Semaphorin 4D induces vaginal epithelial cell apoptosis to control mouse postnatal vaginal tissue remodeling

PubMed Central

ITO, TAKUJI; BAI, TAO; TANAKA, TETSUJI; YOSHIDA, KENJI; UEYAMA, TAKASHI; MIYAJIMA, MASAYASU; NEGISHI, TAKAYUKI; KAWASAKI, TAKAHIKO; TAKAMATSU, HYOTA; KIKUTANI, HITOSHI; KUMANOGOH, ATSUSHI; YUKAWA, KAZUNORI

2015-01-01

The opening of the mouse vaginal cavity to the skin is a postnatal tissue remodeling process that occurs at approximately five weeks of age for the completion of female genital tract maturation at puberty. The tissue remodeling process is primarily composed of a hormonally triggered apoptotic process predominantly occurring in the epithelium of the distal section of the vaginal cavity. However, the detailed mechanism underlying the apoptotic induction remains to be elucidated. In the present study, it was observed that the majority of BALB/c mice lacking the class 4 semaphorin, semaphorin 4D (Sema4D), developed imperforate vagina and hydrometrocolpos resulting in a perpetually unopened vaginal cavity regardless of a normal estrogen level comparable with that in wild-type (WT) mice. Administration of β-estradiol to infant Sema4D-deficient (Sema4D−/−) mice did not induce precocious vaginal opening, which was observed in WT mice subjected to the same β-estradiol administration, excluding the possibility that the closed vaginal phenotype was due to insufficient estrogen secretion at the time of vaginal opening. In order to assess the role of Sema4D in the postnatal vaginal tissue remodeling process, the expression of Sema4D and its receptor, plexin-B1, was examined as well as the level of apoptosis in the vaginal epithelia of five-week-old WT and Sema4D−/− mice. Immunohistochemical analyses confirmed the localization of Sema4D and plexin-B1 in the mouse vaginal epithelia. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunohistochemistry detecting activated caspase-3 revealed significantly fewer apoptotic cells in situ in the vaginal mucosa of five-week-old Sema4D−/− mice compared with WT mice. The addition of recombinant Sema4D to Sema4D−/− vaginal epithelial cells in culture significantly enhanced apoptosis of the vaginal epithelial cells, demonstrating the apoptosis-inducing activity of Sema4D. The experimental reduction of

Intestinal epithelial cell-specific RARα depletion results in aberrant epithelial cell homeostasis and underdeveloped immune system.

PubMed

Jijon, H B; Suarez-Lopez, L; Diaz, O E; Das, S; De Calisto, J; Yaffe, M B; Pittet, M J; Mora, J R; Belkaid, Y; Xavier, R J; Villablanca, E J

2018-05-01

Retinoic acid (RA), a dietary vitamin A metabolite, is crucial in maintaining intestinal homeostasis. RA acts on intestinal leukocytes to modulate their lineage commitment and function. Although the role of RA has been characterized in immune cells, whether intestinal epithelial cells (IECs) rely on RA signaling to exert their immune-regulatory function has not been examined. Here we demonstrate that lack of RA receptor α (RARα) signaling in IECs results in deregulated epithelial lineage specification, leading to increased numbers of goblet cells and Paneth cells. Mechanistically, lack of RARα resulted in increased KLF4 + goblet cell precursors in the distal bowel, whereas RA treatment inhibited klf4 expression and goblet cell differentiation in zebrafish. These changes in secretory cells are associated with increased Reg3g, reduced luminal bacterial detection, and an underdeveloped intestinal immune system, as evidenced by an almost complete absence of lymphoid follicles and gut resident mononuclear phagocytes. This underdeveloped intestinal immune system shows a decreased ability to clear infection with Citrobacter rodentium. Collectively, our findings indicate that epithelial cell-intrinsic RARα signaling is critical to the global development of the intestinal immune system.

Nasal epithelial cells as surrogates for bronchial epithelial cells in airway inflammation studies.

PubMed

McDougall, Catherine M; Blaylock, Morgan G; Douglas, J Graham; Brooker, Richard J; Helms, Peter J; Walsh, Garry M

2008-11-01

The nose is an attractive source of airway epithelial cells, particularly in populations in which bronchoscopy may not be possible. However, substituting nasal cells for bronchial epithelial cells in the study of airway inflammation depends upon comparability of responses, and evidence for this is lacking. Our objective was to determine whether nasal epithelial cell inflammatory mediator release and receptor expression reflect those of bronchial epithelial cells. Paired cultures of undifferentiated nasal and bronchial epithelial cells were obtained from brushings from 35 subjects, including 5 children. Cells were subject to morphologic and immunocytochemical assessment. Mediator release from resting and cytokine-stimulated cell monolayers was determined, as was cell surface receptor expression. Nasal and bronchial cells had identical epithelial morphology and uniform expression of cytokeratin 19. There were no differences in constitutive expression of CD44, intercellular adhesion molecule-1, alphavbeta3, and alphavbeta5. Despite significantly higher constitutive release of IL-8, IL-6, RANTES (regulated on activation, normal T cell expressed and secreted), and matrix metalloproteinase (MMP)-9 from nasal compared with bronchial cells, the increments in release of all studied mediators in response to stimulation with IL-1beta and TNF-alpha were similar, and there were significant positive correlations between nasal and bronchial cell secretion of IL-6, RANTES, vascular endothelial growth factor, monocyte chemoattractant protein-1, MMP-9, and tissue inhibitor of metalloproteinase-1. Despite differences in absolute mediator levels, the responses of nasal and bronchial epithelial cells to cytokine stimulation were similar, expression of relevant surface receptors was comparable, and there were significant correlations between nasal and bronchial cell mediator release. Therefore, nasal epithelial cultures constitute an accessible surrogate for studying lower airway

Expression Pattern of the Pro-apoptotic Gene PAR-4 During the Morphogenesis of MCF-10A Human Mammary Epithelial Cells.

PubMed

de Bessa Garcia, Simone A; Pereira, Michelly C; Nagai, Maria A

2010-12-21

The histological organization of the mammary gland involves a spatial interaction of epithelial and myoepithelial cells with the specialized basement membrane (BM), composed of extra-cellular matrix (ECM) proteins, which is disrupted during the tumorigenic process. The interactions between mammary epithelial cells and ECM components play a major role in mammary gland branching morphogenesis. Critical signals for mammary epithelial cell proliferation, differentiation, and survival are provided by the ECM proteins. Three-dimensional (3D) cell culture was developed to establish a system that simulates several features of the breast epithelium in vivo; 3D cell culture of the spontaneously immortalized cell line, MCF10A, is a well-established model system to study breast epithelial cell biology and morphogenesis. Mammary epithelial cells grown in 3D form spheroids, acquire apicobasal polarization, and form lumens that resemble acini structures, processes that involve cell death. Using this system, we evaluated the expression of the pro-apoptotic gene PAWR (PKC apoptosis WT1 regulator; also named PAR-4, prostate apoptosis response-4) by immunofluorescence and quantitative real time PCR (qPCR). A time-dependent increase in PAR-4 mRNA expression was found during the process of MCF10A acinar morphogenesis. Confocal microscopy analysis also showed that PAR-4 protein was highly expressed in the MCF10A cells inside the acini structure. During the morphogenesis of MCF10A cells in 3D cell culture, the cells within the lumen showed caspase-3 activation, indicating apoptotic activity. PAR-4 was only partially co-expressed with activated caspase-3 on these cells. Our results provide evidence, for the first time, that PAR-4 is differentially expressed during the process of MCF10A acinar morphogenesis.

CD8+ T cells induce thyroid epithelial cell hyperplasia and fibrosis.

PubMed

Yu, Shiguang; Fang, Yujiang; Sharav, Tumenjargal; Sharp, Gordon C; Braley-Mullen, Helen

2011-02-15

CD8(+) T cells can be important effector cells in autoimmune inflammation, generally because they can damage target cells by cytotoxicity. This study shows that activated CD8(+) T cells induce thyroid epithelial cell hyperplasia and proliferation and fibrosis in IFN-γ(-/-) NOD.H-2h4 SCID mice in the absence of CD4(+) T cells. Because CD8(+) T cells induce proliferation rather than cytotoxicity of target cells, these results describe a novel function for CD8(+) T cells in autoimmune disease. In contrast to the ability of purified CD8(+) T cells to induce thyrocyte proliferation, CD4(+) T cells or CD8 T cell-depleted splenocytes induced only mild thyroid lesions in SCID recipients. T cells in both spleens and thyroids highly produce TNF-α. TNF-α promotes proliferation of thyrocytes in vitro, and anti-TNF-α inhibits development of thyroid epithelial cell hyperplasia and proliferation in SCID recipients of IFN-γ(-/-) splenocytes. This suggests that targeting CD8(+) T cells and/or TNF-α may be effective for treating epithelial cell hyperplasia and fibrosis.

Effect of β-glucan on MUC4 and MUC5B expression in human airway epithelial cells.

PubMed

Kim, Yong-Dae; Bae, Chang Hoon; Song, Si-Youn; Choi, Yoon Seok

2015-08-01

β-Glucan is found in the cell walls of fungi, bacteria, and some plant tissues, and is detected by the innate immune system. Furthermore, this recognition is known to worsen respiratory symptoms in patients with allergic and inflammatory airway diseases. However, the means by which β-glucan affects the secretion of major mucins by human airway epithelial cells has not been elucidated. Therefore, in this study, the effect and signaling pathway of β-glucan on mucins MUC4 and MUC5B were investigated in human airway epithelial cells. In NCI-H292 cells and human normal nasal epithelial cells, the effect and signaling pathway of β-glucan on MUC4 and MUC5B expression were investigated using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme immunoassay, and immunoblot analysis with specific inhibitors and small interfering RNA (siRNA). β-Glucan increased MUC4 and MUC5B expression and activated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). SB203580 (a p38 MAPK inhibitor) and pyrrolidine dithiocarbamate (PDTC; a NF-κB inhibitor) inhibited β-glucan-induced MUC4 and MUC5B expression. In addition, siRNA knockdown of p38 MAPK blocked β-glucan-induced MUC4 and MUC5B mRNA expression and β-glucan-activated phosphorylation of NF-κB. Furthermore, Toll-like receptor 4 (TLR4) mRNA expression was increased by β-glucan, and siRNA knockdown of TLR4 blocked β-glucan-induced MUC4 and MUC5B mRNA expression and β-glucan-activated phosphorylation of p38 MAPK and NF-κB. These results demonstrate that in human airway epithelial cells β-glucan induces MUC4 and MUC5B expression via the TLR4-p38 MAPK-NF-κB signaling pathway. © 2015 ARS-AAOA, LLC.

Nasal Epithelial Cells as Surrogates for Bronchial Epithelial Cells in Airway Inflammation Studies

PubMed Central

McDougall, Catherine M.; Blaylock, Morgan G.; Douglas, J. Graham; Brooker, Richard J.; Helms, Peter J.; Walsh, Garry M.

2008-01-01

The nose is an attractive source of airway epithelial cells, particularly in populations in which bronchoscopy may not be possible. However, substituting nasal cells for bronchial epithelial cells in the study of airway inflammation depends upon comparability of responses, and evidence for this is lacking. Our objective was to determine whether nasal epithelial cell inflammatory mediator release and receptor expression reflect those of bronchial epithelial cells. Paired cultures of undifferentiated nasal and bronchial epithelial cells were obtained from brushings from 35 subjects, including 5 children. Cells were subject to morphologic and immunocytochemical assessment. Mediator release from resting and cytokine-stimulated cell monolayers was determined, as was cell surface receptor expression. Nasal and bronchial cells had identical epithelial morphology and uniform expression of cytokeratin 19. There were no differences in constitutive expression of CD44, intercellular adhesion molecule-1, αvβ3, and αvβ5. Despite significantly higher constitutive release of IL-8, IL-6, RANTES (regulated on activation, normal T cell expressed and secreted), and matrix metalloproteinase (MMP)-9 from nasal compared with bronchial cells, the increments in release of all studied mediators in response to stimulation with IL-1β and TNF-α were similar, and there were significant positive correlations between nasal and bronchial cell secretion of IL-6, RANTES, vascular endothelial growth factor, monocyte chemoattractant protein-1, MMP-9, and tissue inhibitor of metalloproteinase-1. Despite differences in absolute mediator levels, the responses of nasal and bronchial epithelial cells to cytokine stimulation were similar, expression of relevant surface receptors was comparable, and there were significant correlations between nasal and bronchial cell mediator release. Therefore, nasal epithelial cultures constitute an accessible surrogate for studying lower airway inflammation. PMID

[Effect of resveratrol on expression of TLR4 and inflammatory factors in gingival epithelial cells under high glucose environment].

PubMed

Lv, Jia-Shu; Jiang, Xue-Wei; Zhang, Yan; Zhen, Lei

2017-02-01

Through a study of the molecular mechanism of the effect of resveratrol(RSV) on expression of TLR4 and inflammatory factors in gingival epithelial cells under high glucose environment, the therapeutic effect and molecular mechanism of resveratrol on periodontitis in patients with diabetes mellitus was investigated. Gingival epithelial cells were cultured in vitro; according to the way of action, the cultured cells were divided into control group, high glucose group(HG) and HG+RSV group. The mRNA expression of TLR4 was detected by PCR; The third generation of gingival epithelial cells were pre-treated with or without RSV for 24 h under high glucose conditions, and subsequently treated with LPS at 100 ng/mL for 2 h. ELISA was used to detect the secretion of IL-1 beta, IL-6, IL-8 and TNF- alpha; the activation of TLR4 downstream signaling molecules NF-κB p65, p38 MAPK, and STAT3 was determined by Western blot. SPSS17.0 software package was used for statistical analysis. RSV could reverse the increase of TLR4 level in gingival epithelial cells in high glucose medium.LPS markedly increased the expression and secretion of IL-1β, IL-6, IL-8, and TNF-α in GECs cultured in high glucose medium, which was partly blocked in the presence of RSV. Furthermore, Western blot results showed that RSV significantly suppressed the phosphorylation of TLR4 downstream factors NF-κB p65, p38MAPK, and STAT3. RSV reduces inflammatory cytokine secretion in gingival epithelial cells, through negative regulation of TLR4 signaling pathway.

Modulation of epithelial sodium channel trafficking and function by sodium 4-phenylbutyrate in human nasal epithelial cells.

PubMed

Prulière-Escabasse, Virginie; Planès, Carole; Escudier, Estelle; Fanen, Pascale; Coste, André; Clerici, Christine

2007-11-23

Sodium 4-phenylbutyrate (4-PBA) has been shown to correct the cellular trafficking of several mutant or nonmutant plasma membrane proteins such as cystic fibrosis transmembrane conductance regulator through the expression of 70-kDa heat shock proteins. The objective of the study was to determine whether 4-PBA may influence the functional expression of epithelial sodium channels (ENaC) in human nasal epithelial cells (HNEC). Using primary cultures of HNEC, we demonstrate that 4-PBA (5 mm for 6 h) markedly stimulated amiloride-sensitive sodium channel activity and that this was related to an increased abundance of alpha-, beta-, and gamma-ENaC subunits in the apical membrane. The increase in ENaC cell surface expression (i) was due to insertion of newly ENaC subunits as determined by brefeldin A experiments and (ii) was not associated with cell surface retention of ENaC subunits because endocytosis of ENaC subunits was unchanged. In addition, we find that ENaC co-immunoprecipitated with the heat shock protein constitutively expressed Hsc70, that has been reported to modulate ENaC trafficking, and that 4-PBA decreased Hsc70 protein level. Finally, we report that in cystic fibrosis HNEC obtained from two cystic fibrosis patients, 4-PBA increased functional expression of ENaC as demonstrated by the increase in amiloride-sensitive sodium transport and in alpha-, beta-, and gamma-ENaC subunit expression in the apical membrane. Our results suggest that in HNEC, 4-PBA increases the functional expression of ENaC through the insertion of new alpha-, beta-, and gamma-ENaC subunits into the apical membrane and also suggest that 4-PBA could modify ENaC trafficking by reducing Hsc70 protein expression.

Role of medullary progenitor cells in epithelial cell migration and proliferation

PubMed Central

Chen, Dong; Chen, Zhiyong; Zhang, Yuning; Park, Chanyoung; Al-Omari, Ahmed

2014-01-01

This study is aimed at characterizing medullary interstitial progenitor cells and to examine their capacity to induce tubular epithelial cell migration and proliferation. We have isolated a progenitor cell side population from a primary medullary interstitial cell line. We show that the medullary progenitor cells (MPCs) express CD24, CD44, CXCR7, CXCR4, nestin, and PAX7. MPCs are CD34 negative, which indicates that they are not bone marrow-derived stem cells. MPCs survive >50 passages, and when grown in epithelial differentiation medium develop phenotypic characteristics of epithelial cells. Inner medulla collecting duct (IMCD3) cells treated with conditioned medium from MPCs show significantly accelerated cell proliferation and migration. Conditioned medium from PGE2-treated MPCs induce tubule formation in IMCD3 cells grown in 3D Matrigel. Moreover, most of the MPCs express the pericyte marker PDGFR-b. Our study shows that the medullary interstitium harbors a side population of progenitor cells that can differentiate to epithelial cells and can stimulate tubular epithelial cell migration and proliferation. The findings of this study suggest that medullary pericyte/progenitor cells may play a critical role in collecting duct cell injury repair. PMID:24808539

Role of Corneal Stromal Cells on Epithelial Cell Function during Wound Healing

PubMed Central

Kowtharapu, Bhavani S.; Murín, Radovan; Jünemann, Anselm G. M.; Stachs, Oliver

2018-01-01

Following injury, corneal stromal keratocytes transform into repair-phenotype of activated stromal fibroblasts (SFs) and participate in wound repair. Simultaneously, ongoing bi-directional communications between corneal stromal-epithelial cells also play a vital role in mediating the process of wound healing. Factors produced by stromal cells are known to induce proliferation, differentiation, and motility of corneal epithelial cells, which are also subsequently the main processes that occur during wound healing. In this context, the present study aims to investigate the effect of SFs conditioned medium (SFCM) on corneal epithelial cell function along with substance P (SP). Antibody microarrays were employed to profile differentially expressed cell surface markers and cytokines in the presence of SFCM and SP. Antibody microarray data revealed enhanced expression of the ITGB1 in corneal epithelial cells following stimulation with SP whereas SFCM induced abundant expression of IL-8, ITGB1, PD1L1, PECA1, IL-15, BDNF, ICAM1, CD8A, CD44 and NTF4. All these proteins have either direct or indirect roles in epithelial cell growth, movement and adhesion related signaling cascades during tissue regeneration. We also observed activation of MAPK signaling pathway along with increased expression of focal adhesion kinase (FAK), paxillin, vimentin, β-catenin and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Additionally, epithelial-to-mesenchymal transition (EMT) regulating transcription factors Slug and ZEB1 expression were enhanced in the presence of SFCM. SP enriched the expression of integrin subunits α4, α5, αV, β1 and β3 whereas SFCM increased α4, α5, αV, β1 and β5 integrin subunits. We also observed increased expression of Serpin E1 following SP and SFCM treatment. Wound healing scratch assay revealed enhanced migration of epithelial cells following the addition of SFCM. Taken together, we conclude that SFCM-mediated sustained activation of ZEB1

4-Methoxyestradiol-induced oxidative injuries in human lung epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng Yahsin; Chang, Louis W.; Cheng Lichuan

2007-05-01

Epidemiological studies indicated that people exposed to dioxins were prone to the development of lung diseases including lung cancer. Animal studies demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased liver tumors and promoted lung metaplasia in females. Metabolic changes in 17{beta}-estradiol (E{sub 2}) resulted from an interaction between TCDD and E{sub 2} could be associated with gender difference. Previously, we reported that methoxylestradiols (MeOE{sub 2}), especially 4-MeOE{sub 2}, accumulated in human lung cells (BEAS-2B) co-treated with TCDD and E{sub 2}. In the present study, we demonstrate unique accumulation of 4-MeOE{sub 2}, as a result of TCDD/E{sub 2} interaction and revealed its bioactivity inmore » human lung epithelial cell line (H1355). 4-Methoxyestradiol treatment significantly decreased cell growth and increased mitotic index. Elevation of ROS and SOD activity, with a concomitant decrease in the intracellular GSH/GSSG ratio, was also detected in 4-MeOE{sub 2}-treated cells. Quantitative comet assay showed increased oxidative DNA damage in the 4-MeOE{sub 2}-treated H1355 cells, which could be significantly reduced by the anti-oxidant N-acetylcysteine (NAC). However, inhibition of cell growth and increase in mitotic arrest induced by 4-MeOE{sub 2} were unaffected by NAC. We concluded that 4-MeOE{sub 2} accumulation resulting from TCDD and E{sub 2} interaction would contribute to the higher vulnerability on lung pathogenesis in females when exposed to TCDD.« less

Extremely stringent activation of p16INK4a prevents immortalization of uterine cervical epithelial cells without human papillomavirus oncogene expression

PubMed Central

Hang, Su; Tiwari, Agnes F.Y.; Ngan, Hextan Y.S.; Yip, Yim-Ling; Cheung, Annie L.M.; Tsao, Sai Wah; Deng, Wen

2016-01-01

Cervical epithelial cell immortalization with defined genetic factors without viral oncogenes has never been reported. Here we report that HPV-negative cervical epithelial cells failed to be immortalized by telomerase activation or the combination of p53 knockdown and telomerase activation. Under those conditions, p16INK4a expression was always elevated during the late stage of limited cell lifespan, suggesting that cervical epithelial cells possess an intrinsic property of uniquely stringent activation of p16INK4a, which may offer an explanation for the rarity of HPV-negative cervical cancer. Combining p16INK4a knockdown with telomerase activation resulted in efficient immortalization of HPV-negative cervical epithelial cells under ordinary culture conditions. Compared with the HPV16-E6E7-immortalized cell lines derived from the same primary cell sources, the novel HPV-negative immortalized cell lines had lower degrees of chromosomal instability, maintained more sensitive p53/p21 response to DNA damage, exhibited more stringent G2 checkpoint function, and were more resistant to replication-stress-induced genomic instability. The newly immortalized HPV-negative cervical epithelial cell lines were non-tumorigenic in nude mice. The cell lines can be used not only as much-needed HPV-negative non-malignant cell models but also as starting models that can be genetically manipulated in a stepwise fashion to investigate the roles of defined genetic alterations in the development of HPV-negative cervical cancer. PMID:27344169

Epithelial-mesenchymal transition in breast epithelial cells treated with cadmium and the role of Snail.

PubMed

Wei, Zhengxi; Shan, Zhongguo; Shaikh, Zahir A

2018-04-01

Epidemiological and experimental studies have implicated cadmium (Cd) with breast cancer. In breast epithelial MCF10A and MDA-MB-231 cells, Cd has been shown to promote cell growth. The present study examined whether Cd also promotes epithelial-mesenchymal transition (EMT), a hallmark of cancer progression. Human breast epithelial cells consisting of non-cancerous MCF10A, non-metastatic HCC 1937 and HCC 38, and metastatic MDA-MB-231 were treated with 1 or 3 μM Cd for 4 weeks. The MCF10A epithelial cells switched to a more mesenchymal-like morphology, which was accompanied by a decrease in the epithelial marker E-cadherin and an increase in the mesenchymal markers N-cadherin and vimentin. In both non-metastatic HCC 1937 and HCC 38 cells, treatment with Cd decreased the epithelial marker claudin-1. In addition, E-cadherin also decreased in the HCC 1937 cells. Even the mesenchymal-like MDA-MB-231 cells exhibited an increase in the mesenchymal marker vimentin. These changes indicated that prolonged treatment with Cd resulted in EMT in both normal and cancer-derived breast epithelial cells. Furthermore, both the MCF10A and MDA-MB-231 cells labeled with Zcad, a dual sensor for tracking EMT, demonstrated a decrease in the epithelial marker E-cadherin and an increase in the mesenchymal marker ZEB-1. Treatment of cells with Cd significantly increased the level of Snail, a transcription factor involved in the regulation of EMT. However, the Cd-induced Snail expression was completely abolished by actinomycin D. Luciferase reporter assay indicated that the expression of Snail was regulated by Cd at the promotor level. Snail was essential for Cd-induced promotion of EMT in the MDA-MB-231 cells, as knockdown of Snail expression blocked Cd-induced cell migration. Together, these results indicate that Cd promotes EMT in breast epithelial cells and does so by modulating the transcription of Snail. Copyright © 2018 Elsevier Inc. All rights reserved.

Release of HIV-1 sequestered in the vesicles of oral and genital mucosal epithelial cells by epithelial-lymphocyte interaction

PubMed Central

Yasen, Aizezi; Herrera, Rossana; Rosbe, Kristina

2017-01-01

Oropharyngeal mucosal epithelia of fetuses/neonates/infants and the genital epithelia of adults play a critical role in HIV-1 mother-to-child transmission and sexual transmission of virus, respectively. To study the mechanisms of HIV-1 transmission through mucosal epithelium, we established polarized tonsil, cervical and foreskin epithelial cells. Analysis of HIV-1 transmission through epithelial cells showed that approximately 0.05% of initially inoculated virions transmigrated via epithelium. More than 90% of internalized virions were sequestered in the endosomes of epithelial cells, including multivesicular bodies (MVBs) and vacuoles. Intraepithelial HIV-1 remained infectious for 9 days without viral release. Release of sequestered intraepithelial HIV-1 was induced by the calcium ionophore ionomycin and by cytochalasin D, which increase intracellular calcium and disrupt the cortical actin of epithelial cells, respectively. Cocultivation of epithelial cells containing HIV-1 with activated peripheral blood mononuclear cells and CD4+ T lymphocytes led to the disruption of epithelial cortical actin and spread of virus from epithelial cells to lymphocytes. Treatment of epithelial cells with proinflammatory cytokines tumor necrosis factor-alpha and interferon gamma also induced reorganization of cortical actin and release of virus. Inhibition of MVB formation by small interfering RNA (siRNA)-mediated silencing of its critical protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) expression reduced viral sequestration in epithelial cells and its transmission from epithelial cells to lymphocytes by ~60–70%. Furthermore, inhibition of vacuole formation of epithelial cells by siRNA-inactivated rabankyrin-5 expression also significantly reduced HIV-1 sequestration in epithelial cells and spread of virus from epithelial cells to lymphocytes. Interaction of the intercellular adhesion molecule-1 of epithelial cells with the function-associated antigen-1

Intestinal Epithelial Cells Modulate Antigen-Presenting Cell Responses to Bacterial DNA

PubMed Central

Campeau, J. L.; Salim, S. Y.; Albert, E. J.; Hotte, N.

2012-01-01

Intestinal epithelial cells and antigen-presenting cells orchestrate mucosal innate immunity. This study investigated the role of bacterial DNA in modulating epithelial and bone marrow-derived antigen-presenting cells (BM-APCs) and subsequent T-lymphocyte responses. Murine MODE-K epithelial cells and BM-APCs were treated with DNA from either Bifidobacterium breve or Salmonella enterica serovar Dublin directly and under coculture conditions with CD4+ T cells. Apical stimulation of MODE-K cells with S. Dublin DNA enhanced secretion of cytokines from underlying BM-APCs and induced interleukin-17 (IL-17) and gamma interferon (IFN-γ) secretion from CD4+ T cells. Bacterial DNA isolated from either strain induced maturation and increased cytokine secretion from BM-APCs. Conditioned medium from S. Dublin-treated MODE-K cells elicited an increase in cytokine secretion similar to that seen for S. Dublin DNA. Treatment of conditioned medium from MODE-K cells with RNase and protease prevented the S. Dublin-induced increased cytokine secretion. Oral feeding of mice with B. breve DNA resulted in enhanced levels of colonic IL-10 and transforming growth factor β (TGFβ) compared with what was seen for mice treated with S. Dublin DNA. In contrast, feeding mice with S. Dublin DNA increased levels of colonic IL-17 and IL-12p70. T cells from S. Dublin DNA-treated mice secreted high levels of IL-12 and IFN-γ compared to controls and B. breve DNA-treated mice. These results demonstrate that intestinal epithelial cells are able to modulate subsequent antigen-presenting and T-cell responses to bacterial DNA with pathogenic but not commensal bacterial DNA inducing effector CD4+ T lymphocytes. PMID:22615241

Eosinophils Promote Epithelial to Mesenchymal Transition of Bronchial Epithelial Cells

PubMed Central

Toda, Masaaki; Miyake, Yasushi; Matsushima, Yuki; Matsumoto, Takahiro; Boveda-Ruiz, Daniel; Gil-Bernabe, Paloma; Nagao, Mizuho; Sugimoto, Mayumi; Hiraguchi, Yukiko; Tokuda, Reiko; Naito, Masahiro; Takagi, Takehiro; D'Alessandro-Gabazza, Corina N.; Suga, Shigeru; Kobayashi, Tetsu; Fujisawa, Takao; Taguchi, Osamu; Gabazza, Esteban C.

2013-01-01

Eosinophilic inflammation and remodeling of the airways including subepithelial fibrosis and myofibroblast hyperplasia are characteristic pathological findings of bronchial asthma. Epithelial to mesenchymal transition (EMT) plays a critical role in airway remodelling. In this study, we hypothesized that infiltrating eosinophils promote airway remodelling in bronchial asthma. To demonstrate this hypothesis we evaluated the effect of eosinophils on EMT by in vitro and in vivo studies. EMT was assessed in mice that received intra-tracheal instillation of mouse bone marrow derived eosinophils and in human bronchial epithelial cells co-cultured with eosinophils freshly purified from healthy individuals or with eosinophilic leukemia cell lines. Intra-tracheal instillation of eosinophils was associated with enhanced bronchial inflammation and fibrosis and increased lung concentration of growth factors. Mice instilled with eosinophils pre-treated with transforming growth factor(TGF)-β1 siRNA had decreased bronchial wall fibrosis compared to controls. EMT was induced in bronchial epithelial cells co-cultured with human eosinophils and it was associated with increased expression of TGF-β1 and Smad3 phosphorylation in the bronchial epithelial cells. Treatment with anti-TGF-β1 antibody blocked EMT in bronchial epithelial cells. Eosinophils induced EMT in bronchial epithelial cells, suggesting their contribution to the pathogenesis of airway remodelling. PMID:23700468

Regulation of the membrane mucin Muc4 in corneal epithelial cells by proteosomal degradation and TGF-beta.

PubMed

Lomako, Joseph; Lomako, Wieslawa M; Carothers Carraway, Coralie A; Carraway, Kermit L

2010-04-01

MUC4 is a heterodimeric membrane mucin, composed of a mucin subunit ASGP-1 (MUC4alpha) and a transmembrane subunit ASGP-2 (MUC4beta), which has been implicated in the protection of epithelial cell surfaces. In the rat stratified corneal epithelium Muc4 is found predominantly in the most superficial cell layers. Since previous studies in other tissues have shown that Muc4 is regulated by TGF-beta via a proteosomal degradation mechanism, we investigated the regulation of corneal Muc4 in stratified cultures of corneal epithelial cells. Application of proteosome or processing inhibitors led to increases in levels of Muc4, particularly in the basal and intermediate levels of the stratified cultures. These changes were accompanied by increases in Muc4 ubiquitination, chaperone association and incorporation into intracellular aggresomes. In contrast, treatment with TGF-beta resulted in reduced levels of Muc4, which were reversed by proteosome inhibition. The results support a model in which Muc4 precursor is synthesized in all layers of the corneal epithelium, but Muc4 is degraded in basal and intermediate layers by a proteosomal mechanism at least partly dependent on TGF-beta inhibition of Muc4 processing. J. Cell. Physiol. 223: 209-214, 2010. (c) 2009 Wiley-Liss, Inc.

Zinc transport by respiratory epithelial cells and interaction with iron homeostasis.

PubMed

Deng, Zhongping; Dailey, Lisa A; Soukup, Joleen; Stonehuerner, Jacqueline; Richards, Judy D; Callaghan, Kimberly D; Yang, Funmei; Ghio, Andrew J

2009-10-01

Despite recurrent exposure to zinc through inhalation of ambient air pollution particles, relatively little information is known about the homeostasis of this metal in respiratory epithelial cells. We describe zinc uptake and release by respiratory epithelial cells and test the postulate that Zn(2+) transport interacts with iron homeostasis in these same cells. Zn(2+) uptake after 4 and 8 h of exposure to zinc sulfate was concentration- and time-dependent. A majority of Zn(2+) release occurred in the 4 h immediately following cell exposure to ZnSO(4). Regarding metal importers, mRNA for Zip1 and Zip2 showed no change after respiratory epithelial cell exposure to zinc while mRNA for divalent metal transporter (DMT)1 increased. Western blot assay for DMT1 protein supported an elevated expression of this transport protein following zinc exposure. RT-PCR confirmed mRNA for the metal exporters ZnT1 and ZnT4 with the former increasing after ZnSO(4). Cell concentrations of ferritin increased with zinc exposure while oxidative stress, measured as lipid peroxides, was decreased supporting an anti-oxidant function for Zn(2+). Increased DMT1 expression, following pre-incubations of respiratory epithelial cells with TNF-alpha, IFN-gamma, and endotoxin, was associated with significantly decreased intracellular zinc transport. Finally, incubations of respiratory epithelial cells with both zinc sulfate and ferric ammonium citrate resulted in elevated intracellular concentrations of both metals. We conclude that exposure to zinc increases iron uptake by respiratory epithelial cells. Elevations in cell iron can possibly affect an increased expression of DMT1 and ferritin which function to diminish oxidative stress. Comparable to other metal exposures, changes in iron homeostasis may contribute to the biological effects of zinc in specific cells and tissues.

Cell competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes.

PubMed

Kon, Shunsuke; Ishibashi, Kojiro; Katoh, Hiroto; Kitamoto, Sho; Shirai, Takanobu; Tanaka, Shinya; Kajita, Mihoko; Ishikawa, Susumu; Yamauchi, Hajime; Yako, Yuta; Kamasaki, Tomoko; Matsumoto, Tomohiro; Watanabe, Hirotaka; Egami, Riku; Sasaki, Ayana; Nishikawa, Atsuko; Kameda, Ikumi; Maruyama, Takeshi; Narumi, Rika; Morita, Tomoko; Sasaki, Yoshiteru; Enoki, Ryosuke; Honma, Sato; Imamura, Hiromi; Oshima, Masanobu; Soga, Tomoyoshi; Miyazaki, Jun-Ichi; Duchen, Michael R; Nam, Jin-Min; Onodera, Yasuhito; Yoshioka, Shingo; Kikuta, Junichi; Ishii, Masaru; Imajo, Masamichi; Nishida, Eisuke; Fujioka, Yoichiro; Ohba, Yusuke; Sato, Toshiro; Fujita, Yasuyuki

2017-05-01

Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis.

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

Pre-existing Epithelial Diversity in Normal Human Livers: A Tissue-tethered Cytometric Analysis in Portal/Periportal Epithelial Cells

PubMed Central

Isse, Kumiko; Lesniak, Andrew; Grama, Kedar; Maier, John; Specht, Susan; Castillo-Rama, Marcela; Lunz, John; Roysam, Badrinath; Michalopoulos, George; Demetris, Anthony J.

2012-01-01

Routine light microscopy identifies two distinct epithelial cell populations in normal human livers: hepatocytes and biliary epithelial cells (BEC). Considerable epithelial diversity, however, arises during disease states when a variety of hepatocyte-BEC hybrid cells appear. This has been attributed to activation and differentiation of putative hepatic progenitor cells (HPC) residing in the Canals of Hering and/or metaplasia of pre-existing mature epithelial cells. A novel analytic approach consisting of multiplex labeling, high resolution whole slide imaging (WSI), and automated image analysis was used to determine if more complex epithelial cell phenotypes pre-existed in normal adult human livers, which might provide an alternative explanation for disease-induced epithelial diversity. “Virtually digested” WSI enabled quantitative cytometric analyses of individual cells displayed in a variety of formats (e.g. scatter plots) while still tethered to the WSI and tissue structure. We employed biomarkers specifically-associated with mature epithelial forms (HNF4α for hepatocytes, CK19 and HNF1β for BEC) and explored for the presence of cells with hybrid biomarker phenotypes. Results showed abundant hybrid cells in portal bile duct BEC, canals of Hering, and immediate periportal hepatocytes. These bi-potential cells likely serve as a reservoir for the epithelial diversity of ductular reactions, appearance of hepatocytes in bile ducts, and the rapid and fluid transition of BEC to hepatocytes, and vice versa. Conclusion Novel imaging and computational tools enable increased information extraction from tissue samples and quantify the considerable pre-existent hybrid epithelial diversity in normal human liver. This computationally-enabled tissue analysis approach offers much broader potential beyond the results presented here. PMID:23150208

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

Epithelial-mesenchymal transition abolishes the susceptibility of polarized epithelial cell lines to measles virus.

PubMed

Shirogane, Yuta; Takeda, Makoto; Tahara, Maino; Ikegame, Satoshi; Nakamura, Takanori; Yanagi, Yusuke

2010-07-02

Measles virus (MV), an enveloped negative-strand RNA virus, remains a major cause of morbidity and mortality in developing countries. MV predominantly infects immune cells by using signaling lymphocyte activation molecule (SLAM; also called CD150) as a receptor, but it also infects polarized epithelial cells, forming tight junctions in a SLAM-independent manner. Although the ability of MV to infect polarized epithelial cells is thought to be important for its transmission, the epithelial cell receptor for MV has not been identified. A transcriptional repressor, Snail, induces epithelial-mesenchymal transition (EMT), in which epithelial cells lose epithelial cell phenotypes, such as adherens and tight junctions. In this study, EMT was induced by expressing Snail in a lung adenocarcinoma cell line, II-18, which is highly susceptible to wild-type MV. Snail-expressing II-18 cells lost adherens and tight junctions. Microarray analysis confirmed the induction of EMT in II-18 cells and suggested a novel function of Snail in protein degradation and distribution. Importantly, wild-type MV no longer entered EMT-induced II-18 cells, suggesting that the epithelial cell receptor is down-regulated by the induction of EMT. Other polarized cell lines, NCI-H358 and HT-29, also lost susceptibility to wild-type MV when EMT was induced. However, the complete formation of tight junctions rather reduced MV entry into HT-29 cells. Taken together, these data suggest that the unidentified epithelial cell receptor for MV is involved in the formation of epithelial intercellular junctions.

Epithelial stem cells are formed by small-particles released from particle-producing cells

PubMed Central

Kong, Wuyi; Zhu, Xiao Ping; Han, Xiu Juan; Nuo, Mu; Wang, Hong

2017-01-01

Recent spatiotemporal report demonstrated that epidermal stem cells have equal potential to divide or differentiate, with no asymmetric cell division observed. Therefore, how epithelial stem cells maintain lifelong stem-cell support still needs to be elucidated. In mouse blood and bone marrow, we found a group of large cells stained strongly for eosin and containing coiled-tubing-like structures. Many were tightly attached to each other to form large cellular clumps. After sectioning, these large cell-clumps were composed of not cells but numerous small particles, however with few small “naked” nuclei. The small particles were about 2 to 3 μm in diameter and stained dense red for eosin, so they may be rich in proteins. Besides the clumps composed of small particles, we identified clumps formed by fusion of the small particles and clumps of newly formed nucleated cells. These observations suggest that these small particles further fused and underwent cellularization. E-cadherin was expressed in particle-fusion areas, some “naked” nuclei and the newly formed nucleated cells, which suggests that these particles can form epithelial cells via fusion and nuclear remodeling. In addition, we observed similar-particle fusion before epithelial cellularization in mouse kidney ducts after kidney ischemia, which suggests that these particles can be released in the blood and carried to the target tissues for epithelial-cell regeneration. Oct4 and E-cadherin expressed in the cytoplasmic areas in cells that were rich in protein and mainly located in the center of the cellular clumps, suggesting that these newly formed cells have become tissue-specific epithelial stem cells. Our data provide evidence that these large particle-producing cells are the origin of epithelial stem cells. The epithelial stem cells are newly formed by particle fusion. PMID:28253358

USP17 is upregulated in osteosarcoma and promotes cell proliferation, metastasis, and epithelial-mesenchymal transition through stabilizing SMAD4.

PubMed

Song, Chenyang; Liu, Wenge; Li, Jiandong

2017-07-01

USP17 is upregulated in several cancers, indicating that USP17 might play essential functions in tumor development. However, the function of USP17 in osteosarcoma is still unknown. Our work aimed to investigate the function of USP17 in osteosarcoma. We found that the expression of USP17 was upregulated in osteosarcoma tissues and cell lines, including MG-63 and U2OS. Several functional experiments, such as colony formation analysis, Cell Counting Kit-8 assay, wound healing analysis, and transwell assay, showed that USP17 promoted cell proliferation, migration, and invasion. Moreover, we found that USP17 facilitated migration and invasion through promoting epithelial-mesenchymal transition. SMAD4 has been found to regulate epithelial-mesenchymal transition, co-immunopurification, and glutathione S-transferase pull-down analysis demonstrated that USP17 interacted with SMAD4. Furthermore, USP17 stabilized SMAD4 through its deubiquitinase activity. In conclusion, this study shows that USP17 enhances osteosarcoma cell proliferation and invasion through stabilizing SMAD4.

Transplantation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in Macular Degeneration.

PubMed

Mehat, Manjit S; Sundaram, Venki; Ripamonti, Caterina; Robson, Anthony G; Smith, Alexander J; Borooah, Shyamanga; Robinson, Martha; Rosenthal, Adam N; Innes, William; Weleber, Richard G; Lee, Richard W J; Crossland, Michael; Rubin, Gary S; Dhillon, Baljean; Steel, David H W; Anglade, Eddy; Lanza, Robert P; Ali, Robin R; Michaelides, Michel; Bainbridge, James W B

2018-06-05

Transplantation of human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells offers the potential for benefit in macular degeneration. Previous trials have reported improved visual acuity (VA), but lacked detailed analysis of retinal structure and function in the treated area. Phase 1/2 open-label dose-escalation trial to evaluate safety and potential efficacy (clinicaltrials.gov identifier, NCT01469832). Twelve participants with advanced Stargardt disease (STGD1), the most common cause of macular degeneration in children and young adults. Subretinal transplantation of up to 200 000 hESC-derived RPE cells with systemic immunosuppressive therapy for 13 weeks. The primary end points were the safety and tolerability of hESC-derived RPE cell administration. We also investigated evidence of the survival of transplanted cells and measured retinal structure and function using microperimetry and spectral-domain OCT. Focal areas of subretinal hyperpigmentation developed in all participants in a dose-dependent manner in the recipient retina and persisted after withdrawal of systemic immunosuppression. We found no evidence of uncontrolled proliferation or inflammatory responses. Borderline improvements in best-corrected VA in 4 participants either were unsustained or were matched by a similar improvement in the untreated contralateral eye. Microperimetry demonstrated no evidence of benefit at 12 months in the 12 participants. In one instance at the highest dose, localized retinal thinning and reduced sensitivity in the area of hyperpigmentation suggested the potential for harm. Participant-reported quality of life using the 25-item National Eye Institute Visual Function Questionnaire indicated no significant change. Subretinal hyperpigmentation is consistent with the survival of viable transplanted hESC-derived RPE cells, but may reflect released pigment in their absence. The findings demonstrate the value of detailed analysis of spatial correlation of

The status of intercellular junctions in established lens epithelial cell lines.

