCs), potent producers of Interleukin-22 (IL-22) after intestinal injury3,4, increased the growth of murine small intestine (SI) organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both murine and human intestinal organoids, increasing proliferation, and promoting ISC expansion. IL-22 induced Stat3 phosphorylation in Lgr5+ ISCs, and Stat3 was critical for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after murine allogeneic bone marrow transplantation (BMT) enhanced recovery of ISCs, increased epithelial regeneration, and reduced intestinal pathology and mortality from graft vs. host disease (GVHD). Atoh1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independent of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support intestinal epithelium, activating ISCs to promote regeneration. PMID:26649819

  9. Notch Signaling in Meibomian Gland Epithelial Cell Differentiation

    PubMed Central

    Gidfar, Sanaz; Afsharkhamseh, Neda; Sanjari, Sara; Djalilian, Ali R.

    2016-01-01

    Purpose Notch1 was previously shown to play a critical role in murine meibomian gland function and maintenance. In this study, we have examined the expression and activation of Notch pathway in human meibomian gland epithelial cells in vitro. Methods An immortalized human meibomian gland epithelial cell (HMGEC) line was cultured under proliferative and differentiative conditions. Expression of Notch receptors and ligands were evaluated by quantitative PCR and Western blot. The effect of Notch inhibition and induction on oil production was also assessed. Results Human meibomian gland epithelial cell expressed Notch1, Notch2, Notch3, Jagged1, Jagged2, Delta-like 1, and Delta-like 3. The level of cleaved (activated) Notch1 strongly increased with differentiation. The expression of Notch3 was inversely correlated with proliferation. Induction and inhibition of Notch1 led to an increase and decrease in the amount of oil production, respectively. Conclusions Notch signaling appears to play an important role in human meibomian gland epithelial differentiation and oil production. This may provide a potential therapeutic pathway for treating meibomian gland dysfunction. PMID:26943148

  10. Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration.

    PubMed

    Lindemans, Caroline A; Calafiore, Marco; Mertelsmann, Anna M; O'Connor, Margaret H; Dudakov, Jarrod A; Jenq, Robert R; Velardi, Enrico; Young, Lauren F; Smith, Odette M; Lawrence, Gillian; Ivanov, Juliet A; Fu, Ya-Yuan; Takashima, Shuichiro; Hua, Guoqiang; Martin, Maria L; O'Rourke, Kevin P; Lo, Yuan-Hung; Mokry, Michal; Romera-Hernandez, Monica; Cupedo, Tom; Dow, Lukas E; Nieuwenhuis, Edward E; Shroyer, Noah F; Liu, Chen; Kolesnick, Richard; van den Brink, Marcel R M; Hanash, Alan M

    2015-12-24

    Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch and epidermal growth factor (EGF) signals supporting Lgr5(+) crypt base columnar ISCs for normal epithelial maintenance. However, little is known about the regulation of the ISC compartment after tissue damage. Using ex vivo organoid cultures, here we show that innate lymphoid cells (ILCs), potent producers of interleukin-22 (IL-22) after intestinal injury, increase the growth of mouse small intestine organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both mouse and human intestinal organoids, increasing proliferation and promoting ISC expansion. IL-22 induced STAT3 phosphorylation in Lgr5(+) ISCs, and STAT3 was crucial for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after mouse allogeneic bone marrow transplantation enhanced the recovery of ISCs, increased epithelial regeneration and reduced intestinal pathology and mortality from graft-versus-host disease. ATOH1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independently of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support the intestinal epithelium, activating ISCs to promote regeneration. PMID:26649819

  11. Structural and functional analysis of endosomal compartments in epithelial cells.

    PubMed

    Bay, Andres E Perez; Schreiner, Ryan; Rodriguez-Boulan, Enrique

    2015-01-01

    Epithelial cells display segregated early endosomal compartments, termed apical sorting endosomes and basolateral sorting endosomes, that converge into a common late endosomal-lysosomal degradative compartment and common recycling endosomes (CREs). Unlike recycling endosomes of nonpolarized cells, CREs have the ability to sort apical and basolateral plasma membrane proteins into distinct apical and basolateral recycling routes, utilizing mechanisms similar to those employed by the trans Golgi network in the biosynthetic pathway. The apical recycling route includes an additional compartment, the apical recycling endosomes, consisting of multiple vesicles bundled around the basal body. Recent evidence indicates that, in addition to their role in internalizing ligands and recycling their receptors back to the cell surface, endosomal compartments act as intermediate stations in the biosynthetic routes to the plasma membrane. Here we review methods employed by our laboratory to study the endosomal compartments of epithelial cells and their multiple trafficking roles. PMID:26360040

  12. Epithelial-Mesenchymal Transition and Cell Cooperativity in Metastasis

    PubMed Central

    Tsuji, Takanori; Ibaragi, Soichiro; Hu, Guo-fu

    2009-01-01

    The role of epithelial-mesenchymal transition (EMT) in metastasis remains to be controversial. EMT has been postulated as an absolute requirement for tumor invasion and metastasis. Three different models including incomplete EMT, mesenchymal-epithelial transition (MET), and collective migration have been proposed for the role of EMT in cancer invasion and metastasis. However, skepticism remains as to whether EMT truly occurs during caner progression, and if it does, whether it plays an indispensible role in metastasis. Our recent findings suggest that EMT cells are responsible for degrading the surrounding matrix to enable invasion and intravasation of both EMT and non-EMT cells. Only non-EMT cell that have entered the blood stream are able to reestablish colonies in the secondary sites. Here we discuss an alternative model for the role of EMT in cancer metastasis in which EMT and non-EMT cells cooperate to complete the entire process of spontaneous metastasis. PMID:19738043

  13. Culture and immortalization of pancreatic ductal epithelial cells.

    PubMed

    Lawson, Terence; Ouellette, Michel; Kolar, Carol; Hollingsworth, Michael

    2005-01-01

    Some populations of the epithelial cells from the duct and ductular network of the mammalian pancreas have been isolated and maintained in vitro for up to 3 mo. These cells express many of the surface factors that are unique to them in vivo. They also retain significant drug- and carcinogen-metabolizing capacity in vitro. In this chapter we review the progression of the methods for the isolation, culture and maintenance in vitro for these cells from the earliest when only duct/ductular fragments were obtainable to the current ones which provide epithelial cells. The critical steps in the isolation process are identified and strategies are provided to facilitate these steps. These include the selection of tissue digestive enzymes, the importance of extensive mincing before culture and the importance of roles of some co-factors used in the culture medium. PMID:15542901

  14. Inactivation of Rb in stromal fibroblasts promotes epithelial cell invasion.

    PubMed

    Pickard, Adam; Cichon, Ann-Christin; Barry, Anna; Kieran, Declan; Patel, Daksha; Hamilton, Peter; Salto-Tellez, Manuel; James, Jacqueline; McCance, Dennis J

    2012-07-18

    Stromal-derived growth factors are required for normal epithelial growth but are also implicated in tumour progression. We have observed inactivation of the retinoblastoma protein (Rb), through phosphorylation, in cancer-associated fibroblasts in oro-pharyngeal cancer specimens. Rb is well known for its cell-autonomous effects on cancer initiation and progression; however, cell non-autonomous functions of Rb are not well described. We have identified a cell non-autonomous role of Rb, using three-dimensional cultures, where depletion of Rb in stromal fibroblasts enhances invasive potential of transformed epithelia. In part, this is mediated by upregulation of keratinocyte growth factor (KGF), which is produced by the depleted fibroblasts. KGF drives invasion of epithelial cells through induction of MMP1 expression in an AKT- and Ets2-dependent manner. Our data identify that stromal fibroblasts can alter the invasive behaviour of the epithelium, and we show that altered expression of KGF can mediate these functions. PMID:22643222

  15. Cytotoxic Action of Serratia marcescens Hemolysin on Human Epithelial Cells

    PubMed Central

    Hertle, Ralf; Hilger, Martina; Weingardt-Kocher, Sandra; Walev, Iwan

    1999-01-01

    Incubation of human epithelial cells with nanomolar concentrations of chromatographically purified Serratia marcescens hemolysin (ShlA) caused irreversible vacuolation and subsequent lysis of the cells. Vacuolation differed from vacuole formation by Helicobacter pylori VacA. Sublytic doses of ShlA led to a reversible depletion of intracellular ATP. Restoration to the initial ATP level was presumably due to the repair of the toxin damage and was inhibited by cycloheximide. Pores formed in epithelial cells and fibroblasts without disruption of the plasma membrane, and the pores appeared to be considerably smaller than those observed in artificial lipid membranes and in erythrocytes and did not allow the influx of propidium iodide or trypan blue. All cytotoxic effects induced by isolated recombinant ShlA were also obtained with exponentially growing S. marcescens cells. The previously suggested role of the hemolysin in the pathogenicity of S. marcescens is supported by these data. PMID:9916096

  16. COMPARISON OF PM-INDUCED GENE EXPRESSION PROFILES BETWEEN BRONCHIAL EPITHELIAL CELLS AND NASAL EPITHELIAL CELLS IN HUMAN

    EPA Science Inventory

    Epidemiologic studies have linked exposures to particulate matter (PM) and increased pulmonary mortality and morbidity. Bronchial epithelial cells (BEC) are the primary target of PM. PM exposure induces a wide array of biological responses in BEC. Primary human BEC, however, need...

  17. Circulating progenitor epithelial cells traffic via CXCR4/CXCL12 in response to airway injury.

    PubMed

    Gomperts, Brigitte N; Belperio, John A; Rao, P Nagesh; Randell, Scott H; Fishbein, Michael C; Burdick, Marie D; Strieter, Robert M

    2006-02-01

    Recipient airway epithelial cells are found in human sex-mismatched lung transplants, implying that circulating progenitor epithelial cells contribute to the repair of the airway epithelium. Markers of circulating progenitor epithelial cells and mechanisms for their trafficking remain to be elucidated. We demonstrate that a population of progenitor epithelial cells exists in the bone marrow and the circulation of mice that is positive for the early epithelial marker cytokeratin 5 (CK5) and the chemokine receptor CXCR4. We used a mouse model of sex-mismatched tracheal transplantation and found that CK5+ circulating progenitor epithelial cells contribute to re-epithelialization of the airway and re-establishment of the pseudostratified epithelium. The presence of CXCL12 in tracheal transplants provided a mechanism for CXCR4+ circulating progenitor epithelial cell recruitment to the airway. Depletion of CXCL12 resulted in the epithelium defaulting to squamous metaplasia, which was derived solely from the resident tissue progenitor epithelial cells. Our findings demonstrate that CK5+CXCR4+ cells are markers of circulating progenitor epithelial cells in the bone marrow and circulation and that CXCR4/CXCL12-mediated recruitment of circulating progenitor epithelial cells is necessary for the re-establishment of a normal pseudostratified epithelium after airway injury. These findings support a novel paradigm for the development of squamous metaplasia of the airway epithelium and for developing therapeutic strategies for circulating progenitor epithelial cells in airway diseases. PMID:16424223

  18. Cancer cells remodel themselves and vasculature to overcome the endothelial barrier.

    PubMed

    Shenoy, Anitha K; Lu, Jianrong

    2016-10-01

    Metastasis refers to the spread of cancer cells from a primary tumor to distant organs mostly via the bloodstream. During the metastatic process, cancer cells invade blood vessels to enter circulation, and later exit the vasculature at a distant site. Endothelial cells that line blood vessels normally serve as a barrier to the movement of cells into or out of the blood. It is thus critical to understand how metastatic cancer cells overcome the endothelial barrier. Epithelial cancer cells acquire increased motility and invasiveness through epithelial-to-mesenchymal transition (EMT), which enables them to move toward vasculature. Cancer cells also express a variety of adhesion molecules that allow them to attach to vascular endothelium. Finally, cancer cells secrete or induce growth factors and cytokines to actively prompt vascular hyperpermeability that compromises endothelial barrier function and facilitates transmigration of cancer cells through the vascular wall. Elucidation of the mechanisms underlying metastatic dissemination may help develop new anti-metastasis therapeutics. PMID:25449784

  19. Midbody remnant licenses primary cilia formation in epithelial cells.

    PubMed

    Ott, Carolyn M

    2016-08-01

    Tethered midbody remnants dancing across apical microvilli, encountering the centrosome, and beckoning forth a cilium-who would have guessed this is how polarized epithelial cells coordinate the end of mitosis and the beginning of ciliogenesis? New evidence from Bernabé-Rubio et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201601020) supports this emerging model. PMID:27482049

  20. Review: Corneal epithelial stem cells, their niche and wound healing

    PubMed Central

    2013-01-01

    Stem cells emerged as a concept during the second half of 19th century, first as a theoretical entity, but then became one of the most promising research fields in cell biology. This work describes the most important characteristics of adult stem cells, including the experimental criteria used to identify them, and discusses current knowledge that led to the proposal that stem cells existed in different parts of the eye, such as the retina, lens, conjunctiva, corneal stroma, Descemet’s membrane, and the subject of this review: the corneal epithelium. Evidence includes results that support the presence of corneal epithelial stem cells at the limbus, as well as the major obstacles to isolating them as pure cell populations. Part of this review describes the variation in the basement membrane composition between the limbus and the central cornea, to show the importance of the corneal stem cell niche, its structure, and the participation of extracellular matrix (ECM) components in regulating corneal stem cell compartment. Results obtained by various laboratories suggest that the extracellular matrix plays a central role in regulating stem cell commitment, corneal differentiation, and participation in corneal wound healing, in addition to other environmental signals such as cytokines and growth factors. The niche could define cell division patterns in corneal stem cell populations, establishing whether stem cells divide asymmetrically or symmetrically. Characterization and understanding of the factors that regulate corneal epithelial stem cells should open up new paths for developing new therapies and strategies for accelerating and improving corneal wound healing. PMID:23901244

  1. C/EBPα and the Vitamin D Receptor Cooperate in the Regulation of Cathelicidin in Lung Epithelial Cells.

    PubMed

    Dhawan, Puneet; Wei, Ran; Sun, Cheng; Gombart, Adrian F; Koeffler, H Phillip; Diamond, Gill; Christakos, Sylvia

    2015-02-01

    1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) and the vitamin D receptor (VDR) have been reported to have an important role in the regulation of innate immunity. We earlier reported that the antimicrobial peptide cathelicidin is induced by 1,25(OH)2D3 in normal human bronchial epithelial cells with a resultant increase in antimicrobial activity against airway pathogens. In this study, we demonstrate that C/EBP alpha (C/EBPα) is a potent enhancer of human cathelicidin antimicrobial peptide (CAMP) gene transcription in human lung epithelial cells. In addition we found that C/EBPα functionally cooperates with VDR in the regulation of CAMP transcription. A C/EBP binding site was identified at -627/-619 within the CAMP promoter, adjacent to the vitamin D response element (VDRE; -615/-600). Mutation of this site markedly attenuated the transcriptional response to C/EBPα as well as to 1,25(OH)2D3, further indicating cooperation between these two factors in the regulation of CAMP. ChIP analysis using 1,25(OH)2D3 treated human lung epithelial cells showed C/EBPα and VDR binding to the CAMP promoter. C/EBPα has previously been reported to cooperate with Brahma (Brm), an ATPase that is component of the SWI/SNF chromatin remodeling complex. We found that dominant negative Brm significantly inhibited C/EBPα as well as 1,25(OH)2D3 mediated induction of CAMP transcription, suggesting the functional involvement of Brm. These findings define novel mechanisms involving C/EBPα, SWI/SNF, and 1,25(OH)2D3 in the regulation of CAMP in lung epithelial cells. These mechanisms of enhanced activation of the CAMP gene in lung epithelial cells suggest potential candidates for the development of modulators of innate immune responses for adjunct therapy in the treatment of airway infections. PMID:25078430

  2. Vangl2 Regulates E-Cadherin in Epithelial Cells

    PubMed Central

    Nagaoka, Tadahiro; Inutsuka, Ayumu; Begum, Khadiza; hafiz, Khandakar musabbir bin; Kishi, Masashi

    2014-01-01

    E-cadherin belongs to the classic cadherin subfamily of calcium-dependent cell adhesion molecules and is crucial for the formation and function of epithelial adherens junctions. In this study, we demonstrate that Vangl2, a vertebrate regulator of planar cell polarity (PCP), controls E-cadherin in epithelial cells. E-cadherin co-immunoprecipitates with Vangl2 from embryonic kidney extracts, and this association is also observed in transfected fibroblasts. Vangl2 enhances the internalization of E-cadherin when overexpressed. Conversely, the quantitative ratio of E-cadherin exposed to the cell surface is increased in cultured renal epithelial cells derived from Vangl2Lpt/+ mutant mice. Interestingly, Vangl2 is also internalized through protein traffic involving Rab5- and Dynamin-dependent endocytosis. Taken together with recent reports regarding the transport of Frizzled3, MMP14 and nephrin, these results suggest that one of the molecular functions of Vangl2 is to enhance the internalization of specific plasma membrane proteins with broad selectivity. This function may be involved in the control of intercellular PCP signalling or in the PCP-related rearrangement of cell adhesions. PMID:25373475

  3. Sef Regulates Epithelial-Mesenchymal Transition in Breast Cancer Cells.

    PubMed

    He, Qing; Gong, Yan; Gower, Lindsey; Yang, Xuehui; Friesel, Robert E

    2016-10-01

    Sef (similar expression to fgf), also know as IL17RD, is a transmembrane protein shown to inhibit fibroblast growth factor signaling in developmental and cancer contexts; however, its role as a tumor suppressor remains to be fully elucidated. Here, we show that Sef regulates epithelial-mesenchymal transition (EMT) in breast cancer cell lines. Sef expression was highest in the normal breast epithelial cell line MCF10A, intermediate expression in MCF-7 cells and lowest in MDA-MB-231 cells. Knockdown of Sef increased the expression of genes associated with EMT, and promoted cell migration, invasion, and a fibroblastic morphology of MCF-7 cells. Overexpression of Sef inhibited the expression of EMT marker genes and inhibited cell migration and invasion in MCF-7 cells. Induction of EMT in MCF10A cells by TGF-β and TNF-α resulted in downregulation of Sef expression concomitant with upregulation of EMT gene expression and loss of epithelial morphology. Overexpression of Sef in MCF10A cells partially blocked cytokine-induced EMT. Sef was shown to block β-catenin mediated luciferase reporter activity and to cause a decrease in the nuclear localization of active β-catenin. Furthermore, Sef was shown to co-immunoprecipitate with β-catenin. In a mouse orthotopic xenograft model, Sef overexpression in MDA-MB-231 cells slowed tumor growth and reduced expression of EMT marker genes. Together, these data indicate that Sef plays a role in the negative regulation of EMT in a β-catenin dependent manner and that reduced expression of Sef in breast tumor cells may be permissive for EMT and the acquisition of a more metastatic phenotype. J. Cell. Biochem. 117: 2346-2356, 2016. © 2016 Wiley Periodicals, Inc. PMID:26950413

  4. Molecular mechanisms and functional implications of polarized actin remodeling at the T cell immunological synapse

    PubMed Central

    Le Floc’h, Audrey; Huse, Morgan

    2014-01-01

    Transient, specialized cell-cell interactions play a central role in leukocyte function by enabling specific intercellular communication in the context of a highly dynamic systems level response. The dramatic structural changes required for the formation of these contacts are driven by rapid and precise cytoskeletal remodeling events. In recent years, the immunological synapse that forms between a T lymphocyte and its antigen-presenting target cell has emerged as an important model system for understanding immune cell interactions. In this review, we discuss how regulators of the cortical actin cytoskeleton control synaptic architecture and in this way specify T cell function. PMID:25355055

  5. Alveolar epithelial type II cell: defender of the alveolus revisited

    PubMed Central

    Fehrenbach, Heinz

    2001-01-01

    In 1977, Mason and Williams developed the concept of the alveolar epithelial type II (AE2) cell as a defender of the alveolus. It is well known that AE2 cells synthesise, secrete, and recycle all components of the surfactant that regulates alveolar surface tension in mammalian lungs. AE2 cells influence extracellular surfactant transformation by regulating, for example, pH and [Ca2+] of the hypophase. AE2 cells play various roles in alveolar fluid balance, coagulation/fibrinolysis, and host defence. AE2 cells proliferate, differentiate into AE1 cells, and remove apoptotic AE2 cells by phagocytosis, thus contributing to epithelial repair. AE2 cells may act as immunoregulatory cells. AE2 cells interact with resident and mobile cells, either directly by membrane contact or indirectly via cytokines/growth factors and their receptors, thus representing an integrative unit within the alveolus. Although most data support the concept, the controversy about the character of hyperplastic AE2 cells, reported to synthesise profibrotic factors, proscribes drawing a definite conclusion today. PMID:11686863

  6. Generation of Stratified Squamous Epithelial Progenitor Cells from Mouse Induced Pluripotent Stem Cells

    PubMed Central

    Yoshida, Satoru; Yasuda, Miyuki; Miyashita, Hideyuki; Ogawa, Yoko; Yoshida, Tetsu; Matsuzaki, Yumi; Tsubota, Kazuo; Okano, Hideyuki; Shimmura, Shigeto

    2011-01-01

    Background Application of induced pluripotent stem (iPS) cells in regenerative medicine will bypass ethical issues associated with use of embryonic stem cells. In addition, patient-specific IPS cells can be useful to elucidate the pathophysiology of genetic disorders, drug screening, and tailor-made medicine. However, in order to apply iPS cells to mitotic tissue, induction of tissue stem cells that give rise to progeny of the target organ is required. Methodology/Principal Findings We induced stratified epithelial cells from mouse iPS cells by co-culture with PA6 feeder cells (SDIA-method) with use of BMP4. Clusters of cells positive for the differentiation markers KRT1 or KRT12 were observed in KRT14-positive colonies. We successfully cloned KRT14 and p63 double-positive stratified epithelial progenitor cells from iPS-derived epithelial cells, which formed stratified epithelial sheets consisting of five- to six-polarized epithelial cells in vitro. When these clonal cells were cultured on denuded mouse corneas, a robust stratified epithelial layer was observed with physiological cell polarity including high levels of E-cadherin, p63 and K15 expression in the basal layer and ZO-1 in the superficial layer, recapitulating the apico-basal polarity of the epithelium in vivo. Conclusions/Significance These results suggest that KRT14 and p63 double-positive epithelial progenitor cells can be cloned from iPS cells in order to produce polarized multilayer epithelial cell sheets. PMID:22174914

  7. Cytokeratin changes in cell culture systems of epithelial cells isolated from oral mucosa: a short review.

    PubMed

    Gasparoni, Alberto; Squier, Christopher Alan; Fonzi, Luciano

    2005-01-01

    In the past three decades, many studies have analyzed ultrastructural and molecular markers of differentiation in squamous stratified epithelial tissues. In these tissues, epithelial cells migrating from the basal layer to the upper layers undergo drastic changes, which involve membrane-associated proteins, DNA synthesis, phenotypic aspects, lipid composition, and cytoskeletal components. Cytoskeletal components include a large and heterogeneous group, including intermediate filaments, components of the cornified envelope, and of the stratum corneum. When grown in mono- and multilayer cell cultures, epithelial cells isolated from the oral mucosa may reproduce many of the biochemical and morphological aspects of epithelial tissue in vivo. In the present paper, we examine phenotypic changes, development of suprabasal layer, and Involucrin expression occurring in differentiating oral epithelial cells, based on literature review and original data. PMID:16277157

  8. Spatiotemporal regulation of chemical reaction kinetics of cell surface molecules by active remodeling of cortical actin

    NASA Astrophysics Data System (ADS)

    Bhattacharyya, Bhaswati; Chaudhuri, Abhishek; Gowrishankar, Kripa; Mayor, Satyajit; Rao, Madan

    2010-03-01

    Cell surface proteins such as lipid tethered GPI-anchored proteins and Ras-proteins are distributed as monomers and nanoclusters on the surface of living cells. Recent work from our laboratory suggests that the spatial distribution and dynamics of formation and breakup of these nanoclusters is controlled by the active remodeling dynamics of the underlying cortical actin. To explain these observations, we propose a novel mechanism of nanoclustering, involving the transient binding to and advection along constitutively occuring ``asters'' of cortical actin. Here we study the consequences of such active actin based clustering, in the context of chemical reactions involving conformational changes of cell surface proteins. We find that active remodeling of cortical actin, can give rise to a dramatic increase in the reaction efficiency and output levels. In general, such actin driven clustering of membrane proteins could be a cellular mechanism to spatiotemporally regulate and amplify local chemical reaction rates, in the context of signalling and endocytosis.

  9. Temporal Monitoring of Differentiated Human Airway Epithelial Cells Using Microfluidics

    PubMed Central

    Blume, Cornelia; Reale, Riccardo; Held, Marie; Millar, Timothy M.; Collins, Jane E.; Davies, Donna E.; Morgan, Hywel; Swindle, Emily J.

