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Sample records for epithelial gene expression

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

    PubMed Central

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

    2009-01-01

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

  2. Gene expression in epithelial cells in response to pneumovirus infection

    PubMed Central

    Domachowske, Joseph B; Bonville, Cynthia A; Rosenberg, Helene F

    2001-01-01

    Respiratory syncytial virus (RSV) and pneumonia virus of mice (PVM) are viruses of the family Paramyxoviridae, subfamily pneumovirus, which cause clinically important respiratory infections in humans and rodents, respectively. The respiratory epithelial target cells respond to viral infection with specific alterations in gene expression, including production of chemoattractant cytokines, adhesion molecules, elements that are related to the apoptosis response, and others that remain incompletely understood. Here we review our current understanding of these mucosal responses and discuss several genomic approaches, including differential display reverse transcription-polymerase chain reaction (PCR) and gene array strategies, that will permit us to unravel the nature of these responses in a more complete and systematic manner. PMID:11686888

  3. Stability of Reference Gene Expression After Porcine Sapelovirus Infection in Porcine Intestinal Epithelial Cells.

    PubMed

    Huang, Yong; Chen, Yabing; Sun, Huan; Lan, Daoliang

    2016-01-01

    Intestinal epithelial cells, which serve as the first physical barrier to protect intestinal tract from external antigens, have an important role in the local innate immunity. Screening of reference genes that have stable expression levels after viral infection in porcine intestinal epithelial cells is critical for ensuring the reliability of the expression analysis on anti-infection genes in porcine intestinal epithelial cells. In this study, nine common reference genes in pigs, including ACTB, B2M, GAPDH, HMBS, SDHA, HPRT1, TBP, YWHAZ, and RPL32, were chosen as the candidate reference genes. Porcine sapelovirus (PSV) was used as a model virus to infect porcine intestinal epithelial cell line (IPEC-J2). The expression stability of the nine genes was assessed by the geNorm, NormFinder, and BestKeeper software. Moreover, RefFinder program was used to evaluate the analytical results of above three softwares, and a relative expression experiment of selected target gene was used to verify the analysis results. The comprehensive results indicated that the gene combination of TBP and RPL32 has the most stable expression, which could be considered as an appropriate reference gene for research on gene expression after PSV infection in IPEC-J2cells. The results provided essential data for expression analysis of anti-infection genes in porcine intestinal epithelial cells. PMID:27092424

  4. Epithelial expression and chromosomal location of human TLE genes: Implications for notch signaling and neoplasia

    SciTech Connect

    Liu, Yanling; Dehni, Ghassan; Stifani, S.

    1996-01-01

    The TLE genes are the human homologues of Drosophila groucho, a member of the Notch signaling pathway. This pathway controls a number of different cell-fate choices in invertebrates and vertebrates. We are interested in investigating the functions of the TLE gene family during epithelial determination and carcinogenesis. We show that expression of individual TLE genes correlates with immature epithelial cells that are progressing toward their terminally differentiated state, suggesting a role during epithelial differentiation. In both normal tissues and conditions resulting from incorrect or incomplete maturation events, such as metaplastic and neoplastic transformations, TLE expression is elevated and coincides with Notch expression, implicating these molecules in the maintenance of the undifferentiated state in epithelial cells. We also show that TLE1 and TLE2 are organized in a tandem array at chromosomal location 19p13.3, while TLE3 maps to 15q22. 26 refs., 4 figs.

  5. Gene expression analysis uncovers novel Hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells

    PubMed Central

    Zhou, Xiaobo; Qiu, Weiliang; Sathirapongsasuti, J. Fah.; Cho, Michael H.; Mancini, John D.; Lao, Taotao; Thibault, Derek M.; Litonjua, Gus; Bakke, Per S.; Gulsvik, Amund; Lomas, David A.; Beaty, Terri H.; Hersh, Craig P.; Anderson, Christopher; Geigenmuller, Ute; Raby, Benjamin A.; Rennard, Stephen I.; Perrella, Mark A.; Choi, Augustine M.K.; Quackenbush, John; Silverman, Edwin K.

    2013-01-01

    Hedgehog Interacting Protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis. PMID:23459001

  6. UV-induced changes in cell cycle and gene expression within rabbit lens epithelial cells

    SciTech Connect

    Sidjanin, D.; Grdina, D.; Woloschak, G.E.

    1994-11-01

    Damage to lens epithelial cells is a probable initiation process in cataract formation induced by ultraviolet radiation. These experiments investigated the ability of 254 nm radiation on cell cycle progression and gene expression in rabbit lens epithelial cell line N/N1003A. No changes in expression of c-fos, c-jun, alpha- tubulin, or vimentin was observed following UV exposure. Using flow cytometry, an accumulation of cells in G1/S phase of the cell cycle 1 hr following exposure. The observed changes in gene expression, especially the decreased histone transcripts reported here may play a role in UV induced inhibition of cell cycle progression.

  7. Gene expression correlations in human cancer cell lines define molecular interaction networks for epithelial phenotype.

    PubMed

    Kohn, Kurt W; Zeeberg, Barry M; Reinhold, William C; Pommier, Yves

    2014-01-01

    Using gene expression data to enhance our knowledge of control networks relevant to cancer biology and therapy is a challenging but urgent task. Based on the premise that genes that are expressed together in a variety of cell types are likely to functions together, we derived mutually correlated genes that function together in various processes in epithelial-like tumor cells. Expression-correlated genes were derived from data for the NCI-60 human tumor cell lines, as well as data from the Broad Institute's CCLE cell lines. NCI-60 cell lines that selectively expressed a mutually correlated subset of tight junction genes served as a signature for epithelial-like cancer cells. Those signature cell lines served as a seed to derive other correlated genes, many of which had various other epithelial-related functions. Literature survey yielded molecular interaction and function information about those genes, from which molecular interaction maps were assembled. Many of the genes had epithelial functions unrelated to tight junctions, demonstrating that new function categories were elicited. The most highly correlated genes were implicated in the following epithelial functions: interactions at tight junctions (CLDN7, CLDN4, CLDN3, MARVELD3, MARVELD2, TJP3, CGN, CRB3, LLGL2, EPCAM, LNX1); interactions at adherens junctions (CDH1, ADAP1, CAMSAP3); interactions at desmosomes (PPL, PKP3, JUP); transcription regulation of cell-cell junction complexes (GRHL1 and 2); epithelial RNA splicing regulators (ESRP1 and 2); epithelial vesicle traffic (RAB25, EPN3, GRHL2, EHF, ADAP1, MYO5B); epithelial Ca(+2) signaling (ATP2C2, S100A14, BSPRY); terminal differentiation of epithelial cells (OVOL1 and 2, ST14, PRSS8, SPINT1 and 2); maintenance of apico-basal polarity (RAB25, LLGL2, EPN3). The findings provide a foundation for future studies to elucidate the functions of regulatory networks specific to epithelial-like cancer cells and to probe for anti-cancer drug targets. PMID:24940735

  8. Cigarette Smoke Modulates Expression of Human Rhinovirus-Induced Airway Epithelial Host Defense Genes

    PubMed Central

    Proud, David; Hudy, Magdalena H.; Wiehler, Shahina; Zaheer, Raza S.; Amin, Minaa A.; Pelikan, Jonathan B.; Tacon, Claire E.; Tonsaker, Tabitha O.; Walker, Brandie L.; Kooi, Cora; Traves, Suzanne L.; Leigh, Richard

    2012-01-01

    Human rhinovirus (HRV) infections trigger acute exacerbations of chronic obstructive pulmonary disease (COPD) and asthma. The human airway epithelial cell is the primary site of HRV infection and responds to infection with altered expression of multiple genes, the products of which could regulate the outcome to infection. Cigarette smoking aggravates asthma symptoms, and is also the predominant risk factor for the development and progression of COPD. We, therefore, examined whether cigarette smoke extract (CSE) modulates viral responses by altering HRV-induced epithelial gene expression. Primary cultures of human bronchial epithelial cells were exposed to medium alone, CSE alone, purified HRV-16 alone or to HRV-16+ CSE. After 24 h, supernatants were collected and total cellular RNA was isolated. Gene array analysis was performed to examine mRNA expression. Additional experiments, using real-time RT-PCR, ELISA and/or western blotting, validated altered expression of selected gene products. CSE and HRV-16 each induced groups of genes that were largely independent of each other. When compared to gene expression in response to CSE alone, cells treated with HRV+CSE showed no obvious differences in CSE-induced gene expression. By contrast, compared to gene induction in response to HRV-16 alone, cells exposed to HRV+CSE showed marked suppression of expression of a number of HRV-induced genes associated with various functions, including antiviral defenses, inflammation, viral signaling and airway remodeling. These changes were not associated with altered expression of type I or type III interferons. Thus, CSE alters epithelial responses to HRV infection in a manner that may negatively impact antiviral and host defense outcomes. PMID:22808255

  9. The multidrug resistance and cystic fibrosis genes have complementary patterns of epithelial expression.

    PubMed Central

    Trezise, A E; Romano, P R; Gill, D R; Hyde, S C; Sepúlveda, F V; Buchwald, M; Higgins, C F

    1992-01-01

    The cystic fibrosis gene product, CFTR, and the multidrug resistance P-glycoprotein (encoded by the MDR1 gene) are structurally related proteins and both are associated with epithelial chloride channel activities. We have compared their cell-specific expression in the rat by in situ hybridization. In all tissues examined the two genes were found to have complementary patterns of expression, demonstrating exquisite regulation in both cell-specific and temporal fashions. Additionally, a switch in expression from one gene to the other was observed in certain tissues. For example, expression in the intestine switches from CFTR to MDR1 as the cells migrate across the crypt-villus boundary. A switch from CFTR to MDR1 expression was also observed in the uterine epithelium upon pregnancy. These data suggest that CFTR and P-glycoprotein serve analogous roles in epithelial cells and provide additional evidence that P-glycoprotein has a physiological role in regulating epithelial cell volume. The patterns of expression suggest that the regulation of these two genes is coordinately controlled. Images PMID:1385112

  10. Macrophages regulate expression of α1,2-fucosyltransferase genes in human endometrial epithelial cells.

    PubMed

    Nakamura, Hitomi; Jasper, Melinda J; Hull, M Louise; Aplin, John D; Robertson, Sarah A

    2012-04-01

    The epithelial cell surface of the endometrium undergoes substantial biochemical changes to allow embryo attachment and implantation in early pregnancy. We hypothesized that tissue macrophages influence these events to promote uterine receptivity. To investigate the role of macrophages in regulating epithelial cell expression of genes linked to glycan-mediated embryo adhesion, Ishikawa, RL95-2 and HEC1A endometrial epithelial cells were cultured alone or with unactivated or lipopolysaccharide-activated monocytic U937 cells, separated using transwell inserts. Expression of mRNAs encoding two α1,2-fucosyltransferases (FUT1, FUT2) was increased in all three epithelial cell lines following co-culture with U937 cells, and was associated with increased fucosylation of cell surface glycoproteins detected using lectins from Ulex europaeus (UEA-1) and Dolichos biflorus (DBA). FUT1 induction by U937 cells also occurred in primary endometrial epithelial cells collected in luteal but not proliferative phase. Activation of the interleukin-6 (IL6)/leukemia inhibitory factor (LIF) cytokine signaling pathway with phosphorylation of STAT3 and elevated SOCS3 mRNA expression was evident in epithelial cells stimulated by U937 co-culture. Several recombinant macrophage-secreted cytokines exerted stimulatory or inhibitory effects on FUT1 and FUT2 mRNA expression, and the macrophage-derived cytokine LIF partially replicated the effects of U937 cells on both FUT1 and FUT2 expression and UEA-1 and DBA lectin reactivity in Ishikawa cells. These results suggest that macrophage-derived factors including LIF might facilitate development of an implantation-receptive endometrium by regulating surface glycan structures in epithelial cells. Abnormal phenotypes or altered abundance of uterine macrophages could contribute to the pathophysiology of primary unexplained infertility in women. PMID:22053055

  11. Differential gene expression in normal and transformed human mammary epithelial cells in response to oxidative stress

    PubMed Central

    Cortes, Diego F; Sha, Wei; Hower, Valerie; Blekherman, Greg; Laubenbacher, Reinhard; Akman, Steven; Torti, Suzy V; Shulaev, Vladimir

    2011-01-01

    Oxidative stress plays a key role in breast carcinogenesis. To investigate whether normal and malignant breast epithelial cells differ in their responses to oxidative stress, we examined the global gene expression profiles of three cell types, representing cancer progression from a normal to a malignant stage, under oxidative stress. Normal human mammary epithelial cells (HMEC), an immortalized cell line (HMLER-1), and a tumorigenic cell line (HMLER-5), were exposed to increased levels of reactive oxygen species (ROS) by treatment with glucose oxidase. Functional analysis of the metabolic pathways enriched with differentially expressed genes demonstrates that normal and malignant breast epithelial cells diverge substantially in their response to oxidative stress. While normal cells exhibit the up-regulation of antioxidant mechanisms, cancer cells are unresponsive to the ROS insult. However, the gene expression response of normal HMEC cells under oxidative stress is comparable to that of the malignant cells under normal conditions, indicating that altered redox status is persistent in breast cancer cells, which makes them resistant to increased generation of ROS. This study discusses some of the possible adaptation mechanisms of breast cancer cells under persistent oxidative stress that differentiate them from the response to acute oxidative stress in normal mammary epithelial cells. PMID:21397008

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

    PubMed

    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

  13. 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

  14. Milk fat globule is an alternative to mammary epithelial cells for gene expression analysis in buffalo.

    PubMed

    Chen, Qiuming; Wu, Yanjun; Zhang, Mingyuan; Xu, Wenwen; Guo, Xiaoping; Yan, Xueyu; Deng, Haiying; Jiang, Qinyang; Yang, Xiurong; Lan, Ganqiu; Guo, Yafen; Qin, Guangsheng; Jiang, Hesheng

    2016-05-01

    Owing to the difficulty in obtaining mammary gland tissue from lactating animals, it is difficult to test the expression levels of genes in mammary gland. The aim of the current study was to identify if milk fat globule (MFG) in buffalo milk was an alternative to mammary gland (MG) and milk somatic cell (MSC) for gene expression analysis. Six buffalos in late lactation were selected to collect MFG and MSC, and then MG was obtained by surgery. MFG was stained with acridine orange to successfully visualise RNA and several cytoplasmic crescents in MFG. The total RNA in MFG was successfully isolated and the integrity was assessed by agarose gel electrophoresis. We analysed the cellular components in MFG, MG and MSC through testing the expression of cell-specific genes by qRT-PCR. The results showed that adipocyte-specific gene (AdipoQ) and leucocyte-specific genes (CD43, CSF1 and IL1α) in MFG were not detected, whereas epithelial cell marker genes (Keratin 8 and Keratin 18) in MFG were higher than in MSC and lower than in MG, fibroblast marker gene (vimentin) in MFG was significantly lower than in MG and MSC, milk protein genes (LALBA, BLG and CSN2) and milk fat synthesis-related genes (ACC, BTN1A1, FABP3 and FAS) in MFG were higher than in MG and MSC. In conclusion, the total RNA in MFG mainly derives from mammary epithelial cells and can be used to study the functional gene expression of mammary epithelial cells. PMID:27032540

  15. Increased GADD gene expression in human colon epithelial cells exposed to deoxycholate.

    PubMed

    Scott, David W; Mutamba, Sophia; Hopkins, Robin G; Loo, George

    2005-01-01

    The colonic epithelium is often exposed to high concentrations of secondary bile acids, which stresses the epithelial cells, leading potentially to activation of stress-response genes. To examine this possibility in vitro, the purpose of this study was to determine if expression of certain growth arrest and DNA damage-inducible genes (GADD) is upregulated in human colonic epithelial cells exposed to deoxycholate (DOC). DNA macroarray screening of a small cluster of stress/apoptosis-related genes in DOC-treated HCT-116 colonocytes revealed clearly higher expression of only GADD45, which was confirmed by gene-specific relative RT-PCR analysis. Subsequently, it was found that DOC also increased GADD34 mRNA expression. However, mRNA expression of GADD153 was increased most markedly in DOC-treated HCT-116 colonocytes, which express wild-type p53. However, the upregulation of GADD34, GADD45, and GADD153 mRNA expression apparently did not require p53, based on the finding that DOC increased expression of all three GADD genes in HCT-15 colonocytes, which express mutant p53. In further studying GADD153 in particular, the effect of DOC on GADD153 mRNA was prevented by actinomycin-D (Act-D), but not by antioxidants or MAPK inhibitors. DOC also caused GADD153 protein to be expressed in close parallel with increased GADD153 mRNA expression. Induction of GADD153 protein by DOC was prevented by either anisomycin or cycloheximide. These findings suggest that DOC-induced upregulation of GADD153 mRNA expression occurred at the level of transcription without involving reactive oxygen species and MAPK signaling, and that the expression of GADD153 protein was due also to translation of pre-existing, and not just newly synthesized, mRNA. PMID:15316935

  16. cis-acting elements that confer lung epithelial cell expression of the CC10 gene.

    PubMed

    Stripp, B R; Sawaya, P L; Luse, D S; Wikenheiser, K A; Wert, S E; Huffman, J A; Lattier, D L; Singh, G; Katyal, S L; Whitsett, J A

    1992-07-25

    To define cis-acting genetic elements responsible for cell-specific transcriptional regulation of the CC10 gene, DNA sequences spanning nucleotides -2338 to +49 of the rat CC10 gene were linked to a reporter gene coding for chloramphenicol acetyltransferase (CAT). In transient expression assays, CC10 sequences were capable of restricting CAT expression to a human lung adenocarcinoma cell line similar to pulmonary Clara cells. Transgenic mice harboring the hybrid RtCC10-CAT construct expressed high levels of CAT activity specifically within protein extracts of lung and trachea. Transcripts for the CAT reporter gene colocalized with those for the endogenous murine CC10 gene within the airways of transgenic mice. Functional analysis of deletion mutants identified stimulatory, inhibitory, and cell type-specific transcriptional regulatory elements. The results of gel retention and DNaseI protection assays suggest that a transcriptional stimulatory region located between -320 and -175, and a cell type-specific regulatory element located between -175 and +49, result from a series of protein-DNA interactions occurring at -220 to -205 and -128 to -86, respectively. Lung epithelial specific transcriptional regulatory elements described herein will be useful for expression of chimeric genes within epithelial cells lining the trachea, bronchi, and bronchioles of mice. PMID:1634515

  17. Proteasome dysfunction inhibits surfactant protein gene expression in lung epithelial cells: mechanism of inhibition of SP-B gene expression.

    PubMed

    Das, Aparajita; Boggaram, Vijayakumar

    2007-01-01

    Surfactant proteins maintain lung function through their actions to reduce alveolar surface tension and control of innate immune responses in the lung. The ubiquitin proteasome pathway is responsible for the degradation of majority of intracellular proteins in eukaryotic cells, and proteasome dysfunction has been linked to the development of neurodegenerative, cardiac, and other diseases. Proteasome function is impaired in interstitial lung diseases associated with surfactant protein C (SP-C) mutation mapping to the BRICHOS domain located in the proSP-C protein. In this study we determined the effects of proteasome inhibition on surfactant protein expression in H441 and MLE-12 lung epithelial cells to understand the relationship between proteasome dysfunction and surfactant protein gene expression. Proteasome inhibitors lactacystin and MG132 reduced the levels of SP-A, SP-B, and SP-C mRNAs in a concentration-dependent manner in H441 and MLE-12 cells. In H441 cells, lactacystin and MG132 inhibition of SP-B mRNA was associated with similar decreases in SP-B protein, and the inhibition was due to inhibition of gene transcription. Proteasome inhibitors decreased thyroid transcription factor-1 (TTF-1)/Nkx2.1 DNA binding activity, and the reduced TTF-1 DNA binding activity was due to reduced expression levels of TTF-1 protein. These data indicated that the ubiquitin proteasome pathway is essential for the maintenance of surfactant protein gene expression and that disruption of this pathway inhibits surfactant protein gene expression via reduced expression of TTF-1 protein. PMID:16905641

  18. Histone Deacetylase Inhibitor Induces the Expression of Select Epithelial Genes in Mouse Utricle Sensory Epithelia-Derived Progenitor Cells

    PubMed Central

    Wang, Jue

    2014-01-01

    Abstract Mouse utricle sensory epithelial cell–derived progenitor cells (MUCs), which have hair cell progenitor and mesenchymal features via epithelial-to-mesenchymal transition (EMT) as previously described, provide a potential approach for hair cell regeneration via cell transplantation. In this study, we treated MUCs with trichostatin A (TSA) to determine whether histone deacetylase inhibitor is able to stimulate the expression of epithelial genes in MUCs, an essential step for guiding mesenchymal-like MUCs to become sensory epithelial cells. After 72 h of TSA treatment, MUCs acquired epithelial-like features, which were indicated by increased expression of epithelial markers such as Cdh1, Krt18, and Dsp. Additionally, TSA decreased the expression of mesenchymal markers, including Zeb1, Zeb2, Snai1, and Snai2, and prosensory genes Lfng, Six1, and Dlx5. Moreover, the expression of the hair cell genes Atoh1 and Myo6 was increased in TSA-treated MUCs. We also observed significantly decreased expression of Hdac2 and Hdac3 in TSA-treated MUCs. However, no remarkable change was detected in protein expression using immunofluorescence, indicating that TSA-induced HDAC inhibition may contribute to the initial stage of the mesenchymal-to-epithelial phenotypic change. In the future, more work is needed to induce hair cell regeneration using inner ear tissue–derived progenitors to achieve an entire mesenchymal-to-epithelial transition. PMID:24945595

  19. Expression analysis of immune response genes in fish epithelial cells following ranavirus infection.

    PubMed

    Holopainen, Riikka; Tapiovaara, Hannele; Honkanen, Jarno

    2012-06-01

    Ranaviruses (family Iridoviridae) are a growing threat to fish and amphibian populations worldwide. The immune response to ranavirus infection has been studied in amphibians, but little is known about the responses elicited in piscine hosts. In this study, the immune response and apoptosis induced by ranaviruses were investigated in fish epithelial cells. Epithelioma papulosum cyprini (EPC) cells were infected with four different viral isolates: epizootic haematopoietic necrosis virus (EHNV), frog virus 3 (FV3), European catfish virus (ECV) and doctor fish virus (DFV). Quantitative real-time PCR (qPCR) assays were developed to measure the mRNA expression of immune response genes during ranavirus infection. The target genes included tumour necrosis factor α (TNF-α), interleukin-1β (IL-1β), β2-microglobulin (β2M), interleukin-10 (IL-10) and transforming growth factor β (TGF-β). All ranaviruses elicited changes in immune gene expression. EHNV and FV3 caused a strong pro-inflammatory response with an increase in the expression of both IL-1β and TNF-α, whereas ECV and DFV evoked transient up-regulation of regulatory cytokine TGF-β. Additionally, all viral isolates induced increased β2M expression as well as apoptosis in the EPC cells. Our results indicate that epithelial cells can serve as an in vitro model for studying the mechanisms of immune response in the piscine host in the first stages of ranavirus infection. PMID:22452879

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

    PubMed Central

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

    2015-01-01

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

  1. Changes in the gene expression pattern of cytokeratins in human respiratory epithelial cells during culture.

    PubMed

    Endres, Michaela; Leinhase, Iris; Kaps, Christian; Wentges, Marek; Unger, Manja; Olze, Heidi; Ringe, Jochen; Sittinger, Michael; Rotter, Nicole

    2005-05-01

    The replacement of extensive tracheal defects resulting from intensive care medicine, trauma or large resections is still challenged by the re-epithelialization of an autologous or alloplastic trachea replacement. Therefore, this study was performed to investigate the potential of culture-expanded human respiratory epithelial cells (hREC) to regenerate a functional epithelium for tracheal tissue engineering. hREC from seven male nasal turbinates were freshly isolated, expanded on a collagenous matrix and subsequently cultured in high-density multi-layers to allow epithelial differentiation. The composition of epithelial cells in native respiratory epithelial tissue and culture-expanded hREC was analyzed by histological staining with Alcian blue and by immunohistochemical staining of cytokeratin pairs CK1/10 and CK5/14 with the antibodies 34betaE12 and CD44v6. Differentiation of culture-expanded hREC was further characterized by gene expression analysis of cytokeratins CK5, CK13, CK14 and CK18 using semi-quantitative real-time RT-PCR technique. Histological and immunohistochemical staining of culture-expanded hREC demonstrated basal cells covering the collagenous matrix. These cells formed a cellular multi-layer, which was composed of a basal layer of undifferentiated basal cells and an upper layer of cells differentiating along the squamous metaplasia and ciliated cell lineage. Lineage development of culture-expanded hREC was further documented by the induction of cytokeratins CK13 and CK18. Our results suggest that culture-expanded hREC have the potential to colonize collagen-coated biomaterials and to regenerate epithelial cell types for tracheal tissue engineering. PMID:15549340

  2. Meta-Analysis of Gene Expression Signatures Defining the Epithelial to Mesenchymal Transition during Cancer Progression

    PubMed Central

    Gröger, Christian J.; Grubinger, Markus; Waldhör, Thomas; Vierlinger, Klemens; Mikulits, Wolfgang

    2012-01-01

    The epithelial to mesenchymal transition (EMT) represents a crucial event during cancer progression and dissemination. EMT is the conversion of carcinoma cells from an epithelial to a mesenchymal phenotype that associates with a higher cell motility as well as enhanced chemoresistance and cancer stemness. Notably, EMT has been increasingly recognized as an early event of metastasis. Numerous gene expression studies (GES) have been conducted to obtain transcriptome signatures and marker genes to understand the regulatory mechanisms underlying EMT. Yet, no meta-analysis considering the multitude of GES of EMT has been performed to comprehensively elaborate the core genes in this process. Here we report the meta-analysis of 18 independent and published GES of EMT which focused on different cell types and treatment modalities. Computational analysis revealed clustering of GES according to the type of treatment rather than to cell type. GES of EMT induced via transforming growth factor-β and tumor necrosis factor-α treatment yielded uniformly defined clusters while GES of models with alternative EMT induction clustered in a more complex fashion. In addition, we identified those up- and downregulated genes which were shared between the multitude of GES. This core gene list includes well known EMT markers as well as novel genes so far not described in this process. Furthermore, several genes of the EMT-core gene list significantly correlated with impaired pathological complete response in breast cancer patients. In conclusion, this meta-analysis provides a comprehensive survey of available EMT expression signatures and shows fundamental insights into the mechanisms that are governing carcinoma progression. PMID:23251436

  3. Tamoxifen Induces Expression of Immune Response-Related Genes in Cultured Normal Human Mammary Epithelial Cells

    PubMed Central

    Schild-Hay, Laura J.; Leil, Tarek A.; Divi, Rao L.; Olivero, Ofelia, A.; Weston, Ainsley; Poirier, Miriam C.

    2008-01-01

    Use of tamoxifen (TAM) is associated with a 50% reduction in breast cancer incidence and an increase in endometrial cancer incidence. Here, we documented TAM-induced gene expression changes in cultured normal human mammary epithelial cells (NHMEC strains numbered 5, 16 and 40), established from tissue taken at reduction mammoplasty from 3 individuals. Cells exposed to 0, 10 or 50 μM TAM for 48 hours were evaluated for (E)-α-(deoxyguanosin-N2-yl)-tamoxifen (dG-N2-TAM) adduct formation by TAM-DNA (DNA modified with dG-N2-TAM) chemiluminescence immunoassay (CIA), gene expression changes using NCI DNA-oligonucleotide microarray, and real time (RT)-PCR. At 48 hr, cells exposed to 10 μM and 50 μM TAM were 85.6% and 48.4% viable, respectively, and there were no measurable dG-N2-TAM adducts. For microarray, cells were exposed to 10 μM TAM and genes with expression changes of ≥ 3-fold were as follows: thirteen genes up-regulated and one down-related for strain 16; seventeen genes up-regulated for strain 5; and eleven genes up-regulated for strain 40. Interferon-inducible genes (IFITM1, IFIT1, IFNA1, MXI and GIP3), and a potassium ion channel (KCNJ1) were up-regulated in all 3 strains. No significant expression changes were found for genes related to estrogen or xenobiotic metabolism. RT-PCR revealed up-regulation of interferon α (IFNA1) and confirmed the TAM-induced up-regulation of the genes identified by microarray, with the exception of GIP3 and MX1, which were not up-regulated in strain 40. Induction of interferon-related genes in the three NHMEC strains suggests that, in addition to hormonal effects, TAM exposure may enhance immune response in normal breast tissue. PMID:19155303

  4. Exposure to zearalenone mycotoxin alters in vitro porcine intestinal epithelial cells by differential gene expression.

    PubMed

    Taranu, Ionelia; Braicu, Cornelia; Marin, Daniela Eliza; Pistol, Gina Cecilia; Motiu, Monica; Balacescu, Loredana; Beridan Neagoe, Ioana; Burlacu, Radu

    2015-01-01

    The gut represents the main route of intoxication with mycotoxins. To evaluate the effect and the underlying molecular changes that occurred when the intestine is exposed to zearalenone, a Fusarium sp mycotoxin, porcine epithelial cells (IPEC-1) were treated with 10μM of ZEA for 24h and analysed by microarray using Gene Spring GX v.11.5. Our results showed that 10μM of ZEA did not affect cell viability, but can increase the expression of toll like receptors (TLR1-10) and of certain cytokines involved in inflammation (TNF-α, IL-1β, IL-6, IL-8, MCP-1, IL-12p40, CCL20) or responsible for the recruitment of immune cells (IL-10, IL-18). Microarray results identified 190 genes significantly and differentially expressed, of which 70% were up-regulated. ZEA determined the over expression of ITGB5 gene, essential against the attachment and adhesion of ETEC to porcine jejunal cells and of TFF2 implicated in mucosal protection. An up-regulation of glutathione peroxidase enzymes (GPx6, GPx2, GPx1) was also observed. Upon ZEA challenge, genes like GTF3C4 responsible for the recruitment of polymerase III and initiation of tRNA transcription in eukaryotes and STAT5B were significantly higher induced. The up-regulation of CD97 gene and the down-regulation of tumour suppressor genes (DKK-1, PCDH11X and TC531386) demonstrates the carcinogenic potential of ZEA. PMID:25455459

  5. Differential gene expression profiling of porcine epithelial cells infected with three enterotoxigenic Escherichia coli strains

    PubMed Central

    2012-01-01

    Background Enterotoxigenic Escherichia coli (ETEC) is one of the most important pathogenic bacteria causing severe diarrhoea in human and pigs. In ETEC strains, the fimbrial types F4 and F18 are commonly found differently colonized within the small intestine and cause huge economic losses in the swine industry annually worldwide. To address the underlying mechanism, we performed a transcriptome study of porcine intestinal epithelial cells (IPEC-J2) with and without infection of three representative ETEC strains. Results A total 2443, 3493 and 867 differentially expressed genes were found in IPEC-J2 cells infected with F4ab ETEC (CF4ab), with F4ac ETEC (CF4ac) and with F18ac ETEC (CF18ac) compared to the cells without infection (control), respectively. The number of differentially expressed genes between CF4ab and CF4ac, CF4ab and CF18ac, and CF4ac and CF18ac were 77, 1446 and 1629, respectively. The gene ontology and pathway analysis showed that the differentially expressed genes in CF4abvs control are significantly involved in cell-cycle progress and amino acid metabolism, while the clustered terms of the differentially expressed genes in CF4acvs control comprise immune, inflammation and wounding response and apoptosis as well as cell cycle progress and proteolysis. Differentially expressed genes between CF18acvs control are mainly involved in cell-cycle progression and immune response. Furthermore, fundamental differences were observed in expression levels of immune-related genes among the three ETEC treatments, especially for the important pro-inflammatory molecules, including IL-6, IL-8, TNF-α, CCL20, CXCL2 etc. Conclusions The discovery in this study provides insights into the interaction of porcine intestinal epithelial cells with F4 ETECs and F18 ETEC, respectively. The genes induced by ETECs with F4 versus F18 fimbriae suggest why ETEC with F4 may be more virulent compared to F18 which seems to elicit milder effects. PMID:22823589

  6. Live Imaging and Gene Expression Analysis in Zebrafish Identifies a Link between Neutrophils and Epithelial to Mesenchymal Transition

    PubMed Central

    Freisinger, Christina M.; Huttenlocher, Anna

    2014-01-01

    Chronic inflammation is associated with epithelial to mesenchymal transition (EMT) and cancer progression however the relationship between inflammation and EMT remains unclear. Here, we have exploited zebrafish to visualize and quantify the earliest events during epithelial cell transformation induced by oncogenic HRasV12. Live imaging revealed that expression of HRasV12 in the epidermis results in EMT and chronic neutrophil and macrophage infiltration. We have developed an in vivo system to probe and quantify gene expression changes specifically in transformed cells from chimeric zebrafish expressing oncogenic HRasV12 using translating ribosomal affinity purification (TRAP). We found that the expression of genes associated with EMT, including slug, vimentin and mmp9, are enriched in HRasV12 transformed epithelial cells and that this enrichment requires the presence of neutrophils. An early signal induced by HRasV12 in epithelial cells is the expression of il-8 (cxcl8) and we found that the chemokine receptor, Cxcr2, mediates neutrophil but not macrophage recruitment to the transformed cells. Surprisingly, we also found a cell autonomous role for Cxcr2 signaling in transformed cells for both neutrophil recruitment and EMT related gene expression associated with Ras transformation. Taken together, these findings implicate both autocrine and paracrine signaling through Cxcr2 in the regulation of inflammation and gene expression in transformed epithelial cells. PMID:25372289

  7. Expression of epithelial cell iron-related genes upon infection by Neisseria meningitidis.

    PubMed

    Bonnah, Robert A; Muckenthaler, Martina U; Carlson, Hanqian; Minana, Belen; Enns, Caroline A; Hentze, Matthias W; So, Magdalene

    2004-05-01

    Infection by the obligate human pathogens Neisseria meningitidis (MC) and Neisseria gonorrhoeae (GC) reduces the expression of host epithelial cell transferrin receptor 1 (TfR-1) (Bonnah et al., 2000, Cellular Microbiology 2: 207-218). In addition, the rate and pattern of TfR-1 cycling is altered, leading to diminished uptake of Tf-iron by infected host cells. As Tf-iron is important for maintaining iron homeostasis in the eukaryotic cell, these findings raised the possibility that Neisseria infection might affect further pathways of epithelial cell iron metabolism. We used a specialized cDNA microarray platform, the 'IronChip', to investigate the expression of genes involved in iron transport, storage and regulation. We show that mRNA expression of several host genes involved in iron homeostasis is altered. Surprisingly, the general mRNA expression profile of infected cells closely resembled that of uninfected cells grown in an iron-limited environment. An important exception to this profile is TfR-1, the mRNA level of which is strongly reduced. Low TfR-1 expression may be explained in part by decreased activity of the iron-regulatory proteins (IRPs) in MC-infected cells, which may result in the destabilization of TfR-1 mRNA. Intriguingly, low IRP activity contrasts with the decrease in H-ferritin protein levels in infected cells. This finding suggests that low IRP activity may be responsible in part for the decrease in TfR-1 mRNA levels. A discussion of these novel findings in relation to MC infection and virulence is provided. PMID:15056217

  8. Comparative Analysis of Human Conjunctival and Corneal Epithelial Gene Expression with Oligonucleotide Microarrays

    PubMed Central

    Turner, Helen C.; Budak, Murat T.; Murat Akinci, M. A.; Wolosin, J. Mario

    2010-01-01

    Purpose To determine global mRNA expression levels in corneal and conjunctival epithelia and identify transcripts that exhibit preferential tissue expression. Methods cDNA samples derived from human conjunctival and corneal epithelia were hybridized in three independent experiments to a commercial oligonucleotide array representing more than 22,000 transcripts. The resultant signal intensities and microarray software transcript present/absent calls were used in conjunction with the local pooled error (LPE) statistical method to identify transcripts that are preferentially or exclusively expressed in one of the two tissues at significant levels (expression >1% of the β-actin level). EASE (Expression Analysis Systematic Explorer software) was used to identify biological systems comparatively overrepresented in either epithelium. Immuno-, and cytohistochemistry was performed to validate or expand on selected results of interest. Results The analysis identified 332 preferential and 93 exclusive significant corneal epithelial transcripts. The corresponding numbers of conjunctival epithelium transcripts were 592 and 211, respectively. The overrepresented biological processes in the cornea were related to cell adhesion and oxiredox equilibria and cytoprotection activities. In the conjunctiva, the biological processes that were most prominent were related to innate immunity and melanogenesis. Immunohistochemistry for antigen-presenting cells and melanocytes was consistent with these gene signatures. The transcript comparison identified a substantial number of genes that have either not been identified previously or are not known to be highly expressed in these two epithelia, including testican-1, ECM1, formin, CRTAC1, and NQO1 in the cornea and, in the conjunctiva, sPLA2-IIA, lipocalin 2, IGFBP3, multiple MCH class II proteins, and the Na-Pi cotransporter type IIb. Conclusions Comparative gene expression profiling leads to the identification of many biological processes

  9. Expression of epithelial-mesenchymal transition-related genes increases with copy number in multiple cancer types.

    PubMed

    Zhao, Min; Liu, Yining; Qu, Hong

    2016-04-26

    Epithelial-mesenchymal transition (EMT) is a cellular process through which epithelial cells transform into mesenchymal cells. EMT-implicated genes initiate and promote cancer metastasis because mesenchymal cells have greater invasive and migration capacities than epithelial cells. In this pan-cancer analysis, we explored the relationship between gene expression changes and copy number variations (CNVs) for EMT-implicated genes. Based on curated 377 EMT-implicated genes from the literature, we identified 212 EMT-implicated genes associated with more frequent copy number gains (CNGs) than copy number losses (CNLs) using data from The Cancer Genome Atlas (TCGA). Then by correlating these CNV data with TCGA gene expression data, we identified 71 EMT-implicated genes with concordant CNGs and gene up-regulation in 20 or more tumor samples. Of those, 14 exhibited such concordance in over 110 tumor samples. These 14 genes were predominantly apoptosis regulators, which may implies that apoptosis is critical during EMT. Moreover, the 71 genes with concordant CNG and up-regulation were largely involved in cellular functions such as phosphorylation cascade signaling. This is the first observation of concordance between CNG and up-regulation of specific genes in hundreds of samples, which may indicate that somatic CNGs activate gene expression by increasing the gene dosage. PMID:27029057

  10. Neutrophil elastase in respiratory epithelial lining fluid of individuals with cystic fibrosis induces interleukin-8 gene expression in a human bronchial epithelial cell line.

    PubMed Central

    Nakamura, H; Yoshimura, K; McElvaney, N G; Crystal, R G

    1992-01-01

    The respiratory manifestations of cystic fibrosis (CF) are characterized by neutrophil-dominated airway inflammation. Since a variety of inflammatory stimuli are capable of inducing bronchial epithelial cells to express the gene for IL-8, a cytokine that attracts and activates neutrophils, mediators in respiratory epithelial lining fluid (ELF) of CF individuals might induce IL-8 production by epithelial cells, thus recruiting neutrophils to the airways. BET-1A human bronchial epithelial cells at rest or incubated with normal ELF showed little IL-8 gene expression, but after incubation with CF ELF, a marked increase in IL-8 transcript levels was observed. CF ELF contained high levels of neutrophil elastase (NE) and various serine protease inhibitors prevented CF ELF from inducing IL-8 gene expression in BET-1A cells, suggesting that NE was the dominant inducer for IL-8 production in CF ELF. The addition of purified NE caused BET-1A cells to increase IL-8 gene transcription with accumulation of mRNA transcripts and to release IL-8-like neutrophil chemotactic activity. These observations suggest a self-perpetuating inflammatory process on the CF bronchial surface where NE released by neutrophils induced the bronchial epithelium to secrete IL-8, which in turn recruits additional neutrophils to the bronchial surface. Images PMID:1569186

  11. Regulation of sucrase-isomaltase gene expression in human intestinal epithelial cells by inflammatory cytokines.

    PubMed

    Ziambaras, T; Rubin, D C; Perlmutter, D H

    1996-01-12

    Using metabolic labeling techniques in human intestinal epithelial cell lines in tissue culture and in situ hybridization techniques in normal and inflamed (Crohn's) intestine, recent studies have shown that there is synthesis of acute phase proteins in enterocytes. Moreover, these studies have shown that acute phase protein biosynthesis in enterocytes is regulated by inflammatory cytokines in a manner characteristic of the physiologic acute phase response. In the course of these studies it was noticed that one inflammatory cytokine, interleukin-6 (IL-6), mediated selective down-regulation of the enterocyte-specific, differentiation-dependent integral membrane protein sucrase-isomaltase (SI) in the Caco2 intestinal epithelial cell line. In the current study we examined the effect of several other inflammatory cytokines interleukin-1 (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and interferon gamma (IFN gamma) on synthesis of SI in Caco2 cells, examined the possibility that inflammatory cytokines affect the synthesis of other enterocyte integral membrane proteins using lactase as a prototype, and examined the possibility that SI gene expression was down-regulated in villous enterocytes in vivo during the local inflammatory response of Crohn's disease. The results show that IL-6 and IFN gamma each mediate a decrease and TNF alpha mediates an increase in synthesis of SI in Caco2 cells. The magnitude of down-regulation by IL-6 and IFN gamma is significantly greater than the up-regulation by TNF alpha. IL-1 beta has no effect on synthesis of SI. Synthesis of lactase is not affected by any of the cytokines. There is a marked specific decrease in SI gene expression in villous enterocytes in acutely inflamed Crohn's ileum as compared to adjacent uninflamed ileum and normal ileum. Taken together, these data show that inflammatory cytokines have specific and selective effects on the expression of the brush border hydrolase SI in tissue culture and in vivo and

  12. SMARCA4 regulates gene expression and higher-order chromatin structure in proliferating mammary epithelial cells.

    PubMed

    Barutcu, A Rasim; Lajoie, Bryan R; Fritz, Andrew J; McCord, Rachel P; Nickerson, Jeffrey A; van Wijnen, Andre J; Lian, Jane B; Stein, Janet L; Dekker, Job; Stein, Gary S; Imbalzano, Anthony N

    2016-09-01

    The packaging of DNA into chromatin plays an important role in transcriptional regulation and nuclear processes. Brahma-related gene-1 SMARCA4 (also known as BRG1), the essential ATPase subunit of the mammalian SWI/SNF chromatin remodeling complex, uses the energy from ATP hydrolysis to disrupt nucleosomes at target regions. Although the transcriptional role of SMARCA4 at gene promoters is well-studied, less is known about its role in higher-order genome organization. SMARCA4 knockdown in human mammary epithelial MCF-10A cells resulted in 176 up-regulated genes, including many related to lipid and calcium metabolism, and 1292 down-regulated genes, some of which encode extracellular matrix (ECM) components that can exert mechanical forces and affect nuclear structure. ChIP-seq analysis of SMARCA4 localization and SMARCA4-bound super-enhancers demonstrated extensive binding at intergenic regions. Furthermore, Hi-C analysis showed extensive SMARCA4-mediated alterations in higher-order genome organization at multiple resolutions. First, SMARCA4 knockdown resulted in clustering of intra- and inter-subtelomeric regions, demonstrating a novel role for SMARCA4 in telomere organization. SMARCA4 binding was enriched at topologically associating domain (TAD) boundaries, and SMARCA4 knockdown resulted in weakening of TAD boundary strength. Taken together, these findings provide a dynamic view of SMARCA4-dependent changes in higher-order chromatin organization and gene expression, identifying SMARCA4 as a novel component of chromatin organization. PMID:27435934

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

    PubMed Central

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

    2016-01-01

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

  14. ABCA Transporter Gene Expression and Poor Outcome in Epithelial Ovarian Cancer

    PubMed Central

    Hedditch, Ellen L.; Gao, Bo; Russell, Amanda J.; Lu, Yi; Emmanuel, Catherine; Beesley, Jonathan; Johnatty, Sharon E.; Chen, Xiaoqing; Harnett, Paul; George, Joshy; Williams, Rebekka T.; Flemming, Claudia; Lambrechts, Diether; Despierre, Evelyn; Lambrechts, Sandrina; Vergote, Ignace; Karlan, Beth; Lester, Jenny; Orsulic, Sandra; Walsh, Christine; Fasching, Peter; Beckmann, Matthias W.; Ekici, Arif B.; Hein, Alexander; Matsuo, Keitaro; Hosono, Satoyo; Nakanishi, Toru; Yatabe, Yasushi; Pejovic, Tanja; Bean, Yukie; Heitz, Florian; Harter, Philipp; du Bois, Andreas; Schwaab, Ira; Hogdall, Estrid; Kjaer, Susan K.; Jensen, Allan; Hogdall, Claus; Lundvall, Lene; Engelholm, Svend Aage; Brown, Bob; Flanagan, James; Metcalf, Michelle D; Siddiqui, Nadeem; Sellers, Thomas; Fridley, Brooke; Cunningham, Julie; Schildkraut, Joellen; Iversen, Ed; Weber, Rachel P.; Berchuck, Andrew; Goode, Ellen; Bowtell, David D.; Chenevix-Trench, Georgia; deFazio, Anna; Norris, Murray D.; MacGregor, Stuart; Haber, Michelle; Henderson, Michelle J.

    2014-01-01

    Background ATP-binding cassette (ABC) transporters play various roles in cancer biology and drug resistance, but their association with outcomes in serous epithelial ovarian cancer (EOC) is unknown. Methods The relationship between clinical outcomes and ABC transporter gene expression in two independent cohorts of high-grade serous EOC tumors was assessed with real-time quantitative polymerase chain reaction, analysis of expression microarray data, and immunohistochemistry. Associations between clinical outcomes and ABCA transporter gene single nucleotide polymorphisms were tested in a genome-wide association study. Impact of short interfering RNA–mediated gene suppression was determined by colony forming and migration assays. Association with survival was assessed with Kaplan–Meier analysis and log-rank tests. All statistical tests were two-sided. Results Associations with outcome were observed with ABC transporters of the “A” subfamily, but not with multidrug transporters. High-level expression of ABCA1, ABCA6, ABCA8, and ABCA9 in primary tumors was statistically significantly associated with reduced survival in serous ovarian cancer patients. Low levels of ABCA5 and the C-allele of rs536009 were associated with shorter overall survival (hazard ratio for death = 1.50; 95% confidence interval [CI] =1.26 to 1.79; P = 6.5e−6). The combined expression pattern of ABCA1, ABCA5, and either ABCA8 or ABCA9 was associated with particularly poor outcome (mean overall survival in group with adverse ABCA1, ABCA5 and ABCA9 gene expression = 33.2 months, 95% CI = 26.4 to 40.1; vs 55.3 months in the group with favorable ABCA gene expression, 95% CI = 49.8 to 60.8; P = .001), independently of tumor stage or surgical debulking status. Suppression of cholesterol transporter ABCA1 inhibited ovarian cancer cell growth and migration in vitro, and statin treatment reduced ovarian cancer cell migration. Conclusions Expression of ABCA transporters was associated with poor

  15. In vivo gene expression profiling of human intestinal epithelial cells: analysis by laser microdissection of formalin fixed tissues

    PubMed Central

    George, Michael D; Wehkamp, Jan; Kays, Robert J; Leutenegger, Christian M; Sabir, Sadiah; Grishina, Irina; Dandekar, Satya; Bevins, Charles L

    2008-01-01

    Background The small intestinal epithelium mediates vital functions of nutrient absorption and host defense. The spatial organization of the epithelial cells along the crypt-villus axis segregates them into regions of specialized function. However, the differences in transcriptional programming and the molecular machinery that governs the migration, adhesion, and differentiation of intestinal epithelial cell lineages in humans remain under-explored. To increase our understanding of these mechanisms, we have evaluated gene expression patterns of ileal epithelial cells isolated by laser capture microdissection from either the villus epithelial or crypt cell regions of healthy human small intestinal mucosa. Expression profiles in villus and crypt epithelium were determined by DNA microarray, quantitative real-time PCR, and immunohistochemistry based methods. The expression levels of selected epithelial biomarkers were also compared between gastrointestinal tissues. Results Previously established biomarkers as well as a novel and distinct set of genes believed to be linked to epithelial cell motility, adhesion, and differentiation were found to be enriched in each of the two corresponding cell populations (GEO accession: GSE10629). Additionally, high baseline expression levels of innate antimicrobials, alpha defensin 5 (HD5) and regenerating islet-derived 3 alpha (Reg3A), were detected exclusively within the small bowel crypt, most notably in the ileum in comparison to other sites along the gastrointestinal tract. Conclusion The elucidation of differential gene expression patterns between crypt and villus epithelial cell lineages in human ileal tissue provides novel insights into the molecular machinery that mediates their functions and spatial organization. Moreover, our findings establish an important framework of knowledge for future investigations of human gastrointestinal diseases. PMID:18457593

  16. Gene expression profile of human lung epithelial cells chronically exposed to single-walled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Chen, Dongquan; Stueckle, Todd A.; Luanpitpong, Sudjit; Rojanasakul, Yon; Lu, Yongju; Wang, Liying

    2015-01-01

    A rapid increase in utility of engineered nanomaterials, including carbon nanotubes (CNTs), has raised a concern over their safety. Based on recent evidence from animal studies, pulmonary exposure of CNTs may lead to nanoparticle accumulation in the deep lung without effective clearance which could interact with local lung cells for a long period of time. Physicochemical similarities of CNTs to asbestos fibers may contribute to their asbestos-like carcinogenic potential after long-term exposure, which has not been well addressed. More studies are needed to identify and predict the carcinogenic potential and mechanisms for promoting their safe use. Our previous study reported a long-term in vitro exposure model for CNT carcinogenicity and showed that 6-month sub-chronic exposure of single-walled carbon nanotubes (SWCNT) causes malignant transformation of human lung epithelial cells. In addition, the transformed cells induced tumor formation in mice and exhibited an apoptosis resistant phenotype, a key characteristic of cancer cells. Although the potential role of p53 in the transformation process was identified, the underlying mechanisms of oncogenesis remain largely undefined. Here, we further examined the gene expression profile by using genome microarrays to profile molecular mechanisms of SWCNT oncogenesis. Based on differentially expressed genes, possible mechanisms of SWCNT-associated apoptosis resistance and oncogenesis were identified, which included activation of pAkt/p53/Bcl-2 signaling axis, increased gene expression of Ras family for cell cycle control, Dsh-mediated Notch 1, and downregulation of apoptotic genes BAX and Noxa. Activated immune responses were among the major changes of biological function. Our findings shed light on potential molecular mechanisms and signaling pathways involved in SWCNT oncogenic potential.

  17. Effects of fescue toxicosis induced by endophyte-infected tall fescue seed on forestomach epithelial gene expression in Angus steers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A previous report demonstrated that steers exposed to an endophyte-infected tall fescue seed extract had altered rumen epithelial blood flow and decreased ruminal flux of VFA. Thus, this study was conducted to determine whether there are differences in gene expression related to VFA absorption betwe...