PubMed

Dave, Alpana; Craig, Jamie E; Sharma, Shiwani

2012-01-01

Cataract is the major cause of vision-related disability worldwide. Mutations in the crystallin genes are the most common known cause of inherited congenital cataract. Mutations in the genes associated with intercellular contacts, such as Nance-Horan Syndrome (NHS) and Ephrin type A receptor-2 (EPHA2), are other recognized causes of congenital cataract. The EPHA2 gene has been also associated with age-related cataract, suggesting that intercellular junctions are important in not only lens development, but also in maintaining lens transparency. The purpose of this study was to analyze the expression and localization of the key cell junction and cytoskeletal proteins, and of NHS and EPHA2, in established lens epithelial cell lines to determine their suitability as model epithelial systems for the functional investigation of genes involved in intercellular contacts and implicated in cataract. The expression and subcellular localization of occludin and zona occludens protein-1 (ZO-1), which are associated with tight junctions; E-cadherin, which is associated with adherence junctions; and the cytoskeletal actin were analyzed in monolayers of a human lens epithelial cell line (SRA 01/04) and a mouse lens epithelial cell line (αTN4). In addition, the expression and subcellular localization of the NHS and EPHA2 proteins were analyzed in these cell lines. Protein or mRNA expression was respectively determined by western blotting or reverse transcription-polymerase chain reaction (RT-PCR), and localization was determined by immunofluorescence labeling. Human SRA 01/04 and mouse αTN4 lens epithelial cells expressed either the proteins of interest or their encoding mRNA. Occludin, ZO-1, and NHS proteins localized to the cellular periphery, whereas E-cadherin, actin, and EPHA2 localized in the cytoplasm in these cell lines. The human SRA 01/04 and mouse αTN4 lens epithelial cells express the key junctional proteins. The localization patterns of these proteins suggest that

Characterizing mutagenesis in the hprt gene of rat alveolar epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Driscoll, K.E.; Deyo, L.C.; Howard, B.W.

1995-12-31

A clonal selection assay was developed for mutation in the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene of rat alveolar epithelial cells. Studies were conducted to establish methods for isolation and long-term culture of rat alveolar epithelial cells. When isolated by pronase digestion purified on a Nycodenz gradient and cultured in media containing 7.5% fetal bovine serum (FBS), pituitary extract, EGF, insulin, and IGF-1, rat alveolar epithelial cells could be maintained in culture for several weeks with cell doubling times of 2-4 days. The rat alveolar epithelial cell cultures were exposed in vitro to the mutagens ethylnitrosourea (ENU) and H{sub 2}O{sub 2},more » and mutation in the hprt gene was selected for by culture in the presence of the toxic purine analog, 6-thioguanine (6TG). In vitro exposure to ENU or H{sub 2}O produced a dose-dependent increase in hprt mutation frequency in the alveolar epithelial cells. To determine if the assay system could be used to evaluate mutagenesis in alveolar type II cells after in vivo mutagen or carcinogen exposure, cells were isolated from rats treated previously with ENU or {alpha}-quartz. A significant increase in hprt mutation frequency was detected in alveolar epithelial cells obtained from rats exposed to ENU or {alpha}-quartz; the latter observation is the first demonstration that crystalline silica exposure is mutagenic in vivo. In summary, these studies show that rat alveolar epithelial cells isolated by pronase digestion and Nycodenz separation techniques and cultured in a defined media can be used in a clonal selection assay for mutation in the hprt gene. This assay demonstrates that ENU and H{sub 2}O{sub 2} in vitro and ENU and {alpha}-quartz in vivo are mutagenic for rat alveolar epithelial cells. This model should be useful for investigating the genotoxic effects of chemical and physical agents on an important lung cell target for neoplastic transformation. 41 refs., 4 figs., 3 tabs.« less

Activation of deltaF508 CFTR in a cystic fibrosis respiratory epithelial cell line by 4-phenylbutyrate, genistein and CPX.

PubMed

Andersson, C; Roomans, G M

2000-05-01

The cellular basis of cystic fibrosis (CF) is a defect in a cyclic adenosine monophosphate (cAMP)-activated chloride channel (CF transmembrane conductance regulator) in epithelial cells that leads to decreased chloride ion transport and impaired water transport across the cell membrane. This study investigated whether it was possible to activate the defective chloride channel in cystic fibrosis respiratory epithelial cells with 4-phenylbutyrate (4PBA), genistein and 8-cyclopentyl-1,3-dipropylxanthine (CPX). The CF bronchial epithelial cell line CFBE41o-, which expresses the deltaF508 mutation, was treated with these agents and loss of Cl-, indicating Cl- efflux, measured by X-ray microanalysis. 8-bromo-cAMP alone did not induce Cl- efflux in CFBE41o- cells, but after incubation with 4PBA a significant efflux of Cl- occurred. Stimulation of cells with a combination of genistein and cAMP also induced Cl- efflux, whereas a combination of pretreatment with 4PBA and a combined stimulation with genistein and cAMP induced an even larger Cl- efflux. Cl- efflux could also be stimulated by CPX, but this effect was not enhanced by 4PBA pretreatment. The deltaF508 mutation leads to impaired processing of the cystic fibrosis transmembrane conductance regulator. The increased efflux of chloride after 4-phenylbutyrate treatment can be explained by the fact that 4-phenylbutyrate allows the deltaF508 cystic fibrosis transmembrane conductance regulator to escape degradation and to be transported to the cell surface. Genistein and 8-cyclopentyl-1,3-dipropylxanthine act by stimulating chloride ion efflux by increasing the probability of the cystic fibrosis transmembrane conductance regulator being open. The combination of 4-phenylbutyrate and genistein may be useful in a potential pharmacological therapy for cystic fibrosis patients with the deltaF508 mutation.

Pax8 modulates the expression of Wnt4 that is necessary for the maintenance of the epithelial phenotype of thyroid cells

PubMed Central

2014-01-01

Background The transcription factor Pax8 is expressed during thyroid development and is involved in the morphogenesis of the thyroid gland and maintenance of the differentiated phenotype. In particular, Pax8 has been shown to regulate genes that are considered markers of thyroid differentiation. Recently, the analysis of the gene expression profile of FRTL-5 differentiated thyroid cells after the silencing of Pax8 identified Wnt4 as a novel target. Like the other members of the Wnt family, Wnt4 has been implicated in several developmental processes including regulation of cell fate and patterning during embryogenesis. To date, the only evidence on Wnt4 in thyroid concerns its down-regulation necessary for the progression of thyroid epithelial tumors. Results Here we demonstrate that Pax8 is involved in the transcriptional modulation of Wnt4 gene expression directly binding to its 5’-flanking region, and that Wnt4 expression in FRTL-5 cells is TSH-dependent. Interestingly, we also show that in thyroid cells a reduced expression of Wnt4 correlates with the alteration of the epithelial phenotype and that the overexpression of Wnt4 in thyroid cancer cells is able to inhibit cellular migration. Conclusions We have identified and characterized a functional Pax8 binding site in the 5’-flanking region of the Wnt4 gene and we show that Pax8 modulates the expression of Wnt4 in thyroid cells. Taken together, our results suggest that in thyroid cells Wnt4 expression correlates with the integrity of the epithelial phenotype and is reduced when this integrity is perturbed. In the end, we would like to suggest that the overexpression of Wnt4 in thyroid cancer cells is able to revert the mesenchymal phenotype. PMID:25270402

Fibrin glue inhibits migration of ocular surface epithelial cells

PubMed Central

Yeung, A M; Faraj, L A; McIntosh, O D; Dhillon, V K; Dua, H S

2016-01-01

Purpose Fibrin glue has been used successfully in numerous ophthalmic surgical procedures. Recently, fibrin glue has been used in limbal stem cell transplantation to reduce both operative time and to negate the need for sutures. The aim of this study was to determine the effects of fibrin glue on epithelial cell migration in vitro. Methods Corneoscleral rims were split to retain the epithelial layer, Bowman's layer, and anterior stroma. Rims were cut into eight equal-sized pieces and were placed directly on culture plates or affixed with fibrin glue. Rims were maintained in culture for 25 days and epithelial cell growth was monitored. Cells were photographed to measure area or growth and immunofluorescence staining of explants for fibrin was performed. Results Explants that were glued demonstrated significantly delayed epithelial cell growth and migration as compared with explants without glue. By day 16, all fibrin glue had dissolved and coincided with onset of cell growth from glued explants. Cell growth commenced between days 3 and 4 for control explants without glue and around days 14–16 for explants with fibrin glue. Conclusions Fibrin glue delays epithelial cell migration by acting as a physical barrier and can potentially interfere with explant-derived limbal epithelial cell migration on to the corneal surface. We propose that glue should be used to attach the conjunctival frill of the limbal explant but care should be taken to ensure that the glue does not wrap around the explant if used to secure the explant as well. Strategic use of glue, to attach the recessed conjunctiva, can be advantageous in delaying conjunctival cell migration and reducing the need for sequential sector conjunctival epitheliectomy. PMID:27367746

MUC4 mucin-induced epithelial to mesenchymal transition: a novel mechanism for metastasis of human ovarian cancer cells.

PubMed

Ponnusamy, M P; Lakshmanan, I; Jain, M; Das, S; Chakraborty, S; Dey, P; Batra, S K

2010-10-21

The acquisition of invasiveness in ovarian cancer (OC) is accompanied by the process of epithelial-to-mesenchymal transition (EMT). The MUC4 mucin is overexpressed in ovarian tumors and has a role in the invasiveness of OC cells. The present study was aimed at evaluating the potential involvement of MUC4 in the metastasis of OC cells by inducing EMT. Ectopic overexpression of MUC4 in OC cells (SKOV3-MUC4) resulted in morphological alterations along with a decreased expression of epithelial markers (E-cadherin and cytokeratin (CK)-18) and an increased expression of mesenchymal markers (N-cadherin and vimentin) compared with the control cells (SKOV3-vector). Also, pro-EMT transcription factors TWIST1, TWIST2 and SNAIL showed an upregulation in SKOV3-MUC4 cells. We further investigated the pathways upstream of N-cadherin, such as focal adhesion kinase (FAK), MKK7, JNK1/2 and c-Jun, which were also activated in the SKOV3-MUC4 cells compared with SKOV3-vector cells. Inhibition of phospho-FAK (pFAK) and pJNK1/2 decreased N-cadherin expression in the MUC4-overexpressing cells, which further led to a significant decrease in cellular motility. Knockdown of N-cadherin decreased the activation of extracellular signal-regulated kinase-1/2 (ERK1/2), AKT and matrix metalloproteinase 9 (MMP9), and inhibited the motility in the SKOV3-MUC4 cells. Upon in vivo tumorigenesis and metastasis analysis, the SKOV3-MUC4 cells produced significantly larger tumors and demonstrated a higher incidence of metastasis to distance organs (peritoneal wall, colon, intestine, stomach, lymph nodes, liver and diaphragm). Taken together, our study reveals a novel role for MUC4 in inducing EMT through the upregulation of N-cadherin and promoting metastasis of OC cells.

Effect of doxycycline on epithelial-mesenchymal transition via the p38/Smad pathway in respiratory epithelial cells.

PubMed

Shin, Jae-Min; Kang, Ju-Hyung; Lee, Seoung-Ae; Park, Il-Ho; Lee, Heung-Man

2017-03-01

Doxycycline has antibacterial and anti-inflammatory effects, and it also suppresses collagen biosynthesis. This study aimed to confirm the effects and mechanism of doxycycline on transforming growth factor (TGF) beta 1 induced epithelial-mesenchymal transition and cell migration in A549 and primary nasal epithelial cells. A 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay and phalloidin-fluorescein isothiocyanate staining were used to evaluate cytotoxicity and cellular morphologic changes. Western blot and immunofluorescence staining were used to determine the expression levels of E-cadherin, vimentin, alpha-smooth muscle actin, fibronectin, phosphorylated Smad2/3, and mitogen-activated protein kinases. Scratch and transwell migration assays were used to assess cellular migration ability. Doxycycline (0-10 μg/mL) had no significant cytotoxic effects in A549 and primary nasal epithelial cells. Increased expression of mesenchymal markers, including vimentin, alpha-smooth muscle actin, and fibronectin in TGF beta 1 induced A549 cells were downregulated by doxycycline treatment. In contrast, E-cadherin expression was upregulated in TGF beta 1 induced A549 cells. An in vitro cell migration assay showed that doxycycline also inhibited the ability of TGF beta 1 induced migration. Doxycycline treatment suppressed the activation of Smad2/3 and p38, whereas its inhibitory effects were similar to each element-specific inhibitor in A549 and primary nasal epithelial cells. Doxycycline inhibited TGF beta 1 induced epithelial-to-mesenchymal transition and migration by targeting Smad2/3 and p38 signal pathways in respiratory epithelial cells.

deltaNp63 Has a Role in Maintaining Epithelial Integrity in Airway Epithelium

PubMed Central

Arason, Ari Jon; Jonsdottir, Hulda R.; Halldorsson, Skarphedinn; Benediktsdottir, Berglind Eva; Bergthorsson, Jon Thor; Ingthorsson, Saevar; Baldursson, Olafur; Sinha, Satrajit; Gudjonsson, Thorarinn; Magnusson, Magnus K.

2014-01-01

The upper airways are lined with a pseudostratified bronchial epithelium that forms a barrier against unwanted substances in breathing air. The transcription factor p63, which is important for stratification of skin epithelium, has been shown to be expressed in basal cells of the lungs and its ΔN isoform is recognized as a key player in squamous cell lung cancer. However, the role of p63 in formation and maintenance of bronchial epithelia is largely unknown. The objective of the current study was to determine the expression pattern of the ΔN and TA isoforms of p63 and the role of p63 in the development and maintenance of pseudostratified lung epithelium in situ and in culture. We used a human bronchial epithelial cell line with basal cell characteristics (VA10) to model bronchial epithelium in an air-liquid interface culture (ALI) and performed a lentiviral-based silencing of p63 to characterize the functional and phenotypic consequences of p63 loss. We demonstrate that ΔNp63 is the major isoform in the human lung and its expression was exclusively found in the basal cells lining the basement membrane of the bronchial epithelium. Knockdown of p63 affected proliferation and migration of VA10 cells and facilitated cellular senescence. Expression of p63 is critical for epithelial repair as demonstrated by wound healing assays. Importantly, generation of pseudostratified VA10 epithelium in the ALI setup depended on p63 expression and goblet cell differentiation, which can be induced by IL-13 stimulation, was abolished by the p63 knockdown. After knockdown of p63 in primary bronchial epithelial cells they did not proliferate and showed marked senescence. We conclude that these results strongly implicate p63 in the formation and maintenance of differentiated pseudostratified bronchial epithelium. PMID:24533135

deltaNp63 has a role in maintaining epithelial integrity in airway epithelium.

PubMed

Arason, Ari Jon; Jonsdottir, Hulda R; Halldorsson, Skarphedinn; Benediktsdottir, Berglind Eva; Bergthorsson, Jon Thor; Ingthorsson, Saevar; Baldursson, Olafur; Sinha, Satrajit; Gudjonsson, Thorarinn; Magnusson, Magnus K

2014-01-01

The upper airways are lined with a pseudostratified bronchial epithelium that forms a barrier against unwanted substances in breathing air. The transcription factor p63, which is important for stratification of skin epithelium, has been shown to be expressed in basal cells of the lungs and its ΔN isoform is recognized as a key player in squamous cell lung cancer. However, the role of p63 in formation and maintenance of bronchial epithelia is largely unknown. The objective of the current study was to determine the expression pattern of the ΔN and TA isoforms of p63 and the role of p63 in the development and maintenance of pseudostratified lung epithelium in situ and in culture. We used a human bronchial epithelial cell line with basal cell characteristics (VA10) to model bronchial epithelium in an air-liquid interface culture (ALI) and performed a lentiviral-based silencing of p63 to characterize the functional and phenotypic consequences of p63 loss. We demonstrate that ΔNp63 is the major isoform in the human lung and its expression was exclusively found in the basal cells lining the basement membrane of the bronchial epithelium. Knockdown of p63 affected proliferation and migration of VA10 cells and facilitated cellular senescence. Expression of p63 is critical for epithelial repair as demonstrated by wound healing assays. Importantly, generation of pseudostratified VA10 epithelium in the ALI setup depended on p63 expression and goblet cell differentiation, which can be induced by IL-13 stimulation, was abolished by the p63 knockdown. After knockdown of p63 in primary bronchial epithelial cells they did not proliferate and showed marked senescence. We conclude that these results strongly implicate p63 in the formation and maintenance of differentiated pseudostratified bronchial epithelium.

FABP4 induces asthmatic airway epithelial barrier dysfunction via ROS-activated FoxM1.

PubMed

Wu, Gaohui; Yang, Liteng; Xu, Yi; Jiang, Xiaohong; Jiang, Xiaomin; Huang, Lisha; Mao, Ling; Cai, Shaoxi

2018-01-01

Functional abnormal airway epithelial cells, along with activated inflammatory cells, resulting in chronic airway inflammation, are considered as the characteristic of asthma. Fatty Acid Binding Protein 4 (FABP4) takes part in glucose and lipid homeostasis, and also have an important role in allergic airway inflammation. However, whether FABP4 influence barrier function of airway epithelial cells is unknown. In vivo, a HDM-induced murine model of asthma was obtained to assessed airway inflammation and protein expression of E-cadherin and Forkhead Box M1 (FoxM1). In vitro, 16-HBE was cultured and was treated with hrFABP4, siFABP4, FABPF4 inhibitor BMS, or FoxM1 inhibitor RCM-1. IL-4, IL-5, and IL-13 level was determined by ELISA. Transepithelial electrical resistance (TER), paracellular permeability and E-cadherin-special immunofluorescence were measured to value airway epithelial barrier function. Intracellular ROS production was determined by DCF-DA fluorescence. FABP4 inhibitor BMS alleviate airway inflammation and destruction of E-cad in allergic mouse. Treatment with HDM or hrFABP4 aggravated inflammatory response, damaged airway epithelial barrier, which could be inhibited by siFABP4 and BMS. Treatment with HDM or hrFABP4 also enhanced levels of FoxM1, and Inhibited FoxM1 suppressed HDM- and hrFABP4-induced inflammation and airway epithelial barrier dysfunction. In addition, H 2 O 2 promoted FoxM1 expression, HDM and hrFABP4 induced-FoxM1 could be inhibited by NAC, leading to decreased inflammation and improved airway epithelial barrier. Upregulated ROS induced by FABP4 was of significance in activating FoxM1 leading to airway inflammation and epithelial barrier dysfunction. Copyright © 2017 Elsevier Inc. All rights reserved.

The status of intercellular junctions in established lens epithelial cell lines

PubMed Central

Dave, Alpana; Craig, Jamie E.

2012-01-01

Purpose Cataract is the major cause of vision-related disability worldwide. Mutations in the crystallin genes are the most common known cause of inherited congenital cataract. Mutations in the genes associated with intercellular contacts, such as Nance-Horan Syndrome (NHS) and Ephrin type A receptor-2 (EPHA2), are other recognized causes of congenital cataract. The EPHA2 gene has been also associated with age-related cataract, suggesting that intercellular junctions are important in not only lens development, but also in maintaining lens transparency. The purpose of this study was to analyze the expression and localization of the key cell junction and cytoskeletal proteins, and of NHS and EPHA2, in established lens epithelial cell lines to determine their suitability as model epithelial systems for the functional investigation of genes involved in intercellular contacts and implicated in cataract. Methods The expression and subcellular localization of occludin and zona occludens protein-1 (ZO-1), which are associated with tight junctions; E-cadherin, which is associated with adherence junctions; and the cytoskeletal actin were analyzed in monolayers of a human lens epithelial cell line (SRA 01/04) and a mouse lens epithelial cell line (αTN4). In addition, the expression and subcellular localization of the NHS and EPHA2 proteins were analyzed in these cell lines. Protein or mRNA expression was respectively determined by western blotting or reverse transcription-polymerase chain reaction (RT–PCR), and localization was determined by immunofluorescence labeling. Results Human SRA 01/04 and mouse αTN4 lens epithelial cells expressed either the proteins of interest or their encoding mRNA. Occludin, ZO-1, and NHS proteins localized to the cellular periphery, whereas E-cadherin, actin, and EPHA2 localized in the cytoplasm in these cell lines. Conclusions The human SRA 01/04 and mouse αTN4 lens epithelial cells express the key junctional proteins. The localization

Murine epithelial cells: isolation and culture.

PubMed

Davidson, Donald J; Gray, Michael A; Kilanowski, Fiona M; Tarran, Robert; Randell, Scott H; Sheppard, David N; Argent, Barry E; Dorin, Julia R

2004-08-01

We describe an air-liquid interface primary culture method for murine tracheal epithelial cells on semi-permeable membranes, forming polarized epithelia with a high transepithelial resistance, differentiation to ciliated and secretory cells, and physiologically appropriate expression of key genes and ion channels. We also describe the isolation of primary murine nasal epithelial cells for patch-clamp analysis, generating polarised cells with physiologically appropriate distribution and ion channel expression. These methods enable more physiologically relevant analysis of murine airway epithelial cells in vitro and ex vivo, better utilisation of transgenic mouse models of human pulmonary diseases, and have been approved by the European Working Group on CFTR expression.

Rho GTPases and Regulation of Cell Migration and Polarization in Human Corneal Epithelial Cells

PubMed Central

Hou, Aihua; Toh, Li Xian; Gan, Kah Hui; Lee, Khee Jin Ryan; Manser, Edward; Tong, Louis

2013-01-01

Purpose Epithelial cell migration is required for regeneration of tissues and can be defective in a number of ocular surface diseases. This study aimed to determine the expression pattern of Rho family small G-proteins in human corneal epithelial cells to test their requirement in directional cell migration. Methods Rho family small G-protein expression was assessed by reverse transcription-polymerase chain reaction. Dominant-inhibitory constructs encoding Rho proteins or Rho protein targeting small interfering RNA were transfected into human corneal epithelial large T antigen cells, and wound closure rate were evaluated by scratch wounding assay, and a complementary non-traumatic cell migration assay. Immunofluorescence staining was performed to study cell polarization and to assess Cdc42 downstream effector. Results Cdc42, Chp, Rac1, RhoA, TC10 and TCL were expressed in human corneal epithelial cells. Among them, Cdc42 and TCL were found to significantly affect cell migration in monolayer scratch assays. These results were confirmed through the use of validated siRNAs directed to Cdc42 and TCL. Scramble siRNA transfected cells had high percentage of polarized cells than Cdc42 or TCL siRNA transfected cells at the wound edge. We showed that the Cdc42-specific effector p21-activated kinase 4 localized predominantly to cell-cell junctions in cell monolayers, but failed to translocate to the leading edge in Cdc42 siRNA transfected cells after monolayer wounding. Conclusion Rho proteins expressed in cultured human corneal epithelial cells, and Cdc42, TCL facilitate two-dimensional cell migration in-vitro. Although silencing of Cdc42 and TCL did not noticeably affect the appearance of cell adhesions at the leading edge, the slower migration of these cells indicates both GTP-binding proteins play important roles in promoting cell movement of human corneal epithelial cells. PMID:24130842

Effect of phoshpodiesterase 4 (PDE4) inhibibtors on eotaxin expression in humen bronchial epithelial cells.

PubMed

Paplinska, M; Chazan, R; Grubek-Jaworska, H

2011-06-01

The increasing number of eosinophils into bronchoaelvolar space is observed during noninfectious inflammatory lung diseases. Eotaxins (eotaxin-1/CCL11, eotaxin-2/CCL24, eotaxin-3/CCL26) are the strongest chemotactic agents for eosinophils. Inhibitors of phosphodiesterase 4 (PDE4), the enzyme decomposing cAMP, are anti-inflammatory agents which act through cAMP elevation and inhibit numerous steps of allergic inflammation. The effect of PDE4 inhibitors on eotaxin expression is not known in details. The aim of our study was to evaluate the influence of PDE4 inhibitors: rolipram and RO-20-1724 on expression of eotaxins in bronchial epithelial cell line BEAS-2B. Cells were preincubated with PDE4 inhibitors or dexamethasone for 1 hour and then stimulated with IL-4 or IL-13 alone or in combination with TNF-α. After 48 hours eotaxin protein level was measured by ELISA and mRNA level by real time PCR. PDE4 inhibitors decreased CCL11 and CCL26 expression only in cultures co-stimulated with TNF-α. In cultures stimulated with IL-4 and TNF-α rolipram and RO-20-1724 diminished CCL11 mRNA expression by 34 and 37%, respectively, and CCL26 by 43 and 47%. In cultures stimulated with IL-13 and TNF-α rolipram and RO-20-1724 decreased expression of both eotaxins by about 50%. These results were confirmed at the protein level. The effect of PDE4 inhibitors on eotaxin expression in BEAS-2B cells, in our experimental conditions, depends on TNF-α contribution.

Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4{sup +} intestinal intraepithelial lymphocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hatano, Ryo; Yamada, Kiyoshi; Iwamoto, Taku

2013-06-14

Highlights: •Small intestinal epithelial cells (sIECs). •sIECs are able to induce antigen specific proliferation of CD4{sup +} IELs. •sIECs induce markedly enhanced IFN-γ secretion by CD4{sup +} IELs. •Induction of enhanced IFN-γ secretion by sIECs is uniquely observed in CD4{sup +} IELs. -- Abstract: Small intestinal epithelial cells (sIECs) express major histocompatibility complex class II molecules even in a normal condition, and are known to function as antigen presenting cells (APCs) at least in vitro. These findings raised the possibility that sIECs play an important role in inducing immune responses against luminal antigens, especially those of intestinal intraepithelial lymphocytes (IELs)more » and lamina propria lymphocytes (LPLs). We herein showed that antigenic stimulation with sIECs induced markedly greater secretion of interferon-gamma (IFN-γ) by CD4{sup +} IELs, but not interleukin (IL)-4, IL-10 and IL-17 although the proliferative response was prominently lower than that with T cell-depleted splenic APCs. In contrast, no enhanced IFN-γ secretion by CD4{sup +} LPLs and primed splenic CD4{sup +} T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4{sup +} IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo.« less

Candida albicans triggers interleukin-8 secretion by oral epithelial cells.

PubMed

Dongari-Bagtzoglou, A; Kashleva, H

2003-04-01

Oropharyngeal candidiasis is a frequent opportunistic infection associated with immunocompromised hosts. Candida albicans is the principal species responsible for this infection. Production of interleukin-8 (IL-8), by oral epithelial cells can be expected to play a major role in the recruitment and activation of professional phagocytes at the infected site. The purpose of this study was to determine whether C. albicans triggers secretion of IL-8 by oral epithelial cells in vitro and investigate mechanisms of host cell-fungal interactions that trigger such responses. Oral epithelial cell lines (SCC4, SCC15, and OKF6/TERT-2) as well as primary gingival epithelial cells were used. Epithelial cells were cocultured with C. albicans, strains SC5314, ATCC28366 or ATCC32077, for 24-48 hr, and supernatants were analyzed for IL-8 content by ELISA. A germination-deficient mutant (efg1/efg1 cph1/cph1), otherwise isogenic to strain SC5314, was used to assess the requirement for germination in triggering IL-8 responses. In order to ascertain whether direct contact of yeast with host cells is required to trigger cytokine production, epithelial cells were separated from yeast using cell culture inserts. To test whether IL-8 secretion is dependent on IL-1alpha activity, epithelial cells were challenged with viable C. albicans in the presence or absence of neutralizing anti-IL-1alpha antibody or IL-1ra, and IL-8 secretion was measured in the supernatants. All cell lines and primary cultures responded to C. albicans with an increase in IL-8 secretion. IL-8 responses were contact-dependent, strain-specific, required yeast viability and germination into hyphae, and were in part autoregulated by IL-1alpha. Copyright 2003 Elsevier Science Ltd.

Intestinal Epithelial Toll-Like Receptor 4 Signaling Affects Epithelial Function and Colonic Microbiota and Promotes a Risk for Transmissible Colitis

PubMed Central

Dheer, Rishu; Santaolalla, Rebeca; Davies, Julie M.; Lang, Jessica K.; Phillips, Matthew C.; Pastorini, Cristhine; Vazquez-Pertejo, Maria T.

2016-01-01

Evidence obtained from gene knockout studies supports the role of Toll-like receptor 4 (TLR4) in intestinal inflammation and microbiota recognition. Increased epithelial TLR4 expression is observed in patients with inflammatory bowel disease. However, little is known of the effect of increased TLR4 signaling on intestinal homeostasis. Here, we examined the effect of increased TLR4 signaling on epithelial function and microbiota by using transgenic villin-TLR4 mice that overexpress TLR4 in the intestinal epithelium. Our results revealed that villin-TLR4 mice are characterized by increases in the density of mucosa-associated bacteria and bacterial translocation. Furthermore, increased epithelial TLR4 signaling was associated with an impaired epithelial barrier, altered expression of antimicrobial peptide genes, and altered epithelial cell differentiation. The composition of the colonic luminal and mucosa-associated microbiota differed between villin-TLR4 and wild-type (WT) littermates. Interestingly, WT mice cohoused with villin-TLR4 mice displayed greater susceptibility to acute colitis than singly housed WT mice did. The results of this study suggest that epithelial TLR4 expression shapes the microbiota and affects the functional properties of the epithelium. The changes in the microbiota induced by increased epithelial TLR4 signaling are transmissible and exacerbate dextran sodium sulfate-induced colitis. Together, our findings imply that host innate immune signaling can modulate intestinal bacteria and ultimately the host's susceptibility to colitis. PMID:26755160

Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis.

PubMed

Krtolica, Ana; Ortiz de Solorzano, Carlos; Lockett, Stephen; Campisi, Judith

2002-10-01

To demonstrate that senescent fibroblasts stimulate the proliferation and neoplastic transformation of premalignant epithelial cells (Krtolica et al.: Proc Natl Acad Sci USA 98:12072-12077, 2001), we developed methods to quantify the proliferation of epithelial cells cocultured with fibroblasts. We stained epithelial-fibroblast cocultures with the fluorescent DNA-intercalating dye 4,6-diamidino-2-phenylindole (DAPI), or expressed green fluorescent protein (GFP) in the epithelial cells, and then cultured them with fibroblasts. The cocultures were photographed under an inverted microscope with appropriate filters, and the fluorescent images were captured with a digital camera. We modified an image analysis program to selectively recognize the smaller, more intensely fluorescent epithelial cell nuclei in DAPI-stained cultures and used the program to quantify areas with DAPI fluorescence generated by epithelial nuclei or GFP fluorescence generated by epithelial cells in each field. Analysis of the image areas with DAPI and GFP fluorescences produced nearly identical quantification of epithelial cells in coculture with fibroblasts. We confirmed these results by manual counting. In addition, GFP labeling permitted kinetic studies of the same coculture over multiple time points. The image analysis-based quantification method we describe here is an easy and reliable way to monitor cells in coculture and should be useful for a variety of cell biological studies. Copyright 2002 Wiley-Liss, Inc.

Growth of Normal Mouse Vaginal Epithelial Cells in and on Collagen Gels

NASA Astrophysics Data System (ADS)

Iguchi, Taisen; Uchima, Francis-Dean A.; Ostrander, Patricia L.; Bern, Howard A.

1983-06-01

Sustained growth in primary culture of vaginal epithelial cells from ovariectomized adult BALB/cCrg1 mice embedded within or seeded on collagen gel matrix was achieved in a serum-free medium composed of Ham's F-12 medium/Dulbecco's modified Eagle's medium, 1:1 (vol/vol), supplemented with insulin, bovine serum albumin fraction V, epidermal growth factor, cholera toxin, and transferrin. Three-dimensional growth of vaginal epithelial cells occurred inside the collagen gel matrix. Cell numbers increased 4- to 8-fold in collagen gel and about 4-fold on collagen gel after 9-10 days in culture. The effect of 17β -estradiol (0.00018-180 nM in gel or 0.018-180 nM on gel) and diethylstilbestrol (DES; 0.0186-186 nM in gel) on the growth of vaginal epithelial cells was examined. The addition of estrogen did not enhance the growth of vaginal epithelial cells during this time period either in the complete medium or in a suboptimal medium. Cultures on floating collagen gels in the serum-free medium are composed of 1-3 cell layers with superficial cornification. Estrogen does not appear to be a direct mitogen for vaginal epithelial cells, at least in this system.

Cell confinement controls centrosome positioning and lumen initiation during epithelial morphogenesis

PubMed Central

Rodríguez-Fraticelli, Alejo E.; Auzan, Muriel; Alonso, Miguel A.; Bornens, Michel

2012-01-01

Epithelial organ morphogenesis involves sequential acquisition of apicobasal polarity by epithelial cells and development of a functional lumen. In vivo, cells perceive signals from components of the extracellular matrix (ECM), such as laminin and collagens, as well as sense physical conditions, such as matrix stiffness and cell confinement. Alteration of the mechanical properties of the ECM has been shown to promote cell migration and invasion in cancer cells, but the effects on epithelial morphogenesis have not been characterized. We analyzed the effects of cell confinement on lumen morphogenesis using a novel, micropatterned, three-dimensional (3D) Madin-Darby canine kidney cell culture method. We show that cell confinement, by controlling cell spreading, limits peripheral actin contractility and promotes centrosome positioning and lumen initiation after the first cell division. In addition, peripheral actin contractility is mediated by master kinase Par-4/LKB1 via the RhoA–Rho kinase–myosin II pathway, and inhibition of this pathway restores lumen initiation in minimally confined cells. We conclude that cell confinement controls nuclear–centrosomal orientation and lumen initiation during 3D epithelial morphogenesis. PMID:22965908

DA-6034 Induces [Ca(2+)]i Increase in Epithelial Cells.

PubMed

Yang, Yu-Mi; Park, Soonhong; Ji, Hyewon; Kim, Tae-Im; Kim, Eung Kweon; Kang, Kyung Koo; Shin, Dong Min

2014-04-01

DA-6034, a eupatilin derivative of flavonoid, has shown potent effects on the protection of gastric mucosa and induced the increases in fluid and glycoprotein secretion in human and rat corneal and conjunctival cells, suggesting that it might be considered as a drug for the treatment of dry eye. However, whether DA-6034 induces Ca(2+) signaling and its underlying mechanism in epithelial cells are not known. In the present study, we investigated the mechanism for actions of DA-6034 in Ca(2+) signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated Ca(2+)-activated Cl(-) channels (CaCCs) and increased intracellular calcium concentrations ([Ca(2+)]i) in primary cultured human conjunctival cells. DA-6034 also increased [Ca(2+)]i in mouse salivary gland cells and human corneal epithelial cells. [Ca(2+)]i increase of DA-6034 was dependent on the Ca(2+) entry from extracellular and Ca(2+) release from internal Ca(2+) stores. Interestingly, these effects of DA-6034 were related to ryanodine receptors (RyRs) but not phospholipase C/inositol 1,4,5-triphosphate (IP3) pathway and lysosomal Ca(2+) stores. These results suggest that DA-6034 induces Ca(2+) signaling via extracellular Ca(2+) entry and RyRs-sensitive Ca(2+) release from internal Ca(2+) stores in epithelial cells.

S100A4 amplifies TGF-β-induced epithelial-mesenchymal transition in a pleural mesothelial cell line.

PubMed

Ning, Qian; Li, Feiyan; Wang, Lei; Li, Hong; Yao, Yan; Hu, Tinghua; Sun, Zhongmin

2018-02-01

Pleural fibrosis can dramatically lower the quality of life. Numerous studies have reported that epithelial-mesenchymal transition (EMT) regulated by transforming growth factor-β (TGF-β) is involved in fibrosis. However, the molecular mechanism is inadequately understood. Fibroblast-specific protein-1 (S100A4) is a target of TGF-β signaling. In our previous study, we have reported that S100A4 is highly expressed in pleural fibrosis. Thus, we suggest that S100A4 took part in the TGF-β-induced EMT in pleural fibrosis. In this study, we determined the expression of S100A4 and EMT-related markers in Met-5A cells (pleural mesothelial cells) treated with TGF-β or TGF-β inhibitor by real-time PCR and western blot. In order to explore the role of S100A4, we used siRNA to knock down the expression of S100A4 in cell model. We found that the expression of epithelial cell marker was decreased and the mesenchymal cell marker increased with S100A4 upregulation after treatment with TGF-β. Moreover, the changes of EMT-related event were restricted when the expression of S100A4 was knocked down. Conversely, S100A4 can partially rescue the EMT-related expression changes induced by TGF-β inhibitor. These findings suggest that S100A4 expression is induced by the TGF-β pathway, and silencing S100A4 expression can inhibit the process of TGF-β-induced EMT. © American Federation for Medical Research (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

The epithelial-mesenchymal transition generates cells with properties of stem cells.

PubMed

Mani, Sendurai A; Guo, Wenjun; Liao, Mai-Jing; Eaton, Elinor Ng; Ayyanan, Ayyakkannu; Zhou, Alicia Y; Brooks, Mary; Reinhard, Ferenc; Zhang, Cheng Cheng; Shipitsin, Michail; Campbell, Lauren L; Polyak, Kornelia; Brisken, Cathrin; Yang, Jing; Weinberg, Robert A

2008-05-16

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We here report that the induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the acquisition of mesenchymal traits and in the expression of stem-cell markers. Furthermore, we show that those cells have an increased ability to form mammospheres, a property associated with mammary epithelial stem cells. Independent of this, stem cell-like cells isolated from HMLE cultures form mammospheres and express markers similar to those of HMLEs that have undergone an EMT. Moreover, stem-like cells isolated either from mouse or human mammary glands or mammary carcinomas express EMT markers. Finally, transformed human mammary epithelial cells that have undergone an EMT form mammospheres, soft agar colonies, and tumors more efficiently. These findings illustrate a direct link between the EMT and the gain of epithelial stem cell properties.

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

Distinct effects of EGFR inhibitors on epithelial- and mesenchymal-like esophageal squamous cell carcinoma cells.

PubMed

Yoshioka, Masahiro; Ohashi, Shinya; Ida, Tomomi; Nakai, Yukie; Kikuchi, Osamu; Amanuma, Yusuke; Matsubara, Junichi; Yamada, Atsushi; Miyamoto, Shin'ichi; Natsuizaka, Mitsuteru; Nakagawa, Hiroshi; Chiba, Tsutomu; Seno, Hiroshi; Muto, Manabu

2017-08-01

Epidermal growth factor receptor (EGFR) plays a pivotal role in the pathophysiology of esophageal squamous cell carcinoma (ESCC). However, the clinical effects of EGFR inhibitors on ESCC are controversial. This study sought to identify the factors determining the therapeutic efficacy of EGFR inhibitors in ESCC cells. Immortalized-human esophageal epithelial cells (EPC2-hTERT), transformed-human esophageal epithelial cells (T-Epi and T-Mes), and ESCC cells (TE-1, TE-5, TE-8, TE-11, TE-11R, and HCE4) were treated with the EGFR inhibitors erlotinib or cetuximab. Inhibitory effects on cell growth were assessed by cell counting or cell-cycle analysis. The expression levels of genes and proteins such as involucrin and cytokeratin13 (a squamous differentiation marker), E-cadherin, and vimentin were evaluated by real-time polymerase chain reaction or western blotting. To examine whether mesenchymal phenotype influenced the effects of EGFR inhibitors, we treated T-Epi cells with TGF-β1 to establish a mesenchymal phenotype (mesenchymal T-Epi cells). We then compared the effects of EGFR inhibitors on parental T-Epi cells and mesenchymal T-Epi cells. TE-8 (mesenchymal-like ESCC cells)- or TE-11R (epithelial-like ESCC cells)-derived xenograft tumors in mice were treated with cetuximab, and the antitumor effects of EGFR inhibitors were evaluated. Cells were classified as epithelial-like or mesenchymal-like phenotypes, determined by the expression levels of E-cadherin and vimentin. Both erlotinib and cetuximab reduced cell growth and the ratio of cells in cell-cycle S phase in epithelial-like but not mesenchymal-like cells. Additionally, EGFR inhibitors induced squamous cell differentiation (defined as increased expression of involucrin and cytokeratin13) in epithelial-like but not mesenchymal-like cells. We found that EGFR inhibitors did not suppress the phosphorylation of EGFR in mesenchymal-like cells, while EGFR dephosphorylation was observed after treatment with EGFR

β2 adrenergic agonist suppresses eosinophil-induced epithelial-to-mesenchymal transition of bronchial epithelial cells.

PubMed

Kainuma, Keigo; Kobayashi, Tetsu; D'Alessandro-Gabazza, Corina N; Toda, Masaaki; Yasuma, Taro; Nishihama, Kota; Fujimoto, Hajime; Kuwabara, Yu; Hosoki, Koa; Nagao, Mizuho; Fujisawa, Takao; Gabazza, Esteban C

2017-05-02

Epithelial-mesenchymal transition is currently recognized as an important mechanism for the increased number of myofibroblasts in cancer and fibrotic diseases. We have already reported that epithelial-mesenchymal transition is involved in airway remodeling induced by eosinophils. Procaterol is a selective and full β 2 adrenergic agonist that is used as a rescue of asthmatic attack inhaler form and orally as a controller. In this study, we evaluated whether procaterol can suppress epithelial-mesenchymal transition of airway epithelial cells induced by eosinophils. Epithelial-mesenchymal transition was assessed using a co-culture system of human bronchial epithelial cells and primary human eosinophils or an eosinophilic leukemia cell line. Procaterol significantly inhibited co-culture associated morphological changes of bronchial epithelial cells, decreased the expression of vimentin, and increased the expression of E-cadherin compared to control. Butoxamine, a specific β 2 -adrenergic antagonist, significantly blocked changes induced by procaterol. In addition, procaterol inhibited the expression of adhesion molecules induced during the interaction between eosinophils and bronchial epithelial cells, suggesting the involvement of adhesion molecules in the process of epithelial-mesenchymal transition. Forskolin, a cyclic adenosine monophosphate-promoting agent, exhibits similar inhibitory activity of procaterol. Overall, these observations support the beneficial effect of procaterol on airway remodeling frequently associated with chronic obstructive pulmonary diseases.

Rebamipide increases the mucin-like glycoprotein production in corneal epithelial cells.

PubMed

Takeji, Yasuhiro; Urashima, Hiroki; Aoki, Akihiro; Shinohara, Hisashi

2012-06-01

Dry eye is a multifactorial disease of tears and the ocular surface due to tear deficiency or excessive tear evaporation. Tear film instability is due to a disturbance in ocular surface mucin leading to a dysfunction of mucin, resulting in dry eye. In this study, we examined the effect of rebamipide, an anti-ulcer agent, on glycoconjugate production, as an indicator of mucin-like glycoprotein in cultured corneal epithelial cells. Further, we investigated the effect of rebamipide on the gene expression of membrane-associated mucins. Confluent cultured human corneal epithelial cells were incubated with rebamipide for 24 h. The glycoconjugate content in the supernatant and the cell extracts was measured by wheat germ agglutinin-enzyme-linked lectin assay combined gel-filtration method. In the experiment on mucin gene expression, cultured human corneal epithelial cells were collected at 0, 3, 6, and 12 h after administration of rebamipide. Real-time quantitative polymerase chain reaction was used to analyze the quantity of MUC1, MUC 4, and MUC16 gene expression. Rebamipide significantly increased the glycoconjugate contents in the supernatant and cell extract. In the mucin gene expression in the cells, rebamipide increased MUC1 and MUC4 gene expression, but did not increase MUC16 gene expression. Rebamipide promoted glycoconjugate, which has a property as a mucin-like glycoprotein, in human corneal epithelial cells. The increased production was mediated by MUC1 and MUC4 gene expression.

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.

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

Migration of guinea pig airway epithelial cells in response to bombesin analogues.