    2015-01-01

    The airway epithelium is exposed to a variety of harmful agents during breathing and appropriate cellular responses are essential to maintain tissue homeostasis. Recent evidence has highlighted the contribution of epithelial barrier dysfunction in the development of many chronic respiratory diseases. Despite intense research efforts, the responses of the airway barrier to environmental agents are not fully understood, mainly due to lack of suitable in vitro models that recapitulate the complex in vivo situation accurately. Using an interdisciplinary approach, we describe a novel dynamic 3D in vitro model of the airway epithelium, incorporating fully differentiated primary human airway epithelial cells at the air-liquid interface and a basolateral microfluidic supply of nutrients simulating the interstitial flow observed in vivo. Through combination of the microfluidic culture system with an automated fraction collector the kinetics of cellular responses by the airway epithelium to environmental agents can be analysed at the early phases for the first time and with much higher sensitivity compared to common static in vitro models. Following exposure of primary differentiated epithelial cells to pollen we show that CXCL8/IL–8 release is detectable within the first 2h and peaks at 4–6h under microfluidic conditions, a response which was not observed in conventional static culture conditions. Such a microfluidic culture model is likely to have utility for high resolution temporal profiling of toxicological and pharmacological responses of the airway epithelial barrier, as well as for studies of disease mechanisms. PMID:26436734

  10. Temporal Monitoring of Differentiated Human Airway Epithelial Cells Using Microfluidics.

    PubMed

    Blume, Cornelia; Reale, Riccardo; Held, Marie; Millar, Timothy M; Collins, Jane E; Davies, Donna E; Morgan, Hywel; Swindle, Emily J

    2015-01-01

    The airway epithelium is exposed to a variety of harmful agents during breathing and appropriate cellular responses are essential to maintain tissue homeostasis. Recent evidence has highlighted the contribution of epithelial barrier dysfunction in the development of many chronic respiratory diseases. Despite intense research efforts, the responses of the airway barrier to environmental agents are not fully understood, mainly due to lack of suitable in vitro models that recapitulate the complex in vivo situation accurately. Using an interdisciplinary approach, we describe a novel dynamic 3D in vitro model of the airway epithelium, incorporating fully differentiated primary human airway epithelial cells at the air-liquid interface and a basolateral microfluidic supply of nutrients simulating the interstitial flow observed in vivo. Through combination of the microfluidic culture system with an automated fraction collector the kinetics of cellular responses by the airway epithelium to environmental agents can be analysed at the early phases for the first time and with much higher sensitivity compared to common static in vitro models. Following exposure of primary differentiated epithelial cells to pollen we show that CXCL8/IL-8 release is detectable within the first 2h and peaks at 4-6h under microfluidic conditions, a response which was not observed in conventional static culture conditions. Such a microfluidic culture model is likely to have utility for high resolution temporal profiling of toxicological and pharmacological responses of the airway epithelial barrier, as well as for studies of disease mechanisms. PMID:26436734

  11. Retinoic acid promotes primary fetal alveolar epithelial type II cell proliferation and differentiation to alveolar epithelial type I cells.

    PubMed

    Gao, Rui-wei; Kong, Xiang-yong; Zhu, Xiao-xi; Zhu, Guo-qing; Ma, Jin-shuai; Liu, Xiu-xiang

    2015-05-01

    Retinoic acid (RA) plays an important role in lung development and maturation. Many stimuli can induce alveolar epithelial cell damage which will result in the injury of lung parenchyma. The aim of this study was to observe the effect of RA on the proliferation and differentiation of primary fetal alveolar epithelial type II cells (fAECIIs). Primary fAECIIs were isolated from fetal rats at 19 d of gestation and purified by a differential centrifugation and adhesion method. The cells were randomly divided into control (dimethyl sulfoxide, DMSO) and RA groups. Cell proliferation, viability, apoptosis, cycle, and expression of target protein were examined at 24, 48, and 72 h. We found that the proliferation and viability of cells in the RA-exposed group significantly increased compared with the DMSO control group. The proportion (%) of cells in the G2 and S phases in the RA group was significantly higher than that in control group cells. The proportion (%) of both early apoptotic cells and late apoptotic cells decreased significantly in cells exposed to RA compared with cells exposed to DMSO. RA significantly enhanced the expression of aquaporin 5 (AQP5). The expression level of pulmonary surfactant C (SPC) was elevated after cells were exposed to RA for 24 and 72 h but was inhibited when cells were exposed to RA for 48 h. These results suggest that RA promotes fAECII proliferation by improving cell viability, promoting S phase entry and inhibiting apoptosis and RA promotes fAECIIs differentiation to alveolar epithelial type I cells (AECIs). PMID:25515249

  12. Human airway xenograft models of epithelial cell regeneration.

    PubMed

    Puchelle, E; Peault, B

    2000-01-01

    Regeneration and restoration of the airway epithelium after mechanical, viral or bacterial injury have a determinant role in the evolution of numerous respiratory diseases such as chronic bronchitis, asthma and cystic fibrosis. The study in vivo of epithelial regeneration in animal models has shown that airway epithelial cells are able to dedifferentiate, spread, migrate over the denuded basement membrane and progressively redifferentiate to restore a functional respiratory epithelium after several weeks. Recently, human tracheal xenografts have been developed in immunodeficient severe combined immunodeficiency (SCID) and nude mice. In this review we recall that human airway cells implanted in such conditioned host grafts can regenerate a well-differentiated and functional human epithelium; we stress the interest in these humanized mice in assaying candidate progenitor and stem cells of the human airway mucosa. PMID:11667974

  13. Cell wall remodeling in mycorrhizal symbiosis: a way towards biotrophism

    PubMed Central

    Balestrini, Raffaella; Bonfante, Paola

    2014-01-01

    Cell walls are deeply involved in the molecular talk between partners during plant and microbe interactions, and their role in mycorrhizae, i.e., the widespread symbiotic associations established between plant roots and soil fungi, has been investigated extensively. All mycorrhizal interactions achieve full symbiotic functionality through the development of an extensive contact surface between the plant and fungal cells, where signals and nutrients are exchanged. The exchange of molecules between the fungal and the plant cytoplasm takes place both through their plasma membranes and their cell walls; a functional compartment, known as the symbiotic interface, is thus defined. Among all the symbiotic interfaces, the complex intracellular interface of arbuscular mycorrhizal (AM) symbiosis has received a great deal of attention since its first description. Here, in fact, the host plasma membrane invaginates and proliferates around all the developing intracellular fungal structures, and cell wall material is laid down between this membrane and the fungal cell surface. By contrast, in ectomycorrhizae (ECM), where the fungus grows outside and between the root cells, plant and fungal cell walls are always in direct contact and form the interface between the two partners. The organization and composition of cell walls within the interface compartment is a topic that has attracted widespread attention, both in ecto- and endomycorrhizae. The aim of this review is to provide a general overview of the current knowledge on this topic by integrating morphological observations, which have illustrated cell wall features during mycorrhizal interactions, with the current data produced by genomic and transcriptomic approaches. PMID:24926297

  14. The Roles of SNF2/SWI2 Nucleosome Remodeling Enzymes in Blood Cell Differentiation and Leukemia

    PubMed Central

    Prasad, Punit; Lennartsson, Andreas; Ekwall, Karl

    2015-01-01

    Here, we review the role of sucrose nonfermenting (SNF2) family enzymes in blood cell development. The SNF2 family comprises helicase-like ATPases, originally discovered in yeast, that can remodel chromatin by changing chromatin structure and composition. The human genome encodes 30 different SNF2 enzymes. SNF2 family enzymes are often part of multisubunit chromatin remodeling complexes (CRCs), which consist of noncatalytic/auxiliary subunit along with the ATPase subunit. However, blood cells express a limited set of SNF2 ATPases that are necessary to maintain the pool of hematopoietic stem cells (HSCs) and drive normal blood cell development and differentiation. The composition of CRCs can be altered by the association of specific auxiliary subunits. Several auxiliary CRC subunits have specific functions in hematopoiesis. Aberrant expressions of SNF2 ATPases and/or auxiliary CRC subunit(s) are often observed in hematological malignancies. Using large-scale data from the International Cancer Genome Consortium (ICGC) we observed frequent mutations in genes encoding SNF2 helicase-like enzymes and auxiliary CRC subunits in leukemia. Hence, orderly function of SNF2 family enzymes is crucial for the execution of normal blood cell developmental program, and defects in chromatin remodeling caused by mutations or aberrant expression of these proteins may contribute to leukemogenesis. PMID:25789315

  15. Chromatin remodeling by cell cycle stage-specific extracts from Physarum polycephalum.

    PubMed

    Thiriet, C; Hayes, J J

    1999-03-01

    Remodeling of chromatin is an essential process allowing the establishment of specific genetic programs. The slime mold Physarum polycephalum presents the attractive advantage of natural synchrony of the cell cycle in several million nuclei. Whole-cell extracts prepared at precise stages during the cell cycle were tested for the ability to induce remodeling in erythrocyte nuclei as monitored by microscopy, protamine competition assays, micrococcal nuclease digestions, and release of histone H5. Extracts derived from two specific cell cycle stages caused opposite types of changes in erythrocyte nuclei. An increase in chromatin compaction was imparted by extracts prepared during S-phase while extracts harvested at the end of G2-phase caused increases in nuclear volume, DNA accessibility, and release of linker histone. We also found that late G2 extracts had the ability to alter the DNase I digestion profile of mononucleosomes reconstituted in vitro in a classical nucleosomes remodeling assay. The relevance of these finding to the Physarum cell cycle is discussed. PMID:10219572

  16. Transcriptional Regulation of Tlr11 Gene Expression in Epithelial Cells*

    PubMed Central

    Cai, Zhenyu; Shi, Zhongcheng; Sanchez, Amir; Zhang, Tingting; Liu, Mingyao; Yang, Jianghua; Wang, Fen; Zhang, Dekai

    2009-01-01

    As sensors of invading microorganisms, Toll-like receptors (TLRs) are expressed not only on macrophages and dendritic cells (DCs) but also on epithelial cells. In the TLR family, Tlr11 appears to have the unique feature in that it is expressed primarily on epithelial cells, although it is also expressed on DCs and macrophages. Here, we demonstrate that transcription of the Tlr11 gene is regulated through two cis-acting elements, one Ets-binding site and one interferon regulatory factor (IRF)-binding site. The Ets element interacts with the epithelium-specific transcription factors, ESE-1 and ESE-3, and the IRF motif interacts with IRF-8. Thus, Tlr11 expression on epithelial cells is regulated by the transcription factors that are presumably distinct from transcription factors that regulate the expression of TLRs in innate immune cells such as macrophages and DCs. Our results imply that the distinctive transcription regulatory machinery for TLRs on epithelium may represent a promising new avenue for the development of epithelia-specific therapeutic interventions. PMID:19801549

  17. Differentiation capacity of epithelial cells in the sponge Suberites domuncula.

    PubMed

    Schröder, Heinz C; Perović-Ottstadt, Sanja; Wiens, Matthias; Batel, Renato; Müller, Isabel M; Müller, Werner E G

    2004-05-01

    Sponges (phylum Porifera) represent the oldest metazoans. Their characteristic metazoan adhesion molecules and transcription factors enable them to establish a complex "Bauplan"; three major differentiated cell types (epithelial cells, skeletal cells/sclerocytes, and contractile cells) can be distinguished. Since no molecular markers are as yet available to distinguish these somatic cells or the corresponding embryonic cells from which they originate, we have selected the following three genes for their characterization: noggin (a signaling molecule in development), a caspase that encodes an apoptotic molecule, and silicatein. Silicatein is an enzyme that is involved in the synthesis of siliceous spicules and can hence be considered as a marker for scleroblasts. We have used the demosponge Suberites domuncula as a model system. During the hatching of the gemmules (asexual reproduction bodies) of S. domuncula, the expression of both noggin and caspase increases, whereas no transcripts for silicatein can be detected, irrespective of the presence of silicate or ferric iron (Fe3+) in the medium. In contrast, in adult specimens, silicate/Fe3+ cause an increased expression of these genes. In situ analysis has revealed that the first cells that express noggin, caspase, and silicatein lie in the epithelial layer of the pinacoderm. In a later phase, the noggin- and silicatein-positive cells migrate into the mesohyl, where they are found in association with spicules. Thus, the pinacoderm of sponges contains cells that have a differentiating capacity and from which somatic cells, such as skeletal cells/sclerocytes, derive. PMID:15024642

  18. Radiogenic transformation of human mammary epithelial cells in vitro

    NASA Technical Reports Server (NTRS)

    Yang, T. C.; Georgy, K. A.; Tavakoli, A.; Craise, L. M.; Durante, M.

    1996-01-01

    Cancer induction by space radiations is a major concern for manned space exploration. Accurate assessment of radiation risk at low doses requires basic understanding of mechanism(s) of radiation carcinogenesis. For determining the oncogenic effects of ionizing radiation in human epithelial cells, we transformed a mammary epithelial cell line (185B5), which was immortalized by benzo(a)pyrene, with energetic heavy ions and obtained several transformed clones. These transformed cells showed growth properties on Matrigel similar to human mammary tumor cells. To better understand the mechanisms of radiogenic transformation of human cells, we systematically examined the alterations in chromosomes and cancer genes. Among 16 autosomes examined for translocations, by using fluorescence in situ hybridization (FISH) technique, chromosomes 3, 12, 13, 15, 16, and 18 appeared to be normal in transformed cells. Chromosomes 1, 4, 6, 8, and 17 in transformed cells, however, showed patterns different from those in nontransformed cells. Southern blot analyses indicated no detectable alterations in myc, ras, Rb, or p53 genes. Further studies of chromosome 17 by using in situ hybridization with unique sequence p53 gene probe and a centromere probe showed no loss of p53 gene in transformed cells. Experimental results from cell fusion studies indicated that the transforming gene(s) is recessive. The role of genomic instability and tumor suppressor gene(s) in radiogenic transformation of human breast cells remains to be identified.

  19. miR-8 modulates cytoskeletal regulators to influence cell survival and epithelial organization in Drosophila wings.

    PubMed

    Bolin, Kelsey; Rachmaninoff, Nicholas; Moncada, Kea; Pula, Katharine; Kennell, Jennifer; Buttitta, Laura

    2016-04-01

    The miR-200 microRNA family plays important tumor suppressive roles. The sole Drosophila miR-200 ortholog, miR-8 plays conserved roles in Wingless, Notch and Insulin signaling - pathways linked to tumorigenesis, yet homozygous null animals are viable and often appear morphologically normal. We observed that wing tissues mosaic for miR-8 levels by genetic loss or gain of function exhibited patterns of cell death consistent with a role for miR-8 in modulating cell survival in vivo. Here we show that miR-8 levels impact several actin cytoskeletal regulators that can affect cell survival and epithelial organization. We show that loss of miR-8 can confer resistance to apoptosis independent of an epithelial to mesenchymal transition while the persistence of cells expressing high levels of miR-8 in the wing epithelium leads to increased JNK signaling, aberrant expression of extracellular matrix remodeling proteins and disruption of proper wing epithelial organization. Altogether our results suggest that very low as well as very high levels of miR-8 can contribute to hallmarks associated with cancer, suggesting approaches to increase miR-200 microRNAs in cancer treatment should be moderate. PMID:26902111

  20. Nucleus Morphometry in Cultured Epithelial Cells Correlates with Phenotype.

    PubMed

    Khan, Ayyad Z; Utheim, Tor P; Jackson, Catherine J; Reppe, Sjur; Lyberg, Torstein; Eidet, Jon R

    2016-06-01

    Phenotype of cultured ocular epithelial transplants has been shown to affect clinical success rates following transplantation to the cornea. The purpose of this study was to evaluate the relationship between cell nucleus morphometry and phenotype in three types of cultured epithelial cells. This study provides knowledge for the development of a non-invasive method of determining the phenotype of cultured epithelium before transplantation. Cultured human conjunctival epithelial cells (HCjE), human epidermal keratinocytes (HEK), and human retinal pigment epithelial cells (HRPE) were analyzed by quantitative immunofluorescence. Assessments of nucleus morphometry and nucleus-to-cytoplasm ratio (N/C ratio) were performed using ImageJ. Spearman's correlation coefficient was employed for statistical analysis. Levels of the proliferation marker PCNA in HCjE, HEK, and HRPE correlated positively with nuclear area. Nuclear area correlated significantly with levels of the undifferentiated cell marker ABCG2 in HCjE. Bmi1 levels, but not p63α levels, correlated significantly with nuclear area in HEK. The N/C ratio did not correlate significantly with any of the immunomarkers in HCjE (ABCG2, CK7, and PCNA) and HRPE (PCNA). In HEK, however, the N/C ratio was negatively correlated with levels of the undifferentiated cell marker CK14 and positively correlated with Bmi1 expression. The size of the nuclear area correlated positively with proliferation markers in all three epithelia. Morphometric indicators of phenotype in cultured epithelia can be identified using ImageJ. Conversely, the N/C ratio did not show a uniform relationship with phenotype in HCjE, HEK, or HRPE. N/C ratio therefore, may not be a useful morphometric marker for in vitro assessment of phenotype in these three epithelia. PMID:27329312

  1. Zinc modulates cytokine-induced lung epithelial cell barrier permeability.

    PubMed

    Bao, Shenying; Knoell, Daren L

    2006-12-01

    Apoptosis plays a causative role in acute lung injury in part due to epithelial cell loss. We recently reported that zinc protects the lung epithelium during inflammatory stress whereas depletion of intracellular zinc enhances extrinsic apoptosis. In this investigation, we evaluated the relationship between zinc, caspase-3, and cell-to-cell contact via proteins that form the adherens junction complex. Cell adhesion proteins are directly responsible for formation of the mechanical barrier of the lung epithelium. We hypothesized that exposure to inflammatory cytokines, in conjunction with zinc deprivation, would induce caspase-3, leading to degradation of junction proteins, loss of cell-to-cell contact, and compromised barrier function. Primary human upper airway and type I/II alveolar epithelial cultures were obtained from multiple donors and exposed to inflammatory stimuli that provoke extrinsic apoptosis in addition to depletion of intracellular zinc. We observed that zinc deprivation combined with tumor necrosis factor-alpha, interferon-gamma, and Fas receptor ligation accelerates caspase-3 activation, proteolysis of E-cadherin and beta-catenin, and cellular apoptosis, leading to increased paracellular leak across monolayers of both upper airway and alveolar lung epithelial cultures. Zinc supplementation inhibited apoptosis and paracellular leak, whereas caspase inhibition was less effective. We conclude that zinc is a vital factor in the lung epithelium that protects against death receptor-mediated apoptosis and barrier dysfunction. Furthermore, our findings suggest that although caspase-3 inhibition reduces lung epithelial apoptosis it does not prevent mechanical dysfunction. These findings facilitate future studies aimed at developing therapeutic strategies to prevent acute lung injury. PMID:16844947

  2. Borrelia burgdorferi bind to epithelial cell proteoglycans.

    PubMed Central

    Isaacs, R D

    1994-01-01

    Borrelia burgdorferi adhere to mammalian cells in vitro but neither the ligand(s) nor the receptor(s) has (have) been clearly established. Using an in vitro attachment-inhibition assay, a B. burgdorferi attachment mechanism has been identified. Heparin, heparan sulfate, and dermatan sulfate reduced the attachment of virulent B. burgdorferi strain 297 to HeLa cells by approximately 60%. In addition, virulent, but not avirulent, B. burgdorferi strains B31, N40, and HB19 demonstrated heparin attachment-inhibition. Attachment to Chinese hamster ovary cells deficient in heparan sulfate proteoglycans was reduced by 68% compared to attachment to wild-type cells and was identical to attachment at maximum heparin inhibition to the wild-type cells. Pretreatment of HeLa cell monolayers with heparitinase, heparinase, and chondroitinase ABC, but not with chondroitinase AC, reduced borrelial attachment by approximately 50%. A moderately high affinity, low copy number, promiscuous B. burgdorferi glycosaminoglycan receptor was demonstrated by equilibrium binding studies. A 39-kD polypeptide, purified by heparin affinity chromatography from Triton X-100 extracts derived from virulent borrelia, was a candidate for this receptor. These studies indicate that one mode of B. burgdorferi attachment to eukaryotic cells is mediated by a borrelial glycosaminoglycan receptor attaching to surface-exposed proteoglycans on mammalian cells. Images PMID:8113413

  3. Abl Kinases Regulate HGF/Met Signaling Required for Epithelial Cell Scattering, Tubulogenesis and Motility

    PubMed Central

    Li, Ran; Knight, Jennifer F.; Park, Morag; Pendergast, Ann Marie

    2015-01-01

    Tight regulation of receptor tyrosine kinases (RTKs) is crucial for normal development and homeostasis. Dysregulation of RTKs signaling is associated with diverse pathological conditions including cancer. The Met RTK is the receptor for hepatocyte growth factor (HGF) and is dysregulated in numerous human tumors. Here we show that Abl family of non-receptor tyrosine kinases, comprised of Abl (ABL1) and Arg (ABL2), are activated downstream of the Met receptor, and that inhibition of Abl kinases dramatically suppresses HGF-induced cell scattering and tubulogenesis. We uncover a critical role for Abl kinases in the regulation of HGF/Met-dependent RhoA activation and RhoA-mediated actomyosin contractility and actin cytoskeleton remodeling in epithelial cells. Moreover, treatment of breast cancer cells with Abl inhibitors markedly decreases Met-driven cell migration and invasion. Notably, expression of a transforming mutant of the Met receptor in the mouse mammary epithelium results in hyper-activation of both Abl and Arg kinases. Together these data demonstrate that Abl kinases link Met activation to Rho signaling and Abl kinases are required for Met-dependent cell scattering, tubulogenesis, migration, and invasion. Thus, inhibition of Abl kinases might be exploited for the treatment of cancers driven by hyperactivation of HGF/Met signaling. PMID:25946048

  4. Vinculin is required for cell polarization, migration, and extracellular matrix remodeling in 3D collagen.

    PubMed

    Thievessen, Ingo; Fakhri, Nikta; Steinwachs, Julian; Kraus, Viola; McIsaac, R Scott; Gao, Liang; Chen, Bi-Chang; Baird, Michelle A; Davidson, Michael W; Betzig, Eric; Oldenbourg, Rudolf; Waterman, Clare M; Fabry, Ben

    2015-11-01

    Vinculin is filamentous (F)-actin-binding protein enriched in integrin-based adhesions to the extracellular matrix (ECM). Whereas studies in 2-dimensional (2D) tissue culture models have suggested that vinculin negatively regulates cell migration by promoting cytoskeleton-ECM coupling to strengthen and stabilize adhesions, its role in regulating cell migration in more physiologic, 3-dimensional (3D) environments is unclear. To address the role of vinculin in 3D cell migration, we analyzed the morphodynamics, migration, and ECM remodeling of primary murine embryonic fibroblasts (MEFs) with cre/loxP-mediated vinculin gene disruption in 3D collagen I cultures. We found that vinculin promoted 3D cell migration by increasing directional persistence. Vinculin was necessary for persistent cell protrusion, cell elongation, and stable cell orientation in 3D collagen, but was dispensable for lamellipodia formation, suggesting that vinculin-mediated cell adhesion to the ECM is needed to convert actin-based cell protrusion into persistent cell shape change and migration. Consistent with this finding, vinculin was necessary for efficient traction force generation in 3D collagen without affecting myosin II activity and promoted 3D collagen fiber alignment and macroscopical gel contraction. Our results suggest that vinculin promotes directionally persistent cell migration and tension-dependent ECM remodeling in complex 3D environments by increasing cell-ECM adhesion and traction force generation. PMID:26195589

  5. Osteoclasts control reactivation of dormant myeloma cells by remodelling the endosteal niche.

    PubMed

    Lawson, Michelle A; McDonald, Michelle M; Kovacic, Natasa; Hua Khoo, Weng; Terry, Rachael L; Down, Jenny; Kaplan, Warren; Paton-Hough, Julia; Fellows, Clair; Pettitt, Jessica A; Neil Dear, T; Van Valckenborgh, Els; Baldock, Paul A; Rogers, Michael J; Eaton, Colby L; Vanderkerken, Karin; Pettit, Allison R; Quinn, Julian M W; Zannettino, Andrew C W; Phan, Tri Giang; Croucher, Peter I

    2015-01-01

    Multiple myeloma is largely incurable, despite development of therapies that target myeloma cell-intrinsic pathways. Disease relapse is thought to originate from dormant myeloma cells, localized in specialized niches, which resist therapy and repopulate the tumour. However, little is known about the niche, and how it exerts cell-extrinsic control over myeloma cell dormancy and reactivation. In this study, we track individual myeloma cells by intravital imaging as they colonize the endosteal niche, enter a dormant state and subsequently become activated to form colonies. We demonstrate that dormancy is a reversible state that is switched 'on' by engagement with bone-lining cells or osteoblasts, and switched 'off' by osteoclasts remodelling the endosteal niche. Dormant myeloma cells are resistant to chemotherapy that targets dividing cells. The demonstration that the endosteal niche is pivotal in controlling myeloma cell dormancy highlights the potential for targeting cell-extrinsic mechanisms to overcome cell-intrinsic drug resistance and prevent disease relapse. PMID:26632274

  6. βA3/A1-crystallin in astroglial cells regulates retinal vascular remodeling during development

    PubMed Central

    Sinha, Debasish; Klise, Andrew; Sergeev, Yuri; Hose, Stacey; Bhutto, Imran A.; Hackler, Laszlo; Malpic-llanos, Tanya; Samtani, Sonia; Grebe, Rhonda; Goldberg, Morton F.; Hejtmancik, J. Fielding; Nath, Avindra; Zack, Donald J.; Fariss, Robert N.; McLeod, D. Scott; Sundin, Olof; Broman, Karl W.; Lutty, Gerard A.; Zigler, J. Samuel

    2016-01-01

    Vascular remodeling is a complex process critical to development of the mature vascular system. Astrocytes are known to be indispensable for initial formation of the retinal vasculature; our studies with the Nuc1 rat provide novel evidence that these cells are also essential in the retinal vascular remodeling process. Nuc1 is a spontaneous mutation in the Sprague–Dawley rat originally characterized by nuclear cataracts in the heterozygote and microphthalmia in the homozygote. We report here that the Nuc1 allele results from mutation of the βA3/A1-crystallin gene, which in the neural retina is expressed only in astrocytes. We demonstrate striking structural abnormalities in Nuc1 astrocytes with profound effects on the organization of intermediate filaments. While vessels form in the Nuc1 retina, the subsequent remodeling process required to provide a mature vascular network is deficient. Our data implicate βA3/A1-crystallin as an important regulatory factor mediating vascular patterning and remodeling in the retina. PMID:17931883

  7. Antagonism of Stem Cell Factor/c-kit Signaling Attenuates Neonatal Chronic Hypoxia-Induced Pulmonary Vascular Remodeling

    PubMed Central

    Young, Karen C; Torres, Eneida; Hehre, Dorothy; Wu, Shu; Suguihara, Cleide; Hare, Joshua M.