  18. Effect of Growth Factors on the Proliferation and Gene Expression of Human Meibomian Gland Epithelial Cells

    PubMed Central

    Liu, Shaohui; Kam, Wendy R.; Ding, Juan; Hatton, Mark P.; Sullivan, David A.

    2013-01-01

    Purpose. We hypothesize that growth factors, including epidermal growth factor (EGF) and bovine pituitary extract (BPE), induce proliferation, but not differentiation (e.g., lipid accumulation), of human meibomian gland epithelial cells. We also hypothesize that these actions involve a significant upregulation of genes linked to cell cycle processes, and a significant downregulation of genes associated with differentiation. Our objective was to test these hypotheses. Methods. Immortalized human meibomian gland and conjunctival epithelial cells were cultured for varying time periods in the presence or absence of EGF, BPE, EGF + BPE, or serum, followed by cell counting, neutral lipid staining, or RNA isolation for molecular biological procedures. Results. Our studies show that growth factors stimulate a significant, time-dependent proliferation of human meibomian gland epithelial cells. These effects are associated with a significant upregulation of genes linked to cell cycle, DNA replication, ribosomes, and translation, and a significant decrease in those related to cell differentiation, tissue development, lipid metabolic processes, and peroxisome proliferator-activated receptor signaling. Serum-induced differentiation, but not growth factor-related proliferation, elicits a pronounced lipid accumulation in human meibomian gland epithelial cells. This lipogenic response is unique, and is not duplicated by human conjunctival epithelial cells. Conclusions. Our results demonstrate that EGF and BPE stimulate human meibomian gland epithelial cells to proliferate. Further, our findings show that action is associated with an upregulation of cell cycle and translation ontologies, and a downregulation of genetic pathways linked to differentiation and lipid biosynthesis. PMID:23493293

  19. Identification of global gene expression differences between human lens epithelial and cortical fiber cells reveals specific genes and their associated pathways important for specialized lens cell functions

    PubMed Central

    Hawse, John R.; DeAmicis-Tress, Candida; Cowell, Tracy L.; Kantorow, Marc

    2005-01-01

    Purpose: In order to identify specific genes that may play important roles in maintaining the specialized functions of lens epithelial and fiber cells, we have analyzed the global gene expression profiles of these two cell types in the human lens. This analysis will also reveal those genes that are exclusively expressed in the epithelial and cortical fiber cells and those genes that may play important roles in the differentiation of epithelial cells to mature fiber cells. Methods: Oligonucleotide microarray hybridization was used to analyze the expression profiles of 22,215 genes between adult (average age greater than 56 years) human lens epithelial and cortical fiber cells. The expression levels of selected genes were further compared by semi-quantitative RT-PCR and selected genes were functionally clustered into common categories using the EASE bioinformatics software package. Results: Analysis of three separate microarray hybridizations revealed 1,196 transcripts that exhibit increased expression and 1,278 transcripts that exhibit decreased expression at the 2 fold or greater level between lens epithelial cells and cortical fiber cells on all three of the arrays analyzed. Of these, 222 transcripts exhibited increased expression and 135 transcripts exhibited decreased expression by an average of 5 fold or greater levels on all three arrays. Semi-quantitative RT-PCR analysis of 21 randomly selected genes revealed identical expression patterns as those detected by microarray hybridization indicating that the microarray data are accurate. Functional clustering of the identified gene expression patterns using the EASE program revealed a wide variety of biological pathways that exhibited altered expression patterns between the two cell types including mRNA processing, cell adhesion, cell proliferation, translation, protein folding, oxidative phosphorylation, and apoptosis, among others. Conclusions: These data reveal novel and previously identified gene expression

  20. Gene Expression Profiling of the Paracrine Effects of Uterine Natural Killer Cells on Human Endometrial Epithelial Cells

    PubMed Central

    Gong, Xin; Chen, Zhenzhen; Liu, Yanxia; Lu, Qiudan; Jin, Zhe

    2014-01-01

    The endometrium contains a population of immune cells that undergo changes during implantation and pregnancy. The majority of these cells are uterine natural killer (uNK) cells; however, it is unclear how these cells interact with endometrial epithelial cells. Therefore, we investigated the paracrine effects of the uNK cell-secretion medium on the gene expression profile of endometrial epithelial cells in vitro through microarray analysis. Our results, which were verified by qRT-PCR and western blot, revealed that soluble factors from uNK cells alter the gene expression profiles of epithelial cells. The upregulated genes included interleukin-15 (IL-15) and interleukin-15 receptor alpha (IL-15RA), which result in a loop that stimulates uNK cell proliferation. In addition, vascular endothelial growth factor C (VEGF-C) and chemokine (C-X-C motif) ligand 10 (CXCL-10) were also determined to be upregulated in epithelial cells, which suggests that uNK cells work synergistically with epithelial cells to support implantation and pregnancy. In addition, oriental herbal medicines have been used to treat infertility since ancient times; however, we failed to find that Zi Dan Yin can regulate these endometrial paracrine effects. PMID:24790599

  1. Microarray Analysis of Gene Expression Alteration in Human Middle Ear Epithelial Cells Induced by Asian Sand Dust

    PubMed Central

    Go, Yoon Young; Park, Moo Kyun; Kwon, Jee Young; Seo, Young Rok; Chae, Sung-Won

    2015-01-01

    Objectives The primary aim of this study is to evaluate the gene expression profile of Asian sand dust (ASD)-treated human middle ear epithelial cell (HMEEC) using microarray analysis. Methods The HMEEC was treated with ASD (400 µg/mL) and total RNA was extracted for microarray analysis. Molecular pathways among differentially expressed genes were further analyzed. For selected genes, the changes in gene expression were confirmed by real-time polymerase chain reaction. Results A total of 1,274 genes were differentially expressed by ASD. Among them, 1,138 genes were 2 folds up-regulated, whereas 136 genes were 2 folds down-regulated. Up-regulated genes were mainly involved in cellular processes, including apoptosis, cell differentiation, and cell proliferation. Down-regulated genes affected cellular processes, including apoptosis, cell cycle, cell differentiation, and cell proliferation. The 10 genes including ADM, CCL5, EDN1, EGR1, FOS, GHRL, JUN, SOCS3, TNF, and TNFSF10 were identified as main modulators in up-regulated genes. A total of 11 genes including CSF3, DKK1, FOSL1, FST, TERT, MMP13, PTHLH, SPRY2, TGFBR2, THBS1, and TIMP1 acted as main components of pathway associated with 2-fold down regulated genes. Conclusion We identified the differentially expressed genes in ASD-treated HMEEC. Our work indicates that air pollutant like ASD, may play an important role in the pathogenesis of otitis media. PMID:26622952

  2. Analysis of global gene expression changes in human bronchial epithelial cells exposed to spores of the allergenic fungus, Alternaria alternata

    PubMed Central

    Babiceanu, M. C.; Howard, B. A.; Rumore, A. C.; Kita, H.; Lawrence, C. B.

    2013-01-01

    Exposure and sensitivity to ubiquitous airborne fungi such as Alternaria alternata have long been implicated in the development, onset, and exacerbation of chronic allergic airway disorders. This present study is the first to investigate global changes in host gene expression during the interaction of cultured human bronchial epithelial cells and live Alternaria spores. In in vitro experiments human bronchial epithelial cells (BEAS-2B) were exposed to spores or media alone for 24 h. RNA was collected from three biological replicates per treatment and was used to assess changes in gene expression patterns using Affymetrix Human Genome U133 Plus 2.0 Arrays. In cells treated with Alternaria spores compared to controls, 613 probe sets representing 460 individual genes were found differentially expressed (p ≤ 0.05). In this set of 460 statistically significant, differentially expressed genes, 397 genes were found to be up-regulated and 63 were down-regulated. Of these 397 up-regulated genes, 156 genes were found to be up-regulated ≥2 fold. Interestingly, none of the 63 down-regulated genes were found differentially expressed at ≤−2 fold. Differentially expressed genes were identified following statistical analysis and subsequently used for pathway and network evaluation. Interestingly, many cytokine and chemokine immune response genes were up-regulated with a particular emphasis on interferon-inducible genes. Genes involved in cell death, retinoic acid signaling, and TLR3 response pathways were also significantly up-regulated. Many of the differentially up-regulated genes have been shown in other systems to be associated with innate immunity, inflammation and/or allergic airway diseases. This study now provides substantial information for further investigating specific genes and innate immune system pathways activated by Alternaria in the context of allergic airway diseases. PMID:23882263

  3. Lactobacilli Reduce Helicobacter pylori Attachment to Host Gastric Epithelial Cells by Inhibiting Adhesion Gene Expression.

    PubMed

    de Klerk, Nele; Maudsdotter, Lisa; Gebreegziabher, Hanna; Saroj, Sunil D; Eriksson, Beatrice; Eriksson, Olaspers Sara; Roos, Stefan; Lindén, Sara; Sjölinder, Hong; Jonsson, Ann-Beth

    2016-05-01

    The human gastrointestinal tract, including the harsh environment of the stomach, harbors a large variety of bacteria, of which Lactobacillus species are prominent members. The molecular mechanisms by which species of lactobacilli interfere with pathogen colonization are not fully characterized. In this study, we aimed to study the effect of lactobacillus strains upon the initial attachment of Helicobacter pylori to host cells. Here we report a novel mechanism by which lactobacilli inhibit adherence of the gastric pathogen H. pylori In a screen with Lactobacillus isolates, we found that only a few could reduce adherence of H. pylori to gastric epithelial cells. Decreased attachment was not due to competition for space or to lactobacillus-mediated killing of the pathogen. Instead, we show that lactobacilli act on H. pylori directly by an effector molecule that is released into the medium. This effector molecule acts on H. pylori by inhibiting expression of the adhesin-encoding gene sabA Finally, we verified that inhibitory lactobacilli reduced H. pylori colonization in an in vivo model. In conclusion, certain Lactobacillus strains affect pathogen adherence by inhibiting sabA expression and thereby reducing H. pylori binding capacity. PMID:26930708

  4. Gene Expression Profile and Toxic Effects in Human Bronchial Epithelial Cells Exposed to Zearalenone

    PubMed Central

    So, Mei Yu; Tian, ZhiPeng; Phoon, Yong Shian; Sha, Sha; Antoniou, Michael N.; Zhang, JiangWen; Wu, Rudolf S. S.; Tan-Un, Kian C

    2014-01-01

    Zearalenone (ZEA), a mycoestrogen produced by Fusarium fungal species, is mainly found in cereal crops such as maize, wheat and barley. Although ZEA has been reported to be present in air, little is known about the health risk or the molecular basis of action when lung cells are exposed to ZEA. As ZEA has a similar structure to estrogen, its potential risk as an endocrine disrupting chemical (EDC) has thus aroused both environmental and public health concerns. The purpose of this study is to identify the responses and underlying molecular changes that occur when human bronchial epithelial BEAS-2B cells are exposed to ZEA. Differential gene expression profiles were identified in cells that were treated with 40 µM ZEA for 6 h and 24 h by high-throughput microarray analysis using Affymetrix Human Gene 2.0 GeneChip. The array results showed that after ZEA treatment, 262 genes at 6 h and 1073 genes at 24 h were invovled in the differential regulation. Pathway analysis revealed that diverse cellular processes were affected when lung cells were exposed to ZEA resulting in impaired response to DNA damage, cell cycle arrest, down-regulation of inflammatory responses and alterations of epigenetic marks. Results of further experiments indicated that 40 µM ZEA decreased cell viability, induced apoptosis and promoted reactive oxygen species (ROS) generation in a time-dependent manner. Immuno-suppressive effects of ZEA were further revealed through the suppression of lipopolysaccharide (LPS)-induced expression of pro-inflammatory cytokines (IL-6, IL-8 and IL-1β). Interestingly, the level of global DNA methylation was markedly decreased after 24 h exposure to ZEA. Collectively, these observations suggested that a broad range of toxic effects are elicited by ZEA. Particularly, ROS may play a pivotal role in ZEA-induced cell death. These adverse effects observed in lung cells suggest that exposure to ZEA may increase susceptibility of lung cells to diseases and required further

  5. Differential Regulation of Gene Expression of Alveolar Epithelial Cell Markers in Human Lung Adenocarcinoma-Derived A549 Clones

    PubMed Central

    Kondo, Hiroshi; Miyoshi, Keiko; Sakiyama, Shoji; Tangoku, Akira; Noma, Takafumi

    2015-01-01

    Stem cell therapy appears to be promising for restoring damaged or irreparable lung tissue. However, establishing a simple and reproducible protocol for preparing lung progenitor populations is difficult because the molecular basis for alveolar epithelial cell differentiation is not fully understood. We investigated an in vitro system to analyze the regulatory mechanisms of alveolus-specific gene expression using a human alveolar epithelial type II (ATII) cell line, A549. After cloning A549 subpopulations, each clone was classified into five groups according to cell morphology and marker gene expression. Two clones (B7 and H12) were further analyzed. Under serum-free culture conditions, surfactant protein C (SPC), an ATII marker, was upregulated in both H12 and B7. Aquaporin 5 (AQP5), an ATI marker, was upregulated in H12 and significantly induced in B7. When the RAS/MAPK pathway was inhibited, SPC and thyroid transcription factor-1 (TTF-1) expression levels were enhanced. After treatment with dexamethasone (DEX), 8-bromoadenosine 3′5′-cyclic monophosphate (8-Br-cAMP), 3-isobutyl-1-methylxanthine (IBMX), and keratinocyte growth factor (KGF), surfactant protein B and TTF-1 expression levels were enhanced. We found that A549-derived clones have plasticity in gene expression of alveolar epithelial differentiation markers and could be useful in studying ATII maintenance and differentiation. PMID:26167183

  6. Multifocal Epithelial Hyperplasia of Oral Cavity Expressing HPV 16 Gene: A Rare Entity

    PubMed Central

    Prabhat, M. P. V.; Raja Lakshmi, Chintamaneni; Sai Madhavi, N.; Bhavana, Sujana Mulk; Sarat, Gummadapu; Ramamohan, Kodali

    2013-01-01

    Focal epithelial hyperplasia is a rare contagious disease caused by human papilloma virus. Usually HPV involves either cutaneous or mucosal surfaces, whereas concomitant mucocutaneous involvement is extremely rare. We report such a unique case of multifocal epithelial hyperplasia involving multiple sites of oral cavity along with skin lesions in a 65-year-old female. We also discuss the probable multifactorial etiology and variable clinical presentations of the lesions, including evidence of HPV 16 expression, as detected by polymerase chain reaction. The present report illustrates the need for careful examination and prompt diagnosis of the disease, as it might be associated with high risk genotypes such as HPV 16 and 18. PMID:24455323

  7. Tumour necrosis factor-alpha up-regulates decay-accelerating factor gene expression in human intestinal epithelial cells.

    PubMed Central

    Andoh, A; Fujiyama, Y; Sumiyoshi, K; Sakumoto, H; Okabe, H; Bamba, T

    1997-01-01

    The increased expression of decay-accelerating factor (DAF) has been detected in intestinal epithelial cells at the inflamed mucosa. In this study, we examined the effects of tumour necrosis factor (TNF)-alpha on DAF expression in three intestinal epithelial cell lines. DAF mRNA expression was evaluated by Northern blot analysis, and DAF protein expression was analysed by biotin labelling and immunoprecipitation. TNF-alpha induced a marked increase in DAF mRNA and protein expression in HT-29, T84 and Caco-2 cells. In HT-29 cells, the effects of TNF-a on DAF mRNA accumulation were observed in a dose-dependent manner; DAF mRNA accumulation reached a maximum at 3-6 hr, and then gradually decreased. These effects of TNF-alpha required de novo protein synthesis. Messenger RNA stability studies suggested that TNF-alpha partially regulated DAF gene expression by a posttranscriptional mechanism. Moreover, the combination of TNF-alpha and interleukin (IL)-4 induced an additive increase in DAF mRNA accumulation in HT-29 and T84 cells. In human intestinal epithelial cells, TNF-alpha acts as a potent inducer of DAF mRNA expression, indicating an important role for TNF-alpha in the regulation of DAF expression at the inflamed mucosa. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 Figure 7 Figure 8 PMID:9155641

  8. NME genes in epithelial morphogenesis

    PubMed Central

    2012-01-01

    The NME family of genes encodes highly conserved multifunctional proteins that have been shown to participate in nucleic acid metabolism, energy homeostasis, cell signaling, and cancer progression. Some family members, particularly isoforms 1 and 2, have attracted extensive interests because of their potential anti-metastasis activity. Unfortunately, there have been few consensus mechanistic explanations for this critical function because of the numerous molecular functions ascribed to these proteins, including nucleoside diphosphate kinase, protein kinase, nuclease, transcription factor, growth factor, among others. In addition, different studies showed contradictory prognostic correlations between NME expression levels and tumor progression in clinical samples. Thus, analyses using pliable in vivo systems have become critical for unraveling at least some aspects of the complex functions of this family of genes. Recent works using the Drosophila genetic system have suggested a role for NME in regulating epithelial cell motility and morphogenesis, which has also been demonstrated in mammalian epithelial cell culture. This function is mediated by promoting internalization of growth factor receptors in motile epithelial cells, and the adherens junction components such as E-cadherin and β-catenin in epithelia that form the tissue linings. Interestingly, NME genes in epithelial cells appear to function in a defined range of expression levels. Either down-regulation or over-expression can perturb epithelial integrity, resulting in different aspects of epithelial abnormality. Such biphasic functions provide a plausible explanation for the documented anti-metastatic activity and the suspected oncogenic function. This review summarizes these recent findings and discusses their implications. PMID:21336542

  9. Oxidative Stress Regulates CFTR Gene Expression in Human Airway Epithelial Cells through a Distal Antioxidant Response Element

    PubMed Central

    Zhang, Zhaolin; Leir, Shih-Hsing

    2015-01-01

    Cystic fibrosis transmembrane conductance regulator gene (CFTR) expression in human airway epithelial cells involves the recruitment of distal cis-regulatory elements, which are associated with airway-selective DNase hypersensitive sites at −44 kb and −35 kb from the gene. The −35-kb site encompasses an enhancer that is regulated by the immune mediators interferon regulatory factor 1 and 2 and by nuclear factor Y. Here we investigate the −44-kb element, which also has enhancer activity in vitro in airway epithelial cells but is inactive in intestinal epithelial cells. This site contains an antioxidant response element (ARE) that plays a critical role in its function in airway cell lines and primary human bronchial epithelial cells. The natural antioxidant sulforaphane (SFN) induces nuclear translocation of nuclear factor, erythroid 2-like 2 (Nrf2), a transcription factor that regulates genes with AREs in their promoters, many of which are involved in response to injury. Under normal conditions, the −44-kb ARE is occupied by the repressor BTB and CNC homology 1, basic leucine zipper transcription factor (Bach1), and v-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog K (MafK) heterodimers. After 2 hours of SFN treatment, Nrf2 displaces these repressive factors and activates CFTR expression. Site-directed mutagenesis shows that both the ARE and an adjacent NF-κB binding site are required for activation of the –44-kb element in airway epithelial cells. Moreover, this element is functionally linked to the −35-kb enhancer in modulating CFTR expression in response to environmental stresses in the airway. PMID:25259561

  10. The luxS Gene of Streptococcus pyogenes Regulates Expression of Genes That Affect Internalization by Epithelial Cells

    PubMed Central

    Marouni, Mehran J.; Sela, Shlomo

    2003-01-01

    The gram-positive pathogen Streptococcus pyogenes was recently reported to possess a homologue of the luxS gene that is responsible for the production of autoinducer 2, which participates in quorum sensing of both gram-positive and gram-negative bacteria. To test the effect of LuxS on streptococcal internalization, a LuxS mutant was constructed in strain SP268, an invasive M3 serotype. Functional analysis of the mutant revealed that it was internalized by HEp-2 cells with higher efficiency than the wild type (wt). Several genes, including hasA (hyaluronic acid synthesis), speB (streptococcal pyrogenic exotoxin B), and csrR (capsule synthesis regulator), a part of a two-component regulatory system, are known to affect the internalization of strain SP268 (J. Jadoun, O. Eyal, and S. Sela, Infect. Immun. 70:462-469, 2002). Therefore, the expression of these genes in the mutant and in the wt was examined. LuxS mutation significantly reduced the mRNA level of speB and increased the mRNA level of emm3. No substantial effect was observed on transcription of hasA and csrR. Yet less hyaluronic acid capsule was expressed in the mutant. Further analysis revealed that luxS is under the regulation of the two-component global regulator CsrR. Our results indicate that LuxS activity in strain SP268 plays an important role in the expression of virulence factors associated with epithelial cell internalization. PMID:14500483

  11. The luxS gene of Streptococcus pyogenes regulates expression of genes that affect internalization by epithelial cells.

    PubMed

    Marouni, Mehran J; Sela, Shlomo

    2003-10-01

    The gram-positive pathogen Streptococcus pyogenes was recently reported to possess a homologue of the luxS gene that is responsible for the production of autoinducer 2, which participates in quorum sensing of both gram-positive and gram-negative bacteria. To test the effect of LuxS on streptococcal internalization, a LuxS mutant was constructed in strain SP268, an invasive M3 serotype. Functional analysis of the mutant revealed that it was internalized by HEp-2 cells with higher efficiency than the wild type (wt). Several genes, including hasA (hyaluronic acid synthesis), speB (streptococcal pyrogenic exotoxin B), and csrR (capsule synthesis regulator), a part of a two-component regulatory system, are known to affect the internalization of strain SP268 (J. Jadoun, O. Eyal, and S. Sela, Infect. Immun. 70:462-469, 2002). Therefore, the expression of these genes in the mutant and in the wt was examined. LuxS mutation significantly reduced the mRNA level of speB and increased the mRNA level of emm3. No substantial effect was observed on transcription of hasA and csrR. Yet less hyaluronic acid capsule was expressed in the mutant. Further analysis revealed that luxS is under the regulation of the two-component global regulator CsrR. Our results indicate that LuxS activity in strain SP268 plays an important role in the expression of virulence factors associated with epithelial cell internalization. PMID:14500483

  12. Expression of acetylcholine receptor genes in human thymic epithelial cells: implications for myasthenia gravis.

    PubMed

    Wakkach, A; Guyon, T; Bruand, C; Tzartos, S; Cohen-Kaminsky, S; Berrih-Aknin, S

    1996-10-15

    The intrathymic presence of the muscle acetylcholine receptor (AChR) is controversial, and the nature of the cell(s) expressing it is unclear. We thus analyzed the molecular expression of muscle AChR in human thymi. mRNA studies indicated that the two isoforms (P3A+ and P3A-) of the alpha-subunit were present in thymic extracts and in cultured thymic epithelial cells (TEC), while expression in thymocytes was low and not consistently detectable. The amount of mRNA coding for the alpha-subunit, evaluated by means of quantitative PCR, was about 20 times less in TEC than in muscle, and was similar in TEC from normal subjects and from patients with myasthenia gravis (MG). The beta- and epsilon-subunits present in adult AChR were also expressed in TEC (but not in thymocytes), while the embryonic subunit (gamma) was absent. In TEC cultures, the AChR alpha- and epsilon-subunit mRNA levels were down-regulated by forskolin, as also observed in the TE671 rhabdomyosarcoma cell line, suggesting similar regulation of AChR subunits in thymus and muscle. Protein expression was evidenced on TEC (but not on thymocytes), by Western blotting as well as by immunofluorescence, thus demonstrating AChR expression on human thymic epithelial cells. There was no difference in the expression of AChR between TEC from MG patients and controls, meaning that the expression of AChR subunits alone is not sufficient to explain the onset of MG. PMID:8871679

  13. Indications for distinct pathogenic mechanisms of asbestos and silica through gene expression profiling of the response of lung epithelial cells

    PubMed Central

    Perkins, Timothy N.; Peeters, Paul M.; Shukla, Arti; Arijs, Ingrid; Dragon, Julie; Wouters, Emiel F.M.; Reynaert, Niki L.; Mossman, Brooke T.

    2015-01-01

    Occupational and environmental exposures to airborne asbestos and silica are associated with the development of lung fibrosis in the forms of asbestosis and silicosis, respectively. However, both diseases display distinct pathologic presentations, likely associated with differences in gene expression induced by different mineral structures, composition and bio-persistent properties. We hypothesized that effects of mineral exposure in the airway epithelium may dictate deviating molecular events that may explain the different pathologies of asbestosis versus silicosis. Using robust gene expression-profiling in conjunction with in-depth pathway analysis, we assessed early (24 h) alterations in gene expression associated with crocidolite asbestos or cristobalite silica exposures in primary human bronchial epithelial cells (NHBEs). Observations were confirmed in an immortalized line (BEAS-2B) by QRT-PCR and protein assays. Utilization of overall gene expression, unsupervised hierarchical cluster analysis and integrated pathway analysis revealed gene alterations that were common to both minerals or unique to either mineral. Our findings reveal that both minerals had potent effects on genes governing cell adhesion/migration, inflammation, and cellular stress, key features of fibrosis. Asbestos exposure was most specifically associated with aberrant cell proliferation and carcinogenesis, whereas silica exposure was highly associated with additional inflammatory responses, as well as pattern recognition, and fibrogenesis. These findings illustrate the use of gene-profiling as a means to determine early molecular events that may dictate pathological processes induced by exogenous cellular insults. In addition, it is a useful approach for predicting the pathogenicity of potentially harmful materials. PMID:25351596

  14. Gene Expression in Wilms’ Tumor Mimics the Earliest Committed Stage in the Metanephric Mesenchymal-Epithelial Transition

    PubMed Central

    Li, Chi-Ming; Guo, Meirong; Borczuk, Alain; Powell, Charles A.; Wei, Michelle; Thaker, Harshwardhan M.; Friedman, Richard; Klein, Ulf; Tycko, Benjamin

    2002-01-01

    Wilms’ tumor (WT) has been considered a prototype for arrested cellular differentiation in cancer, but previous studies have relied on selected markers. We have now performed an unbiased survey of gene expression in WTs using oligonucleotide microarrays. Statistical criteria identified 357 genes as differentially expressed between WTs and fetal kidneys. This set contained 124 matches to genes on a microarray used by Stuart and colleagues (Stuart RO, Bush KT, Nigam SK: Changes in global gene expression patterns during development and maturation of the rat kidney. Proc Natl Acad Sci USA 2001, 98:5649–5654) to establish genes with stage-specific expression in the developing rat kidney. Mapping between the two data sets showed that WTs systematically overexpressed genes corresponding to the earliest stage of metanephric development, and underexpressed genes corresponding to later stages. Automated clustering identified a smaller group of 27 genes that were highly expressed in WTs compared to fetal kidney and heterologous tumor and normal tissues. This signature set was enriched in genes encoding transcription factors. Four of these, PAX2, EYA1, HBF2, and HOXA11, are essential for cell survival and proliferation in early metanephric development, whereas others, including SIX1, MOX1, and SALL2, are predicted to act at this stage. SIX1 and SALL2 proteins were expressed in the condensing mesenchyme in normal human fetal kidneys, but were absent (SIX1) or reduced (SALL2) in cells at other developmental stages. These data imply that the blastema in WTs has progressed to the committed stage in the mesenchymal-epithelial transition, where it is partially arrested in differentiation. The WT-signature set also contained the Wnt receptor FZD7, the tumor antigen PRAME, the imprinted gene NNAT and the metastasis-associated transcription factor E1AF. PMID:12057921

  15. Profiling follicle stimulating hormone-induced gene expression changes in normal and malignant human ovarian surface epithelial cells.

    PubMed

    Ho, Shuk-Mei; Lau, Kin-Mang; Mok, Samuel Chi-Ho; Syed, Viqar

    2003-07-01

    Epidemiological data have implicated the pituitary gonadotropin follicle stimulating hormone (FSH) as both a risk factor for and a protective agent against epithelial ovarian cancer. Yet, little is known about how this hormone could play such opposing roles in ovarian carcinogenesis. Complementary DNA microarrays containing 2400 named genes were used to examine FSH-induced gene expression changes in ovarian cancer (OC) and immortalized normal human ovarian surface epithelial (HOSE) cell lines. Two-way t-statistics analyses of array data identified two distinct sets of FSH-regulated genes in HOSE and in established OC cell lines established from patients (OVCA cell lines). Among the HOSE cell lines, FSH increased expression of 57% of the 312 genes and downregulated 43%. In contrast, FSH diminished expression of 92% of the 177 genes in the OVCA cell lines. All but 18 of the genes affected by FSH in HOSE cell lines were different from those altered in OVCA cell lines. Among the 18 overlapping genes, nine genes exhibited the same direction of change following FSH challenge, while the other nine showed discordance in response between HOSE and OVCA cell lines. The FSH-induced differential expression of seven out of nine genes was confirmed by real-time RT-PCR. Gene-specific antisense oligonuleotides (ODNs) were used to inhibit the expression of genes encoding GTPase activating protein (rap1GAP), neogenin, and restin in HOSE and OVCA cells. Antisense ODNs to neogenin and restin, but not an antisense ODN to rap1GAP, were effective in inhibiting OVCA cell growth, diminishing proliferating cell nuclear antigen expression, and increasing caspase 3 activities. Furthermore, the ODN to rap1GAP was further shown to be ineffective in altering migration properties of OVCA cell lines. HOSE cell proliferation was not affected by treatment with any of the antisense ODNs. In summary, gene profiling data reveal for the first time that FSH may exert different biological actions on OVCA

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

    EPA Science Inventory

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

  17. The Effect of Estradiol and Progesterone on Toll Like Receptor Gene Expression in A Human Fallopian Tube Epithelial Cell Line

    PubMed Central

    Zandieh, Zahra; Amjadi, Fatemehsadat; Ashrafi, Mahnaz; Aflatoonian, Abbas; Fazeli, Alireza; Aflatoonian, Reza

    2016-01-01

    Objective Toll like receptors (TLRs) are one of the main components of the innate im- mune system. It has been reported that expression of these receptors are altered in the female reproductive tract (FRT) during menstrual cycle. Here we used a fallopian tube epithelial cell line (OE-E6/E7) to evaluate the effect of two sex hormones in modulating TLR expression. Materials and Methods In this experimental study, initially TLR gene expression in OE- E6/E7 cells was evaluated and compared with that of fallopian tube tissue using quanti- tative real time-polymerase chain reaction (qRT-PCR) and immunostaining. Thereafter, OE-E6/E7 cells were cultured with different concentrations of estradiol and progesterone, and combination of both. qRT-PCR was performed to reveal any changes in expression of TLR genes as a result of hormonal treatment. Results TLR1-10 genes were expressed in human fallopian tube tissue. TLR1-6 genes and their respective proteins were expressed in the OE-E6/E7 cell line. Although estradiol and progesterone separately had no significant effect on TLR expression, their combined treatment altered the expression of TLRs in this cell line. Also, the pattern of TLR expres- sion in preovulation (P), mensturation (M) and window of implantation (W) were the same for all TLRs with no significant differences between P, M and W groups. Conclusion These data show the significant involvement of the combination of es- tradiol and progesterone in modulation of TLR gene expression in this human fal- lopian tube cell line. Further experiments may reveal the regulatory mechanism and signalling pathway behind the effect of sex hormones in modulating TLRs in the hu- man FRT. PMID:26862527

  18. Butyrate induces profound changes in gene expression related to multiple signal pathways in bovine kidney epithelial cells

    PubMed Central

    Li, Robert W; Li, CongJun

    2006-01-01

    Background Global gene expression profiles of bovine kidney epithelial cells regulated by sodium butyrate were investigated with high-density oligonucleotide microarrays. The bovine microarray with 86,191 distinct 60mer oligonucleotides, each with 4 replicates, was designed and produced with Maskless Array Synthesizer technology. These oligonucleotides represent approximately 45,383 unique cattle sequences. Results 450 genes significantly regulated by butyrate with a median False Discovery Rate (FDR) = 0 % were identified. The majority of these genes were repressed by butyrate and associated with cell cycle control. The expression levels of 30 selected genes identified by the microarray were confirmed using real-time PCR. The results from real-time PCR positively correlated (R = 0.867) with the results from the microarray. Conclusion This study presented the genes related to multiple signal pathways such as cell cycle control and apoptosis. The profound changes in gene expression elucidate the molecular basis for the pleiotropic effects of butyrate on biological processes. These findings enable better recognition of the full range of beneficial roles butyrate may play during cattle energy metabolism, cell growth and proliferation, and possibly in fighting gastrointestinal pathogens. PMID:16972989

  19. Actin-Mediated Gene Expression Depends on RhoA and Rac1 Signaling in Proximal Tubular Epithelial Cells

    PubMed Central

    Giehl, Klaudia; Keller, Christof; Muehlich, Susanne; Goppelt-Struebe, Margarete

    2015-01-01

    Morphological alterations of cells can lead to modulation of gene expression. An essential link is the MKL1-dependent activation of serum response factor (SRF), which translates changes in the ratio of G- and F-actin into mRNA transcription. SRF activation is only partially characterized in non-transformed epithelial cells. Therefore, the impact of GTPases of the Rho family and changes in F-actin structures were analyzed in renal proximal tubular epithelial cells. Activation of SRF signaling was compared to the regulation of a known MKL1/SRF target gene, connective tissue growth factor (CTGF). In the human proximal tubular cell line HKC-8 overexpression of two actin mutants either favoring or preventing the formation of F-actin fibers regulated SRF-mediated transcription as well as CTGF expression. Only overexpression of constitutively active RhoA activated SRF-dependent gene expression whereas no effect was detected upon overexpression of Rac1 mutants. To elucidate the functional role of Rho kinases as downstream mediators of RhoA, pharmacological inhibition and genetic inhibition by transient siRNA knock down were compared. Upon stimulation with lysophosphatidic acid (LPA) Rho kinase inhibitors partially suppressed SRF-mediated transcription, whereas interference with Rho kinase expression by siRNA reduced activation of SRF, but barely affected CTGF expression. Together with the partial inhibition of CTGF expression by the pharmacological inhibitors Y27432 and H1154, Rho kinases seem to be less important in mediating RhoA signaling related to CTGF expression in HKC-8 epithelial cells. Short term pharmacological inhibition of Rac1 activity by EHT1864 reduced SRF-dependent CTGF expression in HKC-8 cells, but was overcome by a stimulatory effect after prolonged incubation after 4-6 h. Similarly, human primary cells of proximal but not of distal tubular origin showed inhibitory as well as stimulatory effects of Rac1 inhibition. Thus, RhoA signaling activates MKL1-SRF

  20. Lipoprotein Lipase, Tissue Expression and Effects on Genes Related to Fatty Acid Synthesis in Goat Mammary Epithelial Cells

    PubMed Central

    Zhao, Wang-Sheng; Hu, Shi-Liang; Yu, Kang; Wang, Hui; Wang, Wei; Loor, Juan; Luo, Jun

    2014-01-01

    Lipoprotein lipase (LPL) serves as a central factor in hydrolysis of triacylglycerol and uptake of free fatty acids from the plasma. However, there are limited data concerning the action of LPL on the regulation of milk fat synthesis in goat mammary gland. In this investigation, we describe the cloning and sequencing of the LPL gene from Xinong Saanen dairy goat mammary gland, along with a study of its phylogenetic relationships. Sequence analysis showed that goat LPL shares similarities with other species including sheep, bovine, human and mouse. LPL mRNA expression in various tissues determined by RT-qPCR revealed the highest expression in white adipose tissue, with lower expression in heart, lung, spleen, rumen, small intestine, mammary gland, and kidney. Expression was almost undetectable in liver and muscle. The expression profiles of LPL gene in mammary gland at early, peak, mid, late lactation, and the dry period were also measured. Compared with the dry period, LPL mRNA expression was markedly greater at early lactation. However, compared with early lactation, the expression was lower at peak lactation and mid lactation. Despite those differences, LPL mRNA expression was still greater at peak, mid, and late lactation compared with the dry period. Using goat mammary epithelial cells (GMEC), the in vitro knockdown of LPL via shRNA or with Orlistat resulted in a similar degree of down-regulation of LPL (respectively). Furthermore, knockdown of LPL was associated with reduced mRNA expression of SREBF1, FASN, LIPE and PPARG but greater expression of FFAR3. There was no effect on ACACA expression. Orlistat decreased expression of LIPE, FASN, ACACA, and PPARG, and increased FFAR3 and SREBF1 expression. The pattern of LPL expression was similar to the changes in milk fat percentage in lactating goats. Taken together, results suggest that LPL may play a crucial role in fatty acid synthesis. PMID:25501331

  1. Global gene expression and the role of sigma factors in Neisseria gonorrhoeae in interactions with epithelial cells.

    PubMed

    Du, Ying; Lenz, Jonathan; Arvidson, Cindy Grove

    2005-08-01

    Like many bacterial pathogens, Neisseria gonorrhoeae must adapt to environmental changes in order to successfully colonize and proliferate in a new host. Modulation of gene expression in response to environmental signals is an efficient mechanism used by bacteria to achieve this goal. Using DNA microarrays and a tissue culture model for gonococcal infection, we examined global changes in gene expression in N. gonorrhoeae in response to adherence to host cells. Among those genes induced upon adherence to human epithelial cells in culture was rpoH, which encodes a homolog of the heat shock sigma factor, sigma(32) (RpoH), as well as genes of the RpoH regulon, groEL and groES. Attempts to construct an rpoH null mutant in N. gonorrhoeae were unsuccessful, suggesting that RpoH is essential for viability of N. gonorrhoeae. The extracytoplasmic sigma factor, RpoE (sigma(E)), while known to regulate rpoH in other bacteria, was found not to be necessary for the up-regulation of rpoH in gonococci upon adherence to host cells. To examine the role of RpoH in host cell interactions, an N. gonorrhoeae strain conditionally expressing rpoH was constructed. The results of our experiments showed that while induction of rpoH expression is not necessary for adherence of gonococci to epithelial cells, it is important for the subsequent invasion step, as gonococci depleted for rpoH invade cells two- to threefold less efficiently than a wild-type strain. Taken together, these results indicate that sigma(32), but not sigma(E), is important for the response of gonococci in the initial steps of an infection. PMID:16040997

  2. Gene expression profiling and pathway analysis of human bronchial epithelial cells exposed to airborne particulate matter collected from Saudi Arabia

    SciTech Connect

    Sun, Hong; Shamy, Magdy; Kluz, Thomas; Muñoz, Alexandra B.; Zhong, Mianhua; Laulicht, Freda; Alghamdi, Mansour A.; Khoder, Mamdouh I.; Chen, Lung-Chi; Costa, Max

    2012-12-01

    Epidemiological studies have established a positive correlation between human mortality and increased concentration of airborne particulate matters (PM). However, the mechanisms underlying PM related human diseases, as well as the molecules and pathways mediating the cellular response to PM, are not fully understood. This study aims to investigate the global gene expression changes in human cells exposed to PM{sub 10} and to identify genes and pathways that may contribute to PM related adverse health effects. Human bronchial epithelial cells were exposed to PM{sub 10} collected from Saudi Arabia for 1 or 4 days, and whole transcript expression was profiled using the GeneChip human gene 1.0 ST array. A total of 140 and 230 genes were identified that significantly changed more than 1.5 fold after PM{sub 10} exposure for 1 or 4 days, respectively. Ingenuity Pathway Analysis revealed that different exposure durations triggered distinct pathways. Genes involved in NRF2-mediated response to oxidative stress were up-regulated after 1 day exposure. In contrast, cells exposed for 4 days exhibited significant changes in genes related to cholesterol and lipid synthesis pathways. These observed changes in cellular oxidative stress and lipid synthesis might contribute to PM related respiratory and cardiovascular disease. -- Highlights: ► PM exposure modulated gene expression and associated pathways in BEAS-2B cells. ► One-day exposure to PM induced genes involved in responding to oxidative stress. ► 4-day exposure to PM changed genes associated to cholesterol and lipid synthesis.

  3. Gene expression profile of duodenal epithelial cells in response to chronic metabolic acidosis.

    PubMed

    Wongdee, Kannikar; Teerapornpuntakit, Jarinthorn; Riengrojpitak, Suda; Krishnamra, Nateetip; Charoenphandhu, Narattaphol

    2009-01-01

    Chronic metabolic acidosis (CMA) affects ion transport, permeability, and metabolism of the intestinal absorptive cells. Most effects of CMA on the intestine are long-term adaptations at genomic level. To identify the CMA-regulated genes, the Illumina's microarray featuring high-performance BeadArray technology was performed on RNA samples from the rat duodenal epithelial cells exposed to long-standing acidemia. After 21 days of CMA, we found 423 transcripts upregulated and 261 transcripts downregulated. Gene ontology analysis suggested effects of CMA on cellular processes, such as cell adhesion, proliferation, fuel metabolism, and biotransformation. Interestingly, 27 upregulated transcripts (e.g., Aqp1, Cacnb1, Atp1a2, Kcnab2, and Slc2a1) and 13 downregulated transcripts (e.g., Slc17a7, Slc9a4, and Slc30a3) are involved in the absorption of water, ions, and nutrients. Some upregulated genes, such as Slc38a5 and Slc1a7 encoding glutamine transporters, may be parts of the total body adaptation to alleviate negative nitrogen balance. Therefore, the present results provided a novel genome-wide information for further investigations of the mechanism of CMA effect on the intestine. PMID:18979233

  4. Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice

    NASA Astrophysics Data System (ADS)

    Lemaire-Vieille, Catherine; Schulze, Tobias; Podevin-Dimster, Valérie; Follet, Jérome; Bailly, Yannick; Blanquet-Grossard, Françoise; Decavel, Jean-Pierre; Heinen, Ernst; Cesbron, Jean-Yves

    2000-05-01

    The expression of the cellular form of the prion protein (PrPc) gene is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. The identification of the cell types expressing PrPc is necessary to understanding how the agent replicates and spreads from peripheral sites to the central nervous system. To determine the nature of the cell types expressing PrPc, a green fluorescent protein reporter gene was expressed in transgenic mice under the control of 6.9 kb of the bovine PrP gene regulatory sequences. It was shown that the bovine PrP gene is expressed as two populations of mRNA differing by alternative splicing of one 115-bp 5' untranslated exon in 17 different bovine tissues. The analysis of transgenic mice showed reporter gene expression in some cells that have been identified as expressing PrP, such as cerebellar Purkinje cells, lymphocytes, and keratinocytes. In addition, expression of green fluorescent protein was observed in the plexus of the enteric nervous system and in a restricted subset of cells not yet clearly identified as expressing PrP: the epithelial cells of the thymic medullary and the endothelial cells of both the mucosal capillaries of the intestine and the renal capillaries. These data provide valuable information on the distribution of PrPc at the cellular level and argue for roles of the epithelial and endothelial cells in the spread of infection from the periphery to the brain. Moreover, the transgenic mice described in this paper provide a model that will allow for the study of the transcriptional activity of the PrP gene promoter in response to scrapie infection.

  5. Strategies for Profiling Single Mouse Intestinal Epithelial Cells by Targeted Gene Expression

    PubMed Central

    McDowell, W.; Box, A.; Staehling, K.; Wang, F.; Li, L.; Zueckert-Gaudenz, K.

    2014-01-01

    Targeted gene expression profiling of single cells permits the study of heterogeneity in cell populations. Here, a pool of mouse intestinal crypt-base CD44+/GRP78- cells was collected by fluorescence activated cell sorting. Aliquots were either loaded onto Fluidigm's C1 System for microfluidic cell capture and cDNA synthesis in nanoliter volumes, or flow-sorted directly into individual PCR plate wells for cDNA synthesis in microliter volumes. The pre-amplified cDNAs were transferred to the BioMark System for EvaGreen real-time PCR. The two sample preparation methods were compared by expression analysis of 86 genes, using Fluidigm's SINGuLAR R-scripts. After outlier identification, gene expression values from 42% of the “C1” and 92% of the “flow” wells were retained. For 55 of the genes, expression was measured in both the “C1” and “flow” cells. Genes with a high variance in expression likely stemming from the sample preparation method and/or unspecific amplification were removed. Hierarchical clustering on the remaining data revealed gene clusters that contributed to the expected Lgr5hi and Lgr5lo intestinal stem cell (ISC) populations as well as a small population of differentiated cells. The subpopulations could be defined by either method. However, as ISCs quickly undergo apoptosis at room temperature, the use of the C1 System provided no clear advantage over the direct sorting of the fragile cells into lysis/RT reaction buffer. Specifically, the C1 quality control step to verify the number of captured cells and cell viability was omitted to accelerate processing.

  6. Gene expression profiling of the effects of organic dust in lung epithelial and THP-1 cells reveals inductive effects on inflammatory and immune response genes.

    PubMed

    Boggaram, Vijay; Loose, David S; Gottipati, Koteswara R; Natarajan, Kartiga; Mitchell, Courtney T

    2016-04-01

    The intensification and concentration of animal production operations expose workers to high levels of organic dusts in the work environment. Exposure to organic dusts is a risk factor for the development of acute and chronic respiratory symptoms and diseases. Lung epithelium plays important roles in the control of immune and inflammatory responses to environmental agents to maintain lung health. To better understand the effects of organic dust on lung inflammatory responses, we characterized the gene expression profiles of A549 alveolar and Beas2B bronchial epithelial and THP-1 monocytic cells influenced by exposure to poultry dust extract by DNA microarray analysis using Illumina Human HT-12 v4 Expression BeadChip. We found that A549 alveolar and Beas2B bronchial epithelial and THP-1 cells responded with unique changes in the gene expression profiles with regulation of genes encoding inflammatory cytokines, chemokines, and other inflammatory proteins being common to all the three cells. Significantly induced genes included IL-8, IL-6, IL-1β, ICAM-1, CCL2, CCL5, TLR4, and PTGS2. Validation by real-time qRT-PCR, ELISA, Western immunoblotting, and immunohistochemical staining of lung sections from mice exposed to dust extract validated DNA microarray results. Pathway analysis indicated that dust extract induced changes in gene expression influenced functions related to cellular growth and proliferation, cell death and survival, and cellular development. These data show that a broad range of inflammatory mediators produced in response to poultry dust exposure can modulate lung immune and inflammatory responses. This is the first report on organic dust induced changes in expression profiles in lung epithelial and THP-1 monocytic cells. PMID:26884459

  7. Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

    SciTech Connect

    Medina, D.; Oborn, C.J. ); Li, M.L.; Bissell, M.J. )

    1987-09-01

    The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.

  8. Analysis of the gene expression profile in response to human epididymis protein 4 in epithelial ovarian cancer cells.

    PubMed

    Zhu, Liancheng; Guo, Qian; Jin, Shan; Feng, Huilin; Zhuang, Huiyu; Liu, Cong; Tan, Mingzi; Liu, Juanjuan; Li, Xiao; Lin, Bei

    2016-09-01

    Currently, there are emerging multiple studies on human epididymis protein 4 (HE4) in ovarian cancer. HE4 possesses higher sensitivity and specificity than CA125 in the confirmative early diagnosis for ovarian cancer. Although much attention has been given to explore its clinical application, research of the basic mechanisms of HE4 in ovarian cancer are still unclear. In the present study, we provide fundamental data to identify full-scale differentially expressed genes (DEGs) in response to HE4 by use of human whole-genome microarrays in human epithelial ovarian cancer cell line ES-2 following overexpression and silencing of HE4. We found that a total of 717 genes were upregulated and 898 genes were downregulated in the HE4-overexpressing cells vs. the HE4-Mock cells, and 166 genes were upregulated and 285 were downregulated in the HE4-silenced cells vs. the HE4-Mock cells. An overlap of 16 genes consistently upregulated and 8 genes downregulated in response to HE4 were noted. These DEGs were involved in MAPK, steroid biosynthesis, cell cycle, the p53 hypoxia pathway, and focal adhesion pathways. Interaction network analysis predicted that the genes participated in the regulatory connection. Highly differential expression of the FOXA2, SERPIND1, BDKRD1 and IL1A genes was verified by quantitative real-time PCR in 4 cell line samples. Finally, SERPIND1 (HCII) was validated at the protein level by immunohistochemistry in 107 paraffin-embedded ovarian tissues. We found that SERPIND1 may act as a potential oncogene in the development of ovarian cancer. The present study displayed the most fundamental and full-scale data to show DEGs in response to HE4. These identified genes may provide a theoretical basis for investigations of the underlying molecular mechanism of HE4 in ovarian cancer. PMID:27430660

  9. Transgenic Expression of miR-222 Disrupts Intestinal Epithelial Regeneration by Targeting Multiple Genes Including Frizzled-7

    PubMed Central

    Chung, Hee Kyoung; Chen, Yu; Rao, Jaladanki N; Liu, Lan; Xiao, Lan; Turner, Douglas J; Yang, Peixin; Gorospe, Myriam; Wang, Jian-Ying

    2015-01-01

    Defects in intestinal epithelial integrity occur commonly in various pathologies. miR-222 is implicated in many aspects of cellular function and plays an important role in several diseases, but its exact biological function in the intestinal epithelium is underexplored. We generated mice with intestinal epithelial tissue-specific overexpression of miR-222 to investigate the function of miR-222 in intestinal physiology and diseases in vivo. Transgenic expression of miR-222 inhibited mucosal growth and increased susceptibility to apoptosis in the small intestine, thus leading to mucosal atrophy. The miR-222–elevated intestinal epithelium was vulnerable to pathological stress, since local overexpression of miR-222 not only delayed mucosal repair after ischemia/reperfusion-induced injury, but also exacerbated gut barrier dysfunction induced by exposure to cecal ligation and puncture. miR-222 overexpression also decreased expression of the Wnt receptor Frizzled-7 (FZD7), cyclin-dependent kinase 4 and tight junctions in the mucosal tissue. Mechanistically, we identified the Fzd7 messenger ribonucleic acid (mRNA) as a novel target of miR-222 and found that [miR-222/Fzd7 mRNA] association repressed Fzd7 mRNA translation. These results implicate miR-222 as a negative regulator of normal intestinal epithelial regeneration and protection by downregulating expression of multiple genes including the Fzd7. Our findings also suggest a novel role of increased miR-222 in the pathogenesis of mucosal growth inhibition, delayed healing and barrier dysfunction. PMID:26252186

  10. Vulnerability of the human airway epithelium to hyperoxia. Constitutive expression of the catalase gene in human bronchial epithelial cells despite oxidant stress.