PubMed

Kim, J S; McKinnis, V S; White, S R

1997-03-01

Bombesin-like peptides within neuroepithelial cells elicit proliferation of normal and malignant airway epithelial cells. It is not clear that these peptides also elicit epithelial cell migration, a necessary component of airway repair after injury. We studied the effects of the bombesin analogues, gastrin releasing peptide (GRP) and neuromedin B (NMB), on guinea pig tracheal epithelial cell (GPTEC) migration. Primary GPTEC were allowed to migrate through 8-microm-pore gelatin-coated filters for 6 h in a chemotaxis chamber, after which the number of migrated cells per 10 high power fields (10 hpf) were counted. Both neuropeptides elicited migration of GPTEC: 24.8 +/- 4.5 cells for 10(-11) M NMB (P < 0.001 versus control, n = 4) and 16.8 +/- 1.2 cells for 10(-12) M GRP (P < 0.001 versus control, n = 8). Migration was attenuated substantially by a bombesin receptor antagonist. To investigate further the relationship of migration through a filter to the repair of a damaged epithelium, we studied the repair of epithelial cells by video microscopy. A 0.3- to 0.5-microm2 wound was created in a confluent monolayer of GPTEC, and wound closure was followed over 24 h. There was no significant acceleration in the rate of repair of GRP- or NMB-stimulated monolayers compared to control. These data demonstrate that GRP and NMB elicit migration of airway epithelial cells but may not play a significant role in the early repair of the airway epithelium in culture.

Invasion of Human Oral Epithelial Cells by Prevotella intermedia

PubMed Central

Dorn, Brian R.; Leung, K.-P.; Progulske-Fox, Ann

1998-01-01

Invasion of oral epithelial cells by pathogenic oral bacteria may represent an important virulence factor in the progression of periodontal disease. Here we report that a clinical isolate of Prevotella intermedia, strain 17, was found to invade a human oral epithelial cell line (KB), whereas P. intermedia 27, another clinical isolate, and P. intermedia 25611, the type strain, were not found to invade the cell line. Invasion was quantified by the recovery of viable bacteria following a standard antibiotic protection assay and observed by electron microscopy. Cytochalasin D, cycloheximide, monodansylcadaverine, and low temperature (4°C) inhibited the internalization of P. intermedia 17. Antibodies raised against P. intermedia type C fimbriae and against whole cells inhibited invasion, but the anti-type-C-fimbria antibody inhibited invasion to a greater extent than the anti-whole-cell antibody. This work provides evidence that at least one strain of P. intermedia can invade an oral epithelial cell line and that the type C fimbriae and a cytoskeletal rearrangement are required for this invasion. PMID:9826397

Role of human epididymis protein 4 in chemoresistance and prognosis of epithelial ovarian cancer.

PubMed

Lee, Seungho; Choi, Seowon; Lee, Yookyung; Chung, Donghae; Hong, Suntaek; Park, Nohhyun

2017-01-01

Human epididymis protein 4 (HE4) is a novel biomarker for epithelial ovarian cancer. This study was designed to evaluate the role of HE4 in chemo-response against anti-cancer drugs and prognosis of epithelial ovarian cancer. HE4-depleted cells and HE4-overexpressing cells were generated. The effect of HE4 gene silencing and overexpression was examined using a cell viability assay after exposure to chemotherapeutic agents and the signaling pathway. We studied the expression of HE4 in ovarian cancer tissue and the prognostic significance. Cytoplasmic staining was graded for intensity and percentage of positive cells. The grades were multiplied to determine an H-score. Knockdown of HE4 in OVCAR-3 cells resulted in reduction in cell growth and increased sensitivity to paclitaxel and cisplatin compared to control cells. This effect originated from the decreased activation of cell-growth-related signaling, such as AKT and Erk mediated by epidermal growth factor (EGF), while overexpression of HE4 resulted in enhanced cell growth and suppressed the anti-tumorigenic activity of paclitaxel. Activation of AKT and Erk pathways was enhanced in HE4-overexpressing cells compared to control cells. Based on the results of multivariate analysis, the risk of death was significantly higher in patients with an H-score > 4. HE4 induces chemoresistance against anti-cancer drugs and activates the AKT and Erk pathways to enhance tumor survival. HE4 expression in ovarian cancer tissue is associated with a worse prognosis for epithelial ovarian cancer patients. © 2016 Japan Society of Obstetrics and Gynecology.

MIG-6 negatively regulates STAT3 phosphorylation in uterine epithelial cells

PubMed Central

Yoo, Jung-Yoon; Yang, Woo Sub; Lee, Jae Hee; Kim, Byung Gak; Broaddus, Russell R.; Lim, Jeong M.; Kim, Tae Hoon; Jeong, Jae-Wook

2017-01-01

Endometrial cancer is the most common malignancy of the female genital tract. Progesterone (P4) has been used for several decades in endometrial cancer treatment, especially in women who wish to retain fertility. However, it is unpredictable which patients will respond to P4 treatment and which may have a P4 resistant cancer. Therefore, identifying the mechanism of P4 resistance is essential to improve the therapies for endometrial cancer. Mitogen-inducible gene 6 (Mig-6) is a critical mediator of progesterone receptor (PGR) action in the uterus. In order to study the function of Mig-6 in P4 resistance, we generated a mouse model in which we specifically ablated Mig-6 in uterine epithelial cells using Sprr2f-cre mice (Sprr2fcre+Mig-6f/f). Female mutant mice develop endometrial hyperplasia due to aberrant phosphorylation of STAT3 and proliferation of the endometrial epithelial cells. The results from our immunoprecipitation and cell culture experiments showed that MIG-6 inhibited phosphorylation of STAT3 via protein interactions. Our previous study showed P4 resistance in mice with Mig-6 ablation in Pgr positive cells (Pgrcre/+Mig-6f/f). However, Sprr2fcre+Mig-6f/f mice were P4 responsive. P4 treatment significantly decreased STAT3 phosphorylation and epithelial proliferation in the uterus of mutant mice. We showed that Mig-6 has an important function of tumor suppressor via inhibition of STAT3 phosphorylation in uterine epithelial cells and the anti-tumor effects of P4 are mediated by the endometrial stroma. This data helps to develop a new signaling pathway in the regulation of steroid hormones in the uterus, and to overcome P4 resistance in human reproductive diseases, such as endometrial cancer. PMID:28925396

Myb permits multilineage airway epithelial cell differentiation

PubMed Central

Pan, Jie-hong; Adair-Kirk, Tracy L.; Patel, Anand C.; Huang, Tao; Yozamp, Nicholas S.; Xu, Jian; Reddy, E. Premkumar; Byers, Derek E.; Pierce, Richard A.; Holtzman, Michael J.; Brody, Steven L.

2014-01-01

The epithelium of the pulmonary airway is specially differentiated to provide defense against environmental insults, but also subject to dysregulated differentiation that results in lung disease. The current paradigm for airway epithelial differentiation is a one-step program whereby a p63+ basal epithelial progenitor cell generates a ciliated or secretory cell lineage, but the cue for this transition and whether there are intermediate steps is poorly defined. Here we identify transcription factor Myb as a key regulator that permits early multilineage differentiation of airway epithelial cells. Myb+ cells were identified as p63− and therefore distinct from basal progenitor cells, but were still negative for markers of differentiation. Myb RNAi treatment of primary-culture airway epithelial cells and Myb gene deletion in mice resulted in a p63− population with failed maturation of Foxj1+ ciliated cells, as well as Scbg1a1+ and Muc5ac+ secretory cells. Consistent with these findings, analysis of whole genome expression of Myb-deficient cells identified Myb-dependent programs for ciliated and secretory cell differentiation. Myb+ cells were rare in human airways but were increased in regions of ciliated cells and mucous cell hyperplasia in samples from subjects with chronic obstructive pulmonary disease. Together, the results show that a p63− Myb+ population of airway epithelial cells represents a distinct intermediate stage of differentiation that is required under normal conditions and may be heightened in airway disease. PMID:25103188

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

Establishment and characterization of three immortal bovine muscular epithelial cell lines.

PubMed

Jin, Xun; Lee, Joong-Seob; Kwak, Sungwook; Lee, Soo-Yeon; Jung, Ji-Eun; Kim, Tae-Kyung; Xu, Chenxiong; Hong, Zhongshan; Li, Zhehu; Kim, Sun-Myung; Pian, Xumin; Lee, Dong-Hee; Yoon, Jong-Taek; You, Seungkwon; Choi, Yun-Jaie; Kim, Huunggee

2006-02-28

We have established three immortal bovine muscular epithelial (BME) cell lines, one spontaneously immortalized (BMES), the second SV40LT-mediated (BMEV) and the third hTERT-mediated (BMET). The morphology of the three immortal cell lines was similar to that of early passage primary BME cells. Each of the immortal cell lines made cytokeratin, a typical epithelial marker. BMET grew faster than the other immortal lines and the BME cells, in 10% FBS-DMEM medium, whereas neither the primary cells nor the three immortal cell lines grew in 0.5% FBS-DMEM. The primary BME cells and the immortal cell lines, with the exception of BMES, made increasing amounts of p53 protein when treated with doxorubicin, a DNA damaging agent. On the other hand, almost half of the cells in populations of the three immortal cell lines may lack p16(INK4a) regulatory function, compared to primary BME cells that were growth arrested by enforced expression of p16(INK4a). In soft-agar assays, the primary cells and immortal cell lines proved to be less transformed in phenotype than HeLa cells. The three immortal epithelial-type cell lines reported here are the first cell lines established from muscle tissue of bovine or other species.

Epithelial Cells in Urine: MedlinePlus Lab Test Information

MedlinePlus

... page: https://medlineplus.gov/labtests/epithelialcellsinurine.html Epithelial Cells in Urine To use the sharing features on ... page, please enable JavaScript. What is an Epithelial Cells in Urine Test? Epithelial cells are a type ...

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. © 2016 International Federation for Cell Biology.

Silk Film Topography Directs Collective Epithelial Cell Migration

PubMed Central

Rosenblatt, Mark I.

2012-01-01

The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization. PMID:23185573

Slit2 ameliorates renal inflammation and fibrosis after hypoxia-and lipopolysaccharide-induced epithelial cells injury in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Xiangjun; Yao, Qisheng, E-mail: yymcyqs@126.com; Sun, Xinbo

Hypoxic acute kidney injury (AKI) is often incompletely repaired and leads to chronic kidney disease (CKD), which is characterized by tubulointerstitial inflammation and fibrosis. The Slit2 family of secreted glycoproteins is expressed in the kidney, it has been shown to exert an anti-inflammatory activity and prevent ischemic renal injury in vivo. However, whether Slit2 reduces renal fibrosis and inflammation after hypoxic and inflammatory epithelial cells injury in vitro remains unknown. In this study, we aimed to evaluate whether Slit2 ameliorated fibrosis and inflammation in two renal epithelial cells line challenged with hypoxia and lipopolysaccharide (LPS). Renal epithelial cells were treatedmore » with hypoxia and LPS to induce cell injury. Hoechst staining and Western blot analysis was conducted to examine epithelial cells injury. Immunofluorescence staining and Western blot analysis was performed to evaluate tubulointerstitial fibrosis. Real-time polymerase chain reaction (PCR) tested the inflammatory factor interleukin (IL)−1β and tumor necrosis factor (TNF)-α, and Western blot analysis determined the hypoxia-inducible factor (HIF)−1α, Toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB. Results revealed that hypoxia induced epithelial cells apoptosis, inflammatory factor IL-1β and TNF-α release and tubulointerstitial fibrosis. LPS could exacerbate hypoxia -induced epithelial cells apoptosis, IL-1β and TNF-α release and fibrosis. Slit2 reduced the expression of fibronectin, the rate of epithelial cell apoptosis, and the expression of inflammatory factor. Slit2 could also inhibit the expression of TLR4 and NF-κB, but not the expression of HIF-1α. Therefore, Slit2 attenuated inflammation and fibrosis after LPS- and hypoxia-induced epithelial cells injury via the TLR4/NF-κB signaling pathway, but not depending on the HIF-1α signaling pathway. - Highlights: • Slit2 ameliorates inflammation after hypoxia-and LPS-induced epithelial cells

Estradiol Increases Mucus Synthesis in Bronchial Epithelial Cells

PubMed Central

Tam, Anthony; Wadsworth, Samuel; Dorscheid, Delbert; Man, Shu-Fan Paul; Sin, Don D.

2014-01-01

Airway epithelial mucus hypersecretion and mucus plugging are prominent pathologic features of chronic inflammatory conditions of the airway (e.g. asthma and cystic fibrosis) and in most of these conditions, women have worse prognosis compared with male patients. We thus investigated the effects of estradiol on mucus expression in primary normal human bronchial epithelial cells from female donors grown at an air liquid interface (ALI). Treatment with estradiol in physiological ranges for 2 weeks caused a concentration-dependent increase in the number of PAS-positive cells (confirmed to be goblet cells by MUC5AC immunostaining) in ALI cultures, and this action was attenuated by estrogen receptor beta (ER-β) antagonist. Protein microarray data showed that nuclear factor of activated T-cell (NFAT) in the nuclear fraction of NHBE cells was increased with estradiol treatment. Estradiol increased NFATc1 mRNA and protein in ALI cultures. In a human airway epithelial (1HAE0) cell line, NFATc1 was required for the regulation of MUC5AC mRNA and protein. Estradiol also induced post-translational modification of mucins by increasing total fucose residues and fucosyltransferase (FUT-4, -5, -6) mRNA expression. Together, these data indicate a novel mechanism by which estradiol increases mucus synthesis in the human bronchial epithelium. PMID:24964096

Evidence from a Mouse Model That Epithelial Cell Migration and Mesenchymal-Epithelial Transition Contribute to Rapid Restoration of Uterine Tissue Integrity during Menstruation

PubMed Central

Cousins, Fiona L.; Murray, Alison; Esnal, Arantza; Gibson, Douglas A.; Critchley, Hilary O. D.; Saunders, Philippa T. K.

2014-01-01

Background In women dynamic changes in uterine tissue architecture occur during each menstrual cycle. Menses, characterised by the shedding of the upper functional layer of the endometrium, is the culmination of a cascade of irreversible changes in tissue function including stromal decidualisation, inflammation and production of degradative enzymes. The molecular mechanisms that contribute to the rapid restoration of tissue homeostasis at time of menses are poorly understood. Methodology A modified mouse model of menses was developed to focus on the events occurring within the uterine lining during endometrial shedding/repair. Decidualisation, vaginal bleeding, tissue architecture and cell proliferation were evaluated at 4, 8, 12, and 24 hours after progesterone (P4) withdrawal; mice received a single injection of bromodeoxyuridine (BrdU) 90 mins before culling. Expression of genes implicated in the regulation of mesenchymal to epithelial transition (MET) was determined using a RT2 PCR profiler array, qRTPCR and bioinformatic analysis. Principal Findings Mice exhibited vaginal bleeding between 4 and 12 hours after P4 withdrawal, concomitant with detachment of the decidualised cell mass from the basal portion of the endometrial lining. Immunostaining for BrdU and pan cytokeratin revealed evidence of epithelial cell proliferation and migration. Cells that appeared to be in transition from a mesenchymal to an epithelial cell identity were identified within the stromal compartment. Analysis of mRNAs encoding genes expressed exclusively in the epithelial or stromal compartments, or implicated in MET, revealed dynamic changes in expression, consistent with a role for reprogramming of mesenchymal cells so that they could contribute to re-epithelialisation. Conclusions/Significance These studies have provided novel insights into the cellular processes that contribute to re-epithelialisation post-menses implicating both epithelial cell migration and mesenchymal cell

Evidence from a mouse model that epithelial cell migration and mesenchymal-epithelial transition contribute to rapid restoration of uterine tissue integrity during menstruation.

PubMed

Cousins, Fiona L; Murray, Alison; Esnal, Arantza; Gibson, Douglas A; Critchley, Hilary O D; Saunders, Philippa T K

2014-01-01

In women dynamic changes in uterine tissue architecture occur during each menstrual cycle. Menses, characterised by the shedding of the upper functional layer of the endometrium, is the culmination of a cascade of irreversible changes in tissue function including stromal decidualisation, inflammation and production of degradative enzymes. The molecular mechanisms that contribute to the rapid restoration of tissue homeostasis at time of menses are poorly understood. A modified mouse model of menses was developed to focus on the events occurring within the uterine lining during endometrial shedding/repair. Decidualisation, vaginal bleeding, tissue architecture and cell proliferation were evaluated at 4, 8, 12, and 24 hours after progesterone (P4) withdrawal; mice received a single injection of bromodeoxyuridine (BrdU) 90 mins before culling. Expression of genes implicated in the regulation of mesenchymal to epithelial transition (MET) was determined using a RT2 PCR profiler array, qRTPCR and bioinformatic analysis. Mice exhibited vaginal bleeding between 4 and 12 hours after P4 withdrawal, concomitant with detachment of the decidualised cell mass from the basal portion of the endometrial lining. Immunostaining for BrdU and pan cytokeratin revealed evidence of epithelial cell proliferation and migration. Cells that appeared to be in transition from a mesenchymal to an epithelial cell identity were identified within the stromal compartment. Analysis of mRNAs encoding genes expressed exclusively in the epithelial or stromal compartments, or implicated in MET, revealed dynamic changes in expression, consistent with a role for reprogramming of mesenchymal cells so that they could contribute to re-epithelialisation. These studies have provided novel insights into the cellular processes that contribute to re-epithelialisation post-menses implicating both epithelial cell migration and mesenchymal cell differentiation in restoration of an intact epithelial cell layer. These

A novel closed cell culture device for fabrication of corneal epithelial cell sheets.

PubMed

Nakajima, Ryota; Kobayashi, Toyoshige; Moriya, Noboru; Mizutani, Manabu; Kan, Kazutoshi; Nozaki, Takayuki; Saitoh, Kazuo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

2015-11-01

Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets. Copyright © 2012 John Wiley & Sons, Ltd.

Fatty acid-binding protein 4 (FABP4) and FABP5 modulate cytokine production in the mouse thymic epithelial cells.

PubMed

Adachi, Yasuhiro; Hiramatsu, Sumie; Tokuda, Nobuko; Sharifi, Kazem; Ebrahimi, Majid; Islam, Ariful; Kagawa, Yoshiteru; Koshy Vaidyan, Linda; Sawada, Tomoo; Hamano, Kimikazu; Owada, Yuji

2012-09-01

Thymic stromal cells, including cortical thymic epithelial cells (cTEC) produce many humoral factors, such as cytokines and eicosanoids to modulate thymocyte homeostasis, thereby regulating the peripheral immune responses. In this study, we identified fatty acid-binding protein (FABP4), an intracellular fatty acid chaperone, in the mouse thymus, and examined its role in the control of cytokine production in comparison with FABP5. By immunofluorescent staining, FABP4(+) cells enclosing the thymocytes were scattered throughout the thymic cortex with a spatial difference from the FABP5(+) cell that were distributed widely throughout the cTEC. The FABP4(+) cells were immunopositive for MHC class II, NLDC145 and cytokeratin 8, and were identified as part of cTEC. The FABP4(+) cells were identified as thymic nurse cells (TNC), a subpopulation of cTEC, by their active phagocytosis of apoptotic thymocytes. Furthermore, FABP4 expression was confirmed in the isolated TNC at the gene and protein levels. To explore the function of FABP in TNC, TSt-4/DLL1 cells stably expressing either FABP4 or FABP5 were established and the gene expressions of various cytokines were examined. The gene expression of interleukin (IL)-7 and IL-18 was increased both in FABP4 and FABP5 over-expressing cells compared with controls, and moreover, the increase in their expressions by adding of stearic acids was significantly enhanced in the FABP4 over-expressing cells. These data suggest that both FABPs are involved in the maintenance of T lymphocyte homeostasis through the modulation of cytokine production, which is possibly regulated by cellular fatty acid-mediated signaling in TEC, including TNC.

Characterization of a continuous feline mammary epithelial cell line susceptible to feline epitheliotropic viruses.

PubMed

Pesavento, Patricia; Liu, Hongwei; Ossiboff, Robert J; Stucker, Karla M; Heymer, Anna; Millon, Lee; Wood, Jason; van der List, Deborah; Parker, John S L

2009-04-01

Mucosal epithelial cells are the primary targets for many common viral pathogens of cats. Viral infection of epithelia can damage or disrupt the epithelial barrier that protects underlying tissues. In vitro cell culture systems are an effective means to study how viruses infect and disrupt epithelial barriers, however no true continuous or immortalized feline epithelial cell culture lines are available. A continuous cell culture of feline mammary epithelial cells (FMEC UCD-04-2) that forms tight junctions with high transepithelial electrical resistance (>2000Omegacm(-1)) 3-4 days after reaching confluence was characterized. In addition, it was shown that FMECs are susceptible to infection with feline calicivirus (FCV), feline herpesvirus (FHV-1), feline coronavirus (FeCoV), and feline panleukopenia virus (FPV). These cells will be useful for studies of feline viral disease and for in vitro studies of feline epithelia.

[BIOCOMPATIBILITY OF POLY-LACTIDE-CO-GLYCOLIDE/COLLAGEN TYPE I SCAFFOLD WITH RAT VAGINAL EPITHELIAL CELLS].

PubMed

Li, Yachai; Huang, Xianghua; Zhang, Mingle; Li, Yanan; Chen, Yexing; Jia, Jingfei

2015-09-01

To explore the biocompatibility of the poly-lactide-co-glycolide (PLGA)/collagen type I scaffold with rat vaginal epithelial cells, and the feasibility of using PLGA/collagen type I as scaffold to reconstruct vagina by the tissue engineering. PLGA/collagen type I scaffold was prepared with PLGA covered polylysine and collagen type I. The vaginal epithelial cells of Sprague Dawley rat of 10-12 weeks old were cultured by enzyme digestion method. The vaginal epithelial cells of passage 2 were cultured in the leaching liquor of scaffold for 48 hours to detect its cytotoxicity by MTT. The vaginal epithelial cells were inoculated on the PLGA/collagen type I scaffold (experimental group) and PLGA scaffold (control group) to calculate the cell adhesion rate. Epithelial cells-scaffold complexes were implanted subcutaneously on the rat back. At 2, 4, and 8 weeks after implantation, the epithelial cells-scaffold complexes were harvested to observe the cell growth by HE staining and immunohistochemical analysis. The epithelial cells-scaffold complexes were transplanted to reconstruct vagina in 6 rats with vaginal defect. After 3 and 6 months, the vaginal length was measured and the appearance was observed. The neovagina tissues were harvested for histological evaluation after 6 months. The epithelial cells grew and proliferated well in the leaching liquor of PLGA/collagen type I scaffold, and the cytotoxicity was at grade 1. The cell adhesion rate on the PLGA/collagen type I scaffold was 71.8%±9.2%, which significantly higher than that on the PLGA scaffold (63.4%±5.7%) (t=2.195, P=0.005). The epithelial cells could grow and adhere to the PLGA/collagen type I scaffolds. At 2 weeks after implanted subcutaneously, the epithelial cells grew and proliferated in the pores of scaffolds, and the fibroblasts were observed. At 4 weeks, 1-3 layers epithelium formed on the surface of scaffold. At 8 weeks, the epithelial cells increased and arranged regularly, which formed the membrane

Epithelial-to-mesenchymal transition in penile squamous cell carcinoma.

PubMed

Masferrer, Emili; Ferrándiz-Pulido, Carla; Masferrer-Niubò, Magalí; Rodríguez-Rodríguez, Alfredo; Gil, Inmaculada; Pont, Antoni; Servitje, Octavi; García de Herreros, Antonio; Lloveras, Belen; García-Patos, Vicenç; Pujol, Ramon M; Toll, Agustí; Hernández-Muñoz, Inmaculada

2015-02-01

Epithelial-to-mesenchymal transition is a phenomenon in epithelial tumors that involves loss of intercellular adhesion, mesenchymal phenotype acquisition and enhanced migratory potential. While the epithelial-to-mesenchymal transition process has been extensively linked to metastatic progression of squamous cell carcinoma, studies of the role of epithelial-to-mesenchymal transition in squamous cell carcinoma containing high risk human papillomaviruses are scarce. Moreover, to our knowledge epithelial-to-mesenchymal transition involvement in human penile squamous cell carcinoma, which can arise through transforming HPV infections or independently of HPV, has not been investigated. We evaluated the presence of epithelial-to-mesenchymal transition markers and their relationship to HPV in penile squamous cell carcinoma. We assessed the expression of E-cadherin, vimentin and the epithelial-to-mesenchymal transition related transcription factors Twist, Zeb1 and Snail by immunohistochemical staining in 64 penile squamous cell carcinoma cases. HPV was detected by polymerase chain reaction amplification. Simultaneous loss of membranous E-cadherin expression and vimentin over expression were noted in 43.5% of penile squamous cell carcinoma cases. HPV was significantly associated with loss of membranous E-cadherin but not with epithelial-to-mesenchymal transition. Recurrence and mortality rates were significantly higher in cases showing epithelial-to-mesenchymal transition. Our findings indicate that in penile squamous cell carcinoma epithelial-to-mesenchymal transition is associated with poor prognosis but not with the presence of HPV. Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Epithelial cells as alternative human biomatrices for comet assay.

PubMed

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.

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

Aggregatibacter actinomycetemcomitans regulates the expression of integrins and reduces cell adhesion via integrin α5 in human gingival epithelial cells.

PubMed

Kochi, Shinsuke; Yamashiro, Keisuke; Hongo, Shoichi; Yamamoto, Tadashi; Ugawa, Yuki; Shimoe, Masayuki; Kawamura, Mari; Hirata-Yoshihara, Chiaki; Ideguchi, Hidetaka; Maeda, Hiroshi; Takashiba, Shogo

2017-12-01

Gingival epithelial cells form a physiological barrier against bacterial invasion. Excessive bacterial invasion destroys the attachment between the tooth surface and the epithelium, resulting in periodontitis. Integrins play a significant role in cell attachment; therefore, we hypothesized that bacterial infection might decrease the expressions of these integrins in gingival epithelial cells, resulting in reduced cell adhesion. Immortalized human gingival epithelial cells were co-cultured with Aggregatibacter actinomycetemcomitans Y4 (Aa Y4), and the gene expression levels of IL-8, proliferating cell nuclear antigen (PCNA), and integrins (α2, α3, α5, β4, and β6) were measured using quantitative reverse transcription polymerase chain reaction. Expression of PCNA and integrins, except integrin α5, was significantly downregulated, while expression of IL-8 and integrin α5 was significantly upregulated in the cells co-cultured with Aa Y4. The number of adherent cells significantly decreased when co-cultured with Aa Y4, as determined using cell adhesion assays. In the cells co-cultured with Aa Y4 and an integrin α5 neutralizing antibody, there was no effect on the expression of IL-8 and PCNA, while the expressions of integrins α2, α3, β4, and β6, and the number of adherent cells did not decrease. The number of invading bacteria in the cells was reduced in the presence of the antibody and increased in the presence of TLR2/4 inhibitor. Therefore, integrin α5 might be involved in Aa Y4 invasion into gingival epithelial cells, and the resulting signal transduction cascade reduces cell adhesion by decreasing the expression of integrins, while the TLR2/4 signaling cascade regulates IL-8 expression.

Establishment of immortal swine kidney epithelial cells.

PubMed

Kwak, Sungwook; Jung, Ji-Eun; Jin, Xun; Kim, Sun-Myung; Kim, Tae-Kyung; Lee, Joong-Seob; Lee, Soo-Yeon; Pian, Xumin; You, Seungkwon; Kim, Hyunggee; Choi, Yun-Jaie

2006-01-01

Using normal swine kidney epithelial (SKE) cells that were shown to be senescent at passages 12 to 14, we have established one lifespan-extended cell line and two lifespan-extended cell lines by exogenous introduction of the human catalytic subunit of telomerase (hTERT) and simian virus 40 large T-antigen (SV40LT), all of which maintain epithelial morphology and express cytokeratin, a marker of epithelial cells. SV40LT- and hTERT-transduced immortal cell lines appeared to be smaller and exhibited more uniform morphology relative to primary and spontaneously immortalized SKE cells. We determined the in vitro lifespan of primary SKE cells using a standard 3T6 protocol. There were two steps of the proliferation barrier at 12 and 20, in which a majority of primary SKE cells appeared enlarged, flattened, vacuolated, and ss-galactosidase-positive, all phenotypical characteristics of senescent cells. Lifespan-extended SKE cells were eventually established from most of the cellular foci, which is indicative of spontaneous cellular conversion at passage 23. Beyond passage 25, the rate of population doubling of the established cells gradually increased. At passage 30, immortal cell lines grew faster than primary counterpart cells in 10% FBS-DMEM culture conditions, and only SV40LT-transduced immortal cells grew faster than primary and other SKE immortal cells in 0.5% FBS-DMEM. These lifespan-extended SKE cell lines failed to grow in an anchorage-independent manner in soft-agar dishes. Hence, three immortalized swine kidney epithelial cells that are not transformed would be valuable biological tools for virus propagation and basic kidney epithelial cell research.

Lactobacillus acidophilus contributes to a healthy environment for vaginal epithelial cells.

PubMed

Pi, Woojin; Ryu, Jae-Sook; Roh, Jaesook

2011-09-01

Lactobacillus species in the female genital tract are thought to act as a barrier to infection. Several studies have demonstrated that lactobacilli can adhere to vaginal epithelial cells. However, little is known about how the adherence of lactobacilli to vaginal epithelial cells affects the acidity, cell viability, or proliferation of the lactobacilli themselves or those of vaginal epithelial cells. Lactobacillus acidophilus was co-cultured with immortalized human vaginal epithelial cells (MS74 cell line), and the growth of L. acidophilus and the acidity of the culture medium were measured. MS74 cell density and viability were also assessed by counting cell numbers and observing the cell attachment state. L. acidophilus showed exponential growth for the first 6 hr until 9 hr, and the pH was maintained close to 4.0-5.0 at 24 hr after culture, consistent with previous studies. The growth curve of L. acidophilus or the pH values were relatively unaffected by co-culture with MS74 cells, confirming that L. acidophilus maintains a low pH in the presence of MS74 cells. This co-culture model could therefore potentially be used to mimic vaginal conditions for future in vitro studies. On the other hand, MS74 cells co-cultured with L. acidophilus more firmly attached to the culture plate, and a higher number of cells were present compared to cells cultured in the absence of L. acidophilus. These results indicate that L. acidophilus increases MS74 cell proliferation and viability, suggesting that lactobacilli may contribute to the healthy environment for vaginal epithelial cells.

Human Urinary Epithelial Cells as a Source of Engraftable Hepatocyte-Like Cells Using Stem Cell Technology.

PubMed

Sauer, Vanessa; Tchaikovskaya, Tatyana; Wang, Xia; Li, Yanfeng; Zhang, Wei; Tar, Krisztina; Polgar, Zsuzsanna; Ding, Jianqiang; Guha, Chandan; Fox, Ira J; Roy-Chowdhury, Namita; Roy-Chowdhury, Jayanta

2016-12-13

Although several types of somatic cells have been reprogrammed into induced pluripotent stem cells (iPSCs) and then differentiated to hepatocyte-like cells (iHeps), the method for generating such cells from renal tubular epithelial cells shed in human urine and transplanting them into animal livers has not been described systematically. We report reprogramming of human urinary epithelial cells into iPSCs and subsequent hepatic differentiation, followed by a detailed characterization of the newly generated iHeps. The epithelial cells were reprogrammed into iPSCs by delivering the pluripotency factors OCT3/4, SOX2, KLF4, and MYC using methods that do not involve transgene integration, such as nucleofection of episomal (oriP/EBNA-1) plasmids or infection with recombinant Sendai viruses. After characterization of stable iPSC lines, a three-step differentiation toward hepatocytes was performed. The iHeps expressed a large number of hepatocyte-preferred genes, including nuclear receptors that regulate genes involved in cholesterol homeostasis, bile acid transport, and detoxification. MicroRNA profile of the iHeps largely paralleled that of primary human hepatocytes. The iHeps engrafted into the livers of Scid mice transgenic for mutant human SERPINA1 after intrasplenic injection. Thus, urine is a readily available source for generating human iHeps that could be potentially useful for disease modeling, pharmacological development, and regenerative medicine.

Oligosaccharide receptor mimics inhibit Legionella pneumophila attachment to human respiratory epithelial cells.

PubMed

Thomas, Richard J; Brooks, Tim J

2004-02-01

Legionnaire's disease is caused by the intracellular pathogen Legionella pneumophila, presenting as an acute pneumonia. Attachment is the key step during infection, often relying on an interaction between host cell oligosaccharides and bacterial adhesins. Inhibition of this interaction by receptor mimics offers possible novel therapeutic treatments. L. pneumophila attachment to the A549 cell line was significantly reduced by treatment with tunicamycin (73.6%) and sodium metaperiodate (63.7%). This indicates the importance of cell surface oligosaccharide chains in adhesion. A number of putative anti-adhesion compounds inhibited attachment to the A549 and U937 cell lines. The most inhibitory compounds were polymeric saccharides, GalNAcbeta1-4Gal, Galbeta1-4GlcNAc and para-nitrophenol. These compounds inhibited adhesion to a range of human respiratory cell lines, including nasal epithelial, bronchial epithelial and alveolar epithelial cell lines and the human monocytic cell line, U937. Some eukaryotic receptors for L. pneumophila were determined to be the glycolipids, asialo-GM1 and asialo-GM2 that contain the inhibitory saccharide moiety, GalNAcbeta1-4Gal. The identified compounds have the potential to be used as novel treatments for Legionnaire's disease.

Characterization of newly established bovine intestinal epithelial cell line.

PubMed

Miyazawa, Kohtaro; Hondo, Tetsuya; Kanaya, Takashi; Tanaka, Sachi; Takakura, Ikuro; Itani, Wataru; Rose, Michael T; Kitazawa, Haruki; Yamaguchi, Takahiro; Aso, Hisashi

2010-01-01

Membranous epithelial cells (M cells) of the follicle-associated epithelium in Peyer's patches have a high capacity for transcytosis of several viruses and microorganisms. Here, we report that we have successfully established a bovine intestinal epithelial cell line (BIE cells) and developed an in vitro M cell model. BIE cells have a cobblestone morphology and microvilli-like structures, and strongly express cell-to-cell junctional proteins and cytokeratin, which is a specific intermediate filament protein of epithelial cells. After co-culture with murine intestinal lymphocytes or treatment with supernatant from bovine PBMC cultured with IL-2, BIE cells acquired the ability of transcytosis. Therefore, BIE cells have typical characteristics of bovine intestinal epithelial cells and also have the ability to differentiate into an M cell like linage. In addition, our results indicate that contact between immune cells and epithelial cells may not be absolutely required for the differentiation of M cells. We think that BIE cells will be useful for studying the transport mechanisms of various pathogens and also the evaluation of drug delivery via M cells.

The bovine endometrial epithelial cells promote the differentiation of trophoblast stem-like cells to binucleate trophoblast cells.

PubMed

Li, Xiawei; Li, Zhiying; Hou, Dongxia; Zhao, Yuhang; Wang, Chen; Li, Xueling

2016-12-01

Endometrial epithelial cells (EECs) cultured in vitro are valuable tools for investigating embryo implantation and trophoblast differentiation. In this study, we have established the bovine EECs and trophoblast stem-like (TS) coculture system, and used it to investigate the binucleate cell formation of ungulates. The EECs was derived from the uterine horn ipsilateral to the corpus luteum by using collagenase I and deoxyribonuclease I, which exhibited typical epithelial morphology and were expressing bovine uterine epithelial marker such as IFNAR1, IFNAR2, Erα, PGR, ESR1 and KRT18. The cells immunostained positively by epithelial and trophectoderm marker cytokeratin 18 (KRT18) and stromal marker vimentin antibodies, and the KRT18 positive cells reached 99 %. The EECs can be cultured for up to 20 passages in vitro with no significant morphology changes and uterine epithelial marker gene expression alteration. The bTS cells were established in a dual inhibitor system and exhibited typical trophoblast stem cell characteristics. When bTS cells were cultured with EECs, the bTS cells adhered to the EECs as adhering to feeder cells. Binucleate cells began appearing on day 4 of coculture and reached approximately 18.47 % of the differentiated cells. Quantitative real-time PCR or immunofluorescence analyses were performed on bTS cells cocultured at day 6 and day 12. The results showed that the expression level of KRT18 was down-regulated while the expression level of trophoblast differentiation marker MASH2, HAND1, GCM1 and CDX2 was up-regulated in bTS cells. In conclusion, bovine EECs can be obtained from the uterine horn ipsilateral to the corpus luteum via treatment with collagenase I and deoxyribonuclease I, and the EECs-bTS cells coculture system presents an ideal tool for studying the differentiation of bTS cells to trophoblast binucleate cells.

Characterization of immortalized human mammary epithelial cell line HMEC 2.6.

PubMed

Joshi, Pooja S; Modur, Vishnu; Cheng, JiMing; Robinson, Kathy; Rao, Krishna

2017-10-01

Primary human mammary epithelial cells have a limited life span which makes it difficult to study them in vitro for most purposes. To overcome this problem, we have developed a cell line that was immortalized using defined genetic elements, and we have characterized this immortalized non-tumorigenic human mammary epithelial cell line to establish it as a potential model system. human mammary epithelial cells were obtained from a healthy individual undergoing reduction mammoplasty at SIU School of Medicine. The cells were transduced with CDK4R24C followed by transduction with human telomerase reverse transcriptase. Post all manipulation, the cells displayed a normal cell cycle phase distribution and were near diploid in nature, which was confirmed by flow cytometry and karyotyping. In vitro studies showed that the cells were anchorage dependent and were non-invasive in nature. The cell line expressed basal epithelial markers such as cytokeratin 7, CD10, and p63 and was negative for the expression of estrogen receptor and progesterone receptor. Upon G-band karyotyping, the cell line displayed the presence of a few cytogenic abnormalities, including trisomy 20 and trisomy 7, which are also commonly present in other immortalized mammary cell lines. Furthermore, the benign nature of these cells was confirmed by multiple in vitro and in vivo experiments. Therefore, we think that this cell line could serve as a good model to understand the molecular mechanisms involved in the development and progression of breast cancer and to also assess the effect of novel therapeutics on human mammary epithelial cells.

CTCF-Mediated and Pax6-Associated Gene Expression in Corneal Epithelial Cell-Specific Differentiation

PubMed Central

Tsui, Shanli; Wang, Jie; Wang, Ling; Dai, Wei; Lu, Luo

2016-01-01

Background The purpose of the study is to elicit the epigenetic mechanism involving CCCTC binding factor (CTCF)-mediated chromatin remodeling that regulates PAX6 gene interaction with differentiation-associated genes to control corneal epithelial differentiation. Methods Cell cycle progression and specific keratin expressions were measured to monitor changes of differentiation-induced primary human limbal stem/progenitor (HLS/P), human corneal epithelial (HCE) and human telomerase-immortalized corneal epithelial (HTCE) cells. PAX6-interactive and differentiation-associated genes in chromatin remodeling mediated by the epigenetic factor CTCF were detected by circular chromosome conformation capture (4C) and ChIP (Chromatin immunoprecipitation)-on-chip approaches, and verified by FISH (Fluorescent in situ hybridization). Furthermore, CTCF activities were altered by CTCF-shRNA to study the effect of CTCF on mediating interaction of Pax6 and differentiation-associated genes in corneal epithelial cell fate. Results Our results demonstrated that differentiation-induced human corneal epithelial cells expressed typical corneal epithelial characteristics including morphological changes, increased keratin12 expression and G0/G1 accumulations. Expressions of CTCF and PAX6 were suppressed and elevated following the process of differentiation, respectively. During corneal epithelial cell differentiation, differentiation-induced RCN1 and ADAM17 were found interacting with PAX6 in the process of CTCF-mediated chromatin remodeling detected by 4C and verified by ChIP-on-chip and FISH. Diminished CTCF mRNA with CTCF-shRNA in HTCE cells weakened the interaction of PAX6 gene in controlling RCN1/ADAM17 and enhanced early onset of the genes in cell differentiation. Conclusion Our results explain how epigenetic factor CTCF-mediated chromatin remodeling regulates interactions between eye-specific PAX6 and those genes that are induced/associated with cell differentiation to modulate

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

Differential Alteration in Intestinal Epithelial Cell Expression of Toll-Like Receptor 3 (TLR3) and TLR4 in Inflammatory Bowel Disease

PubMed Central

Cario, Elke; Podolsky, Daniel K.

2000-01-01

Initiation and perpetuation of the inflammatory intestinal responses in inflammatory bowel disease (IBD) may result from an exaggerated host defense reaction of the intestinal epithelium to endogenous lumenal bacterial flora. Intestinal epithelial cell lines constitutively express several functional Toll-like receptors (TLRs) which appear to be key regulators of the innate response system. The aim of this study was to characterize the expression pattern of TLR2, TLR3, TLR4, and TLR5 in primary intestinal epithelial cells from patients with IBD. Small intestinal and colonic biopsy specimens were collected from patients with IBD (Crohn's disease [CD], ulcerative colitis [UC]) and controls. Non-IBD specimens were assessed by immunofluorescence histochemistry using polyclonal antibodies specific for TLR2, TLR3, TLR4, and TLR5. Primary intestinal epithelial cells (IEC) of normal mucosa constitutively expressed TLR3 and TLR5, while TLR2 and TLR4 were only barely detectable. In active IBD, the expression of TLR3 and TLR4 was differentially modulated in the intestinal epithelium. TLR3 was significantly downregulated in IEC in active CD but not in UC. In contrast, TLR4 was strongly upregulated in both UC and CD. TLR2 and TLR5 expression remained unchanged in IBD. These data suggest that IBD may be associated with distinctive changes in selective TLR expression in the intestinal epithelium, implying that alterations in the innate response system may contribute to the pathogenesis of these disorders. PMID:11083826

Trans, trans-2,4-decadienal induced cell proliferation via p27 pathway in human bronchial epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Y.-C.; Lin Pinpin

2008-04-01

Lung cancer is the leading cause of cancer deaths worldwide. Epidemiological studies have shown that exposure to cooking oil fumes (COF) is a risk factor for lung cancer. Trans, trans-2,4-decadienal (tt-DDE), a dienaldehyde, is abundant in heated oils and COF. Previously, we found that long-term exposure (45 days) to a sub-lethal dose (1 {mu}M) of tt-DDE significantly increased growth of human bronchial epithelial cells (BEAS-2B). Aims of this study are to understand the mechanism of tt-DDE-induced cell proliferation and possible protective effects of antioxidant, vitamin C and N-acetylcysteine (NAC) in BEAS-2B cells. Utilizing the real-time RT-PCR and Western immunoblotting, wemore » found that p27 mRNA and protein levels were significantly increased by 1 {mu}M tt-DDE treatment. Co-treatment with vitamin C or NAC partially prevented tt-DDE-induced cell proliferation. In addition, the downstream targets of p27, including CDK4, cyclin D{sub 1} and phosphorylated-Rb proteins, increased in 1 {mu}M tt-DDE-treated cells and these changes were prevented by NAC co-treatment. Therefore, these results suggest that tt-DDE increased cell proliferation via inhibition of p27 expression, increase in CDK4/cyclin D{sub 1} protein accumulation and enhancement of Rb phosphorylation. Increased cell proliferation is considered as the early stages of lung carcinogenesis. Administration of antioxidants may prevent COF-associated lung carcinogenesis.« less

Down-regulation of Transducin-Like Enhancer of Split protein 4 in hepatocellular carcinoma promotes cell proliferation and epithelial-Mesenchymal-Transition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Xiao-cai; Xiao, Cui-cui; Li, Hua

Background: Transducin-Like Enhancer of Split protein 4 (TLE4) has been reported to be involved in some subsets of acute myeloid leukemia and colorectal cancer. In the present study, we aimed to explore the role of TLE4 in tumorigenesis and cancer progression in hepatocellular carcinoma (HCC). Methods: The expression pattern of TLE4 in HCC was determined by Western-blot and qRT-PCR, gain-of-function and loss-of-function was used to explore the biological role of TLE4 in HCC cells. A xenograft model was established to confirm its effects on proliferation. Results: The protein expression levels of TLE4 were significantly down-regulated in HCC tissues compared tomore » matched adjacent normal liver tissues. In vitro, down-regulation of TLE4 in Huh7 or SMMC-7721 promoted cell proliferation and ectopical expression of TLE4 in Hep3B or Bel-7404 suppressed cell proliferation. In addition, the cell colony formation ability was enhanced after down-regulation of TLE4 expression in Huh-7 but suppressed after over-expression in Hep3B. Furthermore, down-regulation of TLE4 increased the cell invasion ability, as well as increased the expression level of Vimentin and decreased that of E-cadherin, indicating a phenotype of epithelial-mesenchymal transition (EMT) in HCC cells. On the contrary, ectopical expression of TLE4 in HCC cells decreased the cell invasion ability and inhibited EMT. In vivo, compared to control group, xenograft tumor volumes were significantly decreased in TLE4 overexpression group. Conclusions: These results demonstrated that TLE4 might play important regulatory roles in cellular proliferation and EMT process in HCC. - Highlights: • TLE4 is significantly down-regulated in HCC samples. • Down regulated of TLE4 in HCC cells promotes cell proliferation. • Down regulated of TLE4 in HCC cells promotes epithelial-to-mesenchymal transition.« less

Profiling human breast epithelial cells using single cell RNA sequencing identifies cell diversity.