    2015-01-01

    Background Accumulating evidence suggests that c-kit positive cells are present in the remodeled pulmonary vasculature bed of patients with pulmonary hypertension (PH). Whether stem cell factor (SCF)/ c-kit regulated pathways potentiate pulmonary vascular remodeling is unknown. Here, we tested the hypothesis that attenuated c-kit signaling would decrease chronic hypoxia-induced pulmonary vascular remodeling by decreasing pulmonary vascular cell mitogenesis. Methods Neonatal FVB/NJ mice treated with non-immune IgG (PL), or c-kit neutralizing antibody (ACK2) as well as c-kit mutant mice (WBB6F1- Kit W− v/ +) and their congenic controls, were exposed to normoxia (FiO2=0.21) or hypoxia (FiO2=0.12) for two weeks. Following this exposure, right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH), pulmonary vascular cell proliferation and remodeling were evaluated. Results As compared to chronically hypoxic controls, c-kit mutant mice had decreased RVSP, RVH, pulmonary vascular remodeling and proliferation. Consistent with these findings, administration of ACK2 to neonatal mice with chronic hypoxia-induced PH decreased RVSP, RVH, pulmonary vascular cell proliferation and remodeling. This attenuation in PH was accompanied by decreased extracellular signal-regulated protein kinase (ERK) 1/2 activation. Conclusion SCF/c-kit signaling may potentiate chronic hypoxia-induced vascular remodeling by modulating ERK activation. Inhibition of c-kit activity may be a potential strategy to alleviate PH. PMID:26705118

  8. CCL20, (gamma)(delta) T cells, and IL-22 in corneal epithelial healing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    After corneal epithelial abrasion, leukocytes and platelets rapidly enter the corneal stroma, and CCR6 (+) IL-17(+) gamma delta T cells migrate into the epithelium. Gamma delta T-cell-deficient (TCRd(-/-)) mice have significantly reduced inflammation and epithelial wound healing. Epithelial CCL20 mR...

  9. Mesenchymal precursor cells maintain the differentiation and proliferation potentials of breast epithelial cells

    PubMed Central

    2014-01-01

    Introduction Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. Not surprisingly, aberrant stromal-epithelial interactions contribute to malignancies. Studies of the cellular and molecular mechanisms underlying these interactions require ex vivo experimental model systems that recapitulate the complexity of human tissue without compromising the differentiation and proliferation potentials of human primary cells. Methods We isolated and characterized human breast epithelial and mesenchymal precursors from reduction mammoplasty tissue and tagged them with lentiviral vectors. We assembled heterotypic co-cultures and compared mesenchymal and epithelial cells to cells in corresponding monocultures by analyzing growth, differentiation potentials, and gene expression profiles. Results We show that heterotypic culture of non-immortalized human primary breast epithelial and mesenchymal precursors maintains their proliferation and differentiation potentials and constrains their growth. We further describe the gene expression profiles of stromal and epithelial cells in co-cultures and monocultures and show increased expression of the tumor growth factor beta (TGFβ) family member inhibin beta A (INHBA) in mesenchymal cells grown as co-cultures compared with monocultures. Notably, overexpression of INHBA in mesenchymal cells increases colony formation potential of epithelial cells, suggesting that it contributes to the dynamic reciprocity between breast mesenchymal and epithelial cells. Conclusions The described heterotypic co-culture system will prove useful for further characterization of the molecular mechanisms mediating interactions between human normal or neoplastic breast epithelial cells and the stroma, and will provide a framework to test the relevance of the ever-increasing number of oncogenomic alterations identified in human breast cancer. PMID:24916766

  10. Effect of mesenchymal stem cells on inhibiting airway remodeling and airway inflammation in chronic asthma.

    PubMed

    Ge, Xiahui; Bai, Chong; Yang, Jianming; Lou, Guoliang; Li, Qiang; Chen, Ruohua

    2013-07-01

    Previous studies proved that bone marrow-derived mesenchymal stem cells (BMSCs) could improve a variety of immune-mediated disease by its immunomodulatory properties. In this study, we investigated the effect on airway remodeling and airway inflammation by administrating BMSCs in chronic asthmatic mice. Forty-eight female BALB/c mice were randomly distributed into PBS group, BMSCs treatment group, BMSCs control group, and asthmatic group. The levels of cytokine and immunoglobulin in serum and bronchoalveolar lavage fluid were detected by enzyme-linked immunosorbent assay. The number of CD4(+) CD25(+) regulatory T cells and morphometric analysis was determined by flow cytometry, hematoxylin-eosin, immunofluorescence staining, periodic-acid Schiff, and masson staining, respectively. We found that airway remodeling and airway inflammation were evident in asthmatic mice. Moreover, low level of IL-12 and high levels of IL-13, IL-4, OVA-specific IgG1, IgE, and IgG2a and the fewer number of CD4(+) CD25(+) regulatory T cells were present in asthmatic group. However, transplantation of BMSCs significantly decreased airway inflammation and airway remodeling and level of IL-4, OVA-specific IgE, and OVA-specific IgG1, but elevated level of IL-12 and the number of CD4 + CD25 + regulatory T cells in asthma (P < 0.05). However, BMSCs did not contribute to lung regeneration and had no significant effect on levels of IL-10, IFN-Y, and IL-13. In our study, BMSCs engraftment prohibited airway inflammation and airway remodeling in chronic asthmatic group. The beneficial effect of BMSCs might involved the modulation imbalance cytokine toward a new balance Th1-Th2 profiles and up-regulation of protective CD4 + CD25 + regulatory T cells in asthma, but not contribution to lung regeneration. PMID:23334934

  11. Tbx2 and Tbx3 Regulate the Dynamics of Cell Proliferation during Heart Remodeling

    PubMed Central

    Ribeiro, Inês; Kawakami, Yasuhiko; Büscher, Dirk; Raya, Ángel; Rodríguez-León, Joaquín; Morita, Masanobu; Rodríguez Esteban, Concepción; Izpisúa Belmonte, Juan Carlos

    2007-01-01

    Background The heart forms from a linear tube that is subject to complex remodeling during embryonic development. Hallmarks of this remodeling are the looping of the heart tube and the regionalization into chamber and non-chamber myocardium. Cardiomyocytes in the future chamber myocardium acquire different cellular and physiological characteristics through activation of a chamber-specific genetic program, which is in part mediated by T-box genes. Methodology/Principal Finding We characterize two new zebrafish T-box transcription factors, tbx3b and tbx2a, and analyze their role during the development of the atrioventricular canal. Loss- and gain-of-function analyses demonstrate that tbx3b and tbx2a are necessary to repress the chamber-genetic program in the non-chamber myocardium. We also show that tbx3b and tbx2a are required to control cell proliferation in the atrioventricular canal and that misregulation of cell proliferation in the heart tube influences looping. Furthermore, we characterize the heart phenotype of a novel Tbx3 mutation in mice and show that both the control of cell proliferation and the repression of chamber-specific genetic program in the non-chamber myocardium are conserved roles of Tbx3 in this species. Conclusions/Significance Taken together, our results uncover an evolutionarily conserved role of Tbx2/3 transcription factors during remodeling of the heart myocardium and highlight the importance of controlling cell proliferation as a driving force of morphogenesis. PMID:17460765

  12. Efficacy of several candidate protein biomarkers in the differentiation of vaginal from buccal epithelial cells.

    PubMed

    Simons, Joanne L; Vintiner, Sue K

    2012-11-01

    Currently, there is no accurate method to differentiate vaginal epithelial cells from buccal epithelial cells in biological samples typically encountered in forensic casework. This study tested the expression of a selection of candidate proteins in buccal and vaginal epithelial cells. We investigated six candidate biomarkers, such as loricrin, vimentin, stratifin, cytokeratin 4, cytokeratin 13, small proline-rich protein 2, and involucrin, using Western blot analysis on whole protein extracts and immunohistochemistry (IHC) on intact cells in an attempt to identify cell-specific markers that would differentiate these cells by microscopy. Involucrin, loricrin, and stratifin showed differential expression during Western blot analysis and were carried through to IHC. Although proteins unique to vaginal epithelial cells and buccal epithelial cells were not identified from among the proteins tested, the increased expression levels of two proteins, loricrin and stratifin in vaginal cells, when compared to buccal cells, do provide encouraging results in the search for epithelial cell-specific markers. PMID:22612601

  13. XB130 promotes bronchioalveolar stem cell and Club cell proliferation in airway epithelial repair and regeneration

    PubMed Central

    Toba, Hiroaki; Wang, Yingchun; Bai, Xiaohui; Zamel, Ricardo; Cho, Hae-Ra; Liu, Hongmei; Lira, Alonso; Keshavjee, Shaf; Liu, Mingyao

    2015-01-01

    Proliferation of bronchioalveolar stem cells (BASCs) is essential for epithelial repair. XB130 is a novel adaptor protein involved in the regulation of epithelial cell survival, proliferation and migration through the PI3K/Akt pathway. To determine the role of XB130 in airway epithelial injury repair and regeneration, a naphthalene-induced airway epithelial injury model was used with XB130 knockout (KO) mice and their wild type (WT) littermates. In XB130 KO mice, at days 7 and 14, small airway epithelium repair was significantly delayed with fewer number of Club cells (previously called Clara cells). CCSP (Club cell secreted protein) mRNA expression was also significantly lower in KO mice at day 7. At day 5, there were significantly fewer proliferative epithelial cells in the KO group, and the number of BASCs significantly increased in WT mice but not in KO mice. At day 7, phosphorylation of Akt, GSK-3β, and the p85α subunit of PI3K was observed in airway epithelial cells in WT mice, but to a much lesser extent in KO mice. Microarray data also suggest that PI3K/Akt-related signals were regulated differently in KO and WT mice. An inhibitory mechanism for cell proliferation and cell cycle progression was suggested in KO mice. XB130 is involved in bronchioalveolar stem cell and Club cell proliferation, likely through the PI3K/Akt/GSK-3β pathway. PMID:26360608

  14. Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

    PubMed Central

    Joshi, Sagar D.; Davidson, Lance A.

    2010-01-01

    Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis. PMID:20498627

  15. Interaction exists between matriptase inhibitors and intestinal epithelial cells.

    PubMed

    Pászti-Gere, Erzsebet; Barna, Réka Fanni; Ujhelyi, Gabriella; Steinmetzer, Torsten

    2016-10-01

    The type II trypsin-like transmembrane serine protease matriptase, is mainly expressed in epithelial cells and one of the key regulators in the formation and maintenance of epithelial barrier integrity. Therefore, we have studied the inhibition of matriptase in a non-transformed porcine intestinal IPEC-J2 cell monolayer cultured on polyester membrane inserts by the non-selective 4-(2-aminoethyl)-benzosulphonylfluoride (AEBSF) and four more selective 3-amidinophenylalanine-derived matriptase inhibitors. It was found that suppression of matriptase activity by MI-432 and MI-460 led to decreased transepithelial electrical resistance (TER) of the cell monolayer and to an enhanced transport of fluorescently labelled dextran, a marker for paracellular transport between apical and basolateral compartments. To this date this is the first report in which the inhibition of matriptase activity by synthetic inhibitors has been correlated to a reduced barrier integrity of a non-cancerous IPEC-J2 epithelial cell monolayer in order to describe interaction between matriptase activity and intestinal epithelium in vitro. PMID:26118419

  16. Blood group glycolipids as epithelial cell receptors for Candida albicans.

    PubMed Central

    Cameron, B J; Douglas, L J

    1996-01-01

    The role of glycosphingolipids as possible epithelial cell receptors for Candida albicans was examined by investigating the binding of biotinylated yeasts to lipids extracted from human buccal epithelial cells and separated on thin-layer chromatograms. Binding was visualized by the addition of 125I-streptavidin followed by autoradiography. Five C. albicans strains thought from earlier work to have a requirement for fucose-containing receptors all bound to the same three components in the lipid extract. A parallel chromatogram overlaid with biotinylated Ulex europaeus lectin, which is a fucose-binding lectin with a specificity for the H blood group antigen, showed that two of these glycosphingolipids carried this antigenic determinant. Preparations of crude and purified adhesin (a protein with a size of 15.7 kDa which lacked cysteine residues) from one of the strains also bound to these same two components. The third glycosphingolipid, which bound whole cells but neither preparation of adhesin, was recognized by Helix pomatia lectin, indicating that it contained N-acetylgalactosamine, possibly in the form of the A blood group antigen. Overlay assays with a sixth strain of C. albicans (GDH 2023) revealed a completely different binding pattern of four receptors, each of which contained N-acetylglucosamine. These results confirm earlier predictions about the receptor specificity of the strains made on the basis of adhesion inhibition studies and indicate that blood group antigens can act as epithelial cell receptors for C. albicans. PMID:8641797

  17. Photodynamic treatment of lens epithelial cells for cataract surgery

    NASA Astrophysics Data System (ADS)

    Lingua, Robert W.; Parel, Jean-Marie A.; Simon, Gabriel; Li, Kam

    1991-06-01

    Photodynamic therapy (PDT) eiiploying Dihematopor*iyrin ethers (DHE) (Photofrin II) at pharmacologic lvels, has been denonstrate3 to kill rabbit lens epithelial cells, in vivo. This in vitro study, reports on the minimal necessary parameters for rabbit lens epithelial cell death. Explants of rabbit lenses were incubated in various concentrations of DHE (1O,, 100, 500, 1000 ug/ml) for 1, 2, or 5 minutes. 30 to 120 Joules/an of collimated 514.5 nm Argon laser light re delivered to the locier concentrations of 10, 50, and 100 ug,'ml DHE treated cells. One hundre1 fifteen explants were treated, in all. Higher concentrations of DHE alone (500 and 1000 ug/ml) were sufficient to induce cellular swelling. Lower concentrations required light for cellular effect. Trypan blue staining revealed cell death at these minimal pa9ieters: DHE 50 ug/ml, incubation 1 minute, 514.5 r Argon light 1.0 Watt/an for 30 sec (30 Joules) . In future studies, these rameters will be tested in vivo, for their ability to eliminate lens epithelial proliferation after cataract surgery.

  18. Regulation of local immunity by airway epithelial cells.

    PubMed

    Mayer, Anja K; Dalpke, Alexander H

    2007-01-01

    Epithelial cells are the first line of defense against invading microbial pathogens. They are important contributors to innate mucosal immunity and generate various and sophisticated anti-microbial defense mechanisms, including the formation of a tight barrier and secretion of anti-microbial substances as well as inflammatory mediators. To provide these active defense mechanisms, epithelial cells functionally express various pattern-recognition receptors. Toll-like receptors have been shown to recognize conserved microbial patterns mediating inducible activation of innate immunity. Mucosal surfaces, however, are prone to contact with pathogenic as well as non-pathogenic microbes and, therefore, immune-recognition principles have to be strictly regulated to avoid uncontrolled permanent activation. This review will focus on mechanisms by which epithelial cells regulate mucosal immune responses, thus creating an organ-specific microenvironment. This includes local adaptations in microbial recognition, regulation of local immune homeostasis, and modulation of antigen-presenting cells and adaptive immune responses. These regulatory mechanisms serve the special needs of controlled microbial recognition in mucosal compartments. PMID:18060372

  19. Dynamin-mediated endocytosis is required for tube closure, cell intercalation, and biased apical expansion during epithelial tubulogenesis in the Drosophila ovary.

    PubMed

    Peters, Nathaniel C; Berg, Celeste A

    2016-01-01

    Most metazoans are able to grow beyond a few hundred cells and to support differentiated tissues because they elaborate multicellular, epithelial tubes that are indispensable for nutrient and gas exchange. To identify and characterize the cellular behaviors and molecular mechanisms required for the morphogenesis of epithelial tubes (i.e., tubulogenesis), we have turned to the D. melanogaster ovary. Here, epithelia surrounding the developing egg chambers first pattern, then form and extend a set of simple, paired, epithelial tubes, the dorsal appendage (DA) tubes, and they create these structures in the absence of cell division or cell death. This genetically tractable system lets us assess the relative contributions that coordinated changes in cell shape, adhesion, orientation, and migration make to basic epithelial tubulogenesis. We find that Dynamin, a conserved regulator of endocytosis and the cytoskeleton, serves a key role in DA tubulogenesis. We demonstrate that Dynamin is required for distinct aspects of DA tubulogenesis: DA-tube closure, DA-tube-cell intercalation, and biased apical-luminal cell expansion. We provide evidence that Dynamin promotes these processes by facilitating endocytosis of cell-cell and cell-matrix adhesion complexes, and we find that precise levels and sub-cellular distribution of E-Cadherin and specific Integrin subunits impact DA tubulogenesis. Thus, our studies identify novel morphogenetic roles (i.e., tube closure and biased apical expansion), and expand upon established roles (i.e., cell intercalation and adhesion remodeling), for Dynamin in tubulogenesis. PMID:26542010

  20. *Iron accumulation in bronchial epithelial cells is dependent on concurrent sodium transport

    EPA Science Inventory

    Airway epithelial cells prevent damaging effects of extracellular iron by taking up the metal and sequestering it within intracellular ferritin. Epithelial iron transport is associated with transcellular movement of other cations including changes in the expression or activity of...

  1. Extracellular matrix remodeling by dynamic strain in a three-dimensional tissue-engineered human airway wall model.

    PubMed

    Choe, Melanie M; Sporn, Peter H S; Swartz, Melody A

    2006-09-01

    Airway wall remodeling is a hallmark of asthma, characterized by subepithelial thickening and extracellular matrix (ECM) remodeling. Mechanical stress due to hyperresponsive smooth muscle cells may contribute to this remodeling, but its relevance in a three-dimensional environment (where the ECM plays an important role in modulating stresses felt by cells) is unclear. To characterize the effects of dynamic compression in ECM remodeling in a physiologically relevant three-dimensional environment, a tissue-engineered human airway wall model with differentiated bronchial epithelial cells atop a collagen gel containing lung fibroblasts was used. Lateral compressive strain of 10 or 30% at 1 or 60 cycles per hour was applied using a novel straining device. ECM remodeling was assessed by immunohistochemistry and zymography. Dynamic strain, particularly at the lower magnitude, induced airway wall remodeling, as indicated by increased deposition of types III and IV collagen and increased secretion of matrix metalloproteinase-2 and -9. These changes paralleled increased myofibroblast differentiation and were fibroblast-dependent. Furthermore, the spatial pattern of type III collagen deposition correlated with that of myofibroblasts; both were concentrated near the epithelium and decreased diffusely away from the surface, indicating some epithelial control of the remodeling response. Thus, in a physiologically relevant three-dimensional model of the bronchial wall, dynamic compressive strain induced tissue remodeling that mimics many features of remodeling seen in asthma, in the absence of inflammation and dependent on epithelial-fibroblast signaling. PMID:16601241

  2. Novel human bronchial epithelial cell lines for cystic fibrosis research

    PubMed Central

    Fulcher, M. L.; Gabriel, S. E.; Olsen, J. C.; Tatreau, J. R.; Gentzsch, M.; Livanos, E.; Saavedra, M. T.; Salmon, P.; Randell, S. H.

    2009-01-01

    Immortalization of human bronchial epithelial (hBE) cells often entails loss of differentiation. Bmi-1 is a protooncogene that maintains stem cells, and its expression creates cell lines that recapitulate normal cell structure and function. We introduced Bmi-1 and the catalytic subunit of telomerase (hTERT) into three non-cystic fibrosis (CF) and three ΔF508 homozygous CF primary bronchial cell preparations. This treatment extended cell life span, although not as profoundly as viral oncogenes, and at passages 14 and 15, the new cell lines had a diploid karyotype. Ussing chamber analysis revealed variable transepithelial resistances, ranging from 200 to 1,200 Ω·cm2. In the non-CF cell lines, short-circuit currents were stimulated by forskolin and inhibited by CFTR(inh)-172 at levels mostly comparable to early passage primary cells. CF cell lines exhibited no forskolin-stimulated current and minimal CFTR(inh)-172 response. Amiloride-inhibitable and UTP-stimulated currents were present, but at lower and higher amplitudes than in primary cells, respectively. The cells exhibited a pseudostratified morphology, with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells, and occasional ciliated cells. CF and non-CF cell lines produced similar levels of IL-8 at baseline and equally increased IL-8 secretion in response to IL-1β, TNF-α, and the Toll-like receptor 2 agonist Pam3Cys. Although they have lower growth potential and more fastidious growth requirements than viral oncogene transformed cells, Bmi-1/hTERT airway epithelial cell lines will be useful for several avenues of investigation and will help fill gaps currently hindering CF research and therapeutic development. PMID:18978040

  3. Epithelial Cell Polarity Determinant CRB3 in Cancer Development

    PubMed Central

    Li, Pingping; Mao, Xiaona; Ren, Yu; Liu, Peijun

    2015-01-01

    Cell polarity, which is defined as asymmetry in cell shape, organelle distribution and cell function, is essential in numerous biological processes, including cell growth, cell migration and invasion, molecular transport, and cell fate. Epithelial cell polarity is mainly regulated by three conserved polarity protein complexes, the Crumbs (CRB) complex, partitioning defective (PAR) complex and Scribble (SCRIB) complex. Research evidence has indicated that dysregulation of cell polarity proteins may play an important role in cancer development. Crumbs homolog 3 (CRB3), a member of the CRB complex, may act as a cancer suppressor in mouse kidney epithelium and mouse mammary epithelium. In this review, we focus on the current data available on the roles of CRB3 in cancer development. PMID:25552927

  4. Clonal analysis of limbal epithelial stem cell populations.

    PubMed

    Schlötzer-Schrehardt, Ursula

    2013-01-01

    While convincing data clearly suggest the presence of stem cells in the basal limbal epithelium in vivo, testing the proliferation, self-renewal, and differentiation capacity of stem cells relies on the development of methodologies that allow for their isolation and extensive propagation in vitro. Clonal analysis involving differentiation between short-lived transient cell clones and long-lived stem cell clones is an invaluable technique to identify stem cells in vitro, and allows cells to be expanded over multiple passages. This chapter describes a protocol for the isolation, expansion, and clonal analysis of limbal epithelial stem cells. The cultivation method described may be essential for long-term restoration of the damaged ocular surface in patients with limbal stem cell deficiency. PMID:23690004

  5. Measles virus breaks through epithelial cell barriers to achieve transmission

    PubMed Central

    Takeda, Makoto

    2008-01-01

    Measles is a highly contagious disease that causes immunosuppression in patients. Measles virus infection has been thought to begin in the respiratory epithelium and then spread to lymphoid tissue. In this issue of the JCI, Leonard et al. provide data to suggest an alternative model of measles virus pathogenesis (see the related article beginning on page 2448). In human primary epithelial cells and rhesus monkeys in vivo, the authors show that initial infection of respiratory epithelium is not necessary for the virus to enter the host but that viral entry into epithelial cells via interaction of the virus with a receptor located on the basolateral side of the epithelium is required for viral shedding into the airway and subsequent transmission. PMID:18568081

  6. Oral epithelial cell responses to multispecies microbial biofilms.

    PubMed

    Peyyala, R; Kirakodu, S S; Novak, K F; Ebersole, J L

    2013-03-01

    This report describes the use of a novel model of multispecies biofilms to stimulate profiles of cytokines/chemokines from oral epithelial cells that contribute to local inflammation in the periodontium. Streptococcus gordonii (Sg)/S. oralis (So)/S. sanguinis (Ss) and Sg/Fusobacterium nucleatum (Fn)/Porphyromonas gingivalis (Pg) biofilms elicited significantly elevated levels of IL-1α and showed synergistic stimulatory activity compared with an additive effect of the 3 individual bacteria. Only the Sg/Actinomyces naeslundii (An)/Fn multispecies biofilms elicited IL-6 levels above those of control. IL-8 was a primary response to the Sg/An/Fn biofilms, albeit the level was not enhanced compared with a predicted composite level from the monospecies challenges. These results represent some of the first data documenting alterations in profiles of oral epithelial cell responses to multispecies biofilms. PMID:23300185

  7. CXCL12 expression by healthy and malignant ovarian epithelial cells

    PubMed Central

    2011-01-01

    Background CXCL12 has been widely reported to play a biologically relevant role in tumor growth and spread. In epithelial ovarian cancer (EOC), CXCL12 enhances tumor angiogenesis and contributes to the immunosuppressive network. However, its prognostic significance remains unclear. We thus compared CXCL12 status in healthy and malignant ovaries, to assess its prognostic value. Methods Immunohistochemistry was used to analyze CXCL12 expression in the reproductive tracts, including the ovaries and fallopian tubes, of healthy women, in benign and borderline epithelial tumors, and in a series of 183 tumor specimens from patients with advanced primary EOC enrolled in a multicenter prospective clinical trial of paclitaxel/carboplatin/gemcitabine-based chemotherapy (GINECO study). Univariate COX model analysis was performed to assess the prognostic value of clinical and biological variables. Kaplan-Meier methods were used to generate progression-free and overall survival curves. Results Epithelial cells from the surface of the ovary and the fallopian tubes stained positive for CXCL12, whereas the follicles within the ovary did not. Epithelial cells in benign, borderline and malignant tumors also expressed CXCL12. In EOC specimens, CXCL12 immunoreactivity was observed mostly in epithelial tumor cells. The intensity of the signal obtained ranged from strong in 86 cases (47%) to absent in 18 cases (<10%). This uneven distribution of CXCL12 did not reflect the morphological heterogeneity of EOC. CXCL12 expression levels were not correlated with any of the clinical parameters currently used to determine EOC prognosis or with HER2 status. They also had no impact on progression-free or overall survival. Conclusion Our findings highlight the previously unappreciated constitutive expression of CXCL12 on healthy epithelia of the ovary surface and fallopian tubes, indicating that EOC may originate from either of these epithelia. We reveal that CXCL12 production by malignant