    PubMed

    Yoo, J H; Erzurum, S C; Hay, J G; Lemarchand, P; Crystal, R G

    1994-01-01

    Although catalase is a major intracellular antioxidant, the expression of the human catalase gene appears to be limited in the airway epithelium, making these cells vulnerable to oxidant stress. The basis for this limited gene expression was examined by evaluation of the expression of the endogenous gene in human bronchial epithelial cells in response to hyperoxia. Hyperoxia failed to upregulate endogenous catalase gene expression, in contrast to a marked increase in expression of the heat shock protein gene. Sequence analysis of 1.7 kb of the 5'-flanking region of the human catalase gene showed features of a "house-keeping" gene (no TATA box, high GC content, multiple CCAAT boxes, and transcription start sites). Transfection of human bronchial epithelial cells with fusion genes composed of various lengths of the catalase 5'-flanking region and luciferase as a reporter gene showed low level constitutive promoter activity that did not change after exposure to hyperoxia. Importantly, using a replication-deficient recombinant adenoviral vector containing the human catalase cDNA, levels of catalase were significantly increased in human airway epithelial cells and this was associated with increased survival of the cells when exposed to hyperoxia. These observations provide a basis for understanding the sensitivity of the human airway epithelium to oxidant stress and a strategy for protecting the epithelium from such injury. PMID:8282800

  11. Cross-species comparison of genomewide gene expression profiles reveals induction of hypoxia-inducible factor-responsive genes in iron-deprived intestinal epithelial cells

    PubMed Central

    Hu, Zihua; Gulec, Sukru

    2010-01-01

    Molecular mechanisms mediating the induction of metal ion homeostasis-related genes in the mammalian intestine during iron deficiency remain unknown. To elucidate relevant regulatory pathways, genomewide gene expression profiles were determined in fully differentiated human intestinal epithelial (Caco-2) cells. Cells were deprived of iron (or not) for 6 or 18 h, and Gene Chip analyses were subsequently performed (Affymetrix). More than 2,000 genes were differentially expressed; genes related to monosaccharide metabolism, regulation of gene expression, hypoxia, and cell death were upregulated, while those related to mitotic cell cycle were downregulated. A large proportion of induced genes are hypoxia responsive, and promoter enrichment analyses revealed a statistical overrepresentation of hypoxia response elements (HREs). Immunoblot experiments demonstrated a >60-fold increase in HIF2α protein abundance in iron-deprived cells; HIF1α levels were unchanged. Furthermore, comparison of the Caco-2 cell data set with a Gene Chip data set from iron-deficient rat intestine revealed 29 common upregulated genes; the majority are hypoxia responsive, and their promoters are enriched for HREs. We conclude that the compensatory response of the intestinal epithelium to iron deprivation relates to hypoxia and that stabilization of HIF2α may be the primary event mediating metabolic and morphological changes observed during iron deficiency. PMID:20702690

  12. Extracellular matrix-dependent tissue-specific gene expression in mammary epithelial cells requires both physical and biochemical signal transduction

    SciTech Connect

    Roskelley, C.D.; Desprez, P.Y.; Bissell, M.J. )

    1994-12-20

    Extracellular matrix (ECM) profoundly influences the growth and differentiation of the mammary gland epithelium, both in culture and in vivo. Utilizing a clonal population of mouse mammary epithelial cells that absolutely requires an exogenous ECM for function, we developed a rapid assay to study signal transduction by ECM. Two components of the cellular response to a basement membrane overlay that result in the expression of the milk protein [beta]-casein were defined. The first component of this response involves a rounding and clustering of the cells that can be physically mimicked by plating the cells on a nonadhesive substratum. The second component is biochemical in nature, and it is associated with [beta][sub 1] integrin clustering and increased tyrosine phosphorylation. The second component is initiated in a morphology-independent manner, but the proper translation of this biochemical signal into a functional response requires cell rounding and cell clustering. Thus, physical and biochemical signal transduction events contribute to the ECM-dependent regulation of tissue-specific gene expression in mouse mammary epithelial cells. 44 refs., 6 figs.

  13. Early changes in gene expression induced by blue light irradiation of A2E-laden retinal pigment epithelial cells

    PubMed Central

    van der Burght, Barbro W.; Hansen, Morten; Olsen, Jørgen; Zhou, Jilin; Wu, Yalin; Nissen, Mogens H.; Sparrow, Janet R.

    2016-01-01

    Purpose Accumulation of bisretinoids as lipofuscin in retinal pigment epithelial (RPE) cells is implicated in the pathogenesis of some blinding diseases including age-related macular degeneration (AMD). To identify genes whose expression may change under conditions of bisretinoid accumulation, we investigated the differential gene expression in RPE cells that had accumulated the lipofuscin fluorophore A2E and were exposed to blue light (430 nm). Methods A2E-laden RPE cells were exposed to blue light (A2E/430 nm) at various time intervals. Cell death was quantified using Dead Red staining, and RNA levels for the entire genome was determined using DNA microarrays (Affymetrix GeneChip Human Genome 2.0 Plus). Array results for selected genes were confirmed by real-time reverse-transcriptase polymerase chain reaction. Results Principal component analysis revealed that the A2E-laden RPE cells irradiated with blue light were clearly distinguishable from the control samples. We found differential regulation of genes belonging to the following functional groups: transcription factors, stress response, apoptosis and immune response. Among the last mentioned were downregulation of four genes that coded for proteins that have an inhibitory effect on the complement cascade: (complement factor H, complement factor H-related 1, complement factor I and vitronectin) and of two belonging to the classical pathway (complement component 1, s subcomponent and complement component 1, r subcomponent). Conclusion This study demonstrates that blue light irradiation of A2E-laden RPE cells can alter the transcription of genes belonging to different functional pathways including stress response, apoptosis and the immune response. We suggest that these molecules may be associated to the pathogenesis of AMD and can potentially serve as future therapeutic targets. PMID:23742627

  14. Detection of Epstein-Barr virus genome and latent infection gene expression in normal epithelia, epithelial dysplasia, and squamous cell carcinoma of the oral cavity.

    PubMed

    Kikuchi, Kentaro; Noguchi, Yoshihiro; de Rivera, Michelle Wendoline Garcia-Niño; Hoshino, Miyako; Sakashita, Hideaki; Yamada, Tsutomu; Inoue, Harumi; Miyazaki, Yuji; Nozaki, Tadashige; González-López, Blanca Silvia; Ide, Fumio; Kusama, Kaoru

    2016-03-01

    A relationship between Epstein-Barr virus (EBV) infection and cancer of lymphoid and epithelial tissues such as Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma (NPC), gastric carcinoma, and oral cancer has been reported. EBV is transmitted orally and infects B cells and epithelial cells. However, it has remained uncertain whether EBV plays a role in carcinogenesis of oral mucosal tissue. In the present study, we detected the EBV genome and latent EBV gene expression in normal mucosal epithelia, epithelial dysplasia, and oral squamous cell carcinoma (OSCC) to clarify whether EBV is involved in carcinogenesis of the oral cavity. We examined 333 formalin-fixed, paraffin-embedded tissue samples (morphologically normal oral mucosa 30 samples, gingivitis 32, tonsillitis 17, oral epithelial dysplasia 83, OSCC 150, and NPC 21). EBV latent infection genes (EBNA-2, LMP-1) were detected not only in OSCC (50.2 %, 10.7 %) but also in severe epithelial dysplasia (66.7 %, 44.4 %), mild to moderate epithelial dysplasia (43.1 %, 18.5 %), gingivitis (78.1 %, 21.9 %), and normal mucosa (83.3 %, 23.3 %). Furthermore, the intensity of EBV latent infection gene expression (EBER, LMP-1) was significantly higher in severe epithelial dysplasia (94.4 %, 72.2 %) than in OSCC (34.7 %, 38.7 %). These results suggest that EBV latent infection genes and their increased expression in severe epithelial dysplasia might play an important role in the dysplasia-carcinoma sequence in the oral cavity. PMID:26449822

  15. Gene expression regulation in retinal pigment epithelial cells induced by viral RNA and viral/bacterial DNA

    PubMed Central

    Brosig, Anton; Kuhrt, Heidrun; Wiedemann, Peter; Kohen, Leon; Bringmann, Andreas

    2015-01-01

    Purpose The pathogenesis of age-related macular degeneration (AMD) is associated with systemic and local inflammation. Various studies suggested that viral or bacterial infection may aggravate retinal inflammation in the aged retina. We compared the effects of synthetic viral RNA (poly(I:C)) and viral/bacterial DNA (CpG-ODN) on the expression of genes known to be involved in the development of AMD in retinal pigment epithelial (RPE) cells. Methods Cultured human RPE cells were stimulated with poly(I:C; 500 µg/ml) or CpG-ODN (500 nM). Alterations in gene expression and protein secretion were determined with real-time RT–PCR and ELISA, respectively. Phosphorylation of signal transduction molecules was revealed by western blotting. Results Poly(I:C) induced gene expression of the pattern recognition receptor TLR3, transcription factors (HIF-1α, p65/NF-κB), the angiogenic factor bFGF, inflammatory factors (IL-1β, IL-6, TNFα, MCP-1, MIP-2), and complement factors (C5, C9, CFB). Poly(I:C) also induced phosphorylation of ERK1/2 and p38 MAPK proteins, and the secretion of bFGF and TNFα from the cells. CpG-ODN induced moderate gene expression of transcription factors (p65/NF-κB, NFAT5) and complement factors (C5, C9), while it had no effect on the expression of various TLR, angiogenic factor, and inflammatory factor genes. The activities of various signal transduction pathways and transcription factors were differentially involved in mediating the poly(I:C)-induced transcriptional activation of distinct genes. Conclusions The widespread effects of viral RNA, and the restricted effects of viral/bacterial DNA, on the gene expression pattern of RPE cells may suggest that viral RNA rather than viral/bacterial DNA induces physiologic alterations of RPE cells, which may aggravate inflammation in the aged retina. The data also suggest that selective inhibition of distinct signal transduction pathways or individual transcription factors may not be effective to inhibit

  16. Airway Epithelial Cells are the Site of Expression of a Mammalian Antimicrobial Peptide Gene

    NASA Astrophysics Data System (ADS)

    Diamond, Gill; Jones, Douglas E.; Bevins, Charles L.

    1993-05-01

    We previously reported the isolation and characterization of a broad-spectrum antimicrobial peptide from the bovine tracheal mucosa, which we called tracheal antimicrobial peptide (TAP). We now show the TAP gene is expressed throughout the adult conducting airway, from nasal to bronchiolar tissue, but not in tissues other than airway mucosa, as determined by Northern blot analysis. In situ hybridization of airway sections localizes TAP mRNA to columnar cells of the pseudostratified epithelium. We report the structural organization of the TAP gene and show that TAP is a member of a large family of related sequences with high nucleotide identity in the 5'exon. The data support the hypothesis that antimicrobial peptides contribute to host defense of the respiratory tract.

  17. Transcriptome profiling reveals novel gene expression signatures and regulating transcription factors of TGFβ-induced epithelial-to-mesenchymal transition.

    PubMed

    Du, Liutao; Yamamoto, Shota; Burnette, Barry L; Huang, Danshang; Gao, Kun; Jamshidi, Neema; Kuo, Michael D

    2016-08-01

    Dysregulated epithelial to mesenchymal transition (EMT) in cancer cells endows invasive and metastatic properties upon cancer cells that favor successful colonization of distal target organs and therefore play a critical role in transforming early-stage carcinomas into invasive malignancies. EMT has also been associated with tumor recurrence and drug resistance and cancer stem cell initiation. Therefore, better understanding of the mechanisms behind EMT could ultimately contribute to the development of novel prognostic approaches and individualized therapies that specifically target EMT processes. As an effort to characterize the central transcriptome changes during EMT, we have developed a Transforming growth factor (TGF)-beta-based in vitro EMT model and used it to profile EMT-related gene transcriptional changes in two different cell lines, a non-small cell lung cancer cell line H358, and a breast cell line MCF10a. After 7 days of TGF-beta/Oncostatin M (OSM) treatment, changes in cell morphology to a mesenchymal phenotype were observed as well as concordant EMT-associated changes in mRNA and protein expression. Further, increased motility was noted and flow cytometry confirmed enrichment in cancer stem cell-like populations. Microarray-based differential expression analysis identified an EMT-associated gene expression signature which was confirmed by RT-qPCR and which significantly overlapped with a previously published EMT core signature. Finally, two novel EMT-regulating transcription factors, IRF5 and LMCD1, were identified and independently validated. PMID:27318801

  18. Specific Glycosylation of Membrane Proteins in Epithelial Ovarian Cancer Cell Lines: Glycan Structures Reflect Gene Expression and DNA Methylation Status *

    PubMed Central

    Anugraham, Merrina; Jacob, Francis; Nixdorf, Sheri; Everest-Dass, Arun Vijay; Heinzelmann-Schwarz, Viola; Packer, Nicolle H.

    2014-01-01

    Epithelial ovarian cancer is the fifth most common cause of cancer in women worldwide bearing the highest mortality rate among all gynecological cancers. Cell membrane glycans mediate various cellular processes such as cell signaling and become altered during carcinogenesis. The extent to which glycosylation changes are influenced by aberrant regulation of gene expression is nearly unknown for ovarian cancer and remains crucial in understanding the development and progression of this disease. To address this effect, we analyzed the membrane glycosylation of non-cancerous ovarian surface epithelial (HOSE 6.3 and HOSE 17.1) and serous ovarian cancer cell lines (SKOV 3, IGROV1, A2780, and OVCAR 3), the most common histotype among epithelial ovarian cancers. N-glycans were released from membrane glycoproteins by PNGase F and analyzed using nano-liquid chromatography on porous graphitized carbon and negative-ion electrospray ionization mass spectrometry (ESI-MS). Glycan structures were characterized based on their molecular masses and tandem MS fragmentation patterns. We identified characteristic glycan features that were unique to the ovarian cancer membrane proteins, namely the “bisecting N-acetyl-glucosamine” type N-glycans, increased levels of α 2–6 sialylated N-glycans and “N,N′-diacetyl-lactosamine” type N-glycans. These N-glycan changes were verified by examining gene transcript levels of the enzymes specific for their synthesis (MGAT3, ST6GAL1, and B4GALNT3) using qRT-PCR. We further evaluated the potential epigenetic influence on MGAT3 expression by treating the cell lines with 5-azacytidine, a DNA methylation inhibitor. For the first time, we provide evidence that MGAT3 expression may be epigenetically regulated by DNA hypomethylation, leading to the synthesis of the unique “bisecting GlcNAc” type N-glycans on the membrane proteins of ovarian cancer cells. Linking the observation of specific N-glycan substructures and their complex association

  19. Specific glycosylation of membrane proteins in epithelial ovarian cancer cell lines: glycan structures reflect gene expression and DNA methylation status.

    PubMed

    Anugraham, Merrina; Jacob, Francis; Nixdorf, Sheri; Everest-Dass, Arun Vijay; Heinzelmann-Schwarz, Viola; Packer, Nicolle H

    2014-09-01

    Epithelial ovarian cancer is the fifth most common cause of cancer in women worldwide bearing the highest mortality rate among all gynecological cancers. Cell membrane glycans mediate various cellular processes such as cell signaling and become altered during carcinogenesis. The extent to which glycosylation changes are influenced by aberrant regulation of gene expression is nearly unknown for ovarian cancer and remains crucial in understanding the development and progression of this disease. To address this effect, we analyzed the membrane glycosylation of non-cancerous ovarian surface epithelial (HOSE 6.3 and HOSE 17.1) and serous ovarian cancer cell lines (SKOV 3, IGROV1, A2780, and OVCAR 3), the most common histotype among epithelial ovarian cancers. N-glycans were released from membrane glycoproteins by PNGase F and analyzed using nano-liquid chromatography on porous graphitized carbon and negative-ion electrospray ionization mass spectrometry (ESI-MS). Glycan structures were characterized based on their molecular masses and tandem MS fragmentation patterns. We identified characteristic glycan features that were unique to the ovarian cancer membrane proteins, namely the "bisecting N-acetyl-glucosamine" type N-glycans, increased levels of α 2-6 sialylated N-glycans and "N,N'-diacetyl-lactosamine" type N-glycans. These N-glycan changes were verified by examining gene transcript levels of the enzymes specific for their synthesis (MGAT3, ST6GAL1, and B4GALNT3) using qRT-PCR. We further evaluated the potential epigenetic influence on MGAT3 expression by treating the cell lines with 5-azacytidine, a DNA methylation inhibitor. For the first time, we provide evidence that MGAT3 expression may be epigenetically regulated by DNA hypomethylation, leading to the synthesis of the unique "bisecting GlcNAc" type N-glycans on the membrane proteins of ovarian cancer cells. Linking the observation of specific N-glycan substructures and their complex association with epigenetic

  20. Gene delivery and expression in human retinal pigment epithelial cells: effects of synthetic carriers, serum, extracellular matrix and viral promoters.

    PubMed

    Urtti, A; Polansky, J; Lui, G M; Szoka, F C

    2000-01-01

    Non-viral gene therapy is a potential treatment to many incurable retinal diseases. To fulfill this promise, plasmid DNA must be delivered to the retinal target cells. We evaluated the efficacy of synthetic DNA complexing compounds in transfecting primary human retinal pigment epithelial (RPE) cells in vitro. Fetal human RPE cells were cultured with or without extracellular matrix (ECM), produced using calf corneal endothelial cells. Plasmids encoding nuclear localizing beta galactosidase or luciferase (pRSVLuc, pCLuc4, pSV2Luc) were complexed in water at various +/- charge ratios using cationic lipids (Lipofectin, DOTAP, DOGS), polyethylene imines (25 and 750 kDa), and with degraded 6th generation starburst polyamidoamine dendrimers. Luciferase was quantified using a luminometric assay and beta galactosidase with X-gal staining. Toxicities of transfections were evaluated with the MTT-assay. Using beta galactosidase as the reporter gene naked DNA did not transfect RPE cells at measurable levels whereas 1-5% of the cells expressed histochemically detectable amounts of the gene after transfection with cationic lipid DNA complexes. In RPE cells, Rous sarcoma virus and cytomegalovirus (CMV) were more efficient promoters than SV40 in driving luciferase expression, and CMV was chosen for further experiments. At optimal complex charge ratios, expression levels of luciferase were > 10(9) light units/mg protein after transfection using dendrimers and PEI25, while transfection mediated with the other carriers resulted in luciferase expression levels of 10(7)-10(9) light units/mg protein or less. In general, dendrimers and large molecular weight PEI were less toxic than cationic lipids or PEI25 to RPE cells. Serum and ECM decreased gene expression to the RPE cells with all carriers. Despite low percentage of transfected cells the transgene expression per RPE cell is high, important feature in the retinal tissue with small dimensions, in particular in the case of secreted gene

  1. ECM and FGF-dependent assay of embryonic submandibular gland epithelial morphogenesis: investigating growth factor/matrix regulation of gene expression during development

    PubMed Central

    Rebustini, Ivan T.; Hoffman, Matthew P.

    2012-01-01

    Epithelial-mesenchyme interactions during organogenesis are regulated by dynamic and reciprocal interactions between growth factors and extracellular matrix (ECM) components. Mouse embryonic submandibular gland (SMG) epithelium, isolated from its endogenous mesenchyme, undergoes branching morphogenesis when cultured ex vivo in a basement membrane extract in serum-free medium with growth factor stimulation. The resulting three-dimensional epithelial morphogenesis in the defined culture system makes this a useful model to analyze cell-cell and cell-matrix interactions, growth factor-mediated signaling and gene expression, proliferation, apoptosis, migration, lumen formation, and epithelial morphogenesis in a primary organ culture system. SMG epithelial culture is robust, reproducible, uses small amounts of reagents, and changes in gene expression are measured by real-time PCR using a limited amount of embryonic tissue. In this chapter, we describe a detailed protocol for isolating primary embryonic SMG epithelium and setting up an ECM and growth factor-dependent, serum-free assay of epithelial morphogenesis, with subsequent analysis of gene expression by real-time PCR. PMID:19247608

  2. DIFFERENTIAL GENE EXPRESSION PROFILES IN RAT TRACHAEL EPITHELIAL (RTE) CELLS IN RESPONSE TO COMBUSTION-SOURCE PARTICULATE MATTER (PM) AND VANADIUM (V) A PRIMARY METAL CONSTITUENT

    EPA Science Inventory

    Differential gene expression profiles in rat tracheal epithelial (RTE) cells in response to combustion-source particulate matter (PM) and vanadium (V) a primary metal constituent
    Srikanth S. Nadadur, Janice A. Dye and Daniel L. Costa, US EPA, ORD, NHEERL (ETD, Pulmonary Toxico...

  3. GENE EXPRESSION PROFILING OF NORMAL HUMAN BRONCHIAL EPITHELIAL CELLS EXPOSED TO TRIVALENT ARSENICALS AND DIMETHYLTHIOARSINIC ACID

    EPA Science Inventory

    Lung is a major target for arsenic carcinogenesis in humans. However, the carcinogenic mode of action of arsenicals is unknown. We investigated, in human bronchial epithelial (BEAS2B) cells, the effects of inorganic arsenic (iAsIII), monomethylarsonous acid (MMAIII), dimethylarsi...

  4. CHANGES IN GENE EXPRESSION DURING DIFFERENTIATION OF CULTURED HUMAN PRIMARY BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Primary airway epithelial cell cultures are a useful tool for the in vitro study of normal bronchial cell differentiation and function, airway disease mechanisms, and pathogens and toxin response. Growth of these cells at an air-liquid interface for several days results in the f...

  5. MicroRNA-16 suppresses epithelial-mesenchymal transition‑related gene expression in human glioma.

    PubMed

    Wang, Qin; Li, Xu; Zhu, Yu; Yang, Ping

    2014-12-01

    Glioma is one of the most prevalent types of brain tumor and is associated with the highest mortality rate of all CNS cancers. Epithelial‑mesenchymal transition (EMT) has been recognized as an important factor in tumor metastasis. Previously, it has been demonstrated that microRNA-16 (miR-16) has an important role in tumor metastasis in human cancer cell lines. However, the role of miR-16 in epithelial‑mesenchymal transition of human glioma cells remains unclear. In the present study, U87 and U251 glioma cell lines overexpressing miR-16 were established and it was identified that miR-16 suppressed invasion, adhesion, cell cycle, production of interleukin (IL)-6, IL-8 and transforming growth factor-β, and EMT-related gene expression, including vimentin, β-catenin and E-cadherin in miR-16 overexpressing U87 and U251 glioma cells. Furthermore, miR-16 suppressed EMT mainly through the downregulation of p-FAK and p-Akt expression, and nuclear factor-κB and Slug transcriptional activity. Therefore, miR-16 may be an important therapeutic target and predictor for glioma therapy. PMID:25242314

  6. Transcriptional regulation of SP-B gene expression by nitric oxide in H441 lung epithelial cells.

    PubMed

    Boggaram, Vijay; Chandru, Hemakumar; Gottipati, Koteswara Rao; Thakur, Vijayander; Das, Aparajita; Berhane, Kiflu

    2010-08-01

    Surfactant protein B (SP-B) is essential for the surface tension-lowering function of pulmonary surfactant. Surfactant dysfunction and reduced SP-B levels are associated with elevated nitric oxide (NO) in inflammatory lung diseases, such as acute respiratory distress syndrome. We previously found that NO donors decreased SP-B expression in H441 and MLE-12 lung epithelial cells by reducing SP-B promoter activity. In this study, we determined the roles of DNA elements and interacting transcription factors necessary for NO inhibition of SP-B promoter activity in H441 cells. We found that the NO donor diethylenetriamine-nitric oxide adduct (DETA-NO) decreased SP-B promoter thyroid transcription factor 1 (TTF-1), hepatocyte nuclear factor 3 (HNF-3), and Sp1 binding activities but increased activator protein 1 (AP-1) binding activity. DETA-NO decreased TTF-1, but not Sp1, levels, suggesting that reduced TTF-1 expression contributes to reduced TTF-1 binding activity. Lack of effect on Sp1 levels suggested that DETA-NO inhibits Sp1 binding activity per se. Overexpression of Sp1, but not TTF-1, blocked DETA-NO inhibition of SP-B promoter activity. DETA-NO inhibited SP-B promoter induction by exogenous TTF-1 without altering TTF-1 levels. DETA-NO decreased TTF-1 mRNA levels and gene transcription rate, indicating that DETA-NO inhibits TTF-1 expression at the transcriptional level. We conclude that NO inhibits SP-B promoter by decreasing TTF-1, Sp1, and HNF-3 binding activities and increasing AP-1 binding activity. NO inhibits TTF-1 levels and activity to decrease SP-B expression. NO inhibition of SP-B expression could be a mechanism by which surfactant dysfunction occurs in inflammatory lung diseases. PMID:20418387

  7. 3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression.

    PubMed

    Pinto, Sheena; Stark, Hans-Jürgen; Martin, Iris; Boukamp, Petra; Kyewski, Bruno

    2015-01-01

    Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance. Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80(lo), Aire-negative into CD80(hi), Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80(hi) mTECs. PMID:26275017

  8. Staphylococcus aureus and Lipopolysaccharide Modulate Gene Expressions of Drug Transporters in Mouse Mammary Epithelial Cells Correlation to Inflammatory Biomarkers.

    PubMed

    Yagdiran, Yagmur; Tallkvist, Jonas; Artursson, Karin; Oskarsson, Agneta

    2016-01-01

    Inflammation in the mammary gland (mastitis) is the most common disease in dairy herds worldwide, often caused by the pathogens Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Little is known about the effects of mastitis on drug transporters and the impact on transporter-mediated excretion of drugs into milk. We used murine mammary epithelial HC11 cells, after lactogenic differentiation into a secreting phenotype, and studied gene expressions of ABC- and SLC- transporters after treatment of cells with S. aureus and lipopolysaccharide, an endotoxin secreted by E. coli. The studied transporters were Bcrp, Mdr1, Mrp1, Oatp1a5, Octn1 and Oct1. In addition, Csn2, the gene encoding β-casein, was analyzed. As biomarkers of the inflammatory response, gene expressions of the cytokines Il6 and Tnfα and the chemokine Cxcl2 were determined. Our results show that S. aureus and LPS treatment of cells, at non-cytotoxic concentrations, induced an up-regulation of Mdr1 and of the inflammatory biomarkers, except that Tnfα was not affected by lipopolysaccharide. By simple regression analysis we could demonstrate statistically significant positive correlations between each of the transporters with each of the inflammatory biomarkers in cells treated with S. aureus. The coefficients of determination (R2) were 0.7-0.9 for all but one correlation. After treatment of cells with lipopolysaccharide, statistically significant correlations were only found between Mdr1 and the two parameters Cxcl2 and Il6. The expression of Csn2 was up-regulated in cells treated with S. aureus, indicating that the secretory function of the cells was not impaired. The strong correlation in gene expressions between transporters and inflammatory biomarkers may suggest a co-regulation and that the transporters have a role in the transport of cytokines and chemokines. Our results demonstrate that transporters in mammary cells can be affected by infection, which may have an impact on transport

  9. Medullary Epithelial Cells of the Human Thymus Express a Highly Diverse Selection of Tissue-specific Genes Colocalized in Chromosomal Clusters

    PubMed Central

    Gotter, Jörn; Brors, Benedikt; Hergenhahn, Manfred; Kyewski, Bruno

    2004-01-01

    Promiscuous expression of tissue-specific self-antigens in the thymus imposes T cell tolerance and protects from autoimmune diseases, as shown in animal studies. Analysis of promiscuous gene expression in purified stromal cells of the human thymus at the single and global gene level documents the species conservation of this phenomenon. Medullary thymic epithelial cells overexpress a highly diverse set of genes (>400) including many tissue-specific antigens, disease-associated autoantigens, and cancer-germline genes. Although there are no apparent structural or functional commonalities among these genes and their products, they cluster along chromosomes. These findings have implications for human autoimmune diseases, immuno-therapy of tumors, and the understanding of the nature of this unorthodox regulation of gene expression. PMID:14734521

  10. Gene expression of epithelial glucose transporters: the role of diabetes mellitus.

    PubMed

    Dominguez, J H; Song, B; Maianu, L; Garvey, W T; Qulali, M

    1994-11-01

    The functions of absorption of dietary glucose by the small intestine and reabsorption of filtered glucose by the renal proximal tubule are strikingly similar in their organization and in the way they adapt to uncontrolled diabetes mellitus. In both cases, transepithelial glucose and Na+ fluxes are augmented. The epithelial adaptations to hyperglycemia of uncontrolled diabetes are accomplished by increasing the glucose transport surface area and the number of the efflux glucose transporter GLUT2 located in the basolateral membrane. The signals that modify the size of the epithelium and the overexpression of basolateral GLUT2 are not known. It was speculated that high glucose levels and enhanced Na+ flux may be important factors in the signaling event that culminates in a renal and intestinal epithelium that is modified to transport higher rates of glucose against a higher extracellular level of glucose. PMID:7873742

  11. REDOX regulation of IL-13 signaling in intestinal epithelial cells: usage of alternate pathways mediates distinct gene expression patterns

    PubMed Central

    Mandal, Debasmita; Fu, Pingfu; Levine, Alan D.

    2010-01-01

    In the classic view interleukin-13 (IL-13) binds to a heterodimer protein complex of the IL-13Rα1 and IL-4Rα chains and signals through a janus kinase 1 (JAK1)-signal transducer and activator of transcription 6 (STAT6) mechanism. We recently reported that IL-13 also signals through the IL-13Rα2 chain initiating all three mitogen activated protein kinase (MAPK) pathways, and the relative expression of IL-13Rα1 and IL-13Rα2 modulates one another’s transduction pathway. Therefore we investigated whether generation of reactive oxygen species (ROS) as second messengers may serve as a common nexus between these two pathways emanating from the individual IL-13 receptor chains in intestinal epithelial cells (IEC). IL-13 stimulates intracellular ROS synthesis within 5 min via IL-13Rα1-JAK1-STAT6- and IL-13Rα2-MEK1/2-ERK1/2-dependent activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-1 (NOX-1). IL-13-induced ROS generation in turn positively regulates phosphorylation of ERK1/2 and STAT6, yielding a feed forward amplification loop. IL-13 also stimulates the stable, long-term gene expression of two other NADPH oxidases, NOX-4 and DUOX-2, which along with constitutive NOX-1, might facilitate elevated, continuous production of ROS in IL-13-activated IEC. The contribution of each signal transduction pathway initiated by IL-13 engagement to such biological functions as wound healing, inflammation, and apoptosis was mapped for representative, responsive genes. Distinct usage patterns were observed, demonstrating that not only is IL-13 signal transduction through STAT6, MAPK, and ROS regulated in both an antagonistic and cyclic fashion, but each pathway also plays a specific role in modulating the wound healing and anti-apoptotic capabilities of the intestinal epithelium. PMID:20570727

  12. Interaction of mouse mammary epithelial cells with collagen substrata: regulation of casein gene expression and secretion

    SciTech Connect

    Lee, E.Y.H.P.; Lee, W.H.; Kaetzel, C.S.; Parry, G.; Bissell, M.J.

    1985-03-01

    Mouse mammary epithelial cells (MMEC) secrete certain milk proteins only when cultured on floating collagen gels. The authors demonstrate that modulation of milk proteins by substrata is manifested at several regulatory levels; (i) cells cultured on floating collagen gels have 3- to 10-fold more casein mRNA than cells cultured on plastic or attached collagen gels. (ii) Cells on the latter two flat substrata, nevertheless, synthesize a significant amount of caseins, indicating that the remaining mRNA is functional. (iii) Cells on all substrata are inducible for casein mRNA and casein proteins by prolactin, but the extent of induction is greater on collagen than that on plastic - i.e., the substratum confers an altered degree of inducibility. (iv) Cells on all substrata synthesize casein proteins at rates proportional to the amount of casein mRNA, but the newly synthesized caseins in cells on plastic are degraded intracellularly, whereas those synthesized by cells on floating gels are secreted into the medium. (v) Cells on all substrata examined lose virtually all mRNA for whey acidic protein despite the fact that this mRNA is abundant in the mammary gland itself; the authors conclude that additional, as-yet-unknown, factors are necessary for synthesis and secretion of whey acidic protein in culture.

  13. Association and Virulence Gene Expression Vary among Serotype III Group B Streptococcus Isolates following Exposure to Decidual and Lung Epithelial Cells

    PubMed Central

    Korir, Michelle L.; Knupp, David; LeMerise, Kathryn; Boldenow, Erica; Loch-Caruso, Rita; Aronoff, David M.

    2014-01-01

    Group B Streptococcus (GBS) causes severe disease in neonates, the elderly, and immunocompromised individuals. GBS species are highly diverse and can be classified by serotype and multilocus sequence typing. Sequence type 17 (ST-17) strains cause invasive neonatal disease more frequently than strains of other STs. Attachment and invasion of host cells are key steps in GBS pathogenesis. We investigated whether four serotype III strains representing ST-17 (two strains), ST-19, and ST-23 differ in their abilities to attach to and invade both decidual cells and lung epithelial cells. Virulence gene expression following host cell association and exposure to amnion cells was also tested. The ST-17 strains differed in their abilities to attach to and invade decidual cells, whereas there were no differences with lung epithelial cells. The ST-19 and ST-23 strains, however, attached to and invaded decidual cells less than both ST-17 strains. Although the ST-23 strain attached to lung epithelial cells better than ST-17 and -19 strains, none of the strains effectively invaded the lung epithelial cells. Notably, the association with host cells resulted in the differential expression of several virulence genes relative to basal expression levels. Similar expression patterns of some genes were observed regardless of cell type used. Collectively, these results show that GBS strains differ in their abilities to attach to distinct host cell types and express key virulence genes that are relevant to the disease process. Enhancing our understanding of pathogenic mechanisms could aid in the identification of novel therapeutic targets or vaccine candidates that could potentially decrease morbidity and mortality associated with neonatal infections. PMID:25135682

  14. Downregulation of expression of mater genes SOX9, FOXA2, and GATA4 in pancreatic cancer cells stimulated with TGFβ1 epithelial-mesenchymal transition.

    PubMed

    Kondratyeva, L G; Sveshnikova, A A; Grankina, E V; Chernov, I P; Kopantseva, M R; Kopantzev, E P; Sverdlov, E D

    2016-07-01

    We show characteristic morphological changes corresponding to epithelial-mesenchymal transition (EMT) program fulfillment in PANC1 cell line stimulated with TGFβ1. Our results support downregulation of E-cadherin protein. We show 5- and 28-fold increase in SNAI1 and SNAI2 expression levels and 25- and 15-fold decrease in CDH1 and KRT8 expression levels, respectively, which confirms the EMT-program fulfillment. We demonstrate downregulation of expression of pancreatic master genes SOX9, FOXA2, and GATA4 (2-, 5-, and 4-fold, respectively) and absence of significant changes in HES1, NR5A2, and GATA6 expression levels in the cells stimulated with TGFβ1. Our results indicate the absence of induction of expression of PTF1A, PDX1, HNF1b, NEUROG3, RPBJL, NKX6.1, and ONECUT1 genes, which are inactive in PANC1 cell line after the EMT stimulated by TGFβ1. PMID:27599506

  15. A novel dissociative steroid VBP15 reduces MUC5AC gene expression in airway epithelial cells but lacks the GRE mediated transcriptional properties of dexamethasone.

    PubMed

    Garvin, Lindsay M; Chen, Yajun; Damsker, Jesse M; Rose, Mary C

    2016-06-01

    Overproduction of secretory mucins contributes to morbidity/mortality in inflammatory lung diseases. Inflammatory mediators directly increase expression of mucin genes, but few drugs have been shown to directly repress mucin gene expression. IL-1β upregulates the MUC5AC mucin gene in part via the transcription factors NFκB while the glucocorticoid Dexamethasone (Dex) transcriptionally represses MUC5AC expression by Dex-activated GR binding to two GRE cis-sites in the MUC5AC promoter in lung epithelial cells. VBP compounds (ReveraGen BioPharma) maintain anti-inflammatory activity through inhibition of NFκB but exhibit reduced GRE-mediated transcriptional properties associated with adverse side-effects and thus have potential to minimize harmful side effects of long-term steroid therapy in inflammatory lung diseases. We investigated VBP15 efficacy as an anti-mucin agent in two types of airway epithelial cells and analyzed the transcription factor activity and promoter binding associated with VBP15-induced MUC5AC repression. VBP15 reduced MUC5AC mRNA abundance in a dose- and time-dependent manner similar to Dex in the presence or absence of IL-1β in A549 and differentiated human bronchial epithelial cells. Repression was abrogated in the presence of RU486, demonstrating a requirement for GR in the VBP15-induced repression of MUC5AC. Inhibition of NFκB activity resulted in reduced baseline expression of MUC5AC indicating that constitutive activity maintains MUC5AC production. Chromatin immunoprecipitation analysis demonstrated lack of GR and of p65 (NFκB) binding to composite GRE domains in the MUC5AC promoter following VBP15 exposure of cells, in contrast to Dex. These data demonstrate that VBP15 is a novel anti-mucin agent that mediates the reduction of MUC5AC gene expression differently than the classical glucocorticoid, Dex. PMID:27133900

  16. The urologic epithelial stem cell database (UESC) – a web tool for cell type-specific gene expression and immunohistochemistry images of the prostate and bladder

    PubMed Central

    Pascal, Laura E; Deutsch, Eric W; Campbell, David S; Korb, Martin; True, Lawrence D; Liu, Alvin Y

    2007-01-01

    Background Public databases are crucial for analysis of high-dimensional gene and protein expression data. The Urologic Epithelial Stem Cells (UESC) database is a public database that contains gene and protein information for the major cell types of the prostate, prostate cancer cell lines, and a cancer cell type isolated from a primary tumor. Similarly, such information is available for urinary bladder cell types. Description Two major data types were archived in the database, protein abundance localization data from immunohistochemistry images, and transcript abundance data principally from DNA microarray analysis. Data results were organized in modules that were made to operate independently but built upon a core functionality. Gene array data and immunostaining images for human and mouse prostate and bladder were made available for interrogation. Data analysis capabilities include: (1) CD (cluster designation) cell surface protein data. For each cluster designation molecule, a data summary allows easy retrieval of images (at multiple magnifications). (2) Microarray data. Single gene or batch search can be initiated with Affymetrix Probeset ID, Gene Name, or Accession Number together with options of coalescing probesets and/or replicates. Conclusion Databases are invaluable for biomedical research, and their utility depends on data quality and user friendliness. UESC provides for database queries and tools to examine cell type-specific gene expression (normal vs. cancer), whereas most other databases contain only whole tissue expression datasets. The UESC database provides a valuable tool in the analysis of differential gene expression in prostate cancer genes in cancer progression. PMID:18072977

  17. A combined blood based gene expression and plasma protein abundance signature for diagnosis of epithelial ovarian cancer - a study of the OVCAD consortium

    PubMed Central

    2013-01-01

    Background The immune system is a key player in fighting cancer. Thus, we sought to identify a molecular ‘immune response signature’ indicating the presence of epithelial ovarian cancer (EOC) and to combine this with a serum protein biomarker panel to increase the specificity and sensitivity for earlier detection of EOC. Methods Comparing the expression of 32,000 genes in a leukocytes fraction from 44 EOC patients and 19 controls, three uncorrelated shrunken centroid models were selected, comprised of 7, 14, and 6 genes. A second selection step using RT-qPCR data and significance analysis of microarrays yielded 13 genes (AP2A1, B4GALT1, C1orf63, CCR2, CFP, DIS3, NEAT1, NOXA1, OSM, PAPOLG, PRIC285, ZNF419, and BC037918) which were finally used in 343 samples (90 healthy, six cystadenoma, eight low malignant potential tumor, 19 FIGO I/II, and 220 FIGO III/IV EOC patients). Using new 65 controls and 224 EOC patients (thereof 14 FIGO I/II) the abundances of six plasma proteins (MIF, prolactin, CA125, leptin, osteopondin, and IGF2) was determined and used in combination with the expression values from the 13 genes for diagnosis of EOC. Results Combined diagnostic models using either each five gene expression and plasma protein abundance values or 13 gene expression and six plasma protein abundance values can discriminate controls from patients with EOC with Receiver Operator Characteristics Area Under the Curve values of 0.998 and bootstrap .632+ validated classification errors of 3.1% and 2.8%, respectively. The sensitivities were 97.8% and 95.6%, respectively, at a set specificity of 99.6%. Conclusions The combination of gene expression and plasma protein based blood derived biomarkers in one diagnostic model increases the sensitivity and the specificity significantly. Such a diagnostic test may allow earlier diagnosis of epithelial ovarian cancer. PMID:23551967

  18. Next-Generation Sequencing Identifies TGF-β1-Associated Gene Expression Profiles in Renal Epithelial Cells Reiterated in Human Diabetic Nephropathy

    PubMed Central

    Brennan, Eoin P.; Morine, Melissa J.; Walsh, David W.; Roxburgh, Sarah A.; Lindenmeyer, Maja T.; Brazil, Derek P.; Gaora, Peadar Ó.; Roche, Helen M.; Sadlier, Denise M.; Cohen, Clemens D.; Godson, Catherine; Martin, Finian

    2012-01-01

    Transforming growth factor-beta (TGF-β1) is implicated in the onset and progression of renal fibrosis and diabetic nephropathy (DN), leading to a loss of epithelial characteristics of tubular cells. The transcriptional profile of renal tubular epithelial cells stimulated with TGF-β1 was assessed using RNA-Seq, with 2027 differentially expressed genes identified. Promoter analysis of transcription factor binding sites in the TGF-β1 responsive gene set predicted activation of multiple transcriptional networks, including NFκB. Comparison of RNA-Seq with microarray data from identical experimental conditions identified low abundance transcripts exclusive to RNA-Seq data. We compared these findings to human disease by analyzing transcriptomic data from renal biopsies of patients with DN versus control groups, identifying a shared subset of 179 regulated genes. ARK5, encoding an AMP-related kinase, and TGFBI - encoding transforming growth factor, beta-induced protein were induced by TGF-β1 and also upregulated in human DN. Suppression of ARK5 attenuated fibrotic responses of renal epithelia to TGF-β1 exposure; and silencing of TGFBI induced expression of the epithelial cell marker – E-cadherin. We identified low abundance transcripts in sequence data and validated expression levels of several transcripts (ANKRD56, ENTPD8) in tubular enriched kidney biopsies of DN patients versus living donors. In conclusion, we have defined a TGF-β1-driven pro-fibrotic signal in renal epithelial cells that is also evident in the DN renal transcriptome. PMID:22266139

  19. Isolation and functional characterization of buffalo (Bubalus bubalis) β-casein promoter for driving mammary epithelial cell-specific gene expression.

    PubMed

    Ganguli, Nirmalya; Ganguli, Nilanjana; Usmani, Abul; Majumdar, Subeer S

    2015-03-20

    Therapeutic proteins are produced in microbes, mammalian cell lines, and body fluids by applying recombinant DNA technology. They are required for compensating the deficiency of essential proteins in patients. Animal bioreactors producing such valuable bio-pharmaceuticals in body fluids have lately emerged as efficient and cost-effective expression systems. Promoters, along with other regulatory elements of genes coding for milk proteins, have been cloned from few species for directing the expression of desired proteins in the milk of farm animals. However, buffaloes, which are the second largest source of milk production in the world, have remained unexplored for such use. Since mammary epithelial cell-specific β-casein is the most abundantly expressed protein found in buffalo milk, we have isolated the promoter region and the transcriptional regulatory element along with exon 1, Intron 1 and partial exon 2 of the β-casein gene from the genome of the Indian river buffalo (Bubalus bubalis) and have characterized the same (GenBank accession no. KF612339). Mammary epithelial cells of buffalo and human (MCF7) expressed Enhanced green fluorescent protein (EGFP) upon transfection with the construct where egfp was cloned under the β-casein promoter. Transfected HEK-293 cells failed to express EGFP. Transgenic female mice generated using this construct expressed EGFP in the milk gland during lactation, without leaky expression in any other organs. This promoter also drove expression of recombinant human Interferonγ suggesting its use for expressing recombinant bio-pharmaceuticals in the milk of buffalo or other farm animals. Additionally, this may also allow breast gland-specific gene expression for remediation of breast gland-associated diseases. PMID:25678138

  20. Promegestone (R5020) and mifepristone (RU486) both function as progestational agonists of human glycodelin gene expression in isolated human epithelial cells.

    PubMed

    Taylor, R N; Savouret, J F; Vaisse, C; Vigne, J L; Ryan, I; Hornung, D; Seppälä, M; Milgrom, E

    1998-11-01

    One of the most abundant protein products of human secretory endometrium is glycodelin, a glycoprotein previously referred to as PP14. Although the precise function of this protein is unknown, its unique glycosylation pattern is believed to affect immunomodulatory activity during human embryonic implantation and inhibition of sperm-egg binding after ovulation. Having confirmed the expression of glycodelin in secretory endometrial glands, we used purified endometrial epithelial cell cultures to demonstrate the hormonal regulation of glycodelin synthesis and secretion. The findings were corroborated by transiently transfecting glycodelin gene promoter-reporter constructs into human epithelioid HeLa and Ishikawa cells. Our results indicate that glycodelin protein production by endometrial epithelial cells is directly up-regulated 4- to 9-fold by progestins and antiprogestins in vitro. Transcriptional regulation of the glycodelin gene promoter expressed in HeLa cells is progesterone receptor-dependent. As observed in the primary endometrial cells, progestins and antiprogestins both act as agonists on the in vitro expression of this endometrial gene. Our findings provide insight into the regulation of this abundant endometrial protein and raise interesting questions about the physical nature of the interaction of agonist- and antagonist-bound progesterone receptors with the glycodelin gene promoter. PMID:9814484

  1. Whole-Genome Analysis of Temporal Gene Expression during Early Transdifferentiation of Human Lung Alveolar Epithelial Type 2 Cells In Vitro

    PubMed Central

    Johansson, Helena Morales; Newman, Donna R.; Sannes, Philip L.

    2014-01-01

    It is generally accepted that the surfactant-producing pulmonary alveolar epithelial type II (AT2) cell acts as the progenitor of the type I (AT1) cell, but the regulatory mechanisms involved in this relationship remain the subject of active investigation. While previous studies have established a number of specific markers that are expressed during transdifferentiation from AT2 to AT1 cells, we hypothesized that additional, previously unrecognized, signaling pathways and relevant cellular functions are transcriptionally regulated at early stages of AT2 transition. In this study, a discovery-based gene expression profile analysis was undertaken of freshly isolated human AT2 (hAT2) cells grown on extracellular matrix (ECM) substrata known to either support (type I collagen) or retard (Matrigel) the early transdifferentiation process into hAT1-like cells over the first three days. Cell type-specific expression patterns analyzed by Illumina Human HT-12 BeadChip yielded over 300 genes that were up- or down-regulated. Candidate genes significantly induced or down-regulated during hAT2 transition to hAT1-like cells compared to non-transitioning hAT2 cells were identified. Major functional groups were also recognized, including those of signaling and cytoskeletal proteins as well as genes of unknown function. Expression of established signatures of hAT2 and hAT1 cells, such as surfactant proteins, caveolin-1, and channels and transporters, was confirmed. Selected novel genes further validated by qRT-PCR, protein expression analysis, and/or cellular localization included SPOCK2, PLEKHO1, SPRED1, RAB11FIP1, PTRF/CAVIN-1 and RAP1GAP. These results further demonstrate the utility of genome-wide analysis to identify relevant, novel cell type-specific signatures of early ECM-regulated alveolar epithelial transdifferentiation processes in vitro. PMID:24690998

  2. dsRNA-induced changes in gene expression profiles of primary nasal and bronchial epithelial cells from patients with asthma, rhinitis and controls

    PubMed Central

    2014-01-01

    Background Rhinovirus infections are the most common cause of asthma exacerbations. The complex responses by airway epithelium to rhinovirus can be captured by gene expression profiling. We hypothesized that: a) upper and lower airway epithelium exhibit differential responses to double-stranded RNA (dsRNA), and b) that this is modulated by the presence of asthma and allergic rhinitis. Objectives Identification of dsRNA-induced gene expression profiles of primary nasal and bronchial epithelial cells from the same individuals and examining the impact of allergic rhinitis with and without concomitant allergic asthma on expression profiles. Methods This study had a cross-sectional design including 18 subjects: 6 patients with allergic asthma with concomitant rhinitis, 6 patients with allergic rhinitis, and 6 healthy controls. Comparing 6 subjects per group, the estimated false discovery rate was approximately 5%. RNA was extracted from isolated and cultured primary epithelial cells from nasal biopsies and bronchial brushings stimulated with dsRNA (poly(I:C)), and analyzed by microarray (Affymetrix U133+ PM Genechip Array). Data were analysed using R and the Bioconductor Limma package. Overrepresentation of gene ontology groups were captured by GeneSpring GX12. Results In total, 17 subjects completed the study successfully (6 allergic asthma with rhinitis, 5 allergic rhinitis, 6 healthy controls). dsRNA-stimulated upper and lower airway epithelium from asthma patients demonstrated significantly fewer induced genes, exhibiting reduced down-regulation of mitochondrial genes. The majority of genes related to viral responses appeared to be similarly induced in upper and lower airways in all groups. However, the induction of several interferon-related genes (IRF3, IFNAR1, IFNB1, IFNGR1, IL28B) was impaired in patients with asthma. Conclusions dsRNA differentially changes transcriptional profiles of primary nasal and bronchial epithelial cells from patients with allergic

  3. Bovine DNase I: gene organization, mRNA expression, and changes in the topological distribution of the protein during apoptosis in lens epithelial cells.

    PubMed

    De María, Alicia; Arruti, Cristina

    2003-12-19

    Genomic DNA sequencing and alignment with the known DNase I mRNA showed that the bovine gene consists of 9 exons and that only the last 8 encode the protein, since initial ATG was found at exon II. RT-PCR was used to identify DNase I mRNA in lens epithelium in vivo and in cultured epithelial cells. We found DNase I transcripts having the same nucleotide sequence as the pancreas form and others lacking almost all exon V. The lens protein presented a slightly higher relative molecular weight than the pancreatic enzyme. Lens DNase I was located in secretory pathway organelles and excluded from the nucleus. Nevertheless, in apoptotic lens epithelial cells in vitro, DNase I translocated to the nucleus and co-localized with TUNEL positive chromatin aggregates. These results indicate that cells in the lens epithelium constitutively express DNase I, and suggest a direct involvement of this nuclease in the final phases of chromatin degradation. PMID:14680812

  4. Tumor necrosis factor-α mediates interactions between macrophages and epithelial cells underlying proinflammatory gene expression induced by particulate matter.