PubMed

Nguyen, Quy H; Pervolarakis, Nicholas; Blake, Kerrigan; Ma, Dennis; Davis, Ryan Tevia; James, Nathan; Phung, Anh T; Willey, Elizabeth; Kumar, Raj; Jabart, Eric; Driver, Ian; Rock, Jason; Goga, Andrei; Khan, Seema A; Lawson, Devon A; Werb, Zena; Kessenbrock, Kai

2018-05-23

Breast cancer arises from breast epithelial cells that acquire genetic alterations leading to subsequent loss of tissue homeostasis. Several distinct epithelial subpopulations have been proposed, but complete understanding of the spectrum of heterogeneity and differentiation hierarchy in the human breast remains elusive. Here, we use single-cell mRNA sequencing (scRNAseq) to profile the transcriptomes of 25,790 primary human breast epithelial cells isolated from reduction mammoplasties of seven individuals. Unbiased clustering analysis reveals the existence of three distinct epithelial cell populations, one basal and two luminal cell types, which we identify as secretory L1- and hormone-responsive L2-type cells. Pseudotemporal reconstruction of differentiation trajectories produces one continuous lineage hierarchy that closely connects the basal lineage to the two differentiated luminal branches. Our comprehensive cell atlas provides insights into the cellular blueprint of the human breast epithelium and will form the foundation to understand how the system goes awry during breast cancer.

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...

[Primary culture of human normal epithelial cells].

PubMed

Tang, Yu; Xu, Wenji; Guo, Wanbei; Xie, Ming; Fang, Huilong; Chen, Chen; Zhou, Jun

2017-11-28

The traditional primary culture methods of human normal epithelial cells have disadvantages of low activity of cultured cells, the low cultivated rate and complicated operation. To solve these problems, researchers made many studies on culture process of human normal primary epithelial cell. In this paper, we mainly introduce some methods used in separation and purification of human normal epithelial cells, such as tissue separation method, enzyme digestion separation method, mechanical brushing method, red blood cell lysis method, percoll layered medium density gradient separation method. We also review some methods used in the culture and subculture, including serum-free medium combined with low mass fraction serum culture method, mouse tail collagen coating method, and glass culture bottle combined with plastic culture dish culture method. The biological characteristics of human normal epithelial cells, the methods of immunocytochemical staining, trypan blue exclusion are described. Moreover, the factors affecting the aseptic operation, the conditions of the extracellular environment, the conditions of the extracellular environment during culture, the number of differential adhesion, and the selection and dosage of additives are summarized.

Characterization of cultivated murine lacrimal gland epithelial cells

PubMed Central

Kobayashi, Shinya; Kawashima, Motoko; Okada, Naoko; Mishima, Kenji; Saito, Ichiro; Ito, Masataka; Shimmura, Shigeto; Tsubota, Kazuo

2012-01-01

Purpose To date, mouse lacrimal gland epithelial cells have been cultured successfully but only in cases involving newborn mouse lacrimal glands. In this work, we attempted to cultivate and characterize adult mouse lacrimal gland epithelial cells. Methods Lacrimal glands were removed from newborn mice (C57B/6) and isolated lacrimal gland epithelial cells were seeded onto tissue culture treated or low adherent culture dishes in Cnt-07 culture medium with or without cholera toxin. Cultivated cells were characterized by immunostaining with pan-cytokeratin, α-smooth muscle actin, and lactoferrin antibodies. Lacrimal gland cells from 7-week-old green fluorescent protein (GFP) and non-GFP (C57B/6) mice were mixed and seeded onto uncoated dishes to assess sphere-forming efficiency. Cells were also seeded onto 3T3 cell feeder layers to assess colony forming efficiency. Results Lacrimal gland epithelial cells were selectively cultured with cholera toxin, and cell type was verified by pan-cytokeratin and α-smooth muscle actin immunostaining. Sphere formation from single cells of adult mice was observed using specific medium and low adherent culture dishes. These cells could also undergo colony formation on 3T3 feeder cells. Conclusions Adult mouse lacrimal gland epithelial cells were successfully cultivated in cholera toxin-containing medium, and were observed to form spheres from single cells. PMID:22665974

Overexpression of the Ubiquilin-4 (UBQLN4) is Associated with Cell Cycle Arrest and Apoptosis in Human Normal Gastric Epithelial Cell Lines GES-1 Cells by Activation of the ERK Signaling Pathway

PubMed Central

Huang, Shengkai; Dong, Xin; Wang, Jia; Ding, Jie; Li, Yan; Li, Dongdong; Lin, Hong; Wang, Wenjie; Zhao, Mei

2018-01-01

Background Ubiquilin-4 (UBQLN4) is a component of the ubiquitin-proteasome system and regulates the degradation of many proteins implicated in pathological conditions. The aim of this study was to determine the role of UBQLN4 in regulating the proliferation and survival of the normal gastric epithelial cell line GES-1. Material/Methods We constructed GES-1 lines stably overexpressing UBQLN4 by lentiviral infection. Cell proliferation, apoptosis, and the cell cycle were analyzed using the MTT assay and flow cytometric assays. Phosphorylation of ERK, JNK, p38, and expression of cyclin D1 were detected by western blot analysis. Results Overexpression of UBQLN4 significantly reduced proliferation and induced G2/M phase arrest and apoptosis in GES-1 cells. Moreover, upregulation of UBQLN4 increased the expression of cyclin D1 and phosphorylated ERK, but not JNK or p38. Conclusions These data suggest that UBQLN4 may induce cell cycle arrest and apoptosis via activation of the ERK pathway and upregulation of cyclin D1 in GES-1 cells. PMID:29807370

Dragon enhances BMP signaling and increases transepithelial resistance in kidney epithelial cells.

PubMed

Xia, Yin; Babitt, Jodie L; Bouley, Richard; Zhang, Ying; Da Silva, Nicolas; Chen, Shanzhuo; Zhuang, Zhenjie; Samad, Tarek A; Brenner, Gary J; Anderson, Jennifer L; Hong, Charles C; Schneyer, Alan L; Brown, Dennis; Lin, Herbert Y

2010-04-01

The neuronal adhesion protein Dragon acts as a bone morphogenetic protein (BMP) coreceptor that enhances BMP signaling. Given the importance of BMP signaling in nephrogenesis and its putative role in the response to injury in the adult kidney, we studied the localization and function of Dragon in the kidney. We observed that Dragon localized predominantly to the apical surfaces of tubular epithelial cells in the thick ascending limbs, distal convoluted tubules, and collecting ducts of mice. Dragon expression was weak in the proximal tubules and glomeruli. In mouse inner medullary collecting duct (mIMCD3) cells, Dragon generated BMP signals in a ligand-dependent manner, and BMP4 is the predominant endogenous ligand for the Dragon coreceptor. In mIMCD3 cells, BMP4 normally signaled through BMPRII, but Dragon enhanced its signaling through the BMP type II receptor ActRIIA. Dragon and BMP4 increased transepithelial resistance (TER) through the Smad1/5/8 pathway. In epithelial cells isolated from the proximal tubule and intercalated cells of collecting ducts, we observed coexpression of ActRIIA, Dragon, and BMP4 but not BMPRII. Taken together, these results suggest that Dragon may enhance BMP signaling in renal tubular epithelial cells and maintain normal renal physiology.

Amphiregulin suppresses epithelial cell apoptosis in lipopolysaccharide-induced lung injury in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ogata-Suetsugu, Saiko; Yanagihara, Toyoshi; Hamada, Naoki

Background and objective: As a member of the epidermal growth factor family, amphiregulin contributes to the regulation of cell proliferation. Amphiregulin was reported to be upregulated in damaged lung tissues in patients with chronic obstructive pulmonary disease and asthma and in lung epithelial cells in a ventilator-associated lung injury model. In this study, we investigated the effect of amphiregulin on lipopolysaccharide (LPS)-induced acute lung injury in mice. Methods: Acute lung injury was induced by intranasal instillation of LPS in female C57BL/6 mice, and the mice were given intraperitoneal injections of recombinant amphiregulin or phosphate-buffered saline 6 and 0.5 h before andmore » 3 h after LPS instillation. The effect of amphiregulin on apoptosis and apoptotic pathways in a murine lung alveolar type II epithelial cell line (LA-4 cells) were examined using flow cytometry and western blotting, respectively. Results: Recombinant amphiregulin suppressed epithelial cell apoptosis in LPS-induced lung injury in mice. Western blotting revealed that amphiregulin suppressed epithelial cell apoptosis by inhibiting caspase-8 activity. Conclusion: Amphiregulin signaling may be a therapeutic target for LPS-induced lung injury treatment through its prevention of epithelial cell apoptosis. - Highlights: • Amphiregulin suppresses epithelial cell apoptosis in LPS-induced lung injury in mice. • The mechanism relies on inhibiting caspase-8 activity. • Amphiregulin signaling may be a therapeutic target for LPS-induced lung injury.« less

Cell death at the intestinal epithelial front line.

PubMed

Delgado, Maria Eugenia; Grabinger, Thomas; Brunner, Thomas

2016-07-01

The intestinal epithelium represents the largest epithelial surface in our body. This single-cell-layer epithelium mediates important functions in the absorption of nutrients and in the maintenance of barrier function, preventing luminal microorganisms from invading the body. Due to its constant regeneration the intestinal epithelium is a tissue not only with very high proliferation rates but also with very prominent physiological and pathophysiological cell death induction. The normal physiological differentiation and maturation of intestinal epithelial cells leads to their shedding and apoptotic cell death within a few days, without disturbing the epithelial barrier integrity. In contrast excessive intestinal epithelial cell death induced by irradiation, drugs and inflammation severely impairs the vital functions of this tissue. In this review we discuss cell death processes in the intestinal epithelium in health and disease, with special emphasis on cell death triggered by the tumour necrosis factor receptor family. © 2015 FEBS.

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. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Epithelial stem cells and intestinal cancer.

PubMed

Tan, Shawna; Barker, Nick

2015-06-01

The mammalian intestine is comprised of an epithelial layer that serves multiple functions in order to maintain digestive activity as well as intestinal homeostasis. This epithelial layer contains highly proliferative stem cells which facilitate its characteristic rapid regeneration. How these stem cells contribute to tissue repair and normal homeostasis are actively studied, and while we have a greater understanding of the molecular mechanisms and cellular locations that underlie stem cell regulation in this tissue, much still remains undiscovered. This review describes epithelial stem cells in both intestinal and non-intestinal tissues, as well as the strategies that have been used to further characterize the cells. Through a discussion of the current understanding of intestinal self-renewal and tissue regeneration in response to injury, we focus on how dysregulation of critical signaling pathways results in potentially oncogenic aberrations, and highlight issues that should be addressed in order for effective intestinal cancer therapies to be devised. Copyright © 2014 Elsevier Ltd. All rights reserved.

Expression of a fms-related oncogene in carcinogen-induced neoplastic epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walker, C.; Nettesheim, P.; Barrett, J.C.

1987-04-01

Following carcinogen exposure in vitro, normal rat tracheal epithelial cells are transformed in a multistage process in which the cultured cells become immortal and ultimately, neoplastic. Five cell lines derived from tumors produced by neoplastically transformed rat tracheal epithelial cells were examined for the expression of 11 cellular oncogenes previously implicated in pulmonary or epithelial carcinogenesis. RNA homologous to fms was expressed at a level 5-19 times higher than normal tracheal epithelial cells in three of five of the tumor-derived lines. All three lines expressing high levels of fms-related RNA gave rise to invasive tumors of epithelial origin when injectedmore » into nude mice. Increased expression of the fms-related mRNA was not due to gene amplification, and no gene rearrangement was detected by Southern analyses. RNA blot analysis using a 3' v-fms probe detected a 9.5-kilobase message in the three tumor-derived lines, whereas both normal rat aveolar macrophages and the human choriocarcinoma line BeWo expressed a fms transcript of approx. = 4 kilobases. The authors conclude from these data that the gene expressed as a 9.5-kilobase transcript in these neoplastic epithelial cells is a member of a fms-related gene family but may be distinct from the gene that encodes the macrophage colony-stimulating factor (CSF-1) receptor.« less

Helicobacter pylori induces vascular endothelial growth factor production in gastric epithelial cells through hypoxia-inducible factor-1α-dependent pathway.

PubMed

Kang, Min-Jung; Song, Eun-Jung; Kim, Bo-Yeon; Kim, Dong-Jae; Park, Jong-Hwan

2014-12-01

Although Helicobacter pylori have been known to induce vascular endothelial growth factor (VEGF) production in gastric epithelial cells, the precise mechanism for cellular signaling is incompletely understood. In this study, we investigated the role of bacterial virulence factor and host cellular signaling in VEGF production of H. pylori-infected gastric epithelial cells. We evaluated production of VEGF, activation of nuclear factor nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinases (MAPKs) and hypoxia-inducible factor-1α (HIF-1α) stabilization in gastric epithelial cells infected with H. pylori WT or isogenic mutants deficient in type IV secretion system (T4SS). H. pylori induced VEGF production in gastric epithelial cells via both T4SS-dependent and T4SS-independent pathways, although T4SS-independent pathway seems to be the dominant signaling. The inhibitor assay implicated that activation of NF-κB and MAPKs is dispensable for H. pylori-induced VEGF production in gastric epithelial cells. H. pylori led to HIF-1α stabilization in gastric epithelial cells independently of T4SS, NF-κB, and MAPKs, which was essential for VEGF production in these cells. N-acetyl-cysteine (NAC), a reactive oxygen species (ROS) inhibitor, treatment impaired H. pylori-induced HIF-1α stabilization and VEGF production in gastric epithelial cells. We defined the important role of ROS-HIF-1α axis in VEGF production of H. pylori-infected gastric epithelial cells, and bacterial T4SS has a minor role in H. pylori-induced VEGF production of gastric epithelial cells. © 2014 John Wiley & Sons Ltd.

Cytokeratin 5 positive cells represent a therapy resistant subpopulation in epithelial ovarian cancer

PubMed Central

Corr, Bradley R.; Finlay-Schultz, Jessica; Rosen, Rachel B.; Qamar, Lubna; Post, Miriam D.; Behbakht, Kian; Spillman, Monique A.; Sartorius, Carol A.

2015-01-01

Objective Cytokeratin 5 (CK5) is an epithelial cell marker implicated in stem and progenitor cell activity in glandular reproductive tissues and endocrine and chemotherapy resistance in estrogen receptor (ER)+ breast cancer. The goal of this study was to determine the prevalence of CK5 expression in ovarian cancer and the response of CK5+ cell populations to cisplatin therapy. Materials and Methods CK5 expression was evaluated in two ovarian tissue microarrays, representing 137 neoplasms, and six ovarian cancer cell lines. Cell lines were treated with IC50 cisplatin and the prevalence of CK5+ cells pre- and post-treatment determined. Proliferation of CK5+ vs. CK5− cell populations was determined using bromodeoxyuridine (BrdU) incorporation. Chemotherapy induced apoptosis in CK5+ vs. CK5− cells was measured using immunohistochemical staining for cleaved caspase-3. Results CK5 was expressed in 39.3% (42/107) of epithelial ovarian cancers with a range of 1-80% positive cells. Serous and endometrioid histologic subtypes had the highest percentage of CK5+ specimens. CK5 expression correlated with ER positivity (38/42 CK5+ tumors were also ER+). CK5 was expressed in 5/6 overall and 4/4 ER+ epithelial ovarian cancer cell lines ranging from 2.4-52.7% positive cells. CK5+ compared to CK5− cells were slower proliferating. The prevalence of CK5+ cells increased following 48 hour cisplatin treatment in 4/5 cell lines tested. CK5+ compared to CK5− ovarian cancer cells were more resistant to cisplatin induced apoptosis. Conclusions CK5 is expressed in a significant proportion of epithelial ovarian cancers and represents a slower proliferating, chemoresistant subpopulation that may warrant co-targeting in combination therapy. PMID:26495758

Functional MUC4 suppress epithelial-mesenchymal transition in lung adenocarcinoma metastasis.

PubMed

Gao, Liuwei; Liu, Jun; Zhang, Bin; Zhang, Hua; Wang, Daowei; Zhang, Tiemei; Liu, Yang; Wang, Changli

2014-02-01

The mucin MUC4 is a high molecular weight membrane-bound transmembrane glycoprotein that is frequently detected in invasive and metastatic cancer. The overexpression of MUC4 is associated with increased risks for several types of cancer. However, the functional role of MUC4 is poorly understood in lung adenocarcinoma. Using antisense-MUC4-RNA transfected adenocarcinoma cells, we discovered that the loss of MUC4 expression results in epithelial-mesenchymal transition (EMT). We found morphological alterations and the repression of the epithelial marker E-cadherin in transfected cells. Additionally, the loss of MUC4 caused the upregulation of the mesenchymal marker vimentin compared to control cells. Using a MUC4-knockdown versus control LTEP xenograft mice model (129/sv mice), we also found that EMT happened in lung tissues of MUC4-knockdown-LTEP xenograft mice. Moreover, antisense-MUC4-RNA transfected cells had a significantly increased cellular migration ability in vitro. The loss of MUC4 also occurred in lung adenocarcinoma patients with lymph node metastases. We further investigated MUC4 and found that it plays a critical role in regulating EMT by modulating β-catenin. Taken together, our study reveals a novel role for MUC4 in suppressing EMT and suggests that the assessment of MUC4 may function as a prognostic biomarker and could be a potential therapeutic target for lung adenocarcinoma metastasis.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malizia, Andrea P.; Lacey, Noreen; Walls, Dermot

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-canonicalmore » 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.« less

Anti-inflammatory effects of embelin in A549 cells and human asthmatic airway epithelial tissues.

PubMed

Lee, In-Seung; Cho, Dong-Hyuk; Kim, Ki-Suk; Kim, Kang-Hoon; Park, Jiyoung; Kim, Yumi; Jung, Ji Hoon; Kim, Kwanil; Jung, Hee-Jae; Jang, Hyeung-Jin

2018-02-01

Allergic asthma is the most common type in asthma, which is defined as a chronic inflammatory disease of the lung. In this study, we investigated whether embelin (Emb), the major component of Ardisia japonica BL. (AJB), exhibits anti-inflammatory effects on allergic asthma via inhibition of NF-κB activity using A549 cells and asthmatic airway epithelial tissues. Inflammation was induced in A549 cells, a human airway epithelial cell line, by IL-1β (10 ng/ml) treatment for 4 h. The effects of Emb on NF-κB activity and COX-2 protein expression in inflamed airway epithelial cells and human asthmatic airway epithelial tissues were analyzed via western blot. The secretion levels of NF-κB-mediated cytokines/chemokines, including IL-4, 6, 9, 13, TNF-α and eotaxin, were measured by a multiplex assay. Emb significantly blocked NF-κB activity in IL-1β-treated A549 cells and human asthmatic airway epithelial tissues. COX-2 expression was also reduced in both IL-1β-treated A549 cells and asthmatic tissues Emb application. Emb significantly reduced the secretion of IL-4, IL-6 and eotaxin in human asthmatic airway epithelial tissues by inhibiting activity of NF-κB. The results of this study suggest that Emb may be used as an anti-inflammatory agent via inhibition of NF-κB and related cytokines.

Telomerase-immortalized non-malignant human prostate epithelial cells retain the properties of multipotent stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Hongzhen; Zhou Jianjun; Miki, Jun

2008-01-01

Understanding prostate stem cells may provide insight into the origin of prostate cancer. Primary cells have been cultured from human prostate tissue but they usually survive only 15-20 population doublings before undergoing senescence. We report here that RC-170N/h/clone 7 cells, a clonal cell line from hTERT-immortalized primary non-malignant tissue-derived human prostate epithelial cell line (RC170N/h), retain multipotent stem cell properties. The RC-170N/h/clone 7 cells expressed a human embryonic stem cell marker, Oct-4, and potential prostate epithelial stem cell markers, CD133, integrin {alpha}2{beta}1{sup hi} and CD44. The RC-170N/h/clone 7 cells proliferated in KGM and Dulbecco's Modified Eagle Medium with 10% fetalmore » bovine serum and 5 {mu}g/ml insulin (DMEM + 10% FBS + Ins.) medium, and differentiated into epithelial stem cells that expressed epithelial cell markers, including CK5/14, CD44, p63 and cytokeratin 18 (CK18); as well as the mesenchymal cell markers, vimentin, desmin; the neuron and neuroendocrine cell marker, chromogranin A. Furthermore the RC170 N/h/clone 7 cells differentiated into multi tissues when transplanted into the sub-renal capsule and subcutaneously of NOD-SCID mice. The results indicate that RC170N/h/clone 7 cells retain the properties of multipotent stem cells and will be useful as a novel cell model for studying the mechanisms of human prostate stem cell differentiation and transformation.« less

Mesenchymal Stromal Cell-Derived Interleukin-6 Promotes Epithelial-Mesenchymal Transition and Acquisition of Epithelial Stem-Like Cell Properties in Ameloblastoma Epithelial Cells.

PubMed

Jiang, Chunmiao; Zhang, Qunzhou; Shanti, Rabie M; Shi, Shihong; Chang, Ting-Han; Carrasco, Lee; Alawi, Faizan; Le, Anh D

2017-09-01

Epithelial-mesenchymal transition (EMT), a biological process associated with cancer stem-like or cancer-initiating cell formation, contributes to the invasiveness, metastasis, drug resistance, and recurrence of the malignant tumors; it remains to be determined whether similar processes contribute to the pathogenesis and progression of ameloblastoma (AM), a benign but locally invasive odontogenic neoplasm. Here, we demonstrated that EMT- and stem cell-related genes were expressed in the epithelial islands of the most common histologic variant subtype, the follicular AM. Our results revealed elevated interleukin (IL)-6 signals that were differentially expressed in the stromal compartment of the follicular AM. To explore the stromal effect on tumor pathogenesis, we isolated and characterized both mesenchymal stromal cells (AM-MSCs) and epithelial cells (AM-EpiCs) from follicular AM and demonstrated that, in in vitro culture, AM-MSCs secreted a significantly higher level of IL-6 as compared to the counterpart AM-EpiCs. Furthermore, both in vitro and in vivo studies revealed that exogenous and AM-MSC-derived IL-6 induced the expression of EMT- and stem cell-related genes in AM-EpiCs, whereas such effects were significantly abrogated either by a specific inhibitor of STAT3 or ERK1/2, or by knockdown of Slug gene expression. These findings suggest that AM-MSC-derived IL-6 promotes tumor-stem like cell formation by inducing EMT process in AM-EpiCs through STAT3 and ERK1/2-mediated signaling pathways, implying a role in the etiology and progression of the benign but locally invasive neoplasm. Stem Cells 2017;35:2083-2094. © 2017 AlphaMed Press.

Quantitative 4D analyses of epithelial folding during Drosophila gastrulation.

PubMed

Khan, Zia; Wang, Yu-Chiun; Wieschaus, Eric F; Kaschube, Matthias

2014-07-01

Understanding the cellular and mechanical processes that underlie the shape changes of individual cells and their collective behaviors in a tissue during dynamic and complex morphogenetic events is currently one of the major frontiers in developmental biology. The advent of high-speed time-lapse microscopy and its use in monitoring the cellular events in fluorescently labeled developing organisms demonstrate tremendous promise in establishing detailed descriptions of these events and could potentially provide a foundation for subsequent hypothesis-driven research strategies. However, obtaining quantitative measurements of dynamic shapes and behaviors of cells and tissues in a rapidly developing metazoan embryo using time-lapse 3D microscopy remains technically challenging, with the main hurdle being the shortage of robust imaging processing and analysis tools. We have developed EDGE4D, a software tool for segmenting and tracking membrane-labeled cells using multi-photon microscopy data. Our results demonstrate that EDGE4D enables quantification of the dynamics of cell shape changes, cell interfaces and neighbor relations at single-cell resolution during a complex epithelial folding event in the early Drosophila embryo. We expect this tool to be broadly useful for the analysis of epithelial cell geometries and movements in a wide variety of developmental contexts. © 2014. Published by The Company of Biologists Ltd.

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. © 2014 AlphaMed Press.

Mechanical stretch triggers rapid epithelial cell division through Piezo1.

PubMed

Gudipaty, S A; Lindblom, J; Loftus, P D; Redd, M J; Edes, K; Davey, C F; Krishnegowda, V; Rosenblatt, J

2017-03-02

Despite acting as a barrier for the organs they encase, epithelial cells turn over at some of the fastest rates in the body. However, epithelial cell division must be tightly linked to cell death to preserve barrier function and prevent tumour formation. How does the number of dying cells match those dividing to maintain constant numbers? When epithelial cells become too crowded, they activate the stretch-activated channel Piezo1 to trigger extrusion of cells that later die. However, it is unclear how epithelial cell division is controlled to balance cell death at the steady state. Here we show that mammalian epithelial cell division occurs in regions of low cell density where cells are stretched. By experimentally stretching epithelia, we find that mechanical stretch itself rapidly stimulates cell division through activation of the Piezo1 channel. To stimulate cell division, stretch triggers cells that are paused in early G2 phase to activate calcium-dependent phosphorylation of ERK1/2, thereby activating the cyclin B transcription that is necessary to drive cells into mitosis. Although both epithelial cell division and cell extrusion require Piezo1 at the steady state, the type of mechanical force controls the outcome: stretch induces cell division, whereas crowding induces extrusion. How Piezo1-dependent calcium transients activate two opposing processes may depend on where and how Piezo1 is activated, as it accumulates in different subcellular sites with increasing cell density. In sparse epithelial regions in which cells divide, Piezo1 localizes to the plasma membrane and cytoplasm, whereas in dense regions in which cells extrude, it forms large cytoplasmic aggregates. Because Piezo1 senses both mechanical crowding and stretch, it may act as a homeostatic sensor to control epithelial cell numbers, triggering extrusion and apoptosis in crowded regions and cell division in sparse regions.

Genetics and epithelial cell dysfunction in cystic fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

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.

Measles Virus Infection of Epithelial Cells in the Macaque Upper Respiratory Tract Is Mediated by Subepithelial Immune Cells

PubMed Central

Ludlow, Martin; Lemon, Ken; de Vries, Rory D.; McQuaid, Stephen; Millar, Emma L.; van Amerongen, Geert; Yüksel, Selma; Verburgh, R. Joyce; Osterhaus, Albert D. M. E.; Duprex, W. Paul

2013-01-01

Measles virus (MV), one of the most contagious viruses infecting humans, causes a systemic infection leading to fever, immune suppression, and a characteristic maculopapular rash. However, the specific mechanism or mechanisms responsible for the spread of MV into the respiratory epithelium in the late stages of the disease are unknown. Here we show the crucial role of PVRL4 in mediating the spread of MV from immune to epithelial cells by generating a PVRL4 “blind” recombinant wild-type MV and developing a novel in vitro coculture model of B cells with primary differentiated normal human bronchial epithelial cells. We utilized the macaque model of measles to analyze virus distribution in the respiratory tract prior to and at the peak of MV replication. Expression of PVRL4 was widespread in both the lower and upper respiratory tract (URT) of macaques, indicating MV transmission can be facilitated by more than only epithelial cells of the trachea. Analysis of tissues collected at early time points after experimental MV infection demonstrated the presence of MV-infected lymphoid and myeloid cells contacting respiratory tract epithelium in the absence of infected epithelial cells, suggesting that these immune cells seed the infection in vivo. Thereafter, lateral cell-to-cell spread of MV led to the formation of large foci of infected cells in the trachea and high levels of MV infection in the URT, particularly in the nasal cavity. These novel findings have important implications for our understanding of the high transmissibility of measles. PMID:23365435

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

2015-06-04

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.

The ZO-1–associated Y-box factor ZONAB regulates epithelial cell proliferation and cell density

PubMed Central

Balda, Maria S.; Garrett, Michelle D.; Matter, Karl

2003-01-01

Epithelial tight junctions regulate paracellular permeability, restrict apical/basolateral intramembrane diffusion of lipids, and have been proposed to participate in the control of epithelial cell proliferation and differentiation. Previously, we have identified ZO-1–associated nucleic acid binding proteins (ZONAB), a Y-box transcription factor whose nuclear localization and transcriptional activity is regulated by the tight junction–associated candidate tumor suppressor ZO-1. Now, we found that reduction of ZONAB expression using an antisense approach or by RNA interference strongly reduced proliferation of MDCK cells. Transfection of wild-type or ZONAB-binding fragments of ZO-1 reduced proliferation as well as nuclear ZONAB pools, indicating that promotion of proliferation by ZONAB requires its nuclear accumulation. Overexpression of ZONAB resulted in increased cell density in mature monolayers, and depletion of ZONAB or overexpression of ZO-1 reduced cell density. ZONAB was found to associate with cell division kinase (CDK) 4, and reduction of nuclear ZONAB levels resulted in reduced nuclear CDK4. Thus, our data indicate that tight junctions can regulate epithelial cell proliferation and cell density via a ZONAB/ZO-1–based pathway. Although this regulatory process may also involve regulation of transcription by ZONAB, our data suggest that one mechanism by which ZONAB and ZO-1 influence proliferation is by regulating the nuclear accumulation of CDK4. PMID:12566432

Epithelial transformation by KLF4 requires Notch1 but not canonical Notch1 signaling

PubMed Central

Liu, Zhaoli; Teng, Lihong; Bailey, Sarah K.; Frost, Andra R.; Bland, Kirby I.; LoBuglio, Albert F.; Ruppert, J. Michael; Lobo-Ruppert, Susan M.

2009-01-01

The transcription factors Notch1 and KLF4 specify epithelial cell fates and confer stem cell properties. suggesting a functional relationship, each gene can act to promote or suppress tumorigenesis in a context-dependent manner, and alteration of KLF4 or Notch pathway genes in mice gives rise to similar phenotypes. Activation of a conditional allele of KLF4 in RK3E epithelial cells rapidly induces expression of Notch1 mRNA and the active, intracellular form of Notch1. KLF4-induced transformation was suppressed by knockdown of endogenous Notch1 using siRNA or an inhibitor of γ-secretase. Chromatin immunoprecipitation assay shows that KLF4 binds to the proximal Notch1 promoter in human mammary epithelial cells, and siRNA-mediated suppression of KLF4 in human mammary cancer cells results in reduced expression of Notch1. Furthermore, KLF4 and Notch1 expression are correlated in primary human breast tumors (N = 89; pearson analysis, r > 0.5, p < 0.0001). Like KLF4, Notch1 was previously shown to induce transformation of rat cells immortalized with adenovirus E1A, similar to RK3E cells. We therefore compared the signaling requirements for Notch1- or KLF4-induced malignant transformation of RK3E. As expected, transformation by Notch1 was suppressed by dominant-negative CSL or MaML1, inhibitors of canonical Notch1 signaling. However, these inhibitors did not suppress transformation by KLF4. Therefore, while KLF4-induced transformation requires Notch1, canonical Notch1 signaling is not required, and Notch1 may signal through a distinct pathway in cells with increased KLF4 activity. These results suggest that KLF4 could contribute to breast tumor progression by activating synthesis of Notch1 and by promoting signaling through a non-canonical Notch1 pathway. PMID:19717984

Cell reintegration: Stray epithelial cells make their way home.

PubMed

Wilson, Tyler J; Bergstralh, Dan T

2017-06-01

Ongoing work shows that misplaced epithelial cells have the capacity to reintegrate back into tissue layers. This movement appears to underlie tissue stability and may also control aspects of tissue structure. A recent study reveals that cell reintegration in at least one tissue, the Drosophila follicular epithelium, is based on adhesion molecules that line lateral cell surfaces. In this article we will review these observations, discuss their implications for epithelial tissue development and maintenance, and identify future directions for study. © 2017 WILEY Periodicals, Inc.

The Effect of Ozone on Colonic Epithelial Cells.

PubMed

Himuro, Hidetomo

2018-05-21

Due to its strong oxidation activity, ozone has been well known to kill bacteria and exert toxic effects on human tissues. At the same time, ozone is being used for the treatment of diseases such as inflammatory bowel disease in some European countries. However, the use of ozone for therapeutic purposes, despite its strong toxic effects, remains largely unexplored. Interestingly, we found that intrarectal administration of ozone gas induced transient colonic epithelial cell damage characterized by the impairment of cell survival pathways involved in DNA replication, cell cycle, and mismatch repair. However, the damaged cells were rapidly extruded from the epithelial layer, and appeared to immediately stimulate turnover of the epithelial layer in the colon. Therefore, it is possible that ozone gas is able to trigger damage-induced rapid regeneration of intestinal epithelial cells, and that this explains why ozone does not cause harmful or persistent damage in the colon.

Efficient Immortalization of Primary Nasopharyngeal Epithelial Cells for EBV Infection Study

PubMed Central

Yip, Yim Ling; Pang, Pei Shin; Deng, Wen; Tsang, Chi Man; Zeng, Musheng; Hau, Pok Man; Man, Cornelia; Jin, Yuesheng; Yuen, Anthony Po Wing; Tsao, Sai Wah

2013-01-01

Nasopharyngeal carcinoma (NPC) is common among southern Chinese including the ethnic Cantonese population living in Hong Kong. Epstein-Barr virus (EBV) infection is detected in all undifferentiated type of NPC in this endemic region. Establishment of stable and latent EBV infection in premalignant nasopharyngeal epithelial cells is an early event in NPC development and may contribute to its pathogenesis. Immortalized primary nasopharyngeal epithelial cells represent an important tool for investigation of EBV infection and its tumorigenic potential in this special type of epithelial cells. However, the limited availability and small sizes of nasopharyngeal biopsies have seriously restricted the establishment of primary nasopharyngeal epithelial cells for immortalization. A reliable and effective method to immortalize primary nasopharyngeal epithelial cells will provide unrestricted materials for EBV infection studies. An earlier study has reported that Bmi-1 expression could immortalize primary nasopharyngeal epithelial cells. However, its efficiency and actions in immortalization have not been fully characterized. Our studies showed that Bmi-1 expression alone has limited ability to immortalize primary nasopharyngeal epithelial cells and additional events are often required for its immortalization action. We have identified some of the key events associated with the immortalization of primary nasopharyngeal epithelial cells. Efficient immortalization of nasopharyngeal epithelial cells could be reproducibly and efficiently achieved by the combined actions of Bmi-1 expression, activation of telomerase and silencing of p16 gene. Activation of MAPK signaling and gene expression downstream of Bmi-1 were detected in the immortalized nasopharyngeal epithelial cells and may play a role in immortalization. Furthermore, these newly immortalized nasopharyngeal epithelial cells are susceptible to EBV infection and supported a type II latent EBV infection program characteristic

M2 polarization of macrophages facilitates arsenic-induced cell transformation of lung epithelial cells

PubMed Central

Li, Hui; Dai, Lu; Frank, Jacqueline A.; Peng, Shaojun; Wang, Siying; Chen, Gang

2017-01-01

The alterations in microenvironment upon chronic arsenic exposure may contribute to arsenic-induced lung carcinogenesis. Immune cells, such as macrophages, play an important role in mediating the microenvironment in the lungs. Macrophages carry out their functions after activation. There are two activation status for macrophages: classical (M1) or alternative (M2); the latter is associated with tumorigenesis. Our previous work showed that long-term arsenic exposure induces transformation of lung epithelial cells. However, the crosstalk between epithelial cells and macrophages upon arsenic exposure has not been investigated. In this study, using a co-culture system in which human lung epithelial cells are cultured with macrophages, we determined that long-term arsenic exposure polarizes macrophages towards M2 status through ROS generation. Co-culture with epithelial cells further enhanced the polarization of macrophages as well as transformation of epithelial cells, while blocking macrophage M2 polarization decreased the transformation. In addition, macrophage M2 polarization decreased autophagy activity, which may account for increased cell transformation of epithelial cells with co-culture of macrophages. PMID:28423485

Fabrication of corneal epithelial cell sheets maintaining colony-forming cells without feeder cells by oxygen-controlled method.

PubMed

Nakajima, Ryota; Takeda, Shizu

2014-01-01

The use of murine 3T3 feeder cells needs to be avoided when fabricating corneal epithelial cell sheets for use in treating ocular surface diseases. However, the expression level of the epithelial stem/progenitor cell marker, p63, is down-regulated in feeder-free culture systems. In this study, in order to fabricate corneal epithelial cell sheets that maintain colony-forming cells without using any feeder cells, we investigated the use of an oxygen-controlled method that was developed previously to fabricate cell sheets efficiently. Rabbit limbal epithelial cells were cultured under hypoxia (1-10% O2) and under normoxia during stratification after reaching confluence. Multilayered corneal epithelial cell sheets were fabricated using an oxygen-controlled method, and immunofluorescence analysis showed that cytokeratin 3 and p63 was expressed in appropriate localization in the cell sheets. The colony-forming efficiency of the cell sheets fabricated by the oxygen-controlled method without feeder cells was significantly higher than that of cell sheets fabricated under 20% O2 without feeder cells. These results indicate that the oxygen-controlled method has the potential to achieve a feeder-free culture system for fabricating corneal epithelial cell sheets for corneal regeneration. Copyright © 2013 Elsevier Ltd. All rights reserved.

Airway epithelial cell response to human metapneumovirus infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bao, X.; Liu, T.; Spetch, L.

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 typemore » 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.« less

Cytotoxicity and Induction of Inflammation by Pepsin in Acid in Bronchial Epithelial Cells

PubMed Central

Bathoorn, Erik; Daly, Paul; Gaiser, Birgit; Sternad, Karl; Poland, Craig; MacNee, William; Drost, Ellen M.

2011-01-01

Introduction. Gastroesophageal reflux has been associated with chronic inflammatory diseases and may be a cause of airway remodelling. Aspiration of gastric fluids may cause damage to airway epithelial cells, not only because acidity is toxic to bronchial epithelial cells, but also since it contains digestive enzymes, such as pepsin. Aim. To study whether pepsin enhances cytotoxicity and inflammation in airway epithelial cells, and whether this is pH-dependent. Methods. Human bronchial epithelial cells were exposed to increasing pepsin concentrations in varying acidic milieus, and cell proliferation and cytokine release were assessed. Results. Cell survival was decreased by pepsin exposure depending on its concentration (F = 17.4) and pH level of the medium (F = 6.5) (both P < 0.01). Pepsin-induced interleukin-8 release was greater at lower pH (F = 5.1; P < 0.01). Interleukin-6 induction by pepsin was greater at pH 1.5 compared to pH 2.5 (mean difference 434%; P = 0.03). Conclusion. Pepsin is cytotoxic to bronchial epithelial cells and induces inflammation in addition to acid alone, dependent on the level of acidity. Future studies should assess whether chronic aspiration causes airway remodelling in chronic inflammatory lung diseases. PMID:21785693

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

Eukaryotic Translation Initiation Factor 4E Is a Feed-Forward Translational Coactivator of Transforming Growth Factor β Early Protransforming Events in Breast Epithelial Cells

PubMed Central

Decarlo, Lindsey; Mestel, Celine; Barcellos-Hoff, Mary-Helen

2015-01-01

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed early in breast cancers in association with disease progression and reduced survival. Much remains to be understood regarding the role of eIF4E in human cancer. We determined, using immortalized human breast epithelial cells, that elevated expression of eIF4E translationally activates the transforming growth factor β (TGF-β) pathway, promoting cell invasion, a loss of cell polarity, increased cell survival, and other hallmarks of early neoplasia. Overexpression of eIF4E is shown to facilitate the selective translation of integrin β1 mRNA, which drives the translationally controlled assembly of a TGF-β receptor signaling complex containing α3β1 integrins, β-catenin, TGF-β receptor I, E-cadherin, and phosphorylated Smad2/3. This receptor complex acutely sensitizes nonmalignant breast epithelial cells to activation by typically substimulatory levels of activated TGF-β. TGF-β can promote cellular differentiation or invasion and transformation. As a translational coactivator of TGF-β, eIF4E confers selective mRNA translation, reprogramming nonmalignant cells to an invasive phenotype by reducing the set point for stimulation by activated TGF-β. Overexpression of eIF4E may be a proinvasive facilitator of TGF-β activity. PMID:25986608

Human induced pluripotent stem cell differentiation and direct transdifferentiation into corneal epithelial-like cells

PubMed Central

Cieślar-Pobuda, Artur; Rafat, Mehrdad; Knoflach, Viktoria; Skonieczna, Magdalena; Hudecki, Andrzej; Małecki, Andrzej; Urasińska, Elżbieta; Ghavami, Seaid; Łos, Marek J.

2016-01-01

The corneal epithelium is maintained by a small pool of tissue stem cells located at the limbus. Through certain injuries or diseases this pool of stem cells may get depleted. This leads to visual impairment. Standard treatment options include autologous or allogeneic limbal stem cell (LSC) transplantation, however graft rejection and chronic inflammation lowers the success rate over long time. Induced pluripotent stem (iPS) cells have opened new possibilities for treating various diseases with patient specific cells, eliminating the risk of immune rejection. In recent years, several protocols have been developed, aimed at the differentiation of iPS cells into the corneal epithelial lineage by mimicking the environmental niche of limbal stem cells. However, the risk of teratoma formation associated with the use of iPS cells hinders most applications from lab into clinics. Here we show that the differentiation of iPS cells into corneal epithelial cells results in the expression of corneal epithelial markers showing a successful differentiation, but the process is long and the level of gene expression for the pluripotency markers does not vanish completely. Therefore we set out to determine a direct transdifferentiation approach to circumvent the intermediate state of pluripotency (iPS-stage). The resulting cells, obtained by direct transdifferentiation of fibroblasts into limbal cells, exhibited corneal epithelial cell morphology and expressed corneal epithelial markers. Hence we shows for the first time a direct transdifferentiation of human dermal fibroblasts into the corneal epithelial lineage that may serve as source for corneal epithelial cells for transplantation approaches. PMID:27275539

The Contribution of the Airway Epithelial Cell to Host Defense.