  8. Cellular and molecular remodelling of a host cell for vertical transmission of bacterial symbionts.

    PubMed

    Luan, Jun-Bo; Shan, Hong-Wei; Isermann, Philipp; Huang, Jia-Hsin; Lammerding, Jan; Liu, Shu-Sheng; Douglas, Angela E

    2016-06-29

    Various insects require intracellular bacteria that are restricted to specialized cells (bacteriocytes) and are transmitted vertically via the female ovary, but the transmission mechanisms are obscure. We hypothesized that, in the whitefly Bemisia tabaci, where intact bacteriocytes (and not isolated bacteria) are transferred to oocytes, the transmission mechanism would be evident as cellular and molecular differences between the nymph (pre-adult) and adult bacteriocytes. We demonstrate dramatic remodelling of bacteriocytes at the developmental transition from nymph to adulthood. This transition involves the loss of cell-cell adhesion, high division rates to constant cell size and onset of cell mobility, enabling the bacteriocytes to crawl to the ovaries. These changes are accompanied by cytoskeleton reorganization and changes in gene expression: genes functioning in cell-cell adhesion display reduced expression and genes involved in cell division, cell motility and endocytosis/exocytosis have elevated expression in adult bacteriocytes, relative to nymph bacteriocytes. This study demonstrates, for the first time, how developmentally orchestrated remodelling of gene expression and correlated changes in cell behaviour underpin the capacity of bacteriocytes to mediate the vertical transmission and persistence of the symbiotic bacteria on which the insect host depends. PMID:27358364

  9. Connecting Mechanics and Bone Cell Activities in the Bone Remodeling Process: An Integrated Finite Element Modeling

    PubMed Central

    Hambli, Ridha

    2014-01-01

    Bone adaptation occurs as a response to external loadings and involves bone resorption by osteoclasts followed by the formation of new bone by osteoblasts. It is directly triggered by the transduction phase by osteocytes embedded within the bone matrix. The bone remodeling process is governed by the interactions between osteoblasts and osteoclasts through the expression of several autocrine and paracrine factors that control bone cell populations and their relative rate of differentiation and proliferation. A review of the literature shows that despite the progress in bone remodeling simulation using the finite element (FE) method, there is still a lack of predictive models that explicitly consider the interaction between osteoblasts and osteoclasts combined with the mechanical response of bone. The current study attempts to develop an FE model to describe the bone remodeling process, taking into consideration the activities of osteoclasts and osteoblasts. The mechanical behavior of bone is described by taking into account the bone material fatigue damage accumulation and mineralization. A coupled strain–damage stimulus function is proposed, which controls the level of autocrine and paracrine factors. The cellular behavior is based on Komarova et al.’s (2003) dynamic law, which describes the autocrine and paracrine interactions between osteoblasts and osteoclasts and computes cell population dynamics and changes in bone mass at a discrete site of bone remodeling. Therefore, when an external mechanical stress is applied, bone formation and resorption is governed by cells dynamic rather than adaptive elasticity approaches. The proposed FE model has been implemented in the FE code Abaqus (UMAT routine). An example of human proximal femur is investigated using the model developed. The model was able to predict final human proximal femur adaptation similar to the patterns observed in a human proximal femur. The results obtained reveal complex spatio-temporal bone

  10. USP22 promotes epithelial-mesenchymal transition via the FAK pathway in pancreatic cancer cells.

    PubMed

    Ning, Zhen; Wang, Aman; Liang, Jinxiao; Xie, Yunpeng; Liu, Jiwei; Yan, Qiu; Wang, Zhongyu

    2014-10-01

    Epithelial-mesenchymal transition (EMT) contributes to the occurrence and development of tumors, particularly to the promotion of tumor invasion and metastasis. As a newly discovered ubiquitin hydrolase family member, USP22 plays a key role in the malignant transformation of tumors and the regulation of the cell cycle. However, recent studies on USP22 have primarily focused on its role in cell cycle regulation, and the potential mechanism underlying the promotion of tumor invasion and metastasis by abnormal USP22 expression has not been reported. Our studies revealed that the overexpression of USP22 in PANC-1 cells promoted Ezrin redistribution and phosphorylation and cytoskeletal remodeling, upregulated expression of the transcription factors Snail and ZEB1 to promote EMT, and increased cellular invasion and migration. In contrast, blockade of USP22 expression resulted in the opposite effects. In addition, the focal adhesion kinase (FAK) signaling pathway was shown to play a key role in the process of EMT induction in PANC-1 cells by USP22. Thus, the present study suggests that USP22 acts as a regulatory protein for EMT in pancreatic cancer, which may provide a new approach for the targeted therapy of pancreatic cancer. PMID:25070659

  11. Molecular Mechanisms of Malignant Transformation by Low Dose Cadmium in Normal Human Bronchial Epithelial Cells

    PubMed Central

    Kluz, Thomas; Cohen, Lisa; Shen, Steven S.; Costa, Max

    2016-01-01

    Cadmium is a carcinogenic metal, the mechanisms of which are not fully understood. In this study, human bronchial epithelial cells were transformed with sub-toxic doses of cadmium (0.01, 0.05, and 0.1 μM) and transformed clones were characterized for gene expression changes using RNA-seq, as well as other molecular measurements. 440 genes were upregulated and 47 genes were downregulated in cadmium clones relative to control clones over 1.25-fold. Upregulated genes were associated mostly with gene ontology terms related to embryonic development, immune response, and cell movement, while downregulated genes were associated with RNA metabolism and regulation of transcription. Several embryonic genes were upregulated, including the transcription regulator SATB2. SATB2 is critical for normal skeletal development and has roles in gene expression regulation and chromatin remodeling. Small hairpin RNA knockdown of SATB2 significantly inhibited growth in soft agar, indicating its potential as a driver of metal-induced carcinogenesis. An increase in oxidative stress and autophagy was observed in cadmium clones. In addition, the DNA repair protein O6-methylguanine-DNA-methyltransferase was depleted by transformation with cadmium. MGMT loss caused significant decrease in cell viability after treatment with the alkylating agent temozolomide, demonstrating diminished capacity to repair such damage. Results reveal various mechanisms of cadmium-induced malignant transformation in BEAS-2B cells including upregulation of SATB2, downregulation of MGMT, and increased oxidative stress. PMID:27186882

  12. Ivermectin Inhibits Growth of Chlamydia trachomatis in Epithelial Cells

    PubMed Central

    Pettengill, Matthew A.; Lam, Verissa W.; Ollawa, Ikechukwu; Marques-da-Silva, Camila; Ojcius, David M.

    2012-01-01

    Ivermectin is currently approved for treatment of both clinical and veterinary infections by nematodes, including Onchocerca cervicalis in horses and Onchocerca volvulus in humans. However, ivermectin has never been shown to be effective against bacterial pathogens. Here we show that ivermectin also inhibits infection of epithelial cells by the bacterial pathogen, Chlamydia trachomatis, at doses that could be envisioned clinically for sexually-transmitted or ocular infections by Chlamydia. PMID:23119027

  13. Ivermectin inhibits growth of Chlamydia trachomatis in epithelial cells.

    PubMed

    Pettengill, Matthew A; Lam, Verissa W; Ollawa, Ikechukwu; Marques-da-Silva, Camila; Ojcius, David M

    2012-01-01

    Ivermectin is currently approved for treatment of both clinical and veterinary infections by nematodes, including Onchocerca cervicalis in horses and Onchocerca volvulus in humans. However, ivermectin has never been shown to be effective against bacterial pathogens. Here we show that ivermectin also inhibits infection of epithelial cells by the bacterial pathogen, Chlamydia trachomatis, at doses that could be envisioned clinically for sexually-transmitted or ocular infections by Chlamydia. PMID:23119027

  14. Sensitivity of proliferating human breast epithelial cells to hypotonic treatment

    SciTech Connect

    Goldstone, S.E.; Stanyon, R.; Lan, S.

    1982-12-01

    An assay for colony-forming cells of breast epithelia derived from normal and malignant surgical specimens is described using an IMR 90 fibroblast feeder layer. Their radiosensitivity (DO: 120-172) is consistent with the proliferative origin of the colonies. Distilled water inhibits proliferation of a proportion of the colony-forming cells after a 1-minute exposure. Continued detection of colonies after 10 minutes of exposure indicates that it is an inefficient way of completely eradicating proliferating epithelial cells of normal and malignant origin.

  15. Somatic embryogenesis - Stress-induced remodeling of plant cell fate.

    PubMed

    Fehér, Attila

    2015-04-01

    Plants as sessile organisms have remarkable developmental plasticity ensuring heir continuous adaptation to the environment. An extreme example is somatic embryogenesis, the initiation of autonomous embryo development in somatic cells in response to exogenous and/or endogenous signals. In this review I briefly overview the various pathways that can lead to embryo development in plants in addition to the fertilization of the egg cell and highlight the importance of the interaction of stress- and hormone-regulated pathways during the induction of somatic embryogenesis. Somatic embryogenesis can be initiated in planta or in vitro, directly or indirectly, and the requirement for dedifferentiation as well as the way to achieve developmental totipotency in the various systems is discussed in light of our present knowledge. The initiation of all forms of the stress/hormone-induced in vitro as well as the genetically provoked in planta somatic embryogenesis requires extensive and coordinated genetic reprogramming that has to take place at the chromatin level, as the embryogenic program is under strong epigenetic repression in vegetative plant cells. Our present knowledge on chromatin-based mechanisms potentially involved in the somatic-to-embryogenic developmental transition is summarized emphasizing the potential role of the chromatin to integrate stress, hormonal, and developmental pathways leading to the activation of the embryogenic program. The role of stress-related chromatin reorganization in the genetic instability of in vitro cultures is also discussed. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity. PMID:25038583

  16. Role of mitochondrial remodeling in programmed cell death in Drosophila melanogaster.

    PubMed

    Goyal, Gaurav; Fell, Brennan; Sarin, Apurva; Youle, Richard J; Sriram, V

    2007-05-01

    The role of mitochondria in Drosophila programmed cell death remains unclear, although certain gene products that regulate cell death seem to be evolutionarily conserved. We find that developmental programmed cell death stimuli in vivo and multiple apoptotic stimuli ex vivo induce dramatic mitochondrial fragmentation upstream of effector caspase activation, phosphatidylserine exposure, and nuclear condensation in Drosophila cells. Unlike genotoxic stress, a lipid cell death mediator induced an increase in mitochondrial contiguity prior to fragmentation of the mitochondria. Using genetic mutants and RNAi-mediated knockdown of drp-1, we find that Drp-1 not only regulates mitochondrial fission in normal cells, but mediates mitochondrial fragmentation during programmed cell death. Mitochondria in drp-1 mutants fail to fragment, resulting in hyperplasia of tissues in vivo and protection of cells from multiple apoptotic stimuli ex vivo. Thus, mitochondrial remodeling is capable of modifying the propensity of cells to undergo death in Drosophila. PMID:17488630

  17. Human alveolar epithelial type II cells in primary culture.

    PubMed

    Mao, Pu; Wu, Songling; Li, Jianchun; Fu, Wei; He, Weiqun; Liu, Xiaoqing; Slutsky, Arthur S; Zhang, Haibo; Li, Yimin

    2015-02-01

    Alveolar epithelial type II (AEII) cells are a key structure and defender in the lung but also are the targets in many lung diseases, including acute respiratory distress syndrome, ventilator-induced lung injury, and pulmonary fibrosis. We sought to establish an optimized method for high yielding and long maintenance of characteristics of primary human AEII cells to facilitate the investigation of the mechanisms of lung diseases at the cellular and molecular levels. Adult human peripheral normal lung tissues of oncologic patients undergoing lung resection were collected. The AEII cells were isolated and identified by the expression of pro-surfactant protein (SP)C, epithelial sodium channel (αENaC) and cytokeratin (CK)-8, the lamellar bodies specific for AEII cells, and confirmed by the histology using electron microscopy. The phenotype of AEII cells was characterized by the expression of surfactant proteins (SP-A, SP-B, SP-C, SP-D), CK-8, KL-6, αENaC, and aquaporin (AQP)-3, which was maintained over 20 days. The biological activity of the primary human AEII cells producing SP-C, cytokines, and intercellular adhesion molecule-1 was vigorous in response to stimulation with tumor necrosis factor-α. We have modified previous methods and optimized a method for isolation of high purity and long maintenance of the human AEII cell phenotype in primary culture. This method provides an important tool for studies aiming at elucidating the molecular mechanisms of lung diseases exclusively in AEII cells. PMID:25677546

  18. Human alveolar epithelial type II cells in primary culture

    PubMed Central

    Mao, Pu; Wu, Songling; Li, Jianchun; Fu, Wei; He, Weiqun; Liu, Xiaoqing; Slutsky, Arthur S; Zhang, Haibo; Li, Yimin

    2015-01-01

    Alveolar epithelial type II (AEII) cells are a key structure and defender in the lung but also are the targets in many lung diseases, including acute respiratory distress syndrome, ventilator-induced lung injury, and pulmonary fibrosis. We sought to establish an optimized method for high yielding and long maintenance of characteristics of primary human AEII cells to facilitate the investigation of the mechanisms of lung diseases at the cellular and molecular levels. Adult human peripheral normal lung tissues of oncologic patients undergoing lung resection were collected. The AEII cells were isolated and identified by the expression of pro-surfactant protein (SP)C, epithelial sodium channel (αENaC) and cytokeratin (CK)-8, the lamellar bodies specific for AEII cells, and confirmed by the histology using electron microscopy. The phenotype of AEII cells was characterized by the expression of surfactant proteins (SP-A, SP-B, SP-C, SP-D), CK-8, KL-6, αENaC, and aquaporin (AQP)-3, which was maintained over 20 days. The biological activity of the primary human AEII cells producing SP-C, cytokines, and intercellular adhesion molecule-1 was vigorous in response to stimulation with tumor necrosis factor-α. We have modified previous methods and optimized a method for isolation of high purity and long maintenance of the human AEII cell phenotype in primary culture. This method provides an important tool for studies aiming at elucidating the molecular mechanisms of lung diseases exclusively in AEII cells. PMID:25677546

  19. TCDD alters medial epithelial cell differentiation during palatogenesis

    SciTech Connect

    Abbott, B.D.; Birnbaum, L.S. )

    1989-06-15

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widely distributed, persistent environmental contaminant that is teratogenic in mice, where it induces hydronephrosis and cleft palate. The incidence of clefting has been shown to be dose dependent after exposure on either gestation Day (GD) 10 or 12, although the embryo is more susceptible on GD 12. TCDD-exposed palatal shelves meet but do not fuse, and programmed cell death of the medial epithelial cells is inhibited. The mechanism of action through which TCDD alters the program of medial cell development has not been examined in earlier studies, and it is not known whether the mechanism is the same regardless of the dose or developmental stage of exposure. In this study, C57BL/6N mice, a strain sensitive to TCDD, were dosed orally on GD 10 or 12 with 0, 6, 12, 24, or 30 micrograms/kg body wt, in 10 ml corn oil/kg. Embryonic palatal shelves were examined on GD 14, 15, or 16. The degree of palatal closure, epithelial surface morphology, and cellular ultrastructure, the incorporation of (3H)TdR, the expression of EGF receptors, and the binding of 125I-EGF were assessed. After exposure on GD 10 or 12, TCDD altered the differentiation pathway of the medial epithelial cells. The palatal shelves were of normal size and overall morphology, but fusion of the medial epithelia of the opposing shelves did not occur. TCDD prevented programmed cell death of the medial peridermal cells. The expression of EGF receptors by medial cells continued through Day 16 and the receptors were able to bind ligand. The medial cells differentiated into a stratified, squamous, keratinizing epithelium. The shift in phenotype to an oral-like epithelium occurred after exposure on either GD 10 or 12. At the lower dose (6 micrograms/kg), fewer cleft palates were produced, but those shelves which did respond had a fully expressed shift in differentiation.

  20. A Molecular Switch for the Orientation of Epithelial Cell Polarization

    PubMed Central

    Bryant, David M.; Roignot, Julie; Datta, Anirban; Overeem, Arend W.; Kim, Minji; Yu, Wei; Peng, Xiao; Eastburn, Dennis J.; Ewald, Andrew J.; Werb, Zena; Mostov, Keith E.

    2014-01-01

    SUMMARY The formation of epithelial tissues containing lumens requires not only the apical-basolateral polarization of cells, but also the coordinated orientation of this polarity such that the apical surfaces of neighboring cells all point toward the central lumen. Defects in extracellular matrix (ECM) signaling lead to inverted polarity so that the apical surfaces face the surrounding ECM. We report a molecular switch mechanism controlling polarity orientation. ECM signals through a β1-integrin/FAK/p190RhoGAP complex to down-regulate a RhoA/ROCK/Ezrin pathway at the ECM interface. PKCβII phosphorylates the apical identity-promoting Podocalyxin/NHERF1/Ezrin complex, removing Podocalyxin from the ECM-abutting cell surface and initiating its transcytosis to an apical membrane initiation site for lumen formation. Inhibition of this switch mechanism results in the retention of Podocalyxin at the ECM interface and the development instead of collective front-rear polarization and motility. Thus, ECM-derived signals control the morphogenesis of epithelial tissues by controlling the collective orientation of epithelial polarization. PMID:25307480

  1. Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells.

    PubMed Central

    Corbeil, L B; Hodgson, J L; Jones, D W; Corbeil, R R; Widders, P R; Stephens, L R

    1989-01-01

    Adherence of Tritrichomonas foetus to bovine vaginal epithelial cells (VECs) in vitro was investigated with fresh washed bovine VECs and log-phase cultures of T. foetus. Observation under phase-contrast microscopy showed that T. foetus usually adhered first by the posterior flagellum and later by the body. Significantly more keratinized squamous epithelial cells were detected with attached parasites than nonkeratinized round epithelial cells. The optimal pH range for attachment was 6.0 to 7.5, with peak attachment at pH 6.5 for squamous VECs. Surface-reactive bovine antiserum to T. foetus prevented adherence to bovine squamous VECs. Inhibition of adherence occurred at nonagglutinating, nonimmobilizing serum dilutions. Antiserum fractions enriched for immunoglobulin G1 inhibited adherence, but fractions enriched for immunoglobulin G2 did not. The inhibitory antiserum was specific for several medium- to high-molecular-weight membrane antigens as detected in Western blots (immunoblots). The ability of surface-reactive antibodies to prevent adherence and to agglutinate and immobilize T. foetus indicates that they may be protective. Images PMID:2471692

  2. Concise review: Insights from normal bone remodeling and stem cell-based therapies for bone repair.

    PubMed

    Khosla, Sundeep; Westendorf, Jennifer J; Mödder, Ulrike I

    2010-12-01

    There is growing interest in the use of mesenchymal stem cells for bone repair. As a major reason for normal bone remodeling is the removal of fatigue microcracks, advances in our understanding of this process may inform approaches to enhance fracture healing. Increasing evidence now indicates that physiological bone remodeling occurs in close proximity to blood vessels and that these vessels carry perivascular stem cells that differentiate into osteoblasts. Similarly, fracture healing is critically dependent on the ingrowth of blood vessels not only for a nutrient supply but also for the influx of osteoblasts. A number of animal and human studies have now shown the potential benefit of bone marrow-derived mesenchymal stem cells in enhancing bone repair. However, as in other tissues, the question of whether these cells improve fracture healing directly by differentiating into osteoblasts or indirectly by secreting paracrine factors that recruit blood vessels and the accompanying perivascular stem cells remains a major unresolved issue. Moreover, CD34+ cells, which are enriched for endothelial/hematopoietic cells, have also shown efficacy in various bone repair models, at least in part due to the induction of angiogenesis and recruitment of host progenitor cells. Thus, mesenchymal and nonmesenchymal stem/progenitor cells are attractive options for bone repair. It is possible that they contribute directly to bone repair, but it is also likely that they express paracrine factors in the appropriate amounts and combinations that promote and sustain the healing process. PMID:20960512

  3. Cell elongation is key to in silico replication of in vitro vasculogenesis and subsequent remodeling

    PubMed Central

    Merks, Roeland M.H.; Brodsky, Sergey V.; Goligorksy, Michael S.; Newman, Stuart A.; Glazier, James A.

    2008-01-01

    Vasculogenesis, the de novo growth of the primary vascular network from initially dispersed endothelial cells, is the first step in the development of the circulatory system in vertebrates. In the first stages of vasculogenesis, endothelial cells elongate and form a network-like structure, called the primary capillary plexus, which subsequently remodels, with the size of the vacancies between ribbons of endothelial cells coarsening over time. To isolate such intrinsic morphogenetic ability of endothelial cells from its regulation by long-range guidance cues and additional cell types, we use an in vitro model of human umbilical vein endothelial cells (HUVEC) in Matrigel. This quasi-two-dimensional endothelial cell culture model would most closely correspond to vasculogenesis in flat areas of the embryo like the yolk sac. Several studies have used continuum mathematical models to explore in vitro vasculogenesis: such models describe cell ensembles but ignore the endothelial cells’ shapes and active surface fluctuations. While these models initially reproduce vascular-like morphologies, they eventually stabilize into a disconnected pattern of vascular “islands.” Also, they fail to reproduce temporally correct network coarsening. Using a cell-centered computational model, we show that the endothelial cells’ elongated shape is key to correct spatiotemporal in silico replication of stable vascular network growth. We validate our simulation results against HUVEC cultures using time-resolved image analysis and find that our simulations quantitatively reproduce in vitro vasculogenesis and subsequent in vitro remodeling. PMID:16325173

  4. 5-Hydroxymethylcytosine Remodeling Precedes Lineage Specification during Differentiation of Human CD4(+) T Cells.