    PubMed

    Musah, Sadiatu; DeJarnett, Natasha; Hoyle, Gary W

    2012-09-28

    Ambient particulate matter (PM) exposure is known to have adverse effects on respiratory health, but the underlying mechanisms remain obscure. We tested the hypothesis that macrophages and epithelial cells synergize to produce maximal cytokine release in response to PM exposure, thereby promoting inflammatory responses. We developed a co-culture model using MLE-12 (mouse lung epithelial) cells and RAW 264.7 (mouse monocyte/macrophage) cells. MLE-12 cells produced KC (Cxcl1) but not tumor necrosis factor-α (TNF), and KC was upregulated only at high levels of urban particulate matter (UPM; NIST 1648a). RAW 264.7 cells produced TNF but not KC, and TNF production was increased by treatment with UPM. In contrast, KC production was upregulated by co-culture of MLE-12 and RAW 264.7 cells, and it was further increased by treatment with a concentration of UPM that had no effect on MLE-12 cells alone. Multiplex cytokine assay revealed a similar pattern of synergistic production of MIG (Cxcl9) and IP-10 (Cxcl10) in co-cultures in response to UPM. TNF was implicated as mediating the synergistic increase in KC production because TNF upregulated KC production in MLE-12 cells, and UPM-induced KC production in co-cultures could be inhibited by a TNF blocking antibody. Intratracheal instillation of UPM into both wild-type and TNF receptor knockout mice resulted in increased TNF production in lavage fluid and increased TNF mRNA expression in cells recovered from lavage fluid. Additionally, UPM instillation into wild-type mice resulted in increased neutrophils and KC in lavage fluid, and these were inhibited in UPM-exposed TNF receptor knockout mice. These results are consistent with a model in which PM activates TNF production in macrophages which in turn stimulates epithelial cells to produce proinflammatory cytokines such as KC. The findings suggest a potential mechanism by which inhaled PM induces inflammation in the lung. PMID:22634322

  5. Luteolin inhibited the gene expression, production and secretion of MUC5AC mucin via regulation of nuclear factor kappa B signaling pathway in human airway epithelial cells.

    PubMed

    Lee, Hyun Jae; Seo, Hyo-Seok; Ryu, Jiho; Yoon, Yong Pill; Park, Su Hyun; Lee, Choong Jae

    2015-04-01

    Luteolin, a flavonoidal compound derived from Lonicera japonica Thunb. and Chrysanthemum indicum L., has been reported to show anti-inflammatory, anti-oxidative and anti-carcinogenic effects. In this study, we investigated whether luteolin significantly affects the secretion, production and gene expression of airway mucin. Confluent NCI-H292 cells were pretreated with luteolin for 30 min and then stimulated with EGF (epidermal growth factor) or PMA (phorbol 12-myristate 13-acetate) for 24 h or the indicated periods. The MUC5AC mucin gene expression was measured by RT-PCR. Production and secretion of MUC5AC mucin protein were measured by ELISA. To elucidate the action mechanism of luteolin, effect of luteolin on PMA-induced NF-κB signaling pathway was investigated by western blot analysis. The results were as follows: (1) Luteolin inhibited the secretion of MUC5AC mucin protein induced by EGF or PMA; (2) Luteolin inhibited the production of MUC5AC mucin protein and the expression of MUC5AC mucin gene induced by EGF or PMA; (3) Luteolin inhibited PMA-induced phosphorylation and degradation of inhibitory kappa Bα (IκBα); (4) Luteolin inhibited PMA-induced phosphorylation and nuclear translocation of nuclear factor kappa B (NF-κB) p65. This result suggests that luteolin can regulate the secretion, production and gene expression of mucin by acting on airway epithelial cells via regulation of NF-kB signaling pathway. PMID:25285988

  6. Homeodomain-interacting protein kinase 2, a novel autoimmune regulator interaction partner, modulates promiscuous gene expression in medullary thymic epithelial cells.

    PubMed

    Rattay, Kristin; Claude, Janine; Rezavandy, Esmail; Matt, Sonja; Hofmann, Thomas G; Kyewski, Bruno; Derbinski, Jens

    2015-02-01

    Promiscuous expression of a plethora of tissue-restricted Ags (TRAs) by medullary thymic epithelial cells (mTECs) plays an essential role in T cell tolerance. Although the cellular mechanisms by which promiscuous gene expression (pGE) imposes T cell tolerance have been well characterized, the underlying molecular mechanisms remain poorly understood. The autoimmune regulator (AIRE) is to date the only validated molecule known to regulate pGE. AIRE is part of higher-order multiprotein complexes, which promote transcription, elongation, and splicing of a wide range of target genes. How AIRE and its partners mediate these various effects at the molecular level is still largely unclear. Using a yeast two-hybrid screen, we searched for novel AIRE-interacting proteins and identified the homeodomain-interacting protein kinase 2 (HIPK2) as a novel partner. HIPK2 partially colocalized with AIRE in nuclear bodies upon cotransfection and in human mTECs in situ. Moreover, HIPK2 phosphorylated AIRE in vitro and suppressed the coactivator activity of AIRE in a kinase-dependent manner. To evaluate the role of Hipk2 in modulating the function of AIRE in vivo, we compared whole-genome gene signatures of purified mTEC subsets from TEC-specific Hipk2 knockout mice with control mice and identified a small set of differentially expressed genes. Unexpectedly, most differentially expressed genes were confined to the CD80(lo) mTEC subset and preferentially included AIRE-independent TRAs. Thus, although it modulates gene expression in mTECs and in addition affects the size of the medullary compartment, TEC-specific HIPK2 deletion only mildly affects AIRE-directed pGE in vivo. PMID:25552543

  7. Assessment of gene expression of intracellular calcium channels, pumps and exchangers with epidermal growth factor-induced epithelial-mesenchymal transition in a breast cancer cell line

    PubMed Central

    2013-01-01

    Background Epithelial-mesenchymal transition (EMT) is a process implicated in cancer metastasis that involves the conversion of epithelial cells to a more mesenchymal and invasive cell phenotype. In breast cancer cells EMT is associated with altered store-operated calcium influx and changes in calcium signalling mediated by activation of cell surface purinergic receptors. In this study, we investigated whether MDA-MB-468 breast cancer cells induced to undergo EMT exhibit changes in mRNA levels of calcium channels, pumps and exchangers located on intracellular calcium storing organelles, including the Golgi, mitochondria and endoplasmic reticulum (ER). Methods Epidermal growth factor (EGF) was used to induce EMT in MDA-MB-468 breast cancer cells. Serum-deprived cells were treated with EGF (50 ng/mL) for 12 h and gene expression was assessed using quantitative RT-PCR. Results and conclusions These data reveal no significant alterations in mRNA levels of the Golgi calcium pump secretory pathway calcium ATPases (SPCA1 and SPCA2), or the mitochondrial calcium uniporter (MCU) or Na+/Ca2+ exchanger (NCLX). However, EGF-induced EMT was associated with significant alterations in mRNA levels of specific ER calcium channels and pumps, including (sarco)-endoplasmic reticulum calcium ATPases (SERCAs), and inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RYR) calcium channel isoforms. The most prominent change in gene expression between the epithelial and mesenchymal-like states was RYR2, which was enriched 45-fold in EGF-treated MDA-MB-468 cells. These findings indicate that EGF-induced EMT in breast cancer cells may be associated with major alterations in ER calcium homeostasis. PMID:23890218

  8. Expression of the DNase encoded by the BGLF5 gene of Epstein-Barr virus in nasopharyngeal carcinoma epithelial cells.

    PubMed

    Sbih-Lammali, F; Berger, F; Busson, P; Ooka, T

    1996-08-01

    In contrast with most Epstein-Barr virus (EBV)-infected healthy carriers, nasopharyngeal carcinoma patients frequently have increased serum levels of antibodies directed against EBV-DNase. These antibodies are potentially interesting serological markers for the diagnosis and the follow-up of nasopharyngeal carcinoma (NPC). In this context, it is important to determine whether malignant EBV-infected cells are the source of significant amounts of EBV-DNase contributing to antigenic stimulation. Therefore EBV-DNase expression has been investigated in several NPC specimens. A significant expression of this viral enzyme was demonstrated in both fresh biopsies and transplanted tumor lines. The DNase isolated from tumor has a molecular weight varying between 52 and 60 kDa and its activity eluted from a single-stranded DNA affinity column was specifically inhibited by both NPC sera and the rabbit polyclonal antibody against EBV-DNase. The enzyme activity was functional in the presence of 300 mM KCl, with which cellular DNases are completely inhibited. The DNase was mainly localized in epithelial tumor cells of both NPC biopsies and nude mice-derived NPC cells. PMID:8806488

  9. HOXA9 is Underexpressed in Cervical Cancer Cells and its Restoration Decreases Proliferation, Migration and Expression of Epithelial-to-Mesenchymal Transition Genes.

    PubMed

    Alvarado-Ruiz, Liliana; Martinez-Silva, Maria Guadalupe; Torres-Reyes, Luis Alberto; Pina-Sanchez, Patricia; Ortiz-Lazareno, Pablo; Bravo-Cuellar, Alejandro; Aguilar-Lemarroy, Adriana; Jave-Suarez, Luis Felipe

    2016-01-01

    HOX transcription factors are evolutionarily conserved in many different species and are involved in important cellular processes such as morphogenesis, differentiation, and proliferation. They have also recently been implicated in carcinogenesis, but their precise role in cancer, especially in cervical cancer (CC), remains unclear. In this work, using microarray assays followed by the quantitative polymerase chain reaction (qPCR), we found that the expression of 25 HOX genes was downregulated in CC derived cell lines compared with nontumorigenic keratinocytes. In particular, the expression of HOXA9 was observed as down-modulated in CCderived cell lines. The expression of HOXA9 has not been previously reported in CC, or in normal keratinocytes of the cervix. We found that normal CC from women without cervical lesions express HOXA9; in contrast, CC cell lines and samples of biopsies from women with CC showed significantly diminished HOXA9 expression. Furthermore, we found that methylation at the first exon of HOXA9 could play an important role in modulating the expression of this gene. Exogenous restoration of HOXA9 expression in CC cell lines decreased cell proliferation and migration, and induced an epithelial-like phenotype. Interestingly, the silencing of human papilloma virus (HPV) E6 and E7 oncogenes induced expression of HOXA9. In conclusion, controlling HOXA9 expression appears to be a necessary step during CC development. Further studies are needed to delineate the role of HOXA9 during malignant progression and to afford more insights into the relationship between downmodulation of HOXA9 and viral HPV oncoprotein expression during cercical cancer development. PMID:27039722

  10. Infection of human airway epithelial cells by different subtypes of Dobrava-Belgrade virus reveals gene expression patterns corresponding to their virulence potential.

    PubMed

    Witkowski, Peter T; Bourquain, Daniel; Bankov, Katrin; Auste, Brita; Dabrowski, Piotr W; Nitsche, Andreas; Krüger, Detlev H; Schaade, Lars

    2016-06-01

    Dobrava-Belgrade virus (DOBV) is a pathogen causing hemorrhagic fever with renal syndrome in Europe. Virulence and case fatality rate are associated with virus genotype; however the reasons for these differences are not well understood. In this work we present virus-specific effects on the gene expression profiles of human lung epithelial cells (A549) infected with different genotypes of DOBV (Dobrava, Kurkino, and Sochi), as well as the low-virulent Tula virus (TULV). The data was collected by whole-genome gene expression microarrays and confirmed by quantitative real-time PCR. Despite their close genetic relationship, the expression profiles induced by infection with different hantaviruses are significantly varying. Major differences were observed in regulation of immune response genes, which were especially induced by highly virulent DOBV genotypes Dobrava and Sochi in contrast to less virulent DOBV-Kurkino and TULV. This work gives first insights into the differences of virus - host interactions of DOBV on genotype level. PMID:27058765

  11. The effect of heat stress on gene expression and synthesis of heat-shock and milk proteins in bovine mammary epithelial cells.

    PubMed

    Hu, Han; Zhang, Yangdong; Zheng, Nan; Cheng, Jianbo; Wang, Jiaqi

    2016-01-01

    In this study, bovine mammary epithelial cells were used to study stress responses after cells were exposed to 42°C for 0.5, 1, 3, 5, 8 or 12 h, and 38°C as control. The transcription of the genes (HSP27, HSP70 and HSP90) of heat shock protein (Hsp) was significantly enhanced under heat stress (HS). The peak transcription of HSP70 was 14 times the control at 1 h. Expression of proteins Hsp27 and Hsp70 was gradually increased under HS, with rapid deposition of Hsp70 in epithelial cells. The major milk protein genes of β-casein (CSN2) and butyrophilin (BTN1A1) were down-regulated and the synthesis of total caseins was decreased. After the cells were under HS (42°C) for 1 or 5 h, the cells were cultured at 38°C for 1, 6, 12 or 24 h for recovery. When the cells were cultured at 38°C for 24 h after HS for 1 h, the transcription of HSP70, HSP90, CSN2 and BTN reached normal levels. Our results suggest that HS initiated Hsp synthesis and decreased the milk protein synthesis. Hsp70 is extremely sensitive to HS and mainly responsible for mammary cell protection from HS. PMID:26467738

  12. Analysis of the coding sequence and expression of the coiled-coil α-helical rod protein 1 gene in normal and neoplastic epithelial cervical cells

    PubMed Central

    PACHOLSKA-BOGALSKA, JOANNA; MYGA-NOWAK, MAGDALENA; CIEPŁUCH, KATARZYNA; JÓZEFIAK, AGATA; KWAŒNIEWSKA, ANNA; GOźDZICKA-JÓZEFIAK, ANNA

    2012-01-01

    The role of the CCHCR1 (coiled-coil α-helical rod protein 1) protein in the cell is poorly understood. It is thought to be engaged in processes such as proliferation and differentiation of epithelial cells, tissue-specific gene transcription and steroidogenesis. It is supposed to participate in keratinocyte transformation. It has also been found that this protein interacts with the E2 protein of human papilloma virus type 16 (HPV16). The oncogenic HPV forms, such as HPV16, are known to be necessary but not sufficient agents in the development of cervical carcinoma. In the present study, the CCHCR1 gene coding sequence and its expression was analyzed in normal, precancerous and cervical cancer cells. Changes in the non-coding region were found in 20.3% of the examined probes from women with cervical cancer or precancerous lesions and in 16.67% of the control probes. Most of the detected changes were single nucleotide polymorphisms (SNPs). Changes in the coding region were found in 22.8% of the probes with cervical cancer and in 16.67% of the control probes and all of them were SNPs. The level of CCHCR1 transcripts was determined using the real-time PCR method and the highest gene expression was detected in the H-SIL group and slightly decreased in the cervical carcinoma cells, compared with the control probes. It suggests that CCHCR1 could have a role in the process of cervical epithelial cell transformation, but this suggestion must be confirmed experimentally. PMID:22218424

  13. Effects of ruminal doses of sucrose, lactose, and corn starch on ruminal fermentation and expression of genes in ruminal epithelial cells.

    PubMed

    Oba, M; Mewis, J L; Zhining, Z

    2015-01-01

    The objective was to evaluate effects of a ruminal dose of sucrose, lactose, and corn starch on ruminal fermentation and expression of genes in ruminal epithelial cells. Six ruminally cannulated nonlactating nonpregnant Holstein cows (body weight=725±69.6kg) were assigned to treatments in a 3×3 Latin square design with 7-d periods; 1d for data and sample collection followed by a 6-d washout period. Cows were fed a diet containing whole-crop barley silage and dry ground corn, and dietary neutral detergent fiber and crude protein contents were 41.8 and 13.2% [dry matter (DM) basis], respectively. Treatment was a pulse-dose of sucrose, lactose, and corn starch (3.0, 3.0, and 2.85kg of DM, respectively; providing similar amounts of hexose across the treatments) through the ruminal cannulas. All treatments were given with alfalfa silage (1.75kg DM) to prevent acute rumen acidosis. Rumen pH was continuously monitored, and rumen fluid was sampled at 0, 30, 60, 90, 120, 150, and 180min after the dose. In addition, ruminal papillae were sampled from the ventral sac at 180min after the dose. Ruminal dosing with sucrose and lactose, compared with corn starch, increased ruminal total volatile fatty acid concentration and molar proportion of butyrate from 60 to 180min after the dose, and expression of genes for sodium hydrogen exchanger isoforms 1 and 2, and ATPase isoform 1 in ruminal epithelial cells. Ruminal dosing with sucrose, compared with lactose and corn starch, decreased rumen pH from 120 to 180min after the dose and molar proportion of acetate in ruminal fluid from 60 to 150min after the dose, and increased molar proportion of propionate in ruminal fluid from 60 to 150min, and expression of genes involved in butyrate metabolism (3-hydroxy-3-methylglutaryl-coenzyme A synthase isoform 1) and anion exchange across ruminal apical cell membrane (putative anion transporter isoform 1). These results suggest that replacing dietary starch with sugars may affect ruminal

  14. Immunohistochemical analyses of cell cycle progression and gene expression of biliary epithelial cells during liver regeneration after partial hepatectomy of the mouse

    PubMed Central

    Fukuda, Tatsuya; Fukuchi, Tomokazu; Yagi, Shinomi; Shiojiri, Nobuyoshi

    2015-01-01

    The liver has a remarkable regeneration capacity, and, after surgical removal of its mass, the remaining tissue undergoes rapid regeneration through compensatory growth of its constituent cells. Although hepatocytes synchronously proliferate under the control of various signaling molecules from neighboring cells, there have been few detailed analyses on how biliary cells regenerate for their cell population after liver resection. The present study was undertaken to clarify how biliary cells regenerate after partial hepatectomy of mice through extensive analyses of their cell cycle progression and gene expression using immunohistochemical and RT-PCR techniques. When expression of PCNA, Ki67 antigen, topoisomerase IIα and phosphorylated histone H3, which are cell cycle markers, was immunohistochemically examined during liver regeneration, hepatocytes had a peak of the S phase and M phase at 48–72 h after resection. By contrast, biliary epithelial cells had much lower proliferative activity than that of hepatocytes, and their peak of the S phase was delayed. Mitotic figures were rarely detectable in biliary cells. RT-PCR analyses of gene expression of biliary markers such as Spp1 (osteopontin), Epcam and Hnf1b demonstrated that they were upregulated during liver regeneration. Periportal hepatocytes expressed some of biliary markers, including Spp1 mRNA and protein. Some periportal hepatocytes had downregulated expression of HNF4α and HNF1α. Gene expression of Notch signaling molecules responsible for cell fate decision of hepatoblasts to biliary cells during development was upregulated during liver regeneration. Notch signaling may be involved in biliary regeneration. PMID:26633692

  15. Downregulation of hepatocyte nuclear factor-4{alpha} and its role in regulation of gene expression by TGF-{beta} in mammary epithelial cells

    SciTech Connect

    Ishikawa, Fumihiro; Nose, Kiyoshi; Shibanuma, Motoko

    2008-06-10

    We found that a specific isoform of hepatocyte nuclear factor 4{alpha} (HNF-4{alpha}), HNF-4{alpha}8, was expressed in mouse mammary epithelial NMuMG cells, and that its expression was repressed by TGF-{beta}. The repression was interfered by dominant negative forms of activin receptor-like kinase 5 (ALK5) and Smad3, and sensitive to cycloheximide, suggesting the involvement of additional protein(s) as well as ALK5 and Smad3 in the repression. Further study showed that high mobility group A2 (HMGA2), which is reported to be directly upregulated by Smads, repressed HNF-4{alpha}8 expression. Therefore, it is likely that HMGA2 mediates the downregulation of HNF-4{alpha}8 downstream of ALK5 and Smads To determine the significance of the downregulation of HNF-4{alpha}8 in TGF-{beta} signaling, we performed DNA microarray analysis and extracted a subgroup of TGF-{beta}1-regulated genes, including tenascin C and tissue inhibitor of metalloproteinase 3 (TIMP-3), whose regulation by TGF-{beta}1 was attenuated by forced expression of HNF-4{alpha}8. HMGA2 has recently emerged as a transcriptional organizer of TGF-{beta} signaling, regulating several key factors involved in epithelial-mesenchymal transition (EMT). In this study, we identified an isoform of HNF-4{alpha} as a new target downstream of HMGA2 and assigned a new role to HNF-4{alpha} in the TGF-{beta} signaling/transcriptional cascade driven by ALK5/Smad/HMGA2 and associated with the malignant transformation of cells.

  16. Abundance of ruminal bacteria, epithelial gene expression, and systemic biomarkers of metabolism and inflammation are altered during the peripartal period in dairy cows.

    PubMed

    Minuti, A; Palladino, A; Khan, M J; Alqarni, S; Agrawal, A; Piccioli-Capelli, F; Hidalgo, F; Cardoso, F C; Trevisi, E; Loor, J J

    2015-12-01

    Seven multiparous Holstein cows with a ruminal fistula were used to investigate the changes in rumen microbiota, gene expression of the ruminal epithelium, and blood biomarkers of metabolism and inflammation during the transition period. Samples of ruminal digesta, biopsies of ruminal epithelium, and blood were obtained during -14 through 28d in milk (DIM). A total of 35 genes associated with metabolism, transport, inflammation, and signaling were evaluated by quantitative reverse transcription-PCR. Among metabolic-related genes, expression of HMGCS2 increased gradually from -14 to a peak at 28 DIM, underscoring its central role in epithelial ketogenesis. The decrease of glucose and the increase of nonesterified fatty acids and β-hydroxybutyrate in the blood after calving confirmed the state of negative energy balance. Similarly, increases in bilirubin and decreases in albumin concentrations after calving were indicative of alterations in liver function and inflammation. Despite those systemic signs, lower postpartal expression of TLR2, TLR4, CD45, and NFKB1 indicated the absence of inflammation within the epithelium. Alternatively, these could reflect an adaptation to react against inducers of the immune system arising in the rumen (e.g., bacterial endotoxins). The downregulation of RXRA, INSR, and RPS6KB1 between -14 and 10 DIM indicated a possible increase in insulin resistance. However, the upregulation of IRS1 during the same time frame could serve to restore sensitivity to insulin of the epithelium as a way to preserve its proliferative capacity. The upregulation of TGFB1 from -14 and 10 DIM coupled with upregulation of both EGFR and EREG from 10 to 28 DIM indicated the existence of 2 waves of epithelial proliferation. However, the downregulation of TGFBR1 from -14 through 28 DIM indicated some degree of cell proliferation arrest. The downregulation of OCLN and TJP1 from -14 to 10 DIM indicated a loss of tight-junction integrity. The gradual upregulation of

  17. Expression analysis of Matrix Metalloproteinase-9 in epithelialized and non-epithelialized apical periodontitis lesions

    PubMed Central

    Carneiro, Everdan; Menezes, Renato; Garlet, Gustavo Pompermaier; Garcia, Roberto Brandão; Bramante, Clóvis Monteiro; Figueira, Rita; Sogayar, Mari; Granjeiro, José Mauro

    2009-01-01

    OBJECTIVE To determine the expression of matrix metalloproteinase-9 (MMP-9) in apical periodontitis lesions. STUDY DESIGN Nineteen epithelialized and eighteen non-epithelialized apical periodontitis lesions were collected after periapical surgery. After histological processing, serial sectioning, H&E staining and microscopic analysis, 10 epithelialized and 10 non-epithelialized lesions were selected for immunohistochemical analysis for MMP-9 and CD 68. At least 1/3 of each specimen was frozen at −70°C for further mRNA isolation and reverse transcription into cDNA for Real-Time-PCR procedures. The relative expression of a target gene was determined in comparison with reference genes (GAPDH, HPRT, β-actin and BCRP). RESULTS Polymorphonuclear neutrophils, macrophages and lymphocytes were stained for MMP-9 in both types of lesions, and when present, epithelial cells were also stained. The number and the ratio of MMP-9+/total cells were greater in non-epithelialized than epithelialized lesions (p=0.0001) and showed a positive correlation to CD68+/total cells (p=0.045). No significant differences were observed for MMP-9 mRNA expression between ephithelized and non-ephithelized lesions. However, when compared to healthy periapical ligaments, both types of lesions presented increased MMP-9 expression (p<0.0001). CONCLUSION The present data suggest the participation of several inflammatory cells, mainlly CD68+ cells, in the MMP-9 expression in apical periodontitis lesions. MMP-9 could be actively enroled in the ECM degradation in apical periodontitis lesions. PMID:18926740

  18. Wild-type and IL10-null mice have differential colonic epithelial gene expression responses to dietary supplementation with synbiotic Bifidobacterium animalis subspecies lactis and inulin.

    PubMed

    Kuo, Shiu-Ming; Chan, Wan-Chun; Hu, Zihua

    2014-03-01

    Prebiotic plus probiotic (synbiotic) supplementations promote fermentation and have shown anti-inflammatory activity in colonic epithelium. However, in many instances, patients with inflammatory bowel disease (IBD) have demonstrated adverse effects after prebiotic supplementation at a dose well tolerated by normal individuals. To test the hypothesis that the host inflammation affects the colonic epithelial response to increased fermentation, the gene expression of colonic epithelium was analyzed. In a 1-way experimental design to test the effect of supplements in wild-type mice using the standard diet formulated by the American Institute of Nutrition (AIN-93G) as the control diet, fermentable fiber inulin (5%) in the absence or presence of the probiotic Bifidobacterium animalis subspecies lactis (Bb12) (10(8) CFU/kg diet) showed limited effects on gene expression as determined by whole-genome microarray. Bb12 supplementation alone was known not to increase fermentation and here instead significantly upregulated genes in nucleic acid metabolic processes. The effects of the synbiotic diet were then determined in mice exposed to LPS-induced inflammation in a 2-way experimental design testing the effect of diet and LPS. The microarray and quantitative reverse transcription-polymerase chain reaction analyses on the wild-type mice revealed that LPS-induced changes in the colonic epithelium were 4- to 10-fold less in the synbiotic diet group compared with the control diet group. Unlike the wild-type mice, anti-inflammatory cytokine interleukin 10 (IL10)-null mice (susceptible to IBD) given the synbiotic diet, compared with those given the control diet, had 3- to 40-fold increased expression of inflammation-related genes such as Cxcl1 (chemokine C-X-C motif ligand 1) and S100a9 (S100 calcium binding protein A9) in the absence and presence of LPS exposure. These contrasting intestinal epithelial responses to increased fermentation in wild-type and IL10-null mice are similar

  19. Operator Dependent Choice of Prostate Cancer Biopsy Has Limited Impact on a Gene Signature Analysis for the Highly Expressed Genes IGFBP3 and F3 in Prostate Cancer Epithelial Cells

    PubMed Central

    Peng, Zhuochun; Andersson, Karl; Lindholm, Johan; Bodin, Inger; Pramana, Setia; Pawitan, Yudi; Nistér, Monica; Nilsson, Sten; Li, Chunde

    2014-01-01

    Background Predicting the prognosis of prostate cancer disease through gene expression analysis is receiving increasing interest. In many cases, such analyses are based on formalin-fixed, paraffin embedded (FFPE) core needle biopsy material on which Gleason grading for diagnosis has been conducted. Since each patient typically has multiple biopsy samples, and since Gleason grading is an operator dependent procedure known to be difficult, the impact of the operator's choice of biopsy was evaluated. Methods Multiple biopsy samples from 43 patients were evaluated using a previously reported gene signature of IGFBP3, F3 and VGLL3 with potential prognostic value in estimating overall survival at diagnosis of prostate cancer. A four multiplex one-step qRT-PCR test kit, designed and optimized for measuring the signature in FFPE core needle biopsy samples was used. Concordance of gene expression levels between primary and secondary Gleason tumor patterns, as well as benign tissue specimens, was analyzed. Results The gene expression levels of IGFBP3 and F3 in prostate cancer epithelial cell-containing tissue representing the primary and secondary Gleason patterns were high and consistent, while the low expressed VGLL3 showed more variation in its expression levels. Conclusion The assessment of IGFBP3 and F3 gene expression levels in prostate cancer tissue is independent of Gleason patterns, meaning that the impact of operator's choice of biopsy is low. PMID:25296164

  20. Deep sequencing-based analysis of gene expression in bovine mammary epithelial cells after Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae infection.

    PubMed

    Xiu, L; Fu, Y B; Deng, Y; Shi, X J; Bian, Z Y; Ruhan, A; Wang, X

    2015-01-01

    The goal of this study was to characterize the transcriptome of primary bovine mammalian epithelial cells (pBMECs) and to identify candidate genes for response and resistance to Staphylococcus aureus (strain S108), Escherichia coli (strain E23), and Klebsiella pneumoniae (strain K96) infection. Using Solexa sequencing, approximately 4.9 million total sequence tags were obtained from each of the three infected libraries and the control library. Gene Ontology (GO) analysis of the S108-infected pBMECs showed differentially expressed genes (DEGs) were significantly involved in metabolic processes. In E23-infected pBMECs, DEGs were predominantly associated with cell death and programmed cell death GO terms, while in K96-infected pBMECs, DEGs were primarily involved in metabolic processes and in utero embryonic development. Analysis of the cluster of orthologous groups of proteins showed that the S108-infected, E23-infected and K96-infected pBMECs were significantly involved in "Translation, ribosomal structure and biogenesis", "General function prediction only" and "Replication, recombination and repair". The transcriptome sequences were also annotated for KEGG orthology, and it was found that DEGs in S108-infected pBMECs were significantly involved in oxidative phosphorylation and Parkinson's disease. The clustered pathway terms of the DEGs of the E23-infected pBMECs were found to involve the NOD-like receptor signaling pathway and oxidative phosphorylation, while those of the K96-infected pBMECs were primarily involved in oxidative phosphorylation and apoptosis. Our results have identified a number of immune-related genes that showed changes in gene expression after bacterial infection, and provided insight into the interactions between pBMECs and the bacteria. PMID:26681042

  1. Testing gene therapy vectors in human primary nasal epithelial cultures

    PubMed Central

    Cao, Huibi; Ouyang, Hong; Ip, Wan; Du, Kai; Duan, Wenming; Avolio, Julie; Wu, Jing; Duan, Cathleen; Yeger, Herman; Bear, Christine E; Gonska, Tanja; Hu, Jim; Moraes, Theo J

    2015-01-01

    Cystic fibrosis (CF) results from mutations in the CF transmembrane conductance regulator (CFTR) gene, which codes for a chloride/bicarbonate channel in the apical epithelial membranes. CFTR dysfunction results in a multisystem disease including the development of life limiting lung disease. The possibility of a cure for CF by replacing defective CFTR has led to different approaches for CF gene therapy; all of which ultimately have to be tested in preclinical model systems. Primary human nasal epithelial cultures (HNECs) derived from nasal turbinate brushing were used to test the efficiency of a helper-dependent adenoviral (HD-Ad) vector expressing CFTR. HD-Ad-CFTR transduction resulted in functional expression of CFTR at the apical membrane in nasal epithelial cells obtained from CF patients. These results suggest that HNECs can be used for preclinical testing of gene therapy vectors in CF. PMID:26730394

  2. Testing gene therapy vectors in human primary nasal epithelial cultures.

    PubMed

    Cao, Huibi; Ouyang, Hong; Ip, Wan; Du, Kai; Duan, Wenming; Avolio, Julie; Wu, Jing; Duan, Cathleen; Yeger, Herman; Bear, Christine E; Gonska, Tanja; Hu, Jim; Moraes, Theo J

    2015-01-01

    Cystic fibrosis (CF) results from mutations in the CF transmembrane conductance regulator (CFTR) gene, which codes for a chloride/bicarbonate channel in the apical epithelial membranes. CFTR dysfunction results in a multisystem disease including the development of life limiting lung disease. The possibility of a cure for CF by replacing defective CFTR has led to different approaches for CF gene therapy; all of which ultimately have to be tested in preclinical model systems. Primary human nasal epithelial cultures (HNECs) derived from nasal turbinate brushing were used to test the efficiency of a helper-dependent adenoviral (HD-Ad) vector expressing CFTR. HD-Ad-CFTR transduction resulted in functional expression of CFTR at the apical membrane in nasal epithelial cells obtained from CF patients. These results suggest that HNECs can be used for preclinical testing of gene therapy vectors in CF. PMID:26730394

  3. Basal and copper-induced expression of metallothionein isoform 1,2 and 3 genes in epithelial cancer cells: The role of tumor suppressor p53.

    PubMed

    Ostrakhovitch, E A; Song, Y P; Cherian, M G

    2016-05-01

    Metallothioneins (MTs) are a ubiquitous low-molecular weight, cysteine rich proteins with a high affinity for metal ions. The expression and induction of MTs have been associated with protection against DNA damage, oxidative stress, and apoptosis. Our past research had shown that p53 is an important factor in metal regulation of MTs. The present study was undertaken to explore further the interrelationship between p53 and MTs. We investigated whether silencing of p53 could affect expression pattern of basal and copper induced metallothioneins. The silencing of wild-type p53 (wt-p53) in epithelial breast cancer MCF7 cells affected the basal level of MT-2A RNA, whereas the levels of MT-1A and MT-1X RNA remained largely unchanged. The expression of MT-3 was undetectable in MCF7 with either functional or silenced p53. MCF7 cells with silenced wt-p53 failed to upregulate MT-2A in response to copper and showed a reduced sensitivity toward copper induced cell apoptotic death. Similarly in MCF7-E6 and MDA-MB-231 cells, the presence of inactive/mutated p53 halted MT-1A and MT-2A gene expression in response to copper. Constitutive expression of MT-3 RNA was detectable in the presence of mutated p53 (mtp53). Transient transfection of MDA-MB-231 cells with wt-p53 enabled copper induced upregulation of both MT-1A and MT-2A but not basal level of MT-2A, MT-1E, MT-1X and MT-3. Inactivation of p53 in HepG2 cells amplified the basal expression of studied MT isoforms, including MT-3, as well as copper-induced mRNA expression of MTs except MT-1H and MT-3. Presented data demonstrate a direct relation between p53 and MT-1A and MT-2A and they also indicate that wt-p53 might be a negative regulator of MT-3 in epithelial cancer cells. PMID:27049123

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

    PubMed

    Zheng, Yue-Mao; He, Xiao-Ying

    2010-12-01

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

  5. Downregulation of gangliotetraosylceramide and β1,3-galactosyltransferase-4 gene expression by Smads during transforming growth factor β-induced epithelial-mesenchymal transition.

    PubMed

    Guo, Jia; Song, Bo; Li, Xiang; Hε, Chenhui; Yang, Ganglong; Yang, Xiaomin; Guan, Feng

    2015-03-01

    The process of epithelial‑mesenchymal transition (EMT) is essential for the proliferation and metastasis of tumor cells. Our previous study demonstrated that the expression of gangliotetraosylceramide (Gg4) and the transcription of UDP‑Gal: β1,3-galactosyltransferase‑4 (β3GalT4), a gene which controls the expression of Gg4, are reduced during transforming growth factor (TGF)‑β‑induced EMT in normal murine mammary gland (NMuMG) cells. The present study revealed that the transcription level of β3GalT4 in patients with breast cancer was decreased compared with healthy control subjects. However, the molecular basis underlying these effects remains to be elucidated. Analysis of the β3GalT4 promoter sequence revealed a putative Smad‑binding element (SBE) of Smad4, which is a transcriptional factor in TGFβ responses and forms a complex with Smad3. To clarify the association between the Smad3/4 complex and the β3GalT4 gene during EMT in NMuMG cells, an electrophoretic mobility shift assay and a chromatin immunoprecipitation assay were performed. The Smad3/4 complex was found to bind directly to the SBE of the β3GalT4 promoter. Overexpression of Smad3 and Smad4 through stable transfection had no notable effect on cell phenotype, but did alter the expression of various EMT protein markers. Treatment with TGFβ reduced the expression of Gg4 and the mRNA levels of the β3GalT4 gene in Smad3‑ and Smad4‑overexpressing cells compared with vector‑transfected cells. Expression of the epithelial markers E‑cadherin and β‑catenin decreased in parallel with the reduction in Gg4. These findings suggested that the activated Smad3/4 complex downregulated the expression of Gg4 and the β3GalT4 gene through translocation into the nucleus and binding to the β3GalT4 promoter. PMID:25384497

  6. Amelotin gene expression is temporarily being upregulated at the initiation of apoptosis induced by TGFβ1 in mouse gingival epithelial cells.

    PubMed

    Nakayama, Yohei; Matsui, Sari; Noda, Keisuke; Yamazaki, Mizuho; Iwai, Yasunobu; Matsumura, Hiroyoshi; Izawa, Takashi; Tanaka, Eiji; Ganss, Bernhard; Ogata, Yorimasa

    2016-10-01

    Amelotin (AMTN) is expressed and secreted by ameloblasts in the maturation stage of amelogenesis and persist with low levels in the junctional epithelium (JE) of erupted teeth. The purpose of this study is to investigate the transcriptional regulation of the AMTN gene by transforming growth factor beta1 (TGFβ1) in gingival epithelial (GE1) cells in the apoptosis phase. Apoptosis was evaluated by the fragmentation of chromosomal DNA and TUNEL staining. A real-time PCR was carried out to examine the AMTN mRNA levels induced by TGFβ1 and Smad3 overexpression. Transient transfection analyses were completed using the various lengths of mouse AMTN gene promoter constructs with or without TGFβ1. Chromatin immunoprecipitation (ChIP) assays were performed to investigate the Smad3 bindings to the AMTN gene promoter by TGFβ1. TGFβ1-induced apoptosis in GE1 cells were detected at 24 and 48 h by DNA fragmentation and TUNEL staining. AMTN mRNA levels increased at 6 h and reached maximum at 24 h in GE1 cells. Luciferase activities of the mouse AMTN gene promoter constructs were induced by TGFβ1. The results of the ChIP assays showed that there was an increase in Smad3 binding to Smad-binding element (SBE)#1 and SBE#2 after stimulation by TGFβ1. Immunohistochemical localization of AMTN was detected in the JE, and the AMTN protein levels in Smad3-deficient mice were decreased compared with wild-type mice. AMTN mRNA levels were induced at the initiation of apoptosis by TGFβ1, which mediated through the Smad3 bindings to SBEs in the mouse AMTN gene promoter. PMID:27502207

  7. RNA interference-mediated knockdown of CD49e (α5 integrin chain) in human thymic epithelial cells modulates the expression of multiple genes and decreases thymocyte adhesion

    PubMed Central

    2010-01-01

    Background The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation. Developing thymocytes interact with thymic microenvironment in a defined spatial order. A component of thymic microenvironment, the thymic epithelial cells, is crucial for the maturation of T-lymphocytes through cell-cell contact, cell matrix interactions and secretory of cytokines/chemokines. There is evidence that extracellular matrix molecules play a fundamental role in guiding differentiating thymocytes in both cortical and medullary regions of the thymic lobules. The interaction between the integrin α5β1 (CD49e/CD29; VLA-5) and fibronectin is relevant for thymocyte adhesion and migration within the thymic tissue. Our previous results have shown that adhesion of thymocytes to cultured TEC line is enhanced in the presence of fibronectin, and can be blocked with anti-VLA-5 antibody. Results Herein, we studied the role of CD49e expressed by the human thymic epithelium. For this purpose we knocked down the CD49e by means of RNA interference. This procedure resulted in the modulation of more than 100 genes, some of them coding for other proteins also involved in adhesion of thymocytes; others related to signaling pathways triggered after integrin activation, or even involved in the control of F-actin stress fiber formation. Functionally, we demonstrated that disruption of VLA-5 in human TEC by CD49e-siRNA-induced gene knockdown decreased the ability of TEC to promote thymocyte adhesion. Such a decrease comprised all CD4/CD8-defined thymocyte subsets. Conclusion Conceptually, our findings unravel the complexity of gene regulation, as regards key genes involved in the heterocellular cell adhesion between developing thymocytes and the major component of the thymic microenvironment, an interaction that is a mandatory event for proper intrathymic T cell differentiation. PMID:21210968

  8. Lutein and zeaxanthin supplementation reduces photo-oxidative damage and modulates the expression of inflammation-related genes in retinal pigment epithelial cells

    PubMed Central

    Bian, Qingning; Gao, Shasha; Zhou, Jilin; Qin, Jian; Taylor, Allen; Johnson, Elizabeth J.; Tang, Guangwen; Sparrow, Janet R.; Gierhart, Dennis; Shang, Fu

    2012-01-01

    Oxidative damage and inflammation are related to the pathogenesis of age-related macular degeneration (AMD). Epidemiologic studies suggest that insufficient dietary lutein and zeaxanthin intake or lower serum zeaxanthin levels are associated with increased risk for AMD. The objective of this work is to test the protective effects of lutein and zeaxanthin against photo-oxidative damage to retinal pigment epithelial cells (RPE) and oxidation-induced changes in expression of inflammation-related genes. To mimic lipofuscin-mediated photo-oxidation in vivo, we used ARPE-19 cells that accumulated A2E, a lipofuscin fluorophore and photosensitizer, as a model system to investigate the effects of lutein and zeaxanthin supplementation. The data show that supplementation with lutein or zeaxanthin in the medium resulted in accumulation of lutein or zeaxanthin in the RPE cells. The concentrations of lutein and zeaxanthin in the cells were 2–14-fold of that detected in the medium, indicating that ARPE-19 cells actively take up lutein or zeaxanthin. As compared with untreated cells, exposure of A2E-containing RPE to blue light resulted in a 40–60% decrease in proteasome activity, a 50–80% decrease in expression of CFH and MCP-1, and an ~ 20-fold increase in expression of IL-8. The photo-oxidation-induced changes in expression of MCP-1, IL-8 and CFH were similar to those caused by chemical inhibition of the proteasome, suggesting that inactivation of the proteasome is involved in the photo-oxidation-induced alteration in expression of these inflammation-related genes. Incubation of the A2E-containing RPE with lutein or zeaxanthin prior to blue light exposure significantly attenuated the photo-oxidation-induced inactivation of the proteasome and photo-oxidation induced changes in expression of MCP-1, IL-8, and CFH. Together, these data indicate that lutein or zeaxanthin modulates inflammatory responses in cultured RPE in response to photo-oxidation. Protecting the proteasome

  9. Sp3 regulates fas expression in lung epithelial cells.

    PubMed Central

    Pang, H; Miranda, K; Fine, A

    1998-01-01

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

  10. Transcriptome sequencing reveals e-cigarette vapor and mainstream-smoke from tobacco cigarettes activate different gene expression profiles in human bronchial epithelial cells.

    PubMed

    Shen, Yifei; Wolkowicz, Michael J; Kotova, Tatyana; Fan, Lonjiang; Timko, Michael P

    2016-01-01

    Electronic cigarettes (e-cigarettes) generate an aerosol vapor (e-vapor) thought to represent a less risky alternative to main stream smoke (MSS) of conventional tobacco cigarettes. RNA-seq analysis was used to examine the transcriptomes of differentiated human bronchial epithelial (HBE) cells exposed to air, MSS from 1R5F tobacco reference cigarettes, and e-vapor with and without added nicotine in an in vitro air-liquid interface model for cellular exposure. Our results indicate that while e-vapor does not elicit many of the cell toxicity responses observed in MSS-exposed HBE cells, e-vapor exposure is not benign, but elicits discrete transcriptomic signatures with and without added nicotine. Among the cellular pathways with the most significantly enriched gene expression following e-vapor exposure are the phospholipid and fatty acid triacylglycerol metabolism pathways. Our data suggest that alterations in cellular glycerophopholipid biosynthesis are an important consequences of e-vapor exposure. Moreover, the presence of nicotine in e-vapor elicits a cellular response distinct from e-vapor alone including alterations of cytochrome P450 function, retinoid metabolism, and nicotine catabolism. These studies establish a baseline for future analysis of e-vapor and e-vapor additives that will better inform the FDA and other governmental bodies in discussions of the risks and future regulation of these products. PMID:27041137

  11. Transcriptome sequencing reveals e-cigarette vapor and mainstream-smoke from tobacco cigarettes activate different gene expression profiles in human bronchial epithelial cells

    PubMed Central

    Shen, Yifei; Wolkowicz, Michael J.; Kotova, Tatyana; Fan, Lonjiang; Timko, Michael P.

    2016-01-01

    Electronic cigarettes (e-cigarettes) generate an aerosol vapor (e-vapor) thought to represent a less risky alternative to main stream smoke (MSS) of conventional tobacco cigarettes. RNA-seq analysis was used to examine the transcriptomes of differentiated human bronchial epithelial (HBE) cells exposed to air, MSS from 1R5F tobacco reference cigarettes, and e-vapor with and without added nicotine in an in vitro air-liquid interface model for cellular exposure. Our results indicate that while e-vapor does not elicit many of the cell toxicity responses observed in MSS-exposed HBE cells, e-vapor exposure is not benign, but elicits discrete transcriptomic signatures with and without added nicotine. Among the cellular pathways with the most significantly enriched gene expression following e-vapor exposure are the phospholipid and fatty acid triacylglycerol metabolism pathways. Our data suggest that alterations in cellular glycerophopholipid biosynthesis are an important consequences of e-vapor exposure. Moreover, the presence of nicotine in e-vapor elicits a cellular response distinct from e-vapor alone including alterations of cytochrome P450 function, retinoid metabolism, and nicotine catabolism. These studies establish a baseline for future analysis of e-vapor and e-vapor additives that will better inform the FDA and other governmental bodies in discussions of the risks and future regulation of these products. PMID:27041137

  12. Gene expression profiles of bovine mammary epithelial cells and association with milk composition traits using RNA-seq

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In most recent years, RNA Sequencing is rapidly emerging as the major quantitative transcriptome profiling system. Elucidation of the bovine mammary gland transcriptome with RNA-seq is essential for identifying candidate genes for milk composition traits in dairy cattle. Here we used massive paralle...

  13. Transporter gene expression in lactating and nonlactating human mammary epithelial cells using real-time reverse transcription-polymerase chain reaction.

    PubMed

    Alcorn, J; Lu, X; Moscow, J A; McNamara, P J

    2002-11-01

    Transporter-mediated processes in the lactating mammary gland may explain the significant accumulation of certain drugs in breast milk. The purpose of this study was to identify potential candidate drug transport proteins involved in drug accumulation in milk. Quantitative reverse transcription-polymerase chain reaction methods were developed to determine the relative RNA levels of 30 different drug transporter genes. Transporter gene RNA levels in lactating mammary epithelial cells (MEC) purified from pooled fresh breast milk samples were compared with levels in nonlactating MEC, liver, and kidney tissue. Transcripts were detected in lactating MEC for OCT1, OCT3, OCTN1, OCTN2, OATP-A, OATP-B, OATP-D, OATP-E, MRP1, MRP2, MRP5, MDR1, CNT1, CNT3, ENT1, ENT3, NCBT1, PEPT1, and PEPT2. No transcripts were detected for OCT2, OAT1, OAT2, OAT3, OAT4, OATP-C, MRP3, MRP4, CNT2, ENT2, and NCBT2. Lactating MEC demonstrated more than 4-fold higher RNA levels of OCT1, OCTN1, PEPT2, CNT1, CNT3, and ENT3, and more than 4-fold lower RNA levels of MDR1 and OCTN2 relative to nonlactating MEC. Lactating MEC showed significantly higher RNA levels of CNT3 relative to liver and kidney, increased PEPT2 RNA levels relative to liver, and increased OATP-A RNA levels relative to kidney. These data imply CNT3 may play a specialized role in nucleoside accumulation in milk and may identify an important role for PEPT2 and OATP-A transporters at the lactating mammary epithelium. Furthermore, transporters expressed in lactating MEC identify a potential role for these transporters in drug disposition at the mammary gland. PMID:12388627

  14. Moderate folate depletion modulates the expression of selected genes involved in cell cycle, intracellular signaling, and folate uptake in human colonic epithelial cell lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Folate deficiency may affect gene expression by disrupting DNA methylation patterns or by inducing base substitution, DNA breaks, gene deletions and gene amplification. Changes in expression may explain the inverse relationship observed between folate status and risk of colorectal cancer. Three cell...

  15. Effect of the ratios of unsaturated fatty acids on the expressions of genes related to fat and protein in the bovine mammary epithelial cells.

    PubMed

    Sheng, R; Yan, S M; Qi, L Z; Zhao, Y L

    2015-04-01

    The objective of this study was to evaluate the effects of the different ratios of unsaturated fatty acids (UFAs) (oleic acid, linoleic acid, and linolenic acid) on the cell viability and triacylglycerol (TAG) content, as well as the mRNA expression of the genes related to lipid and protein synthesis in bovine mammary epithelial cells (BMECs). Primary cells were isolated from the mammary glands of Holstein dairy cows and were passaged twice. Afterward, the cells were randomly allocated to six treatments, five UFA-treated groups, and one control group. For all of the treatments, the the fetal bovine serum in the culture solution was replaced with fatty acid-free BSA (1 g/L), and the cells were treated with different ratios of oleic, linoleic, and linolenic acids (0.75:4:1, 1.5:10:1, 2:13.3:1, 3:20:1, and 4:26.7:1) for 48 h, which were group 1 to group 5. The control culture solution contained only fatty acid-free BSA without UFAs (0 μM). The results indicated that the cell viability was not affected by adding different ratios of UFAs, but the accumulation of TAG was significantly influenced by supplementing with different ratios of UFAs. Adding different ratios of UFAs suppressed the expression of ACACA and FASN but had the opposite effect on the abundances of FABP3 and CD36 mRNA. The expression levels of PPARG, SPEBF1, CSN1S1, and CSN3 mRNA in the BMECs were affected significantly after adding different ratios of UFAs. Our results suggested that groups 1, 2, and 3 (0.75:4:1, 1.5:10:1, and 2:13.3:1) had stronger auxo-action on fat synthesis in the BMECs, where group 3 (2:13.3:1) was the best, followed by group 4 (3:20:1). However, group 5 (4:26.7:1) was the worst. Genes related to protein synthesis in the BMECs were better promoted in groups 2 and 3, and group 3 had the strongest auxo-action, whereas the present study only partly examined the regulation of protein synthesis at the transcriptional level; more studies on translation level are needed in the future

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

    PubMed

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

    2010-12-01

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

  17. Differential Effect of Lactobacillus johnsonii BFE 6128 on Expression of Genes Related to TLR Pathways and Innate Immunity in Intestinal Epithelial Cells.

    PubMed

    Seifert, Stephanie; Rodriguez Gómez, Manuel; Watzl, Bernhard; Holzapfel, Wilhelm H; Franz, Charles M A P; Vizoso Pinto, María G

    2010-12-01

    Probiotics have been shown to enhance immune defenses, but their mechanisms of action are only partially understood. We investigated the modulation of signal pathways involved in innate immunity in enterocytes by Lactobacillus johnsonii BFE 6128 isolated from 'Kule naoto', a Maasai traditional fermented milk product. This lactobacillus sensitized HT29 intestinal epithelial cells toward recognition of Salmonella enterica serovar Typhimurium by increasing the IL-8 levels released after challenge with this pathogen and by differentially modulating genes related to toll-like receptor (TLR) pathways and innate immunity. Thus, the modulation of pro-inflammatory mediators and TLR-pathway-related molecules may be an important mechanism contributing to the potential stimulation of innate immunity by lactobacilli at the intestinal epithelial level. PMID:26781315

  18. High-grain feeding causes strong shifts in ruminal epithelial bacterial community and expression of Toll-like receptor genes in goats

    PubMed Central

    Liu, Jun-hua; Bian, Gao-rui; Zhu, Wei-yun; Mao, Sheng-yong

    2015-01-01

    High-grain (HG) feeding used in intensive goat production can affect the physiology of the rumen wall, but the changes induced in the epimural bacterial community and host Toll-like receptors (TLRs) are not well understood. In this study, 10 male goats were randomly allocated to two groups and fed either a hay diet (0% grain; n = 5) or an HG diet (65% grain; n = 5). The changes in the ruminal epithelial bacterial community and expression of TLRs during long-term (7 weeks) HG feeding were determined using pyrosequencing and quantitative real-time polymerase chain reaction. Principal coordinate analysis and analysis of molecular variance (AMOVA) results showed that HG feeding caused a strong shift in bacterial composition and structure. At the genus level, our data revealed that it increased the relative abundance of taxa Butyrivibrio, unclassified Clostridiales, Mogibacterium, unclassified Anaerolineaceae, and Succiniclasticum, and decreased the proportion of unclassified Ruminococcaceae, unclassified Rikenellaceae, unclassified Erysipelotrichaceae, Howardella, and unclassified Neisseriaceae. The HG-fed goats also exhibited upregulation of the relative mRNA expression of TLR2, TLR3, and TLR5 in the rumen epithelium (P < 0.05). Correlation analysis revealed that the increase in TLR expression was associated with changes in the relative abundance of ruminal epithelial bacteria. This study provides a first insight into the adaptive response of ruminal epithelial bacterial populations to HG feeding in goats and shows that these changes were associated with alterations in TLR expression. These findings provide new insight into understanding of host–microbial relationships in ruminants. PMID:25784904

  19. High-grain feeding causes strong shifts in ruminal epithelial bacterial community and expression of Toll-like receptor genes in goats.