PubMed

Stanke, Frauke

2015-01-01

In the context of cystic fibrosis, the epithelial cell has been characterized in terms of its ion transport capabilities. The ability of an epithelial cell to initiate CFTR-mediated chloride and bicarbonate transport has been recognized early as a means to regulate the thickness of the epithelial lining fluid and recently as a means to regulate the pH, thereby determining critically whether or not host defense proteins such as mucins are able to fold appropriately. This review describes how the epithelial cell senses the presence of pathogens and inflammatory conditions, which, in turn, facilitates the activation of CFTR and thus directly promotes pathogens clearance and innate immune defense on the surface of the epithelial cell. This paper summarizes functional data that describes the effect of cytokines, chemokines, infectious agents, and inflammatory conditions on the ion transport properties of the epithelial cell and relates these key properties to the molecular pathology of cystic fibrosis. Recent findings on the role of cystic fibrosis modifier genes that underscore the role of the epithelial ion transport in host defense and inflammation are discussed.

Induction of KLF4 in basal keratinocytes blocks the proliferation-differentiation switch and initiates squamous epithelial dysplasia

PubMed Central

Foster, K. Wade; Liu, Zhaoli; Nail, Clinton D.; Li, Xingnan; Fitzgerald, Thomas J.; Bailey, Sarah K.; Frost, Andra R.; Louro, Iuri D.; Townes, Tim M.; Paterson, Andrew J.; Kudlow, Jeffrey E.; Lobo-Ruppert, Susan M.; Ruppert, J. Michael

2006-01-01

KLF4/GKLF normally functions in differentiating epithelial cells, but also acts as a transforming oncogene in vitro. To examine the role of this zinc finger protein in skin, we expressed the wild-type human allele from inducible and constitutive promoters. When induced in basal keratinocytes KLF4 rapidly abolished the distinctive properties of basal and parabasal epithelial cells. KLF4 caused a transitory apoptotic response and the skin progressed through phases of hyperplasia and dysplasia. By 6 weeks, lesions exhibited nuclear KLF4 and other morphologic and molecular similarities to squamous cell carcinoma in situ. p53 determined the patch size sufficient to establish lesions, as induction in a mosaic pattern produced skin lesions only when p53 was deficient. Compared with p53 wild-type animals, p53 hemizygous animals had early onset of lesions and a pronounced fibrovascular response that included outgrowth of subcutaneous sarcoma. A KLF4-estrogen receptor fusion protein showed tamoxifen-dependent nuclear localization and conditional transformation in vitro. The results suggest that KLF4 can function in the nucleus to induce squamous epithelial dysplasia, and indicate roles for p53 and epithelial-mesenchymal signaling in these early neoplastic lesions. PMID:15674344

Induction of KLF4 in basal keratinocytes blocks the proliferation-differentiation switch and initiates squamous epithelial dysplasia.

PubMed

Foster, K Wade; Liu, Zhaoli; Nail, Clinton D; Li, Xingnan; Fitzgerald, Thomas J; Bailey, Sarah K; Frost, Andra R; Louro, Iuri D; Townes, Tim M; Paterson, Andrew J; Kudlow, Jeffrey E; Lobo-Ruppert, Susan M; Ruppert, J Michael

2005-02-24

KLF4/GKLF normally functions in differentiating epithelial cells, but also acts as a transforming oncogene in vitro. To examine the role of this zinc finger protein in skin, we expressed the wild-type human allele from inducible and constitutive promoters. When induced in basal keratinocytes, KLF4 rapidly abolished the distinctive properties of basal and parabasal epithelial cells. KLF4 caused a transitory apoptotic response and the skin progressed through phases of hyperplasia and dysplasia. By 6 weeks, lesions exhibited nuclear KLF4 and other morphologic and molecular similarities to squamous cell carcinoma in situ. p53 determined the patch size sufficient to establish lesions, as induction in a mosaic pattern produced skin lesions only when p53 was deficient. Compared with p53 wild-type animals, p53 hemizygous animals had early onset of lesions and a pronounced fibrovascular response that included outgrowth of subcutaneous sarcoma. A KLF4-estrogen receptor fusion protein showed tamoxifen-dependent nuclear localization and conditional transformation in vitro. The results suggest that KLF4 can function in the nucleus to induce squamous epithelial dysplasia, and indicate roles for p53 and epithelial-mesenchymal signaling in these early neoplastic lesions.

Regulation of xanthine dehydrogensase gene expression and uric acid production in human airway epithelial cells

PubMed Central

Huff, Ryan D.; Hsu, Alan C-Y.; Nichol, Kristy S.; Jones, Bernadette; Knight, Darryl A.; Wark, Peter A. B.; Hansbro, Philip M.

2017-01-01

Introduction The airway epithelium is a physical and immunological barrier that protects the pulmonary system from inhaled environmental insults. Uric acid has been detected in the respiratory tract and can function as an antioxidant or damage associated molecular pattern. We have demonstrated that human airway epithelial cells are a source of uric acid. Our hypothesis is that uric acid production by airway epithelial cells is induced by environmental stimuli associated with chronic respiratory diseases. We therefore examined how airway epithelial cells regulate uric acid production. Materials and methods Allergen and cigarette smoke mouse models were performed using house dust mite (HDM) and cigarette smoke exposure, respectively, with outcome measurements of lung uric acid levels. Primary human airway epithelial cells isolated from clinically diagnosed patients with asthma and chronic obstructive pulmonary disease (COPD) were grown in submerged cultures and compared to age-matched healthy controls for uric acid release. HBEC-6KT cells, a human airway epithelial cell line, were grown under submerged monolayer conditions for mechanistic and gene expression studies. Results HDM, but not cigarette smoke exposure, stimulated uric acid production in vivo and in vitro. Primary human airway epithelial cells from asthma, but not COPD patients, displayed elevated levels of extracellular uric acid in culture. In HBEC-6KT, production of uric acid was sensitive to the xanthine dehydrogenase (XDH) inhibitor, allopurinol, and the ATP Binding Cassette C4 (ABCC4) inhibitor, MK-571. Lastly, the pro-inflammatory cytokine combination of TNF-α and IFN-γ elevated extracellular uric acid levels and XDH gene expression in HBEC-6KT cells. Conclusions Our results suggest that the active production of uric acid from human airway epithelial cells may be intrinsically altered in asthma and be further induced by pro-inflammatory cytokines. PMID:28863172

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. © 2010 Blackwell Verlag GmbH.

Canine corneal epithelial cells possess a sustained proliferative capacity and generate a spontaneously derived cell line.

PubMed

Morita, Maresuke; Fujita, Naoki; Abe, Momoko; Hayashimoto, Koji; Nakagawa, Takayuki; Nishimura, Ryohei; Tsuzuki, Keiko

2018-06-01

We have previously reported characteristics of canine corneal epithelial cells in vitro and found that canine corneal epithelial cells could maintain their proliferative capacity even after continuous culture without the use of feeder cells and growth promoting additives. The objective of this study was to elucidate proliferative characteristics of canine corneal epithelial cells independent of feeder cells and growth promoting additives, with the aim of developing a spontaneously derived corneal epithelial cell line. Canine and rabbit corneal epithelial cells were harvested from the limbus and cultured with, or without, feeder cells and growth promoting additives, and both were passaged continuously until growth arrest. Canine corneal epithelial cells could proliferate independently, and could be passaged more times than rabbit cells. A canine corneal epithelial cell line, cCEpi, which could be passaged more than 100 times without using feeder cells and growth promoting additives, was established. cCEpi cells maintained a cell morphology close to the primary culture and expressed p63, cytokeratin 15 (K15), and K3. Although changes in colony morphology, shortening of the population doubling time and a heteroploid karyotype were observed, cCEpi was not tumorigenic. Stratified cell sheets cultured from cCEpi were morphologically and immunohistologically similar to sheets cultivated from early passage cells. In conclusion, canine corneal epithelial cells can proliferate independent of feeder cells and growth promoting additives. cCEpi maintains properties similar to normal corneal epithelial cells and could be a useful source for studies in cellular biology and for developing novel therapies. Copyright © 2018 Elsevier Ltd. All rights reserved.

Implantation of Induced Pluripotent Stem Cell-Derived Tracheal Epithelial Cells.

PubMed

Ikeda, Masakazu; Imaizumi, Mitsuyoshi; Yoshie, Susumu; Nakamura, Ryosuke; Otsuki, Koshi; Murono, Shigeyuki; Omori, Koichi

2017-07-01

Compared with using autologous tissue, the use of artificial materials in the regeneration of tracheal defects is minimally invasive. However, this technique requires early epithelialization on the inner side of the artificial trachea. After differentiation from induced pluripotent stem cells (iPSCs), tracheal epithelial tissues may be used to produce artificial tracheas. Herein, we aimed to demonstrate that after differentiation from fluorescent protein-labeled iPSCs, tracheal epithelial tissues survived in nude rats with tracheal defects. Red fluorescent tdTomato protein was electroporated into mouse iPSCs to produce tdTomato-labeled iPSCs. Embryoid bodies derived from these iPSCs were then cultured in differentiation medium supplemented with growth factors, followed by culture on air-liquid interfaces for further differentiation into tracheal epithelium. The cells were implanted with artificial tracheas into nude rats with tracheal defects on day 26 of cultivation. On day 7 after implantation, the tracheas were exposed and examined histologically. Tracheal epithelial tissue derived from tdTomato-labeled iPSCs survived in the tracheal defects. Moreover, immunochemical analyses showed that differentiated tissues had epithelial structures similar to those of proximal tracheal tissues. After differentiation from iPSCs, tracheal epithelial tissues survived in rat bodies, warranting the use of iPSCs for epithelial regeneration in tracheal defects.

The increase of microRNA-21 during lung fibrosis and its contribution to epithelial-mesenchymal transition in pulmonary epithelial cells.

PubMed

Yamada, Mitsuhiro; Kubo, Hiroshi; Ota, Chiharu; Takahashi, Toru; Tando, Yukiko; Suzuki, Takaya; Fujino, Naoya; Makiguchi, Tomonori; Takagi, Kiyoshi; Suzuki, Takashi; Ichinose, Masakazu

2013-09-24

The excess and persistent accumulation of fibroblasts due to aberrant tissue repair results in fibrotic diseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs during idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast differentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that microRNA-21 is up-regulated in myofibroblasts during fibrosis and promotes transforming growth factor-beta signaling by inhibiting Smad7. However, expression changes in microRNA-21 and the role of microRNA-21 in epithelial-mesenchymal transition during lung fibrosis have not yet been defined. Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with idiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung epithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-21 was analyzed using both quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated mouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of transforming growth factor-β, thus generating conditions that enhance epithelial-mesenchymal transition. To investigate the role of microRNA-21 in epithelial-mesenchymal transition, we transfected cells with a microRNA-21 inhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-21, as well as mRNAs of genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using quantitative PCR. The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased expression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased. MicroRNA-21 was significantly upregulated in isolated lung epithelial

Primary Airway Epithelial Cell Gene Editing Using CRISPR-Cas9.

PubMed

Everman, Jamie L; Rios, Cydney; Seibold, Max A

2018-01-01

The adaptation of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated endonuclease 9 (CRISPR-Cas9) machinery from prokaryotic organisms has resulted in a gene editing system that is highly versatile, easily constructed, and can be leveraged to generate human cells knocked out (KO) for a specific gene. While standard transfection techniques can be used for the introduction of CRISPR-Cas9 expression cassettes to many cell types, delivery by this method is not efficient in many primary cell types, including primary human airway epithelial cells (AECs). More efficient delivery in AECs can be achieved through lentiviral-mediated transduction, allowing the CRISPR-Cas9 system to be integrated into the genome of the cell, resulting in stable expression of the nuclease machinery and increasing editing rates. In parallel, advancements have been made in the culture, expansion, selection, and differentiation of AECs, which allow the robust generation of a bulk edited AEC population from transduced cells. Applying these methods, we detail here our latest protocol to generate mucociliary epithelial cultures knocked out for a specific gene from donor-isolated primary human basal airway epithelial cells. This protocol includes methods to: (1) design and generate lentivirus which targets a specific gene for KO with CRISPR-Cas9 machinery, (2) efficiently transduce AECs, (3) culture and select for a bulk edited AEC population, (4) molecularly screen AECs for Cas9 cutting and specific sequence edits, and (5) further expand and differentiate edited cells to a mucociliary airway epithelial culture. The AEC knockouts generated using this protocol provide an excellent primary cell model system with which to characterize the function of genes involved in airway dysfunction and disease.

Proximal location of mouse prostate epithelial stem cells

PubMed Central

Tsujimura, Akira; Koikawa, Yasuhiro; Salm, Sarah; Takao, Tetsuya; Coetzee, Sandra; Moscatelli, David; Shapiro, Ellen; Lepor, Herbert; Sun, Tung-Tien; Wilson, E. Lynette

2002-01-01

Stem cells are believed to regulate normal prostatic homeostasis and to play a role in the etiology of prostate cancer and benign prostatic hyperplasia. We show here that the proximal region of mouse prostatic ducts is enriched in a subpopulation of epithelial cells that exhibit three important attributes of epithelial stem cells: they are slow cycling, possess a high in vitro proliferative potential, and can reconstitute highly branched glandular ductal structures in collagen gels. We propose a model of prostatic homeostasis in which mouse prostatic epithelial stem cells are concentrated in the proximal region of prostatic ducts while the transit-amplifying cells occupy the distal region of the ducts. This model can account for many biological differences between cells of the proximal and distal regions, and has implications for prostatic disease formation. PMID:12082083

Characterization of rabbit limbal epithelial side population cells using RNA sequencing and single-cell qRT-PCR.

PubMed

Kameishi, Sumako; Umemoto, Terumasa; Matsuzaki, Yu; Fujita, Masako; Okano, Teruo; Kato, Takashi; Yamato, Masayuki

2016-05-06

Corneal epithelial stem cells reside in the limbus, a transitional zone between the cornea and conjunctiva, and are essential for maintaining homeostasis in the corneal epithelium. Although our previous studies demonstrated that rabbit limbal epithelial side population (SP) cells exhibit stem cell-like phenotypes with Hoechst 33342 staining, the different characteristics and/or populations of these cells remain unclear. Therefore, in this study, we determined the gene expression profiles of limbal epithelial SP cells by RNA sequencing using not only present public databases but also contigs that were created by de novo transcriptome assembly as references for mapping. Our transcriptome data indicated that limbal epithelial SP cells exhibited a stem cell-like phenotype compared with non-SP cells. Importantly, gene ontology analysis following RNA sequencing demonstrated that limbal epithelial SP cells exhibited significantly enhanced expression of mesenchymal/endothelial cell markers rather than epithelial cell markers. Furthermore, single-cell quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that the limbal epithelial SP population consisted of at least two immature cell populations with endothelial- or mesenchymal-like phenotypes. Therefore, our present results may propose the presence of a novel population of corneal epithelial stem cells distinct from conventional epithelial stem cells. Copyright © 2015 Elsevier Inc. All rights reserved.

CD4+ lymphocytes control gut epithelial apoptosis and mediate survival in sepsis.

PubMed

Stromberg, Paul E; Woolsey, Cheryl A; Clark, Andrew T; Clark, Jessica A; Turnbull, Isaiah R; McConnell, Kevin W; Chang, Katherine C; Chung, Chun-Shiang; Ayala, Alfred; Buchman, Timothy G; Hotchkiss, Richard S; Coopersmith, Craig M

2009-06-01

Lymphocytes help determine whether gut epithelial cells proliferate or differentiate but are not known to affect whether they live or die. Here, we report that lymphocytes play a controlling role in mediating gut epithelial apoptosis in sepsis but not under basal conditions. Gut epithelial apoptosis is similar in unmanipulated Rag-1(-/-) and wild-type (WT) mice. However, Rag-1(-/-) animals have a 5-fold augmentation in gut epithelial apoptosis following cecal ligation and puncture (CLP) compared to septic WT mice. Reconstitution of lymphocytes in Rag-1(-/-) mice via adoptive transfer decreases intestinal apoptosis to levels seen in WT animals. Subset analysis indicates that CD4(+) but not CD8(+), gammadelta, or B cells are responsible for the antiapoptotic effect of lymphocytes on the gut epithelium. Gut-specific overexpression of Bcl-2 in transgenic mice decreases mortality following CLP. This survival benefit is lymphocyte dependent since gut-specific overexpression of Bcl-2 fails to alter survival when the transgene is overexpressed in Rag-1(-/-) mice. Further, adoptively transferring lymphocytes to Rag-1(-/-) mice that simultaneously overexpress gut-specific Bcl-2 results in improved mortality following sepsis. Thus, sepsis unmasks CD4(+) lymphocyte control of gut apoptosis that is not present under homeostatic conditions, which acts as a key determinant of both cellular survival and host mortality.

Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis

PubMed Central

Mizuno, Takako; Sridharan, Anusha; Du, Yina; Guo, Minzhe; Wikenheiser-Brokamp, Kathryn A.; Perl, Anne-Karina T.; Funari, Vincent A.; Gokey, Jason J.; Stripp, Barry R.; Whitsett, Jeffrey A.

2016-01-01

Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial lung disease characterized by airway remodeling, inflammation, alveolar destruction, and fibrosis. We utilized single-cell RNA sequencing (scRNA-seq) to identify epithelial cell types and associated biological processes involved in the pathogenesis of IPF. Transcriptomic analysis of normal human lung epithelial cells defined gene expression patterns associated with highly differentiated alveolar type 2 (AT2) cells, indicated by enrichment of RNAs critical for surfactant homeostasis. In contrast, scRNA-seq of IPF cells identified 3 distinct subsets of epithelial cell types with characteristics of conducting airway basal and goblet cells and an additional atypical transitional cell that contributes to pathological processes in IPF. Individual IPF cells frequently coexpressed alveolar type 1 (AT1), AT2, and conducting airway selective markers, demonstrating “indeterminate” states of differentiation not seen in normal lung development. Pathway analysis predicted aberrant activation of canonical signaling via TGF-β, HIPPO/YAP, P53, WNT, and AKT/PI3K. Immunofluorescence confocal microscopy identified the disruption of alveolar structure and loss of the normal proximal-peripheral differentiation of pulmonary epithelial cells. scRNA-seq analyses identified loss of normal epithelial cell identities and unique contributions of epithelial cells to the pathogenesis of IPF. The present study provides a rich data source to further explore lung health and disease. PMID:27942595

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

Protons sensitize epithelial cells to mesenchymal transition.

PubMed

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.

Chitin-Induced Airway Epithelial Cell Innate Immune Responses Are Inhibited by Carvacrol/Thymol

PubMed Central

Erle, David J.

2016-01-01

Chitin is produced in large amounts by fungi, insects, and other organisms and has been implicated in the pathogenesis of asthma. Airway epithelial cells are in direct contact with environmental particles and serve as the first line of defense against inhaled allergens and pathogens. The potential contributions of airway epithelial cells to chitin-induced asthma remain poorly understood. We hypothesized that chitin directly stimulates airway epithelial cells to release cytokines that promote type 2 immune responses and to induce expression of molecules which are important in innate immune responses. We found that chitin exposure rapidly induced the expression of three key type 2-promoting cytokines, IL-25, IL-33 and TSLP, in BEAS-2B transformed human bronchial epithelial cells and in A549 and H292 lung carcinoma cells. Chitin also induced the expression of the key pattern recognition receptors TLR2 and TLR4. Chitin induced the expression of miR-155, miR-146a and miR-21, each of which is known to up-regulate the expression of pro-inflammatory cytokines. Also the expression of SOCS1 and SHIP1 which are known targets of miR-155 was repressed by chitin treatment. The monoterpene phenol carvacrol (Car) and its isomer thymol (Thy) are found in herbal essential oils and have been shown to inhibit allergic inflammation in asthma models. We found that Car/Thy inhibited the effects of chitin on type 2-promoting cytokine release and on the expression of TLRs, SOCS1, SHIP1, and miRNAs. Car/Thy could also efficiently reduce the protein levels of TLR4, inhibit the increase in TLR2 protein levels in chitin plus Car/Thy-treated cells and increase the protein levels of SHIP1 and SOCS1, which are negative regulators of TLR-mediated inflammatory responses. We conclude that direct effects of chitin on airway epithelial cells are likely to contribute to allergic airway diseases like asthma, and that Car/Thy directly inhibits epithelial cell pro-inflammatory responses to chitin. PMID

Excessive fluoride reduces Foxo1 expression in dental epithelial cells of the rat incisor.

PubMed

Gao, Jianghong; Ruan, Jianping; Gao, Liping

2014-10-01

Enamel fluorosis is characterized by hypomineralization, and forkhead box O1 (Foxo1) is essential for mouse enamel biomineralization. This study investigated the effect of fluoride on Foxo1 expression and its implications for enamel fluorosis. Mandibular incisors were extracted from Sprague Dawley rats treated for 3 months with water containing 0, 50, or 100 p.p.m. F⁻. Immunohistochemistry was used to localize and quantify FOXO1 expression in dental epithelial layer cells of the incisors. The effect of fluoride on expression of Foxo1, kallikrein-4 (Klk4), and amelotin (Amtn) mRNAs was analyzed by real-time RT-PCR, and western blotting was used to measure total and nuclear FOXO1 protein levels in mature dental epithelial cells. The results revealed that nuclear FOXO1 was mainly localized in the transition and the mature ameloblasts and exhibited weaker expression in the rats exposed to fluoride. In addition to the reduced levels of Foxo1, Klk4, and AmtnmRNAs, the protein levels of total and nuclearFOXO1 were decreased in the mature dental epithelial cells exposed to fluoride. Thus, excessive fluoride may have an effect on the expression levels of Foxo1 in dental epithelial cells and thereby affect hypomineralization of the enamel during fluorosis. © 2014 Eur J Oral Sci.

Low p16INK4a Expression in Early Passage Human Prostate Basal Epithelial Cells Enables Immortalization by Telomerase Expression Alone.

PubMed

Graham, Mindy Kim; Principessa, Lorenzo; Antony, Lizamma; Meeker, Alan K; Isaacs, John T

2017-03-01

There are two principal senescence barriers that must be overcome to successfully immortalize primary human epithelial cells in culture, stress-induced senescence, and replicative senescence. The p16 INK4a /retinoblastoma protein (p16/Rb) pathway mediates stress-induced senescence, and is generally upregulated by primary epithelial cells in response to the artificial conditions from tissue culture. Replicative senescence is associated with telomere loss. Following each round of cell division, telomeres progressively shorten. Once telomeres shorten to a critical length, the DNA damage response pathway is activated, and the tumor suppressor p53 pathway triggers replicative senescence. Exogenous expression of telomerase in normal human epithelial cells extends the replicative capacity of cells, and in some cases, immortalizes cells. However reliable immortalization of epithelial cells usually requires telomerase activity coupled with inactivation of the p16/Rb pathway. A lentiviral vector, pLOX-TERT-iresTK (Addgene #12245), containing a CMV promoter upstream of a bicistronic coding cassette that includes loxP sites flanking the catalytic subunit of human telomerase gene (TERT) and herpes simplex virus type-1 thymidine kinase gene (HSV1-tk) was used to transduce normal prostate basal epithelial cells (PrECs) initiated in cell culture from prostate cancer patients undergoing radical prostatectomies. Transduction of early (i.e., <7) passage PrECs with TERT led to successful immortalization. However, attempts to immortalize late (i.e., >7) passage PrECs were unsuccessful. Late passage PrECs, which acquired elevated p16, were unable to overcome the senescence barrier. Immortalized PrECs (TERT-PrECs) retained a normal male karyotype and low p16 expression. Additionally, TERT-PrECs were non-tumorigenic when inoculated into intact male immunodeficient NSG mice. The present studies document that early passage human PrECs have sufficiently low p16 to permit immortalization by

Sequestration of human cytomegalovirus by human renal and mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Twite, Nicolas; Andrei, Graciela; Kummert, Caroline

2014-07-15

Urine and breast milk represent the main routes of human cytomegalovirus (HCMV) transmission but the contribution of renal and mammary epithelial cells to viral excretion remains unclear. We observed that kidney and mammary epithelial cells were permissive to HCMV infection and expressed immediate early, early and late antigens within 72 h of infection. During the first 24 h after infection, high titers of infectious virus were measured associated to the cells and in culture supernatants, independently of de novo synthesis of virus progeny. This phenomenon was not observed in HCMV-infected fibroblasts and suggested the sequestration and the release of HCMVmore » by epithelial cells. This hypothesis was supported by confocal and electron microscopy analyses. The sequestration and progressive release of HCMV by kidney and mammary epithelial cells may play an important role in the excretion of the virus in urine and breast milk and may thereby contribute to HCMV transmission. - Highlights: • Primary renal and mammary epithelial cells are permissive to HCMV infection. • HCMV is sequestered by epithelial cells and this phenomenon does not require viral replication. • HCMV sequestration by epithelial cells is reduced by antibodies and IFN-γ.« less

Cadherin-23 Mediates Heterotypic Cell-Cell Adhesion between Breast Cancer Epithelial Cells and Fibroblasts

PubMed Central

Apostolopoulou, Maria; Ligon, Lee

2012-01-01

In the early stages of breast cancer metastasis, epithelial cells penetrate the basement membrane and invade the surrounding stroma, where they encounter fibroblasts. Paracrine signaling between fibroblasts and epithelial tumor cells contributes to the metastatic cascade, but little is known about the role of adhesive contacts between these two cell types in metastasis. Here we show that MCF-7 breast cancer epithelial cells and normal breast fibroblasts form heterotypic adhesions when grown together in co-culture, as evidenced by adhesion assays. PCR and immunoblotting show that both cell types express multiple members of the cadherin superfamily, including the atypical cadherin, cadherin-23, when grown in isolation and in co-culture. Immunocytochemistry experiments show that cadherin-23 localizes to homotypic adhesions between MCF-7 cells and also to heterotypic adhesions between the epithelial cells and fibroblasts, and antibody inhibition and RNAi experiments show that cadherin-23 plays a role in mediating these adhesive interactions. Finally, we show that cadherin-23 is upregulated in breast cancer tissue samples, and we hypothesize that heterotypic adhesions mediated by this atypical cadherin may play a role in the early stages of metastasis. PMID:22413011

CD4+ lymphocytes control gut epithelial apoptosis and mediate survival in sepsis

PubMed Central

Stromberg, Paul E.; Woolsey, Cheryl A.; Clark, Andrew T.; Clark, Jessica A.; Turnbull, Isaiah R.; McConnell, Kevin W.; Chang, Katherine C.; Chung, Chun-Shiang; Ayala, Alfred; Buchman, Timothy G.; Hotchkiss, Richard S.; Coopersmith, Craig M.

2009-01-01

Lymphocytes help determine whether gut epithelial cells proliferate or differentiate but are not known to affect whether they live or die. Here, we report that lymphocytes play a controlling role in mediating gut epithelial apoptosis in sepsis but not under basal conditions. Gut epithelial apoptosis is similar in unmanipulated Rag-1−/− and wild-type (WT) mice. However, Rag-1−/− animals have a 5-fold augmentation in gut epithelial apoptosis following cecal ligation and puncture (CLP) compared to septic WT mice. Reconstitution of lymphocytes in Rag-1−/− mice via adoptive transfer decreases intestinal apoptosis to levels seen in WT animals. Subset analysis indicates that CD4+ but not CD8+, γδ, or B cells are responsible for the antiapoptotic effect of lymphocytes on the gut epithelium. Gut-specific overexpression of Bcl-2 in transgenic mice decreases mortality following CLP. This survival benefit is lymphocyte dependent since gut-specific overexpression of Bcl-2 fails to alter survival when the transgene is overexpressed in Rag-1−/− mice. Further, adoptively transferring lymphocytes to Rag-1−/− mice that simultaneously overexpress gut-specific Bcl-2 results in improved mortality following sepsis. Thus, sepsis unmasks CD4+ lymphocyte control of gut apoptosis that is not present under homeostatic conditions, which acts as a key determinant of both cellular survival and host mortality.—Stromberg, P. E., Woolsey, C. A., Clark, A. T., Clark, J. A., Turnbull, I. R., McConnell, K. W., Chang, K. C., Chung, C.-S., Ayala, A., Buchman, T. G., Hotchkiss, R. S., Coopersmith, C. M. CD4+ lymphocytes control gut epithelial apoptosis and mediate survival in sepsis. PMID:19158156

Intestinal epithelial cell secretion of RELM-beta protects against gastrointestinal worm infection

USDA-ARS?s Scientific Manuscript database

IL-4 and IL-13 protect against parasitic helminths, but little is known about the mechanism of host protection. We show that IL-4/IL-13 confer immunity against worms by inducing intestinal epithelial cells (IEC) to differentiate into goblet cells that secrete resistin-like molecule beta (RELMB). R...

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.

Effects of 4-META/MMA-TBB Resin at Different Curing Stages on Osteoblasts and Gingival Epithelial Cells.

PubMed

Morotomi, Takahiko; Hirata-Tsuchiya, Shizu; Washio, Ayako; Kitamura, Chiaki

2016-01-01

To assess the effects of different curing stages of 4-META/MMA-TBB resin on osteoblasts and gingival keratinocytes. The MC3T3-E1 murine pre-osteoblastic cell line and GE-1 murine gingival epithelial cell line were cultured with mixtures of Super-Bond C&B at different curing stages, and the cell viability was assessed. The alkaline phosphatase (ALP) activity of the MC3T3-E1 cells was also assessed. The majority of the MC3T3-E1 cells died and showed no ALP activity when cultured with 4-META/MMA-TBB resin during the initial curing phase (1 min of curing). A later curing phase of the 4-META/MMA-TBB resin (7 min of curing) showed cytotoxicity at day 1, but the toxic effect was temporary and the proliferative capacity and ALP activity in the cells were similar to control cells at day 7. Completely cured 4-META/MMA-TBB resin (after 1 or 12 h of curing) did not affect the cell viability or ALP activity of the MC3T3-E1 cells. In contrast, 4-META/MMA-TBB resin showed no effect on the GE-1 cells at any stage of curing. Although 4-META/MMA-TBB resin during the initial curing phase shows toxic effects on MC3T3-E1 cells, that cytotoxicity is minimal at later curing phases. In contrast, neither the uncured nor cured resins affected the GE-1 cells.

Inhibition of experimental ascending urinary tract infection by an epithelial cell-surface receptor analogue

NASA Astrophysics Data System (ADS)

Edén, C. Svanborg; Freter, R.; Hagberg, L.; Hull, R.; Hull, S.; Leffler, H.; Schoolnik, G.

1982-08-01

It has been shown that the establishment of urinary tract infection by Escherichia coli is dependent on attachment of the bacteria to epithelial cells1-4. The attachment involves specific epithelial cell receptors, which have been characterized as glycolipids5-10. Reversible binding to cell-surface mannosides may also be important4,11-13. This suggests an approach to the treatment of infections-that of blocking bacterial attachment with cell membrane receptor analogues. Using E. coli mutants lacking one or other of the two binding specificities (glycolipid and mannose), we show here that glycolipid analogues can block in vitro adhesion and in vivo urinary tract infection.

Epithelial cell kinase-B61: an autocrine loop modulating intestinal epithelial migration and barrier function.

PubMed

Rosenberg, I M; Göke, M; Kanai, M; Reinecker, H C; Podolsky, D K

1997-10-01

Epithelial cell kinase (Eck) is a member of a large family of receptor tyrosine kinases whose functions remain largely unknown. Expression and regulation of Eck and its cognate ligand B61 were analyzed in the human colonic adenocarcinoma cell line Caco-2. Immunocytochemical staining demonstrated coexpression of Eck and B61 in the same cells, suggestive of an autocrine loop. Eck levels were maximal in preconfluent cells. In contrast, B61 levels were barely detectable in preconfluent cells and increased progressively after the cells reached confluence. Caco-2 cells cultured in the presence of added B61 showed a significant reduction in the levels of dipeptidyl peptidase and sucrase-isomaltase mRNA, markers of Caco-2 cell differentiation. Cytokines interleukin-1beta (IL-1beta), basic fibroblast growth factor, IL-2, epidermal growth factor, and transforming growth factor-beta modulated steady-state levels of Eck and B61 mRNA and regulated Eck activation as assessed by tyrosine phosphorylation. Functionally, stimulation of Eck by B61 resulted in increased proliferation, enhanced barrier function, and enhanced restitution of injured epithelial monolayers. These results suggest that the Eck-B61 interaction, a target of regulatory peptides, plays a role in intestinal epithelial cell development, migration, and barrier function, contributing to homeostasis and preservation of continuity of the epithelial barrier.

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

Immunopathology of childhood celiac disease-Key role of intestinal epithelial cells.

PubMed

Pietz, Grzegorz; De, Rituparna; Hedberg, Maria; Sjöberg, Veronika; Sandström, Olof; Hernell, Olle; Hammarström, Sten; Hammarström, Marie-Louise

2017-01-01

Celiac disease is a chronic inflammatory disease of the small intestine mucosa due to permanent intolerance to dietary gluten. The aim was to elucidate the role of small intestinal epithelial cells in the immunopathology of celiac disease in particular the influence of celiac disease-associated bacteria. Duodenal biopsies were collected from children with active celiac disease, treated celiac disease, and clinical controls. Intestinal epithelial cells were purified and analyzed for gene expression changes at the mRNA and protein levels. Two in vitro models for human intestinal epithelium, small intestinal enteroids and polarized tight monolayers, were utilized to assess how interferon-γ, interleukin-17A, celiac disease-associated bacteria and gluten influence intestinal epithelial cells. More than 25 defense-related genes, including IRF1, SPINK4, ITLN1, OAS2, CIITA, HLA-DMB, HLA-DOB, PSMB9, TAP1, BTN3A1, and CX3CL1, were significantly upregulated in intestinal epithelial cells at active celiac disease. Of these genes, 70% were upregulated by interferon-γ via the IRF1 pathway. Most interestingly, IRF1 was also upregulated by celiac disease-associated bacteria. The NLRP6/8 inflammasome yielding CASP1 and biologically active interleukin-18, which induces interferon-γ in intraepithelial lymphocytes, was expressed in intestinal epithelial cells. A key factor in the epithelial reaction in celiac disease appears to be over-expression of IRF1 that could be inherent and/or due to presence of undesirable microbes that act directly on IRF1. Dual activation of IRF1 and IRF1-regulated genes, both directly and via the interleukin-18 dependent inflammasome would drastically enhance the inflammatory response and lead to the pathological situation seen in active celiac disease.

Immunopathology of childhood celiac disease—Key role of intestinal epithelial cells

PubMed Central

Hedberg, Maria; Sjöberg, Veronika; Sandström, Olof; Hernell, Olle; Hammarström, Sten

2017-01-01

Background & Aims Celiac disease is a chronic inflammatory disease of the small intestine mucosa due to permanent intolerance to dietary gluten. The aim was to elucidate the role of small intestinal epithelial cells in the immunopathology of celiac disease in particular the influence of celiac disease-associated bacteria. Methods Duodenal biopsies were collected from children with active celiac disease, treated celiac disease, and clinical controls. Intestinal epithelial cells were purified and analyzed for gene expression changes at the mRNA and protein levels. Two in vitro models for human intestinal epithelium, small intestinal enteroids and polarized tight monolayers, were utilized to assess how interferon-γ, interleukin-17A, celiac disease-associated bacteria and gluten influence intestinal epithelial cells. Results More than 25 defense-related genes, including IRF1, SPINK4, ITLN1, OAS2, CIITA, HLA-DMB, HLA-DOB, PSMB9, TAP1, BTN3A1, and CX3CL1, were significantly upregulated in intestinal epithelial cells at active celiac disease. Of these genes, 70% were upregulated by interferon-γ via the IRF1 pathway. Most interestingly, IRF1 was also upregulated by celiac disease-associated bacteria. The NLRP6/8 inflammasome yielding CASP1 and biologically active interleukin-18, which induces interferon-γ in intraepithelial lymphocytes, was expressed in intestinal epithelial cells. Conclusion A key factor in the epithelial reaction in celiac disease appears to be over-expression of IRF1 that could be inherent and/or due to presence of undesirable microbes that act directly on IRF1. Dual activation of IRF1 and IRF1-regulated genes, both directly and via the interleukin-18 dependent inflammasome would drastically enhance the inflammatory response and lead to the pathological situation seen in active celiac disease. PMID:28934294

In vitro differentiation of human tooth germ stem cells into endothelial- and epithelial-like cells.

PubMed

Doğan, Ayşegül; Demirci, Selami; Şahin, Fikrettin

2015-01-01

Current clinical techniques in dental practice include stem cell and tissue engineering applications. Dental stem cells are promising primary cell source for mainly tooth tissue engineering. Interaction of mesenchymal stem cell with epithelial and endothelial cells is strictly required for an intact tooth morphogenesis. Therefore, it is important to investigate whether human tooth germ stem cells (hTGSCs) derived from wisdom tooth are suitable for endothelial and epithelial cell transformation in dental tissue regeneration approaches. Differentiation into endothelial and epithelial cell lineages were mimicked under defined conditions, confirmed by real time PCR, western blotting and immunocytochemical analysis by qualitative and quantitative methods. HUVECs and HaCaT cells were used as positive controls for the endothelial and epithelial differentiation assays, respectively. Immunocytochemical and western blotting analysis revealed that terminally differentiated cells expressed cell-lineage markers including CD31, VEGFR2, VE-Cadherin, vWF (endothelial cell markers), and cytokeratin (CK)-17, CK-19, EpCaM, vimentin (epithelial cell markers) in significant levels with respect to undifferentiated control cells. Moreover, high expression levels of VEGFR1, VEGFR2, VEGF, CK-18, and CK-19 genes were detected in differentiated endothelial and epithelial-like cells. Endothelial-like cells derived from hTGSCs were cultured on Matrigel, tube-like structure formations were followed as an indication for functional endothelial differentiation. hTGSCs successfully differentiate into various cell types with a broad range of functional abilities using an in vitro approach. These findings suggest that hTGSCs may serve a potential stem cell source for tissue engineering and cell therapy of epithelial and endothelial tissue. © 2014 International Federation for Cell Biology.

Cigarette smoke suppresses Bik to cause epithelial cell hyperplasia and mucous cell metaplasia.

PubMed

Mebratu, Yohannes A; Schwalm, Kurt; Smith, Kevin R; Schuyler, Mark; Tesfaigzi, Yohannes

2011-06-01

Aberrant regulation of airway epithelial cell numbers in airways leads to increased mucous secretions in chronic lung diseases such as chronic bronchitis. Because the Bcl-2 family of proteins is crucial for airway epithelial homeostasis, identifying the players that reduce cigarette smoke (CS)-induced mucous cell metaplasia can help to develop effective therapies. To identify the Bcl-2 family of proteins that play a role in reducing CS-induced mucous cell metaplasia. We screened for dysregulated expression of the Bcl-2 family members. We identified Bik to be significantly reduced in bronchial brushings of patients with chronic epithelial cell hyperplasia compared with nondiseased control subjects. Reduced Bik but increased MUC5AC mRNA levels were also detected when normal human airway epithelial cells (HAECs) were exposed to CS or when autopsy tissues from former smokers with and without chronic bronchitis were compared. Similarly, exposure of C57Bl/6 mice to CS resulted in increased numbers of epithelial and mucous cells per millimeter of basal lamina, along with reduced Bik but increased Muc5ac expression, and this change was sustained even when mice were allowed to recover in filtered air for 8 weeks. Restoring Bik expression significantly suppressed CS-induced mucous cell metaplasia in differentiated primary HAEC cultures and in airways of mice in vivo. Bik blocked nuclear translocation of phospho-ERK1/2 to induce apoptosis of HAECs. The conserved Leu61 within Bik and ERK1/2 activation were essential to induce cell death in hyperplastic mucous cells. These studies show that CS suppresses Bik expression to block airway epithelia cell death and thereby increases epithelial cell hyperplasia in chronic bronchitis.

Arsenic Compromises Conducting Airway Epithelial Barrier Properties in Primary Mouse and Immortalized Human Cell Cultures

PubMed Central

Sherwood, Cara L.; Liguori, Andrew E.; Olsen, Colin E.; Lantz, R. Clark; Burgess, Jefferey L.; Boitano, Scott

2013-01-01

Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb)] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE) cell model we found that both micromolar (3.9 μM) and submicromolar (0.8 μM) arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl) Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-). We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway. PMID:24349408

Neoplastic transformation of human thyroid epithelial cells by ionizing radiation

NASA Astrophysics Data System (ADS)

Herceg, Zdenko

Neoplastic transformation of human thyroid epithelial cells has been investigated following exposure to ionizing radiation in vitro. The effects of radiation type, irradiation regime, and postirradiation passaging were examined using a human thyroid epithelial cell line, designated HToriS, which was previously immortalized with SV40 genome. Exponentially growing HToriS cells were irradiated with graded doses of 137 Cs gamma- and 238pu alpha-irradiation. Cells were irradiated with either a single or multiple doses of 0.5, 1, 2, 3, or 4 Gy gamma-radiation, or single doses of 0.125, 0.25, 0.5, 1, or 1.5 Gy gamma-radiation. Following passaging, the cells were transplanted into the athymic nude mice, and the animals were screened for tumour formation. Statistically significant increases in tumour incidence were obtained with both gamma- and alpha-irradiation and with both single and multiple irradiation regimes as compared with the un-irradiated group. Regardless of radiation type and or radiation regime there appears to be a trend, with increasing doses of radiation, in which tumour incidence increases and reaches a maximum, after which the tumour incidence decreases. Tumours were characterized by histopathological examination as undifferentiated carcinomas. Investigation of expression time following irradiation demonstrated that post-irradiation passaging, generally regarded as a critical step for expression of radiation-induced DNA damage, was not a prerequisite for the neoplastic conversion of irradiated cells with this system. Cell lines were established from the tumours and their identification and characterization carried out. All cell lines established were determined to be derived from the parent HTori3 cells by DNA fingerprinting, karyotype analysis, cytokeratin staining, and SV40 large T-antigen staining. Tumorigenicity of the cell lines was confirmed by retransplantation. Comparison of the morphology in vitro showed that the tumour cell lines retained the

Clathrin-mediated endocytosis and transcytosis of enterotoxigenic Escherichia coli F4 fimbriae in porcine intestinal epithelial cells.

PubMed

Rasschaert, Kristien; Devriendt, Bert; Favoreel, Herman; Goddeeris, Bruno M; Cox, Eric

2010-10-15

Enterotoxigenic Escherichia coli (ETEC) cause severe diarrhea in neonatal and recently weaned piglets. Previously, we demonstrated that oral immunization of F4 receptor positive piglets with purified F4 fimbriae induces a protective F4-specific intestinal immune response. However, in F4 receptor negative animals no F4-specific immune response can be elicited, indicating that the induction of an F4-specific mucosal immune response upon oral immunisation is receptor-dependent. Although F4 fimbriae undergo transcytosis across the intestinal epithelium in vivo, the endocytosis pathways used remain unknown. In the present study, we characterized the internalization of F4 fimbriae in the porcine intestinal epithelial cell line IPEC-J2. The results in the present study demonstrate that F4 fimbriae are internalized through a clathrin-dependent pathway. Furthermore, our results suggest that F4 fimbriae are transcytosed across differentiated IPEC-J2 cells. This receptor-dependent transcytosis of F4 fimbriae may explain the immunogenicity of these fimbriae upon oral administration in vivo. (c) 2010 Elsevier B.V. All rights reserved.

Comparison of para-aminophenol cytotoxicity in rat renal epithelial cells and hepatocytes.