    PubMed

    Nestor, Colm E; Lentini, Antonio; Hägg Nilsson, Cathrine; Gawel, Danuta R; Gustafsson, Mika; Mattson, Lina; Wang, Hui; Rundquist, Olof; Meehan, Richard R; Klocke, Bernward; Seifert, Martin; Hauck, Stefanie M; Laumen, Helmut; Zhang, Huan; Benson, Mikael

    2016-07-12

    5-methylcytosine (5mC) is converted to 5-hydroxymethylcytosine (5hmC) by the TET family of enzymes as part of a recently discovered active DNA de-methylation pathway. 5hmC plays important roles in regulation of gene expression and differentiation and has been implicated in T cell malignancies and autoimmunity. Here, we report early and widespread 5mC/5hmC remodeling during human CD4(+) T cell differentiation ex vivo at genes and cell-specific enhancers with known T cell function. We observe similar DNA de-methylation in CD4(+) memory T cells in vivo, indicating that early remodeling events persist long term in differentiated cells. Underscoring their important function, 5hmC loci were highly enriched for genetic variants associated with T cell diseases and T-cell-specific chromosomal interactions. Extensive functional validation of 22 risk variants revealed potentially pathogenic mechanisms in diabetes and multiple sclerosis. Our results support 5hmC-mediated DNA de-methylation as a key component of CD4(+) T cell biology in humans, with important implications for gene regulation and lineage commitment. PMID:27346350

  5. Elastic properties of epithelial cells probed by atomic force microscopy.

    PubMed

    Brückner, Bastian R; Janshoff, Andreas

    2015-11-01

    Cellular mechanics plays a crucial role in many biological processes such as cell migration, cell growth, embryogenesis, and oncogenesis. Epithelia respond to environmental cues comprising biochemical and physical stimuli through defined changes in cell elasticity. For instance, cells can differentiate between certain properties such as viscoelasticity or topography of substrates by adapting their own elasticity and shape. A living cell is a complex viscoelastic body that not only exhibits a shell architecture composed of a membrane attached to a cytoskeleton cortex but also generates contractile forces through its actomyosin network. Here we review cellular mechanics of single cells in the context of epithelial cell layers responding to chemical and physical stimuli. This article is part of a Special Issue entitled: Mechanobiology. PMID:26193077

  6. Differential Glutamate Metabolism in Proliferating and Quiescent Mammary Epithelial Cells.

    PubMed

    Coloff, Jonathan L; Murphy, J Patrick; Braun, Craig R; Harris, Isaac S; Shelton, Laura M; Kami, Kenjiro; Gygi, Steven P; Selfors, Laura M; Brugge, Joan S

    2016-05-10

    Mammary epithelial cells transition between periods of proliferation and quiescence during development, menstrual cycles, and pregnancy, and as a result of oncogenic transformation. Utilizing an organotypic 3D tissue culture model coupled with quantitative metabolomics and proteomics, we identified significant differences in glutamate utilization between proliferating and quiescent cells. Relative to quiescent cells, proliferating cells catabolized more glutamate via transaminases to couple non-essential amino acid (NEAA) synthesis to α-ketoglutarate generation and tricarboxylic acid (TCA) cycle anaplerosis. As cells transitioned to quiescence, glutamine consumption and transaminase expression were reduced, while glutamate dehydrogenase (GLUD) was induced, leading to decreased NEAA synthesis. Highly proliferative human tumors display high transaminase and low GLUD expression, suggesting that proliferating cancer cells couple glutamine consumption to NEAA synthesis to promote biosynthesis. These findings describe a competitive and partially redundant relationship between transaminases and GLUD, and they reveal how coupling of glutamate-derived carbon and nitrogen metabolism can be regulated to support cell proliferation. PMID:27133130

  7. Structure of Pneumococcal Peptidoglycan Hydrolase LytB Reveals Insights into the Bacterial Cell Wall Remodeling and Pathogenesis*

    PubMed Central

    Bai, Xiao-Hui; Chen, Hui-Jie; Jiang, Yong-Liang; Wen, Zhensong; Huang, Yubin; Cheng, Wang; Li, Qiong; Qi, Lei; Zhang, Jing-Ren; Chen, Yuxing; Zhou, Cong-Zhao

    2014-01-01

    Streptococcus pneumoniae causes a series of devastating infections in humans. Previous studies have shown that the endo-β-N-acetylglucosaminidase LytB is critical for pneumococcal cell division and nasal colonization, but the biochemical mechanism of LytB action remains unknown. Here we report the 1.65 Å crystal structure of the catalytic domain (residues Lys-375–Asp-658) of LytB (termed LytBCAT), excluding the choline binding domain. LytBCAT consists of three structurally independent modules: SH3b, WW, and GH73. These modules form a “T-shaped” pocket that accommodates a putative tetrasaccharide-pentapeptide substrate of peptidoglycan. Structural comparison and simulation revealed that the GH73 module of LytB harbors the active site, including the catalytic residue Glu-564. In vitro assays of hydrolytic activity indicated that LytB prefers the peptidoglycan from the lytB-deficient pneumococci, suggesting the existence of a specific substrate of LytB in the immature peptidoglycan. Combined with in vitro cell-dispersing and in vivo cell separation assays, we demonstrated that all three modules are necessary for the optimal activity of LytB. Further functional analysis showed that the full catalytic activity of LytB is required for pneumococcal adhesion to and invasion into human lung epithelial cells. Structure-based alignment indicated that the unique modular organization of LytB is highly conserved in its orthologs from Streptococcus mitis group and Gemella species. These findings provided structural insights into the pneumococcal cell wall remodeling and novel hints for the rational design of therapeutic agents against pneumococcal growth and thereby the related diseases. PMID:25002590

  8. 293 cells express both epithelial as well as mesenchymal cell adhesion molecules

    PubMed Central

    INADA, MASAKAZU; IZAWA, GENYA; KOBAYASHI, WAKAKO; OZAWA, MASAYUKI

    2016-01-01

    The 293 cell line, used extensively in various types of studies due to the ease with which these cells can be transfected, was thought to be derived by the transformation of primary cultures of human embryonic kidney cells with sheared adenovirus type 5 DNA. Although the 293 cells were assumed to originate from epithelial cells, the exact origin of these cells remains unknown. Previous attempts to characterize these cells combined immunostaining, immunoblot analysis and microarray analysis to demonstrate that 293 cells express neurofilament subunits, α-internexin, and several other proteins typically found in neurons. These findings raised the possibility that the 293 cell line may have originated from human neuronal lineage cells. Contrary to this suggestion, in this study, we found that the 293 cells expressed N-cadherin and vimentin, which are marker proteins expressed in mesenchymal cells. Furthermore, the 293 cells also expressed E-cadherin, cytokeratins 5/8 and desmoglein 2, which are epithelial cell markers. When the cells, primarily cultured from the kidneys of Clawn miniature swine and passaged 10–15 generations [termed porcine kidney epithelial (PKE) cells] were examined, they were found to be positive for the expression of both mesenchymal and epithelial markers. Thus, transformation by adenovirus was not necessary for the cells to express N-cadherin. Occludin and zonula occludens (ZO)-1, two components of tight junctions in epithelial and endothelial cells, were detected in the 293 and the PKE cells. Thus, the findings of the present study demonstrate that 293 cells retain several characteristics of epithelial cells. PMID:27121032

  9. Comparison of functional limbal epithelial stem cell isolation methods.

    PubMed

    López-Paniagua, Marina; Nieto-Miguel, Teresa; de la Mata, Ana; Dziasko, Marc; Galindo, Sara; Rey, Esther; Herreras, José M; Corrales, Rosa M; Daniels, Julie T; Calonge, Margarita

    2016-05-01

    The transplantation of limbal epithelial stem cells (LESCs) cultured in vitro is a great advance in the treatment of patients suffering from LESC deficiency. However, the optimal technique for LESC isolation from a healthy limbal niche has not yet been established. Our aim was to determine which isolation method renders the highest recovery of functional LESCs from the human limbus. To achieve this purpose, we compared limbal primary cultures (LPCs) obtained from explants and cell suspensions on plastic culture plates. Cell morphology was observed by phase contrast and transmission electron microscopy. LESC, corneal epithelial cell, fibroblast, endothelial cell, melanocyte, and dendritic cell markers were analyzed by real time by reverse transcription polymerase chain reaction and/or immunofluorescence. In addition, colony forming efficiency (CFE) and the presence of holoclones, meroclones, and paraclones were studied. We observed that LPC cells obtained from both methods had cuboidal morphology, desmosomes, and prominent intermediate filaments. The expression of LESC markers (K14, K15, ABCG2, p63α) was similar or higher in LPCs established through cell suspensions, except the expression of p63α mRNA, and there were no significant differences in the expression of corneal epithelial markers (K3, K12). Endothelial cell (PECAM), melanocyte (MART-1), and dendritic cell (CD11c) proteins were not detected, while fibroblast-protein (S100A4) was detected in all LPCs. The CFE was significantly higher in LPCs from cell suspensions. Cells from confluent LPCs produced by explants generated only paraclones (100%), while the percentage of paraclones from LPCs established through cell suspensions was 90% and the remaining 10% were meroclones. In conclusion, LPCs established from cell suspensions have a cell population richer in functional LESCs than LPCs obtained from explants. These results suggest that in a clinical situation in which it is possible to choose between either

  10. Role of Stem Cell Factor and Granulocyte-Colony Stimulating Factor in Remodeling during Liver Regeneration

    PubMed Central

    Meng, Fanyin; Francis, Heather; Glaser, Shannon; Han, Yuyan; DeMorrow, Sharon; Stokes, Allison; Staloch, Dustin; Venter, Julie; White, Melanie; Ueno, Yoshiyuki; Reid, Lola M.; Alpini, Gianfranco

    2011-01-01

    Functional pluripotent characteristics have been observed in specific subpopulations of hepatic cells that express some of the known cholangiocyte markers. Although evidence indicates that specific cytokines, granulocyte-macrophage colony stimulating factors (GM-CSF) and stem cell factor (SCF) may be candidate treatments for liver injury, the role of these cytokines in intrahepatic biliary epithelium remodeling is unknown. Thus, our aim was to characterize the specific cytokines that regulate the remodeling potentials of cholangiocytes after 70% partial hepatectomy (PH). The expression of the cytokines and their downstream signaling molecules was studied in rats after 70% PH by immunoblots, and in small and large murine cholangiocyte cultures (SMCCs and LMCCs) by immunocytochemistry and real-time PCR. There was a significant and stable increase in SCF and GM-CSF levels until 7 days after PH. Real-time PCR analysis revealed significant increases of key remodeling molecules, such as S100A4 and miR-181b after SCF plus GM-CSF administration in SMCCs. SMCCs produced significant amounts of soluble and bound SCF and GM-CSF in response to TGF-β. When SMCCs were incubated with TGF-β plus anti–SCF and GM-CSF antibodies, there was a significant decrease in S100A4 expression. Furthermore, treatment of SMCCs with SCF + GM-CSF significantly increased matrix metalloproteinases (MMP-2 and MMP-9) mRNA as well as miR-181b expression along with a reduction of metalloproteinase inhibitor 3 (TIMP-3). The levels of MMP-2, MMP-9 and miR-181b were also up-regulated in rat liver and isolated cholangiocytes after PH. CONCLUSION Our data suggest that altered expression of SCF and GM-CSF following PH can contribute to biliary remodeling (for example post-transplantation) by functional deregulation of activity of key signaling intermediates involved in cell expansion and multipotent differentiation. PMID:21932404

  11. Ethanol stimulation of HIV infection of oral epithelial cells.

    PubMed

    Zheng, Jun; Yang, Otto O; Xie, Yiming; Campbell, Richard; Chen, Irvin S Y; Pang, Shen

    2004-12-01

    Oral mucosal cells can be infected by exogenous HIV during receptive oral sex or breast-feeding. The risk of oral mucosal infection depends on the infection efficiency of the HIV strains present in the oral cavity, the viral titers, and the defense mechanisms in the oral cavity environment. It is expected that alcohol can weaken the host defense mechanism against HIV infection in the oral cavity. We modified an HIV strain, NL4-3, by inserting the enhanced green fluorescent protein gene and used this virus to infect oral epithelial cells obtained from patients. Various concentrations of ethanol (0%-4%) were added to the infected cells. HIV-infected cells were detected by fluorescent microscopy or fluorescence-activated cell sorting. We found that ethanol significantly increases HIV infection of primary oral epithelial cells (POEs). POEs pretreated with 4% ethanol for less than 10 minutes demonstrated 3- to 6-fold higher susceptibility to infection by the CXCR-4 HIV strain NL4-3. Our studies also demonstrated that HIV infects POEs through a gp120-independent mechanism. We tested an HIV CCR5 strain, JRCSF, and also found its infection efficiency to be stimulated by alcohol. Our results indicate that in cell culture conditions, the ranges of concentrations of alcohol that are commercially available are able to stimulate the infection efficiency of HIV in POEs. PMID:15602121

  12. Prion Infection of Epithelial Rov Cells Is a Polarized Event

    PubMed Central

    Paquet, Sophie; Sabuncu, Elifsu; Delaunay, Jean-Louis; Laude, Hubert; Vilette, Didier

    2004-01-01

    During prion infections, the cellular glycosylphosphatidylinositol-anchored glycoprotein PrP is converted into a conformational isoform. This abnormal conformer is thought to recruit and convert the normal cellular PrP into a likeness of itself and is proposed to be the infectious agent. We investigated the distribution of the PrP protein on the surface of Rov cells, an epithelial cell line highly permissive to prion multiplication, and we found that PrP is primarily expressed on the apical side. We further show that prion transmission to Rov cells is much more efficient if infectivity contacts the apical side, indicating that the apical and basolateral sides of Rov cells are not equally competent for prion infection and adding prions to the list of the conventional infectious agents (viruses and bacteria) that infect epithelial cells in a polarized manner. These data raise the possibility that apically expressed PrP may be involved in this polarized process of infection. This would add further support for a crucial role of PrP at the cell surface in prion infection of target cells. PMID:15194791

  13. Kindlin-1 and -2 Have Overlapping Functions in Epithelial Cells

    PubMed Central

    He, Yinghong; Esser, Philipp; Heinemann, Anja; Bruckner-Tuderman, Leena; Has, Cristina

    2011-01-01

    Kindlins are a novel family of intracellular adaptor proteins in integrin-containing focal adhesions. Kindlin-1 and -2 are expressed in the skin, but whether and how they cooperate in adult epithelial cells have remained elusive. We uncovered the overlapping roles of kindlin-1 and -2 in maintaining epithelial integrity and show that the phenotype of kindlin-1-deficient cells can be modulated by regulating kindlin-2 gene expression and vice versa. The experimental evidence is provided by use of human keratinocyte cell lines that express both kindlins, just kindlin-1 or kindlin-2, or none of them. Double deficiency of kindlin-1 and -2 had significant negative effects on focal adhesion formation and actin cytoskeleton organization, cell adhesion, survival, directional migration, and activation of β1 integrin, whereas deficiency of one kindlin only showed variable perturbation of these functions. Cell motility and formation of cell-cell contacts were particularly affected by lack of kindlin-2. These results predict that kindlin-1 and -2 can functionally compensate for each other, at least in part. The high physiologic and pathologic significance of the compensation was emphasized by the discovery of environmental regulation of kindlin-2 expression. UV-B irradiation induced loss of kindlin-2 in keratinocytes. This first example of environmental regulation of kindlin expression has implications for phenotype modulation in Kindler syndrome, a skin disorder caused by kindlin-1 deficiency. PMID:21356350

  14. Cellular and molecular remodelling of a host cell for vertical transmission of bacterial symbionts

    PubMed Central

    Luan, Jun-Bo; Shan, Hong-Wei; Isermann, Philipp; Huang, Jia-Hsin; Lammerding, Jan; Liu, Shu-Sheng; Douglas, Angela E.

    2016-01-01

    Various insects require intracellular bacteria that are restricted to specialized cells (bacteriocytes) and are transmitted vertically via the female ovary, but the transmission mechanisms are obscure. We hypothesized that, in the whitefly Bemisia tabaci, where intact bacteriocytes (and not isolated bacteria) are transferred to oocytes, the transmission mechanism would be evident as cellular and molecular differences between the nymph (pre-adult) and adult bacteriocytes. We demonstrate dramatic remodelling of bacteriocytes at the developmental transition from nymph to adulthood. This transition involves the loss of cell–cell adhesion, high division rates to constant cell size and onset of cell mobility, enabling the bacteriocytes to crawl to the ovaries. These changes are accompanied by cytoskeleton reorganization and changes in gene expression: genes functioning in cell–cell adhesion display reduced expression and genes involved in cell division, cell motility and endocytosis/exocytosis have elevated expression in adult bacteriocytes, relative to nymph bacteriocytes. This study demonstrates, for the first time, how developmentally orchestrated remodelling of gene expression and correlated changes in cell behaviour underpin the capacity of bacteriocytes to mediate the vertical transmission and persistence of the symbiotic bacteria on which the insect host depends. PMID:27358364

  15. Mitochondrial dysfunction remodels one-carbon metabolism in human cells

    PubMed Central

    Bao, Xiaoyan Robert; Ong, Shao-En; Goldberger, Olga; Peng, Jun; Sharma, Rohit; Thompson, Dawn A; Vafai, Scott B; Cox, Andrew G; Marutani, Eizo; Ichinose, Fumito; Goessling, Wolfram; Regev, Aviv; Carr, Steven A; Clish, Clary B; Mootha, Vamsi K

    2016-01-01

    Mitochondrial dysfunction is associated with a spectrum of human disorders, ranging from rare, inborn errors of metabolism to common, age-associated diseases such as neurodegeneration. How these lesions give rise to diverse pathology is not well understood, partly because their proximal consequences have not been well-studied in mammalian cells. Here we provide two lines of evidence that mitochondrial respiratory chain dysfunction leads to alterations in one-carbon metabolism pathways. First, using hypothesis-generating metabolic, proteomic, and transcriptional profiling, followed by confirmatory experiments, we report that mitochondrial DNA depletion leads to an ATF4-mediated increase in serine biosynthesis and transsulfuration. Second, we show that lesioning the respiratory chain impairs mitochondrial production of formate from serine, and that in some cells, respiratory chain inhibition leads to growth defects upon serine withdrawal that are rescuable with purine or formate supplementation. Our work underscores the connection between the respiratory chain and one-carbon metabolism with implications for understanding mitochondrial pathogenesis. DOI: http://dx.doi.org/10.7554/eLife.10575.001 PMID:27307216

  16. Mitochondrial dysfunction remodels one-carbon metabolism in human cells.

    PubMed

    Bao, Xiaoyan Robert; Ong, Shao-En; Goldberger, Olga; Peng, Jun; Sharma, Rohit; Thompson, Dawn A; Vafai, Scott B; Cox, Andrew G; Marutani, Eizo; Ichinose, Fumito; Goessling, Wolfram; Regev, Aviv; Carr, Steven A; Clish, Clary B; Mootha, Vamsi K

    2016-01-01

    Mitochondrial dysfunction is associated with a spectrum of human disorders, ranging from rare, inborn errors of metabolism to common, age-associated diseases such as neurodegeneration. How these lesions give rise to diverse pathology is not well understood, partly because their proximal consequences have not been well-studied in mammalian cells. Here we provide two lines of evidence that mitochondrial respiratory chain dysfunction leads to alterations in one-carbon metabolism pathways. First, using hypothesis-generating metabolic, proteomic, and transcriptional profiling, followed by confirmatory experiments, we report that mitochondrial DNA depletion leads to an ATF4-mediated increase in serine biosynthesis and transsulfuration. Second, we show that lesioning the respiratory chain impairs mitochondrial production of formate from serine, and that in some cells, respiratory chain inhibition leads to growth defects upon serine withdrawal that are rescuable with purine or formate supplementation. Our work underscores the connection between the respiratory chain and one-carbon metabolism with implications for understanding mitochondrial pathogenesis. PMID:27307216

  17. Mesenchymal Stromal Cells Epithelial Transition Induced by Renal Tubular Cells-Derived Extracellular Vesicles

    PubMed Central

    Chiabotto, Giulia; Bruno, Stefania; Collino, Federica

    2016-01-01

    Mesenchymal-epithelial interactions play an important role in renal tubular morphogenesis and in maintaining the structure of the kidney. The aim of this study was to investigate whether extracellular vesicles (EVs) produced by human renal proximal tubular epithelial cells (RPTECs) may induce mesenchymal-epithelial transition of bone marrow-derived mesenchymal stromal cells (MSCs). To test this hypothesis, we characterized the phenotype and the RNA content of EVs and we evaluated the in vitro uptake and activity of EVs on MSCs. MicroRNA (miRNA) analysis suggested the possible implication of the miR-200 family carried by EVs in the epithelial commitment of MSCs. Bone marrow-derived MSCs were incubated with EVs, or RPTEC-derived total conditioned medium, or conditioned medium depleted of EVs. As a positive control, MSCs were co-cultured in a transwell system with RPTECs. Epithelial commitment of MSCs was assessed by real time PCR and by immunofluorescence analysis of cellular expression of specific mesenchymal and epithelial markers. After one week of incubation with EVs and total conditioned medium, we observed mesenchymal-epithelial transition in MSCs. Stimulation with conditioned medium depleted of EVs did not induce any change in mesenchymal and epithelial gene expression. Since EVs were found to contain the miR-200 family, we transfected MSCs using synthetic miR-200 mimics. After one week of transfection, mesenchymal-epithelial transition was induced in MSCs. In conclusion, miR-200 carrying EVs released from RPTECs induce the epithelial commitment of MSCs that may contribute to their regenerative potential. Based on experiments of MSC transfection with miR-200 mimics, we suggested that the miR-200 family may be involved in mesenchymal-epithelial transition of MSCs. PMID:27409796

  18. Computational Modeling Predicts Simultaneous Targeting of Fibroblasts and Epithelial Cells Is Necessary for Treatment of Pulmonary Fibrosis

    PubMed Central

    Warsinske, Hayley C.; Wheaton, Amanda K.; Kim, Kevin K.; Linderman, Jennifer J.; Moore, Bethany B.; Kirschner, Denise E.

    2016-01-01

    Pulmonary fibrosis is pathologic remodeling of lung tissue that can result in difficulty breathing, reduced quality of life, and a poor prognosis for patients. Fibrosis occurs as a result of insult to lung tissue, though mechanisms of this response are not well-characterized. The disease is driven in part by dysregulation of fibroblast proliferation and differentiation into myofibroblast cells, as well as pro-fibrotic mediator-driven epithelial cell apoptosis. The most well-characterized pro-fibrotic mediator associated with pulmonary fibrosis is TGF-β1. Excessive synthesis of, and sensitivity to, pro-fibrotic mediators as well as insufficient production of and sensitivity to anti-fibrotic mediators has been credited with enabling fibroblast accumulation. Available treatments neither halt nor reverse lung damage. In this study we have two aims: to identify molecular and cellular scale mechanisms driving fibroblast proliferation and differentiation as well as epithelial cell survival in the context of fibrosis, and to predict therapeutic targets and strategies. We combine in vitro studies with a multi-scale hybrid agent-based computational model that describes fibroblasts and epithelial cells in co-culture. Within this model TGF-β1 represents a pro-fibrotic mediator and we include detailed dynamics of TGF-β1 receptor ligand signaling in fibroblasts. PGE2 represents an anti-fibrotic mediator. Using uncertainty and sensitivity analysis we identify TGF-β1 synthesis, TGF-β1 activation, and PGE2 synthesis among the key mechanisms contributing to fibrotic outcomes. We further demonstrate that intervention strategies combining potential therapeutics targeting both fibroblast regulation and epithelial cell survival can promote healthy tissue repair better than individual strategies. Combinations of existing drugs and compounds may provide significant improvements to the current standard of care for pulmonary fibrosis. Thus, a two-hit therapeutic intervention strategy

  19. Computational modeling predicts simultaneous targeting of fibroblasts and epithelial cells is necessary for treatment of pulmonary fibrosis

    DOE PAGESBeta

    Warsinske, Hayley C.; Wheaton, Amanda K.; Kim, Kevin K.; Linderman, Jennifer J.; Moore, Bethany B.; Kirschner, Denise E.

    2016-06-23

    Pulmonary fibrosis is pathologic remodeling of lung tissue that can result in difficulty breathing, reduced quality of life, and a poor prognosis for patients. Fibrosis occurs as a result of insult to lung tissue, though mechanisms of this response are not well-characterized. The disease is driven in part by dysregulation of fibroblast proliferation and differentiation into myofibroblast cells, as well as pro-fibrotic mediator-driven epithelial cell apoptosis. The most well-characterized pro-fibrotic mediator associated with pulmonary fibrosis is TGF-β1. Excessive synthesis of, and sensitivity to, pro-fibrotic mediators as well as insufficient production of and sensitivity to anti-fibrotic mediators has been credited withmore » enabling fibroblast accumulation. Available treatments neither halt nor reverse lung damage. In this study we have two aims: to identify molecular and cellular scale mechanisms driving fibroblast proliferation and differentiation as well as epithelial cell survival in the context of fibrosis, and to predict therapeutic targets and strategies. We combine in vitro studies with a multi-scale hybrid agent-based computational model that describes fibroblasts and epithelial cells in co-culture. Within this model TGF-β1 represents a pro-fibrotic mediator and we include detailed dynamics of TGFβ1 receptor ligand signaling in fibroblasts. PGE2 represents an anti-fibrotic mediator. Using uncertainty and sensitivity analysis we identify TGF-β1 synthesis, TGF-β1 activation, and PGE2 synthesis among the key mechanisms contributing to fibrotic outcomes. We further demonstrate that intervention strategies combining potential therapeutics targeting both fibroblast regulation and epithelial cell survival can promote healthy tissue repair better than individual strategies. Combinations of existing drugs and compounds may provide significant improvements to the current standard of care for pulmonary fibrosis. In conclusion, a two-hit therapeutic

  20. Computational Modeling Predicts Simultaneous Targeting of Fibroblasts and Epithelial Cells Is Necessary for Treatment of Pulmonary Fibrosis.