    PubMed

    Liu, Jun-Hua; Bian, Gao-Rui; Zhu, Wei-Yun; Mao, Sheng-Yong

    2015-01-01

    High-grain (HG) feeding used in intensive goat production can affect the physiology of the rumen wall, but the changes induced in the epimural bacterial community and host Toll-like receptors (TLRs) are not well understood. In this study, 10 male goats were randomly allocated to two groups and fed either a hay diet (0% grain; n = 5) or an HG diet (65% grain; n = 5). The changes in the ruminal epithelial bacterial community and expression of TLRs during long-term (7 weeks) HG feeding were determined using pyrosequencing and quantitative real-time polymerase chain reaction. Principal coordinate analysis and analysis of molecular variance (AMOVA) results showed that HG feeding caused a strong shift in bacterial composition and structure. At the genus level, our data revealed that it increased the relative abundance of taxa Butyrivibrio, unclassified Clostridiales, Mogibacterium, unclassified Anaerolineaceae, and Succiniclasticum, and decreased the proportion of unclassified Ruminococcaceae, unclassified Rikenellaceae, unclassified Erysipelotrichaceae, Howardella, and unclassified Neisseriaceae. The HG-fed goats also exhibited upregulation of the relative mRNA expression of TLR2, TLR3, and TLR5 in the rumen epithelium (P < 0.05). Correlation analysis revealed that the increase in TLR expression was associated with changes in the relative abundance of ruminal epithelial bacteria. This study provides a first insight into the adaptive response of ruminal epithelial bacterial populations to HG feeding in goats and shows that these changes were associated with alterations in TLR expression. These findings provide new insight into understanding of host-microbial relationships in ruminants. PMID:25784904

  20. Transient transfection of polarized epithelial monolayers with CFTR and reporter genes using efficacious lipids.

    PubMed

    Tucker, Torry A; Varga, Karoly; Bebok, Zsuzsa; Zsembery, Akos; McCarty, Nael A; Collawn, James F; Schwiebert, Erik M; Schwiebert, Lisa M

    2003-03-01

    Transient transfection of epithelial cells with lipid reagents has been limited because of toxicity and lack of efficacy. In this study, we show that more recently developed lipids transfect nonpolarized human airway epithelial cells with high efficacy and efficiency and little or no toxicity. Because of this success, we hypothesized that these lipids may also allow transient transfection of polarized epithelial monolayers. A panel of reagents was tested for transfer of the reporter gene luciferase (LUC) into polarized monolayers of non-cystic fibrosis (non-CF) and CF human bronchial epithelial cells, MDCK epithelial cell monolayers, and, ultimately, primary non-CF and CF airway epithelial cells. Lipid reagents, which were most successful in initial LUC assays, were also tested for transfer of vectors bearing the reporter gene green fluorescent protein (GFP) and for successful transfection and expression of an epithelial-specific protein, the cystic fibrosis transmembrane conductance regulator (CFTR). Electrophysiological, biochemical, and immunological assays were performed to show successful complementation of an epithelial monolayer with transiently expressed CFTR. We also present findings that help facilitate monolayer formation by these airway epithelial cell lines. Together, these data show that polarized monolayers are transfected transiently with more recently developed lipids, specifically LipofectAMINE PLUS and LipofectAMINE 2000. Transient transfection of epithelial monolayers provides a powerful system in which to express the cDNA of any epithelium-specific protein transiently in a native polarized epithelium to study protein function. PMID:12421695

  1. Dynamic expression of Six family genes in the dental mesenchyme and the epithelial ameloblast stem/progenitor cells during murine tooth development

    PubMed Central

    Nonomura, Koji; Takahashi, Masanori; Wakamatsu, Yoshio; Takano-Yamamoto, Teruko; Osumi, Noriko

    2010-01-01

    Six family transcription factor genes play multiple and crucial roles in the development of the vertebrate sensory system including the eye, olfactory epithelium and otic vesicle, and these genes are highly expressed in the neural crest-derived cranial mesenchymal cells in the mouse embryo. However, expression patterns have yet to be determined for the Six family genes in the developing tooth germ. In this study, we examined expression of six members of the Six family genes in the dental mesenchyme and the dental epithelium of the developing tooth germs in mice by in situ hybridization. We found dynamic expression patterns for Six1, Six2, Six4 and Six5 in the oral epithelium and mesenchymal cells with distinct expression patterns at the early stage before invagination of the dental epithelium. In addition, expression of Six1 and Six4 was observed in the inner enamel epithelium of the incisor and molar tooth germs at the cap stage. Expression of Six5 was maintained in the bell stage tooth germs, and intense expression of Six1 and Six4 was detected not only in the mesenchyme-derived dental follicle but also in the proliferating inner enamel epithelium of the labial cervical loop of the incisor tooth germ. Taken together, our results suggest that dynamic expression of Six family genes represents specific stages of the developing tooth germ. This dynamic expression is embodied in changes in both space and over time, and these changes in expression suggest that Six family genes may participate in tooth germ morphogenesis and the proliferation and/or differentiation of the incisor ameloblast stem/progenitor cells. PMID:19900183

  2. Silencing heme oxygenase-1 gene expression in retinal pigment epithelial cells inhibits proliferation, migration and tube formation of cocultured endothelial cells

    SciTech Connect

    Zhang, Wenjie; Zhang, Xiaomei; Lu, Hong; Matsukura, Makoto; Zhao, Jien; Shinohara, Makoto

    2013-05-10

    Highlights: •HO-1 is highly induced in RPE cells by hypoxia. •Inhibition of HO-1 activity and knockdown of HO-1 expression inhibit VEGF expression in RPE cells under hypoxia. •Knockdown of HO-1 in RPE cells inhibits angiogenesis of endothelial cells in vitro. -- Abstract: Heme oxygenase-1 (HO-1) plays an important role in the vasculature and in the angiogenesis of tumors, wounds and other environments. Retinal pigment epithelial (RPE) cells and choroidal endothelial cells (CECs) are the main cells involved in choroidal neovascularization (CNV), a process in which hypoxia plays an important role. Our aim was to evaluate the role of human RPE-cell HO-1 in the angiogenic activities of cocultured endothelial cells under hypoxia. Small interfering RNA (siRNA) for HO-1 was transfected into human RPE cell line ARPE-19, and zinc protoporphyrin (ZnPP) was used to inhibit HO-1 activity. Knockdown of HO-1 expression and inhibition of HO-1 activity resulted in potent reduction of the expression of vascular endothelial growth factor (VEGF) under hypoxia. Furthermore, knockdown of HO-1 suppressed the proliferation, migration and tube formation of cocultured endothelial cells. These findings indicated that HO-1 might have an angiogenic effect in CNV through modulation of VEGF expression and might be a potential target for treating CNV.

  3. Tyrosine kinase gene rearrangements in epithelial malignancies

    PubMed Central

    Shaw, Alice T.; Hsu, Peggy P.; Awad, Mark M.; Engelman, Jeffrey A.

    2014-01-01

    Chromosomal rearrangements that lead to oncogenic kinase activation are observed in many epithelial cancers. These cancers express activated fusion kinases that drive the initiation and progression of malignancy, and often have a considerable response to small-molecule kinase inhibitors, which validates these fusion kinases as ‘druggable’ targets. In this Review, we examine the aetiologic, pathogenic and clinical features that are associated with cancers harbouring oncogenic fusion kinases, including anaplastic lymphoma kinase (ALK), ROS1 and RET. We discuss the clinical outcomes with targeted therapies and explore strategies to discover additional kinases that are activated by chromosomal rearrangements in solid tumours. PMID:24132104

  4. Differential effect of immune cells on non-pathogenic Gram-negative bacteria-induced nuclear factor-κB activation and pro-inflammatory gene expression in intestinal epithelial cells

    PubMed Central

    Haller, D; Holt, L; Parlesak, A; Zanga, J; Bäuerlein, A; Sartor, R B; Jobin, C

    2004-01-01

    We have previously shown that non-pathogenic Gram negative bacteria induce RelA phosphorylation, nuclear factor (NF)-κB transcriptional activity and pro-inflammatory gene expression in intestinal epithelial cells (IEC) in vivo and in vitro. In this study, we investigated the molecular mechanism of immune-epithelial cell cross-talk on Gram-negative enteric bacteria-induced NF-κB signalling and pro-inflammatory gene expression in IEC using HT-29/MTX as well as CaCO-2 transwell cultures Interestingly, while differentiated HT-29/MTX cells are unresponsive to non-pathogenic Gram negative bacterial stimulation, interleukin-8 (IL-8) mRNA accumulation is strongly induced in Escherichia coli- but not Bacteroides vulgatus-stimulated IEC cocultured with peripheral blood (PBMC) and lamina propria mononuclear cells (LPMC). The presence of PBMC triggered both E. coli- and B. vulgatus-induced mRNA expression of the Toll-like receptor-4 accessory protein MD-2 as well as endogenous IκBα phosphorylation, demonstrating similar capabilities of these bacteria to induce proximal NF-κB signalling. However, B. vulgatus failed to trigger IκBα degradation and NF-κB transcriptional activity in the presence of PBMC. Interestingly, B. vulgatus- and E. coli-derived lipopolysaccharide-induced similar IL-8 mRNA expression in epithelial cells after basolateral stimulation of HT-29/PBMC cocultures. Although luminal enteric bacteria have adjuvant and antigenic properties in chronic intestinal inflammation, PBMC from patients with active ulcerative colitis and Crohn's disease differentially trigger epithelial cell activation in response to E. coli and E. coli-derived LPS. In conclusion, this study provides evidence for a differential regulation of non-pathogenic Gram-negative bacteria-induced NF-κB signalling and IL-8 gene expression in IEC cocultured with immune cells and suggests the presence of mechanisms that assure hyporesponsiveness of the intestinal epithelium to certain commensally

  5. Crocidolite activates NF-kappa B and MIP-2 gene expression in rat alveolar epithelial cells. Role of mitochondrial-derived oxidants.

    PubMed

    Driscoll, K E; Carter, J M; Howard, B W; Hassenbein, D; Janssen, Y M; Mossman, B T

    1998-10-01

    Nuclear factor kappa B (NF-kappa B) is a transcription factor that regulates expression of several genes coding for inflammatory and immunoregulatory proteins including the neutrophil chemotactic cytokine MIP-2. In previous studies we found that crocidolite asbestos activates the nuclear translocation of NF-kappa B as well as MIP-2 gene expression in rat alveolar type II cells. Here we report that both crocidolite-induced NF-kappa B activation of MIP-2 gene expression can be attenuated by the antioxidant tetramethylthiourea, suggesting the dependence of these responses on oxidative stress. Crocidolite exposure of RLE-TN cells also increased production of H2O2, a response that was inhibited by the mitochondrial respiratory chain inhibitor thenoyltrifluoroacetone (TTFA). TTFA treatment of RLE-6TN cells also inhibited crocidolite-induced nuclear translocation of NF-kappa B and MIP-2 gene expression. These results indicate crocidolite exposure of rat alveolar type II cells results in increased production of mitochondrial-derived hydrogen peroxide and that mitochondrial-derived oxidants contribute to crocidolite activation of NF-kappa B and increases in MIP-2 gene expression. PMID:9788893

  6. Ozone enhances diesel exhaust particles (DEP)-induced interleukin-8 (IL-8) gene expression in human airway epithelial cells through activation of nuclear factors- kappaB (NF-kappaB) and IL-6 (NF-IL6).

    PubMed

    Kafoury, Ramzi M; Kelley, James

    2005-12-01

    Ozone, a highly reactive oxidant gas is a major component of photochemical smog. As an inhaled toxicant, ozone induces its adverse effects mainly on the lung. Inhalation of particulate matter has been reported to cause airway inflammation in humans and animals. Furthermore, epidemiological evidence has indicated that exposure to particulate matter (PM[2.5-10]), including diesel exhaust particles (DEP) has been correlated with increased acute and chronic respiratory morbidity and exacerbation of asthma. Previously, exposure to ozone or particulate matter and their effect on the lung have been addressed as separate environmental problems. Ozone and particulate matter may be chemically coupled in the ambient air. In the present study we determined whether ozone exposure enhances DEP effect on interleukin-8 (IL-8) gene expression in human airway epithelial cells. We report that ozone exposure (0.5 ppm x 1 hr) significantly increased DEP-induced IL-8 gene expression in A549 cells (117 +/- 19 pg/ml, n = 6, p < 0.05) as compared to cultures treated with DEP (100 microg/ml x 4 hr) alone (31 +/- 3 pg/ml, n = 6), or cultures exposed to purified air (24 +/- 6 pg/ml, n = 6). The increased DEP-induced IL-8 gene expression following ozone exposure was attributed to ozone-induced increase in the activity of the transcription factors NF-kappaB and NF-IL6. The results of the present study indicate that ozone exposure enhances the toxicity of DEP in human airway epithelial cells by augmenting IL-8 gene expression, a potent chemoattractant of neutrophils in the lung. PMID:16819095

  7. An analysis of glucocorticoid receptor-mediated gene expression in BEAS-2B human airway epithelial cells identifies distinct, ligand-directed, transcription profiles with implications for asthma therapeutics

    PubMed Central

    Joshi, T; Johnson, M; Newton, R; Giembycz, M

    2015-01-01

    Background and Purpose International asthma guidelines recommend that inhaled glucocorticoids be used as a monotherapy in all patients with mild to moderate disease because of their ability to suppress airways inflammation. Current evidence suggests that the therapeutic benefit of glucocorticoids is due to the transactivation and transrepression of anti-inflammatory and pro-inflammatory genes respectively. However, the extent to which clinically relevant glucocorticoids are equivalent in their ability to modulate gene expression is unclear. Experimental Approach A pharmacodynamics investigation of glucocorticoid receptor (GR)-mediated gene transactivation in BEAS-2B human airway epithelial cells was performed using a glucocorticoid response element luciferase reporter coupled with an analysis of glucocorticoid-inducible genes encoding proteins with anti-inflammatory and adverse-effect potential. Key Results Using transactivation as a functionally relevant output, a given glucocorticoid displayed a unique, gene expression ‘fingerprint’ where intrinsic efficacy and GR density were essential determinants. We showed that depending on the gene selected for analysis, a given glucocorticoid can behave as an antagonist, partial agonist, full agonist or even ‘super agonist’. In the likely event that different, tissue-dependent gene expression profiles are reproduced in vivo, then the anti-inflammatory and adverse-effect potential of many glucocorticoids currently available as asthma therapeutics may not be equivalent. Conclusions and Implications The generation of gene expression ‘fingerprints’ in target and off-target human tissues could assist the rational design of GR agonists with improved therapeutic ratios. This approach could identify compounds that are useful in the management of severe asthma and other inflammatory disorders where systemic exposure is desirable. PMID:25393397

  8. Effect of short-chain fatty acids on triacylglycerol accumulation, lipid droplet formation and lipogenic gene expression in goat mammary epithelial cells.

    PubMed

    Sun, Yuting; Luo, Jun; Zhu, Jiangjiang; Shi, Hengbo; Li, Jun; Qiu, Siyuan; Wang, Ping; Loor, Juan J

    2016-02-01

    Short-chain fatty acids (SCFAs) are the major energy sources for ruminants and are known to regulate various physiological functions in other species. However, their roles in ruminant milk fat metabolism are still unclear. In this study, goat mammary gland epithelial cells (GMECs) were treated with 3 mmol/L acetate, propionate or butyrate for 24 h to assess their effects on lipogenesis. Data revealed that the content of triacylglycerol (TAG) and lipid droplet formation were significantly stimulated by propionate and butyrate. The expression of FABP3, SCD1, PPARG, SREBP1, DGAT1, AGPAT6 and ADRP were upregulated by propionate and butyrate treatment. In contrast, the messenger RNA (mRNA) expression of FASN and LXRα was not affected by propionate, but reduced by butyrate. Acetate had no obvious effect on the content of TAG and lipid droplets but increased the mRNA expression of SCD1 and FABP3 in GMECs. Additionally, it was observed that propionate significantly increased the relative content of mono-unsaturated fatty acids (C18:1 and C16:1) at the expense of decreased saturated fatty acids (C16:0 and C18:0). Butyrate and acetate had no significant effect on fatty acid composition. Overall, the results from this work help enhance our understanding of the regulatory role of SCFAs on goat mammary cell lipid metabolism. PMID:26304676

  9. Differential Roles of Hydrogen Peroxide in Adaptive and Inflammatory Gene Expression Induced by Exposure of Human Airway Epithelial Cells to Zn2+

    EPA Science Inventory

    Oxidant stress is believed to play an important role in particulate matter (PM)–mediated toxicity in the respiratory tract. Zinc (Zn2+) is a ubiquitous component of PM that has been shown to induce adverse responses such as inflammatory and adaptive gene expression in airway epit...

  10. Differences in the expression of chromosome 1 genes between lung telocytes and other cells: mesenchymal stem cells, fibroblasts, alveolar type II cells, airway epithelial cells and lymphocytes

    PubMed Central

    Sun, Xiaoru; Zheng, Minghuan; Zhang, Miaomiao; Qian, Mengjia; Zheng, Yonghua; Li, Meiyi; Cretoiu, Dragos; Chen, Chengshui; Chen, Luonan; Popescu, Laurentiu M; Wang, Xiangdong

    2014-01-01

    Telocytes (TCs) are a unique type of interstitial cells with specific, extremely long prolongations named telopodes (Tps). Our previous study showed that TCs are distinct from fibroblasts (Fbs) and mesenchymal stem cells (MSCs) as concerns gene expression and proteomics. The present study explores patterns of mouse TC-specific gene profiles on chromosome 1. We investigated the network of main genes and the potential functional correlations. We compared gene expression profiles of mouse pulmonary TCs, MSCs, Fbs, alveolar type II cells (ATII), airway basal cells (ABCs), proximal airway cells (PACs), CD8+ T cells from bronchial lymph nodes (T-BL) and CD8+ T cells from lungs (T-LL). The functional and feature networks were identified and compared by bioinformatics tools. Our data showed that on TC chromosome 1, there are about 25% up-regulated and 70% down-regulated genes (more than onefold) as compared with the other cells respectively. Capn2, Fhl2 and Qsox1 were over-expressed in TCs compared to the other cells, indicating that biological functions of TCs are mainly associated with morphogenesis and local tissue homoeostasis. TCs seem to have important roles in the prevention of tissue inflammation and fibrogenesis development in lung inflammatory diseases and as modulators of immune cell response. In conclusion, TCs are distinct from the other cell types. PMID:24826900

  11. Endogenous IL-33 is highly expressed in mouse epithelial barrier tissues, lymphoid organs, brain, embryos, and inflamed tissues: in situ analysis using a novel Il-33-LacZ gene trap reporter strain.

    PubMed

    Pichery, Mélanie; Mirey, Emilie; Mercier, Pascale; Lefrancais, Emma; Dujardin, Arnaud; Ortega, Nathalie; Girard, Jean-Philippe

    2012-04-01

    IL-33 (previously known as NF from high endothelial venules) is an IL-1 family cytokine that signals through the ST2 receptor and drives cytokine production in mast cells, basophils, eosinophils, invariant NKT and NK cells, Th2 lymphocytes, and type 2 innate immune cells (natural helper cells, nuocytes, and innate helper 2 cells). Little is known about endogenous IL-33; for instance, the cellular sources of IL-33 in mouse tissues have not yet been defined. In this study, we generated an Il-33-LacZ gene trap reporter strain (Il-33(Gt/Gt)) and used this novel tool to analyze expression of endogenous IL-33 in vivo. We found that the Il-33 promoter exhibits constitutive activity in mouse lymphoid organs, epithelial barrier tissues, brain, and embryos. Immunostaining with anti-IL-33 Abs, using Il-33(Gt/Gt) (Il-33-deficient) mice as control, revealed that endogenous IL-33 protein is highly expressed in mouse epithelial barrier tissues, including stratified squamous epithelia from vagina and skin, as well as cuboidal epithelium from lung, stomach, and salivary gland. Constitutive expression of IL-33 was not detected in blood vessels, revealing the existence of species-specific differences between humans and mice. Importantly, IL-33 protein was always localized in the nucleus of producing cells with no evidence for cytoplasmic localization. Finally, strong expression of the Il-33-LacZ reporter was also observed in inflamed tissues, in the liver during LPS-induced endotoxin shock, and in the lung alveoli during papain-induced allergic airway inflammation. Together, our findings support the possibility that IL-33 may function as a nuclear alarmin to alert the innate immune system after injury or infection in epithelial barrier tissues. PMID:22371395

  12. Epithelial expression of keratinocytes growth factor in oral precancer lesions

    PubMed Central

    Jimson, Sudha; Murali, S.; Zunt, Susan L.; Goldblatt, Lawrence I.; Srinivasan, Mythily

    2016-01-01

    Background: Keratinocyte growth factor (KGF) is a potent epithelial mitogen that acts by binding the KGF receptors (KGFRs) expressed on epithelial cells and regulates proliferation and differentiation. The objective of this study was to investigate the expression of KGF in the epithelium in oral precancer. Materials and Methods: Archival tissues of oral submucous fibrosis (SMF) and leukoplakia were assessed for epithelial KGF expression by immunohistochemistry and real-time quantitative polymerase chain reaction. Results: KGF was predominantly expressed in the basal and parabasal cells in the epithelium of SMF tissues. KGF transcript in the epithelial cells increased with increasing severity of epithelial dysplasia in oral leukoplakia. Conclusion: Although widely reported as a product secreted by the mesenchymal cells, our data suggest that the KGF is also expressed in oral epithelial cells much like the expression in ovarian epithelial cells. Based on the localization of KGF in cells at the epithelial mesenchymal junction and that of the reported presence of KGFR in oral keratinocytes, a potential mechanism involving paracrine and autocrine interactions of KGF and KGFR in early stages of oral precancer is postulated. PMID:27274338

  13. Activation of transcription factor IL-6 (NF-IL-6) and nuclear factor-kappaB (NF-kappaB) by lipid ozonation products is crucial to interleukin-8 gene expression in human airway epithelial cells.

    PubMed

    Kafoury, Ramzi M; Hernandez, Jazmir M; Lasky, Joseph A; Toscano, William A; Friedman, Mitchell

    2007-04-01

    Ozone (O(3)) is a major component of smog and an inhaled toxicant to the lung. O(3) rapidly reacts with the airway epithelial cell membrane phospholipids to generate lipid ozonation products (LOP). 1-Hydroxy-1-hydroperoxynonane (HHP-C9) is an important LOP, produced from the ozonation of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine. This LOP, at a biologically relevant concentration (100 microM), increases the activity of phospholipase C, nuclear factors-kappaB (NF-kappaB), and interleukin-6 (NF-IL-6) and the expression of the inflammatory gene, interleukin-8 (IL-8) in a cultured human bronchial epithelial cell line (BEAS-2B). The signaling pathways of ozone and its biologically-active products are as yet undefined. In the present study, we report that the HHP LOP, HHP-C9 (100 microM x 4 h), activated the expression of IL-8 (218 +/- 26% increase over control, n = 4, P < 0.01) through an apparent interaction between the two transcription factors, NF-kappaB and NF-IL-6. Transfection studies using luciferase reporter assays demonstrated that HHP-C9 induced a significant increase in NF-kappaB-DNA binding activity (37 +/- 7% increase over control, n = 6, P < 0.05). Inhibition of NF-kappaB showed a statistically significant but modest decrease in IL-8 release, which suggested a role for another transcription factor, NF-IL-6. Exposure of BEAS-2B cells to HHP-C9 induced a significant increase in the DNA binding activity of NF-IL-6 (45 +/- 11% increase over control, n = 6, P < 0.05). The results of the present study indicate that NF-IL-6 interacts with NF-kappaB in regulating the expression of IL-8 in cultured human airway epithelial cells exposed to LOP, the biological products of ozone in the lung. PMID:17366569

  14. miR-30 Family Controls Proliferation and Differentiation of Intestinal Epithelial Cell Models by Directing a Broad Gene Expression Program That Includes SOX9 and the Ubiquitin Ligase Pathway*

    PubMed Central

    Sincavage, John; Feinstein, Sydney; Mah, Amanda T.; Simmons, James G.; Lund, P. Kay; Sethupathy, Praveen

    2016-01-01

    Proliferation and differentiation of intestinal epithelial cells (IECs) occur in part through precise regulation of key transcription factors, such as SOX9. MicroRNAs (miRNAs) have emerged as prominent fine-tuners of transcription factor expression and activity. We hypothesized that miRNAs, in part through the regulation of SOX9, may mediate IEC homeostasis. Bioinformatic analyses of the SOX9 3′-UTR revealed highly conserved target sites for nine different miRNAs. Of these, only the miR-30 family members were both robustly and variably expressed across functionally distinct cell types of the murine jejunal epithelium. Inhibition of miR-30 using complementary locked nucleic acids (LNA30bcd) in both human IECs and human colorectal adenocarcinoma-derived Caco-2 cells resulted in significant up-regulation of SOX9 mRNA but, interestingly, significant down-regulation of SOX9 protein. To gain mechanistic insight into this non-intuitive finding, we performed RNA sequencing on LNA30bcd-treated human IECs and found 2440 significantly increased genes and 2651 significantly decreased genes across three time points. The up-regulated genes are highly enriched for both predicted miR-30 targets, as well as genes in the ubiquitin-proteasome pathway. Chemical suppression of the proteasome rescued the effect of LNA30bcd on SOX9 protein levels, indicating that the regulation of SOX9 protein by miR-30 is largely indirect through the proteasome pathway. Inhibition of the miR-30 family led to significantly reduced IEC proliferation and a dramatic increase in markers of enterocyte differentiation. This in-depth analysis of a complex miRNA regulatory program in intestinal epithelial cell models provides novel evidence that the miR-30 family likely plays an important role in IEC homeostasis. PMID:27261459

  15. miR-30 Family Controls Proliferation and Differentiation of Intestinal Epithelial Cell Models by Directing a Broad Gene Expression Program That Includes SOX9 and the Ubiquitin Ligase Pathway.

    PubMed

    Peck, Bailey C E; Sincavage, John; Feinstein, Sydney; Mah, Amanda T; Simmons, James G; Lund, P Kay; Sethupathy, Praveen

    2016-07-29

    Proliferation and differentiation of intestinal epithelial cells (IECs) occur in part through precise regulation of key transcription factors, such as SOX9. MicroRNAs (miRNAs) have emerged as prominent fine-tuners of transcription factor expression and activity. We hypothesized that miRNAs, in part through the regulation of SOX9, may mediate IEC homeostasis. Bioinformatic analyses of the SOX9 3'-UTR revealed highly conserved target sites for nine different miRNAs. Of these, only the miR-30 family members were both robustly and variably expressed across functionally distinct cell types of the murine jejunal epithelium. Inhibition of miR-30 using complementary locked nucleic acids (LNA30bcd) in both human IECs and human colorectal adenocarcinoma-derived Caco-2 cells resulted in significant up-regulation of SOX9 mRNA but, interestingly, significant down-regulation of SOX9 protein. To gain mechanistic insight into this non-intuitive finding, we performed RNA sequencing on LNA30bcd-treated human IECs and found 2440 significantly increased genes and 2651 significantly decreased genes across three time points. The up-regulated genes are highly enriched for both predicted miR-30 targets, as well as genes in the ubiquitin-proteasome pathway. Chemical suppression of the proteasome rescued the effect of LNA30bcd on SOX9 protein levels, indicating that the regulation of SOX9 protein by miR-30 is largely indirect through the proteasome pathway. Inhibition of the miR-30 family led to significantly reduced IEC proliferation and a dramatic increase in markers of enterocyte differentiation. This in-depth analysis of a complex miRNA regulatory program in intestinal epithelial cell models provides novel evidence that the miR-30 family likely plays an important role in IEC homeostasis. PMID:27261459

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

    PubMed

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

    2016-08-01

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

  17. MiR-200a regulates epithelial to mesenchymal transition-related gene expression and determines prognosis in colorectal cancer patients

    PubMed Central

    Pichler, M; Ress, A L; Winter, E; Stiegelbauer, V; Karbiener, M; Schwarzenbacher, D; Scheideler, M; Ivan, C; Jahn, S W; Kiesslich, T; Gerger, A; Bauernhofer, T; Calin, G A; Hoefler, G

    2014-01-01

    Background: MicroRNAs (miRNAs) regulate the biological properties of colorectal cancer (CRC) cells and might serve as potential prognostic factors and therapeutic targets. In this study, we therefore globally profiled miRNAs associated with E-cadherin expression in CRC cells in an attempt to identify miRNAs that are associated with aggressive clinical course in CRC patients. Methods: Two CRC cell lines (Caco-2 and HRT-18) with different E-cadherin expression pattern were profiled for differences in abundance for more than 1000 human miRNAs using microarray technology. One of the most differentially expressed miRNAs, miR-200a was evaluated for its prognostic role in a cohort of 111 patients and independently validated in 217 patients of the Cancer Genome Atlas data set. To further characterise the biological role of miR-200a expression in CRC, in vitro miR-200a inhibition and overexpression were performed and the effects on cellular growth, apoptosis and epithelial–mesenchymal transition (EMT)-related gene expression were explored. Results: In situ hybridisation specifically localised miR-200a in CRC cells. In both cohorts, a low miR-200a expression was associated with poor survival (P<0.05). Multivariate Cox regression analysis identified low levels of miR-200a expression as an independent prognostic factor with respect to cancer-specific survival (HR=2.04, CI=1.28–3.25, P<0.002). Gain and loss of function assays for miR-200a in vitro led to a significantly differential and converse expression of EMT-related genes (P<0.001.) A low expression of miR-200a was also observed in cancer stem cell-enriched spheroid growth conditions (P<0.05). Conclusions: In conclusion, our data suggest that low miR-200a expression is associated with poor prognosis in CRC patients. MiR-200a has a regulatory effect on EMT and is associated with cancer stem cell properties in CRC. PMID:24504363

  18. Synergistic effects of gamma interferon on inflammatory mediators that induce interleukin-6 gene expression and secretion by human retinal pigment epithelial cells.

    PubMed Central

    Nagineni, C N; Detrick, B; Hooks, J J

    1994-01-01

    The retinal pigment epithelial (RPE) cell is a potent regulatory cell within the retina. It helps to maintain normal retinal activity, and following gamma interferon (IFN-gamma) exposure, it may express major histocompatibility complex class II molecules and function as an antigen-presenting cell. Since interleukin-1 (IL-1) and IL-6 are potent cytokines observed in ocular inflammatory processes, we initiated studies to evaluate conditions which enable RPE cells to produce these cytokines. Cultures of human RPE cells from two eye donors were established and characterized, and enzyme immunoassays were employed to screen for IL-1 and IL-6 production. Treatment of RPE cells with lipopolysaccharide (LPS) or recombinant tumor necrosis factor alpha, IL-1, or IFN-gamma resulted in a significant level of secretion of IL-6. In contrast, treatment with recombinant epidermal growth factor, basic fibroblast growth factor, platelet-derived growth factor, or transforming growth factor alpha, or LPS can dramatically augment the secretion of IL-6 by RPE cells. Thus, these inflammatory mediators can act alone or synergistically with IFN-gamma to activate RPE cells and dramatically increase the expression and secretion of IL-6. In contrast, IL-1 was not detected following stimulation with any of the above-mentioned cytokines or LPS. Characterization of IL-6 protein production by RPE cells revealed that 98% of the protein is promptly secreted by the cell, its induction is dependent upon the time and concentration of the stimulant, and the continuous presence of the stimulant is required for IL-6 production. Moreover, Western blot (immunoblot) analysis of secreted proteins revealed that IL-6 was produced in multiple molecular forms.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8556503

  19. Identification of an Alternative Splicing Product of the Otx2 Gene Expressed in the Neural Retina and Retinal Pigmented Epithelial Cells

    PubMed Central

    Kole, Christo; Berdugo, Naomi; Da Silva, Corinne; Aït-Ali, Najate; Millet-Puel, Géraldine; Pagan, Delphine; Blond, Frédéric; Poidevin, Laetitia; Ripp, Raymond; Fontaine, Valérie; Wincker, Patrick; Zack, Donald J.; Sahel, José-Alain; Poch, Olivier; Léveillard, Thierry

    2016-01-01

    To investigate the complexity of alternative splicing in the retina, we sequenced and analyzed a total of 115,706 clones from normalized cDNA libraries from mouse neural retina (66,217) and rat retinal pigmented epithelium (49,489). Based upon clustering the cDNAs and mapping them with their respective genomes, the estimated numbers of genes were 9,134 for the mouse neural retina and 12,050 for the rat retinal pigmented epithelium libraries. This unique collection of retinal of messenger RNAs is maintained and accessible through a web-base server to the whole community of retinal biologists for further functional characterization. The analysis revealed 3,248 and 3,202 alternative splice events for mouse neural retina and rat retinal pigmented epithelium, respectively. We focused on transcription factors involved in vision. Among the six candidates suitable for functional analysis, we selected Otx2S, a novel variant of the Otx2 gene with a deletion within the homeodomain sequence. Otx2S is expressed in both the neural retina and retinal pigmented epithelium, and encodes a protein that is targeted to the nucleus. OTX2S exerts transdominant activity on the tyrosinase promoter when tested in the physiological environment of primary RPE cells. By overexpressing OTX2S in primary RPE cells using an adeno associated viral vector, we identified 10 genes whose expression is positively regulated by OTX2S. We find that OTX2S is able to bind to the chromatin at the promoter of the retinal dehydrogenase 10 (RDH10) gene. PMID:26985665

  20. Identification of an Alternative Splicing Product of the Otx2 Gene Expressed in the Neural Retina and Retinal Pigmented Epithelial Cells.

    PubMed

    Kole, Christo; Berdugo, Naomi; Da Silva, Corinne; Aït-Ali, Najate; Millet-Puel, Géraldine; Pagan, Delphine; Blond, Frédéric; Poidevin, Laetitia; Ripp, Raymond; Fontaine, Valérie; Wincker, Patrick; Zack, Donald J; Sahel, José-Alain; Poch, Olivier; Léveillard, Thierry

    2016-01-01

    To investigate the complexity of alternative splicing in the retina, we sequenced and analyzed a total of 115,706 clones from normalized cDNA libraries from mouse neural retina (66,217) and rat retinal pigmented epithelium (49,489). Based upon clustering the cDNAs and mapping them with their respective genomes, the estimated numbers of genes were 9,134 for the mouse neural retina and 12,050 for the rat retinal pigmented epithelium libraries. This unique collection of retinal of messenger RNAs is maintained and accessible through a web-base server to the whole community of retinal biologists for further functional characterization. The analysis revealed 3,248 and 3,202 alternative splice events for mouse neural retina and rat retinal pigmented epithelium, respectively. We focused on transcription factors involved in vision. Among the six candidates suitable for functional analysis, we selected Otx2S, a novel variant of the Otx2 gene with a deletion within the homeodomain sequence. Otx2S is expressed in both the neural retina and retinal pigmented epithelium, and encodes a protein that is targeted to the nucleus. OTX2S exerts transdominant activity on the tyrosinase promoter when tested in the physiological environment of primary RPE cells. By overexpressing OTX2S in primary RPE cells using an adeno associated viral vector, we identified 10 genes whose expression is positively regulated by OTX2S. We find that OTX2S is able to bind to the chromatin at the promoter of the retinal dehydrogenase 10 (RDH10) gene. PMID:26985665

  1. Epithelial expression of interleukin-37b in inflammatory bowel disease.

    PubMed

    Imaeda, H; Takahashi, K; Fujimoto, T; Kasumi, E; Ban, H; Bamba, S; Sonoda, H; Shimizu, T; Fujiyama, Y; Andoh, A

    2013-06-01

    Interleukin (IL)-37 is a member of the IL-1 cytokine family. We investigated IL-37b expression in the inflamed mucosa of inflammatory bowel disease (IBD) patients. Furthermore, we analysed IL-37b expression in human colonic epithelial cells. The human colonic epithelial cell line T84 and human colonic subepithelial myofibroblasts (SEMFs) were used. IL-37b expression in the IBD mucosa was evaluated by immunohistochemistry. IL-37b mRNA and protein expression were determined by real time-polymerase chain reaction (PCR) and Western blotting, respectively. IL-37b was not detected in the normal colonic mucosa. In the inflamed mucosa of IBD patients, epithelial IL-37b expression was increased markedly. In ulcerative colitis (UC) and Crohn's disease (CD) patients, IL-37b expression was enhanced in the affected mucosa. In the intestinal epithelial cell line T84, the expression of IL-37b mRNA and protein was enhanced by tumour necrosis factor (TNF)-α. This IL-37b induction by TNF-α was mediated by nuclear factor (NF)-κB and activator protein (AP)-1 activation. Furthermore, IL-37b inhibited TNF-α-induced interferon-γ-inducible protein (IP)-10 expression significantly in human colonic SEMFs. Epithelial IL-37b expression was increased in IBD patients, especially UC patients. IL-37b may be involved in the pathophysiology of IBD as an anti-inflammatory cytokine and an inhibitor of both innate and acquired immune responses. PMID:23600829

  2. Cigarette Smoke Enhances the Expression of Profibrotic Molecules in Alveolar Epithelial Cells

    PubMed Central

    Checa, Marco; Hagood, James S.; Velazquez-Cruz, Rafael; Ruiz, Victor; García-De-Alba, Carolina; Rangel-Escareño, Claudia; Urrea, Francisco; Becerril, Carina; Montaño, Martha; García-Trejo, Semiramis; Cisneros Lira, José; Aquino-Gálvez, Arnoldo; Pardo, Annie; Selman, Moisés

    2016-01-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disease of unknown etiology. A growing body of evidence indicates that it may result from an aberrant activation of alveolar epithelium, which induces the expansion of the fibroblast population, their differentiation to myofibroblasts and the excessive accumulation of extracellular matrix. The mechanisms that activate the alveolar epithelium are unknown, but several studies indicate that smoking is the main environmental risk factor for the development of IPF. In this study we explored the effect of cigarette smoke on the gene expression profile and signaling pathways in alveolar epithelial cells. Lung epithelial cell line from human (A549), was exposed to cigarette smoke extract (CSE) for 1, 3, and 5 weeks at 1, 5 and 10% and gene expression was evaluated by complete transcriptome microarrays. Signaling networks were analyzed with the Ingenuity Pathway Analysis software. At 5 weeks of exposure, alveolar epithelial cells acquired a fibroblast-like phenotype. At this time, gene expression profile revealed a significant increase of more than 1000 genes and deregulation of canonical signaling pathways such as TGF-β and Wnt. Several profibrotic genes involved in EMT were over-expressed, and incomplete EMT was observed in these cells, and corroborated in mouse (MLE-12) and rat (RLE-6TN) epithelial cells. The secretion of activated TGF-β1 increased in cells exposed to cigarette smoke, which decreased when the integrin alpha v gene was silenced. These findings suggest that the exposure of alveolar epithelial cells to CSE induces the expression and release of a variety of profibrotic genes, and the activation of TGF-β1, which may explain at least partially, the increased risk of developing IPF in smokers. PMID:26934369

  3. Cigarette Smoke Enhances the Expression of Profibrotic Molecules in Alveolar Epithelial Cells.

    PubMed

    Checa, Marco; Hagood, James S; Velazquez-Cruz, Rafael; Ruiz, Victor; García-De-Alba, Carolina; Rangel-Escareño, Claudia; Urrea, Francisco; Becerril, Carina; Montaño, Martha; García-Trejo, Semiramis; Cisneros Lira, José; Aquino-Gálvez, Arnoldo; Pardo, Annie; Selman, Moisés

    2016-01-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disease of unknown etiology. A growing body of evidence indicates that it may result from an aberrant activation of alveolar epithelium, which induces the expansion of the fibroblast population, their differentiation to myofibroblasts and the excessive accumulation of extracellular matrix. The mechanisms that activate the alveolar epithelium are unknown, but several studies indicate that smoking is the main environmental risk factor for the development of IPF. In this study we explored the effect of cigarette smoke on the gene expression profile and signaling pathways in alveolar epithelial cells. Lung epithelial cell line from human (A549), was exposed to cigarette smoke extract (CSE) for 1, 3, and 5 weeks at 1, 5 and 10% and gene expression was evaluated by complete transcriptome microarrays. Signaling networks were analyzed with the Ingenuity Pathway Analysis software. At 5 weeks of exposure, alveolar epithelial cells acquired a fibroblast-like phenotype. At this time, gene expression profile revealed a significant increase of more than 1000 genes and deregulation of canonical signaling pathways such as TGF-β and Wnt. Several profibrotic genes involved in EMT were over-expressed, and incomplete EMT was observed in these cells, and corroborated in mouse (MLE-12) and rat (RLE-6TN) epithelial cells. The secretion of activated TGF-β1 increased in cells exposed to cigarette smoke, which decreased when the integrin alpha v gene was silenced. These findings suggest that the exposure of alveolar epithelial cells to CSE induces the expression and release of a variety of profibrotic genes, and the activation of TGF-β1, which may explain at least partially, the increased risk of developing IPF in smokers. PMID:26934369

  4. [Expression of Catechol-O-Methyltransferase (Comt), Mineralocorticoid Receptor (Mlr), and Epithelial Sodium Channel (ENaC) Genes in Kidneys of Hypertensive ISIAH Rats at Rest and during Response to Stress].

    PubMed

    Abramova, T O; Smolenskaya, S E; Antonov, E V; Redina, O E; Markel, A L

    2016-02-01

    Emotional stress plays a significant role in the processes of the development of arterial hypertension, especially in the presence of genetic predisposition. The origin and maintenance of hypertensive status during stress development can be activated by the sympathetic nervous system. An increase in sympathetic stimulation can, in turn, result in a change in the functions of kidneys, which provide fluid and electrolyte balance of the organism. A comparative study of the mRNA expression level of catechol-o-methyltransferase (Comt), mineralocorticoid receptor (Mlr), and β-subunit of epithelial sodium channel (β-ENaC) genes was conducted on the kidneys of hypertensive ISIAH rats and normotensive WAG rats at rest and after the effect of emotional stress. The discovered changes in the expression level of the selected genes confirm their involvement in increased sympathetic stimulation of the kidney, along with changes in the function of kidney regulation of fluid and electrolyte balance, which is an important factor of the development of sustained hypertension in the ISIAH rats strain. PMID:27215035

  5. Effects of butyrate on the expression of insulin-like growth factor binding proteins in bovine kidney epithelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sodium butyrate induces cell cycle arrest and apoptosis in bovine kidney epithelial cells primarily via down-regulating cell cycle-related gene expression and enhancing expression of pro-apoptotic genes. The insulin-like growth factor (IGF) system plays an essential role in these processes as well a...

  6. Expression and Function of CD44 in Epithelial Ovarian Carcinoma

    PubMed Central

    Sacks, Joelle D.; Barbolina, Maria V.

    2015-01-01

    CD44, a cell surface glycoprotein, has been increasingly implicated in the pathogenesis and progression of epithelial ovarian cancer, the deadliest gynecologic malignancy in women. Here, we review recent reports on the expression and function of CD44 in epithelial ovarian carcinoma. Further functional data for CD44 in peritoneal adhesion and metastatic progression and its association with stem cells is highlighted. Recent studies utilizing CD44 for therapeutic targeting are also discussed. PMID:26569327

  7. GENE EXPRESSION NETWORKS

    EPA Science Inventory

    "Gene expression network" is the term used to describe the interplay, simple or complex, between two or more gene products in performing a specific cellular function. Although the delineation of such networks is complicated by the existence of multiple and subtle types of intera...

  8. Coassembled nanostructured bioscaffold reduces the expression of proinflammatory cytokines to induce apoptosis in epithelial cancer cells.

    PubMed

    Li, Rui; Pavuluri, Sivapriya; Bruggeman, Kiara; Long, Benjamin M; Parnell, Andrew J; Martel, Anne; Parnell, Steven R; Pfeffer, Frederick M; Dennison, Andrew J C; Nicholas, Kevin R; Barrow, Colin J; Nisbet, David R; Williams, Richard J

    2016-07-01

    The local inflammatory environment of the cell promotes the growth of epithelial cancers. Therefore, controlling inflammation locally using a material in a sustained, non-steroidal fashion can effectively kill malignant cells without significant damage to surrounding healthy cells. A promising class of materials for such applications is the nanostructured scaffolds formed by epitope presenting minimalist self-assembled peptides; these are bioactive on a cellular length scale, while presenting as an easily handled hydrogel. Here, we show that the assembly process can distribute an anti-inflammatory polysaccharide, fucoidan, localized to the nanofibers within the scaffold to create a biomaterial for cancer therapy. We show that it supports healthy cells, while inducing apoptosis in cancerous epithelial cells, as demonstrated by the significant down-regulation of gene and protein expression pathways associated with epithelial cancer progression. Our findings highlight an innovative material approach with potential applications in local epithelial cancer immunotherapy and drug delivery. PMID:26961467

  9. Activation of type III interferon genes by pathogenic bacteria in infected epithelial cells and mouse placenta.

    PubMed

    Bierne, Hélène; Travier, Laetitia; Mahlakõiv, Tanel; Tailleux, Ludovic; Subtil, Agathe; Lebreton, Alice; Paliwal, Anupam; Gicquel, Brigitte; Staeheli, Peter; Lecuit, Marc; Cossart, Pascale

    2012-01-01

    Bacterial infections trigger the expression of type I and II interferon genes but little is known about their effect on type III interferon (IFN-λ) genes, whose products play important roles in epithelial innate immunity against viruses. Here, we studied the expression of IFN-λ genes in cultured human epithelial cells infected with different pathogenic bacteria and in the mouse placenta infected with Listeria monocytogenes. We first showed that in intestinal LoVo cells, induction of IFN-λ genes by L. monocytogenes required bacterial entry and increased further during the bacterial intracellular phase of infection. Other Gram-positive bacteria, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis, also induced IFN-λ genes when internalized by LoVo cells. In contrast, Gram-negative bacteria Salmonella enterica serovar Typhimurium, Shigella flexneri and Chlamydia trachomatis did not substantially induce IFN-λ. We also found that IFN-λ genes were up-regulated in A549 lung epithelial cells infected with Mycobacterium tuberculosis and in HepG2 hepatocytes and BeWo trophoblastic cells infected with L. monocytogenes. In a humanized mouse line permissive to fetoplacental listeriosis, IFN-λ2/λ3 mRNA levels were enhanced in placentas infected with L. monocytogenes. In addition, the feto-placental tissue was responsive to IFN-λ2. Together, these results suggest that IFN-λ may be an important modulator of the immune response to Gram-positive intracellular bacteria in epithelial tissues. PMID:22720036

  10. Gene Expression Patterns in Ovarian Carcinomas

    PubMed Central

    Schaner, Marci E.; Ross, Douglas T.; Ciaravino, Giuseppe; Sørlie, Therese; Troyanskaya, Olga; Diehn, Maximilian; Wang, Yan C.; Duran, George E.; Sikic, Thomas L.; Caldeira, Sandra; Skomedal, Hanne; Tu, I-Ping; Hernandez-Boussard, Tina; Johnson, Steven W.; O'Dwyer, Peter J.; Fero, Michael J.; Kristensen, Gunnar B.; Børresen-Dale, Anne-Lise; Hastie, Trevor; Tibshirani, Robert; van de Rijn, Matt; Teng, Nelson N.; Longacre, Teri A.; Botstein, David; Brown, Patrick O.; Sikic, Branimir I.

    2003-01-01

    We used DNA microarrays to characterize the global gene expression patterns in surface epithelial cancers of the ovary. We identified groups of genes that distinguished the clear cell subtype from other ovarian carcinomas, grade I and II from grade III serous papillary carcinomas, and ovarian from breast carcinomas. Six clear cell carcinomas were distinguished from 36 other ovarian carcinomas (predominantly serous papillary) based on their gene expression patterns. The differences may yield insights into the worse prognosis and therapeutic resistance associated with clear cell carcinomas. A comparison of the gene expression patterns in the ovarian cancers to published data of gene expression in breast cancers revealed a large number of differentially expressed genes. We identified a group of 62 genes that correctly classified all 125 breast and ovarian cancer specimens. Among the best discriminators more highly expressed in the ovarian carcinomas were PAX8 (paired box gene 8), mesothelin, and ephrin-B1 (EFNB1). Although estrogen receptor was expressed in both the ovarian and breast cancers, genes that are coregulated with the estrogen receptor in breast cancers, including GATA-3, LIV-1, and X-box binding protein 1, did not show a similar pattern of coexpression in the ovarian cancers. PMID:12960427

  11. An enhancer from the 8q24 prostate cancer risk region is sufficient to direct reporter gene expression to a subset of prostate stem-like epithelial cells in transgenic mice

    PubMed Central

    Ting, Man-Chun; Liao, Chun-Peng; Yan, Chunli; Jia, Li; Groshen, Susan; Frenkel, Baruch; Roy-Burman, Pradip; Coetzee, Gerhard A.; Maxson, Robert

    2012-01-01

    SUMMARY Regions in the 8q24 gene desert contribute significantly to the risk of prostate cancer and other adult cancers. This region contains several DNA regions with enhancer activity in cultured cells. One such segment, histone acetylation peak 10 (AcP10), contains a risk single nucleotide polymorphism (SNP) that is significantly associated with the pathogenesis of colorectal, prostate and other cancers. The mechanism by which AcP10 influences cancer risk remains unknown. Here we show that AcP10 contains a sequence that is highly conserved across terrestrial vertebrates and is capable in transgenic mice of directing reporter gene expression to a subset of prostate lumenal epithelial cells. These cells include a small population of Nkx3.1-positive cells that persist even after androgen ablation. Castration-resistant Nkx3.1-positive (CARN) cells were shown by others to function both as stem cells and cells of origin of prostate cancer. Our results thus provide a mechanism by which AcP10 could influence prostate cancer risk. PMID:22279083

  12. Superiority of combined phosphodiesterase PDE3/PDE4 inhibition over PDE4 inhibition alone on glucocorticoid- and long-acting β2-adrenoceptor agonist-induced gene expression in human airway epithelial cells.