PubMed

Li, Ying; Bentzley, Catherine M; Tarloff, Joan B

2005-04-01

Several chemicals, including para-aminophenol (PAP), produce kidney damage in the absence of hepatic damage. Selective nephrotoxicity may be related to the ability of the kidney to reabsorb filtered water, thereby raising the intraluminal concentration of toxicants and exposing tubular epithelial cells to higher concentrations than would be present in other tissues. The present experiments tested the hypothesis that hepatocytes and renal epithelial cells exposed to equivalent concentrations of PAP would be equally susceptible to toxicity. Hepatocytes and renal epithelial cells were prepared by collagenase digestion of tissues obtained from female Sprague-Dawley rats. Toxicity was monitored using trypan blue exclusion, oxygen consumption and ATP content. We measured the rate of PAP clearance and formation of PAP-glutathione conjugate by HPLC. We found that renal epithelial cells accumulated trypan blue and showed declines in oxygen consumption and ATP content at significantly lower concentrations of PAP and at earlier time points than hepatocytes. The half-life of PAP in hepatocyte incubations was significantly shorter (0.71+/-0.07 h) than in renal epithelial cell incubations (1.33+/-0.23 h), suggesting that renal epithelial cells were exposed to PAP for longer time periods than hepatocytes. Renal epithelial cells formed significantly less glutathione conjugates of PAP (PAP-SG) than did hepatocytes, consistent with less efficient detoxification of reactive PAP intermediates by renal epithelial cells. Finally, hepatocytes contained significant more reduced glutathione (NPSH) than did renal epithelial cells, possibly explaining the enhanced formation of PAP-SG by this cell population. In conclusion, our data indicates that renal epithelial cells are intrinsically more susceptible to PAP cytotoxicity than are hepatocytes. This enhanced cytotoxicity may be due to longer exposure to PAP and/or reduced detoxification of reactive intermediates due to lower concentrations

[Quantitative characterization of nuclear polymorphism of the epithelial cells in fibroadenomatosis and cancer of the breast].

PubMed

Ganina, K P; Petunin, Iu I; Timoshenko, Ia G

1989-01-01

A method for quantitative analysis of epithelial cell nuclear polymorphism was suggested, viz. identification of general statistical population using Petunin's criterion. This criterion was employed to assess heterogeneity of visible surface of interphase epithelial cell nuclei and to assay nuclear DNA level in fibroadenomatous hyperplasia and cancer of the breast. Heterogeneity index (h), alongside with other parameters, appeared useful for quantitative assessment of the disease: heterogeneity index values ranging 0.1-0.4 point to pronounced heterogeneity of epithelial cell nucleus surface and DNA level, and are suggestive of malignant transformation of tissue, whereas benign proliferation of the epithelium is usually characterized by 0.4 less than h less than or equal to 0.9.

Protein S is inducible by interleukin 4 in T cells and inhibits lymphoid cell procoagulant activity

PubMed Central

Smiley, Stephen T.; Boyer, Sarah N.; Heeb, Mary J.; Griffin, John H.; Grusby, Michael J.

1997-01-01

Extravascular procoagulant activity often accompanies cell-mediated immune responses and systemic administration of pharmacologic anticoagulants prevents cell-mediated delayed-type hypersensitivity reactions. These observations suggest a direct association between coagulation and cell-mediated immunity. The cytokine interleukin (IL)-4 potently suppresses cell-mediated immune responses, but its mechanism of action remains to be determined. Herein we demonstrate that the physiologic anticoagulant protein S is IL-4-inducible in primary T cells. Although protein S was known to inhibit the classic factor Va-dependent prothrombinase assembled by endothelial cells and platelets, we found that protein S also inhibits the factor Va-independent prothrombinase assembled by lymphoid cells. Thus, protein S-mediated down-regulation of lymphoid cell procoagulant activity may be one mechanism by which IL-4 antagonizes cell-mediated immunity. PMID:9326636

Isolation, separation, and characterization of epithelial and connective cells from rat palate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Terranova, Victor Paul

1979-01-01

Epithelial and connective tissue cells were isolated from rat palate by sequential collagenase, hyaluronidase and trypsin digestion of the extracellular matrix. Differences between the two populations were noted with respect to total cell protein, total cell water, proline uptake and incorporation, percent collagen synthesized, effects of parathyroid hormone, metabolism of D-valine and cell density. Basal epithelial cells were subsequently separated from the heterogeneous epithelial cell population on shallow linear density gradients by velocity centrifugation. The type of collagen synthesized by the basal epithelial cells was compared to the type of collagen synthesized by the connective tissue cells by means ofmore » labeled amino acid incorporation ratios. Cells isolated from the epithelial and connective tissue were compared. From these studies it can be concluded that epithelial and connective tissue cells can be isolated from rat palate as viable and distinct populations with respect to the biochemical parameters examined. Furthermore, subpopulations can be separated and biochemically characterized.« less

Effects of keratinocyte growth factor on skin epithelial differentiation of human amnion epithelial cells.

PubMed

Fatimah, Simat Siti; Tan, Geok Chin; Chua, Kienhui; Tan, Ay Eeng; Nur Azurah, Abdul Ghani; Hayati, Abdul Rahman

2013-08-01

The aim of the present study was to determine the effects of KGF on the differentiation of cultured human amnion epithelial cells (HAECs) towards skin keratinocyte. HAECs at passage 1 were cultured in medium HAM's F12: Dulbecco's Modified Eagles Medium (1:1) supplemented with different concentrations of KGF (0, 5, 10, 20, 30 and 50 ng/ml KGF). Dose-response of KGF on HAECs was determined by morphological assessment; growth kinetic evaluation; immunocytochemical analysis; stemness and epithelial gene expression quantification with two step real time RT-PCR. KGF promotes the proliferation of HAECs with maximal effect observed at 10 ng/ml KGF. However, KGF decreased the stemness genes expression: Oct-3/4, Sox-2, Nanog3, Rex-1, FGF-4, FZD-9 and BST-1. KGF also down-regulates epithelial genes expression: CK3, CK18, CK19, Integrin-β1, p63 and involucrin in cultured HAECs. No significant difference on the gene expression was detected for each Nestin, ABCG-2, CK1 and CK14 in KGF-treated HAECs. Immunocytochemical analysis for both control and KGF-treated HAECs demonstrated positive staining against CK14 and CK18 but negative staining against involucrin. The results suggested that KGF stimulates an early differentiation of HAECs towards epidermal cells. Differentiation of KGF-treated HAECs to corneal lineage is unfavourable. Therefore, further studies are needed to elucidate the roles of KGF in the differentiation of HAECs towards skin keratinocytes. Copyright © 2012 Elsevier Ltd and ISBI. All rights reserved.

Nedd4L expression is decreased in ovarian epithelial cancer tissues compared to ovarian non-cancer tissue.

PubMed

Yang, Qiuyun; Zhao, Jinghe; Cui, Manhua; Gi, Shuting; Wang, Wei; Han, Xiaole

2015-12-01

Recent studies have demonstrated that the neural precursor cell expressed, developmentally downregulated 4-like (Nedd4L) gene plays a role in the progression of various cancers. However, reports describing Nedd4L expression in ovarian cancer tissues are limited. A cohort (n = 117) of archival formalin-fixed, paraffin embedded resected normal ovarian epithelial tissues (n = 10), benign ovarian epithelial tumor tissues (n = 10), serous borderline ovarian epithelial tumor tissues (n = 14), mucous borderline ovarian epithelial tumor tissues (n = 11), and invasive ovarian epithelial cancer tissues (n = 72) were assessed for Nedd4L protein expression using immunohistochemistry. Nedd4L protein expression was significantly decreased in invasive ovarian epithelial cancer tissues compared to non-cancer tissues (P < 0.05). Decreased Nedd4L protein expression correlated with clinical stage, pathological grade, lymph node metastasis and survival (P < 0.05). Nedd4L protein expression may be an independent prognostic marker of ovarian cancer development. © 2015 Japan Society of Obstetrics and Gynecology.

Epithelial rotation is preceded by planar symmetry breaking of actomyosin and protects epithelial tissue from cell deformations.

PubMed

Viktorinová, Ivana; Henry, Ian; Tomancak, Pavel

2017-11-01

Symmetry breaking is involved in many developmental processes that form bodies and organs. One of them is the epithelial rotation of developing tubular and acinar organs. However, how epithelial cells move, how they break symmetry to define their common direction, and what function rotational epithelial motions have remains elusive. Here, we identify a dynamic actomyosin network that breaks symmetry at the basal surface of the Drosophila follicle epithelium of acinar-like primitive organs, called egg chambers, and may represent a candidate force-generation mechanism that underlies the unidirectional motion of this epithelial tissue. We provide evidence that the atypical cadherin Fat2, a key planar cell polarity regulator in Drosophila oogenesis, directs and orchestrates transmission of the intracellular actomyosin asymmetry cue onto a tissue plane in order to break planar actomyosin symmetry, facilitate epithelial rotation in the opposite direction, and direct the elongation of follicle cells. In contrast, loss of this rotational motion results in anisotropic non-muscle Myosin II pulses that are disorganized in plane and causes cell deformations in the epithelial tissue of Drosophila eggs. Our work demonstrates that atypical cadherins play an important role in the control of symmetry breaking of cellular mechanics in order to facilitate tissue motion and model epithelial tissue. We propose that their functions may be evolutionarily conserved in tubular/acinar vertebrate organs.

Epithelial rotation is preceded by planar symmetry breaking of actomyosin and protects epithelial tissue from cell deformations

PubMed Central

Henry, Ian; Tomancak, Pavel

2017-01-01

Symmetry breaking is involved in many developmental processes that form bodies and organs. One of them is the epithelial rotation of developing tubular and acinar organs. However, how epithelial cells move, how they break symmetry to define their common direction, and what function rotational epithelial motions have remains elusive. Here, we identify a dynamic actomyosin network that breaks symmetry at the basal surface of the Drosophila follicle epithelium of acinar-like primitive organs, called egg chambers, and may represent a candidate force-generation mechanism that underlies the unidirectional motion of this epithelial tissue. We provide evidence that the atypical cadherin Fat2, a key planar cell polarity regulator in Drosophila oogenesis, directs and orchestrates transmission of the intracellular actomyosin asymmetry cue onto a tissue plane in order to break planar actomyosin symmetry, facilitate epithelial rotation in the opposite direction, and direct the elongation of follicle cells. In contrast, loss of this rotational motion results in anisotropic non-muscle Myosin II pulses that are disorganized in plane and causes cell deformations in the epithelial tissue of Drosophila eggs. Our work demonstrates that atypical cadherins play an important role in the control of symmetry breaking of cellular mechanics in order to facilitate tissue motion and model epithelial tissue. We propose that their functions may be evolutionarily conserved in tubular/acinar vertebrate organs. PMID:29176774

Studies Using an in Vitro Model Show Evidence of Involvement of Epithelial-Mesenchymal Transition of Human Endometrial Epithelial Cells in Human Embryo Implantation*

PubMed Central

Uchida, Hiroshi; Maruyama, Tetsuo; Nishikawa-Uchida, Sayaka; Oda, Hideyuki; Miyazaki, Kaoru; Yamasaki, Akiko; Yoshimura, Yasunori

2012-01-01

Human embryo implantation is a critical multistep process consisting of embryo apposition/adhesion, followed by penetration and invasion. Through embryo penetration, the endometrial epithelial cell barrier is disrupted and remodeled by an unknown mechanism. We have previously developed an in vitro model for human embryo implantation employing the human choriocarcinoma cell line JAR and the human endometrial adenocarcinoma cell line Ishikawa. Using this model we have shown that stimulation with ovarian steroid hormones (17β-estradiol and progesterone, E2P4) and suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, enhances the attachment and adhesion of JAR spheroids to Ishikawa. In the present study we showed that the attachment and adhesion of JAR spheroids and treatment with E2P4 or SAHA individually induce the epithelial-mesenchymal transition (EMT) in Ishikawa cells. This was evident by up-regulation of N-cadherin and vimentin, a mesenchymal cell marker, and concomitant down-regulation of E-cadherin in Ishikawa cells. Stimulation with E2P4 or SAHA accelerated Ishikawa cell motility, increased JAR spheroid outgrowth, and enhanced the unique redistribution of N-cadherin, which was most prominent in proximity to the adhered spheroids. Moreover, an N-cadherin functional blocking antibody attenuated all events but not JAR spheroid adhesion. These results collectively provide evidence suggesting that E2P4- and implanting embryo-induced EMT of endometrial epithelial cells may play a pivotal role in the subsequent processes of human embryo implantation with functional control of N-cadherin. PMID:22174415

Probiotics promote endocytic allergen degradation in gut epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Chun-Hua; Liu, Zhi-Qiang; Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON

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 barriermore » 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.« less

Whey protein hydrolysates decrease IL-8 secretion in lipopolysaccharide (LPS)-stimulated respiratory epithelial cells by affecting LPS binding to Toll-like receptor 4.

PubMed

Iskandar, Michèle M; Dauletbaev, Nurlan; Kubow, Stan; Mawji, Nadir; Lands, Larry C

2013-07-14

Whey proteins (WP) exert anti-inflammatory and antioxidant effects. Hyperbaric pressurisation of whey increases its digestibility and changes the spectrum of peptides released during digestion. We have shown that dietary supplementation with pressurised whey improves nutritional status and systemic inflammation in patients with cystic fibrosis (CF). Both clinical indices are largely affected by airway processes, to which respiratory epithelial cells actively contribute. Here, we tested whether peptides released from the digestion of pressurised whey can attenuate the inflammatory responses of CF respiratory epithelial cells. Hydrolysates of pressurised WP (pWP) and native WP (nWP, control) were generated in vitro and tested for anti-inflammatory properties judged by the suppression of IL-8 production in CF and non-CF respiratory epithelial cell lines (CFTE29o- and 1HAEo-, respectively). We observed that, in both cell lines, pWP hydrolysate suppressed IL-8 production stimulated by lipopolysaccharide (LPS) to a greater magnitude compared with nWP hydrolysate. Neither hydrolysate suppressed IL-8 production induced by TNF-α or IL-1β, suggesting an effect on the Toll-like receptor (TLR) 4 pathway, the cellular sensor for LPS. Further, neither hydrolysate affected TLR4 expression or neutralised LPS. Both pWP and nWP hydrolysates similarly reduced LPS binding to surface TLR4, while pWP tended to more potently increase extracellular antioxidant capacity. (1) anti-inflammatory properties of whey are enhanced by pressurisation; (2) suppression of IL-8 production may contribute to the clinical effects of pressurised whey supplementation on CF; (3) this effect may be partly explained by a combination of reduced LPS binding to TLR4 and enhanced extracellular antioxidant capacity.

Adhesion to the host cell surface is sufficient to mediate Listeria monocytogenes entry into epithelial cells

PubMed Central

Ortega, Fabian E.; Rengarajan, Michelle; Chavez, Natalie; Radhakrishnan, Prathima; Gloerich, Martijn; Bianchini, Julie; Siemers, Kathleen; Luckett, William S.; Lauer, Peter; Nelson, W. James; Theriot, Julie A.

2017-01-01

The intestinal epithelium is the first physiological barrier breached by the Gram-positive facultative pathogen Listeria monocytogenes during an in vivo infection. Listeria monocytogenes binds to the epithelial host cell receptor E-cadherin, which mediates a physical link between the bacterium and filamentous actin (F-actin). However, the importance of anchoring the bacterium to F-actin through E-cadherin for bacterial invasion has not been tested directly in epithelial cells. Here we demonstrate that depleting αE-catenin, which indirectly links E-cadherin to F-actin, did not decrease L. monocytogenes invasion of epithelial cells in tissue culture. Instead, invasion increased due to increased bacterial adhesion to epithelial monolayers with compromised cell–cell junctions. Furthermore, expression of a mutant E-cadherin lacking the intracellular domain was sufficient for efficient L. monocytogenes invasion of epithelial cells. Importantly, direct biotin-mediated binding of bacteria to surface lipids in the plasma membrane of host epithelial cells was sufficient for uptake. Our results indicate that the only requirement for L. monocytogenes invasion of epithelial cells is adhesion to the host cell surface, and that E-cadherin–mediated coupling of the bacterium to F-actin is not required. PMID:28877987

Expression of Pattern Recognition Receptors in Epithelial Cells Around Clinically Healthy Implants and Healthy Teeth.

PubMed

Calcaterra, Roberta; Di Girolamo, Michele; Mirisola, Concetta; Baggi, Luigi

2016-06-01

Gingival epithelial cells have a pivotal role in the recognition of microorganisms and damage-associated molecular pattern molecules and in the regulation of the immune response. The investigation of the behavior of Toll-like receptors (TLRs) and nucleotide oligomerization domain (NOD) like receptors (NLRs) around a healthy implant may help to address the first step of periimplantitis pathogenesis. To investigate by quantitative real-time polymerase chain reaction, the mRNA expressions of TLR2, TLR3, TLR4, TLR5, TLR6, TLR9, NOD1, NOD2, and NLRP3 from gingival epithelial cells of the sulcus around healthy implants and around healthy teeth. Two types of implant-abutment systems with tube-in-tube interface were tested. After 6 months of implant restoration, gingival epithelial cells were obtained from the gingival sulcus around the implants and around the adjacent teeth of 10 patients. Our results did not reach statistical significance among the mRNA expressions of TLR2, TLR3, TLR4, TLR5, TLR6, TLR9, NOD1, NOD2, and NLRP3 in epithelial cells around the implant versus around natural teeth. This study shows that the implant-abutment systems tested did not induce an immune response by the surrounding epithelial cells at 6 months since their positioning, as well as in the adjacent clincally healthy teeth.

Cigarette Smoke Suppresses Bik To Cause Epithelial Cell Hyperplasia and Mucous Cell Metaplasia

PubMed Central

Mebratu, Yohannes A.; Schwalm, Kurt; Smith, Kevin R.; Schuyler, Mark; Tesfaigzi, Yohannes

2011-01-01

Rationale: Aberrant regulation of airway epithelial cell numbers in airways leads to increased mucous secretions in chronic lung diseases such as chronic bronchitis. Because the Bcl-2 family of proteins is crucial for airway epithelial homeostasis, identifying the players that reduce cigarette smoke (CS)-induced mucous cell metaplasia can help to develop effective therapies. Objectives: To identify the Bcl-2 family of proteins that play a role in reducing CS-induced mucous cell metaplasia. Methods: We screened for dysregulated expression of the Bcl-2 family members. Measurements and Main Results: We identified Bik to be significantly reduced in bronchial brushings of patients with chronic epithelial cell hyperplasia compared with nondiseased control subjects. Reduced Bik but increased MUC5AC mRNA levels were also detected when normal human airway epithelial cells (HAECs) were exposed to CS or when autopsy tissues from former smokers with and without chronic bronchitis were compared. Similarly, exposure of C57Bl/6 mice to CS resulted in increased numbers of epithelial and mucous cells per millimeter of basal lamina, along with reduced Bik but increased Muc5ac expression, and this change was sustained even when mice were allowed to recover in filtered air for 8 weeks. Restoring Bik expression significantly suppressed CS-induced mucous cell metaplasia in differentiated primary HAEC cultures and in airways of mice in vivo. Bik blocked nuclear translocation of phospho-ERK1/2 to induce apoptosis of HAECs. The conserved Leu61 within Bik and ERK1/2 activation were essential to induce cell death in hyperplastic mucous cells. Conclusions: These studies show that CS suppresses Bik expression to block airway epithelia cell death and thereby increases epithelial cell hyperplasia in chronic bronchitis. PMID:21317312

In vitro pharmacologic restoration of CFTR-mediated chloride transport with sodium 4-phenylbutyrate in cystic fibrosis epithelial cells containing delta F508-CFTR.

PubMed Central

Rubenstein, R C; Egan, M E; Zeitlin, P L

1997-01-01

The most common cystic fibrosis transmembrane conductance regulator mutation, delta F508-CFTR, is a partially functional chloride channel that is retained in the endoplasmic reticulum and degraded. We hypothesize that a known transcriptional regulator, sodium 4-phenylbutyrate (4PBA), will enable a greater fraction of delta F508-CFTR to escape degradation and appear at the cell surface. Primary cultures of nasal polyp epithelia from CF patients (delta F508 homozygous or heterozygous), or the CF bronchial epithelial cell line IB3-1 (delta F508/W1282X) were exposed to 4PBA for up to 7 d in culture. 4PBA treatment at concentrations of 0.1 and 2 mM resulted in the restoration of forskolin-activated chloride secretion. Protein kinase A-activated, linear, 10 pS chloride channels appeared at the plasma membrane of IB3-1 cells at the tested concentration of 2.5 mM. Treatment of IB3-1 cells with 0.1-1 mM 4PBA and primary nasal epithelia with 5 mM 4PBA also resulted in the appearance of higher molecular mass forms of CFTR consistent with addition and modification of oligosaccharides in the Golgi apparatus, as detected by immunoblotting of whole cell lysates with anti-CFTR antisera. Immunocytochemistry in CF epithelial cells treated with 4PBA was consistent with increasing amounts of delta F508-CFTR. These data indicate that 4PBA is a promising pharmacologic agent for inducing correction of the CF phenotype in CF patients carrying the delta F508 mutation. PMID:9366560

In vitro pharmacologic restoration of CFTR-mediated chloride transport with sodium 4-phenylbutyrate in cystic fibrosis epithelial cells containing delta F508-CFTR.

PubMed

Rubenstein, R C; Egan, M E; Zeitlin, P L

1997-11-15

The most common cystic fibrosis transmembrane conductance regulator mutation, delta F508-CFTR, is a partially functional chloride channel that is retained in the endoplasmic reticulum and degraded. We hypothesize that a known transcriptional regulator, sodium 4-phenylbutyrate (4PBA), will enable a greater fraction of delta F508-CFTR to escape degradation and appear at the cell surface. Primary cultures of nasal polyp epithelia from CF patients (delta F508 homozygous or heterozygous), or the CF bronchial epithelial cell line IB3-1 (delta F508/W1282X) were exposed to 4PBA for up to 7 d in culture. 4PBA treatment at concentrations of 0.1 and 2 mM resulted in the restoration of forskolin-activated chloride secretion. Protein kinase A-activated, linear, 10 pS chloride channels appeared at the plasma membrane of IB3-1 cells at the tested concentration of 2.5 mM. Treatment of IB3-1 cells with 0.1-1 mM 4PBA and primary nasal epithelia with 5 mM 4PBA also resulted in the appearance of higher molecular mass forms of CFTR consistent with addition and modification of oligosaccharides in the Golgi apparatus, as detected by immunoblotting of whole cell lysates with anti-CFTR antisera. Immunocytochemistry in CF epithelial cells treated with 4PBA was consistent with increasing amounts of delta F508-CFTR. These data indicate that 4PBA is a promising pharmacologic agent for inducing correction of the CF phenotype in CF patients carrying the delta F508 mutation.

The Human Airway Epithelial Basal Cell Transcriptome

PubMed Central

Wang, Rui; Zwick, Rachel K.; Ferris, Barbara; Witover, Bradley; Salit, Jacqueline; Crystal, Ronald G.

2011-01-01

Background The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population. Methodology/Principal Findings Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the “human airway basal cell signature” as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. Conclusion/Significance The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of the stem

ELF5 in epithelial ovarian carcinoma tissues and biological behavior in ovarian carcinoma cells.

PubMed

Yan, Hongchao; Qiu, Linglin; Xie, Xiaolei; Yang, He; Liu, Yongli; Lin, Xiaoman; Huang, Hongxiang

2017-03-01

The expression of E74-like factor 5 (ELF5) in epithelial ovarian carcinoma tissues and its effects on biological behavior in ovarian carcinoma cells were assessed in search for a new approach for gene treatment of epithelial ovarian carcinoma. RT-PCR technology was applied to detect the expression of ELF5 mRNA in epithelial ovarian carcinoma (n=49), borderline ovarian epithelial tumor (n=19), benign ovarian epithelial tumor (n=31) and normal ovarian tissues (n=40). Then, we transfected recombinant plasmid pcDNA3.1‑ELF5+EGFP into human ovarian carcinoma SKOV3 cells (recombinant plasmid group) in vitro and screened out stably transfected cells to conduct multiplication culture. Western blot analysis was performed to detect the expression of ELF5 protein in the different groups. Flow cytometry was employed to detect cell apoptosis and cycles. ELF5 mRNA in epithelial ovarian carcinoma and borderline ovarian epithelial tumor tissues were significantly lower (P<0.05) than those in benign ovarian epithelial tumor and normal ovarian tissues. ELF5 protein expression in the cells of recombinant plasmid group was significantly higher compared with empty plasmid and blank control groups. The capacity of cell reproductive recombinant plasmid group at each time point decreased (P<0.05). Flow cytometry detection showed that 67.03% of cells in recombinant plasmid group was blocked in G0/G1 phase (P<0.05), compared with empty plasmid group (37.17%) and blank control group (38.24%). Apoptotic rate of recombinant plasmid group was significantly lower (31.4±1.9%; P<0.05), compared with that of empty plasmid group (9.1±2.2%) and blank control group (8.7±1.5%), and the differences were statistically significant. In conclusion, ELF5 interfered with cell cycle of human ovarian carcinoma SKOV3 cells and promoted apoptosis of human ovarian carcinoma SKOV3 cells inhibiting their growth and invasive capacity; and thus providing a new approach to gene treatment of ovarian carcinoma.

Effects of glucose on lactose synthesis in mammary epithelial cells from dairy cow.

PubMed

Lin, Ye; Sun, Xiaoxu; Hou, Xiaoming; Qu, Bo; Gao, Xuejun; Li, Qingzhang

2016-05-26

Lactose, as the primary osmotic component in milk, is the major determinant of milk volume. Glucose is the primary precursor of lactose. However, the effect of glucose on lactose synthesis in dairy cow mammary glands and the mechanism governing this process are poorly understood. Here we showed that glucose has the ability to induce lactose synthesis in dairy cow mammary epithelial cells, as well as increase cell viability and proliferation. A concentration of 12 mM glucose was the optimum concentration to induce cell growth and lactose synthesis in cultured dairy cow mammary epithelial cells. In vitro, 12 mM glucose enhanced lactose content, along with the expression of genes involved in glucose transportation and the lactose biosynthesis pathway, including GLUT1, SLC35A2, SLC35B1, HK2, β4GalT-I, and AKT1. In addition, we found that AKT1 knockdown inhibited cell growth and lactose synthesis as well as expression of GLUT1, SLC35A2, SLC35B1, HK2, and β4GalT-I. Glucose induces cell growth and lactose synthesis in dairy cow mammary epithelial cells. Protein kinase B alpha acts as a regulator of metabolism in dairy cow mammary gland to mediate the effects of glucose on lactose synthesis.

CD47 regulates renal tubular epithelial cell self-renewal and proliferation following renal ischemia reperfusion.

PubMed

Rogers, Natasha M; Zhang, Zheng J; Wang, Jiao-Jing; Thomson, Angus W; Isenberg, Jeffrey S

2016-08-01

Defects in renal tubular epithelial cell repair contribute to renal ischemia reperfusion injury, cause acute kidney damage, and promote chronic renal disease. The matricellular protein thrombospondin-1 and its receptor CD47 are involved in experimental renal ischemia reperfusion injury, although the role of this interaction in renal recovery is unknown. We found upregulation of self-renewal genes (transcription factors Oct4, Sox2, Klf4 and cMyc) in the kidney of CD47(-/-) mice after ischemia reperfusion injury. Wild-type animals had minimal self-renewal gene expression, both before and after injury. Suggestive of cell autonomy, CD47(-/-) renal tubular epithelial cells were found to increase expression of the self-renewal genes. This correlated with enhanced proliferative capacity compared with cells from wild-type mice. Exogenous thrombospondin-1 inhibited self-renewal gene expression in renal tubular epithelial cells from wild-type but not CD47(-/-) mice, and this was associated with decreased proliferation. Treatment of renal tubular epithelial cells with a CD47 blocking antibody or CD47-targeting small interfering RNA increased expression of some self-renewal transcription factors and promoted cell proliferation. In a syngeneic kidney transplant model, treatment with a CD47 blocking antibody increased self-renewal transcription factor expression, decreased tissue damage, and improved renal function compared with that in control mice. Thus, thrombospondin-1 via CD47 inhibits renal tubular epithelial cell recovery after ischemia reperfusion injury through inhibition of proliferation/self-renewal. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Human airway epithelial cells investigated by atomic force microscopy: A hint to cystic fibrosis epithelial pathology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lasalvia, Maria; Istituto Nazionale di Fisica Nucleare, Sezione di Bari, Bari; Castellani, Stefano

The pathophysiology of cystic fibrosis (CF) airway disease stems from mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, leading to a chronic respiratory disease. Actin cytoskeleton is disorganized in CF airway epithelial cells, likely contributing to the CF-associated basic defects, i.e. defective chloride secretion and sodium/fluid hypersorption. In this work, we aimed to find whether this alteration could be pointed out by means of Atomic Force Microscopy (AFM) investigation, as roughness and Young's elastic module. Moreover, we also sought to determine whether disorganization of actin cytoskeleton is linked to hypersoption of apical fluid. Not only CFBE41o- (CFBE) cells, immortalizedmore » airway epithelial cells homozygous for the F508del CFTR allele, showed a different morphology in comparison with 16HBE14o- (16HBE) epithelial cells, wild-type for CFTR, but also they displayed a lack of stress fibers, suggestive of a disorganized actin cytoskeleton. AFM measurements showed that CFBE cells presented a higher membrane roughness and decreased rigidity as compared with 16HBE cells. CFBE overexpressing wtCFTR became more elongated than the parental CFBE cell line and presented actin stress fibers. CFBE cells absorbed more fluid from the apical compartment. Study of fluid absorption with the F-actin-depolymerizing agent Latrunculin B demonstrated that actin cytoskeletal disorganization increased fluid absorption, an effect observed at higher magnitude in 16HBE than in CFBE cells. For the first time, we demonstrate that actin cytoskeleton disorganization is reflected by AFM parameters in CF airway epithelial cells. Our data also strongly suggest that the lack of stress fibers is involved in at least one of the early step in CF pathophysiology at the levels of the airways, i.e. fluid hypersorption. - Highlights: • CF bronchial epithelial (CFBE) cells show a disorganized actin cytoskeleton. • CFBE cells present high roughness and low rigidity

Endothelial induced EMT in breast epithelial cells with stem cell properties.

PubMed

Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J R; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A; Petersen, Ole William; Magnusson, Magnus K; Gudjonsson, Thorarinn

2011-01-01

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

Endothelial Induced EMT in Breast Epithelial Cells with Stem Cell Properties

PubMed Central

Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J. R.; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A.; Petersen, Ole William; Magnusson, Magnus K.; Gudjonsson, Thorarinn

2011-01-01

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44high/CD24low ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer. PMID:21915264

Choroid plexus epithelial cells express the adhesion protein P-cadherin at cell-cell contacts and syntaxin-4 in the luminal membrane domain.

PubMed

Christensen, Inga Baasch; Mogensen, Esben Nees; Damkier, Helle Hasager; Praetorius, Jeppe

2018-05-01

The choroid plexus epithelial cells (CPECs) belong to a small group of polarized cells, where the Na + -K + -ATPase is expressed in the luminal membrane. The basic polarity of the cells is, therefore, still debated. We investigated the subcellular distribution of an array of proteins known to play fundamental roles either in establishing and maintaining basic cell polarity or in the polarized delivery and recycling of plasma membrane proteins. Immunofluorescence histochemical analysis was applied to determine the subcellular localization of apical and basolateral membrane determinants. Mass spectrometry analysis of CPECs isolated by fluorescence-activated cell sorting was applied to determine the expression of specific forms of the proteins. CPECs mainly express the cell-adhesive P-cadherin, which is localized to the lateral membranes. Proteins belonging to the Crumbs and partitioning defective (Par) protein complexes were all localized to the luminal membrane domain. Par-1 and the Scribble complex were localized to the basolateral membrane domain. Lethal(2) giant larvae homolog 2 (Lgl2) labeling was preferentially observed in the luminal membrane domain. Phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) was immunolocalized to the basolateral membrane domain, while phosphatidylinositol 4,5-bisphosphate (PIP 2 ) staining was most prominent in the luminal membrane domain along with the PIP 3 phosphatase, Pten. The apical target-SNARE syntaxin-3 and the basolateral target-SNARE syntaxin-4 were both localized to the apical membrane domain in CPECs, which lack cellular expression of the clathrin adaptor protein AP-1B for basolateral protein recycling. In conclusion, the CPECs are conventionally polarized, but express P-cadherin at cell-cell contacts, and Lgl2 and syntaxin-4 in the luminal plasma membrane domain.

Epithelial to mesenchymal transition in human endocrine islet cells

PubMed Central

Moreno-Amador, José Luis; Téllez, Noèlia; Marin, Sandra; Aloy-Reverté, Caterina; Semino, Carlos; Nacher, Montserrat

2018-01-01

Background β-cells undergo an epithelial to mesenchymal transition (EMT) when expanded in monolayer culture and give rise to highly proliferative mesenchymal cells that retain the potential to re-differentiate into insulin-producing cells. Objective To investigate whether EMT takes place in the endocrine non-β cells of human islets. Methodology Human islets isolated from 12 multiorgan donors were dissociated into single cells, purified by magnetic cell sorting, and cultured in monolayer. Results Co-expression of insulin and the mesenchymal marker vimentin was identified within the first passage (p1) and increased subsequently (insulin+vimentin+ 7.2±6% at p1; 43±15% at p4). The endocrine non-β-cells did also co-express vimentin (glucagon+vimentin+ 59±1.5% and 93±6%, somatostatin+vimentin+ 16±9.4% and 90±10% at p1 and p4 respectively; PP+vimentin+ 74±14% at p1; 88±12% at p2). The percentage of cells expressing only endocrine markers was progressively reduced (0.6±0.2% insulin+, 0.2±0.1% glucagon+, and 0.3±0.2% somatostatin+ cells at p4, and 0.7±0.3% PP+ cells at p2. Changes in gene expression were also indicated of EMT, with reduced expression of endocrine markers and the epithelial marker CDH-1 (p<0.01), and increased expression of mesenchymal markers (CDH-2, SNAI2, ZEB1, ZEB2, VIM, NT5E and ACTA2; p<0.05). Treatment with the EMT inhibitor A83-01 significantly reduced the percentage of co-expressing cells and preserved the expression of endocrine markers. Conclusions In adult human islets, all four endocrine islet cell types undergo EMT when islet cells are expanded in monolayer conditions. The presence of EMT in all islet endocrine cells could be relevant to design of strategies aiming to re-differentiate the expanded islet cells towards a β-cell phenotype. PMID:29360826

ARP2, a novel pro-apoptotic protein expressed in epithelial prostate cancer LNCaP cells and epithelial ovary CHO transformed cells.

PubMed

Mas-Oliva, Jaime; Navarro-Vidal, Enrique; Tapia-Vieyra, Juana Virginia

2014-01-01

Neoplastic epithelial cells generate the most aggressive types of cancers such as those located in the lung, breast, colon, prostate and ovary. During advanced stages of prostate cancer, epithelial cells are associated to the appearance of androgen-independent tumors, an apoptotic-resistant phenotype that ultimately overgrows and promotes metastatic events. We have previously identified and electrophysiologically characterized a novel Ca(2+)-permeable channel activated during apoptosis in the androgen-independent prostate epithelial cancer cell line, LNCaP. In addition, we reported for the first time the cloning and characterization of this channel-like molecule named apoptosis regulated protein 2 (ARP2) associated to a lethal influx of Ca(2+) in Xenopus oocytes. In the present study, LNCaP cells and Chinese hamster ovary cells (CHO cell line) transfected with arp2-cDNA are induced to undergo apoptosis showing an important impact on cell viability and activation of caspases 3 and 7 when compared to serum deprived grown cells and ionomycin treated cells. The subcellular localization of ARP2 in CHO cells undergoing apoptosis was studied using confocal microscopy. While apoptosis progresses, ARP2 initially localized in the peri-nuclear region of cells migrates with time towards the plasma membrane region. Based on the present results and those of our previous studies, the fact that ARP2 constitutes a novel cation channel is supported. Therefore, ARP2 becomes a valuable target to modulate the influx and concentration of calcium in the cytoplasm of epithelial cancer cells showing an apoptotic-resistant phenotype during the onset of an apoptotic event.

Coreceptors and Their Ligands in Epithelial γδ T Cell Biology

PubMed Central

Witherden, Deborah A.; Johnson, Margarete D.; Havran, Wendy L.

2018-01-01

Epithelial tissues line the body providing a protective barrier from the external environment. Maintenance of these epithelial barrier tissues critically relies on the presence of a functional resident T cell population. In some tissues, the resident T cell population is exclusively comprised of γδ T cells, while in others γδ T cells are found together with αβ T cells and other lymphocyte populations. Epithelial-resident γδ T cells function not only in the maintenance of the epithelium, but are also central to the repair process following damage from environmental and pathogenic insults. Key to their function is the crosstalk between γδ T cells and neighboring epithelial cells. This crosstalk relies on multiple receptor–ligand interactions through both the T cell receptor and accessory molecules leading to temporal and spatial regulation of cytokine, chemokine, growth factor, and extracellular matrix protein production. As antigens that activate epithelial γδ T cells are largely unknown and many classical costimulatory molecules and coreceptors are not used by these cells, efforts have focused on identification of novel coreceptors and ligands that mediate pivotal interactions between γδ T cells and their neighbors. In this review, we discuss recent advances in the understanding of functions for these coreceptors and their ligands in epithelial maintenance and repair processes. PMID:29686687

Mechanisms for cellular NO oxidation and nitrite formation in lung epithelial cells.

PubMed

Zhao, Xue-Jun; Wang, Ling; Shiva, Sruti; Tejero, Jesus; Myerburg, Mike M; Wang, Jun; Frizzell, Sam; Gladwin, Mark T

2013-08-01

Airway lining fluid contains relatively high concentrations of nitrite, and arterial blood levels of nitrite are higher than venous levels, suggesting the lung epithelium may represent an important source of nitrite in vivo. To investigate whether lung epithelial cells possess the ability to convert NO to nitrite by oxidation, and the effect of oxygen reactions on nitrite formation, the NO donor DETA NONOate was incubated with or without A549 cells or primary human bronchial epithelial (HBE) cells for 24 h under normoxic (21% O2) and hypoxic (1% O2) conditions. Nitrite production was significantly increased under all conditions in the presence of A549 or HBE cells, suggesting that both A549 and HBE cells have the capacity to oxidize NO to nitrite even under low-oxygen conditions. The addition of oxyhemoglobin to the A549 cell medium decreased the production of nitrite, consistent with NO scavenging limiting nitrite formation. Heat-denatured A549 cells produced much lower nitrite and nitrate, suggesting an enzymatic activity is required. This NO oxidation activity was highest in membrane-bound proteins with molecular size <100kDa. In addition, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one and cyanide inhibited formation of nitrite in A549 cells. It has been shown that ceruloplasmin (Cp) possesses an NO oxidase and nitrite synthase activity in plasma based on NO oxidation to nitrosonium cation. We observed that Cp is expressed intracellularly in lung epithelial A549 cells and secreted into the medium under basal conditions and during cytokine stimulation. However, an analysis of Cp expression level and activity measured via p-phenylenediamine oxidase activity assay revealed very low activity compared with plasma, suggesting that there is insufficient Cp to contribute to detectable NO oxidation to nitrite in A549 cells. Additionally, Cp levels were knocked down using siRNA by more than 75% in A549 cells, with no significant change in either nitrite or cellular S

Mechanisms for Cellular NO Oxidation and Nitrite Formation in Lung Epithelial Cells

PubMed Central

Zhao, Xue-Jun; Wang, Ling; Shiva, Sruti; Tejero, Jesus; Wang, Jun; Frizzell, Sam; Gladwin, Mark T.

2013-01-01

Airway lining fluid contains relatively high concentrations of nitrite and arterial blood levels of nitrite are higher than venous levels, suggesting the lung epithelium may represent an important source of nitrite in vivo. To investigate whether lung epithelial cells possess the ability to convert NO to nitrite by oxidation, and the effect of oxygen reactions on nitrite formation, the NO donor DETA NONOate was incubated with or without A549 cells or primary human bronchial epithelial (HBE) cells for 24 hrs under normoxic (21% O2) and hypoxic (1% O2) conditions. Nitrite production was significantly increased under all conditions in the presence of A549 or HBE cells, suggesting that both A549 and HBE cells have the capacity to oxidize NO to nitrite even under low oxygen conditions. The addition of oxy-hemoglobin (oxy-Hb) to the A549 cell media decreased the production of nitrite, consistent with NO scavenging limiting nitrite formation. Heat-denatured A549 cells produced much lower nitrite and bitrate, suggesting an enzymatic activity is required. This NO oxidation activity was found to be highest in membrane bound proteins with molecular sizes < 100 kDa. In addition, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one] (ODQ) and cyanide inhibited formation of nitrite in A549 cells. It has been shown that ceruloplasmin (Cp) possesses an NO oxidase and nitrite synthase activity in plasma based on NO oxidation to nitrosonium cation (NO+). We observed that Cp is expressed intracellularly in lung epithelial A549 cells and secreted into medium under basal conditions and during cytokine stimulation. However, an analysis of Cp expression level and activity measured via ρ-phenylenediamine oxidase activity assay revealed very low activity compared with plasma, suggesting that there is insufficient Cp to contribute to detectable NO oxidation to nitrite in A549 cells. Additionally, Cp levels were knocked down using siRNA by more than 75% in A549 cells, with no significant change in

Characterization of primary cultures of adult human epididymis epithelial cells.

PubMed

Leir, Shih-Hsing; Browne, James A; Eggener, Scott E; Harris, Ann

2015-03-01

To establish cultures of epithelial cells from all regions of the human epididymis to provide reagents for molecular approaches to functional studies of this epithelium. Experimental laboratory study. University research institute. Epididymis from seven patients undergoing orchiectomy for suspected testicular cancer without epididymal involvement. Human epididymis epithelial cells harvested from adult epididymis tissue. Establishment of a robust culture protocol for adult human epididymal epithelial cells. Cultures of caput, corpus, and cauda epithelial cells were established from epididymis tissue of seven donors. Cells were passaged up to eight times and maintained differentiation markers. They were also cryopreserved and recovered successfully. Androgen receptor, clusterin, and cysteine-rich secretory protein 1 were expressed in cultured cells, as shown by means of immunofluorescence, Western blot, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The distribution of other epididymis markers was also shown by means of qRT-PCR. Cultures developed transepithelial resistance (TER), which was androgen responsive in the caput but androgen insensitive in the corpus and cauda, where unstimulated TER values were much higher. The results demonstrate a robust in vitro culture system for differentiated epithelial cell types in the caput, corpus, and cauda of the human epididymis. These cells will be a valuable resource for molecular analysis of epididymis epithelial function, which has a pivotal role in male fertility. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Arylamine N-acetyltransferase activity in bronchial epithelial cells and its inhibition by cellular oxidants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dairou, Julien; Petit, Emile; Ragunathan, Nilusha

2009-05-01

Bronchial epithelial cells express xenobiotic-metabolizing enzymes (XMEs) that are involved in the biotransformation of inhaled toxic compounds. The activities of these XMEs in the lung may modulate respiratory toxicity and have been linked to several diseases of the airways. Arylamine N-acetyltransferases (NAT) are conjugating XMEs that play a key role in the biotransformation of aromatic amine pollutants such as the tobacco-smoke carcinogens 4-aminobiphenyl (4-ABP) and {beta}-naphthylamine ({beta}-NA). We show here that functional human NAT1 or its murine counterpart Nat2 are present in different lung epithelial cells i.e. Clara cells, type II alveolar cells and bronchial epithelial cells, thus indicating thatmore » inhaled aromatic amines may undergo NAT-dependent biotransformation in lung epithelium. Exposure of these cells to pathophysiologically relevant amounts of oxidants known to contribute to lung dysfunction, such as H{sub 2}O{sub 2} or peroxynitrite, was found to impair the NAT1/Nat2-dependent cellular biotransformation of aromatic amines. Genetic and non genetic impairment of intracellular NAT enzyme activities has been suggested to compromise the important detoxification pathway of aromatic amine N-acetylation and subsequently to contribute to an exacerbation of untoward effects of these pollutants on health. Our study suggests that oxidative/nitroxidative stress in lung epithelial cells, due to air pollution and/or inflammation, could contribute to local and/or systemic dysfunctions through the alteration of the functions of pulmonary NAT enzymes.« less

GTP-Binding Proteins Inhibit cAMP Activation of Chloride Channels in Cystic Fibrosis Airway Epithelial Cells

NASA Astrophysics Data System (ADS)

Schwiebert, Erik M.; Kizer, Neil; Gruenert, Dieter C.; Stanton, Bruce A.