    PubMed

    Warsinske, Hayley C; Wheaton, Amanda K; Kim, Kevin K; Linderman, Jennifer J; Moore, Bethany B; Kirschner, Denise E

    2016-01-01

    Pulmonary fibrosis is pathologic remodeling of lung tissue that can result in difficulty breathing, reduced quality of life, and a poor prognosis for patients. Fibrosis occurs as a result of insult to lung tissue, though mechanisms of this response are not well-characterized. The disease is driven in part by dysregulation of fibroblast proliferation and differentiation into myofibroblast cells, as well as pro-fibrotic mediator-driven epithelial cell apoptosis. The most well-characterized pro-fibrotic mediator associated with pulmonary fibrosis is TGF-β1. Excessive synthesis of, and sensitivity to, pro-fibrotic mediators as well as insufficient production of and sensitivity to anti-fibrotic mediators has been credited with enabling fibroblast accumulation. Available treatments neither halt nor reverse lung damage. In this study we have two aims: to identify molecular and cellular scale mechanisms driving fibroblast proliferation and differentiation as well as epithelial cell survival in the context of fibrosis, and to predict therapeutic targets and strategies. We combine in vitro studies with a multi-scale hybrid agent-based computational model that describes fibroblasts and epithelial cells in co-culture. Within this model TGF-β1 represents a pro-fibrotic mediator and we include detailed dynamics of TGF-β1 receptor ligand signaling in fibroblasts. PGE2 represents an anti-fibrotic mediator. Using uncertainty and sensitivity analysis we identify TGF-β1 synthesis, TGF-β1 activation, and PGE2 synthesis among the key mechanisms contributing to fibrotic outcomes. We further demonstrate that intervention strategies combining potential therapeutics targeting both fibroblast regulation and epithelial cell survival can promote healthy tissue repair better than individual strategies. Combinations of existing drugs and compounds may provide significant improvements to the current standard of care for pulmonary fibrosis. Thus, a two-hit therapeutic intervention strategy

  1. A MicroRNA Network Dysregulated in Asthma Controls IL-6 Production in Bronchial Epithelial Cells

    PubMed Central

    Louafi, Fethi; Francisco-Garcia, Ana S.; Rupani, Hitasha; Bedke, Nicole; Holgate, Stephen; Howarth, Peter H.; Davies, Donna E.; Sanchez-Elsner, Tilman

    2014-01-01

    MicroRNAs are short non-coding single stranded RNAs that regulate gene expression. While much is known about the effects of individual microRNAs, there is now growing evidence that they can work in co-operative networks. MicroRNAs are known to be dysregulated in many diseases and affect pathways involved in the pathology. We investigated dysregulation of microRNA networks using asthma as the disease model. Asthma is a chronic inflammatory disease of the airways characterized by bronchial hyperresponsiveness and airway remodelling. The airway epithelium is a major contributor to asthma pathology and has been shown to produce an excess of inflammatory and pro-remodelling cytokines such as TGF-β, IL-6 and IL-8 as well as deficient amounts of anti-viral interferons. After performing microRNA arrays, we found that microRNAs -18a, -27a, -128 and -155 are down-regulated in asthmatic bronchial epithelial cells, compared to cells from healthy donors. Interestingly, these microRNAs are predicted in silico to target several components of the TGF-β, IL-6, IL-8 and interferons pathways. Manipulation of the levels of individual microRNAs in bronchial epithelial cells did not have an effect on any of these pathways. Importantly, knock-down of the network of microRNAs miR-18a, -27a, -128 and -155 led to a significant increase of IL-8 and IL-6 expression. Interestingly, despite strong in silico predictions, down-regulation of the pool of microRNAs did not have an effect on the TGF-β and Interferon pathways. In conclusion, using both bioinformatics and experimental tools we found a highly relevant potential role for microRNA dysregulation in the control of IL-6 and IL-8 expression in asthma. Our results suggest that microRNAs may have different roles depending on the presence of other microRNAs. Thus, interpretation of in silico analysis of microRNA function should be confirmed experimentally in the relevant cellular context taking into account interactions with other micro

  2. REMOD: A Tool for Analyzing and Remodeling the Dendritic Architecture of Neural Cells

    PubMed Central

    Bozelos, Panagiotis; Stefanou, Stefanos S.; Bouloukakis, Georgios; Melachrinos, Constantinos; Poirazi, Panayiota

    2016-01-01

    Dendritic morphology is a key determinant of how individual neurons acquire a unique signal processing profile. The highly branched dendritic structure that originates from the cell body, explores the surrounding 3D space in a fractal-like manner, until it reaches a certain amount of complexity. Its shape undergoes significant alterations under various physiological or neuropathological conditions. Yet, despite the profound effect that these alterations can have on neuronal function, the causal relationship between the two remains largely elusive. The lack of a systematic approach for remodeling neural cells and their dendritic trees is a key limitation that contributes to this problem. Such causal relationships can be inferred via the use of large-scale neuronal models whereby the anatomical plasticity of neurons is accounted for, in order to enhance their biological relevance and hence their predictive performance. To facilitate this effort, we developed a computational tool named REMOD that allows the structural remodeling of any type of virtual neuron. REMOD is written in Python and can be accessed through a dedicated web interface that guides the user through various options to manipulate selected neuronal morphologies. REMOD can also be used to extract meaningful morphology statistics for one or multiple reconstructions, including features such as sholl analysis, total dendritic length and area, path length to the soma, centrifugal branch order, diameter tapering and more. As such, the tool can be used both for the analysis and/or the remodeling of neuronal morphologies of any type. PMID:26778971

  3. Luteolin Ameliorates Hypertensive Vascular Remodeling through Inhibiting the Proliferation and Migration of Vascular Smooth Muscle Cells

    PubMed Central

    Su, Jie; Xu, Han-Ting; Yu, Jing-Jing; Gao, Jian-Li; Lei, Jing; Yin, Qiao-Shan; Li, Bo; Pang, Min-Xia; Su, Min-Xia; Mi, Wen-Jia; Chen, Su-Hong; Lv, Gui-Yuan

    2015-01-01

    Objectives. Preliminary researches showed that luteolin was used to treat hypertension. However, it is still unclear whether luteolin has effect on the hypertensive complication such as vascular remodeling. The present study was designed to investigate the effect of luteolin on the hypertensive vascular remodeling and its molecular mechanism. Method and Results. We evaluated the effect of luteolin on aorta thickening of hypertension in spontaneous hypertensive rats (SHRs) and found that luteolin could significantly decrease the blood pressure and media thickness of aorta in vivo. Luteolin could inhibit angiotensin II- (Ang II-) induced proliferation and migration of vascular smooth muscle cells (VSMCs). Dichlorofluorescein diacetate (DCFH-DA) staining result showed that luteolin reduced Ang II-stimulated ROS production in VSMCs. Furthermore, western blot and gelatin zymography results showed that luteolin treatment leaded to a decrease in ERK1/2, p-ERK1/2, p-p38, MMP2, and proliferating cell nuclear antigen (PCNA) protein level. Conclusion. These data support that luteolin can ameliorate hypertensive vascular remodeling by inhibiting the proliferation and migration of Ang II-induced VSMCs. Its mechanism is mediated by the regulation of MAPK signaling pathway and the production of ROS. PMID:26495010

  4. Cell-Envelope Remodeling as a Determinant of Phenotypic Antibacterial Tolerance in Mycobacterium tuberculosis

    PubMed Central

    2016-01-01

    The mechanisms that lead to phenotypic antibacterial tolerance in bacteria remain poorly understood. We investigate whether changes in NaCl concentration toward physiologically higher values affect antibacterial efficacy against Mycobacterium tuberculosis (Mtb), the causal agent of human tuberculosis. Indeed, multiclass phenotypic antibacterial tolerance is observed during Mtb growth in physiologic saline. This includes changes in sensitivity to ethionamide, ethambutol, d-cycloserine, several aminoglycosides, and quinolones. By employing organism-wide metabolomic and lipidomic approaches combined with phenotypic tests, we identified a time-dependent biphasic adaptive response after exposure of Mtb to physiological levels of NaCl. A first rapid, extensive, and reversible phase was associated with changes in core and amino acid metabolism. In a second phase, Mtb responded with a substantial remodelling of plasma membrane and outer lipid membrane composition. We demonstrate that phenotypic tolerance at physiological concentrations of NaCl is the result of changes in plasma and outer membrane lipid remodeling and not changes in core metabolism. Altogether, these results indicate that physiologic saline-induced antibacterial tolerance is kinetically coupled to cell envelope changes and demonstrate that metabolic changes and growth arrest are not the cause of phenotypic tolerance observed in Mtb exposed to physiologic concentrations of NaCl. Importantly, this work uncovers a role for bacterial cell envelope remodeling in antibacterial tolerance, alongside well-documented allterations in respiration, metabolism, and growth rate. PMID:27231718

  5. REMOD: A Tool for Analyzing and Remodeling the Dendritic Architecture of Neural Cells.

    PubMed

    Bozelos, Panagiotis; Stefanou, Stefanos S; Bouloukakis, Georgios; Melachrinos, Constantinos; Poirazi, Panayiota

    2015-01-01

    Dendritic morphology is a key determinant of how individual neurons acquire a unique signal processing profile. The highly branched dendritic structure that originates from the cell body, explores the surrounding 3D space in a fractal-like manner, until it reaches a certain amount of complexity. Its shape undergoes significant alterations under various physiological or neuropathological conditions. Yet, despite the profound effect that these alterations can have on neuronal function, the causal relationship between the two remains largely elusive. The lack of a systematic approach for remodeling neural cells and their dendritic trees is a key limitation that contributes to this problem. Such causal relationships can be inferred via the use of large-scale neuronal models whereby the anatomical plasticity of neurons is accounted for, in order to enhance their biological relevance and hence their predictive performance. To facilitate this effort, we developed a computational tool named REMOD that allows the structural remodeling of any type of virtual neuron. REMOD is written in Python and can be accessed through a dedicated web interface that guides the user through various options to manipulate selected neuronal morphologies. REMOD can also be used to extract meaningful morphology statistics for one or multiple reconstructions, including features such as sholl analysis, total dendritic length and area, path length to the soma, centrifugal branch order, diameter tapering and more. As such, the tool can be used both for the analysis and/or the remodeling of neuronal morphologies of any type. PMID:26778971

  6. Intercellular Protein Transfer from Thymocytes to Thymic Epithelial Cells.

    PubMed

    Wang, Hong-Xia; Qiu, Yu-Rong; Zhong, Xiao-Ping

    2016-01-01

    Promiscuous expression of tissue restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs) is crucial for negative selection of self-reactive T cells to establish central tolerance. Intercellular transfer of self-peptide-MHC complexes from mTECs to thymic dendritic cells (DCs) allows DCs to acquire TRAs, which in turn contributes to negative selection and regulatory T cell generation. However, mTECs are unlikely to express all TRAs, such as immunoglobulins generated only in B cells after somatic recombination, hyper-mutation, or class-switches. We report here that both mTECs and cortical TECs can efficiently acquire not only cell surface but also intracellular proteins from thymocytes. This reveals a previously unappreciated intercellular sharing of molecules from thymocytes to TECs, which may broaden the TRA inventory in mTECs for establishing a full spectrum of central tolerance. PMID:27022746

  7. Intercellular Protein Transfer from Thymocytes to Thymic Epithelial Cells

    PubMed Central

    Wang, Hong-Xia; Qiu, Yu-Rong; Zhong, Xiao-Ping

    2016-01-01

    Promiscuous expression of tissue restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs) is crucial for negative selection of self-reactive T cells to establish central tolerance. Intercellular transfer of self-peptide-MHC complexes from mTECs to thymic dendritic cells (DCs) allows DCs to acquire TRAs, which in turn contributes to negative selection and regulatory T cell generation. However, mTECs are unlikely to express all TRAs, such as immunoglobulins generated only in B cells after somatic recombination, hyper-mutation, or class-switches. We report here that both mTECs and cortical TECs can efficiently acquire not only cell surface but also intracellular proteins from thymocytes. This reveals a previously unappreciated intercellular sharing of molecules from thymocytes to TECs, which may broaden the TRA inventory in mTECs for establishing a full spectrum of central tolerance. PMID:27022746

  8. Plasticity of Airway Epithelial Cell Transcriptome in Response to Flagellin

    PubMed Central

    Clark, Joan G.; Kim, Kyoung-Hee; Basom, Ryan S.; Gharib, Sina A.

    2015-01-01

    Airway epithelial cells (AEC) are critical components of the inflammatory and immune response during exposure to pathogens. AECs in monolayer culture and differentiated epithelial cells in air-liquid interface (ALI) represent two distinct and commonly used in vitro models, yet differences in their response to pathogens have not been investigated. In this study, we compared the transcriptional effects of flagellin on AECs in monolayer culture versus ALI culture using whole-genome microarrays and RNA sequencing. We exposed monolayer and ALI AEC cultures to flagellin in vitro and analyzed the transcriptional response by microarray and RNA-sequencing. ELISA and RT-PCR were used to validate changes in select candidates. We found that AECs cultured in monolayer and ALI have strikingly different transcriptional states at baseline. When challenged with flagellin, monolayer AEC cultures greatly increased transcription of numerous genes mapping to wounding response, immunity and inflammatory response. In contrast, AECs in ALI culture had an unexpectedly muted response to flagellin, both in number of genes expressed and relative enrichment of inflammatory and immune pathways. We conclude that in vitro culturing methods have a dramatic effect on the transcriptional profile of AECs at baseline and after stimulation with flagellin. These differences suggest that epithelial responses to pathogen challenges are distinctly different in culture models of intact and injured epithelium. PMID:25668187

  9. Plasticity of airway epithelial cell transcriptome in response to flagellin.

    PubMed

    Clark, Joan G; Kim, Kyoung-Hee; Basom, Ryan S; Gharib, Sina A

    2015-01-01

    Airway epithelial cells (AEC) are critical components of the inflammatory and immune response during exposure to pathogens. AECs in monolayer culture and differentiated epithelial cells in air-liquid interface (ALI) represent two distinct and commonly used in vitro models, yet differences in their response to pathogens have not been investigated. In this study, we compared the transcriptional effects of flagellin on AECs in monolayer culture versus ALI culture using whole-genome microarrays and RNA sequencing. We exposed monolayer and ALI AEC cultures to flagellin in vitro and analyzed the transcriptional response by microarray and RNA-sequencing. ELISA and RT-PCR were used to validate changes in select candidates. We found that AECs cultured in monolayer and ALI have strikingly different transcriptional states at baseline. When challenged with flagellin, monolayer AEC cultures greatly increased transcription of numerous genes mapping to wounding response, immunity and inflammatory response. In contrast, AECs in ALI culture had an unexpectedly muted response to flagellin, both in number of genes expressed and relative enrichment of inflammatory and immune pathways. We conclude that in vitro culturing methods have a dramatic effect on the transcriptional profile of AECs at baseline and after stimulation with flagellin. These differences suggest that epithelial responses to pathogen challenges are distinctly different in culture models of intact and injured epithelium. PMID:25668187

  10. Differentiation potentials of perivascular cells in the bone tissue remodeling zones under microgravity

    NASA Astrophysics Data System (ADS)

    Rodionova, Natalia; Katkova, Olena

    Adaptive remodeling processes in the skeleton bones occur in the close topographical interconnection with blood capillaries followed by perivascular cells. Radioautographic studies with 3Н- thymidine (Kimmel D.B., Fee W.S., 1980; Rodionova N.V., 1989, 2006) has shown that in osteogenesis zones there is sequential differentiation process of the perivascular cells into osteogenic ones. Using electron microscopy and cytochemistry we studied perivsacular cells in metaphysis of the rats femoral bones under conditions of modeling microgravity (28 days duration) and in femoral bonеs metaphyses of rats flown on board of the space laboratory (Spacelab - 2) It was revealed that population of the perivascular cells is not homogeneous in adaptive zones of the remodeling in both control and test groups (lowering support loading). This population comprises adjacent to endothelium little differentiated forms and isolated cells with differentiation features (specific volume of rough endoplasmic reticulum in cytoplasm is increased). Majority of the perivascular cells in the control group reveals reaction to alkaline phosphatase (marker of the osteogenic differentiation). In little differentiated cells this reaction is registered in nucleolus, nucleous and cytoplasm. In differentiating cells activity of the alkaline phosphatase is also detected on the outer surface of the cellular membrane. Unlike the control group in the bones of animals under microgravitaty reaction to the alkaline phosphatase is registered not for all cells of perivascular population. Part of the differentiating perivascular cells does not contain a product of the reaction. There is also visible trend of individual alkaline phosphatase containing perivascular cells amounts decrease (i.e. osteogenic cells-precursors). Under microgravity some little differentiated perivascular cells reveal destruction signs. Found decrease trend of the alkaline phosphatase containing cells (i.e. osteogenic cells) number in

  11. Hertwig's epithelial root sheath cells do not transform into cementoblasts in rat molar cementogenesis.

    PubMed

    Yamamoto, Tsuneyuki; Takahashi, Shigeru

    2009-12-01

    It is generally accepted that cementoblasts originate in the process of differentiation of the mesenchymal cells of the dental follicle. Recently, a different hypothesis for the origin of cementoblasts has been proposed. Hertwig's epithelial root sheath cells undergo the epithelial-mesenchymal transformation to differentiate into cementoblasts. To elucidate whether the epithelial-mesenchymal transformation occurs in the epithelial sheath, developing rat molars were examined by keratin-vimentin and Runx2 (runt-related transcription factor 2)-keratin double immunostaining. In both acellular and cellular cementogenesis, epithelial sheath and epithelial cells derived from the epithelial sheath expressed keratin, but did not express vimentin or Runx2. Dental follicle cells and cementoblasts, however, expressed vimentin and Runx2, but did not express keratin. No cells showed coexisting keratin-vimentin or Runx2-keratin staining. These findings suggest that there is no intermediate phenotype transforming epithelial to mesenchymal cells, and that epithelial sheath cells do not generate mineralized tissue. This study concludes that the epithelial-mesenchymal transformation does not occur in Hertwig's epithelial root sheath in rat acellular or cellular cementogenesis and that the dental follicle is the origin of cementoblasts, as has been proposed in the original hypothesis. PMID:19716687

  12. TCDD exposure disrupts mammary epithelial cell differentiation and function

    PubMed Central

    Collins, Loretta L.; Lew, Betina J.; Lawrence, B. Paige

    2011-01-01

    Mammary gland growth and differentiation during pregnancy is a developmental process that is sensitive to the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD is a widespread environmental contaminant and a potent ligand for the aryl hydrocarbon receptor (AhR). We demonstrate reduced β-casein protein induction in mouse mammary glands and in cultured SCp2 mammary epithelial cells following exposure to TCDD. SCp2 cells exposed to TCDD also show reduced cell clustering and less alveolar-like structure formation. SCp2 cells express transcriptionally active AhR, and exposure to TCDD induces expression of the AhR target gene CYP1B1. Exposure to TCDD during pregnancy reduced expression of the cell adhesion molecule E-cadherin in the mammary gland and decreased phosphorylation of STAT5, a known regulator of β-casein gene expression. These data provide morphological and molecular evidence that TCDD-mediated AhR activation disrupts structural and functional differentiation of the mammary gland, and present an in vitro model for studying the effects of TCDD on mammary epithelial cell function. PMID:19490989

  13. ASBESTOS-INDUCED ACTIVATION OF CELL SIGNALING PATHWAYS IN HUMAN BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Using respiratory epithelial cells transfected with either superoxide dismutase (SOD) or catalase, the authors tested the hypothesis that the activation of the epidermal growth factor (EGF) receptor signal pathway after asbestos exposure involves an oxidative stress. Western blot...

  14. Epithelial cells from smokers modify dendritic cell responses in the context of influenza infection

    EPA Science Inventory

    Epidemiologic evidence suggests that cigarette smoking is a risk factor for infection with influenza, but the mechanisms underlying this susceptibility remain unknown. To ascertain if airway epithelial cells from smokers demonstrate a decreased ability to orchestrate an influenza...

  15. ATP7B detoxifies silver in ciliated airway epithelial cells

    SciTech Connect

    Ibricevic, Aida; Brody, Steven L.; Youngs, Wiley J.; Cannon, Carolyn L.

    2010-03-15

    Silver is a centuries-old antibiotic agent currently used to treat infected burns. The sensitivity of a wide range of drug-resistant microorganisms to silver killing suggests that it may be useful for treating refractory lung infections. Toward this goal, we previously developed a methylated caffeine silver acetate compound, SCC1, that exhibits broad-spectrum antimicrobial activity against clinical strains of bacteria in vitro and when nebulized to lungs in mouse infection models. Preclinical testing of high concentrations of SCC1 in primary culture mouse tracheal epithelial cells (mTEC) showed selective ciliated cell death. Ciliated cell death was induced by both silver- and copper-containing compounds but not by the methylated caffeine portion of SCC1. We hypothesized that copper transporting P-type ATPases, ATP7A and ATP7B, play a role in silver detoxification in the airway. In mTEC, ATP7A was expressed in non-ciliated cells, whereas ATP7B was expressed only in ciliated cells. The exposure of mTEC to SCC1 induced the trafficking of ATP7B, but not ATP7A, suggesting the presence of a cell-specific silver uptake and detoxification mechanisms. Indeed, the expression of the copper uptake protein CTR1 was also restricted to ciliated cells. A role of ATP7B in silver detoxification was further substantiated when treatment of SCC1 significantly increased cell death in ATP7B shRNA-treated HepG2 cells. In addition, mTEC from ATP7B{sup -/-} mice showed enhanced loss of ciliated cells compared to wild type. These studies are the first to demonstrate a cell type-specific expression of the Ag{sup +}/Cu{sup +} transporters ATP7A, ATP7B, and CTR1 in airway epithelial cells and a role for ATP7B in detoxification of these metals in the lung.

  16. Tissuelike 3D Assemblies of Human Broncho-Epithelial Cells

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J.

    2010-01-01

    Three-dimensional (3D) tissuelike assemblies (TLAs) of human broncho-epithelial (HBE) cells have been developed for use in in vitro research on infection of humans by respiratory viruses. The 2D monolayer HBE cell cultures heretofore used in such research lack the complex cell structures and interactions characteristic of in vivo tissues and, consequently, do not adequately emulate the infection dynamics of in-vivo microbial adhesion and invasion. In contrast, the 3D HBE TLAs are characterized by more-realistic reproductions of the geometrical and functional complexity, differentiation of cells, cell-to-cell interactions, and cell-to-matrix interactions characteristic of human respiratory epithelia. Hence, the 3D HBE TLAs are expected to make it possible to perform at least some of the research in vitro under more-realistic conditions, without need to infect human subjects. The TLAs are grown on collagen-coated cyclodextran microbeads under controlled conditions in a nutrient liquid in the simulated microgravitational environment of a bioreactor of the rotating- wall-vessel type. Primary human mesenchymal bronchial-tracheal cells are used as a foundation matrix, while adult human bronchial epithelial immortalized cells are used as the overlying component. The beads become coated with cells, and cells on adjacent beads coalesce into 3D masses. The resulting TLAs have been found to share significant characteristics with in vivo human respiratory epithelia including polarization, tight junctions, desmosomes, and microvilli. The differentiation of the cells in these TLAs into tissues functionally similar to in vivo tissues is confirmed by the presence of compounds, including villin, keratins, and specific lung epithelium marker compounds, and by the production of tissue mucin. In a series of initial infection tests, TLA cultures were inoculated with human respiratory syncytial viruses and parainfluenza type 3 viruses. Infection was confirmed by photomicrographs that

  17. Role of Periostin in Adhesion and Migration of Bone Remodeling Cells

    PubMed Central

    Cobo, Teresa; Viloria, Cristina G.; Solares, Laura; Fontanil, Tania; González-Chamorro, Elena; De Carlos, Félix; Cobo, Juan; Cal, Santiago; Obaya, Alvaro J.

    2016-01-01

    Periostin is an extracellular matrix protein highly expressed in collagen-rich tissues subjected to continuous mechanical stress. Functionally, periostin is involved in tissue remodeling and its altered function is associated to numerous pathological processes. In orthodontics, periostin plays key roles in the maintenance of dental tissues and it is mainly expressed in those areas where tension or pressing forces are taking place. In this regard, high expression of periostin is essential to promote migration and proliferation of periodontal ligament fibroblasts. However little is known about the participation of periostin in migration and adhesion processes of bone remodeling cells. In this work we employ the mouse pre-osteoblastic MC3T3-E1 and the macrophage-like RAW 264.7 cell lines to overexpress periostin and perform different cell-based assays to study changes in cell behavior. Our data indicate that periostin overexpression not only increases adhesion capacity of MC3T3-E1 cells to different matrix proteins but also hampers their migratory capacity. Changes on RNA expression profile of MC3T3-E1 cells upon periostin overexpression have been also analyzed, highlighting the alteration of genes implicated in processes such as cell migration, adhesion or bone metabolism but not in bone differentiation. Overall, our work provides new evidence on the impact of periostin in osteoblasts physiology. PMID:26809067

  18. Incremental responses to light recorded from pigment epithelial cells and horizontal cells of the cat retina

    PubMed Central

    Steinberg, Roy H.

    1971-01-01

    1. Rod-dependent incremental responses were recorded intracellularly in both pigment epithelial cells and horizontal cells of the cat retina. They were elicited by test flashes which were superimposed on background flashes after a delay. 2. In pigment epithelial cells smaller test responses were produced as background intensity was raised. The incremental sensitivity function was linear for about 1·4 log units, with a slope of 0·86, and the approach of saturation occurred at about 2·5 log td scotopic. 3. The amplitude of pigment epithelial test responses could be estimated from the dark-adapted amplitude—log intensity function obtained with single flashes. Test flashes produced the voltage increment predicted by the slope of this function just above the point on the curve equal to the background intensity. The pigment epithelial response to a test flash, therefore, is the response expected if the background were presented alone and made more intense by the amount of the test flash. 4. Rod-dependent incremental sensitivity functions of horizontal cells closely resembled the ones obtained from pigment epithelial cells. 5. It was concluded that the adaptive effects observed in pigment epithelial cells originated in individual rods. These effects arose from the compressive nature of the dark-adapted amplitude—intensity function. In horizontal cell responses these effects may be modified by the failure of the background response to maintain its initial voltage. PMID:5571955

  19. Epidermal Growth Factor Removal or Tyrphostin AG1478 Treatment Reduces Goblet Cells & Mucus Secretion of Epithelial Cells from Asthmatic Children Using the Air-Liquid Interface Model

    PubMed Central

    Parker, Jeremy C.; Douglas, Isobel; Bell, Jennifer; Comer, David; Bailie, Keith; Skibinski, Grzegorz; Heaney, Liam G.; Shields, Michael D.

    2015-01-01

    Rationale Epithelial remodelling in asthma is characterised by goblet cell hyperplasia and mucus hypersecretion for which no therapies exist. Differentiated bronchial air-liquid interface cultures from asthmatic children display high goblet cell numbers. Epidermal growth factor and its receptor have been implicated in goblet cell hyperplasia. Objectives We hypothesised that EGF removal or tyrphostin AG1478 treatment of differentiating air-liquid interface cultures from asthmatic children would result in a reduction of epithelial goblet cells and mucus secretion. Methods In Aim 1 primary bronchial epithelial cells from non-asthmatic (n = 5) and asthmatic (n = 5) children were differentiated under EGF-positive (10ng/ml EGF) and EGF-negative culture conditions for 28 days. In Aim 2, cultures from a further group of asthmatic children (n = 5) were grown under tyrphostin AG1478, a tyrosine kinase inhibitor, conditions. All cultures were analysed for epithelial resistance, markers of differentiation using immunocytochemistry, ELISA for MUC5AC mucin secretion and qPCR for MUC5AC mRNA. Results In cultures from asthmatic children the goblet cell number was reduced in the EGF negative group (p = 0.01). Tyrphostin AG1478 treatment of cultures from asthmatic children had significant reductions in goblet cells at 0.2μg/ml (p = 0.03) and 2μg/ml (p = 0.003) as well as mucus secretion at 2μg/ml (p = 0.04). Conclusions We have shown in this preliminary study that through EGF removal and tyrphostin AG1478 treatment the goblet cell number and mucus hypersecretion in differentiating air-liquid interface cultures from asthmatic children is significantly reduced. This further highlights the epidermal growth factor receptor as a potential therapeutic target to inhibit goblet cell hyperplasia and mucus hypersecretion in asthma. PMID:26057128

  20. The planar cell polarity protein VANGL2 coordinates remodeling of the extracellular matrix.

    PubMed

    Williams, B Blairanne; Mundell, Nathan; Dunlap, Julie; Jessen, Jason

    2012-07-01

    Understanding how planar cell polarity (PCP) is established, maintained, and coordinated in migrating cell populations is an important area of research with implications for both embryonic morphogenesis and tumor cell invasion. We recently reported that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell surface level of membrane type-1 matrix metalloproteinase (MMP14 or MT1-MMP). Here, we further discuss these findings in terms of extracellular matrix (ECM) remodeling, cell migration, and zebrafish gastrulation. We also demonstrate that VANGL2 function impacts the focal degradation of ECM by human cancer cells including the formation or stability of invadopodia. Together, our findings implicate MMP14 as a downstream effector of VANGL2 signaling and suggest a model whereby the regulation of pericellular proteolysis is a fundamental aspect of PCP in migrating cells. PMID:23060953

  1. The planar cell polarity protein VANGL2 coordinates remodeling of the extracellular matrix

    PubMed Central

    Williams, B. Blairanne; Mundell, Nathan; Dunlap, Julie; Jessen, Jason

    2012-01-01

    Understanding how planar cell polarity (PCP) is established, maintained, and coordinated in migrating cell populations is an important area of research with implications for both embryonic morphogenesis and tumor cell invasion. We recently reported that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell surface level of membrane type-1 matrix metalloproteinase (MMP14 or MT1-MMP). Here, we further discuss these findings in terms of extracellular matrix (ECM) remodeling, cell migration, and zebrafish gastrulation. We also demonstrate that VANGL2 function impacts the focal degradation of ECM by human cancer cells including the formation or stability of invadopodia. Together, our findings implicate MMP14 as a downstream effector of VANGL2 signaling and suggest a model whereby the regulation of pericellular proteolysis is a fundamental aspect of PCP in migrating cells. PMID:23060953

  2. Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche.