    PubMed

    BinMahfouz, Hawazen; Borthakur, Bibhusana; Yan, Dong; George, Tresa; Giembycz, Mark A; Newton, Robert

    2015-01-01

    Glucocorticoids, also known as corticosteroids, induce effector gene transcription as a part of their anti-inflammatory mechanisms of action. Such genomic effects can be significantly enhanced by long-acting β2-adrenoceptor agonists (LABAs) and may contribute to the clinical superiority of inhaled corticosteroid (ICS)/LABA combinations in asthma and chronic obstructive pulmonary disease (COPD) over ICSs alone. Using models of cAMP- and glucocorticoid-induced transcription in human bronchial epithelial BEAS-2B cells, we show that combining inhibitors of phosphodiesterase (PDE) 3 and PDE4 provides greater benefits compared with inhibiting either PDE alone. In respect to cAMP-dependent transcription, inhibitors of PDE3 (siguazodan, cilostazol) and PDE4 (rolipram, GSK256066, roflumilast N-oxide) each sensitized to the LABA, formoterol. This effect was magnified by dual PDE3 and PDE4 inhibition. Siguazodan plus rolipram was also more effective at inducing cAMP-dependent transcription than either inhibitor alone. Conversely, the concentration-response curve describing the enhancement of dexamethasone-induced, glucocorticoid response element-dependent transcription by formoterol was displaced to the left by PDE4, but not PDE3, inhibition. Overall, similar effects were described for bona fide genes, including RGS2, CD200, and CRISPLD2. Importantly, the combination of siguazodan plus rolipram prolonged the duration of gene expression induced by formoterol, dexamethasone, or dexamethasone plus formoterol. This was most apparent for RGS2, a bronchoprotective gene that may also reduce the proinflammatory effects of constrictor mediators. Collectively, these data provide a rationale for the use of PDE3 and PDE4 inhibitors in the treatment of COPD and asthma where they may enhance, sensitize, and prolong the effects of LABA/ICS combination therapies. PMID:25324049

  13. c-Myc regulates proliferation and Fgf10 expression in airway smooth muscle after airway epithelial injury in mouse.

    PubMed

    Volckaert, Thomas; Campbell, Alice; De Langhe, Stijn

    2013-01-01

    During lung development, Fibroblast growth factor 10 (Fgf10), which is expressed in the distal mesenchyme and regulated by Wnt signaling, acts on the distal epithelial progenitors to maintain them and prevent them from differentiating into proximal (airway) epithelial cells. Fgf10-expressing cells in the distal mesenchyme are progenitors for parabronchial smooth muscle cells (PSMCs). After naphthalene, ozone or bleomycin-induced airway epithelial injury, surviving epithelial cells secrete Wnt7b which then activates the PSMC niche to induce Fgf10 expression. This Fgf10 secreted by the niche then acts on a subset of Clara stem cells to break quiescence, induce proliferation and initiate epithelial repair. Here we show that conditional deletion of the Wnt target gene c-Myc from the lung mesenchyme during development does not affect proper epithelial or mesenchymal differentiation. However, in the adult lung we show that after naphthalene-mediated airway epithelial injury c-Myc is important for the activation of the PSMC niche and as such induces proliferation and Fgf10 expression in PSMCs. Our data indicate that conditional deletion of c-Myc from PSMCs inhibits airway epithelial repair, whereas c-Myc ablation from Clara cells has no effect on airway epithelial regeneration. These findings may have important implications for understanding the misregulation of lung repair in asthma and COPD. PMID:23967208

  14. Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells

    PubMed Central

    Nawandar, Dhananjay M.; Wang, Anqi; Makielski, Kathleen; Lee, Denis; Ma, Shidong; Barlow, Elizabeth; Reusch, Jessica; Jiang, Ru; Wille, Coral K.; Greenspan, Deborah; Greenspan, John S.; Mertz, Janet E.; Hutt-Fletcher, Lindsey; Johannsen, Eric C.; Lambert, Paul F.; Kenney, Shannon C.

    2015-01-01

    Epstein-Barr virus (EBV) is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue lesion known as oral hairy leukoplakia (OHL) in immunosuppressed patients. However, the cellular mechanism(s) that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1) promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs) cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells. PMID:26431332

  15. Gene expression technology

    SciTech Connect

    Goeddel, D.V. )

    1990-01-01

    The articles in this volume were assemble to enable the reader to design effective strategies for the expression of cloned genes and cDNAs. More than a compilation of papers describing the multitude of techniques now available for expressing cloned genes, this volume provides a manual that should prove useful for solving the majority of expression problems one likely to encounter. The four major expression systems commonly available to most investigators are stressed: Escherichia coli, Bacillus subtilis, yeast, and mammalian cells. Each of these system has its advantages and disadvantages, details of which are found in Chapter 1 and the strategic overviews for the four major sections of the volume. The papers in each of these sections provide many suggestions on how to proceed if initial expression levels are not sufficient.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  18. Azithromycin differentially affects the IL-13-induced expression profile in human bronchial epithelial cells.

    PubMed

    Mertens, Tinne C J; Hiemstra, Pieter S; Taube, Christian

    2016-08-01

    The T helper 2 (Th2) cytokine interleukin(IL)-13 is a central regulator in goblet cell metaplasia and induces the recently described Th2 gene signature consisting of periostin (POSTN), chloride channel regulator 1 (CLCA1) and serpin B2 (SERPINB2) in airway epithelial cells. This Th2 gene signature has been proposed as a biomarker to classify asthma into Th2-high and Th2-low phenotypes. Clinical studies have shown that the macrolide antibiotic azithromycin reduced clinical symptoms in neutrophilic asthma, but not in the classical Th2-mediated asthma despite the ability of azithromycin to reduce IL-13-induced mucus production. We therefore hypothesize that azithromycin differentially affects the IL-13-induced expression profile. To investigate this, we focus on IL-13-induced mucin and Th2-signature expression in human bronchial epithelial cells and how this combined expression profile is affected by azithromycin treatment. Primary bronchial epithelial cells were differentiated at air liquid interface in presence of IL-13 with or without azithromycin. Azithromycin inhibited IL-13-induced MUC5AC, which was accompanied by inhibition of IL-13-induced CLCA1 and SERPINB2 expression. In contrast, IL-13-induced expression of POSTN was further increased in cells treated with azithromycin. This indicates that azithromycin has a differential effect on the IL-13-induced Th2 gene signature. Furthermore, the ability of azithromycin to decrease IL-13-induced MUC5AC expression may be mediated by a reduction in CLCA1. PMID:27246785

  19. Airway Epithelial miRNA Expression Is Altered in Asthma

    PubMed Central

    Solberg, Owen D.; Ostrin, Edwin J.; Love, Michael I.; Peng, Jeffrey C.; Bhakta, Nirav R.; Nguyen, Christine; Solon, Margaret; Nguyen, Cindy; Barczak, Andrea J.; Zlock, Lorna T.; Blagev, Denitza P.; Finkbeiner, Walter E.; Ansel, K. Mark; Arron, Joseph R.; Erle, David J.

    2012-01-01

    Rationale: Changes in airway epithelial cell differentiation, driven in part by IL-13, are important in asthma. Micro-RNAs (miRNAs) regulate cell differentiation in many systems and could contribute to epithelial abnormalities in asthma. Objectives: To determine whether airway epithelial miRNA expression is altered in asthma and identify IL-13–regulated miRNAs. Methods: We used miRNA microarrays to analyze bronchial epithelial brushings from 16 steroid-naive subjects with asthma before and after inhaled corticosteroids, 19 steroid-using subjects with asthma, and 12 healthy control subjects, and the effects of IL-13 and corticosteroids on cultured bronchial epithelial cells. We used quantitative polymerase chain reaction to confirm selected microarray results. Measurements and Main Results: Most (12 of 16) steroid-naive subjects with asthma had a markedly abnormal pattern of bronchial epithelial miRNA expression by microarray analysis. Compared with control subjects, 217 miRNAs were differentially expressed in steroid-naive subjects with asthma and 200 in steroid-using subjects with asthma (false discovery rate < 0.05). Treatment with inhaled corticosteroids had modest effects on miRNA expression in steroid-naive asthma, inducing a statistically significant (false discovery rate < 0.05) change for only nine miRNAs. qPCR analysis confirmed differential expression of 22 miRNAs that were highly differentially expressed by microarrays. IL-13 stimulation recapitulated changes in many differentially expressed miRNAs, including four members of the miR-34/449 family, and these changes in miR-34/449 family members were resistant to corticosteroids. Conclusions: Dramatic alterations of airway epithelial cell miRNA levels are a common feature of asthma. These alterations are only modestly corrected by inhaled corticosteroids. IL-13 effects may account for some of these alterations, including repression of miR-34/449 family members that have established roles in airway

  20. Gene expression networks.

    PubMed

    Thomas, Reuben; Portier, Christopher J

    2013-01-01

    With the advent of microarrays and next-generation biotechnologies, the use of gene expression data has become ubiquitous in biological research. One potential drawback of these data is that they are very rich in features or genes though cost considerations allow for the use of only relatively small sample sizes. A useful way of getting at biologically meaningful interpretations of the environmental or toxicological condition of interest would be to make inferences at the level of a priori defined biochemical pathways or networks of interacting genes or proteins that are known to perform certain biological functions. This chapter describes approaches taken in the literature to make such inferences at the biochemical pathway level. In addition this chapter describes approaches to create hypotheses on genes playing important roles in response to a treatment, using organism level gene coexpression or protein-protein interaction networks. Also, approaches to reverse engineer gene networks or methods that seek to identify novel interactions between genes are described. Given the relatively small sample numbers typically available, these reverse engineering approaches are generally useful in inferring interactions only among a relatively small or an order 10 number of genes. Finally, given the vast amounts of publicly available gene expression data from different sources, this chapter summarizes the important sources of these data and characteristics of these sources or databases. In line with the overall aims of this book of providing practical knowledge to a researcher interested in analyzing gene expression data from a network perspective, the chapter provides convenient publicly accessible tools for performing analyses described, and in addition describe three motivating examples taken from the published literature that illustrate some of the relevant analyses. PMID:23086841

  1. Expression of IL-4/IL-13 receptors in differentiating human airway epithelial cells.

    PubMed

    White, Steven R; Martin, Linda D; Stern, Randi; Laxman, Bharathi; Marroquin, Bertha A

    2010-11-01

    IL-4 and IL-13 elicit several important responses in airway epithelium including chemokine secretion and mucous secretion that may contribute to airway inflammation, cell migration, and differentiation. These cytokines have overlapping but not identical effector profiles likely due to shared subunits in their receptor complexes. These receptors are variably described in epithelial cells, and the relative expression, localization, and function of these receptors in differentiated and repairing epithelial cells are not clear. We examined IL-4/IL-13 receptor expression and localization in primary airway epithelial cells collected from normal human lungs and grown under conditions yielding both undifferentiated and differentiated cells inclusive of basal, goblet, and ciliated cell phenotypes. Gene expression of the IL-4Rα, IL-2Rγc, IL-13Rα1, and IL-13Rα2 receptor subunits increased with differentiation, but different patterns of localization and protein abundance were seen for each subunit based on both differentiation and the cell subtypes present. Increased expression of receptor subunits observed in more differentiated cells was associated with more substantial functional responses to IL-4 stimulation including increased eotaxin-3 expression and accelerated migration after injury. We demonstrate substantial differences in IL-4/IL-13 receptor subunit expression and responsiveness to IL-4 based on the extent of airway epithelial cell differentiation and suggest that these differences may have functional consequences in airway inflammation. PMID:20729386

  2. Claudin-7 expression induces mesenchymal to epithelial transformation (MET) to inhibit colon tumorigenesis.

    PubMed

    Bhat, A A; Pope, J L; Smith, J J; Ahmad, R; Chen, X; Washington, M K; Beauchamp, R D; Singh, A B; Dhawan, P

    2015-08-27

    In normal colon, claudin-7 is one of the highly expressed claudin proteins and its knockdown in mice results in altered epithelial cell homeostasis and neonatal death. Notably, dysregulation of the epithelial homeostasis potentiates oncogenic transformation and growth. However, the role of claudin-7 in the regulation of colon tumorigenesis remains poorly understood. Using a large colorectal cancer (CRC) patient database and mouse models of colon cancer, we found claudin-7 expression to be significantly downregulated in cancer samples. Most notably, forced claudin-7 expression in poorly differentiated and highly metastatic SW620 colon cancer cells induced epithelial characteristics and inhibited their growth in soft agar and tumor growth in vivo. By contrast, knockdown of claudin-7 in HT-29 or DLD-1 cells induced epithelial-to-mesenchymal transition (EMT), colony formation, xenograft-tumor growth in athymic mice and invasion. Importantly, a claudin-7 signature gene profile generated by overlapping the DEGs (differentially expressed genes in a high-throughput transcriptome analysis using claudin-7-manipulated cells) with human claudin-7 signature genes identified high-risk CRC patients. Furthermore, Rab25, a colon cancer suppressor and regulator of the polarized cell trafficking constituted one of the highly upregulated DEGs in claudin-7 overexpressing cells. Notably, silencing of Rab25 expression counteracted the effects of claudin-7 expression and not only increased proliferation and cell invasion but also increased the expression of p-Src and mitogen-activated protein kinase-extracellular signal-regulated kinase 1/2 that were suppressed upon claudin-7 overexpression. Of interest, CRC cell lines, which exhibited decreased claudin-7 expression, also exhibited promoter DNA hypermethylation, a modification associated with transcriptional silencing. Taken together, our data demonstrate a previously undescribed role of claudin-7 as a colon cancer suppressor and suggest

  3. Differential Expression of Wnt Signaling Molecules Between Pre- and Postmenopausal Endometrial Epithelial Cells Suggests a Population of Putative Epithelial Stem/Progenitor Cells Reside in the Basalis Layer

    PubMed Central

    Nguyen, Hong P. T.; Sprung, Carl N.

    2012-01-01

    The human endometrium undergoes extensive monthly regeneration in response to fluctuating levels of circulating estrogen and progesterone in premenopausal (Pre-M) women. In contrast, postmenopausal (Post-M) endometrium is thin and quiescent with low mitotic activity, similar to the Pre-M endometrial basalis layer. Clonogenic epithelial stem/progenitor (ESP) cells, likely responsible for regenerating endometrial epithelium, have been identified in Pre-M and Post-M endometrium, but their location is unknown. We undertook transcriptional profiling of highly purified epithelial cells from full-thickness Pre-M and Post-M endometrium to identify differentially regulated genes that may indicate a putative ESP cell population resides in the basalis of Pre-M and basalis-like Post-M endometrium. Of 1077 differentially expressed genes identified, the Wnt signaling pathway, important in endometrial development and stem cell regulation, was one of the main gene families detected, including 22 Wnt-associated genes. Twelve genes were validated using quantitative RT-PCR, and all were concordant with microarray data. Immunostaining showed glandular epithelial location of Wnt-regulated genes, Axin-related protein 2 and β-catenin. Axin2 localized to the nucleus of basalis Pre-M and Post-M and cytoplasm of functionalis Pre-M endometrium, suggesting that it regulates β-catenin. Comparison of our Post-M gene profile with published gene microarray datasets revealed similarities to Pre-M basalis epithelial profiles. This differential expression of multiple Wnt-associated genes in human Pre-M and Post-M endometrial epithelial cells and the similar gene profile of Post-M and Pre-M basalis epithelium suggests that a population of putative endometrial ESP may reside in the basalis of Pre-M endometrium, which may be responsible for regenerating glandular epithelium each month. PMID:22474188

  4. Defining suitable reference genes for RT-qPCR analysis on intestinal epithelial cells.

    PubMed

    Sirakov, Maria; Borra, Marco; Cambuli, Francesca Maria; Plateroti, Michelina

    2013-07-01

    The study of the mammalian intestinal epithelium concerns several aspects of cellular and molecular biology. In fact, most of these studies aim to define molecular components or mechanisms related with the control of stemness and the balance between cell proliferation and differentiation in physiopathological conditions. It is worth mentioning that real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) approaches are commonly used, but only a few studies are available regarding suitable reference genes to normalize gene expression data. The present study was designed to validate potential reference genes in freshly isolated proliferating or differentiated epithelial cells from the mouse intestine. We also extended our analysis to the IEC6 intestinal epithelial cells, as a promising model to study intestinal physiopathology in vitro. The stability of six potential reference genes (Hprt1, Ppia, Gapdh, Rplp0, Ppib, and Vil1) has been tested both in epithelial cells isolated from the mouse intestine and in the IEC6 cell line. The software programs-geNorm and Normfinder-were used to obtain an estimation of the expression stability of each gene and, by comparing the results, to identify the most suitable genes for RT-qPCR data normalization. These multiple approaches allowed us to select different suitable reference genes for the correct quantification of mRNAs depending on the differentiated or proliferative nature of the cells. PMID:23292893

  5. HNF4α Regulates Claudin-7 Protein Expression during Intestinal Epithelial Differentiation

    PubMed Central

    Farkas, Attila E.; Hilgarth, Roland S.; Capaldo, Christopher T.; Gerner-Smidt, Christian; Powell, Doris R.; Vertino, Paula M.; Koval, Michael; Parkos, Charles A.; Nusrat, Asma

    2016-01-01

    The intestinal epithelium is a dynamic barrier that maintains the distinct environments of intestinal tissue and lumen. Epithelial barrier function is defined principally by tight junctions, which, in turn, depend on the regulated expression of claudin family proteins. Claudins are expressed differentially during intestinal epithelial cell (IEC) differentiation. However, regulatory mechanisms governing claudin expression during epithelial differentiation are incompletely understood. We investigated the molecular mechanisms regulating claudin-7 during IEC differentiation. Claudin-7 expression is increased as epithelial cells differentiate along the intestinal crypt–luminal axis. By using model IECs we observed increased claudin-7 mRNA and nascent heteronuclear RNA levels during differentiation. A screen for potential regulators of the CLDN7 gene during IEC differentiation was performed using a transcription factor/DNA binding array, CLDN7 luciferase reporters, and in silico promoter analysis. We identified hepatocyte nuclear factor 4α as a regulatory factor that bound endogenous CLDN7 promoter in differentiating IECs and stimulated CLDN7 promoter activity. These findings support a role of hepatocyte nuclear factor 4α in controlling claudin-7 expression during IEC differentiation. PMID:26216285

  6. Biological role and clinical implications of homeobox genes in serous epithelial ovarian cancer.

    PubMed

    Miller, Katherine R; Patel, Jai N; Ganapathi, Mahrukh K; Tait, David L; Ganapathi, Ram N

    2016-06-01

    Homeobox (HOX) genes are a family of transcription factors that are essential regulators of development. HOX genes play important roles in normal reproductive physiology, as well as in the development and progression of serous carcinomas, the predominant and most aggressive subtype of epithelial ovarian cancer (EOC). This review discusses aberrant HOX gene expression in serous EOC and its impact on tumor development and progression. Further identification of HOX target genes may facilitate the development of novel diagnostic and therapeutic strategies to improve the prognosis of patients with serous EOC. PMID:26957480

  7. Phenotypic determination of epithelial appendages: genes, developmental pathways, and evolution.

    PubMed

    Chuong, C M; Noveen, A

    1999-12-01

    Epithelial appendages are derivatives of epithelia that elaborate to form specialized structures and functions. The appendage can protrude out, such as in teeth and feathers, or invaginate in, such as in glands. The epithelia can be ectodermal, such as in hairs, or endodermal, such as in livers. Using feather as a prototype of epithelial appendage, we study the molecular signals involved in the successive stages of epithelial-mesenchymal interactions during morphogenesis. We propose that these form the basics of gene networks, which can be integrated to gene supernetwork and totinetwork. Because the unit of development is molecular pathway rather than single molecule, and the unit of morphogenesis is cell group rather than single cell, we make the analogy between genes/developmental pathways and words/sentences. The study of developmental pathways in epithelial appendage organogenesis will help us to understand the grammar of genes and the basic rules in constructing regulated new growth. This knowledge may contribute to the study of cancer biology (deregulated new growth) and organ regeneration. PMID:10674387

  8. Defective DNA methylation in salivary gland epithelial acini from patients with Sjögren's syndrome is associated with SSB gene expression, anti-SSB/LA detection, and lymphocyte infiltration.

    PubMed

    Konsta, O D; Le Dantec, C; Charras, A; Cornec, D; Kapsogeorgou, E K; Tzioufas, A G; Pers, J O; Renaudineau, Y

    2016-04-01

    The pathogenesis of primary Sjögren's syndrome (pSS) is complex, in part due to DNA methylation abnormalities. This study was undertaken to evaluate the importance of global DNA methylation ((5m)C) as determined in minor salivary glands (MSG) from well characterized pSS patients. Twenty-two pSS patients and ten controls were selected, and MSG were stained with anti-(5m)C, anti-(5m)C/anti-cytokeratin (KRT)19, or with anti-SSB/La antibodies (Ab). The DNA methylation status at the SSB gene promoter P1 and P1' was evaluated by methylation-sensitive restriction enzymes (MSRE) coupled with PCR. The effect of the DNA demethylating drug 5 azacytidine (5-Aza) was tested in the human salivary gland (HSG) cell line. In pSS, the reduction of global DNA methylation ((5m)C) was associated with lymphocyte infiltration, the emergence of (5m)C(low) and KRT19(high) acini, and the detection of circulating anti-SSB/La Ab, but not with disease activity (ESSDAI). Next, treating HSG cells with 5-Aza was effective in inducing SSB expression. Finally in pSS patients positive for anti-SSB/La Ab, we further observed DNA demethylation at the SSB gene promoter P1 with consequent SSB overexpression at both the transcriptional and protein levels in salivary gland epithelial cells. In conclusion, our results highlight the importance of DNA methylation in the pathophysiology of pSS and to the emergence of anti-SSB/La Ab. PMID:26725749

  9. Genes involved in TGFβ1-driven epithelial-mesenchymal transition of renal epithelial cells are topologically related in the human interactome map

    PubMed Central

    Campanaro, Stefano; Picelli, Simone; Torregrossa, Rossella; Colluto, Laura; Ceol, Monica; Del Prete, Dorella; D'Angelo, Angela; Valle, Giorgio; Anglani, Franca

    2007-01-01

    Background Understanding how mesenchymal cells arise from epithelial cells could have a strong impact in unveiling mechanisms of epithelial cell plasticity underlying kidney regeneration and repair. In primary human tubular epithelial cells (HUTEC) under different TGFβ1 concentrations we had observed epithelial-to-mesenchymal transition (EMT) but not epithelial-myofibroblast transdifferentiation. We hypothesized that the process triggered by TGFβ1 could be a dedifferentiation event. The purpose of this study is to comprehensively delineate genetic programs associated with TGFβ1-driven EMT in our in vitro model using gene expression profile on large-scale oligonucleotide microarrays. Results In HUTEC under TGFβ1 stimulus, 977 genes were found differentially expressed. Thirty genes were identified whose expression depended directly on TGFβ1 concentration. By mapping the differentially expressed genes in the Human Interactome Map using Cytoscape software, we identified a single scale-free network consisting of 2630 interacting proteins and containing 449 differentially expressed proteins. We identified 27 hub proteins in the interactome with more than 29 edges incident on them and encoded by differentially expressed genes. The Gene Ontology analysis showed an excess of up-regulated proteins involved in biological processes, such as "morphogenesis", "cell fate determination" and "regulation of development", and the most up-regulated genes belonged to these categories. In addition, 267 genes were mapped to the KEGG pathways and 14 pathways with more than nine differentially expressed genes were identified. In our model, Smad signaling was not the TGFβ1 action effector; instead, the engagement of RAS/MAPK signaling pathway seems mainly to regulate genes involved in the cell cycle and proliferation/apoptosis. Conclusion Our present findings support the hypothesis that context-dependent EMT generated in our model by TGFβ1 might be the outcome of a dedifferentiation

  10. Expression of SDF-1 and CXCR4 transcript variants and CXCR7 in epithelial ovarian cancer

    PubMed Central

    JASZCZYNSKA-NOWINKA, KAROLINA; RUCINSKI, MARCIN; ZIOLKOWSKA, AGNIESZKA; MARKOWSKA, ANNA; MALENDOWICZ, LUDWIK K.

    2014-01-01

    Chemokine stromal cell-derived factor-1 (SDF-1) and its receptors, CXCR4 and CXCR7, have been implicated in epithelial ovarian cancer progression and metastasis. However, limited data are available on the expression levels of SDF-1 and CXCR4 variants and CXCR7 in human epithelial ovarian cancer. The present study aimed to characterize the expression pattern and levels of SDF-1, CXCR4 and CXCR7 in normal human ovaries and epithelial ovarian cancer. The expression of SDF-1 and CXCR4 transcript variants and CXCR7 was determined by quantitative polymerase chain reaction (qPCR). Plasma SDF-1α levels were determined by commercially available EIA kits and cancer antigen 125 (CA 125) levels were quantified by automated microparticle enzyme immunosorbent assay. High expression levels of SDF-1 transcript variant 1 were identified in ovarian cancer and control ovaries. By contrast, in both groups the expression levels of SDF-1 transcript variants 3 and 4 were extremely low. Furthermore, SDF-1 variant 1 levels were notably higher in epithelial ovarian cancer than in control ovaries, while data for the remaining transcripts were similar in both groups. CXCR4 transcript variant 2 and CXCR7 expression levels in normal and neoplastic ovaries were similar. In both groups, CXCR4 transcript variant 2 was not detected. Plasma SDF-1α levels were notably higher in females with epithelial ovarian cancer than in the control ovaries. Elevated levels of blood SDF-1α were found prior to surgery, 6 days after surgery and following completion of the first chemotherapy course. These increases were independent of the type of epithelial ovarian cancer. Our results suggest that the expression of SDF-1 and the genes controlling alternative splicing are elevated in epithelial ovarian cancer, leading to an increased formation of SDF-1 variant 1. Elevated plasma SDF-1α levels in epithelial ovarian cancer patients are not associated with the presence of tumors and/or metastases, however reflect a

  11. Expression of SDF-1 and CXCR4 transcript variants and CXCR7 in epithelial ovarian cancer.

    PubMed

    Jaszczynska-Nowinka, Karolina; Rucinski, Marcin; Ziolkowska, Agnieszka; Markowska, Anna; Malendowicz, Ludwik K

    2014-05-01

    Chemokine stromal cell-derived factor-1 (SDF-1) and its receptors, CXCR4 and CXCR7, have been implicated in epithelial ovarian cancer progression and metastasis. However, limited data are available on the expression levels of SDF-1 and CXCR4 variants and CXCR7 in human epithelial ovarian cancer. The present study aimed to characterize the expression pattern and levels of SDF-1, CXCR4 and CXCR7 in normal human ovaries and epithelial ovarian cancer. The expression of SDF-1 and CXCR4 transcript variants and CXCR7 was determined by quantitative polymerase chain reaction (qPCR). Plasma SDF-1α levels were determined by commercially available EIA kits and cancer antigen 125 (CA 125) levels were quantified by automated microparticle enzyme immunosorbent assay. High expression levels of SDF-1 transcript variant 1 were identified in ovarian cancer and control ovaries. By contrast, in both groups the expression levels of SDF-1 transcript variants 3 and 4 were extremely low. Furthermore, SDF-1 variant 1 levels were notably higher in epithelial ovarian cancer than in control ovaries, while data for the remaining transcripts were similar in both groups. CXCR4 transcript variant 2 and CXCR7 expression levels in normal and neoplastic ovaries were similar. In both groups, CXCR4 transcript variant 2 was not detected. Plasma SDF-1α levels were notably higher in females with epithelial ovarian cancer than in the control ovaries. Elevated levels of blood SDF-1α were found prior to surgery, 6 days after surgery and following completion of the first chemotherapy course. These increases were independent of the type of epithelial ovarian cancer. Our results suggest that the expression of SDF-1 and the genes controlling alternative splicing are elevated in epithelial ovarian cancer, leading to an increased formation of SDF-1 variant 1. Elevated plasma SDF-1α levels in epithelial ovarian cancer patients are not associated with the presence of tumors and/or metastases, however reflect a

  12. Expression of Opacity Proteins Interferes with the Transmigration of Neisseria gonorrhoeae across Polarized Epithelial Cells.

    PubMed

    Stein, Daniel C; LeVan, Adriana; Hardy, Britney; Wang, Liang-Chun; Zimmerman, Lindsey; Song, Wenxia

    2015-01-01

    Neisseria gonorrhoeae (GC) establishes infection at the mucosal surface of the human genital tract, most of which is lined with polarized epithelial cells. GC can cause localized as well as disseminated infections, leading to various complications. GC constantly change their surface structures via phase and antigenic variation, which has been implicated as a means for GC to establish infection at various anatomic locations of male and female genital tracks. However, the exact contribution of each surface molecule to bacterial infectivity remains elusive due to their phase variation. Using a GC derivative that is genetically devoid of all opa genes (MS11∆Opa), this study shows that Opa expression interferes with GC transmigration across polarized human epithelial cells. MS11∆Opa transmigrates across polarized epithelial cells much faster and to a greater extent than MS11Opa+, while adhering at a similar level as MS11Opa+. When MS11Opa+, able to phase vary Opa expression, was inoculated, only those bacteria that turn off Opa expression transmigrate across the polarized epithelial monolayer. Similar to bacteria alone or co-cultured with non-polarized epithelial cells, MS11∆Opa fails to form large microcolonies at the apical surface of polarized epithelial cells. Apical inoculation of MS11Opa+, but not MS11∆Opa, induces the recruitment of the Opa host-cell receptor carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to the apical junction and the vicinity of bacterial adherent sites. Our results suggest that Opa expression limits gonococcal ability to invade into subepithelial tissues by forming tight interactions with neighboring bacteria and by inducing CEACAM redistribution to cell junctions. PMID:26244560

  13. Expression of Opacity Proteins Interferes with the Transmigration of Neisseria gonorrhoeae across Polarized Epithelial Cells

    PubMed Central

    Stein, Daniel C.; LeVan, Adriana; Hardy, Britney; Wang, Liang-Chun; Zimmerman, Lindsey; Song, Wenxia

    2015-01-01

    Neisseria gonorrhoeae (GC) establishes infection at the mucosal surface of the human genital tract, most of which is lined with polarized epithelial cells. GC can cause localized as well as disseminated infections, leading to various complications. GC constantly change their surface structures via phase and antigenic variation, which has been implicated as a means for GC to establish infection at various anatomic locations of male and female genital tracks. However, the exact contribution of each surface molecule to bacterial infectivity remains elusive due to their phase variation. Using a GC derivative that is genetically devoid of all opa genes (MS11∆Opa), this study shows that Opa expression interferes with GC transmigration across polarized human epithelial cells. MS11∆Opa transmigrates across polarized epithelial cells much faster and to a greater extent than MS11Opa+, while adhering at a similar level as MS11Opa+. When MS11Opa+, able to phase vary Opa expression, was inoculated, only those bacteria that turn off Opa expression transmigrate across the polarized epithelial monolayer. Similar to bacteria alone or co-cultured with non-polarized epithelial cells, MS11∆Opa fails to form large microcolonies at the apical surface of polarized epithelial cells. Apical inoculation of MS11Opa+, but not MS11∆Opa, induces the recruitment of the Opa host-cell receptor carcinoembryonic antigen–related cell adhesion molecules (CEACAMs) to the apical junction and the vicinity of bacterial adherent sites. Our results suggest that Opa expression limits gonococcal ability to invade into subepithelial tissues by forming tight interactions with neighboring bacteria and by inducing CEACAM redistribution to cell junctions. PMID:26244560

  14. Regulator of G-protein signalling 2 mRNA is differentially expressed in mammary epithelial subpopulations and over-expressed in the majority of breast cancers

    PubMed Central

    Smalley, Matthew J; Iravani, Marjan; Leao, Maria; Grigoriadis, Anita; Kendrick, Howard; Dexter, Tim; Fenwick, Kerry; Regan, Joseph L; Britt, Kara; McDonald, Sarah; Lord, Christopher J; MacKay, Alan; Ashworth, Alan

    2007-01-01

    Introduction To understand which signalling pathways become deregulated in breast cancer, it is necessary to identify functionally significant gene expression patterns in the stem, progenitor, transit amplifying and differentiated cells of the mammary epithelium. We have previously used the markers 33A10, CD24 and Sca-1 to identify mouse mammary epithelial cell subpopulations. We now investigate the relationship between cells expressing these markers and use gene expression microarray analysis to identify genes differentially expressed in the cell populations. Methods Freshly isolated primary mouse mammary epithelial cells were separated on the basis of staining with the 33A10 antibody and an α-Sca-1 antibody. The populations identified were profiled using gene expression microarray analysis. Gene expression patterns were confirmed on normal mouse and human mammary epithelial subpopulations and were examined in a panel of breast cancer samples and cell lines. Results Analysis of the separated populations demonstrated that Sca-1- 33A10High stained cells were estrogen receptor α (Esr1)- luminal epithelial cells, whereas Sca-1+ 33A10Low/- stained cells were a mix of nonepithelial cells and Esr1+ epithelial cells. Analysis of the gene expression data identified the gene Rgs2 (regulator of G-protein signalling 2) as being highly expressed in the Sca-1- 33A10Low/- population, which included myoepithelial/basal cells. RGS2 has previously been described as a regulator of angiotensin II receptor signalling. Gene expression analysis by quantitative real-time RT-PCR of cells separated on the basis of CD24 and Sca-1 expression confirmed that Rgs2 was more highly expressed in mouse myoepithelial/basal mammary cells than luminal cells. This expression pattern was conserved in normal human breast cells. Functional analysis demonstrated RGS2 to be a modulator of oxytocin receptor signalling. The potential significance of RGS2 expression in breast cancer was demonstrated by semi

  15. CXCL12 expression by healthy and malignant ovarian epithelial cells

    PubMed Central

    2011-01-01

    Background CXCL12 has been widely reported to play a biologically relevant role in tumor growth and spread. In epithelial ovarian cancer (EOC), CXCL12 enhances tumor angiogenesis and contributes to the immunosuppressive network. However, its prognostic significance remains unclear. We thus compared CXCL12 status in healthy and malignant ovaries, to assess its prognostic value. Methods Immunohistochemistry was used to analyze CXCL12 expression in the reproductive tracts, including the ovaries and fallopian tubes, of healthy women, in benign and borderline epithelial tumors, and in a series of 183 tumor specimens from patients with advanced primary EOC enrolled in a multicenter prospective clinical trial of paclitaxel/carboplatin/gemcitabine-based chemotherapy (GINECO study). Univariate COX model analysis was performed to assess the prognostic value of clinical and biological variables. Kaplan-Meier methods were used to generate progression-free and overall survival curves. Results Epithelial cells from the surface of the ovary and the fallopian tubes stained positive for CXCL12, whereas the follicles within the ovary did not. Epithelial cells in benign, borderline and malignant tumors also expressed CXCL12. In EOC specimens, CXCL12 immunoreactivity was observed mostly in epithelial tumor cells. The intensity of the signal obtained ranged from strong in 86 cases (47%) to absent in 18 cases (<10%). This uneven distribution of CXCL12 did not reflect the morphological heterogeneity of EOC. CXCL12 expression levels were not correlated with any of the clinical parameters currently used to determine EOC prognosis or with HER2 status. They also had no impact on progression-free or overall survival. Conclusion Our findings highlight the previously unappreciated constitutive expression of CXCL12 on healthy epithelia of the ovary surface and fallopian tubes, indicating that EOC may originate from either of these epithelia. We reveal that CXCL12 production by malignant

  16. PAX8 Expression in Ovarian Surface Epithelial Cells

    PubMed Central

    Adler, Emily; Mhawech-Fauceglia, Paulette; Gayther, Simon A; Lawrenson, Kate

    2015-01-01

    High-grade serous ovarian carcinoma (HGSOC) is usually diagnosed at a late stage and is associated with poor prognosis. Understanding early stage disease biology is essential in developing clinical biomarkers to detect HGSOC earlier. While recent studies indicate that HGSOCs arise from fallopian tube secretory epithelial cells (FTSECs), a considerable body of evidence also suggests that HGSOC can also arise from ovarian surface epithelial cells (OSECs). PAX8 is overexpressed in HGSOCs and expressed in FTSECs, but there are conflicting reports about PAX8 expression in OSECs. The purpose of this study was to comprehensively characterize PAX8 expression in a large series of OSECs, and to investigate the role of PAX8 in early HGSOC development. PAX8 protein expression was analyzed in the OSECs of 27 normal ovaries and 7 primary OSEC cultures using immunohistochemistry and immunofluorescent cytochemistry. PAX8 mRNA expression was quantified in 66 primary OSEC cultures. Cellular transformation was evaluated in OSECs expressing a PAX8 construct. PAX8 was expressed by 44-71% of OSECs. Calretinin and E-cadherin were frequently co-expressed with PAX8. Expression of PAX8 in OSECs decreased cellular migration (P=0.028), but had no other effects on cellular transformation. In addition, PAX8 expression was significantly increased (P=0.003) in an in vitro stepwise model of neoplastic transformation. In conclusion, PAX8 is frequently expressed by OSECs and endogenous levels of PAX8 expression are non-transforming. These data indicate that in OSECs PAX8 expression may represent a normal state and that OSECs may represent an origin of HGSOCs. PMID:26079312

  17. The CDX2 transcription factor regulates furin expression during intestinal epithelial cell differentiation.

    PubMed

    Gendron, Fernand-Pierre; Mongrain, Sébastien; Laprise, Patrick; McMahon, Stéphanie; Dubois, Claire M; Blais, Mylène; Asselin, Claude; Rivard, Nathalie

    2006-02-01

    CDX2, a member of the caudal family of transcription factors, is involved in enterocyte lineage specification. CDX2 activates many intestine-specific genes, such as sucrase-isomaltase and lactase-phlorizin hydrolase (LPH), and adhesion proteins, namely, LI-cadherin and claudin-2. In this study, we show that the proprotein convertase furin, involved in proteolytic maturation of proprotein substrates including LPH and cell surface proteins, is a CDX2 target. Indeed, expression of the rat furin homolog was induced 1.5-fold, as determined by microarray experiments that compared control with CDX2-expressing intestinal epithelial cells (IEC-6). As determined by transient transfection assays in Caco-2/15 cells, the furin P1 promoter 1.3-kb fragment between SacI and NheI was essential for CDX2 transcriptional activation. Electrophoretic mobility shift/supershift assays followed by site-specific mutagenesis and chromatin immunoprecipitation identified the CDX DNA-binding site (CBS)2 sequence from nt -1827 to -1821 as the major CBS involved in furin P1 promoter activation. Increased furin mRNA and protein expression correlated with both CDX2 expression and intestinal epithelial cell differentiation. In addition, furin mRNAs were detected predominantly in differentiated epithelial cells of the villus, as determined by in situ hybridization. Treatment of Caco-2/15 cells with a furin inhibitor led to inhibition of LPH activity. Morphological differentiation of enterocyte-like features in Caco-2/15 such as epithelial cell polarity and brush-border formation were strongly attenuated by furin inhibition. These results suggest that CDX2 regulates furin expression in intestinal epithelial cells. Furin may be important in modulating the maturation and/or activation of key factors involved in enterocyte differentiation. PMID:16239403

  18. MicroRNA-221 controls expression of intercellular adhesion molecule-1 in epithelial cells in response to Cryptosporidium parvum infection

    PubMed Central

    Gong, Ai-Yu; Hu, Guoku; Zhou, Rui; Liu, Jun; Feng, Yaoyu; Soukup, Garrett A.; Chen, Xian-Ming

    2011-01-01

    Cryptosporidium parvum is a protozoan parasite that infects gastrointestinal epithelial cells and causes diarrheal disease in humans and animals globally. Pathological changes following C. parvum infection include crypt hyperplasia, a modest inflammatory reaction with increased infiltration of lymphocytes into intestinal mucosa. Expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), on infected epithelial cell surfaces may facilitate adhesion and recognition of lymphocytes at infection sites. MicroRNAs (miRNAs) are small RNA molecules of 23 nucleotides that negatively regulate protein-coding gene expression via translational suppression or mRNA degradation. We recently reported that microRNA-221 (miR-221) regulates ICAM-1 translation through targeting the ICAM-1 3′-untranslated region (UTR). In this study, we tested the role of miR-221 in regulating ICAM-1 expression in epithelial cells in response to C. parvum infection using an in vitro model of human biliary cryptosporidiosis. Up-regulation of ICAM-1 at both message and protein levels was detected in epithelial cells following C. parvum infection. Inhibition of ICAM-1 transcription with actinomycin D could only partially block C. parvum-induced ICAM-1 expression at the protein level. Cryptosporidium parvum infection decreased miR-221 expression in infected epithelial cells. When cells were transfected with a luciferase reporter construct covering the miR-221 binding site in the ICAM-1 3′-UTR and then exposed to C. parvum, an enhanced luciferase activity was detected. Transfection of miR-221 precursor abolished C. parvum-stimulated ICAM-1 protein expression. In addition, expression of ICAM-1 on infected epithelial cells facilitated epithelial adherence of co-cultured Jurkat cells. These results indicate that miR-221-mediated translational suppression controls ICAM-1 expression in epithelial cells in response to C. parvum infection. PMID:21236259

  19. Helicobacter pylori induced interleukin-8 expression in gastric epithelial cells is associated with CagA positive phenotype.

    PubMed Central

    Crabtree, J E; Covacci, A; Farmery, S M; Xiang, Z; Tompkins, D S; Perry, S; Lindley, I J; Rappuoli, R

    1995-01-01

    AIMS--To use a range of natural phenotypically variant strains of Helicobacter pylori with disparate CagA and VacA (vacuolating cytotoxin) expression to determine which bacterial factors are more closely associated with epithelial interleukin-8 (IL-8) induction. METHODS--Gastric epithelial cells (AGS and KATO-3) were co-cultured with five H pylori strains which were variously shown to express the cagA gene/CagA protein, VacA and/or to exhibit biological cytotoxicity. Secreted IL-8 was assayed by enzyme leaked immunosorbent assay (ELISA) and IL-8 messenger RNA (mRNA) was assayed using a reverse transcription polymerase chain reaction based technique (RT-PCR). RESULTS--Strains expressing CagA, including a variant strain (D931) which is non-cytotoxic and does not express the VacA protein, were found to upregulate epithelial IL-8 secretion and gene expression. In contrast, strains with no CagA expression, even in the presence of VacA and/or biological cytotoxicity, (G104, BA142), failed to induce IL-8 protein or mRNA above control values. CONCLUSIONS--These results strongly support a role for H pylori CagA or coexpressed factors other than the cytotoxin in upregulation of gastric epithelial IL-8. Increased epithelial IL-8 secretion and concomitant neutrophil chemotaxis and activation in addition to direct cytotoxicity may be an important factor in tissue damage and ulceration. Images PMID:7706517

  20. Genes Regulating Epithelial Polarity Are Critical Suppressors of Esophageal Oncogenesis

    PubMed Central

    Li, Xiu-Min; Wang, Hui; Zhu, Li-Li; Zhao, Run-Zhen; Ji, Hong-Long

    2015-01-01

    Esophageal cancer is an aggressive disease featured by early lymphatic and hematogenous dissemination, and is the sixth leading cause of cancer-related deaths worldwide. The proper formation of apicobasal polarity is essential for normal epithelium physiology and tissue homeostasis, while loss of polarity is a hallmark of cancer development including esophageal oncogenesis. In this review, we summarized the stages of esophageal cancer development associated with the loss or deregulation of epithelial cell apicobasal polarity. Loss of epithelial apicobasal polarity exerts an indispensable role in the initiation of esophageal oncogenesis, tumor progression, and the advancement of tumors from benign to malignant. In particular, we reviewed the involvement of several critical genes, including Lkb1, claudin-4, claudin-7, Par3, Lgl1, E-cadherin, and the Scnn1 gene family. Understanding the role of apicobasal regulators may lead to new paradigms for treatment of esophageal tumors, including improvement of prognostication, early diagnosis, and individually tailored therapeutic interventions in esophageal oncology. PMID:26185530

  1. Expression of epithelial adhesion proteins and integrins in chronic inflammation.

    PubMed Central

    Haapasalmi, K.; Mäkelä, M.; Oksala, O.; Heino, J.; Yamada, K. M.; Uitto, V. J.; Larjava, H.

    1995-01-01

    Epithelial cell behavior in chronic inflammation is poorly characterized. During inflammation of tooth-supporting structures (periodontal disease), increased proliferation of epithelial cells into the inflamed connective tissue stroma is commonly seen. In some areas ulceration and degeneration take place. We studied alterations in the expression of adhesion molecules and integrins during chronic periodontal inflammation. In inflamed tissue, laminin-1 and type IV collagen were still present in the basement membrane and surrounding blood vessels, but they were also found extravascularly in inflamed connective tissue stroma. Type VII collagen and laminin-5 (also known as kalinin, epiligrin, or nicein) were poorly preserved in the basement membrane zone, but both were found in unusual streak-like distributions in the subepithelial connective tissue stroma in inflamed tissue. Both fibronectin and tenascin were substantially decreased in chronically inflamed connective tissue, showing only punctate staining at the basement membrane zone. Integrins of the beta 1 family showed two distinct staining patterns in epithelial cells during chronic inflammation; focal losses of beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1) were found in most areas, while in other areas the entire pocket epithelium was found to be strongly positive for beta 1 integrins. No members of the alpha v integrin family were found in any epithelia studied. Expression of the alpha 6 beta 4 integrin was high in basal cells of healthy tissue, but weak in epithelium associated with chronic inflammation. Chronic inflammation therefore involves alterations in both adhesion proteins and integrins expressed by epithelial cells. Basement membrane components found at abnormal sites in stroma in chronic inflammation might serve as new adhesive ligands for various cell types in inflamed stroma. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:7541610

  2. Trichostatin A inhibits beta-casein expression in mammary epithelial cells

    SciTech Connect

    Pujuguet, Philippe; Radisky, Derek; Levy, Dinah; Lacza, Charlemagne; Bissell, Mina J.

    2002-02-22

    Many aspects of cellular behavior are affected by information derived from association of the extracellular matrix (ECM) and with cell membrane receptors. When cultured in the presence of laminin-containing ECM and prolactin (Prl), normal mammary epithelial cells express the milk protein beta-casein. Previously, we defined the minimal ECM- and Prl-responsive enhancer element BCE-1 from the upstream region of the beta-casein gene. We also found that BCE-1 was only active when stably integrated into chromatin, and that trichostatin A (TSA), a reagent that leads to alterations in chromatin structure, was able to activate the integrated enhancer element. We now show that endogenous b-casein gene, which is controlled by a genetic assembly that is highly similar to that of BCE-1 and which is also activated by incubation in ECM and Prl, is instead inhibited by TSA. We provide evidence that the differing response of b-casein and BCE-1 to TSA is neither due to an unusual effect of TSA on mammary epithelial cells, nor to secondary consequences from the expression of a separate gene, nor to a particular property of the BCE-1 construct. As a component of this investigation, we also showed that ECM could mediate rapid histone deacetylation in mammary epithelial cells. These results are discussed in combination with previous work showing that TSA mediates the differentiation of many types of cancer cells but inhibits differentiation of some nonmalignant cell types.