1992-11-01

Cystic fibrosis (CF) is a genetic disease characterized, in part, by defective regulation of Cl^- secretion by airway epithelial cells. In CF, cAMP does not activate Cl^- channels in the apical membrane of airway epithelial cells. We report here whole-cell patch-clamp studies demonstrating that pertussis toxin, which uncouples heterotrimeric GTP-binding proteins (G proteins) from their receptors, and guanosine 5'-[β-thio]diphosphate, which prevents G proteins from interacting with their effectors, increase Cl^- currents and restore cAMP-activated Cl^- currents in airway epithelial cells isolated from CF patients. In contrast, the G protein activators guanosine 5'-[γ-thio]triphosphate and AlF^-_4 reduce Cl^- currents and inhibit cAMP from activating Cl^- currents in normal airway epithelial cells. In CF cells treated with pertussis toxin or guanosine 5'-[β-thio]diphosphate and in normal cells, cAMP activates a Cl^- conductance that has properties similar to CF transmembrane-conductance regulator Cl^- channels. We conclude that heterotrimeric G proteins inhibit cAMP-activated Cl^- currents in airway epithelial cells and that modulation of the inhibitory G protein signaling pathway may have the therapeutic potential for improving cAMP-activated Cl^- secretion in CF.

Stromal–epithelial cell interactions and alteration of branching morphogenesis in macromastic mammary glands

PubMed Central

Zhong, Aimei; Wang, Guohua; Yang, Jie; Xu, Qijun; Yuan, Quan; Yang, Yanqing; Xia, Yun; Guo, Ke; Horch, Raymund E; Sun, Jiaming

2014-01-01

True macromastia is a rare but disabling condition characterized by massive breast growth. The aetiology and pathogenic mechanisms for this disorder remain largely unexplored because of the lack of in vivo or in vitro models. Previous studies suggested that regulation of epithelial cell growth and development by oestrogen was dependent on paracrine growth factors from the stroma. In this study, a co-culture model containing epithelial and stromal cells was used to investigate the interactions of these cells in macromastia. Epithelial cell proliferation and branching morphogenesis were measured to assess the effect of macromastic stromal cells on epithelial cells. We analysed the cytokines secreted by stromal cells and identified molecules that were critical for effects on epithelial cells. Our results indicated a significant increase in cell proliferation and branching morphogenesis of macromastic and non-macromastic epithelial cells when co-cultured with macromastic stromal cells or in conditioned medium from macromastic stromal cells. Hepatocyte growth factor (HGF) is a key factor in epithelial–stromal interactions of macromastia-derived cell cultures. Blockade of HGF with neutralizing antibodies dramatically attenuated epithelial cell proliferation in conditioned medium from macromastic stromal cells. The epithelial–stromal cell co-culture model demonstrated reliability for studying interactions of mammary stromal and epithelial cells in macromastia. In this model, HGF secreted by macromastic stromal cells was found to play an important role in modifying the behaviour of co-cultured epithelial cells. This model allows further studies to investigate basic cellular and molecular mechanisms in tissue from patients with true breast hypertrophy. PMID:24720804

Proposed comprehensive ototoxicity monitoring program for VA healthcare (COMP-VA)

PubMed Central

Konrad-Martin, Dawn; Reavis, Kelly M.; McMillan, Garnett; Helt, Wendy J.; Dille, Marilyn

2015-01-01

Prevention and rehabilitation of hearing loss and tinnitus, the two most commonly awarded service-connected disabilities, are high priority initiatives in the Department of Veterans Affairs (VA). At least 4,000 Veterans, most with significant hearing loss, will receive cisplatin this year, with more than half sustaining permanent hearing shift and nearly 40% developing new tinnitus. With improved survivability following cancer treatment, Veterans treated with cisplatin are approached with the dual goals of effective treatment and preserved quality of life. This article describes COMP-VA, a comprehensive ototoxicity monitoring program developed for VA patients receiving cisplatin. The program includes an individualized pretreatment prediction model that identifies the likelihood of hearing shift given cisplatin dose and patient factors. It supports both manual and automated hearing testing with a newly developed portable audiometer capable of performing the recommended procedures on the chemotherapy unit during treatment. It also includes objective methods for identifying outer hair cell changes and predicting audiogram changes using distortion-product otoacoustic emissions. We describe this program of evidence-based ototoxicity monitoring protocols using a case example to give the reader an understanding of how this program would be applied, along with a plan for future work to accomplish the final stages of program development. PMID:24805896

Uranium induces oxidative stress in lung epithelial cells

PubMed Central

Periyakaruppan, Adaikkappan; Kumar, Felix; Sarkar, Shubhashish; Sharma, Chidananda S.

2009-01-01

Uranium compounds are widely used in the nuclear fuel cycle, antitank weapons, tank armor, and also as a pigment to color ceramics and glass. Effective management of waste uranium compounds is necessary to prevent exposure to avoid adverse health effects on the population. Health risks associated with uranium exposure includes kidney disease and respiratory disorders. In addition, several published results have shown uranium or depleted uranium causes DNA damage, mutagenicity, cancer and neurological defects. In the current study, uranium toxicity was evaluated in rat lung epithelial cells. The study shows uranium induces significant oxidative stress in rat lung epithelial cells followed by concomitant decrease in the antioxidant potential of the cells. Treatment with uranium to rat lung epithelial cells also decreased cell proliferation after 72 h in culture. The decrease in cell proliferation was attributed to loss of total glutathione and superoxide dismutase in the presence of uranium. Thus the results indicate the ineffectiveness of antioxidant system’s response to the oxidative stress induced by uranium in the cells. PMID:17124605

Emergence of an apical epithelial cell surface in vivo

PubMed Central

Sedzinski, Jakub; Hannezo, Edouard; Tu, Fan; Biro, Maté; Wallingford, John B.

2016-01-01

Epithelial sheets are crucial components of all metazoan animals, enclosing organs and protecting the animal from its environment. Epithelial homeostasis poses unique challenges, as addition of new cells and loss of old cells must be achieved without disrupting the fluid-tight barrier and apicobasal polarity of the epithelium. Several studies have identified cell biological mechanisms underlying extrusion of cells from epithelia, but far less is known of the converse mechanism by which new cells are added. Here, we combine molecular, pharmacological and laser-dissection experiments with theoretical modelling to characterize forces driving emergence of an apical surface as single nascent cells are added to a vertebrate epithelium in vivo. We find that this process involves the interplay between cell-autonomous actin-generated pushing forces in the emerging cell and mechanical properties of neighboring cells. Our findings define the forces driving this cell behavior, contributing to a more comprehensive understanding of epithelial homeostasis. PMID:26766441

Effects of Secreted Mast Cell Mediators on Retinal Pigment Epithelial Cells: Focus on Mast Cell Tryptase.

PubMed

Arai, Rei; Usui-Ouchi, Ayumi; Ito, Yosuke; Mashimo, Keitaro; Murakami, Akira; Ebihara, Nobuyuki

2017-01-01

Numerous mast cells are present in the choroid, but the effects of mast cell mediators on retinal pigment epithelial (RPE) cells are not well understood. We investigated the influence of mast cell mediators on RPE cells in vitro, focusing on tryptase. Expression of receptors was examined by the reverse transcription polymerase chain reaction. We also assessed production of interleukin 8 and vascular endothelial growth factor (VEGF) after RPE cells were stimulated with mast cell mediators by using an antibody array and enzyme-linked immunosorbent assay. Furthermore, we investigated the influence of tryptase on RPE cell migration and integrity by the scratch assay and the transepithelial resistance. RPE cells expressed protease-activated receptor 2 (PAR2), histamine receptor 1, tumor necrosis factor- α (TNF- α ) receptor 1, and CCR 1, 3, 4, 8, and 11. Tryptase, PAR2 agonists, histamine, and TNF- α all enhanced interleukin 8 production by RPE cells, while only tryptase enhanced VEGF production. Tryptase also enhanced expression of phosphorylated extracellular signal-regulated kinases 1/2, resulting in increased migration of RPE cells. However, tryptase did not alter epithelial integrity or the expression of zonula occludens-1 and junctional adhesion molecule-A by RPE cells. Mast cell mediators, especially tryptase, may influence RPE cell inflammation.

Connective Tissue Growth Factor Promotes Pulmonary Epithelial Cell Senescence and Is Associated with COPD Severity.

PubMed

Jang, Jun-Ho; Chand, Hitendra S; Bruse, Shannon; Doyle-Eisele, Melanie; Royer, Christopher; McDonald, Jacob; Qualls, Clifford; Klingelhutz, Aloysius J; Lin, Yong; Mallampalli, Rama; Tesfaigzi, Yohannes; Nyunoya, Toru

2017-04-01

The purpose of this study was to determine whether expression of connective tissue growth factor (CTGF) protein in chronic obstructive pulmonary disease (COPD) is consistent in humans and animal models of COPD and to investigate the role of this protein in lung epithelial cells. CTGF in lung epithelial cells of ex-smokers with COPD was compared with ex-smokers without COPD by immunofluorescence. A total of twenty C57Bl/6 mice and sixteen non-human primates (NHPs) were exposed to cigarette smoke (CS) for 4 weeks. Ten mice of these CS-exposed mice and eight of the CS-exposed NHPs were infected with H3N2 influenza A virus (IAV), while the remaining ten mice and eight NHPs were mock-infected with vehicle as control. Both mRNA and protein expression of CTGF in lung epithelial cells of mice and NHPs were determined. The effects of CTGF overexpression on cell proliferation, p16 protein, and senescence-associated β-galactosidase (SA-β-gal) activity were examined in cultured human bronchial epithelial cells (HBECs). In humans, CTGF expression increased with increasing COPD severity. We found that protein expression of CTGF was upregulated in lung epithelial cells in both mice and NHPs exposed to CS and infected with IAV compared to those exposed to CS only. When overexpressed in HBECs, CTGF accelerated cellular senescence accompanied by p16 accumulation. Both CTGF and p16 protein expression in lung epithelia are positively associated with the severity of COPD in ex-smokers. These findings show that CTGF is consistently expressed in epithelial cells of COPD lungs. By accelerating lung epithelial senescence, CTGF may block regeneration relative to epithelial cell loss and lead to emphysema.

KSA Antigen Ep-CAM Mediates Cell–Cell Adhesion of Pancreatic Epithelial Cells: Morphoregulatory Roles in Pancreatic Islet Development

PubMed Central

Cirulli, V.; Crisa, L.; Beattie, G.M.; Mally, M.I.; Lopez, A.D.; Fannon, A.; Ptasznik, A.; Inverardi, L.; Ricordi, C.; Deerinck, T.; Ellisman, M.; Reisfeld, R.A.; Hayek, A.

1998-01-01

Cell adhesion molecules (CAMs) are important mediators of cell–cell interactions and regulate cell fate determination by influencing growth, differentiation, and organization within tissues. The human pancarcinoma antigen KSA is a glycoprotein of 40 kD originally identified as a marker of rapidly proliferating tumors of epithelial origin. Interestingly, most normal epithelia also express this antigen, although at lower levels, suggesting that a dynamic regulation of KSA may occur during cell growth and differentiation. Recently, evidence has been provided that this glycoprotein may function as an epithelial cell adhesion molecule (Ep-CAM). Here, we report that Ep-CAM exhibits the features of a morphoregulatory molecule involved in the development of human pancreatic islets. We demonstrate that Ep-CAM expression is targeted to the lateral domain of epithelial cells of the human fetal pancreas, and that it mediates calcium-independent cell–cell adhesion. Quantitative confocal immunofluorescence in fetal pancreata identified the highest levels of Ep-CAM expression in developing islet-like cell clusters budding from the ductal epithelium, a cell compartment thought to comprise endocrine progenitors. A surprisingly reversed pattern was observed in the human adult pancreas, displaying low levels of Ep-CAM in islet cells and high levels in ducts. We further demonstrate that culture conditions promoting epithelial cell growth induce upregulation of Ep-CAM, whereas endocrine differentiation of fetal pancreatic epithelial cells, transplanted in nude mice, is associated with a downregulation of Ep-CAM expression. In addition, a blockade of Ep-CAM function by KS1/4 mAb induced insulin and glucagon gene transcription and translation in fetal pancreatic cell clusters. These results indicate that developmentally regulated expression and function of Ep-CAM play a morphoregulatory role in pancreatic islet ontogeny. PMID:9508783

Human Milk Mucin 1 and Mucin 4 Inhibit Salmonella enterica Serovar Typhimurium Invasion of Human Intestinal Epithelial Cells In Vitro123

PubMed Central

Liu, Bo; Yu, Zhuoteng; Chen, Ceng; Kling, David E.; Newburg, David S.

2012-01-01

Many human milk glycans inhibit pathogen binding to host receptors and their consumption by infants is associated with reduced risk of disease. Salmonella infection is more frequent among infants than among the general population, but the incidence is lower in breast-fed babies, suggesting that human milk could contain components that inhibit Salmonella. This study aimed to test whether human milk per se inhibits Salmonella invasion of human intestinal epithelial cells in vitro and, if so, to identify the milk components responsible for inhibition. Salmonella enterica serovar Typhimurium SL1344 (SL1344) invasion of FHs 74 Int and Caco-2 cells were the models of human intestinal epithelium infection. Internalization of fluorescein-5-isothiocyanate–labeled SL1344 into intestinal cells was measured by flow cytometry to quantify infection. Human milk and its fractions inhibited infection; the inhibitory activity localized to the high molecular weight glycans. Mucin 1 and mucin 4 were isolated to homogeneity. At 150 μg/L, a typical concentration in milk, human milk mucin 1 and mucin 4 inhibited SL1344 invasion of both target cell types. These mucins inhibited SL1344 invasion of epithelial cells in a dose-dependent manner. Thus, mucins may prove useful as a basis for developing novel oral prophylactic and therapeutic agents that inhibit infant diseases caused by Salmonella and related pathogens. PMID:22718031

CXCR4/CXCL12 signaling impacts enamel progenitor cell proliferation and motility in the dental stem cell niche

PubMed Central

Otsu, Keishi; Harada, Hidemitsu; Shibata, Shunichi; Obara, Nobuko; Irie, Kazuharu; Taniguchi, Akiyoshi; Nagasawa, Takashi; Aoki, Kazunari; Caliari, Steven R.; Weisgerber, Daniel W.

2015-01-01

Dental stem cells are located at the proximal ends of rodent incisors. These stem cells reside in the dental epithelial stem cell niche, termed the apical bud. We focused on identifying critical features of a chemotactic signal in the niche. Here, we report that CXCR4/CXCL12 signaling impacts enamel progenitor cell proliferation and motility in dental stem cell niche cells. We report cells in the apical bud express CXCR4 mRNA at high levels while expression is restricted in the basal epithelium (BE) and transit-amplifying (TA) cell regions. Furthermore, the CXCL12 ligand is present in mesenchymal cells adjacent to the apical bud. We then performed gain- and loss-of-function analyses to better elucidate the role of CXCR4 and CXCL12. CXCR4-deficient mice contain epithelial cell aggregates, while cell proliferation in mutant incisors was also significantly reduced. We demonstrate in vitro that dental epithelial cells migrate toward sources of CXCL12, whereas knocking down CXCR4 impaired motility and resulted in formation of dense cell colonies. These results suggest that CXCR4 expression may be critical for activation of enamel progenitor cell division and that CXCR4/CXCL12 signaling may control movement of epithelial progenitors from the dental stem cell niche. PMID:26246398

Effects of Weaning on Intestinal Upper Villus Epithelial Cells of Piglets

PubMed Central

Wang, Xiaocheng; Tan, Bie; Li, Tiejun; Yin, Yulong

2016-01-01

The intestinal upper villus epithelial cells represent the differentiated epithelial cells and play key role in digesting and absorbing lumenal nutrients. Weaning stress commonly results in a decrease in villus height and intestinal dysfunction in piglets. However, no study have been conducted to test the effects of weaning on the physiology and functions of upper villus epithelial cells. A total of 40 piglets from 8 litters were weaned at 14 days of age and one piglet from each litter was killed at 0 d (w0d), 1 d (w1d), 3 d (w3d), 5 d (w5d), and 7 d (w7d) after weaning, respectively. The upper villus epithelial cells in mid-jejunum were isolated using the distended intestinal sac method. The expression of proteins in upper villus epithelial cells was analyzed using the isobaric tags for relative and absolute quantification or Western blotting. The expression of proteins involved in energy metabolism, Golgi vesicle transport, protein amino acid glycosylation, secretion by cell, transmembrane transport, ion transport, nucleotide catabolic process, translational initiation, and epithelial cell differentiation and apoptosis, was mainly reduced during the post-weaning period, and these processes may be regulated by mTOR signaling pathway. These results indicated that weaning inhibited various cellular processes in jejunal upper villus epithelial cells, and provided potential new directions for exploring the effects of weaning on the functions of intestine and improving intestinal functions in weaning piglets. PMID:27022727

Regulation of membrane-associated mucins in the human corneal epithelial cells by dexamethasone.

PubMed

Seo, Kyoung Yul; Chung, So-Hyang; Lee, Joon H; Park, Mi Young; Kim, Eung Kweon

2007-07-01

To study the influence of dexamethasone on membrane-associated mucins produced by human corneal epithelial cells. Human corneal epithelial cells were cultured in medium supplemented with various concentrations of dexamethasone (ranging from 10 to 10 M). Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis using monoclonal antibodies specific for human MUC1 (HMFG-1), MUC4 (1G8), and MUC16 (OC125) were performed to evaluate the effect of dexamethasone on membrane-associated mucin expression. The effect of glucocorticoid receptor antagonist (RU38486) on dexamethasone-induced mucin expression was estimated. RT-PCR revealed that MUC1 and MUC16 gene expression were upregulated 48 hours after addition of dexamethasone and that MUC4 gene expression was downregulated in the same condition. Western blot analysis showed that MUC1 and MUC16 proteins were increased after addition of dexamethasone. However, MUC4 was not detected by anti-MUC4 monoclonal antibody (1G8) for ASGP-2 under our conditions. Treatment with RU38486 inhibited the changes of MUC1, MUC4, and MUC16 by dexamethasone; thus, the effect of dexamethasone on mucin expression is mediated by glucocorticoid receptors. This study shows that MUC1, MUC4, and MUC16 are regulated differently by dexamethasone in human corneal epithelial cells. External application of dexamethasone might affect the precorneal mucin.

Contraction and elongation: Mechanics underlying cell boundary deformations in epithelial tissue.

PubMed

Hara, Yusuke

2017-06-01

The cell-cell boundaries of epithelial cells form cellular frameworks at the apical side of tissues. Deformations in these boundaries, for example, boundary contraction and elongation, and the associated forces form the mechanical basis of epithelial tissue morphogenesis. In this review, using data from recent Drosophila studies on cell boundary contraction and elongation, I provide an overview of the mechanism underlying the bi-directional deformations in the epithelial cell boundary, that are sustained by biased accumulations of junctional and apico-medial non-muscle myosin II. Moreover, how the junctional tensions exist on cell boundaries in different boundary dynamics and morphologies are discussed. Finally, some future perspectives on how recent knowledge about single cell boundary-level mechanics will contribute to our understanding of epithelial tissue morphogenesis are discussed. © 2017 Japanese Society of Developmental Biologists.

Fibroblast Growth Factor 2: An Epithelial Ductal Cell Growth Inhibitor That Drops Out in Breast Cancer

DTIC Science & Technology

2009-10-01

AD_________________ Award Number: W81XWH-08-1-0708 TITLE: Fibroblast Growth Factor 2: an...September 2008 – 14 September 2009 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Fibroblast Growth Factor 2: an Epithelial Ductal Cell Growth Inhibitor...9 Fibroblast Growth Factor -2: an Epithelial Ductal Cell Growth Inhibitor that Drops Out in Breast Cancer

Integrin-β4 identifies cancer stem cell-enriched populations of partially mesenchymal carcinoma cells

PubMed Central

Bierie, Brian; Pierce, Sarah E.; Kroeger, Cornelia; Stover, Daniel G.; Pattabiraman, Diwakar R.; Thiru, Prathapan; Liu Donaher, Joana; Reinhardt, Ferenc; Chaffer, Christine L.; Keckesova, Zuzana; Weinberg, Robert A.

2017-01-01

Neoplastic cells within individual carcinomas often exhibit considerable phenotypic heterogeneity in their epithelial versus mesenchymal-like cell states. Because carcinoma cells with mesenchymal features are often more resistant to therapy and may serve as a source of relapse, we sought to determine whether such cells could be further stratified into functionally distinct subtypes. Indeed, we find that a basal epithelial marker, integrin-β4 (ITGB4), can be used to enable stratification of mesenchymal-like triple-negative breast cancer (TNBC) cells that differ from one another in their relative tumorigenic abilities. Notably, we demonstrate that ITGB4+ cancer stem cell (CSC)-enriched mesenchymal cells reside in an intermediate epithelial/mesenchymal phenotypic state. Among patients with TNBC who received chemotherapy, elevated ITGB4 expression was associated with a worse 5-year probability of relapse-free survival. Mechanistically, we find that the ZEB1 (zinc finger E-box binding homeobox 1) transcription factor activity in highly mesenchymal SUM159 TNBC cells can repress expression of the epithelial transcription factor TAp63α (tumor protein 63 isoform 1), a protein that promotes ITGB4 expression. In addition, we demonstrate that ZEB1 and ITGB4 are important in modulating the histopathological phenotypes of tumors derived from mesenchymal TNBC cells. Hence, mesenchymal carcinoma cell populations are internally heterogeneous, and ITGB4 is a mechanistically driven prognostic biomarker that can be used to identify the more aggressive subtypes of mesenchymal carcinoma cells in TNBC. The ability to rapidly isolate and mechanistically interrogate the CSC-enriched, partially mesenchymal carcinoma cells should further enable identification of novel therapeutic opportunities to improve the prognosis for high-risk patients with TNBC. PMID:28270621

Leptin expression in human mammary epithelial cells and breast milk.

PubMed

Smith-Kirwin, S M; O'Connor, D M; De Johnston, J; Lancey, E D; Hassink, S G; Funanage, V L

1998-05-01

Leptin has recently been shown to be produced by the human placenta and potentially plays a role in fetal and neonatal growth. Many functions of the placenta are replaced by the mammary gland in terms of providing critical growth factors for the newborn. In this study, we show that leptin is produced by human mammary epithelial cells as revealed by RT/PCR analysis of total RNA from mammary gland and immunohistochemical staining of breast tissue, cultured mammary epithelial cells, and secretory epithelial cells present in human milk. We also verify that immunoreactive leptin is present in whole milk at 30- to 150-fold higher concentrations than skim milk. We propose that leptin is secreted by mammary epithelial cells in milk fat globules, which partition into the lipid portion of breast milk.

EGF stimulates Mg{sup 2+} influx in mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trapani, Valentina; Arduini, Daniela; Luongo, Francesca

2014-11-28

Highlights: • EGF stimulation potentiates Mg{sup 2+} influx into epithelial cells. • EGF-induced Mg{sup 2+} influx does not depend on the concomitantly induced Ca{sup 2+} signal. • EGF-induced Ca{sup 2+} signal is dependent on the presence of extracellular Mg{sup 2+}. • New players in EGF-mediated signaling might be exploited as therapeutic targets. - Abstract: Magnesium is well established as a fundamental factor that regulates cell proliferation. However, the molecular mechanisms linking mitogenic signals, extracellular magnesium availability and intracellular effectors are still largely unknown. In the present study we sought to determine whether EGF regulates magnesium homeostasis in normal HC11 mammarymore » epithelial cells. To this end, we measured Mg{sup 2+} and Ca{sup 2+} fluxes by confocal imaging in live cells loaded with specific fluorescent ion indicators (Mag-Fluo-4 and Fluo-4, respectively). EGF stimulation induces a rapid and sustained increase in intracellular Mg{sup 2+}, concomitantly with a rise in intracellular calcium. The increase in intracellular Mg{sup 2+} derives from an influx from the extracellular compartment, and does not depend on Ca{sup 2+}. On the contrary, the increase in intracellular Ca{sup 2+} derives from intracellular stores, and is impaired in the absence of extracellular magnesium. Inhibition of the EGF receptor tyrosine kinase by Tyrphostin AG1478 markedly inhibits EGF-induced Mg{sup 2+} and Ca{sup 2+} signals. These findings demonstrate that not only does Mg{sup 2+} influx represent an important step in the physiological response of epithelial cells to EGF, but unexpectedly the EGF-induced Mg{sup 2+} influx is essential for the Ca{sup 2+} signal to occur.« less

Allergen-induced resistin-like molecule-α promotes esophageal epithelial cell hyperplasia in eosinophilic esophagitis

PubMed Central

Mavi, Parm; Niranjan, Rituraj; Dutt, Parmesh; Zaidi, Asifa; Shukla, Jai Shankar; Korfhagen, Thomas

2014-01-01

Resistin-like molecule (Relm)-α is a secreted, cysteine-rich protein belonging to a newly defined family of proteins, including resistin, Relm-β, and Relm-γ. Although resistin was initially defined based on its insulin-resistance activity, the family members are highly induced in various inflammatory states. Earlier studies implicated Relm-α in insulin resistance, asthmatic responses, and intestinal inflammation; however, its function still remains an enigma. We now report that Relm-α is strongly induced in the esophagus in an allergen-challenged murine model of eosinophilic esophagitis (EoE). Furthermore, to understand the in vivo role of Relm-α, we generated Relm-α gene-inducible bitransgenic mice by using lung-specific CC-10 promoter (CC10-rtTA-Relm-α). We found Relm-α protein is significantly induced in the esophagus of CC10-rtTA-Relm-α bitransgenic mice exposed to doxycycline food. The most prominent effect observed by the induction of Relm-α is epithelial cell hyperplasia, basal layer thickness, accumulation of activated CD4+ and CD4− T cell subsets, and eosinophilic inflammation in the esophagus. The in vitro experiments further confirm that Relm-α promotes primary epithelial cell proliferation but has no chemotactic activity for eosinophils. Taken together, our studies report for the first time that Relm-α induction in the esophagus has a major role in promoting epithelial cell hyperplasia and basal layer thickness, and the accumulation of activated CD4+ and CD4− T cell subsets may be responsible for partial esophageal eosinophilia in the mouse models of EoE. Notably, the epithelial cell hyperplasia and basal layer thickness are the characteristic features commonly observed in human EoE. PMID:24994859

Allergen-induced resistin-like molecule-α promotes esophageal epithelial cell hyperplasia in eosinophilic esophagitis.

PubMed

Mavi, Parm; Niranjan, Rituraj; Dutt, Parmesh; Zaidi, Asifa; Shukla, Jai Shankar; Korfhagen, Thomas; Mishra, Anil

2014-09-01

Resistin-like molecule (Relm)-α is a secreted, cysteine-rich protein belonging to a newly defined family of proteins, including resistin, Relm-β, and Relm-γ. Although resistin was initially defined based on its insulin-resistance activity, the family members are highly induced in various inflammatory states. Earlier studies implicated Relm-α in insulin resistance, asthmatic responses, and intestinal inflammation; however, its function still remains an enigma. We now report that Relm-α is strongly induced in the esophagus in an allergen-challenged murine model of eosinophilic esophagitis (EoE). Furthermore, to understand the in vivo role of Relm-α, we generated Relm-α gene-inducible bitransgenic mice by using lung-specific CC-10 promoter (CC10-rtTA-Relm-α). We found Relm-α protein is significantly induced in the esophagus of CC10-rtTA-Relm-α bitransgenic mice exposed to doxycycline food. The most prominent effect observed by the induction of Relm-α is epithelial cell hyperplasia, basal layer thickness, accumulation of activated CD4(+) and CD4(-) T cell subsets, and eosinophilic inflammation in the esophagus. The in vitro experiments further confirm that Relm-α promotes primary epithelial cell proliferation but has no chemotactic activity for eosinophils. Taken together, our studies report for the first time that Relm-α induction in the esophagus has a major role in promoting epithelial cell hyperplasia and basal layer thickness, and the accumulation of activated CD4(+) and CD4(-) T cell subsets may be responsible for partial esophageal eosinophilia in the mouse models of EoE. Notably, the epithelial cell hyperplasia and basal layer thickness are the characteristic features commonly observed in human EoE. Copyright © 2014 the American Physiological Society.

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

Pancreatic stellate cells promote epithelial-mesenchymal transition in pancreatic cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kikuta, Kazuhiro; Masamune, Atsushi, E-mail: amasamune@med.tohoku.ac.jp; Watanabe, Takashi

2010-12-17

Research highlights: {yields} Recent studies have shown that pancreatic stellate cells (PSCs) promote the progression of pancreatic cancer. {yields} Pancreatic cancer cells co-cultured with PSCs showed loose cell contacts and scattered, fibroblast-like appearance. {yields} PSCs decreased the expression of epithelial markers but increased that of mesenchymal markers, along with increased migration. {yields} This study suggests epithelial-mesenchymal transition as a novel mechanism by which PSCs contribute to the aggressive behavior of pancreatic cancer cells. -- Abstract: The interaction between pancreatic cancer cells and pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, is receiving increasing attention. There ismore » accumulating evidence that PSCs promote the progression of pancreatic cancer by increasing cancer cell proliferation and invasion as well as by protecting them from radiation- and gemcitabine-induced apoptosis. Because epithelial-mesenchymal transition (EMT) plays a critical role in the progression of pancreatic cancer, we hypothesized that PSCs promote EMT in pancreatic cancer cells. Panc-1 and SUIT-2 pancreatic cancer cells were indirectly co-cultured with human PSCs isolated from patients undergoing operation for pancreatic cancer. The expression of epithelial and mesenchymal markers was examined by real-time PCR and immunofluorescent staining. The migration of pancreatic cancer cells was examined by scratch and two-chamber assays. Pancreatic cancer cells co-cultured with PSCs showed loose cell contacts and a scattered, fibroblast-like appearance. The expression of E-cadherin, cytokeratin 19, and membrane-associated {beta}-catenin was decreased, whereas vimentin and Snail (Snai-1) expression was increased more in cancer cells co-cultured with PSCs than in mono-cultured cells. The migration of pancreatic cancer cells was increased by co-culture with PSCs. The PSC-induced decrease of E-cadherin expression was not

Uterine epithelial cell proliferation and endometrial hyperplasia: evidence from a mouse model

PubMed Central

Gao, Yang; Li, Shu; Li, Qinglei

2014-01-01

In the uterus, epithelial cell proliferation changes during the estrous cycle and pregnancy. Uncontrolled epithelial cell proliferation results in implantation failure and/or cancer development. Transforming growth factor-β (TGF-β) signaling is a fundamental regulator of diverse biological processes and is indispensable for multiple reproductive functions. However, the in vivo role of TGF-β signaling in uterine epithelial cells remains poorly defined. We have shown that in the uterus, conditional deletion of the Type 1 receptor for TGF-β (Tgfbr1) using anti-Müllerian hormone receptor type 2 (Amhr2) Cre leads to myometrial defects. Here, we describe enhanced epithelial cell proliferation by immunostaining of Ki67 in the uteri of these mice. The aberration culminated in endometrial hyperplasia in aged females. To exclude the potential influence of ovarian steroid hormones, the proliferative status of uterine epithelial cells was assessed following ovariectomy. Increased uterine epithelial cell proliferation was also revealed in ovariectomized Tgfbr1 Amhr2-Cre conditional knockout mice. We further demonstrated that transcript levels for fibroblast growth factor 10 (Fgf10) were markedly up-regulated in Tgfbr1 Amhr2-Cre conditional knockout uteri. Consistently, treatment of primary uterine stromal cells with TGF-β1 significantly reduced Fgf10 mRNA expression. Thus, our findings suggest a potential involvement of TGFBR1-mediated signaling in the regulation of uterine epithelial cell proliferation, and provide genetic evidence supporting the role of uterine epithelial cell proliferation in the pathogenesis of endometrial hyperplasia. PMID:24770950

Impaired airway epithelial cell responses from children with asthma to rhinoviral infection.

PubMed

Kicic, A; Stevens, P T; Sutanto, E N; Kicic-Starcevich, E; Ling, K-M; Looi, K; Martinovich, K M; Garratt, L W; Iosifidis, T; Shaw, N C; Buckley, A G; Rigby, P J; Lannigan, F J; Knight, D A; Stick, S M

2016-11-01

The airway epithelium forms an effective immune and physical barrier that is essential for protecting the lung from potentially harmful inhaled stimuli including viruses. Human rhinovirus (HRV) infection is a known trigger of asthma exacerbations, although the mechanism by which this occurs is not fully understood. To explore the relationship between apoptotic, innate immune and inflammatory responses to HRV infection in airway epithelial cells (AECs) obtained from children with asthma and non-asthmatic controls. In addition, to test the hypothesis that aberrant repair of epithelium from asthmatics is further dysregulated by HRV infection. Airway epithelial brushings were obtained from 39 asthmatic and 36 non-asthmatic children. Primary cultures were established and exposed to HRV1b and HRV14. Virus receptor number, virus replication and progeny release were determined. Epithelial cell apoptosis, IFN-β production, inflammatory cytokine release and epithelial wound repair and proliferation were also measured. Virus proliferation and release was greater in airway epithelial cells from asthmatics but this was not related to the number of virus receptors. In epithelial cells from asthmatic children, virus infection dampened apoptosis, reduced IFN-β production and increased inflammatory cytokine production. HRV1b infection also inhibited wound repair capacity of epithelial cells isolated from non-asthmatic children and exaggerated the defective repair response seen in epithelial cells from asthmatics. Addition of IFN-β restored apoptosis, suppressed virus replication and improved repair of airway epithelial cells from asthmatics but did not reduce inflammatory cytokine production. Collectively, HRV infection delays repair and inhibits apoptotic processes in epithelial cells from non-asthmatic and asthmatic children. The delayed repair is further exaggerated in cells from asthmatic children and is only partially reversed by exogenous IFN-β. © 2016 John Wiley & Sons

Epithelial Cell Rests of Malassez Contain Unique Stem Cell Populations Capable of Undergoing Epithelial–Mesenchymal Transition

PubMed Central

Xiong, Jimin; Mrozik, Krzysztof; Gronthos, Stan

2012-01-01

The epithelial cell rests of Malassez (ERM) are odontogenic epithelial cells located within the periodontal ligament matrix. While their function is unknown, they may support tissue homeostasis and maintain periodontal ligament space or even contribute to periodontal regeneration. We investigated the notion that ERM contain a subpopulation of stem cells that could undergo epithelial–mesenchymal transition and differentiate into mesenchymal stem-like cells with multilineage potential. For this purpose, ERM collected from ovine incisors were subjected to different inductive conditions in vitro, previously developed for the characterization of bone marrow mesenchymal stromal/stem cells (BMSC). We found that ex vivo-expanded ERM expressed both epithelial (cytokeratin-8, E-cadherin, and epithelial membrane protein-1) and BMSC markers (CD44, CD29, and heat shock protein-90β). Integrin α6/CD49f could be used for the enrichment of clonogenic cell clusters [colony-forming units-epithelial cells (CFU-Epi)]. Integrin α6/CD49f-positive-selected epithelial cells demonstrated over 50- and 7-fold greater CFU-Epi than integrin α6/CD49f-negative cells and unfractionated cells, respectively. Importantly, ERM demonstrated stem cell-like properties in their differentiation capacity to form bone, fat, cartilage, and neural cells in vitro. When transplanted into immunocompromised mice, ERM generated bone, cementum-like and Sharpey's fiber-like structures. Additionally, gene expression studies showed that osteogenic induction of ERM triggered an epithelial–mesenchymal transition. In conclusion, ERM are unusual cells that display the morphological and phenotypic characteristics of ectoderm-derived epithelial cells; however, they also have the capacity to differentiate into a mesenchymal phenotype and thus represent a unique stem cell population within the periodontal ligament. PMID:22122577

Brca1 regulates in vitro differentiation of mammary epithelial cells.

PubMed

Kubista, Marion; Rosner, Margit; Kubista, Ernst; Bernaschek, Gerhard; Hengstschläger, Markus

2002-07-18

Murine Brca1 is widely expressed during development in different tissues. Why alterations of BRCA1 lead specifically to breast and ovarian cancer is currently not clarified. Here we show that Brca1 protein expression is upregulated during mammary epithelial differentiation of HC11 cells, during differentiation of C2C12 myoblasts into myotubes and during neuronal differentiation of N1E-115 cells. Ectopic overexpression of BRCA1 and downregulation of endogenous Brca1 expression specifically affect the regulation of mammary epithelial cell differentiation. Accelerated mammary epithelial cell differentiation upon high ectopic BRCA1 expression is not a consequence of the anti-proliferative capacity of this tumor suppressor and independent of functional p53. Overexpression of the BRCA1 variant lacking the large central exon 11 has no effects on mammary epithelial cell differentiation. These data provide new insights into the cellular role of Brca1.

Nicotine transport in lung and non-lung epithelial cells.

PubMed

Takano, Mikihisa; Kamei, Hidetaka; Nagahiro, Machi; Kawami, Masashi; Yumoto, Ryoko

2017-11-01

Nicotine is rapidly absorbed from the lung alveoli into systemic circulation during cigarette smoking. However, mechanism underlying nicotine transport in alveolar epithelial cells is not well understood to date. In the present study, we characterized nicotine uptake in lung epithelial cell lines A549 and NCI-H441 and in non-lung epithelial cell lines HepG2 and MCF-7. Characteristics of [ 3 H]nicotine uptake was studied using these cell lines. Nicotine uptake in A549 cells occurred in a time- and temperature-dependent manner and showed saturation kinetics, with a Km value of 0.31mM. Treatment with some organic cations such as diphenhydramine and pyrilamine inhibited nicotine uptake, whereas treatment with organic cations such as carnitine and tetraethylammonium did not affect nicotine uptake. Extracellular pH markedly affected nicotine uptake, with high nicotine uptake being observed at high pH up to 11.0. Modulation of intracellular pH with ammonium chloride also affected nicotine uptake. Treatment with valinomycin, a potassium ionophore, did not significantly affect nicotine uptake, indicating that nicotine uptake is an electroneutral process. For comparison, we assessed the characteristics of nicotine uptake in another lung epithelial cell line NCI-H441 and in non-lung epithelial cell lines HepG2 and MCF-7. Interestingly, these cell lines showed similar characteristics of nicotine uptake with respect to pH dependency and inhibition by various organic cations. The present findings suggest that a similar or the same pH-dependent transport system is involved in nicotine uptake in these cell lines. A novel molecular mechanism of nicotine transport is proposed. Copyright © 2017 Elsevier Inc. All rights reserved.

Role of salivary epithelial toll-like receptors 2 and 4 in modulating innate immune responses in chronic periodontitis.

PubMed

Swaminathan, V; Prakasam, S; Puri, V; Srinivasan, M

2013-12-01

Chronic periodontitis is initiated by sequential colonization with a broad array of bacteria and is perpetuated by an immune-inflammatory response to the changing biofilm. Host recognition of microbes is largely mediated by toll-like receptors (TLRs), which interact with conserved pathogen-associated molecular patterns. Based on ligand recognition, TLR-2 and TLR-4 interact with most periodontal pathogens. Extracrevicular bacterial reservoirs, such as the oral epithelial cells, contribute to the persistence of periodontitis. Human saliva is a rich source of oral epithelial cells that express functional TLRs. In this study we investigated the role of salivary epithelial cell (SEC) TLR-2 and TLR-4 in patients with generalized chronic periodontitis. Unstimulated whole saliva (UWS) was collected from patients with generalized chronic periodontitis and from healthy individuals after obtaining informed consent. Epithelial cells isolated from each UWS sample were assessed for TLR-2, TLR-4, peptidoglycan recognition protein (PGRP)-3 and PGRP-4 by quantitative real-time PCR. In addition, the SECs were stimulated in vitro with microbial products for up to 24 h. The culture supernatant was assessed for cytokines by ELISA. Stimulation with TLR-2- or TLR-4-specific ligands induced cytokine secretion with differential kinetics and up-regulated TLR2 and TLR4 mRNAs, respectively, in cultures of SECs from patients with periodontitis. In addition, the SECs from patients with periodontitis exhibited reduced PGRP3 and PGRP4 mRNAs, the TLR-responsive genes with antibacterial properties. SECs derived from the UWS of patients with chronic periodontitis are phenotypically distinct and could represent potential resources for assessing the epithelial responses to periodontal pathogens in the course of disease progression and persistence. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Melanin dependent survival of Apergillus fumigatus conidia in lung epithelial cells.

PubMed

Amin, Shayista; Thywissen, Andreas; Heinekamp, Thorsten; Saluz, Hans Peter; Brakhage, Axel A

2014-07-01

Aspergillus fumigatus is the most important air-borne pathogenic fungus of humans. Upon inhalation of conidia, the fungus makes close contact with lung epithelial cells, which only possess low phagocytic activity. These cells are in particular interesting to address the question whether there is some form of persistence of conidia of A. fumigatus in the human host. Therefore, by also using uracil-auxotrophic mutant strains, we were able to investigate the interaction of A549 lung epithelial cells and A. fumigatus conidia in detail for long periods. Interestingly, unlike professional phagocytes, our study showed that the presence of conidial dihydroxynaphthalene (DHN) melanin enhanced the uptake of A. fumigatus conidia by epithelial cells when compared with non-pigmented pksP mutant conidia. Furthermore, conidia of A. fumigatus were able to survive within epithelial cells. This was due to the presence of DHN melanin in the cell wall of conidia, because melanised wild-type conidia showed a higher survival rate inside epithelial cells and led to inhibition of acidification of phagolysosomes. Both effects were not observed for white (non-melanised) conidia of the pksP mutant strain. Moreover, in contrast to pksP mutant conidia, melanised wild-type conidia were able to inhibit the extrinsic apoptotic pathway in A549 lung epithelial cells even for longer periods. The anti-apoptotic effect was not restricted to conidia, because both conidia-derived melanin ghosts (cell-free DHN melanin) and a different type of melanin, dihydroxyphenylalanine (DOPA) melanin, acted anti-apoptotically. Taken together, these data indicate the possibility of melanin-dependent persistence of conidia in lung epithelial cells. Copyright © 2014 Elsevier GmbH. All rights reserved.