    PubMed

    Tamplin, Owen J; Durand, Ellen M; Carr, Logan A; Childs, Sarah J; Hagedorn, Elliott J; Li, Pulin; Yzaguirre, Amanda D; Speck, Nancy A; Zon, Leonard I

    2015-01-15

    Hematopoietic stem and progenitor cells (HSPCs) can reconstitute and sustain the entire blood system. We generated a highly specific transgenic reporter of HSPCs in zebrafish. This allowed us to perform high-resolution live imaging on endogenous HSPCs not currently possible in mammalian bone marrow. Using this system, we have uncovered distinct interactions between single HSPCs and their niche. When an HSPC arrives in the perivascular niche, a group of endothelial cells remodel to form a surrounding pocket. This structure appears conserved in mouse fetal liver. Correlative light and electron microscopy revealed that endothelial cells surround a single HSPC attached to a single mesenchymal stromal cell. Live imaging showed that mesenchymal stromal cells anchor HSPCs and orient their divisions. A chemical genetic screen found that the compound lycorine promotes HSPC-niche interactions during development and ultimately expands the stem cell pool into adulthood. Our studies provide evidence for dynamic niche interactions upon stem cell colonization. PAPERFLICK: PMID:25594182

  3. Changes in the population of perivascular cells in the bone tissue remodeling zones under microgravity

    NASA Astrophysics Data System (ADS)

    Katkova, Olena; Rodionova, Natalia; Shevel, Ivan

    2016-07-01

    Microgravity and long-term hypokinesia induce reduction both in bone mass and mineral saturation, which can lead to the development of osteoporosis and osteopenia. (Oganov, 2003). Reorganizations and adaptive remodeling processes in the skeleton bones occur in the topographical interconnection with blood capillaries and perivascular cells. Radioautographic studies with 3H- thymidine (Kimmel, Fee, 1980; Rodionova, 1989, 2006) have shown that in osteogenesis zones there is sequential differentiation process of the perivascular cells into osteogenic. Hence the study of populations of perivascular stromal cells in areas of destructive changes is actual. Perivascular cells from metaphysis of the rat femoral bones under conditions of modeling microgravity were studied using electron microscopy and cytochemistry (hindlimb unloading, 28 days duration) and biosatellite «Bion-M1» (duration of flight from April 19 till May 19, 2013 on C57, black mice). It was revealed that both control and test groups populations of the perivascular cells are not homogeneous in remodeling adaptive zones. These populations comprise of adjacent to endothelium poorly differentiated forms and isolated cells with signs of differentiation (specific increased volume of rough endoplasmic reticulum in cytoplasm). Majority of the perivascular cells in the control group (modeling microgravity) reveals reaction to alkaline phosphatase (marker of the osteogenic differentiation). In poorly differentiated cells this reaction is registered in nucleolus, nucleous and cytoplasm. In differentiating cells activity of the alkaline phosphatase is also detected on the outer surface of the cellular membrane. Unlike the control group in the bones of experimental animals reaction to the alkaline phosphatase is registered not in all cells of perivascular population. Part of the differentiating perivascular cells does not contain a product of the reaction. Under microgravity some poorly differentiated perivascular

  4. Studies in human skin epithelial cell carcinogenesis

    SciTech Connect

    Lehman, T.A.

    1987-01-01

    Metabolism and DNA adduct formation of benzo(a)pyrene (BP) by human epidermal keratinocytes pretreated with inhibitors or inducer of cytochrame P450 was studied. To study DNA adduct analysis, cultures were pretreated as described above, and then treated with non-radiolabeled BP. DNA was prepared from these cultures, digested to the nucleotide level, and /sup 32/P-postlabeled for adduct analysis. Cultures pretreated with BHA, 7,8-BF or disulfiralm formed significantly fewer BPDE I-dB adducts than non-pretreated cultures, while cultures pretreated with MeBHA formed more BPDE-I-dG adducts. MeBHA increased BP activation and adduct formation inhuman keratinocyte in cultures by inducing a specific isoenzyme of cytochrome P450 which preferentially increases the oxidative metabolism of BP to 7,8 diol BP and 7,8 diol BP to BPDE I. To approximate an in vivo human system, metabolism of BPDE I by human skin xenografts treated with cell cycles modulators was studied. When treated with BPDE I, specific carcinogen-DNA adducts were formed. Separation and identification of these adducts by the /sup 32/P-postlabeling technique indicated that the 7R- and 7S-BPDE I-dG adducts were the major adducts.

  5. Stretching Fibroblasts Remodels Fibronectin and Alters Cancer Cell Migration

    NASA Astrophysics Data System (ADS)

    Ao, Mingfang; Brewer, Bryson M.; Yang, Lijie; Franco Coronel, Omar E.; Hayward, Simon W.; Webb, Donna J.; Li, Deyu

    2015-02-01

    Most investigations of cancer-stroma interactions have focused on biochemical signaling effects, with much less attention being paid to biophysical factors. In this study, we investigated the role of mechanical stimuli on human prostatic fibroblasts using a microfluidic platform that was adapted for our experiments and further developed for both repeatable performance among multiple assays and for compatibility with high-resolution confocal microscopy. Results show that mechanical stretching of normal tissue-associated fibroblasts (NAFs) alters the structure of secreted fibronectin. Specifically, unstretched NAFs deposit and assemble fibronectin in a random, mesh-like arrangement, while stretched NAFs produce matrix with a more organized, linearly aligned structure. Moreover, the stretched NAFs exhibited an enhanced capability for directing co-cultured cancer cell migration in a persistent manner. Furthermore, we show that stretching NAFs triggers complex biochemical signaling events through the observation of increased expression of platelet derived growth factor receptor α (PDGFRα). A comparison of these behaviors with those of cancer-associated fibroblasts (CAFs) indicates that the observed phenotypes of stretched NAFs are similar to those associated with CAFs, suggesting that mechanical stress is a critical factor in NAF activation and CAF genesis.

  6. Stretching Fibroblasts Remodels Fibronectin and Alters Cancer Cell Migration

    PubMed Central

    Ao, Mingfang; Brewer, Bryson M.; Yang, Lijie; Franco Coronel, Omar E.; Hayward, Simon W.; Webb, Donna J.; Li, Deyu

    2015-01-01

    Most investigations of cancer-stroma interactions have focused on biochemical signaling effects, with much less attention being paid to biophysical factors. In this study, we investigated the role of mechanical stimuli on human prostatic fibroblasts using a microfluidic platform that was adapted for our experiments and further developed for both repeatable performance among multiple assays and for compatibility with high-resolution confocal microscopy. Results show that mechanical stretching of normal tissue-associated fibroblasts (NAFs) alters the structure of secreted fibronectin. Specifically, unstretched NAFs deposit and assemble fibronectin in a random, mesh-like arrangement, while stretched NAFs produce matrix with a more organized, linearly aligned structure. Moreover, the stretched NAFs exhibited an enhanced capability for directing co-cultured cancer cell migration in a persistent manner. Furthermore, we show that stretching NAFs triggers complex biochemical signaling events through the observation of increased expression of platelet derived growth factor receptor α (PDGFRα). A comparison of these behaviors with those of cancer-associated fibroblasts (CAFs) indicates that the observed phenotypes of stretched NAFs are similar to those associated with CAFs, suggesting that mechanical stress is a critical factor in NAF activation and CAF genesis. PMID:25660754

  7. MicroRNAs Dynamically Remodel Gastrointestinal Smooth Muscle Cells

    PubMed Central

    Park, Chanjae; Yan, Wei; Ward, Sean M.; Hwang, Sung Jin; Wu, Qiuxia; Hatton, William J.; Park, Jong Kun; Sanders, Kenton M.; Ro, Seungil

    2011-01-01

    Smooth muscle cells (SMCs) express a unique set of microRNAs (miRNAs) which regulate and maintain the differentiation state of SMCs. The goal of this study was to investigate the role of miRNAs during the development of gastrointestinal (GI) SMCs in a transgenic animal model. We generated SMC-specific Dicer null animals that express the reporter, green fluorescence protein, in a SMC-specific manner. SMC-specific knockout of Dicer prevented SMC miRNA biogenesis, causing dramatic changes in phenotype, function, and global gene expression in SMCs: the mutant mice developed severe dilation of the intestinal tract associated with the thinning and destruction of the smooth muscle (SM) layers; contractile motility in the mutant intestine was dramatically decreased; and SM contractile genes and transcriptional regulators were extensively down-regulated in the mutant SMCs. Profiling and bioinformatic analyses showed that SMC phenotype is regulated by a complex network of positive and negative feedback by SMC miRNAs, serum response factor (SRF), and other transcriptional factors. Taken together, our data suggest that SMC miRNAs are required for the development and survival of SMCs in the GI tract. PMID:21533178

  8. Intestinal immune homeostasis is regulated by the crosstalk between epithelial cells and dendritic cells.

    PubMed

    Rimoldi, Monica; Chieppa, Marcello; Salucci, Valentina; Avogadri, Francesca; Sonzogni, Angelica; Sampietro, Gianluca M; Nespoli, Angelo; Viale, Giuseppe; Allavena, Paola; Rescigno, Maria

    2005-05-01

    The control of damaging inflammation by the mucosal immune system in response to commensal and harmful ingested bacteria is unknown. Here we show epithelial cells conditioned mucosal dendritic cells through the constitutive release of thymic stromal lymphopoietin and other mediators, resulting in the induction of 'noninflammatory' dendritic cells. Epithelial cell-conditioned dendritic cells released interleukins 10 and 6 but not interleukin 12, and they promoted the polarization of T cells toward a 'classical' noninflammatory T helper type 2 response, even after exposure to a T helper type 1-inducing pathogen. This control of immune responses seemed to be lost in patients with Crohn disease. Thus, the intimate interplay between intestinal epithelial cells and dendritic cells may help to maintain gut immune homeostasis. PMID:15821737

  9. Stereological Quantification of Cell-Cycle Kinetics and Mobilization of Epithelial Stem Cells during Wound Healing.

    PubMed

    Martínez-Martínez, Eduardo; Uribe-Querol, Eileen; Galván-Hernández, Claudio I; Gutiérrez-Ospina, Gabriel

    2016-01-01

    We describe a stereology method to obtain reliable estimates of the total number of proliferative and migratory epithelial cells after wounding. Using pulse and chase experiments with halogenated thymidine analogs such as iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU), it is possible to track epithelial populations with heterogeneous proliferative characteristics through skin compartments. The stereological and tissue processing methods described here apply widely to address important questions of skin stem-cell biology. PMID:27431250

  10. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    SciTech Connect

    Sidjanin, D.; Grdina, D.; Woloschak, G.E.

    1994-11-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation induced by ultraviolet radiation. These experiments investigated the ability of 254 nm radiation on cell cycle progression and gene expression in rabbit lens epithelial cell line N/N1003A. No changes in expression of c-fos, c-jun, alpha- tubulin, or vimentin was observed following UV exposure. Using flow cytometry, an accumulation of cells in G1/S phase of the cell cycle 1 hr following exposure. The observed changes in gene expression, especially the decreased histone transcripts reported here may play a role in UV induced inhibition of cell cycle progression.

  11. Interleukin-23 Increases Intestinal Epithelial Cell Permeability In Vitro.

    PubMed

    Heinzerling, Nathan P; Donohoe, Deborah; Fredrich, Katherine; Gourlay, David M; Liedel, Jennifer L

    2016-06-01

    Background Breast milk has a heterogeneous composition that differs between mothers and changes throughout the first weeks after birth. The proinflammatory cytokine IL-23 has a highly variable expression in human breast milk. We hypothesize that IL-23 found in human breast milk is biologically active and promotes epithelial barrier dysfunction. Methods The immature rat small intestinal epithelial cell line, IEC-18, was grown on cell inserts or standard cell culture plates. Confluent cultures were exposed to human breast milk with high or low levels of IL-23 and barrier function was measured using a flux of fluorescein isothiocyanate-dextran (FD-70). In addition, protein and mRNA expression of occludin and ZO-1 were measured and immunofluorescence used to stain occludin and ZO-1. Results Exposure to breast milk with high levels of IL-23 caused an increase flux of FD-70 compared with both controls and breast milk with low levels of IL-23. The protein expression of ZO-1 but not occludin was decreased by exposure to high levels of IL-23. These results correlate with immunofluorescent staining of ZO-1 and occludin which show decreased staining of occludin in both the groups exposed to breast milk with high and low IL-23. Conversely, cells exposed to high IL-23 breast milk had little peripheral staining of ZO-1 compared with controls and low IL-23 breast milk. Conclusion IL-23 in human breast milk is biologically active and negatively affects the barrier function of intestinal epithelial cells through the degradation of tight junction proteins. PMID:26007691

  12. Oral microbial biofilm stimulation of epithelial cell responses.

    PubMed

    Peyyala, Rebecca; Kirakodu, Sreenatha S; Novak, Karen F; Ebersole, Jeffrey L

    2012-04-01

    Oral bacterial biofilms trigger chronic inflammatory responses in the host that can result in the tissue destructive events of periodontitis. However, the characteristics of the capacity of specific host cell types to respond to these biofilms remain ill-defined. This report describes the use of a novel model of bacterial biofilms to stimulate oral epithelial cells and profile select cytokines and chemokines that contribute to the local inflammatory environment in the periodontium. Monoinfection biofilms were developed with Streptococcus sanguinis, Streptococcus oralis, Streptococcus gordonii, Actinomyces naeslundii, Fusobacterium nucleatum, and Porphyromonas gingivalis on rigid gas-permeable contact lenses. Biofilms, as well as planktonic cultures of these same bacterial species, were incubated under anaerobic conditions with a human oral epithelial cell line, OKF4, for up to 24h. Gro-1α, IL1α, IL-6, IL-8, TGFα, Fractalkine, MIP-1α, and IP-10 were shown to be produced in response to a range of the planktonic or biofilm forms of these species. P. gingivalis biofilms significantly inhibited the production of all of these cytokines and chemokines, except MIP-1α. Generally, the biofilms of all species inhibited Gro-1α, TGFα, and Fractalkine production, while F. nucleatum biofilms stimulated significant increases in IL-1α, IL-6, IL-8, and IP-10. A. naeslundii biofilms induced elevated levels of IL-6, IL-8 and IP-10. The oral streptococcal species in biofilms or planktonic forms were poor stimulants for any of these mediators from the epithelial cells. The results of these studies demonstrate that oral bacteria in biofilms elicit a substantially different profile of responses compared to planktonic bacteria of the same species. Moreover, certain oral species are highly stimulatory when in biofilms and interact with host cell receptors to trigger pathways of responses that appear quite divergent from individual bacteria. PMID:22266273

  13. Newly identified interfibrillar collagen crosslinking suppresses cell proliferation and remodelling.

    PubMed

    Marelli, Benedetto; Le Nihouannen, Damien; Hacking, S Adam; Tran, Simon; Li, Jingjing; Murshed, Monzur; Doillon, Charles J; Ghezzi, Chiara E; Zhang, Yu Ling; Nazhat, Showan N; Barralet, Jake E

    2015-06-01

    Copper is becoming recognised as a key cation in a variety of biological processes. Copper chelation has been studied as a potential anti-angiogenic strategy for arresting tumour growth. Conversely the delivery of copper ions and complexes in vivo can elicit a pro-angiogenic effect. Previously we unexpectedly found that copper-stimulated intraperitoneal angiogenesis was accompanied by collagen deposition. Here, in hard tissue, not only was healing accelerated by copper, but again enhanced deposition of collagen was detected at 2 weeks. Experiments with reconstituted collagen showed that addition of copper ions post-fibrillogenesis rendered plastically-compressed gels resistant to collagenases, enhanced their mechanical properties and increased the denaturation temperature of the protein. Unexpectedly, this apparently interfibrillar crosslinking was not affected by addition of glucose or ascorbic acid, which are required for crosslinking by advanced glycation end products (AGEs). Fibroblasts cultured on copper-crosslinked gels did not proliferate, whereas those cultured with an equivalent quantity of copper on either tissue culture plastic or collagen showed no effect compared with controls. Although non-proliferative, fibroblasts grown on copper-cross-linked collagen could migrate, remained metabolically active for at least 14 days and displayed a 6-fold increase in Mmps 1 and 3 mRNA expression compared with copper-free controls. The ability of copper ions to crosslink collagen fibrils during densification and independently of AGEs or Fenton type reactions is previously unreported. The effect on MMP susceptibility of collagen and the dramatic change in cell behaviour on this crosslinked ECM may contribute to shedding some light on unexplained phenomena as the apparent benefit of copper complexation in fibrotic disorders or the enhanced collagen deposition in response to localised copper delivery. PMID:25907046

  14. IL-4 attenuates pulmonary epithelial cell-mediated suppression of T cell priming.

    PubMed

    Albrecht, Melanie; Arnhold, Markus; Lingner, Sandra; Mahapatra, Subhashree; Bruder, Dunja; Hansen, Gesine; Dittrich, Anna-Maria

    2012-01-01

    We have previously shown that Th2-polarized airway inflammation facilitates sensitization towards new, protein antigens. In this context, we could demonstrate that IL-4 needs to act on cells of the hematopoetic and the structural compartment in order to facilitate sensitization towards new antigens. We thus aimed to elucidate possible mechanisms of action of IL-4 on structural cells choosing to analyze pulmonary epithelial cells as an important part of the lung's structural system. We used a co-culture system of DC- or APC-dependent in vitro priming of T cells, co-cultivated on a layer of cells of a murine pulmonary epithelial cell line (LA-4) pretreated with or without IL-4. Effects on T cell priming were analyzed via CFSE-dilution and flow cytometric assessment of activation status. Pulmonary epithelial cells suppressed T cell proliferation in vitro but this effect was attenuated by pre-treatment of the epithelial cells with IL-4. Transwell experiments suggest that epithelial-mediated suppression of T cell activation is mostly cell-contact dependent and leads to attenuation in an early naive T cell phenotype. Secretion of soluble factors like TARC, TSLP, GM-CSF and CCL20 by epithelial cells did not change after IL-4 treatment. However, analysis of co-stimulatory expression on pulmonary epithelial cells revealed that pre-treatment of epithelial cells with IL-4 changed expression GITR-L, suggesting a possible mechanism for the effects observed. Our studies provide new insight into the role of IL-4 during the early phases of pulmonary sensitization: The inhibitory activity of pulmonary epithelial cells in homeostasis is reversed in the presence of IL-4, which is secreted in the context of Th2-dominated allergic airway inflammation. This mechanism might serve to explain facilitated sensitization in the clinical context of polysensitization where due to a pre-existing sensitization increased levels of IL-4 in the airways might facilitate T cell priming towards new

  15. Ultrastructural analysis of primary human urethral epithelial cell cultures infected with Neisseria gonorrhoeae.

    PubMed

    Harvey, H A; Ketterer, M R; Preston, A; Lubaroff, D; Williams, R; Apicella, M A

    1997-06-01

    In men with gonococcal urethritis, the urethral epithelial cell is a site of infection. To study the pathogenesis of gonorrhea in this cell type, we have developed a method to culture primary human urethral epithelial cells obtained at the time of urologic surgery. Fluorescent analysis demonstrated that 100% of the cells stained for keratin. Microscopic analyses indicated that these epithelial cells arrayed in a pattern similar to that seen in urethral epithelium. Using immunoelectron and confocal microscopy, we compared the infection process seen in primary cells with events occurring during natural infection of the same cell type in men with gonococcal urethritis. Immunoelectron microscopy studies of cells infected with Neisseria gonorrhoeae 1291 Opa+ P+ showed adherence of organisms to the epithelial cell membrane, pedestal formation with evidence of intimate association between the gonococcal and the epithelial cell membranes, and intracellular gonococci present in vacuoles. Confocal studies of primary urethral epithelial cells showed actin polymerization upon infection. Polyclonal antibodies to the asialoglycoprotein receptor (ASGP-R) demonstrated the presence of this receptor on infected cells in the primary urethral cell culture. In situ hybridization using a fluorescent-labeled probe specific to the ASGP-R mRNA demonstrated this message in uninfected and infected cells. These features were identical to those seen in urethral epithelial cells in exudates from males with gonorrhea. Infection of primary urethral cells in culture mimics events seen in natural infection and will allow detailed molecular analysis of gonococcal pathogenesis in a human epithelial cell which is commonly infected. PMID:9169783

  16. In vitro differentiation of bone marrow mesenchymal stem cells into endometrial epithelial cells in mouse: a proteomic analysis

    PubMed Central

    Cong, Qing; Li, Bin; Wang, Yisheng; Zhang, Wenbi; Cheng, Mingjun; Wu, Zhiyong; Zhang, Xiaoyan; Jiang, Wei; Xu, Congjian

    2014-01-01

    Objective: Mouse bone marrow mesenchymal stem cells (BMSCs) have been demonstrated to differentiate into female endometrial epithelial cells (EECs) in vivo. Our previous studies demonstrated that BMSCs can differentiate in the direction of EECs when co-cultured with endometrial stromal cells in vitro. Here, we obtain and analyse differential proteins and their relevant pathways in the process of BMSCs differentiating into EECs by isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis. Methods: A 0.4-μm pore size indirect co-culture system was established with female mice endometrial stromal cells (EStCs) restricted in the upper Transwell chamber and BMSCs in the lower well plate. After indirect co-culture for several days, the BMSCs were revealed to progressively differentiate towards EECs in vitro. Then, four groups were divided according to different co-culture days with single culture groups of BMSCs as controls. Proteins were detected using iTRAQ based on 2DLC-ESI-MS/MS and data were analysed by bioinformatics. Results: A total number of 311 proteins were detected, of which 210 proteins were identified with relative quantitation. Among them, 107 proteins were differentially expressed with a 1.2-fold change as the benchmark, with 61 up-regulated and 46 down-regulated proteins. Differential proteins CK19 and CK8 were epithelial markers and upregulated. Stromal marker vimentin were downregulated. Top canonical pathways was “remodeling of epithelial adhesions junctions” and “actin cytoskeleton signaling”. Top networks was “cell-to-cell signaling and interaction, tissue development and cellular movement” regulated by ERK/MAPK and α-catenin. Conclusion: To the best of our knowledge, this is the first preliminary study of differential protein expression in the differentiation process of BMSCs into EECs in vitro. We further elucidated BMSCs differentiated in the direction of EECs. In addition, ERK/MAPK and α-catenin played

  17. Isoprenaline induces epithelial-mesenchymal transition in gastric cancer cells.

    PubMed

    Lu, Yan-Jie; Geng, Zhi-Jun; Sun, Xiao-Yan; Li, Yu-Hong; Fu, Xiao-Bing; Zhao, Xiang-Yang; Wei, Bo

    2015-10-01

    The emerging role of stress-related signaling in regulating cancer development and progression has been recognized. However, whether stress serves as a mechanism to promote gastric cancer metastasis is not clear. Here, we show that the β2-AR agonist, isoprenaline, upregulates expression levels of CD44 and CD44v8-10 in gastric cancer cells. CD44, a cancer stem cell-related marker, is expressed at high levels in gastric cancer tissues, which strongly correlates with the occurrence of epithelial-mesenchymal transition (EMT)-associated phenotypes both in vivo and in vitro. Combined with experimental observations in two human gastric cancer cell lines, we found that β2-AR signaling can initiate EMT. It led to an increased expression of mesenchymal markers, such as α-SMA, vimentin, and snail at mRNA and protein levels, and conversely a decrease in epithelial markers, E-cadherin and β-catenin. Isoprenaline stimulation of β2-AR receptors activates the downstream target STAT3, which functions as a positive regulator and mediated the phenotypic switch toward a mesenchymal cell type in gastric cancer cells. Our data provide a mechanistic understanding of the complex signaling cascades involving stress-related hormones and their effects on EMT. In light of our observations, pharmacological interventions targeting β2-AR-STAT3 signaling can potentially be used to ameliorate stress-associated influences on gastric cancer development and progression. PMID:26253173

  18. Niche-induced cell death and epithelial phagocytosis regulate hair follicle stem cell pool.

    PubMed

    Mesa, Kailin R; Rompolas, Panteleimon; Zito, Giovanni; Myung, Peggy; Sun, Thomas Y; Brown, Samara; Gonzalez, David G; Blagoev, Krastan B; Haberman, Ann M; Greco, Valentina