  3. Epithelial-Mesenchymal Transition (EMT) Gene Variants and Epithelial Ovarian Cancer (EOC) Risk.

    PubMed

    Amankwah, Ernest K; Lin, Hui-Yi; Tyrer, Jonathan P; Lawrenson, Kate; Dennis, Joe; Chornokur, Ganna; Aben, Katja K H; Anton-Culver, Hoda; Antonenkova, Natalia; Bruinsma, Fiona; Bandera, Elisa V; Bean, Yukie T; Beckmann, Matthias W; Bisogna, Maria; Bjorge, Line; Bogdanova, Natalia; Brinton, Louise A; Brooks-Wilson, Angela; Bunker, Clareann H; Butzow, Ralf; Campbell, Ian G; Carty, Karen; Chen, Zhihua; Chen, Y Ann; Chang-Claude, Jenny; Cook, Linda S; Cramer, Daniel W; Cunningham, Julie M; Cybulski, Cezary; Dansonka-Mieszkowska, Agnieszka; du Bois, Andreas; Despierre, Evelyn; Dicks, Ed; Doherty, Jennifer A; Dörk, Thilo; Dürst, Matthias; Easton, Douglas F; Eccles, Diana M; Edwards, Robert P; Ekici, Arif B; Fasching, Peter A; Fridley, Brooke L; Gao, Yu-Tang; Gentry-Maharaj, Aleksandra; Giles, Graham G; Glasspool, Rosalind; Goodman, Marc T; Gronwald, Jacek; Harrington, Patricia; Harter, Philipp; Hasmad, Hanis N; Hein, Alexander; Heitz, Florian; Hildebrandt, Michelle A T; Hillemanns, Peter; Hogdall, Claus K; Hogdall, Estrid; Hosono, Satoyo; Iversen, Edwin S; Jakubowska, Anna; Jensen, Allan; Ji, Bu-Tian; Karlan, Beth Y; Jim, Heather; Kellar, Melissa; Kiemeney, Lambertus A; Krakstad, Camilla; Kjaer, Susanne K; Kupryjanczyk, Jolanta; Lambrechts, Diether; Lambrechts, Sandrina; Le, Nhu D; Lee, Alice W; Lele, Shashi; Leminen, Arto; Lester, Jenny; Levine, Douglas A; Liang, Dong; Lim, Boon Kiong; Lissowska, Jolanta; Lu, Karen; Lubinski, Jan; Lundvall, Lene; Massuger, Leon F A G; Matsuo, Keitaro; McGuire, Valerie; McLaughlin, John R; McNeish, Ian; Menon, Usha; Milne, Roger L; Modugno, Francesmary; Moysich, Kirsten B; Ness, Roberta B; Nevanlinna, Heli; Eilber, Ursula; Odunsi, Kunle; Olson, Sara H; Orlow, Irene; Orsulic, Sandra; Weber, Rachel Palmieri; Paul, James; Pearce, Celeste L; Pejovic, Tanja; Pelttari, Liisa M; Permuth-Wey, Jennifer; Pike, Malcolm C; Poole, Elizabeth M; Risch, Harvey A; Rosen, Barry; Rossing, Mary Anne; Rothstein, Joseph H; Rudolph, Anja; Runnebaum, Ingo B; Rzepecka, Iwona K; Salvesen, Helga B; Schernhammer, Eva; Schwaab, Ira; Shu, Xiao-Ou; Shvetsov, Yurii B; Siddiqui, Nadeem; Sieh, Weiva; Song, Honglin; Southey, Melissa C; Spiewankiewicz, Beata; Sucheston-Campbell, Lara; Teo, Soo-Hwang; Terry, Kathryn L; Thompson, Pamela J; Thomsen, Lotte; Tangen, Ingvild L; Tworoger, Shelley S; van Altena, Anne M; Vierkant, Robert A; Vergote, Ignace; Walsh, Christine S; Wang-Gohrke, Shan; Wentzensen, Nicolas; Whittemore, Alice S; Wicklund, Kristine G; Wilkens, Lynne R; Wu, Anna H; Wu, Xifeng; Woo, Yin-Ling; Yang, Hannah; Zheng, Wei; Ziogas, Argyrios; Kelemen, Linda E; Berchuck, Andrew; Schildkraut, Joellen M; Ramus, Susan J; Goode, Ellen L; Monteiro, Alvaro N A; Gayther, Simon A; Narod, Steven A; Pharoah, Paul D P; Sellers, Thomas A; Phelan, Catherine M

    2015-12-01

    Epithelial-mesenchymal transition (EMT) is a process whereby epithelial cells assume mesenchymal characteristics to facilitate cancer metastasis. However, EMT also contributes to the initiation and development of primary tumors. Prior studies that explored the hypothesis that EMT gene variants contribute to epithelial ovarian carcinoma (EOC) risk have been based on small sample sizes and none have sought replication in an independent population. We screened 15,816 single-nucleotide polymorphisms (SNPs) in 296 genes in a discovery phase using data from a genome-wide association study of EOC among women of European ancestry (1,947 cases and 2,009 controls) and identified 793 variants in 278 EMT-related genes that were nominally (P < 0.05) associated with invasive EOC. These SNPs were then genotyped in a larger study of 14,525 invasive-cancer patients and 23,447 controls. A P-value <0.05 and a false discovery rate (FDR) <0.2 were considered statistically significant. In the larger dataset, GPC6/GPC5 rs17702471 was associated with the endometrioid subtype among Caucasians (odds ratio (OR) = 1.16, 95% CI = 1.07-1.25, P = 0.0003, FDR = 0.19), whereas F8 rs7053448 (OR = 1.69, 95% CI = 1.27-2.24, P = 0.0003, FDR = 0.12), F8 rs7058826 (OR = 1.69, 95% CI = 1.27-2.24, P = 0.0003, FDR = 0.12), and CAPN13 rs1983383 (OR = 0.79, 95% CI = 0.69-0.90, P = 0.0005, FDR = 0.12) were associated with combined invasive EOC among Asians. In silico functional analyses revealed that GPC6/GPC5 rs17702471 coincided with DNA regulatory elements. These results suggest that EMT gene variants do not appear to play a significant role in the susceptibility to EOC. PMID:26399219

  4. Mechanical stretch induces lung α-epithelial Na(+) channel expression.

    PubMed

    Mustafa, Shamimunisa B; Isaac, John; Seidner, Steven R; Dixon, Patricia S; Henson, Barbara M; DiGeronimo, Robert J

    2014-10-01

    ABSTRACT During fetal development physiological stretching helps drive lung growth and maturation. At birth, the α-subunit of the alveolar epithelial sodium channel (α-ENaC) is a critical factor in helping to facilitate clearance of lung fluid during the perinatal period. The effects of stretch, however, on α-ENaC expression in the fetal lung have yet to be elucidated. In an effort to explore this question, we used both an in vitro cell culture model that exposes cells to repetitive cyclic stretch (CS) as well as an in vivo preterm animal model of mechanical ventilation (MV). We found that murine lung epithelial (MLE-12) cells exposed to repetitive CS showed a significant rise in α-ENaC mRNA expression. Total and cell-surface protein abundance of α-ENaC were also elevated after 24 h of CS. Stretch-induced increases in α-ENaC expression were suppressed in the presence of either actinomycin D or cycloheximide. Pharmacological inhibition of the extracellular signal-regulated protein kinase (ERK1/2) did not attenuate stretch-induced increases in α-ENaC protein, whereas inhibition of p38 MAPK or c-Jun NH2-terminal kinase (JNK) did. In 29-day preterm rabbits, alveolar stretching secondary to postnatal MV markedly elevated fetal lung α-ENaC expression compared to spontaneously breathing counterparts. In summary, our findings indicate that mechanical stretch promotes α-ENaC expression. PMID:25058750

  5. Analysis of gene regulation in rabbit corneal epithelial cells induced by ultraviolet radiation.

    PubMed

    Stevens, Jacqueline J; Rogers, Christian; Howard, Carolyn B; Moore, Caronda; Chan, Lai-Man

    2005-04-01

    Ultraviolet (UV)-induced cataracts are becoming a major environmental health concern because of the possible decrease in the stratospheric ozone layer. Experiments were designed to isolate gene(s) affected by UV irradiation in rabbit cornea tissues using fluorescent differential display-reverse transcription-polymerase chain reaction (FDDRT-PCR). The epithelial cells were grown in standard medium for 2 or 4 hours post treatment. Cornea epithelial cells were irradiated with UVB for 20 minutes. RNA was extracted and amplified by reverse transcriptase-polymerase chain reaction using poly A+ specific anchoring primers and random arbitrary primers. Polyacrylamide gel electrophoresis revealed several differentially expressed genes in untreated versus UV irradiated cells. Complimentary DNA (cDNA) fragments resulting from fluorescent differentially expressed mRNAs were eluted from the gel and re-amplified. The re-amplified PCR products were cloned directly into the PCR-TRAP cloning system. These data showed that FDDRT-PCR is a useful technique to elucidate UV-regulated gene expressions. Future experiments will involve sequence analysis of cloned inserts. The identification of these genes through sequence analysis could lead to a better understanding of cataract formation via DNA damage and mechanisms of prevention. PMID:16705801

  6. Gene Express Inc.

    PubMed

    Saccomanno, Colette F

    2006-07-01

    Gene Express, Inc. is a technology-licensing company and provider of Standardized Reverse Transcription Polymerase Chain Reaction (StaRT-PCR) services. Designed by and for clinical researchers involved in pharmaceutical, biomarker and molecular diagnostic product development, StaRT-PCR is a unique quantitative and standardized multigene expression measurement platform. StaRT-PCR meets all of the performance characteristics defined by the US FDA as required to support regulatory submissions [101,102] , and by the Clinical Laboratory Improvement Act of 1988 (CLIA) as necessary to support diagnostic testing [1] . A standardized mixture of internal standards (SMIS), manufactured in bulk, provides integrated quality control wherein each native template target gene is measured relative to a competitive template internal standard. Bulk production enables the compilation of a comprehensive standardized database from across multiple experiments, across collaborating laboratories and across the entire clinical development lifecycle of a given compound or diagnostic product. For the first time, all these data are able to be directly compared. Access to such a database can dramatically shorten the time from investigational new drug (IND) to new drug application (NDA), or save time and money by hastening a substantiated 'no-go' decision. High-throughput StaRT-PCR is conducted at the company's automated Standardized Expression Measurement (SEM) Center. Currently optimized for detection on a microcapillary electrophoretic platform, StaRT-PCR products also may be analyzed on microarray, high-performance liquid chromatography (HPLC), or matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) platforms. SEM Center services deliver standardized genomic data--data that will accelerate the application of pharmacogenomic technology to new drug and diagnostic test development and facilitate personalized medicine. PMID:16886903

  7. Cigarette smoke-induced epithelial expression of WNT-5B: implications for COPD.

    PubMed

    Heijink, Irene H; de Bruin, Harold G; Dennebos, Robin; Jonker, Marnix R; Noordhoek, Jacobien A; Brandsma, Corry-Anke; van den Berge, Maarten; Postma, Dirkje S

    2016-08-01

    Wingless/integrase-1 (WNT) signalling is associated with lung inflammation and repair, but its role in chronic obstructive pulmonary disease (COPD) pathogenesis is unclear. We investigated whether cigarette smoke-induced dysregulation of WNT-5B contributes to airway remodelling in COPD.We analysed WNT-5B protein expression in the lung tissue of COPD patients and (non)smoking controls, and investigated the effects of cigarette smoke exposure on WNT-5B expression in COPD and control-derived primary bronchial epithelial cells (PBECs). Additionally, we studied downstream effects of WNT-5B on remodelling related genes fibronectin, matrix metalloproteinase (MMP)-2, MMP-9 and SnaiI in BEAS-2B and air-liquid interface (ALI)-cultured PBECs.We observed that airway epithelial WNT-5B expression is significantly higher in lung tissue from COPD patients than controls. Cigarette smoke extract significantly increased mRNA expression of WNT-5B in COPD, but not control-derived PBECs. Exogenously added WNT-5B augmented the expression of remodelling related genes in BEAS-2B cells, which was mediated by transforming growth factor (TGF)-β/Smad3 signalling. In addition, WNT-5B upregulated the expression of these genes in ALI-cultured PBECs, particularly PBECs from COPD patients.Together, our results provide evidence that exaggerated WNT-5B expression upon cigarette smoke exposure in the bronchial epithelium of COPD patients leads to TGF-β/Smad3-dependent expression of genes related to airway remodelling. PMID:27126693

  8. Differential DNA methylation patterns of homeobox genes in proximal and distal colon epithelial cells.

    PubMed

    Barnicle, Alan; Seoighe, Cathal; Golden, Aaron; Greally, John M; Egan, Laurence J

    2016-04-01

    Region and cell-type specific differences in the molecular make up of colon epithelial cells have been reported. Those differences may underlie the region-specific characteristics of common colon epithelial diseases such as colorectal cancer and inflammatory bowel disease. DNA methylation is a cell-type specific epigenetic mark, essential for transcriptional regulation, silencing of repetitive DNA and genomic imprinting. Little is known about any region-specific variations in methylation patterns in human colon epithelial cells. Using purified epithelial cells and whole biopsies (n= 19) from human subjects, we generated epigenome-wide DNA methylation data (using the HELP-tagging assay), comparing the methylation signatures of the proximal and distal colon. We identified a total of 125 differentially methylated sites (DMS) mapping to transcription start sites of protein-coding genes, most notably several members of the homeobox (HOX) family of genes. Patterns of differential methylation were validated with MassArray EpiTYPER. We also examined DNA methylation in whole biopsies, applying a computational technique to deconvolve variation in methylation within cell types and variation in cell-type composition across biopsies. Including inferred epithelial proportions as a covariate in differential methylation analysis applied to the whole biopsies resulted in greater overlap with the results obtained from purified epithelial cells compared with when the covariate was not included. Results obtained from both approaches highlight region-specific methylation patterns ofHOXgenes in colonic epithelium. Regional variation in methylation patterns has implications for the study of diseases that exhibit regional expression patterns in the human colon, such as inflammatory bowel disease and colorectal cancer. PMID:26812987

  9. Respiratory epithelial cell expression of human transforming growth factor-alpha induces lung fibrosis in transgenic mice.

    PubMed Central

    Korfhagen, T R; Swantz, R J; Wert, S E; McCarty, J M; Kerlakian, C B; Glasser, S W; Whitsett, J A

    1994-01-01

    Increased production of EGF or TGF-alpha by the respiratory epithelial cells has been associated with the pathogenesis of various forms of lung injury. Growth factors and cytokines are thought to act locally, via paracrine and autocrine mechanisms, to stimulate cell proliferation and matrix deposition by interstitial lung cells resulting in pulmonary fibrosis. To test whether TGF-alpha mediates pulmonary fibrotic responses, we have generated transgenic mice expressing human TGF-alpha under control of regulatory regions of the human surfactant protein C (SP-C) gene. Human TGF-alpha mRNA was expressed in pulmonary epithelial cells in the lungs of the transgenic mice. Adult mice bearing the SP-C-TGF-alpha transgene developed severe pulmonary fibrosis. Fibrotic lesions were observed in peribronchial, peribronchiolar, and perivascular regions, as well as subjacent to pleural surfaces. Lesions consisted of fibrous tissue that included groups of epithelial cells expressing endogenous SP-C mRNA, consistent with their identification as distal respiratory epithelial cells. Peripheral fibrotic regions consisted of thickened pleura associated with extensive collagen deposition. Alveolar architecture was disrupted in the transgenic mice with loss of alveoli in the lung parenchyma. Pulmonary epithelial cell expression of TGF-alpha in transgenic mice disrupts alveolar morphogenesis and produces fibrotic lesions mediated by paracrine signaling between respiratory epithelial and interstitial cells of the lung. Images PMID:8163670

  10. Trehalose maintains vitality of mouse epididymal epithelial cells and mediates gene transfer.

    PubMed

    Qu, Bin; Gu, Yihua; Shen, Jian; Qin, Jinzhou; Bao, Jianqiang; Hu, Yuan; Zeng, Wenxian; Dong, Wuzi

    2014-01-01

    In the present study, trehalose was utilized to improve primary culture of mouse epididymal epithelial cells in vitro, and to enhance naked DNA delivery in epididymis in vivo. During the six-day culture, the proliferation activity of the cells in the medium with addition of trehalose was higher than that of those cells cultured in absence of trehalose (p<0.01). To determine the optimal concentration for cell proliferation, a series of trehalose concentrations (0, 60, 120, 180 mM) were tested, and the result indicated that the cell in the medium with 120 mM trehalose showed the highest proliferation potential. The epididymis epithelial cells were cultured in the medium containing 120 mM trehalose upon 16th passage, and they continued expressing markers of epididymal epithelial cell, such as rE-RABP, AR and ER-beta. Our study also indicated that trehalose concentrations of 120-240 mM, especially 180 mM, could effectively enhance DNA delivery into the mouse epididymis epithelial cell in vitro. Moreover, trehalose could induce in vivo expression of exogenous DNA in epididymal epithelial cells and help to internalize plasmid into sperm,which did not influence motility of sperm when the mixture of trehalose (180 mM) and DNA was injected into epididymal lumen through efferent tubule. This study suggested that trehalose, as an effective and safer reagent, could be employed potentially to maintain vitality of mouse epididymal epithelial cells during long-term culture in vitro and to mediate in vitro and in vivo gene transfer. PMID:24651491

  11. Trichostatin A Inhibits β-Casein Expression in Mammary Epithelial Cells

    PubMed Central

    Pujuguet, Philippe; Radisky, Derek; Levy, Dinah; Lacza, Charlemagne; Bissell, Mina J.

    2010-01-01

    Many aspects of cellular behavior are defined by the content of information provided by association of the extracellular matrix (ECM) and with cell membrane receptors. When cultured in the presence of laminin-containing ECM and prolactin (Prl), normal mammary epithelial cells express the milk protein β-casein. We have previously found that the minimal ECM- and Prl-responsive enhancer element BCE-1 was only active when stably integrated into chromatin, and that trichostatin A (TSA), a reagent that leads to alterations in chromatin structure, was able to activate the integrated enhancer element. We now show that endogenous β-casein gene, which is controlled by a genetic assembly that is highly similar to that of BCE-1 and which is also activated by incubation in ECM and Prl, is instead inhibited by TSA. We provide evidence that the differing response of β-casein and BCE-1 to TSA is neither due to an unusual effect of TSA on mammary epithelial cells, nor to secondary consequences from the expression of a separate gene, nor to a particular property of the BCE-1 construct. As a component of this investigation, we also showed that ECM mediated rapid histone deacetylation in mammary epithelial cells. These results are discussed in combination with previous work showing that TSA mediates the differentiation of many types of cancer cells but inhibits differentiation of some nonmalignant cell types. PMID:11746508

  12. Gene Regulation of Intestinal Porcine Epithelial Cells IPEC-J2 Is Dependent on the Site of Deoxynivalenol Toxicological Action

    PubMed Central

    Diesing, Anne-Kathrin; Nossol, Constanze; Ponsuksili, Siriluck; Wimmers, Klaus; Kluess, Jeannette; Walk, Nicole; Post, Andreas; Rothkötter, Hermann-Josef; Kahlert, Stefan

    2012-01-01

    The intestinal epithelial cell layer represents the border between the luminal and systemic side of the gut. The decision between absorption and exclusion of substances is the quintessential function of the gut and varies along the gut axis. Consequently, potentially toxic substances may reach the basolateral domain of the epithelial cell layer via blood stream. The mycotoxin deoxynivalenol (DON) is a Fusarium derived secondary metabolite known to enter the blood stream and displaying a striking toxicity on the basolateral side of polarised epithelial cell layers in vitro. Here we analysed potential mechanisms of apical and basolateral DON toxicity reflected in the gene expression. We used the jejunum-derived, polarised intestinal porcine epithelial cell line IPEC-J2 as an in vitro cell culture model. Luminal and systemic DON challenge of the epithelial cell layer was mimicked by a DON application from the apical or basolateral compartment of membrane inserts for 72 h. We compared the genome-wide gene expression of untreated and DON-treated IPEC-J2 cells with the GeneChip® Porcine Genome Array of Affymetrix. Low basolateral DON (200 ng/mL) application triggered 10 times more gene transcripts in comparison to the corresponding apical application (2539 versus 267) despite the intactness of the challenged cell layer as measured by transepithelial electrical resistance. Analysis of the regulated genes by bioinformatic resource DAVID identified several groups of biochemical pathways modulated by concentration and orientation of DON application. Selected genes representing pathways of the cellular metabolism, information processing and structural design were analysed in detail by quantitative PCR. Our findings clearly show that apical and basolateral challenge of epithelial cell layers trigger different gene response profiles paralleled with a higher susceptibility towards basolateral challenge. The evaluation of toxicological potentials of mycotoxins should take this

  13. Altered aldose reductase gene regulation in cultured human retinal pigment epithelial cells.

    PubMed Central

    Henry, D N; Del Monte, M; Greene, D A; Killen, P D

    1993-01-01

    Aldose reductase (AR2), a putative "hypertonicity stress protein" whose gene is induced by hyperosmolarity, protects renal medullary cells against the interstitial hyperosmolarity of antidiuresis by catalyzing the synthesis of millimolar concentrations of intracellular sorbitol from glucose. Although AR2 gene induction has been noted in a variety of renal and nonrenal cells subjected to hypertonic stress in vitro, the functional significance of AR2 gene expression in cells not normally exposed to a hyperosmolar milieu is not fully understood. The physiological impact of basal AR2 expression in such cells may be limited to hyperglycemic states in which AR2 promotes pathological polyol accumulation, a mechanism invoked in the pathogenesis of diabetic complications. Since AR2 overexpression in the retinal pigment epithelium has been associated with diabetic retinopathy, the regulation of AR2 gene expression and associated changes in sorbitol and myo-inositol were studied in human retinal pigment epithelial cells in culture. The relative abundance of aldehyde reductase (AR1) and AR2 mRNA was quantitated by filter hybridization of RNA from several human retinal pigment epithelial cell lines exposed to hyperglycemic and hyperosmolar conditions in vitro. AR2 but not AR1 mRNA was significantly increased some 11- to 18-fold by hyperosmolarity in several retinal pigment epithelial cell lines. A single cell line with a 15-fold higher basal level of AR2 mRNA than other cell lines tested demonstrated no significant increase in AR2 mRNA in response to hypertonic stress. This cell line demonstrated accelerated and exaggerated production of sorbitol and depletion of myo-inositol upon exposure to 20 mM glucose. Therefore, abnormal AR2 expression may enhance the sensitivity of cells to the biochemical consequences of hyperglycemia potentiating the development of diabetic complications. Images PMID:8349800

  14. Evolution of gene expression after gene amplification.

    PubMed

    Garcia, Nelson; Zhang, Wei; Wu, Yongrui; Messing, Joachim

    2015-05-01

    We took a rather unique approach to investigate the conservation of gene expression of prolamin storage protein genes across two different subfamilies of the Poaceae. We took advantage of oat plants carrying single maize chromosomes in different cultivars, called oat-maize addition (OMA) lines, which permitted us to determine whether regulation of gene expression was conserved between the two species. We found that γ-zeins are expressed in OMA7.06, which carries maize chromosome 7 even in the absence of the trans-acting maize prolamin-box-binding factor (PBF), which regulates their expression. This is likely because oat PBF can substitute for the function of maize PBF as shown in our transient expression data, using a γ-zein promoter fused to green fluorescent protein (GFP). Despite this conservation, the younger, recently amplified prolamin genes in maize, absent in oat, are not expressed in the corresponding OMAs. However, maize can express the oldest prolamin gene, the wheat high-molecular weight glutenin Dx5 gene, even when maize Pbf is knocked down (through PbfRNAi), and/or another maize transcription factor, Opaque-2 (O2) is knocked out (in maize o2 mutant). Therefore, older genes are conserved in their regulation, whereas younger ones diverged during evolution and eventually acquired a new repertoire of suitable transcriptional activators. PMID:25912045

  15. Evolution of Gene Expression after Gene Amplification

    PubMed Central

    Garcia, Nelson; Zhang, Wei; Wu, Yongrui; Messing, Joachim

    2015-01-01

    We took a rather unique approach to investigate the conservation of gene expression of prolamin storage protein genes across two different subfamilies of the Poaceae. We took advantage of oat plants carrying single maize chromosomes in different cultivars, called oat–maize addition (OMA) lines, which permitted us to determine whether regulation of gene expression was conserved between the two species. We found that γ-zeins are expressed in OMA7.06, which carries maize chromosome 7 even in the absence of the trans-acting maize prolamin-box-binding factor (PBF), which regulates their expression. This is likely because oat PBF can substitute for the function of maize PBF as shown in our transient expression data, using a γ-zein promoter fused to green fluorescent protein (GFP). Despite this conservation, the younger, recently amplified prolamin genes in maize, absent in oat, are not expressed in the corresponding OMAs. However, maize can express the oldest prolamin gene, the wheat high-molecular weight glutenin Dx5 gene, even when maize Pbf is knocked down (through PbfRNAi), and/or another maize transcription factor, Opaque-2 (O2) is knocked out (in maize o2 mutant). Therefore, older genes are conserved in their regulation, whereas younger ones diverged during evolution and eventually acquired a new repertoire of suitable transcriptional activators. PMID:25912045

  16. A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion.

    PubMed Central

    Jung, H C; Eckmann, L; Yang, S K; Panja, A; Fierer, J; Morzycka-Wroblewska, E; Kagnoff, M F

    1995-01-01

    Pathogenic bacteria that penetrate the intestinal epithelial barrier stimulate an inflammatory response in the adjacent intestinal mucosa. The present studies asked whether colon epithelial cells can provide signals that are important for the initiation and amplification of an acute mucosal inflammatory response. Infection of monolayers of human colon epithelial cell lines (T84, HT29, Caco-2) with invasive strains of bacteria (Salmonella dublin, Shigella dysenteriae, Yersinia enterocolitica, Listeria monocytogenes, enteroinvasive Escherichia coli) resulted in the coordinate expression and upregulation of a specific array of four proinflammatory cytokines, IL-8, monocyte chemotactic protein-1, GM-CSF, and TNF alpha, as assessed by mRNA levels and cytokine secretion. Expression of the same cytokines was upregulated after TNF alpha or IL-1 stimulation of these cells. In contrast, cytokine gene expression was not altered after infection of colon epithelial cells with noninvasive bacteria or the noninvasive protozoan parasite, G. lamblia. Notably, none of the cell lines expressed mRNA for IL-2, IL-4, IL-5, IL-6, IL-12p40, IFN-gamma, or significant levels of IL-1 or IL-10 in response to the identical stimuli. The coordinate expression of IL-8, MCP-1, GM-CSF and TNF alpha appears to be a general property of human colon epithelial cells since an identical array of cytokines, as well as IL-6, also was expressed by freshly isolated human colon epithelial cells. Since the cytokines expressed in response to bacterial invasion or other proinflammatory agonists have a well documented role in chemotaxis and activation of inflammatory cells, colon epithelial cells appear to be programmed to provide a set of signals for the activation of the mucosal inflammatory response in the earliest phases after microbial invasion. Images PMID:7814646

  17. Serial analysis of gene expression.

    PubMed

    Velculescu, V E; Zhang, L; Vogelstein, B; Kinzler, K W

    1995-10-20

    The characteristics of an organism are determined by the genes expressed within it. A method was developed, called serial analysis of gene expression (SAGE), that allows the quantitative and simultaneous analysis of a large number of transcripts. To demonstrate this strategy, short diagnostic sequence tags were isolated from pancreas, concatenated, and cloned. Manual sequencing of 1000 tags revealed a gene expression pattern characteristic of pancreatic function. New pancreatic transcripts corresponding to novel tags were identified. SAGE should provide a broadly applicable means for the quantitative cataloging and comparison of expressed genes in a variety of normal, developmental, and disease states. PMID:7570003

  18. Epithelial-Mesenchymal Transition (EMT) gene variants and Epithelial Ovarian Cancer (EOC) risk

    PubMed Central

    Amankwah, Ernest K.; Lin, Hui-Yi; Tyrer, Jonathan P.; Lawrenson, Kate; Dennis, Joe; Chornokur, Ganna; Aben, Katja KH.; Anton-Culver, Hoda; Antonenkova, Natalia; Bruinsma, Fiona; Bandera, Elisa V.; Bean, Yukie T.; Beckmann, Matthias W.; Bisogna, Maria; Bjorge, Line; Bogdanova, Natalia; Brinton, Louise A.; Brooks-Wilson, Angela; Bunker, Clareann H.; Butzow, Ralf; Campbell, Ian G.; Carty, Karen; Chen, Zhihua; Chen, Y. Ann; Chang-Claude, Jenny; Cook, Linda S.; Cramer, Daniel W.; Cunningham, Julie M.; Cybulski, Cezary; Dansonka-Mieszkowska, Agnieszka; du Bois, Andreas; Despierre, Evelyn; Dicks, Ed; Doherty, Jennifer A.; Dörk, Thilo; Dürst, Matthias; Easton, Douglas F.; Eccles, Diana M.; Edwards, Robert P.; Ekici, Arif B.; Fasching, Peter A.; Fridley, Brooke L.; Gao, Yu-Tang; Gentry-Maharaj, Aleksandra; Giles, Graham G.; Glasspool, Rosalind; Goodman, Marc T.; Gronwald, Jacek; Harrington, Patricia; Harter, Philipp; Hasmad, Hanis N.; Hein, Alexander; Heitz, Florian; Hildebrandt, Michelle A.T.; Hillemanns, Peter; Hogdall, Claus K.; Hogdall, Estrid; Hosono, Satoyo; Iversen, Edwin S.; Jakubowska, Anna; Jensen, Allan; Ji, Bu-Tian; Karlan, Beth Y.; Jim, Heather; Kellar, Melissa; Kiemeney, Lambertus A.; Krakstad, Camilla; Kjaer, Susanne K.; Kupryjanczyk, Jolanta; Lambrechts, Diether; Lambrechts, Sandrina; Le, Nhu D.; Lee, Alice W.; Lele, Shashi; Leminen, Arto; Lester, Jenny; Levine, Douglas A.; Liang, Dong; Lim, Boon Kiong; Lissowska, Jolanta; Lu, Karen; Lubinski, Jan; Lundvall, Lene; Massuger, Leon F.A.G.; Matsuo, Keitaro; McGuire, Valerie; McLaughlin, John R.; McNeish, Ian; Menon, Usha; Milne, Roger L.; Modugno, Francesmary; Moysich, Kirsten B.; Ness, Roberta B.; Nevanlinna, Heli; Eilber, Ursula; Odunsi, Kunle; Olson, Sara H.; Orlow, Irene; Orsulic, Sandra; Weber, Rachel Palmieri; Paul, James; Pearce, Celeste L.; Pejovic, Tanja; Pelttari, Liisa M.; Permuth-Wey, Jennifer; Pike, Malcolm C.; Poole, Elizabeth M.; Risch, Harvey A.; Rosen, Barry; Rossing, Mary Anne; Rothstein, Joseph H.; Rudolph, Anja; Runnebaum, Ingo B.; Rzepecka, Iwona K.; Salvesen, Helga B.; Schernhammer, Eva; Schwaab, Ira; Shu, Xiao-Ou; Shvetsov, Yurii B.; Siddiqui, Nadeem; Sieh, Weiva; Song, Honglin; Southey, Melissa C.; Spiewankiewicz, Beata; Sucheston-Campbell, Lara; Teo, Soo-Hwang; Terry, Kathryn L.; Thompson, Pamela J.; Thomsen, Lotte; Tangen, Ingvild L.; Tworoger, Shelley S.; van Altena, Anne M.; Vierkant, Robert A.; Vergote, Ignace; Walsh, Christine S.; Wang-Gohrke, Shan; Wentzensen, Nicolas; Whittemore, Alice S.; Wicklund, Kristine G.; Wilkens, Lynne R.; Wu, Anna H.; Wu, Xifeng; Woo, Yin-Ling; Yang, Hannah; Zheng, Wei; Ziogas, Argyrios; Kelemen, Linda E.; Berchuck, Andrew; Schildkraut, Joellen M.; Ramus, Susan J.; Goode, Ellen L.; Monteiro, Alvaro N.A.; Gayther, Simon A.; Narod, Steven A.; Pharoah, Paul D. P.; Sellers, Thomas A.; Phelan, Catherine M.

    2016-01-01

    Introduction Epithelial-mesenchymal transition (EMT) is a process whereby epithelial cells assume mesenchymal characteristics to facilitate cancer metastasis. However, EMT also contributes to the initiation and development of primary tumors. Prior studies that explored the hypothesis that EMT gene variants contribute to EOC risk have been based on small sample sizes and none have sought replication in an independent population. Methods We screened 1254 SNPs in 296 genes in a discovery phase using data from a genome-wide association study of EOC among women of European ancestry (1,947 cases and 2,009 controls) and identified 793 variants in 278 EMT-related genes that were nominally (p<0.05) associated with invasive EOC. These SNPs were then genotyped in a larger study of 14,525 invasive-cancer patients and 23,447 controls. A p-value <0.05 and a false discovery rate (FDR) <0.2 was considered statistically significant. Results In the larger dataset, GPC6/GPC5 rs17702471 was associated with the endometrioid subtype among Caucasians (OR=1.16, 95%CI=1.07–1.25, p=0.0003, FDR=0.19), while F8 rs7053448 (OR=1.69, 95%CI=1.27–2.24, p=0.0003, FDR=0.12), F8 rs7058826 (OR=1.69, 95%CI=1.27–2.24, p=0.0003, FDR=0.12), and CAPN13 rs1983383 (OR=0.79, 95%CI=0.69–0.90, p=0.0005, FDR=0.12) were associated with combined invasive EOC among Asians. In silico functional analyses revealed that GPC6/GPC5 rs17702471 coincided with DNA regulatory elements. Conclusion These results suggest that EMT gene variants do not appear to play a significant role in the susceptibility to EOC. PMID:26399219

  19. A Pituitary Homeobox 2 (Pitx2):microRNA-200a-3p:β-catenin Pathway Converts Mesenchymal Cells to Amelogenin-expressing Dental Epithelial Cells*

    PubMed Central

    Sharp, Thad; Wang, Jianbo; Li, Xiao; Cao, Huojun; Gao, Shan; Moreno, Myriam; Amendt, Brad A.

    2014-01-01

    Pitx2, Wnt/β-catenin signaling, and microRNAs (miRs) play a critical role in the regulation of dental stem cells during embryonic development. In this report, we have identified a Pitx2:β-catenin regulatory pathway involved in epithelial cell differentiation and conversion of mesenchymal cells to amelogenin expressing epithelial cells via miR-200a. Pitx2 and β-catenin are expressed in the labial incisor cervical loop or epithelial stem cell niche, with decreased expression in the differentiating ameloblast cells of the mouse lower incisor. Bioinformatics analyses reveal that miR-200a-3p expression is activated in the pre-ameloblast cells to enhance epithelial cell differentiation. We demonstrate that Pitx2 activates miR-200a-3p expression and miR-200a-3p reciprocally represses Pitx2 and β-catenin expression. Pitx2 and β-catenin interact to synergistically activate gene expression during odontogenesis and miR-200a-3p attenuates their expression and directs differentiation. To understand how this mechanism controls cell differentiation and cell fate, oral epithelial and odontoblast mesenchymal cells were reprogrammed by a two-step induction method using Pitx2 and miR-200a-3p. Conversion to amelogenin expressing dental epithelial cells involved an up-regulation of the stem cell marker Sox2 and proliferation genes and decreased expression of mesenchymal markers. E-cadherin expression was increased as well as ameloblast specific factors. The combination of Pitx2, a regulator of dental stem cells and miR-200a converts mesenchymal cells to a fully differentiated dental epithelial cell type. This pathway and reprogramming can be used to reprogram mesenchymal or oral epithelial cells to dental epithelial (ameloblast) cells, which can be used in tissue repair and regeneration studies. PMID:25122764

  20. VIP17/MAL expression modulates epithelial cyst formation and ciliogenesis

    PubMed Central

    Takiar, Vinita; Mistry, Kavita; Carmosino, Monica; Schaeren-Wiemers, Nicole

    2012-01-01

    The polarized organization of epithelial cells is required for vectorial solute transport and may be altered in renal cystic diseases. Vesicle integral protein of 17 kDa (VIP17/MAL) is involved in apical vesicle transport. VIP17/MAL overexpression in vivo results in renal cystogenesis of unknown etiology. Renal cystogenesis can occur as a consequence of defects of the primary cilium. To explore the role of VIP17/MAL in renal cystogenesis and ciliogenesis, we examined the polarization and ciliary morphology of wild-type and VIP17/MAL overexpressing Madin-Darby canine kidney renal epithelial cells grown in two-dimensional (2D) and three-dimensional (3D) cyst culture. VIP17/MAL is apically localized when expressed in cells maintained in 2D and 3D culture. VIP17/MAL overexpressing cells produce more multilumen cysts compared with controls. While the distributions of basolateral markers are not affected, VIP17/MAL expression results in aberrant sorting of the apical marker gp135 to the primary cilium. VIP17/MAL overexpression is also associated with shortened or absent cilia. Immunofluorescence analysis performed on kidney sections from VIP17/MAL transgenic mice also demonstrates fewer and shortened cilia within dilated lumens (P < 0.01). These studies demonstrate that VIP17/MAL overexpression results in abnormal cilium and cyst development, in vitro and in vivo, suggesting that VIP17/MAL overexpressing mice may develop cysts secondary to a ciliary defect. PMID:22895261

  1. Aberrant Gene Expression in Humans

    PubMed Central

    Yang, Ence; Ji, Guoli; Brinkmeyer-Langford, Candice L.; Cai, James J.

    2015-01-01

    Gene expression as an intermediate molecular phenotype has been a focus of research interest. In particular, studies of expression quantitative trait loci (eQTL) have offered promise for understanding gene regulation through the discovery of genetic variants that explain variation in gene expression levels. Existing eQTL methods are designed for assessing the effects of common variants, but not rare variants. Here, we address the problem by establishing a novel analytical framework for evaluating the effects of rare or private variants on gene expression. Our method starts from the identification of outlier individuals that show markedly different gene expression from the majority of a population, and then reveals the contributions of private SNPs to the aberrant gene expression in these outliers. Using population-scale mRNA sequencing data, we identify outlier individuals using a multivariate approach. We find that outlier individuals are more readily detected with respect to gene sets that include genes involved in cellular regulation and signal transduction, and less likely to be detected with respect to the gene sets with genes involved in metabolic pathways and other fundamental molecular functions. Analysis of polymorphic data suggests that private SNPs of outlier individuals are enriched in the enhancer and promoter regions of corresponding aberrantly-expressed genes, suggesting a specific regulatory role of private SNPs, while the commonly-occurring regulatory genetic variants (i.e., eQTL SNPs) show little evidence of involvement. Additional data suggest that non-genetic factors may also underlie aberrant gene expression. Taken together, our findings advance a novel viewpoint relevant to situations wherein common eQTLs fail to predict gene expression when heritable, rare inter-individual variation exists. The analytical framework we describe, taking into consideration the reality of differential phenotypic robustness, may be valuable for investigating

  2. Epithelial P2X purinergic receptor channel expression and function

    PubMed Central

    Taylor, Amanda L.; Schwiebert, Lisa M.; Smith, Jeffrey J.; King, Chris; Jones, Julie R.; Sorscher, Eric J.; Schwiebert, Erik M.

    1999-01-01

    P2X purinergic receptor (P2XR) channels bind ATP and mediate Ca2+ influx — 2 signals that stimulate secretory Cl– transport across epithelia. We tested the hypotheses that P2XR channels are expressed by epithelia and that P2XRs transduce extracellular ATP signals into stimulation of Cl– transport across epithelia. Electrophysiological data and mRNA analysis of human and mouse pulmonary epithelia and other epithelial cells indicate that multiple P2XRs are broadly expressed in these tissues and that they are active on both apical and basolateral surfaces. Because P2X-selective agonists bind multiple P2XR subtypes, and because P2X agonists stimulate Cl– transport across nasal mucosa of cystic fibrosis (CF) patients as well as across non-CF nasal mucosa, P2XRs may provide novel targets for extracellular nucleotide therapy of CF. PMID:10510328

  3. Identification of human metapneumovirus-induced gene networks in airway epithelial cells by microarray analysis

    SciTech Connect

    Bao, X.; Sinha, M. |; Liu, T.; Hong, C.; Luxon, B.A. |; Garofalo, R.P. ||; Casola, A. ||

    2008-04-25

    Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections in infants, elderly and immunocompromised patients. Little is known about the response to hMPV infection of airway epithelial cells, which play a pivotal role in initiating and shaping innate and adaptive immune responses. In this study, we analyzed the transcriptional profiles of airway epithelial cells infected with hMPV using high-density oligonucleotide microarrays. Of the 47,400 transcripts and variants represented on the Affimetrix GeneChip Human Genome HG-U133 plus 2 array, 1601 genes were significantly altered following hMPV infection. Altered genes were then assigned to functional categories and mapped to signaling pathways. Many up-regulated genes are involved in the initiation of pro-inflammatory and antiviral immune responses, including chemokines, cytokines, type I interferon and interferon-inducible proteins. Other important functional classes up-regulated by hMPV infection include cellular signaling, gene transcription and apoptosis. Notably, genes associated with antioxidant and membrane transport activity, several metabolic pathways and cell proliferation were down-regulated in response to hMPV infection. Real-time PCR and Western blot assays were used to confirm the expression of genes related to several of these functional groups. The overall result of this study provides novel information on host gene expression upon infection with hMPV and also serves as a foundation for future investigations of genes and pathways involved in the pathogenesis of this important viral infection. Furthermore, it can facilitate a comparative analysis of other paramyxoviral infections to determine the transcriptional changes that are conserved versus the one that are specific to individual pathogens.

  4. Bcl-B Expression in Human Epithelial and Nonepithelial Malignancies

    PubMed Central

    Krajewska, Maryla; Kitada, Shinichi; Winter, Jane N.; Variakojis, Daina; Lichtenstein, Alan; Zhai, Dayong; Cuddy, Michael; Huang, Xianshu; Luciano, Frederic; Baker, Cheryl H.; Kim, Hoguen; Shin, Eunah; Kennedy, Susan; Olson, Allen H.; Badzio, Andrzej; Jassem, Jacek; Meinhold-Heerlein, Ivo; Duffy, Michael J.; Schimmer, Aaron D.; Tsao, Ming; Brown, Ewan; Sawyers, Anne; Andreeff, Michael; Mercola, Dan; Krajewski, Stan; Reed, John C.

    2014-01-01

    Purpose Apoptosis plays an important role in neoplastic processes. Bcl-B is an antiapoptotic Bcl-2 family member, which is known to change its phenotype upon binding to Nur77/TR3. The expression pattern of this protein in human malignancies has not been reported. Experimental Design We investigated Bcl-B expression in normal human tissues and several types of human epithelial and nonepithelial malignancy by immunohistochemistry, correlating results with tumor stage, histologic grade, and patient survival. Results Bcl-B protein was strongly expressed in all normal plasma cells but found in only18%of multiple myelomas (n = 133). Bcl-B immunostaining was also present in normal germinal center centroblasts and centrocytes and in approximately half of diffuse large B-cell lymphoma (n =48) specimens, whereas follicular lymphomas (n = 57) did not contain Bcl-B. In breast (n = 119), prostate (n = 66), gastric (n = 180), and colorectal (n = 106) adenocarcinomas, as well as in non – small cell lung cancers (n = 82), tumor-specific overexpression of Bcl-B was observed. Bcl-B expression was associated with variables of poor prognosis, such as high tumor grade in breast cancer (P = 0.009), microsatellite stability (P = 0.0002), and left-sided anatomic location (P = 0.02) of colorectal cancers, as well as with greater incidence of death from prostate cancer (P = 0.005) and shorter survival of patients with small cell lung cancer (P = 0.009). Conversely, although overexpressed in many gastric cancers, Bcl-B tended to correlate with better outcome (P = 0.01) and more differentiated tumor histology (P < 0.0001). Conclusions Tumor-specific alterations in Bcl-B expressionmay define subsets of nonepithelial and epithelial neoplasms with distinct clinical behaviors. PMID:18483366

  5. Vaginal Microbiome and Epithelial Gene Array in Post-Menopausal Women with Moderate to Severe Dryness

    PubMed Central

    Hammond, Jo-Anne; McMillan, Amy; Vongsa, Rebecca; Koenig, David; Gloor, Gregory B.; Reid, Gregor

    2011-01-01

    After menopause, many women experience vaginal dryness and atrophy of tissue, often attributed to the loss of estrogen. An understudied aspect of vaginal health in women who experience dryness due to atrophy is the role of the resident microbes. It is known that the microbiota has an important role in healthy vaginal homeostasis, including maintaining the pH balance and excluding pathogens. The objectives of this study were twofold: first to identify the microbiome of post-menopausal women with and without vaginal dryness and symptoms of atrophy; and secondly to examine any differences in epithelial gene expression associated with atrophy. The vaginal microbiome of 32 post-menopausal women was profiled using Illumina sequencing of the V6 region of the 16S rRNA gene. Sixteen subjects were selected for follow-up sampling every two weeks for 10 weeks. In addition, 10 epithelial RNA samples (6 healthy and 4 experiencing vaginal dryness) were acquired for gene expression analysis by Affymetrix Human Gene array. The microbiota abundance profiles were relatively stable over 10 weeks compared to previously published data on premenopausal women. There was an inverse correlation between Lactobacillus ratio and dryness and an increased bacterial diversity in women experiencing moderate to severe vaginal dryness. In healthy participants, Lactobacillus iners and L. crispatus were generally the most abundant, countering the long-held view that lactobacilli are absent or depleted in menopause. Vaginal dryness and atrophy were associated with down-regulation of human genes involved in maintenance of epithelial structure and barrier function, while those associated with inflammation were up-regulated consistent with the adverse clinical presentation. PMID:22073175

  6. Method of controlling gene expression

    DOEpatents

    Peters, Norman K.; Frost, John W.; Long, Sharon R.

    1991-12-03

    A method of controlling expression of a DNA segment under the control of a nod gene promoter which comprises administering to a host containing a nod gene promoter an amount sufficient to control expression of the DNA segment of a compound of the formula: ##STR1## in which each R is independently H or OH, is described.

  7. Repression of SRF target genes is critical for Myc-dependent apoptosis of epithelial cells

    PubMed Central

    Wiese, Katrin E; Haikala, Heidi M; von Eyss, Björn; Wolf, Elmar; Esnault, Cyril; Rosenwald, Andreas; Treisman, Richard; Klefström, Juha; Eilers, Martin

    2015-01-01

    Oncogenic levels of Myc expression sensitize cells to multiple apoptotic stimuli, and this protects long-lived organisms from cancer development. How cells discriminate physiological from supraphysiological levels of Myc is largely unknown. Here, we show that induction of apoptosis by Myc in breast epithelial cells requires association of Myc with Miz1. Gene expression and ChIP-Sequencing experiments show that high levels of Myc invade target sites that lack consensus E-boxes in a complex with Miz1 and repress transcription. Myc/Miz1-repressed genes encode proteins involved in cell adhesion and migration and include several integrins. Promoters of repressed genes are enriched for binding sites of the serum-response factor (SRF). Restoring SRF activity antagonizes Myc repression of SRF target genes, attenuates Myc-induced apoptosis, and reverts a Myc-dependent decrease in Akt phosphorylation and activity, a well-characterized suppressor of Myc-induced apoptosis. We propose that high levels of Myc engage Miz1 in repressive DNA binding complexes and suppress an SRF-dependent transcriptional program that supports survival of epithelial cells. PMID:25896507

  8. Type II pneumocyte-restricted green fluorescent protein expression after lentiviral transduction of lung epithelial cells.

    PubMed

    Wunderlich, Stephanie; Gruh, Ina; Winkler, Monica E; Beier, Jennifer; Radtke, Kerstin; Schmiedl, Andreas; Groos, Stephanie; Haverich, Axel; Martin, Ulrich

    2008-01-01

    Type II alveolar epithelial (AT2) cell-specific reporter expression has been highly useful in the study of embryology and alveolar regeneration in transgenic mice. Technologies enabling efficient gene transfer and cell type-restricted transgene expression in AT2 cells would allow for correction of AT2 cell-based diseases such as genetic surfactant deficiencies. Moreover, such approaches are urgently required to investigate differentiation of AT2 cells from adult and embryonic stem cells of other species than mouse. Using a human surfactant protein C (SP-C) promoter fragment, we have constructed lentiviral vectors enabling AT2-restricted transgene expression and identification of stem cell-derived AT2 cells. Lung epithelial cell lines M3E3/C3, H441, RLE-6TN, A549, MLE-12, and MLE-15 were characterized at the molecular and ultrastructural levels to identify cell lines useful to assess the cell type specificity of our vector constructs. After transduction, no green fluorescent protein (GFP) expression was observed in nontarget cells including bronchial H441 cells, pulmonary A549 cells, fibroblasts, smooth muscle cells, and endothelial cells. In contrast, and in correlation with endogenous SP-C expression, lentiviral transduction resulted in stable GFP expression in MLE-12 and MLE-15 AT2 cells. In conclusion, we have constructed a lentiviral vector mediating SP-C promoter-dependent GFP expression. Transgene expression strictly corresponds with an AT2 phenotype of the transduced cells. In particular, the generated vector should facilitate local alveolar gene therapy and investigation of alveolar regeneration and stem cell differentiation. PMID:18052721

  9. Expression and distribution of osteopontin in human tissues: widespread association with luminal epithelial surfaces.

    PubMed Central

    Brown, L F; Berse, B; Van de Water, L; Papadopoulos-Sergiou, A; Perruzzi, C A; Manseau, E J; Dvorak, H F; Senger, D R

    1992-01-01

    Osteopontin, a glycoprotein with a glycine-arginine-glycine-aspartate-serine (GRGDS) cell-binding domain, has been described in bone and is also known to be expressed in other organs, particularly kidney. The goal of the present work was to define the distribution of osteopontin synthesis and deposition in a wide variety of normal adult human tissues using a multifaceted approach that included immunohistochemistry, in situ hybridization, and Northern analysis. Immunohistochemical studies have revealed the unexpected finding that osteopontin is deposited as a prominent layer at the luminal surfaces of specific populations of epithelial cells of the gastrointestinal tract, gall bladder, pancreas, urinary and reproductive tracts, lung, breast, salivary glands, and sweat glands. Northern analyses identified gallbladder as a major site of osteopontin gene transcription comparable in magnitude with that of kidney, and immunoblotting identified osteopontin in bile. In situ hybridization localized osteopontin gene transcripts predominantly to the epithelium of a variety of organs as well as to ganglion cells of bowel wall. Osteopontin of epithelial cell origin, like bone-derived osteopontin, promoted GRGDS-dependent cell spreading in attachment assays. We postulate that osteopontin secreted by epithelium binds integrins on luminal surfaces. Collectively, these findings suggest an important role for osteopontin on many luminal epithelial surfaces communicating with the external environment. Images PMID:1421573

  10. Normal and Cystic Fibrosis Human Bronchial Epithelial Cells Infected with Pseudomonas aeruginosa Exhibit Distinct Gene Activation Patterns

    PubMed Central

    Balloy, Viviane; Varet, Hugo; Dillies, Marie-Agnès; Proux, Caroline; Jagla, Bernd; Coppée, Jean-Yves; Tabary, Olivier; Corvol, Harriet; Chignard, Michel; Guillot, Loïc

    2015-01-01

    Background and Aims In cystic fibrosis (CF), Pseudomonas aeruginosa is not eradicated from the lower respiratory tract and is associated with epithelial inflammation that eventually causes tissue damage. To identify the molecular determinants of an effective response to P. aeruginosa infection, we performed a transcriptomic analysis of primary human bronchial epithelial cells from healthy donors (CTRL) 2, 4, and 6 h after induced P. aeruginosa infection. Compared to noninfected cells, infected cells showed changes in gene activity, which were most marked 6 h postinfection and usually consisted in upregulation. Results By comparing for each time point of infection, the transcriptomic response of epithelial cells from CF patients and healthy donors, we identified 851, 638, 667, and 980 differentially expressed genes 0, 2, 4, and 6 h postinfection, respectively. Gene selection followed by bioinformatic analysis showed that most of the differentially expressed genes, either up- or downregulated, were in the protein-binding and catalytic gene-ontology categories. Finally, we established that the protein products of the genes exhibiting the greatest differential upregulation (CSF2, CCL2, TNF, CSF3, MMP1, and MMP10) between CF patients and CTRL were produced in higher amounts by infected cells from CF patients versus CTRL. Conclusions The differentially expressed genes in CF patients may constitute a signature for a detrimental inflammatory response and for an inefficient P. aeruginosa host-cell response. PMID:26485688

  11. Regulation of epithelial sodium channel expression by oestradiol and progestogen in alveolar epithelial cells.

    PubMed

    Luo, Ling; Deng, Jia; Wang, Dao-xin; He, Jing; Deng, Wang

    2015-09-15

    Oestrogen (E) and progestogen (P) exert regulatory effects on the epithelial Na(+) channel (ENaC) in the kidneys and the colon. However, the effects of E and P on the ENaC and on alveolar fluid clearance (AFC) remain unclear, and the mechanisms of action of these hormones are unknown. In this study, we showed that E and/or P administration increased AFC by more than 25% and increased the expression of the α and γ subunits of ENaC by approximately 35% in rats subjected to oleic acid-induced acute lung injury (ALI). A similar effect was observed in the dexamethasone-treated group. Furthermore, E and/or P treatment inhibited 11β-hydroxysteroid dehydrogenase (HSD) type 2 (11β-HSD2) activity, increased corticosterone expression and decreased the serum adrenocorticotrophic hormone (ACTH) levels. These effects were similar to those observed following treatment with carbenoxolone (CBX), a nonspecific HSD inhibitor. Further investigation showed that CBX further significantly increased AFC and α-ENaC expression after treatment with a low dose of E and/or P. In vitro, E or P alone inhibited 11β-HSD2 activity in a dose-dependent manner and increased α-ENaC expression by at least 50%, and E combined with P increased α-ENaC expression by more than 80%. Thus, E and P may augment the expression of α-ENaC, enhance AFC, attenuate pulmonary oedema by inhibiting 11β-HSD2 activity, and increase the active glucocorticoid levels in vivo and in vitro. PMID:26051998

  12. Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells

    NASA Astrophysics Data System (ADS)

    Rich, Devra P.; Anderson, Matthew P.; Gregory, Richard J.; Cheng, Seng H.; Paul, Sucharita; Jefferson, Douglas M.; McCann, John D.; Klinger, Katherine W.; Smith, Alan E.; Welsh, Michael J.