Interaction of the pathogenic mold Aspergillus fumigatus with lung epithelial cells

PubMed Central

Osherov, Nir

2012-01-01

Aspergillus fumigatus is an opportunistic environmental mold that can cause severe allergic responses in atopic individuals and poses a life-threatening risk for severely immunocompromised patients. Infection is caused by inhalation of fungal spores (conidia) into the lungs. The initial point of contact between the fungus and the host is a monolayer of lung epithelial cells. Understanding how these cells react to fungal contact is crucial to elucidating the pathobiology of Aspergillus-related disease states. The experimental systems, both in vitro and in vivo, used to study these interactions, are described. Distinction is made between bronchial and alveolar epithelial cells. The experimental findings suggest that lung epithelial cells are more than just “innocent bystanders” or a purely physical barrier against infection. They can be better described as an active extension of our innate immune system, operating as a surveillance mechanism that can specifically identify fungal spores and activate an offensive response to block infection. This response includes the internalization of adherent conidia and the release of cytokines, antimicrobial peptides, and reactive oxygen species. In the case of allergy, lung epithelial cells can dampen an over-reactive immune response by releasing anti-inflammatory compounds such as kinurenine. This review summarizes our current knowledge regarding the interaction of A. fumigatus with lung epithelial cells. A better understanding of the interactions between A. fumigatus and lung epithelial cells has therapeutic implications, as stimulation or inhibition of the epithelial response may alter disease outcome. PMID:23055997

Characterization of kidney epithelial cells from the Florida manatee, Trichechus manatus latirostris.

PubMed

Sweat JMDunigan, D D; Wright, S D

2001-06-01

The West-Indian manatee, Trichechus manatus latirostris, is a herbivorous marine mammal found in the coastal waters of Florida. Because of their endangered status, animal experimentation is not allowed. Therefore, a cell line was developed and characterized from tissue collected during necropsies of the manatees. A primary cell culture was established by isolating single cells from kidney tissue using both enzymatic and mechanical techniques. Primary manatee kidney (MK) cells were subcultured for characterization. These cells were morphologically similar to the cell lines of epithelial origin. An immunocytochemistry assay was used to localize the cytokeratin filaments common to cells of epithelial origin. At second passage, epithelial-like cells had an average population-doubling time of 48 h, had an optimum seeding density of 5 x 10(3) cells/cm2, and readily attached to plastic culture plates with a high level of seeding efficiency. Although the epithelial-like cells had a rapid growth rate during the first three passages, the cloning potential was low. These cells did not form colonies in agar medium, were serum dependent, had a limited life span of approximately nine passages, and possessed cell-contact inhibition. These data suggest that the cells were finite (noncontinuous growth), did not possess transformed properties, and were of epithelial origin. These cells are now referred to as MK epithelial cells.

Interaction of chitin/chitosan with salivary and other epithelial cells-An overview.

PubMed

Patil, Sharvari Vijaykumar; Nanduri, Lalitha S Y

2017-11-01

Chitin and its deacetylated form, chitosan, have been widely used for tissue engineering of both epithelial and mesenchymal tissues. Epithelial cells characterised by their sheet-like tight cellular arrangement and polarised nature, constitute a major component in various organs and play a variety of roles including protection, secretion and maintenance of tissue homeostasis. Regeneration of damaged epithelial tissues has been studied using biomaterials such as chitin, chitosan, hyaluronan, gelatin and alginate. Chitin and chitosan are known to promote proliferation of various embryonic and adult epithelial cells. However it is not clearly understood how this activity is achieved or what are the mechanisms involved in the chitin/chitosan driven proliferation of epithelial cells. Mechanistic understanding of influence of chitin/chitosan on epithelial cells will guide us to develop more targeted regenerative scaffold/hydrogel systems. Therefore, current review attempts to elicit a mechanistic insight into how chitin and chitosan interact with salivary, mammary, skin, nasal, lung, intestinal and bladder epithelial cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Serratia marcescens internalization and replication in human bladder epithelial cells

PubMed Central

Hertle, Ralf; Schwarz, Heinz

2004-01-01

Background Serratia marcescens, a frequent agent of catheterization-associated bacteriuria, strongly adheres to human bladder epithelial cells in culture. The epithelium normally provides a barrier between lumal organisms and the interstitium; the tight adhesion of bacteria to the epithelial cells can lead to internalization and subsequent lysis. However, internalisation was not shown yet for S. marcescens strains. Methods Elektronmicroscopy and the common gentamycin protection assay was used to assess intracellular bacteria. Via site directed mutagenesis, an hemolytic negative isogenic Serratia strain was generated to point out the importance of hemolysin production. Results We identified an important bacterial factor mediating the internalization of S. marcescens, and lysis of epithelial cells, as the secreted cytolysin ShlA. Microtubule filaments and actin filaments were shown to be involved in internalization. However, cytolysis of eukaryotic cells by ShlA was an interfering factor, and therefore hemolytic-negative mutants were used in subsequent experiments. Isogenic hemolysin-negative mutant strains were still adhesive, but were no longer cytotoxic, did not disrupt the cell culture monolayer, and were no longer internalized by HEp-2 and RT112 bladder epithelial cells under the conditions used for the wild-type strain. After wild-type S. marcescens became intracellular, the infected epithelial cells were lysed by extended vacuolation induced by ShlA. In late stages of vacuolation, highly motile S. marcescens cells were observed in the vacuoles. S. marcescens was also able to replicate in cultured HEp-2 cells, and replication was not dependent on hemolysin production. Conclusion The results reported here showed that the pore-forming toxin ShlA triggers microtubule-dependent invasion and is the main factor inducing lysis of the epithelial cells to release the bacteria, and therefore plays a major role in the development of S. marcescens infections. PMID:15189566

Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype

PubMed Central

Plog, Stephanie; Klymiuk, Nikolai; Binder, Stefanie; Van Hook, Matthew J.; Thoreson, Wallace B.; Gruber, Achim D.; Mundhenk, Lars

2015-01-01

The human CLCA4 (chloride channel regulator, calcium-activated) modulates the intestinal phenotype of cystic fibrosis (CF) patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring null variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype. PMID:26474299

Glucocorticoid receptors in bronchial epithelial cells in asthma.

PubMed

Vachier, I; Chiappara, G; Vignola, A M; Gagliardo, R; Altieri, E; Térouanne, B; Vic, P; Bousquet, J; Godard, P; Chanez, P

1998-09-01

The expression of the glucocorticoid receptor (GR) in untreated or in steroid-dependent asthmatic patients is poorly understood. We therefore studied GR mRNA and protein levels in bronchial biopsies obtained from seven untreated asthmatic patients, seven control volunteers, and seven patients with chronic bronchitis. We also studied in bronchial epithelial cells obtained by brushing from 13 untreated asthmatics, 18 steroid-dependent asthmatics, 11 control volunteers, and 12 patients with chronic bronchitis, GR and heat shock protein 90 kD (hsp90) mRNA as well as the immunoreactivity of GR, intercellular adhesion molecule (ICAM-1), and granulocyte macrophage-colony-stimulating factor (GM-CSF). GR mRNA and protein level was similar in all subject groups in both biopsies and bronchial epithelial cells. Hsp90 mRNA level was also similar in all subject groups. ICAM-1 expression was significantly increased in bronchial epithelial cells from untreated asthmatics, but ICAM-1 was not expressed in those from steroid-dependent asthmatic patients. GM-CSF expression was significantly increased in bronchial epithelial cells from untreated and steroid-dependent asthmatic patients. GR expression within the airways is unaltered by oral long-term steroid treatment in asthma, but the expression of some but not all specific markers for asthma is modified by oral steroid.

In vitro epithelial differentiation of human induced pluripotent stem cells for vocal fold tissue engineering.

PubMed

Imaizumi, Mitsuyoshi; Sato, Yuka; Yang, David T; Thibeault, Susan L

2013-12-01

We determined the feasibility and optimization of differentiating human induced pluripotent stem cells (hiPS) into nonkeratinized stratified squamous epithelial cells for vocal fold engineering. hiPS were cultured and assessed for differentiation in 3 conditions: a 3-dimensional (3D) hyaluronic acid (HA) hydrogel scaffold, a 3D HA hydrogel scaffold with epidermal growth factor (EGF), and a 3D HA hydrogel scaffold cocultured with human vocal fold fibroblasts (hVFF). After 1, 2, and 4 weeks of cultivation, hiPS were selected for histology, immunohistochemistry, and/or transcript expression analysis. At 4 weeks, hiPS cultivated with hVFF or with EGF had significantly decreased levels of Oct 3/4, indicating loss of pluripotency. Immunofluorescence revealed the presence of pancytokeratin and of cytokeratin (CK) 13 and 14 epithelial-associated proteins at 4 weeks after cultivation in hiPS EGF and hiPS hVFF cultures. The transcript expression level of CK14 was significantly increased for hiPS hVFF cultures only and was measured concomitantly with cell morphology that was clearly cohesive and displayed a degree of nuclear polarity suggestive of epithelial differentiation. We found that hiPS cultivated in 3D HA hydrogel with hVFF demonstrated the most robust conversion evidence to date of epithelial differentiation. Further work is necessary to focus on amplification of these progenitors for application in vocal fold regenerative biology.

Fibrosis of Two: Epithelial Cell-Fibroblast Interactions in Pulmonary Fibrosis

PubMed Central

Sakai, Norihiko; Tager, Andrew M.

2013-01-01

Idiopathic pulmonary fibrosis (IPF) is characterized by the progressive and ultimately fatal accumulation of fibroblasts and extracellular matrix in the lung that distorts its architecture and compromises its function. IPF is now thought to result from wound-healing processes that, although initiated to protect the host from injurious environmental stimuli, lead to pathological fibrosis due to these processes becoming aberrant or over-exuberant. Although the environmental stimuli that trigger IPF remain to be identified, recent evidence suggests that they initially injure the alveolar epithelium. Repetitive cycles of epithelial injury and resultant alveolar epithelial cell death provoke the migration, proliferation, activation and myofibroblast differentiation of fibroblasts, causing the accumulation of these cells and the extracellular matrix that they synthesize. In turn, these activated fibroblasts induce further alveolar epithelial cell injury and death, thereby creating a vicious cycle of pro-fibrotic epithelial cell-fibroblast interactions. Though other cell types certainly make important contributions, we focus here on the “pas de deux” (steps of two), or perhaps more appropriate to IPF pathogenesis, the “folie à deux” (madness of two) of epithelial cells and fibroblasts that drives the progression of pulmonary fibrosis. We describe the signaling molecules that mediate the interactions of these cell types in their “fibrosis of two”, including transforming growth factor-β, connective tissue growth factor, sonic hedgehog, prostaglandin E2, angiotensin II and reactive oxygen species. PMID:23499992

Short Chain Fatty Acids (SCFA) Reprogram Gene Expression in Human Malignant Epithelial and Lymphoid Cells

PubMed Central

Astakhova, Lidiia; Ngara, Mtakai; Babich, Olga; Prosekov, Aleksandr; Asyakina, Lyudmila; Dyshlyuk, Lyubov; Midtvedt, Tore; Zhou, Xiaoying; Ernberg, Ingemar; Matskova, Liudmila

2016-01-01

The effect of short chain fatty acids (SCFAs) on gene expression in human, malignant cell lines was investigated, with a focus on signaling pathways. The commensal microbial flora produce high levels of SCFAs with established physiologic effects in humans. The most abundant SCFA metabolite in the human microflora is n-butyric acid. It is well known to activate endogenous latent Epstein-Barr virus (EBV), that was used as a reference read out system and extended to EBV+ epithelial cancer cell lines. N-butyric acid and its salt induced inflammatory and apoptotic responses in tumor cells of epithelial and lymphoid origin. Epithelial cell migration was inhibited. The n-butyric gene activation was reduced by knock-down of the cell membrane transporters MCT-1 and -4 by siRNA. N-butyric acid show biologically significant effects on several important cellular functions, also with relevance for tumor cell phenotype. PMID:27441625

Myosin-X functions in polarized epithelial cells

PubMed Central

Liu, Katy C.; Jacobs, Damon T.; Dunn, Brian D.; Fanning, Alan S.; Cheney, Richard E.

2012-01-01

Myosin-X (Myo10) is an unconventional myosin that localizes to the tips of filopodia and has critical functions in filopodia. Although Myo10 has been studied primarily in nonpolarized, fibroblast-like cells, Myo10 is expressed in vivo in many epithelia-rich tissues, such as kidney. In this study, we investigate the localization and functions of Myo10 in polarized epithelial cells, using Madin-Darby canine kidney II cells as a model system. Calcium-switch experiments demonstrate that, during junction assembly, green fluorescent protein–Myo10 localizes to lateral membrane cell–cell contacts and to filopodia-like structures imaged by total internal reflection fluorescence on the basal surface. Knockdown of Myo10 leads to delayed recruitment of E-cadherin and ZO-1 to junctions, as well as a delay in tight junction barrier formation, as indicated by a delay in the development of peak transepithelial electrical resistance (TER). Although Myo10 knockdown cells eventually mature into monolayers with normal TER, these monolayers do exhibit increased paracellular permeability to fluorescent dextrans. Importantly, knockdown of Myo10 leads to mitotic spindle misorientation, and in three-dimensional culture, Myo10 knockdown cysts exhibit defects in lumen formation. Together these results reveal that Myo10 functions in polarized epithelial cells in junction formation, regulation of paracellular permeability, and epithelial morphogenesis. PMID:22419816

Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells

PubMed Central

Lechuga, Susana; Baranwal, Somesh; Li, Chao; Naydenov, Nayden G.; Kuemmerle, John F.; Dugina, Vera; Chaponnier, Christine; Ivanov, Andrei I.

2014-01-01

Transdifferentiation of epithelial cells into mesenchymal cells and myofibroblasts plays an important role in tumor progression and tissue fibrosis. Such epithelial plasticity is accompanied by dramatic reorganizations of the actin cytoskeleton, although mechanisms underlying cytoskeletal effects on epithelial transdifferentiation remain poorly understood. In the present study, we observed that selective siRNA-mediated knockdown of γ-cytoplasmic actin (γ-CYA), but not β-cytoplasmic actin, induced epithelial-to-myofibroblast transition (EMyT) of different epithelial cells. The EMyT manifested by increased expression of α-smooth muscle actin and other contractile proteins, along with inhibition of genes responsible for cell proliferation. Induction of EMyT in γ-CYA–depleted cells depended on activation of serum response factor and its cofactors, myocardial-related transcriptional factors A and B. Loss of γ-CYA stimulated formin-mediated actin polymerization and activation of Rho GTPase, which appear to be essential for EMyT induction. Our findings demonstrate a previously unanticipated, unique role of γ-CYA in regulating epithelial phenotype and suppression of EMyT that may be essential for cell differentiation and tissue fibrosis. PMID:25143399

Myosin-Va-Dependent Cell-To-Cell Transfer of RNA from Schwann Cells to Axons

PubMed Central

Sotelo, José R.; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José R.; Xu, Lei; Wallrabe, Horst; Calliari, Aldo; Rosso, Gonzalo; Cal, Karina; Mercer, John A.

2013-01-01

To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration. PMID:23626749

Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

PubMed

Sotelo, José R; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José R; Xu, Lei; Wallrabe, Horst; Calliari, Aldo; Rosso, Gonzalo; Cal, Karina; Mercer, John A

2013-01-01

To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.

HIV infection-induced transcriptional program in renal tubular epithelial cells activates a CXCR2-driven CD4+ T-cell chemotactic response.

PubMed

Chen, Ping; Yi, Zhengzi; Zhang, Weijia; Klotman, Mary E; Chen, Benjamin K

2016-07-31

Viral replication and interstitial inflammation play important roles in the pathogenesis of HIV-associated nephropathy. Cell-cell interactions between renal tubule epithelial cells (RTECs) and HIV-infected T cells can trigger efficient virus internalization and viral gene expression by RTEC. To understand how HIV replication initiates HIV-associated nephropathy, we studied the cellular response of RTECs to HIV, examining the transcriptional profiles of primary RTECs exposed to cell-free HIV or HIV-infected T cells. HIV-induced gene expression in hRTECs was examined in vitro by Illumina RNA deep sequencing and revealed an innate response to HIV, which was subclassified by gene ontology biological process terms. Chemokine responses were examined by CD4 T-cell chemotaxis assays. As compared with cell-free virus infection, exposure to HIV-infected T cells elicited a stronger upregulation of inflammatory and immune response genes. A major category of upregulated genes are chemokine/cytokine families involved in inflammation and immune response, including inflammatory cytokines CCL20, IL6 and IL8-related chemokines: IL8, CXCL1, CXCL2, CXCL3, CXCL5 and CXCL6. Supernatants from virus-exposed RTECs contained strong chemoattractant activity on primary CD4 T cells, which was potently blocked by a CXCR2 antagonist that antagonizes IL8-related chemokines. We observed a preferential migration of CXCR2-expressing, central memory CD4 T cells in response to HIV infection of RTECs. Interactions between primary RTECs and HIV-infected T cells result in potent induction of inflammatory response genes and release of cytokines/chemokines from RTECs that can attract additional T cells. Activation of these genes reflects an innate response to HIV by nonimmune cells.

Alarmins from corneal epithelial cells upregulate CCL11 and VCAM-1 in corneal fibroblasts.

PubMed

Fukuda, Ken; Ishida, Waka; Tanaka, Hiroshi; Harada, Yosuke; Matsuda, Akira; Ebihara, Nobuyuki; Fukushima, Atsuki

2013-08-27

Severe ocular allergic diseases are characterized by pronounced conjunctival inflammation triggered by T helper 2 (Th2) cells and corneal epithelial damage induced by eosinophils. To examine the role of alarmins released by damaged corneal epithelial cells in tissue eosinophilia, we investigated the effects of a supernatant derived from necrotic human corneal epithelial (HCE) cells on expression of the chemokine CCL11 (eotaxin) and the adhesion molecule VCAM-1 in human corneal fibroblasts. An alarmin preparation was obtained as the material released from HCE cells after three cycles of freezing and thawing. CCL11 released into culture medium and cell surface expression of VCAM-1 were measured with enzyme-linked immunosorbent assays, and the amounts of CCL11 and VCAM-1 mRNAs were quantitated by reverse transcription and real-time polymerase chain reaction analysis. Signaling by the transcription factor NF-κB was evaluated by immunoblot and immunofluorescence analyses. The combination of the necrotic HCE cell supernatant and either interleukin (IL)-4 or IL-13 induced synergistic increases in CCL11 release, VCAM-1 expression, and the abundance of CCL11 and VCAM-1 mRNAs in corneal fibroblasts. The necrotic HCE cell supernatant also induced NF-κB activation in corneal fibroblasts, whereas an inhibitor of NF-κB and IL-1 receptor antagonist each attenuated CCL11 release induced by the alarmin preparation and either IL-4 or IL-13. Alarmins including IL-1 released from necrotic corneal epithelial cells cooperate with Th2 cytokines to induce CCL11 production and VCAM-1 expression in corneal fibroblasts, and may thereby play an important role in tissue eosinophilia associated with ocular allergic diseases.

Human Rhinovirus Infection of Epithelial Cells Modulates Airway Smooth Muscle Migration.

PubMed

Shariff, Sami; Shelfoon, Christopher; Holden, Neil S; Traves, Suzanne L; Wiehler, Shahina; Kooi, Cora; Proud, David; Leigh, Richard

2017-06-01

Airway remodeling, a characteristic feature of asthma, begins in early life. Recurrent human rhinovirus (HRV) infections are a potential inciting stimulus for remodeling. One component of airway remodeling is an increase in airway smooth muscle cell (ASMC) mass with a greater proximity of the ASMCs to the airway epithelium. We asked whether human bronchial epithelial cells infected with HRV produced mediators that are chemotactic for ASMCs. ASMC migration was investigated using the modified Boyden Chamber and the xCELLigence Real-Time Cell Analyzer (ACEA Biosciences Inc., San Diego, CA). Multiplex bead analysis was used to measure HRV-induced epithelial chemokine release. The chemotactic effects of CCL5, CXCL8, and CXCL10 were also examined. Supernatants from HRV-infected epithelial cells caused ASMC chemotaxis. Pretreatment of ASMCs with pertussis toxin abrogated chemotaxis, as did treatment with formoterol, forskolin, or 8-bromo-cAMP. CCL5, CXCL8, and CXCL10 were the most up-regulated chemokines produced by HRV-infected airway epithelial cells. When recombinant CCL5, CXCL8, and CXCL10 were used at levels found in epithelial supernatants, they induced ASMC chemotaxis similar to that seen with epithelial cell supernatants. When examined individually, CCL5 was the most effective chemokine in causing ASMC migration, and treatment of supernatant from HRV-infected epithelial cells with anti-CCL5 antibodies significantly attenuated ASMC migration. These findings suggest that HRV-induced CCL5 can induce ASMC chemotaxis and thus may contribute to the pathogenesis of airway remodeling in patients with asthma.

Airway epithelial SPDEF integrates goblet cell differentiation and pulmonary Th2 inflammation

PubMed Central

Rajavelu, Priya; Chen, Gang; Xu, Yan; Kitzmiller, Joseph A.; Korfhagen, Thomas R.; Whitsett, Jeffrey A.

2015-01-01

Epithelial cells that line the conducting airways provide the initial barrier and innate immune responses to the abundant particles, microbes, and allergens that are inhaled throughout life. The transcription factors SPDEF and FOXA3 are both selectively expressed in epithelial cells lining the conducting airways, where they regulate goblet cell differentiation and mucus production. Moreover, these transcription factors are upregulated in chronic lung disorders, including asthma. Here, we show that expression of SPDEF or FOXA3 in airway epithelial cells in neonatal mice caused goblet cell differentiation, spontaneous eosinophilic inflammation, and airway hyperresponsiveness to methacholine. SPDEF expression promoted DC recruitment and activation in association with induction of Il33, Csf2, thymic stromal lymphopoietin (Tslp), and Ccl20 transcripts. Increased Il4, Il13, Ccl17, and Il25 expression was accompanied by recruitment of Th2 lymphocytes, group 2 innate lymphoid cells, and eosinophils to the lung. SPDEF was required for goblet cell differentiation and pulmonary Th2 inflammation in response to house dust mite (HDM) extract, as both were decreased in neonatal and adult Spdef–/– mice compared with control animals. Together, our results indicate that SPDEF causes goblet cell differentiation and Th2 inflammation during postnatal development and is required for goblet cell metaplasia and normal Th2 inflammatory responses to HDM aeroallergen. PMID:25866971

Regulation of epithelial and lymphocyte cell adhesion by adenosine deaminase-CD26 interaction.

PubMed Central

Ginés, Silvia; Mariño, Marta; Mallol, Josefa; Canela, Enric I; Morimoto, Chikao; Callebaut, Christian; Hovanessian, Ara; Casadó, Vicent; Lluis, Carmen; Franco, Rafael

2002-01-01

The extra-enzymic function of cell-surface adenosine deaminase (ADA), an enzyme mainly localized in the cytosol but also found on the cell surface of monocytes, B cells and T cells, has lately been the subject of numerous studies. Cell-surface ADA is able to transduce co-stimulatory signals in T cells via its interaction with CD26, an integral membrane protein that acts as ADA-binding protein. The aim of the present study was to explore whether ADA-CD26 interaction plays a role in the adhesion of lymphocyte cells to human epithelial cells. To meet this aim, different lymphocyte cell lines (Jurkat and CEM T) expressing endogenous, or overexpressing human, CD26 protein were tested in adhesion assays to monolayers of colon adenocarcinoma human epithelial cells, Caco-2, which express high levels of cell-surface ADA. Interestingly, the adhesion of Jurkat and CEM T cells to a monolayer of Caco-2 cells was greatly dependent on CD26. An increase by 50% in the cell-to-cell adhesion was found in cells containing higher levels of CD26. Incubation with an anti-CD26 antibody raised against the ADA-binding site or with exogenous ADA resulted in a significant reduction (50-70%) of T-cell adhesion to monolayers of epithelial cells. The role of ADA-CD26 interaction in the lymphocyte-epithelial cell adhesion appears to be mediated by CD26 molecules that are not interacting with endogenous ADA (ADA-free CD26), since SKW6.4 (B cells) that express more cell-surface ADA showed lower adhesion than T cells. Adhesion stimulated by CD26 and ADA is mediated by T cell lymphocyte function-associated antigen. A role for ADA-CD26 interaction in cell-to-cell adhesion was confirmed further in integrin activation assays. FACS analysis revealed a higher expression of activated integrins on T cell lines in the presence of increasing amounts of exogenous ADA. Taken together, these results suggest that the ADA-CD26 interaction on the cell surface has a role in lymphocyte-epithelial cell adhesion. PMID

Observing planar cell polarity in multiciliated mouse airway epithelial cells.

PubMed

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. Copyright © 2015 Elsevier Inc. All rights reserved.

Alveolar epithelial cells undergo epithelial-mesenchymal transition in acute interstitial pneumonia: a case report

PubMed Central

2014-01-01

Background Acute interstitial pneumonia is a rare interstitial lung disease that rapidly progresses to respiratory failure or death. Several studies showed that myofibroblast plays an important role in the evolution of diffuse alveolar damage, which is the typical feature of acute interstitial pneumonia. However, no evidence exists whether alveolar epithelial cells are an additional source of myofibroblasts via epithelial-mesenchymal transition in acute interstitial pneumonia. Case presentation In this report, we present a case of acute interstitial pneumonia in a previously healthy 28-year-old non-smoking woman. Chest high-resolution computed tomography scan showed bilateral and diffusely ground-glass opacification. The biopsy was performed on the fifth day of her hospitalization, and results showed manifestation of acute exudative phase of diffuse alveolar damage characterized by hyaline membrane formation. On the basis of the preliminary diagnosis of acute interstitial pneumonia, high-dose glucocorticoid was used. However, this drug showed poor clinical response and could improve the patient’s symptoms only during the early phase. The patient eventually died of respiratory dysfunction. Histological findings in autopsy were consistent with the late form of acute interstitial pneumonia. Conclusions The results in this study revealed that alveolar epithelial cells underwent epithelial-mesenchymal transition and may be an important origin of myofibroblasts in the progression of acute interstitial pneumonia. Conducting research on the transformation of alveolar epithelial cells into myofibroblasts in the lung tissue of patients with acute interstitial pneumonia may be beneficial for the treatment of this disease. However, to our knowledge, no research has been conducted on this topic. PMID:24755111

Soluble Proteins Produced by Probiotic Bacteria Regulate Intestinal Epithelial Cell Survival and Growth

PubMed Central

YAN, FANG; CAO, HANWEI; COVER, TIMOTHY L.; WHITEHEAD, ROBERT; WASHINGTON, M. KAY; POLK, D. BRENT

2011-01-01

Background & Aims Increased inflammatory cytokine levels and intestinal epithelial cell apoptosis leading to disruption of epithelial integrity are major pathologic factors in inflammatory bowel diseases. The probiotic bacterium Lactobacillus rhamnosus GG (LGG) and factors recovered from LGG broth culture supernatant (LGG-s) prevent cytokine-induced apoptosis in human and mouse intestinal epithelial cells by regulating signaling pathways. Here, we purify and characterize 2 secreted LGG proteins that regulate intestinal epithelial cell antiapoptotic and proliferation responses. Methods LGG proteins were purified from LGG-s, analyzed, and used to generate polyclonal antibodies for immunodepletion of respective proteins from LGG-conditioned cell culture media (CM). Mouse colon epithelial cells and cultured colon explants were treated with purified proteins in the absence or presence of tumor necrosis factor (TNF). Akt activation, proliferation, tissue injury, apoptosis, and caspase-3 activation were determined. Results We purified 2 novel proteins, p75 (75 kilodaltons) and p40 (40 kilodaltons), from LGG-s. Each of these purified protein preparations activated Akt, inhibited cytokine-induced epithelial cell apoptosis, and promoted cell growth in human and mouse colon epithelial cells and cultured mouse colon explants. TNF-induced colon epithelial damage was significantly reduced by p75 and p40. Immunodepletion of p75 and p40 from LGG-CM reversed LGG-CM activation of Akt and its inhibitory effects on cytokine-induced apoptosis and loss of intestinal epithelial cells. Conclusions p75 and p40 are the first probiotic bacterial proteins demonstrated to promote intestinal epithelial homeostasis through specific signaling pathways. These findings suggest that probiotic bacterial components may be useful for preventing cytokine-mediated gastrointestinal diseases. PMID:17258729

Effects of Bisphenol A Metabolite 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene on Lung Function and Type 2 Pulmonary Alveolar Epithelial Cell Growth

PubMed Central

Liu, Shing-Hwa; Su, Chin-Chuan; Lee, Kuan-I; Chen, Ya-Wen

2016-01-01

Bisphenol A (BPA) is recognized as a major pollutant worldwide. 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) is a major active metabolite of BPA. The epidemiological and animal studies have reported that BPA is harmful to lung function. The role of MBP in lung dysfunction after BPA exposure still remains unclear. This study investigated whether MBP would induce lung alveolar cell damage and evaluated the role of MBP in the BPA exposure-induced lung dysfunction. An in vitro type 2 alveolar epithelial cell (L2) model and an ex vivo isolated reperfused rat lung model were used to determine the effects of BPA or MBP on cell growth and lung function. MBP, but not BPA, dose-dependently increased the mean artery pressure (Pa), pulmonary capillary pressure (Pc), pulmonary capillary filtration coefficient (Kfc), and wet/dry weight ratio in isolated reperfused rat lungs. MBP significantly reduced cell viability and induced caspases-3/7 cleavage and apoptosis and increased AMP-activated protein kinas (AMPK) phosphorylation and endoplasmic reticulum (ER) stress-related molecules expression in L2 cells, which could be reversed by AMPK-siRNA transfection. These findings demonstrated for the first time that MBP exposure induced type 2 alveolar cell apoptosis and lung dysfunction through an AMPK-regulated ER stress signaling pathway. PMID:27982077

Effects of Bisphenol A Metabolite 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene on Lung Function and Type 2 Pulmonary Alveolar Epithelial Cell Growth.

PubMed

Liu, Shing-Hwa; Su, Chin-Chuan; Lee, Kuan-I; Chen, Ya-Wen

2016-12-16

Bisphenol A (BPA) is recognized as a major pollutant worldwide. 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) is a major active metabolite of BPA. The epidemiological and animal studies have reported that BPA is harmful to lung function. The role of MBP in lung dysfunction after BPA exposure still remains unclear. This study investigated whether MBP would induce lung alveolar cell damage and evaluated the role of MBP in the BPA exposure-induced lung dysfunction. An in vitro type 2 alveolar epithelial cell (L2) model and an ex vivo isolated reperfused rat lung model were used to determine the effects of BPA or MBP on cell growth and lung function. MBP, but not BPA, dose-dependently increased the mean artery pressure (Pa), pulmonary capillary pressure (Pc), pulmonary capillary filtration coefficient (K fc ), and wet/dry weight ratio in isolated reperfused rat lungs. MBP significantly reduced cell viability and induced caspases-3/7 cleavage and apoptosis and increased AMP-activated protein kinas (AMPK) phosphorylation and endoplasmic reticulum (ER) stress-related molecules expression in L2 cells, which could be reversed by AMPK-siRNA transfection. These findings demonstrated for the first time that MBP exposure induced type 2 alveolar cell apoptosis and lung dysfunction through an AMPK-regulated ER stress signaling pathway.

Uterine epithelial cell proliferation and endometrial hyperplasia: evidence from a mouse model.

PubMed

Gao, Yang; Li, Shu; Li, Qinglei

2014-08-01

In the uterus, epithelial cell proliferation changes during the estrous cycle and pregnancy. Uncontrolled epithelial cell proliferation results in implantation failure and/or cancer development. Transforming growth factor-β (TGF-β) signaling is a fundamental regulator of diverse biological processes and is indispensable for multiple reproductive functions. However, the in vivo role of TGF-β signaling in uterine epithelial cells remains poorly defined. We have shown that in the uterus, conditional deletion of the Type 1 receptor for TGF-β (Tgfbr1) using anti-Müllerian hormone receptor type 2 (Amhr2) Cre leads to myometrial defects. Here, we describe enhanced epithelial cell proliferation by immunostaining of Ki67 in the uteri of these mice. The aberration culminated in endometrial hyperplasia in aged females. To exclude the potential influence of ovarian steroid hormones, the proliferative status of uterine epithelial cells was assessed following ovariectomy. Increased uterine epithelial cell proliferation was also revealed in ovariectomized Tgfbr1 Amhr2-Cre conditional knockout mice. We further demonstrated that transcript levels for fibroblast growth factor 10 (Fgf10) were markedly up-regulated in Tgfbr1 Amhr2-Cre conditional knockout uteri. Consistently, treatment of primary uterine stromal cells with TGF-β1 significantly reduced Fgf10 mRNA expression. Thus, our findings suggest a potential involvement of TGFBR1-mediated signaling in the regulation of uterine epithelial cell proliferation, and provide genetic evidence supporting the role of uterine epithelial cell proliferation in the pathogenesis of endometrial hyperplasia. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Irsogladine maleate regulates gap junctional intercellular communication-dependent epithelial barrier in human nasal epithelial cells.

PubMed

Miyata, Ryo; Nomura, Kazuaki; Kakuki, Takuya; Takano, Ken-Ichi; Kohno, Takayuki; Konno, Takumi; Sawada, Norimasa; Himi, Tetsuo; Kojima, Takashi

2015-04-01

The airway epithelium of the human nasal mucosa acts as the first physical barrier that protects against inhaled substances and pathogens. Irsogladine maleate (IM) is an enhancer of gastric mucosal protective factors via upregulation of gap junctional intercellular communication (GJIC). GJIC is thought to participate in the formation of functional tight junctions. However, the effects of IM on GJIC and the epithelial barrier in human nasal epithelial cells (HNECs) remain unknown. To investigate the effects of IM on GJIC and the tight junctional barrier in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were treated with IM and the GJIC inhibitors oleamide and 18β-GA. Some cells were pretreated with IM before treatment with TLR3 ligand poly(I:C) to examine whether IM prevented the changes via TLR3-mediated signal pathways. In hTERT-HNECs, GJIC blockers reduced the expression of tight junction molecules claudin-1, -4, -7, occludin, tricellulin, and JAM-A. IM induced GJIC activity and enhanced the expression of claudin-1, -4, and JAM-A at the protein and mRNA levels with an increase of barrier function. GJIC blockers prevented the increase of the tight junction proteins induced by IM. Furthermore, IM prevented the reduction of JAM-A but not induction of IL-8 and TNF-α induced by poly(I:C). In conclusion, IM can maintain the GJIC-dependent tight junctional barrier via regulation of GJIC in upper airway nasal epithelium. Therefore, it is possible that IM may be useful as a nasal spray to prevent the disruption of the epithelial barrier by viral infections and exposure to allergens in human nasal mucosa.

Ion pump sorting in polarized renal epithelial cells.

PubMed

Caplan, M J

2001-08-01

The plasma membranes of renal epithelial cells are divided into distinct apical and basolateral domains, which contain different inventories of ion transport proteins. Without this polarity vectorial ion and fluid transport would not be possible. Little is known of the signals and mechanisms that renal epithelial cells use to establish and maintain polarized distributions of their ion transport proteins. Analysis of ion pump sorting reveals that multiple complex signals participate in determining and regulating these proteins' subcellular localizations.

Continuous tooth replacement: the possible involvement of epithelial stem cells.

PubMed

Huysseune, Ann; Thesleff, Irma

2004-06-01

Epithelial stem cells have been identified in integumental structures such as hairs and continuously growing teeth of various rodents, and in the gut. Here we propose the involvement of epithelial stem cells in the continuous tooth replacement that characterizes non-mammalian vertebrates, as exemplified by the zebrafish. Arguments are based on morphological observations of tooth renewal in the zebrafish and on the similarities between molecular control of hair and tooth formation. Dissection of the molecular cascades underlying the regulation of the epithelial stem cell niche might open perspectives for new regenerative treatment strategies in clinical dentistry. Copyright 2004 Wiley Periodicals, Inc.

[Epithelial intestine cells transdifferentiate into bladder urothelium in experiments in vivo].

PubMed

Popov, B K; Zaĭchik, A M; Bud'ko, M B; Zlobina, O V; Tolkunova, E N; Zhidkova, O V; Petrov, N S

2011-01-01

The autoplastic surgery by intestine tissue has been used for reconstructive therapy of the urinary tract since the middle of the last century; however, cell mechanisms of the urothelium engraftment are still obscure. Intestine stem cells possess plasticity and presumably enable after the autoplastic surgery to transdifferentiate into mature cells of urinary tract. Using the preliminary developed in vivo model for evaluation of somatic cells transdifferentiation into urothelium, we have found that the epithelial intestine cells producing Gfp transdifferentiate into the cryoinjured bladder urothelium of the syngenetic C57BL mice. Gfp was detected in the bladder tissue of mice-recipients using reverted polymerase chain reaction, primary fluorescence and immunofluorescence, while colocalization of the Gfp and Her-4 revealing similar to urothelium staining pattern was demonstrated in a few urothelium cells by double immunohistochemical staining of the bladder tissue with specific antibodies. The results obtained suggest that epithelial intestine cells enable to transdifferentiate into bladder urothelium, however the transdifferentiation level is low and presumably can not provide full functional urothelium engraftment in the case of autoplastic bladder surgery by intestine tissue.

MARCKS-related protein regulates cytoskeletal organization at cell-cell and cell-substrate contacts in epithelial cells.

PubMed

Van Itallie, Christina M; Tietgens, Amber Jean; Aponte, Angel; Gucek, Marjan; Cartagena-Rivera, Alexander X; Chadwick, Richard S; Anderson, James M

2018-02-02

Treatment of epithelial cells with interferon-γ and TNF-α (IFN/TNF) results in increased paracellular permeability. To identify relevant proteins mediating barrier disruption, we performed proximity-dependent biotinylation (BioID) of occludin and found that tagging of MARCKS-related protein (MRP; also known as MARCKSL1) increased ∼20-fold following IFN/TNF administration. GFP-MRP was focused at the lateral cell membrane and its overexpression potentiated the physiological response of the tight junction barrier to cytokines. However, deletion of MRP did not abrogate the cytokine responses, suggesting that MRP is not required in the occludin-dependent IFN/TNF response. Instead, our results reveal a key role for MRP in epithelial cells in control of multiple actin-based structures, likely by regulation of integrin signaling. Changes in focal adhesion organization and basal actin stress fibers in MRP-knockout (KO) cells were reminiscent of those seen in FAK-KO cells. In addition, we found alterations in cell-cell interactions in MRP-KO cells associated with increased junctional tension, suggesting that MRP may play a role in focal adhesion-adherens junction cross talk. Together, our results are consistent with a key role for MRP in cytoskeletal organization of cell contacts in epithelial cells. © 2018. Published by The Company of Biologists Ltd.

Obesity Suppresses Cell-Competition-Mediated Apical Elimination of RasV12-Transformed Cells from Epithelial Tissues.

PubMed

Sasaki, Ayana; Nagatake, Takahiro; Egami, Riku; Gu, Guoqiang; Takigawa, Ichigaku; Ikeda, Wataru; Nakatani, Tomoya; Kunisawa, Jun; Fujita, Yasuyuki

2018-04-24

Recent studies have revealed that newly emerging transformed cells are often eliminated from epithelial tissues via cell competition with the surrounding normal epithelial cells. This cancer preventive phenomenon is termed epithelial defense against cancer (EDAC). However, it remains largely unknown whether and how EDAC is diminished during carcinogenesis. In this study, using a cell competition mouse model, we show that high-fat diet (HFD) feeding substantially attenuates the frequency of apical elimination of RasV12-transformed cells from intestinal and pancreatic epithelia. This process involves both lipid metabolism and chronic inflammation. Furthermore, aspirin treatment significantly facilitates eradication of transformed cells from the epithelial tissues in HFD-fed mice. Thus, our work demonstrates that obesity can profoundly influence competitive interaction between normal and transformed cells, providing insights into cell competition and cancer preventive medicine. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Loss of Canonical Smad4 Signaling Promotes KRAS Driven Malignant Transformation of Human Pancreatic Duct Epithelial Cells and Metastasis

PubMed Central

Leung, Lisa; Radulovich, Nikolina; Zhu, Chang-Qi; Wang, Dennis; To, Christine; Ibrahimov, Emin; Tsao, Ming-Sound

2013-01-01

Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer death in North America. Activating KRAS mutations and Smad4 loss occur in approximately 90% and 55% of PDAC, respectively. While their roles in the early stages of PDAC development have been confirmed in genetically modified mouse models, their roles in the multistep malignant transformation of human pancreatic duct cells have not been directly demonstrated. Here, we report that Smad4 represents a barrier in KRAS-mediated malignant transformation of the near normal immortalized human pancreatic duct epithelial (HPDE) cell line model. Marked Smad4 downregulation by shRNA in KRAS G12V expressing HPDE cells failed to cause tumorigenic transformation. However, KRAS-mediated malignant transformation occurred in a new HPDE-TGF-β resistant (TβR) cell line that completely lacks Smad4 protein expression and is resistant to the mito-inhibitory activity of TGF-β. This transformation resulted in tumor formation and development of metastatic phenotype when the cells were implanted orthotopically into the mouse pancreas. Smad4 restoration re-established TGF-β sensitivity, markedly increased tumor latency by promoting apoptosis, and decreased metastatic potential. These results directly establish the critical combination of the KRAS oncogene and complete Smad4 inactivation in the multi-stage malignant transformation and metastatic progression of normal human HPDE cells. PMID:24386371

[Effect of chorionic gonadotropin on thymocyte differentiation in the presence of thymus epithelial cells].

PubMed

Kuklina, E M; Shirshev, S V; Sharova, N I; Iarilin, A A

2003-01-01

We studied the effects of the main placental hormone, chorionic gonadotropin, on differentiation of human thymocytes in vitro in the presence of thymic epithelial cells. It was shown that the hormone at a high dose (100 IU/ml) enhanced the epithelium-induced phenotypic maturation of thymocytes, which is registered by an increased expression of the membrane marker CD3 and transition of CD4+8+ thymocytes in the cells with CD4+8- and CD4-8+ phenotypes. In addition, gonadotropin enhanced the proliferative response of thymocytes to the mitogen during their cultivation with the epithelium. The stimulating effect of the hormone on the epithelium-induced differentiation of thymocytes is mediated by the humoral factors of epithelial cells. In addition, gonadotropin at this dose exerts its own differentiating activity with respect to thymocytes and stimulates their phenotypic and functional maturation in a monoculture.

Asbestos exposure induces alveolar epithelial cell plasticity through MAPK/Erk signaling.

PubMed

Tamminen, Jenni A; Myllärniemi, Marjukka; Hyytiäinen, Marko; Keski-Oja, Jorma; Koli, Katri

2012-07-01

The inhalation of asbestos fibers is considered to be highly harmful, and lead to fibrotic and/or malignant disease. Epithelial-to-mesenchymal transition (EMT) is a common pathogenic mechanism in asbestos associated fibrotic (asbestosis) and malignant lung diseases. The characterization of molecular pathways contributing to EMT may provide new possibilities for prognostic and therapeutic applications. The role of asbestos as an inducer of EMT has not been previously characterized. We exposed cultured human lung epithelial cells to crocidolite asbestos and analyzed alterations in the expression of epithelial and mesenchymal marker proteins and cell morphology. Asbestos was found to induce downregulation of E-cadherin protein levels in A549 lung carcinoma cells in 2-dimensional (2D) and 3D cultures. Similar findings were made in primary small airway epithelial cells cultured in 3D conditions where the cells retained alveolar type II cell phenotype. A549 cells also exhibited loss of cell-cell contacts, actin reorganization and expression of α-smooth muscle actin (α-SMA) in 2D cultures. These phenotypic changes were not associated with increased transforming growth factor (TGF)-β signaling activity. MAPK/Erk signaling pathway was found to mediate asbestos-induced downregulation of E-cadherin and alterations in cell morphology. Our results suggest that asbestos can induce epithelial plasticity, which can be interfered by blocking the MAPK/Erk kinase activity. Copyright © 2012 Wiley Periodicals, Inc.