    2015-06-01

    Tissue homeostasis is achieved through a balance of cell production (growth) and elimination (regression). In contrast to tissue growth, the cells and molecular signals required for tissue regression remain unknown. To investigate physiological tissue regression, we use the mouse hair follicle, which cycles stereotypically between phases of growth and regression while maintaining a pool of stem cells to perpetuate tissue regeneration. Here we show by intravital microscopy in live mice that the regression phase eliminates the majority of the epithelial cells by two distinct mechanisms: terminal differentiation of suprabasal cells and a spatial gradient of apoptosis of basal cells. Furthermore, we demonstrate that basal epithelial cells collectively act as phagocytes to clear dying epithelial neighbours. Through cellular and genetic ablation we show that epithelial cell death is extrinsically induced through transforming growth factor (TGF)-β activation and mesenchymal crosstalk. Strikingly, our data show that regression acts to reduce the stem cell pool, as inhibition of regression results in excess basal epithelial cells with regenerative abilities. This study identifies the cellular behaviours and molecular mechanisms of regression that counterbalance growth to maintain tissue homeostasis. PMID:25849774

  19. Niche induced cell death and epithelial phagocytosis regulate hair follicle stem cell pool

    PubMed Central

    Mesa, Kailin R.; Rompolas, Panteleimon; Zito, Giovanni; Myung, Peggy; Sun, Thomas Yang; Brown, Samara; Gonzalez, David; Blagoev, Krastan B.; Haberman, Ann M.; Greco, Valentina

    2015-01-01

    Summary Tissue homeostasis is achieved through a balance of cell production (growth) and elimination (regression)1,2. Contrary to tissue growth, the cells and molecular signals required for tissue regression remain unknown. To investigate physiological tissue regression, we use the mouse hair follicle, which cycles stereotypically between phases of growth and regression while maintaining a pool of stem cells to perpetuate tissue regeneration3. Here we show by intravital microscopy in live mice4–6 that the regression phase eliminates the majority of the epithelial cells by two distinct mechanisms: terminal differentiation of suprabasal cells and a spatial gradient of apoptosis of basal cells. Furthermore, we demonstrate that basal epithelial cells collectively act as phagocytes to clear dying epithelial neighbors. Through cellular and genetic ablation we show that epithelial cell death is extrinsically induced through TGFβ activation and mesenchymal crosstalk. Strikingly, our data show that regression acts to reduce the stem cell pool as inhibition of regression results in excess basal epithelial cells with regenerative abilities. This study identifies the cellular behaviors and molecular mechanisms of regression that counterbalance growth to maintain tissue homeostasis. PMID:25849774

  20. Cell surface morphology in epithelial malignancy and its precursor lesions.

    PubMed

    Kenemans, P; Davina, J H; de Haan, R W; van der Zanden, P; Vooys, G P; Stolk, J G; Stadhouders, A M

    1981-01-01

    The cell surface organization of cancer cells is of potentially great significance, as it may not only allow (early) diagnosis, but as it may also harbour markers for refined prognosis (degree of oncogenetic and metastatic potential), and targets for selective cancer (chemo- and immuno) therapy. With these aspects in mind, the present review deals with SEM work done on (pre-) malignant cells, both in vivo and in vitro, and in animal models. Attention, however, is focused on human cancer cells. Cancer cells in vitro may lose many of their original malignant characteristics, and show adaptations to culture conditions. Many other factors have been shown to influence cell surface morphology, such as cell cycle, cell contacts, and preparations technique. Cancer cells differ in their surface morphology from normal cells, and have an extra ordinary amount of surface activity. Human malignant epithelial cells show abundant long. pleomorphic microvilli, especially those present in effusions. In squamous epithelium (bladder, cervix) microridge system present on normal superficial cells are progressively replaced by microvilli which increase in number and degree of pleomorphism during experimental and clinical oncogenesis. The question of whether or not the appearance of long. Pleomorphic microvilli reflects an irreversible alteration of the epithelium, and thus provides an early marker of irreversible neoplastic transformation is considered and assessed on the basis of our work with (pre-) malignant cells of the human uterine cervix. Although SEM has contributed significantly to the description of oncogenesis, up to now it has no early diagnostic, prognostic or therapeutic significance. PMID:7199203

  1. Regulation of aryl hydrocarbon receptor-mediated transcription in human retinal pigmented epithelial cells.

    PubMed

    Jin, Hong Lan; Jeong, Kwang Won

    2016-04-01

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor with pleiotropic effects in normal physiology or vascular development, xenobiotic metabolism, and cancer. A previous study has reported that BRG1, a component of the SWI/SNF complex, is a coactivator for AHR and is recruited to the promoter region of the CYP1A1 gene in mouse hepatocytes. Recent data suggest that AHR is also expressed in human retinal pigment epithelial cells (ARPE-19), which play a crucial role in retinal physiology and the visual cycle. Multiple studies have shown that the AHR plays an important role in the pathogenesis of retinal diseases including age-related macular degeneration. However, the mechanism of AHR transcriptional activation in retinal pigment cells has not been reported. Here, we demonstrate that the AHR signaling pathway is active in ARPE-19 cells, as in hepatocytes, but with different target gene specificity. We also found that chromatin remodeling by the BRG1-containing SWI/SNF complex is required for the AHR-mediated expression of target genes in ARPE-19 cells. We identified a novel enhancer region (-12 kb) of the CYP1A1 gene in ARPE-19 cells, to which both AHR and BRG1 are recruited in a ligand-dependent manner. BRG1 is associated with the AHR in ARPE-19 cells, and the C-terminal activation domain of the AHR directly interacts with BRG1. Furthermore, depletion of BRG1 caused a reduction in chromatin accessibility at the CYP1A1 enhancer. These results suggest that ARPE-19 cells possess an AHR-mediated transcription pathway with different target gene specificity, and that BRG1 is required for AHR-mediated transcription in ARPE-19 cells. PMID:26966070

  2. The Molecular Architecture of Cell Adhesion: Dynamic Remodeling Revealed by Videonanoscopy.

    PubMed

    Sergé, Arnauld

    2016-01-01

    The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors, and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation, and metastasis. PMID:27200348

  3. The Molecular Architecture of Cell Adhesion: Dynamic Remodeling Revealed by Videonanoscopy

    PubMed Central

    Sergé, Arnauld

    2016-01-01

    The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors, and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation, and metastasis. PMID:27200348

  4. Intramyocardial stem cell injection in patients with ischemic cardiomyopathy: Functional recovery and reverse remodeling

    PubMed Central

    Williams, Adam R.; Trachtenberg, Barry; Velazquez, Darcy L.; McNiece, Ian; Altman, Peter; Rouy, Didier; Mendizabal, Adam M.; Pattany, Pradip M.; Lopera, Gustavo A.; Fishman, Joel; Zambrano, Juan P.; Heldman, Alan W.; Hare, Joshua M.

    2012-01-01

    Rationale Transcatheter, intramyocardial injections of bone marrow derived cell therapy produces reverse remodeling in large animal models of ischemic cardiomyopathy. Objective We used cardiac magnetic resonance imaging (CMR) in patients with LV dysfunction related to remote myocardial infarction (MI) to test the hypothesis that bone marrow progenitor cell injection cause functional recovery of scarred myocardium and reverse remodeling. Methods and Results Eight patients (age 57.2±13.3) received transendocardial, intramyocardial injection of autologous bone marrow progenitor cells (mononuclear or mesenchymal stem cells) in LV scar and border zone. All patients tolerated the procedure with no serious adverse events. CMR at 1-year demonstrated a decrease in end-diastolic volume (208.7±20.4 vs. 167.4±7.32mL; p=0.03), a trend towards decreased end-systolic volume (142.4±16.5 vs. 107.6±7.4mL; p=0.06), decreased infarct size (p<0.05), and improved regional LV function by peak Ecc in the treated infarct zone (-8.1±1.0 vs. -11.4±1.3; p=0.04). Improvements in regional function were evident at 3 months, while the changes in chamber dimensions were not significant until 6 months. Improved regional function in the infarct zone strongly correlated with reduction of EDV (r2=0.69, p=0.04) and ESV (r2=0.83, p=0.01). Conclusions These data suggest that transcatheter, intramyocardial injections of autologous bone marrow progenitor cells improve regional contractility of a chronic myocardial scar and these changes predict subsequent reverse remodeling. The findings support the potential clinical benefits of this new treatment strategy and ongoing randomized clinical trials. PMID:21415390

  5. Dynamic Reorganization and Enzymatic Remodeling of Type IV Collagen at Cell-Biomaterial Interface.

    PubMed

    Coelho, N M; Llopis-Hernández, V; Salmerón-Sánchez, M; Altankov, G

    2016-01-01

    Vascular basement membrane remodeling involves assembly and degradation of its main constituents, type IV collagen (Col IV) and laminin, which is critical during development, angiogenesis, and tissue repair. Remodeling can also occur at cell-biomaterials interface altering significantly the biocompatibility of implants. Here we describe the fate of adsorbed Col IV in contact with endothelial cells adhering on positively charged NH2 or hydrophobic CH3 substrata, both based on self-assembly monolayers (SAMs) and studied alone or mixed in different proportions. AFM studies revealed distinct pattern of adsorbed Col IV, varying from single molecular deposition on pure NH2 to network-like assembly on mixed SAMs, turning to big globular aggregates on bare CH3. Human umbilical endothelial cells (HUVECs) interact better with Col IV adsorbed as single molecules on NH2 surface and readily rearrange it in fibril-like pattern that coincide with secreted fibronectin fibrils. The cells show flattened morphology and well-developed focal adhesion complexes that are rich on phosphorylated FAK while expressing markedly low pericellular proteolytic activity. Conversely, on hydrophobic CH3 substrata HUVECs showed abrogated spreading and FAK phosphorylation, combined with less reorganization of the aggregated Col IV and significantly increased proteolytic activity. The later involves both MMP-2 and MMP-9, as measured by zymography and FITC-Col IV release. The mixed SAMs support intermediate remodeling activity. Taken together these results show that chemical functionalization combined with Col IV preadsorption provides a tool for guiding the endothelial cells behavior and pericellular proteolytic activity, events that strongly affect the fate of cardiovascular implants. PMID:27567485

  6. Francisella tularensis replicates within alveolar type II epithelial cells in vitro and in vivo following inhalation.

    PubMed

    Hall, Joshua D; Craven, Robin R; Fuller, James R; Pickles, Raymond J; Kawula, Thomas H

    2007-02-01

    Francisella tularensis replicates in macrophages and dendritic cells, but interactions with other cell types have not been well described. F. tularensis LVS invaded and replicated within alveolar epithelial cell lines. Following intranasal inoculation of C57BL/6 mice, Francisella localized to the alveolus and replicated within alveolar type II epithelial cells. PMID:17088343

  7. Isolating Epithelial and Epithelial-to-Mesenchymal Transition Populations from Primary Tumors by Fluorescence-Activated Cell Sorting.

    PubMed

    Aiello, Nicole M; Rhim, Andrew D; Stanger, Ben Z

    2016-01-01

    Transgenic mice that express conditional reporters allow for the isolation of specific cell lineages. These cells can be further stratified by gene expression and collected by fluorescence-activated cell sorting (FACS) for further analysis. Using Cre-recombinase (Cre) technology we have generated a transgenic mouse line termed PKCY in which all pancreatic epithelial cells and therefore all pancreatic cancer cells are constitutively labeled with yellow fluorescent protein (YFP). We have used immunofluorescent staining for E-cadherin to divide the YFP(+) tumor population into epithelial cells (E-cadherin positive) and cells that have undergone an epithelial-to-mesenchymal transition (EMT; E-cadherin negative). This protocol describes how to prepare a tumor sample for FACS, with an emphasis on separating epithelial and EMT populations. These cells can then be used for a number of applications including, but not limited to, the generation of cell lines, gene-expression analysis by quantitative polymerase chain reaction (qPCR) or RNA sequencing, DNA sequencing, chromatin immunoprecipitation, and western blots. PMID:26729901

  8. Intratracheal Bleomycin Causes Airway Remodeling and Airflow Obstruction in Mice

    PubMed Central

    Polosukhin, Vasiliy V.; Degryse, Amber L.; Newcomb, Dawn C.; Jones, Brittany R.; Ware, Lorraine B.; Lee, Jae Woo; Loyd, James E.; Blackwell, Timothy S.; Lawson, William E.

    2014-01-01

    Introduction In addition to parenchymal fibrosis, fibrotic remodeling of the distal airways has been reported in interstitial lung diseases. Mechanisms of airway wall remodeling, which occurs in a variety of chronic lung diseases, are not well defined and current animal models are limited. Methods We quantified airway remodeling in lung sections from subjects with idiopathic pulmonary fibrosis (IPF) and controls. To investigate intratracheal bleomycin as a potential animal model for fibrotic airway remodeling, we evaluated lungs from C57BL/6 mice after bleomycin treatment by histologic scoring for fibrosis and peribronchial inflammation, morphometric evaluation of subepithelial connective tissue volume density, TUNEL assay, and immunohistochemistry for transforming growth factor β1 (TGFβ1), TGFβ2, and the fibroblast marker S100A4. Lung mechanics were determined at 3 weeks post-bleomycin. Results IPF lungs had small airway remodeling with increased bronchial wall thickness compared to controls. Similarly, bleomycin treated mice developed dose-dependent airway wall inflammation and fibrosis and greater airflow resistance after high dose bleomycin. Increased TUNEL+ bronchial epithelial cells and peribronchial inflammation were noted by 1 week, and expression of TGFβ1 and TGFβ2 and accumulation of S100A4+ fibroblasts correlated with airway remodeling in a bleomycin dose-dependent fashion. Conclusions IPF is characterized by small airway remodeling in addition to parenchymal fibrosis, a pattern also seen with intratracheal bleomycin. Bronchial remodeling from intratracheal bleomycin follows a cascade of events including epithelial cell injury, airway inflammation, pro-fibrotic cytokine expression, fibroblast accumulation, and peribronchial fibrosis. Thus, this model can be utilized to investigate mechanisms of airway remodeling. PMID:22394287

  9. Reactivation of Endogenous Genes and Epigenetic Remodeling Are Barriers for Generating Transgene-Free Induced Pluripotent Stem Cells in Pig

    PubMed Central

    Choi, Kwang-Hwan; Park, Jin-Kyu; Son, Dongchan; Hwang, Jae Yeon; Lee, Dong-Kyung; Ka, Hakhyun; Park, Joonghoon; Lee, Chang-Kyu

    2016-01-01

    Cellular reprogramming of committed cells into a pluripotent state can be induced by ectopic expression of genes such as OCT4, SOX2, KLF4, and MYC. Reprogrammed cells can be maintained by activating endogenous pluripotent networks without transgene expression. Although various research groups have attempted to generate pig induced pluripotent stem cells (iPSCs), authentic iPSCs have not be obtained, instead showing dependence on transgene expression. In this study, iPSCs were derived from porcine fetal fibroblasts via drug-inducible vectors carrying human transcription factors (OCT4, SOX2, KLF4, and MYC). Therefore, this study investigated characteristics of iPSCs and reprogramming mechanisms in pig. The iPSCs were stably maintained over an extended period with potential in vitro differentiation into three germ layers. In addition, the pluripotent state of iPSCs was regulated by modulating culture conditions. They showed naive- or primed-like pluripotent states in LIF or bFGF supplemented culture conditions, respectively. However, iPSCs could not be maintained without ectopic expression of transgenes. The cultured iPSCs expressed endogenous transcription factors such as OCT4 and SOX2, but not NANOG (a known gateway to complete reprogramming). Endogenous genes related to mesenchymal-to-epithelial transition (DPPA2, CDH1, EPCAM, and OCLN) were not sufficiently reactivated, as measured by qPCR. DNA methylation analysis for promoters of OCT4, NANOG, and XIST showed that epigenetic reprogramming did not occur in female iPSCs. Based on our results, expression of exogenous genes could not sufficiently activate the essential endogenous genes and remodel the epigenetic milieu to achieve faithful pluripotency in pig. Accordingly, investigating iPSCs could help us improve and develop reprogramming methods by understanding reprogramming mechanisms in pig. PMID:27336671

  10. Reactivation of Endogenous Genes and Epigenetic Remodeling Are Barriers for Generating Transgene-Free Induced Pluripotent Stem Cells in Pig.

    PubMed

    Choi, Kwang-Hwan; Park, Jin-Kyu; Son, Dongchan; Hwang, Jae Yeon; Lee, Dong-Kyung; Ka, Hakhyun; Park, Joonghoon; Lee, Chang-Kyu

    2016-01-01

    Cellular reprogramming of committed cells into a pluripotent state can be induced by ectopic expression of genes such as OCT4, SOX2, KLF4, and MYC. Reprogrammed cells can be maintained by activating endogenous pluripotent networks without transgene expression. Although various research groups have attempted to generate pig induced pluripotent stem cells (iPSCs), authentic iPSCs have not be obtained, instead showing dependence on transgene expression. In this study, iPSCs were derived from porcine fetal fibroblasts via drug-inducible vectors carrying human transcription factors (OCT4, SOX2, KLF4, and MYC). Therefore, this study investigated characteristics of iPSCs and reprogramming mechanisms in pig. The iPSCs were stably maintained over an extended period with potential in vitro differentiation into three germ layers. In addition, the pluripotent state of iPSCs was regulated by modulating culture conditions. They showed naive- or primed-like pluripotent states in LIF or bFGF supplemented culture conditions, respectively. However, iPSCs could not be maintained without ectopic expression of transgenes. The cultured iPSCs expressed endogenous transcription factors such as OCT4 and SOX2, but not NANOG (a known gateway to complete reprogramming). Endogenous genes related to mesenchymal-to-epithelial transition (DPPA2, CDH1, EPCAM, and OCLN) were not sufficiently reactivated, as measured by qPCR. DNA methylation analysis for promoters of OCT4, NANOG, and XIST showed that epigenetic reprogramming did not occur in female iPSCs. Based on our results, expression of exogenous genes could not sufficiently activate the essential endogenous genes and remodel the epigenetic milieu to achieve faithful pluripotency in pig. Accordingly, investigating iPSCs could help us improve and develop reprogramming methods by understanding reprogramming mechanisms in pig. PMID:27336671

  11. Critical role of actin-associated proteins in smooth muscle contraction, cell proliferation, airway hyperresponsiveness and airway remodeling.

    PubMed

    Tang, Dale D

    2015-01-01

    Asthma is characterized by airway hyperresponsiveness and airway remodeling, which are largely attributed to increased airway smooth muscle contractility and cell proliferation. It is known that both chemical and mechanical stimulation regulates smooth muscle contraction. Recent studies suggest that contractile activation and mechanical stretch induce actin cytoskeletal remodeling in smooth muscle. However, the mechanisms that control actin cytoskeletal reorganization are not completely elucidated. This review summarizes our current understanding regarding how actin-associated proteins may regulate remodeling of the actin cytoskeleton in airway smooth muscle. In particular, there is accumulating evidence to suggest that Abelson tyrosine kinase (Abl) plays a critical role in regulating airway smooth muscle contraction and cell proliferation in vitro, and airway hyperresponsiveness and remodeling in vivo. These studies indicate that Abl may be a novel target for the development of new therapy to treat asthma. PMID:26517982

  12. Characterization of an epithelial cell line from bovine mammary gland.

    PubMed

    German, Tania; Barash, Itamar

    2002-05-01

    Elucidation of the bovine mammary gland's unique characteristics depends on obtaining an authentic cell line that will reproduce its function in vitro. Representative clones from bovine mammary cell populations, differing in their attachment capabilities, were cultured. L-1 cells showed strong attachment to the plate, whereas H-7 cells detached easily. Cultures established from these clones were nontumorigenic upon transplantation to an immunodeficient host; they exhibited the epithelial cell characteristics of positive cytokeratin but not smooth muscle actin staining. Both cell lines depended on fetal calf serum for proliferation. They exhibited distinct levels of differentiation on Matrigel in serum-free, insulin-supplemented medium on the basis of their organization and beta-lactoglobulin (BLG) secretion. H-7 cells organized into mammospheres, whereas L-1 cells arrested in a duct-like morphology. In both cell lines, prolactin activated phosphorylation of the signal transducer and activator of transcription, Stat5-a regulator of milk protein gene transcription, and of PHAS-I-an inhibitor of translation initiation in its nonphosphorylated form. De novo synthesis and secretion of BLG were detected in differentiated cultures: in L-1 cells, BLG was dependent on lactogenic hormones for maximal induction but was less stringently controlled than was beta-casein in the mouse CID-9 cell line. L-1 cells also encompassed a near-diploid chromosomal karyotype and may serve as a tool for studying functional characteristics of the bovine mammary gland. PMID:12418925

  13. MicroRNA-146a modulates human bronchial epithelial cell survival in response to the cytokine-induced apoptosis

    SciTech Connect

    Liu Xiangde Nelson, Amy; Wang Xingqi; Kanaji, Nobuhiro; Kim, Miok; Sato, Tadashi; Nakanishi, Masanori; Li Yingji; Sun Jianhong; Michalski, Joel; Patil, Amol; Basma, Hesham; Rennard, Stephen I.

    2009-02-27

    MicroRNA plays an important role in cell differentiation, proliferation and cell death. The current study found that miRNA-146a was up-regulated in human bronchial epithelial cells (HBECs) in response to stimulation by TGF-ss1 plus cytomix (a mixture of IL-1ss, IFN-{gamma} and TNF-{alpha}). TGF-ss1 plus cytomix (TCM) induced apoptosis in HBECs (3.4 {+-} 0.6% of control vs 83.1 {+-} 4.0% of TCM treated cells, p < 0.01), and this was significantly blocked by the miRNA-146a mimic (8.8 {+-} 1.5%, p < 0.01). In contrast, a miRNA-146a inhibitor had only a modest effect on cell survival but appeared to augment the induction of epithelial-mesenchymal transition (EMT) in response to the cytokines. The MicroRNA-146a mimic appears to modulate HBEC survival through a mechanism of up-regulating Bcl-XL and STAT3 phosphorylation, and by this mechanism it could contribute to tissue repair and remodeling.

  14. Novel strategies to enforce an epithelial phenotype in mesenchymal cells.

    PubMed

    Dragoi, Ana-Maria; Swiss, Rachel; Gao, Beile; Agaisse, Hervé

    2014-07-15

    E-cadherin downregulation in cancer cells is associated with epithelial-to-mesenchymal transition (EMT) and metastatic prowess, but the underlying mechanisms are incompletely characterized. In this study, we probed E-cadherin expression at the plasma membrane as a functional assay to identify genes involved in E-cadherin downregulation. The assay was based on the E-cadherin-dependent invasion properties of the intracellular pathogen Listeria monocytogenes. On the basis of a functional readout, automated microscopy and computer-assisted image analysis were used to screen siRNAs targeting 7,000 human genes. The validity of the screen was supported by its definition of several known regulators of E-cadherin expression, including ZEB1, HDAC1, and MMP14. We identified three new regulators (FLASH, CASP7, and PCGF1), the silencing of which was sufficient to restore high levels of E-cadherin transcription. In addition, we identified two new regulators (FBXL5 and CAV2), the silencing of which was sufficient to increase E-cadherin expression at a posttranscriptional level. FLASH silencing regulated the expression of E-cadherin and other ZEB1-dependent genes, through posttranscriptional regulation of ZEB1, but it also regulated the expression of numerous ZEB1-independent genes with functions predicted to contribute to a restoration of the epithelial phenotype. Finally, we also report the identification of siRNA duplexes that potently restored the epithelial phenotype by mimicking the activity of known and putative microRNAs. Our findings suggest new ways to enforce epithelial phenotypes as a general strategy to treat cancer by blocking invasive and metastatic phenotypes associated with EMT. PMID:24845104

  15. Novel strategies to enforce an epithelial phenotype in mesenchymal cells

    PubMed Central

    Dragoi, Ana-Maria; Swiss, Rachel; Gao, Beile; Agaisse, Hervé

    2014-01-01

    E-cadherin downregulation in cancer cells is associated with epithelial-to-mesenchymal transition (EMT) and metastatic prowess, but the underlying mechanisms are incompletely characterized. In this study, we probed E-cadherin expression at the plasma membrane as a functional assay to identify genes involved in E-cadherin downregulation. The assay was based on the E-cadherin-dependent invasion properties of the intracellular pathogen Listeria monocytogenes. On the basis of a functional readout, automated microscopy and computer-assisted image analysis were used to screen siRNAs targeting 7,000 human genes. The validity of the screen was supported by its definion of several known regulators of E-cadherin expression, including ZEB1, HDAC1 and MMP14. We identified three new regulators (FLASH, CASP7 and PCGF1), the silencing of which was sufficient to restore high levels of E-cadherin transcription. Additionally, we identified two new regulators (FBXL5 and CAV2), the silencing of which was sufficient to increase E-cadherin expression at a post-transcriptional level. FLASH silencing regulated the expression of E-cadherin and other ZEB1-dependent genes, through post-transcriptional regulation of ZEB1, but it also regulated the expression of numerous ZEB1-independent genes with functions predicted to contribute to a restoration of the epithelial phenotype. Finally, we also report the identification of siRNA duplexes that potently restored the epithelial phenotype by mimicking the activity of known and putative microRNAs. Our findings suggest new ways to enforce epithelial phenotypes as a general strategy to treat cancer by blocking invasive and metastatic phenotypes associated with EMT. PMID:24845104

  16. Interleukin-1{beta} regulates cell proliferation and activity of extracellular matrix remodelling enzymes in cultured primary pig heart cells

    SciTech Connect

    Zitta, Karina; Brandt, Berenice; Wuensch, Annegret; Meybohm, Patrick; Bein, Berthold; Steinfath, Markus; Scholz, Jens; Albrecht, Martin

    2010-09-03

    Research highlights: {yields} Levels of IL-1{beta} are increased in the pig myocardium after infarction. {yields} Cultured pig heart cells possess IL-1 receptors. {yields} IL-1{beta} increases cell proliferati