    1990-09-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in cultured cystic fibrosis airway epithelial cells and Cl- channel activation assessed in single cells using a fluorescence microscopic assay and the patch-clamp technique. Expression of CFTR, but not of a mutant form of CFTR (ΔF508), corrected the Cl- channel defect. Correction of the phenotypic defect demonstrates a causal relationship between mutations in the CFTR gene and defective Cl- transport which is the hallmark of the disease.

  13. Trehalose Maintains Vitality of Mouse Epididymal Epithelial Cells and Mediates Gene Transfer

    PubMed Central

    Shen, Jian; Qin, Jinzhou; Bao, Jianqiang; Hu, Yuan; Zeng, Wenxian; Dong, Wuzi

    2014-01-01

    In the present study, trehalose was utilized to improve primary culture of mouse epididymal epithelial cells in vitro, and to enhance naked DNA delivery in epididymis in vivo. During the six-day culture, the proliferation activity of the cells in the medium with addition of trehalose was higher than that of those cells cultured in absence of trehalose (p<0.01). To determine the optimal concentration for cell proliferation, a series of trehalose concentrations (0, 60, 120, 180 mM) were tested, and the result indicated that the cell in the medium with 120 mM trehalose showed the highest proliferation potential. The epididymis epithelial cells were cultured in the medium containing 120 mM trehalose upon 16th passage, and they continued expressing markers of epididymal epithelial cell, such as rE-RABP, AR and ER-beta. Our study also indicated that trehalose concentrations of 120–240 mM, especially 180 mM, could effectively enhance DNA delivery into the mouse epididymis epithelial cell in vitro. Moreover, trehalose could induce in vivo expression of exogenous DNA in epididymal epithelial cells and help to internalize plasmid into sperm,which did not influence motility of sperm when the mixture of trehalose (180 mM) and DNA was injected into epididymal lumen through efferent tubule. This study suggested that trehalose, as an effective and safer reagent, could be employed potentially to maintain vitality of mouse epididymal epthetial cells during long-term culture in vitro and to mediate in vitro and in vivo gene transfer. PMID:24651491

  14. [Allergens-induced sensitization alters airway epithelial adhesion molecules expression in mice].

    PubMed

    Zeng, Dan; Tan, Mei-Ling; Xiang, Yang; Qin, Xiao-Qun; Zhu, Li-Ming; Dai, Ai-Guo

    2015-12-25

    To explore the relationship between the epithelial adhesion molecules and immune responses of airway epithelium, we observed the expression of integrin β4 and intercellular adhesion molecule-1 (ICAM-1) in the mice airway epithelium after sensitization with allergens. BALB/c mice were sensitized with intraperitoneal injection of ovalbumin (OVA) or house dust mite (HDM) and then developed airway hyper-responsiveness as determined by barometric whole-body plethysmography. Both OVA and HDM sensitization led to increases of the number of peripheral leukocytes as well as inflammatory cells infiltration in lungs. OVA sensitized mice showed more severe inflammatory cells infiltration than HDM sensitized mice. Immunohistochemistry analysis of mice lung tissues revealed that sensitization with both allergens also led to a decrease of integrin β4 expression and an increase of ICAM-1 expression in airway epithelia. OVA sensitized mice showed a more significant increase of ICAM-1 expression compared with HDM sensitized mice. siRNA mediated silencing of integrin β4 gene in 16HBE cells resulted in an up-regulation of ICAM-1 expression. Our results indicate a possible role of airway epithelial adhesion molecules in allergen-induced airway immune responses. PMID:26701635

  15. Intestinal Epithelial Barrier Disruption through Altered Mucosal MicroRNA Expression in Human Immunodeficiency Virus and Simian Immunodeficiency Virus Infections

    PubMed Central

    Gaulke, Christopher A.; Porter, Matthew; Han, Yan-Hong; Sankaran-Walters, Sumathi; Grishina, Irina; George, Michael D.; Dang, Angeline T.; Ding, Shou-Wei; Jiang, Guochun; Korf, Ian

    2014-01-01

    ABSTRACT Epithelial barrier dysfunction during human immunodeficiency virus (HIV) infection has largely been attributed to the rapid and severe depletion of CD4+ T cells in the gastrointestinal (GI) tract. Although it is known that changes in mucosal gene expression contribute to intestinal enteropathy, the role of small noncoding RNAs, specifically microRNA (miRNA), has not been investigated. Using the simian immunodeficiency virus (SIV)-infected nonhuman primate model of HIV pathogenesis, we investigated the effect of viral infection on miRNA expression in intestinal mucosa. SIV infection led to a striking decrease in the expression of mucosal miRNA compared to that in uninfected controls. This decrease coincided with an increase in 5′-3′-exoribonuclease 2 protein and alterations in DICER1 and Argonaute 2 expression. Targets of depleted miRNA belonged to molecular pathways involved in epithelial proliferation, differentiation, and immune response. Decreased expression of several miRNA involved in maintaining epithelial homeostasis in the gut was localized to the proliferative crypt region of the intestinal epithelium. Our findings suggest that SIV-induced decreased expression of miRNA involved in epithelial homeostasis, disrupted expression of miRNA biogenesis machinery, and increased expression of XRN2 are involved in the development of epithelial barrier dysfunction and gastroenteropathy. IMPORTANCE MicroRNA (miRNA) regulate the development and function of intestinal epithelial cells, and many viruses disrupt normal host miRNA expression. In this study, we demonstrate that SIV and HIV disrupt expression of miRNA in the small intestine during infection. The depletion of several key miRNA is localized to the proliferative crypt region of the gut epithelium. These miRNA are known to control expression of genes involved in inflammation, cell death, and epithelial maturation. Our data indicate that this disruption might be caused by altered expression of mi

  16. Perilla frutescens Leaf Extract Inhibits Mite Major Allergen Der p 2-induced Gene Expression of Pro-Allergic and Pro-Inflammatory Cytokines in Human Bronchial Epithelial Cell BEAS-2B

    PubMed Central

    Liu, Jer-Yuh; Chen, Yi-Ching; Lin, Chun-Hsiang; Kao, Shao-Hsuan

    2013-01-01

    Perilla frutescens has been used in traditional medicine for respiratory diseases due to its anti-bacterial and anti-inflammatory activity. This study aimed to investigate effects of Perilla frutescens leaf extract (PFE) on expression of pro-allergic and pro-inflammatory cytokines in airway epithelial cells exposed to mite major allergen Der p 2 (DP2) and the underlying mechanisms. Our results showed that PFE up to 100 µg/mL had no cytotoxic effect on human bronchial epithelial cell BEAS-2B. Further investigations revealed that PFE dose-dependently diminished mRNA expression of pro-allergic cytokine IL-4, IL-5, IL-13 and GM-CSF, as well as pro-inflammatory cytokine IL-6, IL-8 and MCP-1 in BEAS-2B cells treated with DP2. In parallel to mRNA, the DP-2-elevated levels of the tested cytokines were decreased. Further investigation showed that DP2-indued phosphorylation of p38 MAPK (P38) and JNK, but not Erk1/2, was also suppressed by PFE. In addition, PFE elevated cytosolic IκBα level and decreased nuclear NF-κB level in DP2-stimulated BEAS-2B cells. Taken together, these findings revealed that PFE significantly diminished both mRNA expression and protein levels of pro-allergic and pro-inflammatory cytokines in response to DP2 through inhibition of P38/JNK and NK-κB activation. These findings suggest that PFE should be beneficial to alleviate both allergic and inflammatory responses on airway epithelium in response to aeroallergens. PMID:24204835

  17. Epithelial and organ-related marker expression in pituitary adenomas.

    PubMed

    Cykowski, Matthew D; Takei, Hidehiro; Baskin, David S; Rivera, Andreana L; Powell, Suzanne Z

    2016-08-01

    The histologic expression of epithelial and organ-related immunohistochemical markers in primary sellar region tumors has received little attention to date. This lack of empirical data may lead to mistaken assumptions in the evaluation of sellar region neoplasms. To address this issue, the frequency and specificity of epithelial (cytokeratin 7(CK7), CK20) and organ-related differentiation markers (gross cystic disease fluid protein-15 (GCDFP-15), thyroid transcription factor-1 (TTF-1), Napsin A, paired box 8 (PAX-8), hepatocyte paraffin 1 (HepPar1) and estrogen receptor (ER)) were studied in 40 patients with adenomas comprising five hormonal sub-types. Non-parametric statistical procedures were used to examine associations between marker expression and tumor sub-type. CK7 and CK20 immunoreactivity were seen in 48% and 8% of tumors, respectively, although never in a diffuse pattern. CK20 expression was nearly exclusive to corticotrophs, whereas CK7 frequently highlighted cells with dendritic-type morphology. The specificity of organ-related differentiation markers was 100% (monoclonal Napsin A, GCDFP-15 and TTF-1), 97% (HepPar1 and PAX-8), 90% (polyclonal Napsin A) and 72% (ER); no tumors demonstrated significant co-expression of these organ-related markers with either CK7 or CK20. The first major conclusion of this study is that CK7 staining in adenoma is more frequent than has been previously than has been previously described. CK7 immunoreactive cells often displayed a dendritic-type morphology, including within large macroadenomas, which raises the question as to whether these represent tumor cells with folliculo-stellate cell-type differentiation, as these also have dendritic cell-type morphology and express CK7 in non-neoplastic glands. The second major conclusion, which confirms earlier findings, is that CK20 staining is a very infrequent immunohistochemical finding in adenomas that is virtually limited to corticotrophs and thus is helpful in diagnostic

  18. Gene Expression in Oligodendroglial Tumors

    PubMed Central

    Shaw, Elisabeth J.; Haylock, Brian; Husband, David; du Plessis, Daniel; Sibson, D. Ross; Warnke, Peter C.; Walker, Carol

    2010-01-01

    Background: Oligodendroglial tumors with 1p/19q loss are more likely to be chemosensitive and have longer survival than those with intact 1p/19q, but not all respond to chemotherapy, warranting investigation of the biological basis of chemosensitivity. Methods: Gene expression profiling was performed using amplified antisense RNA from 28 oligodendroglial tumors treated with chemotherapy (26 serial stereotactic biopsy, 2 resection). Expression of differentially expressed genes was validated by real-time PCR. Results: Unsupervised hierarchical clustering showed clustering of multiple samples from the same case in 14/17 cases and identified subgroups associated with tumor grade and 1p/19q status. 176 genes were differentially expressed, 164 being associated with 1p/19q loss (86% not on 1p or 19q). 94 genes differed between responders and non-responders to chemotherapy; 12 were not associated with 1p/19q loss. Significant differential expression was confirmed in 11/13 selected genes. Novel genes associated with response to therapy included SSBP2, GFRA1, FAP and RASD1. IQGAP1, INA, TGIF1, NR2F2 and MYCBP were differentially expressed in oligodendroglial tumors with 1p/19q loss. Conclusion: Gene expression profiling using serial stereotactic biopsies indicated greater homogeneity within tumors than between tumors. Genes associated with 1p/19q status or response were identified warranting further elucidation of their role in oligodendroglial tumors. PMID:20966545

  19. MUC Expression in Gallbladder Epithelial Tissues in Cholesterol-Associated Gallbladder Disease

    PubMed Central

    Yoo, Kyo-Sang; Choi, Ho Soon; Jun, Dae Won; Lee, Hang Lak; Lee, Oh Young; Yoon, Byung Chul; Lee, Kyeong Geun; Paik, Seung Sam; Kim, Yong Seok; Lee, Jin

    2016-01-01

    Background/Aims Gallstone pathogenesis is linked to mucin hypersecretion and bacterial infection. Several mucin genes have been identified in gallbladder epithelial cells (GBECs). We investigated MUC expression in cholesterol-associated gallbladder disease and evaluated the relationship between mucin and bacterial infection. Methods The present study involved 20 patients with cholesterol stones with cholecystitis, five with cholesterol stones with cholesterolosis, six with cholesterol polyps, two with gallbladder cancer, and six controls. Canine GBECs treated with lipopolysaccharide were also studied. MUC3, MUC5AC, MUC5B, and MUC6 antibodies were used for dot/slot immunoblotting and immunohistochemical studies of the gallbladder epithelial tissues, canine GBECs, and bile. Reverse-transcription polymerase chain reaction was performed to evaluate MUC3 and MUC5B expression. Results MUC3, MUC5AC, MUC5B, and MUC6 were expressed in the normal gallbladder epithelium, and of those, MUC3 and MUC5B exhibited the highest expression levels. Greatly increased levels of MUC3 and MUC5B expression were observed in the cholesterol stone group, and slightly increased levels were observed in the cholesterol polyp group; MUC3 and MUC5B mRNA was also upregulated in those groups. Canine GBECs treated with lipopolysaccharide also showed upregulation of MUC3 and MUC5B. Conclusions The mucin genes with the highest expression levels in gallbladder tissue in cholesterol-associated diseases were MUC3 and MUC5B. Cholesterol stones and gallbladder infections were associated with increased MUC3 and MUC5B expression. PMID:27563024

  20. Cell surface expression and biosynthesis of epithelial Na+ channels.

    PubMed Central

    Prince, L S; Welsh, M J

    1998-01-01

    The epithelial Na+ channel (ENaC) complex is composed of three homologous subunits: alpha, beta and gamma. Mutations in ENaC subunits can increase the number of channels on the cell surface, causing a hereditary form of hypertension called Liddle's syndrome, or can decrease channel activity, causing pseudohypoaldosteronism type I, a salt-wasting disease of infancy. To investigate surface expression, we studied ENaC subunits expressed in COS-7 and HEK293 cells. Using surface biotinylation and protease sensitivity, we found that when individual ENaC subunits are expressed alone, they traffic to the cell surface. The subunits are glycosylated with high-mannose oligosaccharides, but seem to have the carbohydrate removed before they reach the cell surface. Moreover, subunits form a complex that cannot be disrupted by several non-ionic detergents. The pattern of glycosylation and detergent solubility/insolubility persists when the N-teminal and C-terminal cytoplasmic regions of ENaC are removed. With co-expression of all three ENaC subunits, the insoluble complex is the predominant species. These results show that ENaC and its family members are unique in their trafficking, biochemical characteristics and post-translational modifications. PMID:9841884

  1. Overexpression of piRNA Pathway Genes in Epithelial Ovarian Cancer

    PubMed Central

    Lim, Shu Ly; Ricciardelli, Carmela; Oehler, Martin K.; De Arao Tan, Izza M. D.; Russell, Darryl; Grützner, Frank

    2014-01-01

    The importance of the Piwi-interacting RNA (piRNA) pathway for germ cell maintenance, genome integrity, DNA methylation and retrotransposon control raises possible roles of this pathway in cancer. Indeed aberrant expression of human PIWI orthologs and Maelstrom has been observed in various cancers. In this study we explored the expression and function of piRNA pathway genes in human ovarian cancer, based on our recent work, which showed widespread expression of piRNA pathway genes in the mammalian. Our work shows that PIWIL1 and MAEL expression is significantly increased in malignant EOC (n = 25) compared to benign tumor tissues (n = 19) and normal ovarian tissue (n = 8). The expression of PIWIL3 is lower in malignant and benign tissues when compared to normal ovary. Sequencing of PIWIL1 transcript revealed that in many tumors deletion of exon 17 leads to the introduction of a premature stop codon in the PIWI domain, likely due to a splicing error. In situ hybridization on tumor sections revealed that L1, PIWIL1, 2 and MAEL are specifically expressed in epithelial cells (cancerous cells) of EOC. Furthermore, PIWIL2 and MAEL are co-expressed in the stromal cells adjacent to tumor cells. Since PIWIL1 and MAEL are up regulated in malignant EOC and expressed in the epithelial cells, we investigated if these two genes affect invasiveness of ovarian cancer cell lines that do not normally express these genes. PIWIL1 and MAEL were transiently over expressed in the ovarian cancer cell line SKOV3, followed by real-time measurements of cell invasiveness. Surprisingly both PIWIL1 and MAEL over expression decreased the invasiveness of SKOV3 cells. Our findings support a growing body of evidence that shows that genes in this pathway are upregulated in cancer. In ovarian cancer we show for the first time that Piwil1 transcript may often be abnormal result in non functional product. In contrast to what has been observed in other cell types, we found that PIWIL1 and

  2. Molecular Pathways: Breaking the Epithelial Cancer Barrier for Chimeric Antigen Receptor and T-cell Receptor Gene Therapy.

    PubMed

    Hinrichs, Christian S

    2016-04-01

    Adoptive transfer of T cells genetically engineered to express a tumor-targeting chimeric antigen receptor (CAR) or T-cell receptor (TCR) can mediate cancer regression in some patients. CARs are synthetic single-chain proteins that use antibody domains to target cell surface antigens. TCRs are natural heterodimeric proteins that can target intracellular antigens through recognition of peptides bound to human leukocyte antigens. CARs have shown promise in B-cell malignancies and TCRs in melanoma, but neither approach has achieved clear success in an epithelial cancer. Treatment of epithelial cancers may be particularly challenging because of a paucity of target antigens expressed by carcinomas and not by important healthy tissues. In addition, epithelial cancers may be protected by inhibitory ligands and soluble factors in the tumor microenvironment. One strategy to overcome these negative regulators is to modulate expression of T-cell genes to enhance intrinsic T-cell function. Programmable nucleases, which can suppress inhibitory genes, and inducible gene expression systems, which can enhance stimulatory genes, are entering clinical testing. Other work is delineating whether control of genes for immune checkpoint receptors (e.g.,PDCD1, CTLA4) and cytokine and TCR signaling regulators (e.g.,CBLB, CISH, IL12, IL15) can increase the antitumor activity of therapeutic T cells.Clin Cancer Res; 22(7); 1559-64. ©2016 AACR. PMID:27037253

  3. CHRFAM7A: a human-specific α7-nicotinic acetylcholine receptor gene shows differential responsiveness of human intestinal epithelial cells to LPS

    PubMed Central

    Dang, Xitong; Eliceiri, Brian P.; Baird, Andrew; Costantini, Todd W.

    2015-01-01

    The human genome contains a unique, distinct, and human-specific α7-nicotinic acetylcholine receptor (α7nAChR) gene [CHRNA7 (gene-encoding α7-nicotinic acetylcholine receptor)] called CHRFAM7A (gene-encoding dup-α7-nicotinic acetylcholine receptor) on a locus of chromosome 15 associated with mental illness, including schizophrenia. Located 5′ upstream from the “wild-type” CHRNA7 gene that is found in other vertebrates, we demonstrate CHRFAM7A expression in a broad range of epithelial cells and sequenced the CHRFAM7A transcript found in normal human fetal small intestine epithelial (FHs) cells to prove its identity. We then compared its expression to CHRNA7 in 11 gut epithelial cell lines, showed that there is a differential response to LPS when compared to CHRNA7, and characterized the CHRFAM7A promoter. We report that both CHRFAM7A and CHRNA7 gene expression are widely distributed in human epithelial cell lines but that the levels of CHRFAM7A gene expression vary up to 5000-fold between different gut epithelial cells. A 3-hour treatment of epithelial cells with 100 ng/ml LPS increased CHRFAM7A gene expression by almost 1000-fold but had little effect on CHRNA7 gene expression. Mapping the regulatory elements responsible for CHRFAM7A gene expression identifies a 1 kb sequence in the UTR of the CHRFAM7A gene that is modulated by LPS. Taken together, these data establish the presence, identity, and differential regulation of the human-specific CHRFAM7A gene in human gut epithelial cells. In light of the fact that CHRFAM7A expression is reported to modulate ligand binding to, and alter the activity of, the wild-type α7nAChR ligand-gated pentameric ion channel, the findings point to the existence of a species-specific α7nAChR response that might regulate gut epithelial function in a human-specific fashion.—Dang, X., Eliceiri, B. P., Baird, A., Costantini, T. W. CHRFAM7A: a human-specific α7-nicotinic acetylcholine receptor gene shows differential

  4. Cationic Surface Modification of PLG Nanoparticles Offers Sustained Gene Delivery to Pulmonary Epithelial Cells

    PubMed Central

    BAOUM, ABDULGADER; DHILLON, NAVNEET; BUCH, SHILPA; BERKLAND, CORY

    2010-01-01

    Biodegradable polymeric nanoparticles are currently being explored as a nonviral gene delivery system; however, many obstacles impede the translation of these nanomaterials. For example, nanoparticles delivered systemically are inherently prone to adsorbing serum proteins and agglomerating as a result of their large surface/volume ratio. What is desired is a simple procedure to prepare nanoparticles that may be delivered locally and exhibit minimal toxicity while improving entry into cells for effectively delivering DNA. The objective of this study was to optimize the formulation of poly(D,L-lactide-co-glycolide) (PLG) nanoparticles for gene delivery performance to a model of the pulmonary epithelium. Using a simple solvent diffusion technique, the chemistry of the particle surface was varied by using different coating materials that adsorb to the particle surface during formation. A variety of cationic coating materials were studied and compared to more conventional surfactants used for PLG nanoparticle fabrication. Nanoparticles (~200 nm) efficiently encapsulated plasmids encoding for luciferase (80–90%) and slowly released the same for 2 weeks. In A549 alveolar lung epithelial cells, high levels of gene expression appeared at day 5 for certain positively charged PLG particles and gene expression was maintained for at least 2 weeks. In contrast, PEI gene expression ended at day 5. PLG particles were also significantly less cytotoxic than PEI suggesting the use of these vehicles for localized, sustained gene delivery to the pulmonary epithelium. PMID:19911425

  5. A novel gene delivery method transduces porcine pancreatic duct epithelial cells.

    PubMed

    Griffin, M A; Restrepo, M S; Abu-El-Haija, M; Wallen, T; Buchanan, E; Rokhlina, T; Chen, Y H; McCray, P B; Davidson, B L; Divekar, A; Uc, A

    2014-02-01

    Gene therapy offers the possibility to treat pancreatic disease in cystic fibrosis (CF), caused by mutations in the CF transmembrane conductance regulator (CFTR) gene; however, gene transfer to the pancreas is untested in humans. The pancreatic disease phenotype is very similar between humans and pigs with CF; thus, CF pigs create an excellent opportunity to study gene transfer to the pancreas. There are no studies showing efficient transduction of pig pancreas with gene-transfer vectors. Our objective is to develop a safe and efficient method to transduce wild-type (WT) porcine pancreatic ducts that express CFTR. We catheterized the umbilical artery of WT newborn pigs and delivered an adeno-associated virus serotype 9 vector expressing green-fluorescent protein (AAV9CMV.sceGFP) or vehicle to the celiac artery, the vessel that supplies major branches to the pancreas. This technique resulted in stable and dose-dependent transduction of pancreatic duct epithelial cells that expressed CFTR. Intravenous (IV) injection of AAV9CMV.sceGFP did not transduce the pancreas. Our technique offers an opportunity to deliver the CFTR gene to the pancreas of CF pigs. The celiac artery can be accessed via the umbilical artery in newborns and via the femoral artery at older ages--delivery approaches that can be translated to humans. PMID:24257348

  6. Aire Expression Is Inherent to Most Medullary Thymic Epithelial Cells during Their Differentiation Program.

    PubMed

    Kawano, Hiroshi; Nishijima, Hitoshi; Morimoto, Junko; Hirota, Fumiko; Morita, Ryoko; Mouri, Yasuhiro; Nishioka, Yasuhiko; Matsumoto, Mitsuru

    2015-12-01

    Aire in medullary thymic epithelial cells (mTECs) plays an important role in the establishment of self-tolerance. Because Aire(+) mTECs appear to be a limited subset, they may constitute a unique lineage(s) among mTECs. An alternative possibility is that all mTECs are committed to express Aire in principle, but Aire expression by individual mTECs is conditional. To investigate this issue, we established a novel Aire reporter strain in which endogenous Aire is replaced by the human AIRE-GFP-Flag tag (Aire/hAGF-knockin) fusion gene. The hAGF reporter protein was produced and retained very efficiently within mTECs as authentic Aire nuclear dot protein. Remarkably, snapshot analysis revealed that mTECs expressing hAGF accounted for >95% of mature mTECs, suggesting that Aire expression does not represent a particular mTEC lineage(s). We confirmed this by generating Aire/diphtheria toxin receptor-knockin mice in which long-term ablation of Aire(+) mTECs by diphtheria toxin treatment resulted in the loss of most mature mTECs beyond the proportion of those apparently expressing Aire. These results suggest that Aire expression is inherent to all mTECs but may occur at particular stage(s) and/or cellular states during their differentiation, thus accounting for the broad impact of Aire on the promiscuous gene expression of mTECs. PMID:26503950

  7. Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants

    PubMed Central

    Gottschalk, Laura B.; Vecchio-Pagan, Briana; Sharma, Neeraj; Han, Sangwoo T.; Franca, Arianna; Wohler, Elizabeth S.; Batista, Denise A.S.; Goff, Loyal A.; Cutting, Garry R.

    2016-01-01

    Background Analysis of the functional consequences and treatment response of rare CFTR variants is challenging due to the limited availability of primary airways cells. Methods A Flp recombination target (FRT) site for stable expression of CFTR was incorporated into an immortalized CF bronchial epithelial cell line (CFBE41o−). CFTR cDNA was integrated into the FRT site. Expression was evaluated by western blotting and confocal microscopy and function measured by short circuit current. RNA sequencing was used to compare the transcriptional profile of the resulting CF8Flp cell line to primary cells and tissues. Results Functional CFTR was expressed from integrated cDNA at the FRT site of the CF8Flp cell line at levels comparable to that seen in native airway cells. CF8Flp cells expressing WT-CFTR have a stable transcriptome comparable to that of primary cultured airway epithelial cells, including genes that play key roles in CFTR pathways. Conclusion CF8Flp cells provide a viable substitute for primary CF airway cells for the analysis of CFTR variants in a native context. PMID:26694805

  8. The proteins secreted by Trichomonas vaginalis and vaginal epithelial cell response to secreted and episomally expressed AP65

    PubMed Central

    Kucknoor, Ashwini S.; Mundodi, Vasanthakrishna; Alderete, John F.

    2007-01-01

    Summary We showed recently that contact of human vaginal epithelial cells (VECs) by Trichomonas vaginalis and incubation with trichomonad proteins in conditioned medium induced expression of VEC genes. We performed 2-D SDS-PAGE followed by MALDI-TOF to identify the major secreted proteins. Based on protein abundance and separation of spots in 2-D gels, 32 major secreted proteins were examined, which gave 19 proteins with accession numbers. These proteins included known secreted cysteine proteinases. In addition, other secreted proteins were enzymes of carbohydrate metabolism, adhesin protein AP65, heat shock proteins, thioredoxin reductase and coronins. We confirmed that the secreted trichomonad proteins induced expression of VEC genes, including interleukin 8 (IL-8), COX-2 and fibronectin. Purified AP65 added to VECs had a pronounced effect only on IL-8 gene expression, which was inhibited in the presence of 12G4 monoclonal antibody to AP65. Moreover, AP65 expressed episomally within epithelial cells was found to enhance the expression of IL-8 and COX-2. This may be the first report of analysis of the secreted proteins of T. vaginalis and of the host epithelial cell response to these proteins and to the prominent adhesin AP65. PMID:17590165

  9. Vitamin D Induces Interleukin-1β Expression: Paracrine Macrophage Epithelial Signaling Controls M. tuberculosis Infection

    PubMed Central

    Verway, Mark; Bouttier, Manuella; Wang, Tian-Tian; Carrier, Marilyn; Calderon, Mario; An, Beum-Soo; Devemy, Emmanuelle; McIntosh, Fiona; Divangahi, Maziar; Behr, Marcel A.; White, John H.

    2013-01-01

    Although vitamin D deficiency is a common feature among patients presenting with active tuberculosis, the full scope of vitamin D action during Mycobacterium tuberculosis (Mtb) infection is poorly understood. As macrophages are the primary site of Mtb infection and are sites of vitamin D signaling, we have used these cells to understand the molecular mechanisms underlying modulation of the immune response by the hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D). We found that the virulent Mtb strain H37Rv elicits a broad host transcriptional response. Transcriptome profiling also revealed that the profile of target genes regulated by 1,25D is substantially altered by infection, and that 1,25D generally boosts infection-stimulated cytokine/chemokine responses. We further focused on the role of 1,25D- and infection-induced interleukin 1β (IL-1β) expression in response to infection. 1,25D enhanced IL-1β expression via a direct transcriptional mechanism. Secretion of IL-1β from infected cells required the NLRP3/caspase-1 inflammasome. The impact of IL-1β production was investigated in a novel model wherein infected macrophages were co-cultured with primary human small airway epithelial cells. Co-culture significantly prolonged survival of infected macrophages, and 1,25D/infection-induced IL-1β secretion from macrophages reduced mycobacterial burden by stimulating the anti-mycobacterial capacity of co-cultured lung epithelial cells. These effects were independent of 1,25D-stimulated autophagy in macrophages but dependent upon epithelial IL1R1 signaling and IL-1β-driven epithelial production of the antimicrobial peptide DEFB4/HBD2. These data provide evidence that the anti-microbial actions of vitamin D extend beyond the macrophage by modulating paracrine signaling, reinforcing its role in innate immune regulation in humans. PMID:23762029

  10. A model of secreting murine mammary epithelial HC11 cells comprising endogenous Bcrp/Abcg2 expression and function.

    PubMed

    Tallkvist, Jonas; Yagdiran, Yagmur; Danielsson, Louise; Oskarsson, Agneta

    2015-04-01

    Breast cancer resistance protein (Bcrp/Abcg2) and multidrug transporter 1 (Mdr1/Abcb1) are efflux proteins located in the apical membrane of mammary epithelial cells (MEC). Bcrp is induced in MEC during gestation and lactation, while Mdr1 is down-regulated during lactation. Numerous drugs and toxic compounds are known to be actively secreted into milk by Bcrp, but most chemicals have not been investigated in this respect, emphasizing the need for functional Bcrp studies in an established cell line with secreting mammary epithelial cells. The present study was undertaken to examine expressions of Bcrp and Mdr1 in mammary epithelial HC11 cells, derived from a mid-gestational murine mammary gland. In addition, Bcrp function was assessed by transport experiments with mitoxantrone (MX) in undifferentiated HC11 cells, in HC11 cells subjected to Bcrp RNA interference (RNAi), as well as in HC11 cells stimulated to differentiate by treatment with lactogenic hormones. Differentiated HC11 cells organized into alveolar-resembling structures and gene expression of the major milk protein β-casein was induced, whereas undifferentiated cells formed monolayers with lower β-casein expression. Bcrp and Mdr1 gene and protein were expressed in both undifferentiated and differentiated HC11 cells. Differentiation of HC11 cells resulted in increased Bcrp protein expression, while Mdr1 gene and protein expressions were reduced. The Bcrp inhibitor elacridar (GF120918) reduced secretion and increased accumulation of MX in both undifferentiated and differentiated HC11 cells. Silencing of the Bcrp gene caused an increased accumulation of MX. The results indicate that the HC11 cell model provides a promising tool to investigate transport of potential Bcrp substrates in mammary epithelial cells. PMID:25791223

  11. Microarray Analysis of Pneumococcal Gene Expression during Invasive Disease

    PubMed Central

    Orihuela, Carlos J.; Radin, Jana N.; Sublett, Jack E.; Gao, Geli; Kaushal, Deepak; Tuomanen, Elaine I.

    2004-01-01

    Streptococcus pneumoniae is a leading cause of invasive bacterial disease. This is the first study to examine the expression of S. pneumoniae genes in vivo by using whole-genome microarrays available from The Institute for Genomic Research. Total RNA was collected from pneumococci isolated from infected blood, infected cerebrospinal fluid, and bacteria attached to a pharyngeal epithelial cell line in vitro. Microarray analysis of pneumococcal genes expressed in these models identified body site-specific patterns of expression for virulence factors, transporters, transcription factors, translation-associated proteins, metabolism, and genes with unknown function. Contributions to virulence predicted for several unknown genes with enhanced expression in vivo were confirmed by insertion duplication mutagenesis and challenge of mice with the mutants. Finally, we cross-referenced our results with previous studies that used signature-tagged mutagenesis and differential fluorescence induction to identify genes that are potentially required by a broad range of pneumococcal strains for invasive disease. PMID:15385455

  12. Human bronchial epithelial cells exposed in vitro to diesel exhaust particles exhibit alterations in cell rheology and cytotoxicity associated with decrease in antioxidant defenses and imbalance in pro- and anti-apoptotic gene expression.

    PubMed

    Seriani, Robson; de Souza, Claudia Emanuele Carvalho; Krempel, Paloma Gava; Frias, Daniela Perroni; Matsuda, Monique; Correia, Aristides Tadeu; Ferreira, Márcia Zotti Justo; Alencar, Adriano Mesquita; Negri, Elnara Marcia; Saldiva, Paulo Hilário Nascimento; Mauad, Thais; Macchione, Mariangela

    2016-05-01

    Diesel exhaust particles (DEPs) from diesel engines produce adverse alterations in cells of the airways by activating intracellular signaling pathways and apoptotic gene overexpression, and also by influencing metabolism and cytoskeleton changes. This study used human bronchial epithelium cells (BEAS-2B) in culture and evaluates their exposure to DEPs (15ug/mL for 1 and 2 h) in order to determine changes to cell rheology (viscoelasticity) and gene expression of the enzymes involved in oxidative stress, apoptosis, and cytotoxicity. BEAS-2B cells exposed to DEPs were found to have a significant loss in stiffness, membrane stability, and mitochondrial activity. The genes involved in apoptosis [B cell lymphoma 2 (BCL-2 and caspase-3)] presented inversely proportional expressions (p = 0.05, p = 0.01, respectively), low expression of the genes involved in antioxidant responses [SOD1 (superoxide dismutase 1); SOD2 (superoxide dismutase 2), and GPx (glutathione peroxidase) (p = 0.01)], along with an increase in cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) (p = 0.01). These results suggest that alterations in cell rheology and cytotoxicity could be associated with oxidative stress and imbalance between pro- and anti-apoptotic genes. PMID:26856867

  13. Common Genetic Variation in Circadian Rhythm Genes and Risk of Epithelial Ovarian Cancer (EOC)

    PubMed Central

    Jim, Heather S.L.; Lin, Hui-Yi; Tyrer, Jonathan P.; Lawrenson, Kate; Dennis, Joe; Chornokur, Ganna; Chen, Zhihua; Chen, Ann Y.; Permuth-Wey, Jennifer; Aben, Katja KH.; Anton-Culver, Hoda; Antonenkova, Natalia; Bruinsma, Fiona; Bandera, Elisa V.; Bean, Yukie T.; Beckmann, Matthias W.; Bisogna, Maria; Bjorge, Line; Bogdanova, Natalia; Brinton, Louise A.; Brooks-Wilson, Angela; Bunker, Clareann H.; Butzow, Ralf; Campbell, Ian G.; Carty, Karen; Chang-Claude, Jenny; Cook, Linda S.; Cramer, Daniel W.; Cunningham, Julie M.; Cybulski, Cezary; Dansonka-Mieszkowska, Agnieszka; du Bois, Andreas; Despierre, Evelyn; Sieh, Weiva; Doherty, Jennifer A.; Dörk, Thilo; Dürst, Matthias; Easton, Douglas F.; Eccles, Diana M.; Edwards, Robert P.; Ekici, Arif B.; Fasching, Peter A.; Fridley, Brooke L.; Gao, Yu-Tang; Gentry-Maharaj, Aleksandra; Giles, Graham G.; Glasspool, Rosalind; Goodman, Marc T.; Gronwald, Jacek; Harter, Philipp; Hasmad, Hanis N.; Hein, Alexander; Heitz, Florian; Hildebrandt, Michelle A.T.; Hillemanns, Peter; Hogdall, Claus K.; Hogdall, Estrid; Hosono, Satoyo; Iversen, Edwin S.; Jakubowska, Anna; Jensen, Allan; Ji, Bu-Tian; Karlan, Beth Y.; Kellar, Melissa; Kiemeney, Lambertus A.; Krakstad, Camilla; Kjaer, Susanne K.; Kupryjanczyk, Jolanta; Vierkant, Robert A.; Lambrechts, Diether; Lambrechts, Sandrina; Le, Nhu D.; Lee, Alice W.; Lele, Shashi; Leminen, Arto; Lester, Jenny; Levine, Douglas A.; Liang, Dong; Lim, Boon Kiong; Lissowska, Jolanta; Lu, Karen; Lubinski, Jan; Lundvall, Lene; Massuger, Leon F.A.G.; Matsuo, Keitaro; McGuire, Valerie; McLaughlin, John R.; McNeish, Ian; Menon, Usha; Milne, Roger L.; Modugno, Francesmary; Thomsen, Lotte; Moysich, Kirsten B.; Ness, Roberta B.; Nevanlinna, Heli; Eilber, Ursula; Odunsi, Kunle; Olson, Sara H.; Orlow, Irene; Orsulic, Sandra; Palmieri Weber, Rachel; Paul, James; Pearce, Celeste L.; Pejovic, Tanja; Pelttari, Liisa M.; Pike, Malcolm C.; Poole, Elizabeth M.; Schernhammer, Eva; Risch, Harvey A.; Rosen, Barry; Rossing, Mary Anne; Rothstein, Joseph H.; Rudolph, Anja; Runnebaum, Ingo B.; Rzepecka, Iwona K.; Salvesen, Helga B.; Schwaab, Ira; Shu, Xiao-Ou; Shvetsov, Yurii B.; Siddiqui, Nadeem; Song, Honglin; Southey, Melissa C.; Spiewankiewicz, Beata; Sucheston-Campbell, Lara; Teo, Soo-Hwang; Terry, Kathryn L.; Thompson, Pamela J.; Tangen, Ingvild L.; Tworoger, Shelley S.; van Altena, Anne M.; Vergote, Ignace; Walsh, Christine S.; Wang-Gohrke, Shan; Wentzensen, Nicolas; Whittemore, Alice S.; Wicklund, Kristine G.; Wilkens, Lynne R.; Wu, Anna H.; Wu, Xifeng; Woo, Yin-Ling; Yang, Hannah; Zheng, Wei; Ziogas, Argyrios; Amankwah, Ernest; Berchuck, Andrew; Schildkraut, Joellen M.; Kelemen, Linda E.; Ramus, Susan J.; Monteiro, Alvaro N.A.; Goode, Ellen L.; Narod, Steven A.; Gayther, Simon A.; Pharoah, Paul D. P.; Sellers, Thomas A.; Phelan, Catherine M.

    2016-01-01

    Disruption in circadian gene expression, whether due to genetic variation or environmental factors (e.g., light at night, shiftwork), is associated with increased incidence of breast, prostate, gastrointestinal and hematologic cancers and gliomas. Circadian genes are highly expressed in the ovaries where they regulate ovulation; circadian disruption is associated with several ovarian cancer risk factors (e.g., endometriosis). However, no studies have examined variation in germline circadian genes as predictors of ovarian cancer risk and invasiveness. The goal of the current study was to examine single nucleotide polymorphisms (SNPs) in circadian genes BMAL1, CRY2, CSNK1E, NPAS2, PER3, REV1 and TIMELESS and downstream transcription factors KLF10 and SENP3 as predictors of risk of epithelial ovarian cancer (EOC) and histopathologic subtypes. The study included a test set of 3,761 EOC cases and 2,722 controls and a validation set of 44,308 samples including 18,174 (10,316 serous) cases and 26,134 controls from 43 studies participating in the Ovarian Cancer Association Consortium (OCAC). Analysis of genotype data from 36 genotyped SNPs and 4600 imputed SNPs indicated that the most significant association was rs117104877 in BMAL1 (OR = 0.79, 95% CI = 0.68–0.90, p = 5.59 × 10−4]. Functional analysis revealed a significant down regulation of BMAL1 expression following cMYC overexpression and increasing transformation in ovarian surface epithelial (OSE) cells as well as alternative splicing of BMAL1 exons in ovarian and granulosa cells. These results suggest that variation in circadian genes, and specifically BMAL1, may be associated with risk of ovarian cancer, likely through disruption of hormonal pathways. PMID:26807442

  14. The expression of gingival epithelial junctions in response to subgingival biofilms

    PubMed Central

    Belibasakis, Georgios N; Kast, Jeannette I; Thurnheer, Thomas; Akdis, Cezmi A; Bostanci, Nagihan

    2015-01-01

    Periodontitis is an infectious inflammatory disease that destroys the tooth-supporting tissues. It is caused by the formation of subgingival biofilms on the surface of the tooth. Characteristic bacteria associated with subgingival biofilms are the Gram-negative anaerobes Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola, collectively known as the “red complex” species. Inter-epithelial junctions ensure the barrier integrity of the gingival epithelium. This may however be disrupted by the biofilm challenge. The aim of this in vitro study was to investigate the effect of subgingival biofilms on the expression of inter-epithelial junctions by gingival epithelia, and evaluate the relative role of the red complex. Multi-layered human gingival epithelial cultures were challenged with a 10-species in vitro subgingival biofilm model, or its variant without the red complex, for 3 h and 24 h. A low-density array microfluidic card platform was then used for analyzing the expression of 62 genes encoding for tight junctions, gap junctions, adherens junctions, and desmosomes. Although there was a limited effect of the biofilms on the expression of tight, adherens and gap junctions, the expression of a number of desmosomal components was affected. In particular, Desmoglein-1 displayed a limited and transient up-regulation in response to the biofilm. In contrast, Desmocollin-2, Desmoplakin and Plakoglobin were down-regulated equally by both biofilm variants, after 24 h. In conclusion, this subgingival biofilm model may down-regulate selected desmosomal junctions in the gingival epithelium, irrespective of the presence of the “red complex.” In turn, this could compromise the structural integrity of the gingival tissue, favoring bacterial invasion and chronic infection. PMID:26305580

  15. Differential transcriptional regulation of IL-8 expression by human airway epithelial cells exposed to diesel exhaust particles

    SciTech Connect

    Tal, Tamara L.; Simmons, Steven O.; Silbajoris, Robert; Dailey, Lisa; Cho, Seung-Hyun; Ramabhadran, Ram; Linak, William; Reed, William; Bromberg, Philip A.; Samet, James M.

    2010-02-15

    Exposure to diesel exhaust particles (DEP) induces inflammatory signaling characterized by MAP kinase-mediated activation of NFkB and AP-1 in vitro and in bronchial biopsies obtained from human subjects exposed to DEP. NFkB and AP-1 activation results in the upregulation of genes involved in promoting inflammation in airway epithelial cells, a principal target of inhaled DEP. IL-8 is a proinflammatory chemokine expressed by the airway epithelium in response to environmental pollutants. The mechanism by which DEP exposure induces IL-8 expression is not well understood. In the current study, we sought to determine whether DEP with varying organic content induces IL-8 expression in lung epithelial cells, as well as, to develop a method to rapidly evaluate the upstream mechanism(s) by which DEP induces IL-8 expression. Exposure to DEP with varying organic content differentially induced IL-8 expression and IL-8 promoter activity human airway epithelial cells. Mutational analysis of the IL-8 promoter was also performed using recombinant human cell lines expressing reporters linked to the mutated promoters. Treatment with a low organic-containing DEP stimulated IL-8 expression by a mechanism that is predominantly NFkB-dependent. In contrast, exposure to high organic-containing DEP induced IL-8 expression independently of NFkB through a mechanism that requires AP-1 activity. Our study reveals that exposure to DEP of varying organic content induces proinflammatory gene expression through multiple specific mechanisms in human airway epithelial cells. The approaches used in the present study demonstrate the utility of a promoter-reporter assay ensemble for identifying transcriptional pathways activated by pollutant exposure.

  16. Expression of β-Defensin Genes in Bovine Alveolar Macrophages

    PubMed Central

    Ryan, Lisa K.; Rhodes, Janice; Bhat, Meenakshi; Diamond, Gill

    1998-01-01

    Bovine alveolar macrophages (BAM) were examined for the expression of β-defensins and to determine whether their expression could be upregulated by bacterial lipopolysaccharide (LPS), as observed with β-defensins expressed in bovine tracheal epithelial cells. Four β-defensins were expressed constitutively in BAM, with bovine neutrophil β-defensin (BNBD)-4 and BNBD-5 being the most predominant. This is the first evidence of β-defensin gene expression in a mature myeloid cell. LPS had no effect on β-defensin expression in BAM, even though tumor necrosis factor alpha (TNF-α) production was induced. Nonbacterial inflammatory particles had little effect on β-defensin gene expression or TNF-α production in BAM. We hypothesize that constitutively expressed β-defensins of alveolar macrophages may have a role in lung host defense. PMID:9453661

  17. [Establishment of goat limbal stem cell strain expressing Venus fluorescent protein and construction of limbal epithelial sheets].

    PubMed

    Yin, Jiqing; Liu, Wenqiang; Liu, Chao; Zhao, Guimin; Zhang, Yihua; Liu, Weishuai; Hua, Jinlian; Dou, Zhongying; Lei, Anmin

    2010-12-01

    The integrity and transparency of cornea plays a key role in vision. Limbal Stem Cells (LSCs) are precursors of cornea, which are responsible for self-renewal and replenishing corneal epithelium. Though it is successful to cell replacement therapy for impairing ocular surface by Limbal Stem Cell Transplantation (LSCT), the mechanism of renew is unclear after LSCT. To real time follow-up the migration and differentiation of corneal transplanted epithelial cells after transplanting, we transfected venus (a fluorescent protein gene) into goat LSCs, selected with G418 and established a stable transfected cell line, named GLSC-V. These cells showed green fluorescence, and which could maintain for at least 3 months. GLSC-V also were positive for anti-P63 and anti-Integrinbeta1 antibody by immunofluorescent staining. We founded neither GLSC-V nor GLSCs expressed keratin3 (k3) and keratinl2 (k12). However, GLSC-V had higher levels in expression of p63, pcna and venus compared with GLSCs. Further, we cultivated the cells on denude amniotic membrane to construct tissue engineered fluorescent corneal epithelial sheets. Histology and HE staining showed that the constructed fluorescent corneal epithelial sheets consisted of 5-6 layers of epithelium. Only the lowest basal cells of fluorescent corneal epithelial sheets expressed P63 analyzed by immunofluorescence, but not superficial epithelial cells. These results showed that our constructed fluorescent corneal epithelial sheets were similar to the normal corneal epithelium in structure and morphology. This demonstrated that they could be transplanted for patents with corneal impair, also may provide a foundation for the study on the mechanisms of corneal epithelial cell regeneration after LSCT. PMID:21387825

  18. NOD2 Expression is Regulated by microRNAs in Colonic Epithelial HCT116 Cells

    PubMed Central

    Chuang, Alice Y.; Chuang, Jim C.; Zhai, Zili; Wu, Feng; Kwon, John H.

    2016-01-01

    Background Crohn's disease (CD) is associated with defective sensing of pathogens in genetically susceptible individuals. Nucleotide-binding oligomerization domain containing 2 (NOD2) mutations in coding regions are strongly linked to CD pathogenesis. Our laboratory has reported that microRNAs (miRNAs) are differentially expressed in CD. However, miRNA regulation of NOD2 remains unknown. This study was designed to determine whether miRNAs regulate NOD2 expression as well as downstream nuclear factor kappaB activation and inflammatory responses in colonic epithelial HCT116 cells. Methods NOD2 and miRNA expression in stimulated HCT116 cells were assessed by quantitative reverse transcription–polymerase chain reaction. Regulation of NOD2 expression by miRNAs was determined by luciferase reporter construct assays and transfection of specific miRNA mimics. Regulation of NOD2 signaling and immune response by miRNAs was assessed by transfection of mimics followed by muramyl dipeptide stimulation. Results Muramyl dipeptide-induced increases in NOD2, interleukin-8, and CXCL3 expression were inversely associated with miRNA expression. Overexpression of miR-192, miR-495, miR-512, and miR-671 suppressed NOD2 expression, muramyl dipeptide-mediated NF-κB activation, and messenger RNA expressions of interleukin-8 and CXCL3 in HCT116 cells. A single-nucleotide polymorphism (rs3135500) located in the NOD2 3′-untranslated region significantly reduced miR-192 effects on NOD2 gene expression. Conclusions To our knowledge, this is the first report demonstrating that miRNAs regulate NOD2 and its signaling pathway. Four miRNAs downregulate NOD2 expression, suppress NF-κB activity, and inhibit interleukin-8 and CXCL3 messenger RNA expression. Treatment of CD with miRNAs may represent a potential anti-inflammatory therapeutic strategy in CD patients with and without NOD2 gene mutations. PMID:24297055

  19. Neisseria gonorrhoeae infection protects human endocervical epithelial cells from apoptosis via expression of host antiapoptotic proteins.

    PubMed

    Follows, S A; Murlidharan, J; Massari, P; Wetzler, L M; Genco, C A

    2009-09-01

    Several microbial pathogens can modulate the host apoptotic response to infection, which may contribute to immune evasion. Various studies have reported that infection with the sexually transmitted disease pathogen Neisseria gonorrhoeae can either inhibit or induce apoptosis. N. gonorrhoeae infection initiates at the mucosal epithelium, and in women, cells from the ectocervix and endocervix are among the first host cells encountered by this pathogen. In this study, we defined the antiapoptotic effect of N. gonorrhoeae infection in human endocervical epithelial cells (End/E6E7 cells). We first established that N. gonorrhoeae strain FA1090B failed to induce cell death in End/E6E7 cells. Subsequently, we demonstrated that stimulation with N. gonorrhoeae protected these cells from staurosporine (STS)-induced apoptosis. Importantly, only End/E6E7 cells incubated with live bacteria and in direct association with N. gonorrhoeae were protected from STS-induced apoptosis, while heat-killed and antibiotic-killed bacteria failed to induce protection. Stimulation of End/E6E7 cells with live N. gonorrhoeae induced NF-kappaB activation and resulted in increased gene expression of the NF-kappaB-regulated antiapoptotic genes bfl-1, cIAP-2, and c-FLIP. Furthermore, cIAP-2 protein levels also increased in End/E6E7 cells incubated with gonococci. Collectively, our results indicate that the antiapoptotic effect of N. gonorrhoeae in human endocervical epithelial cells results from live infection via expression of host antiapoptotic proteins. Securing an intracellular niche through the inhibition of apoptosis may be an important mechanism utilized by N. gonorrhoeae for microbial survival and immune evasion in cervical epithelial cells. PMID:19546192

  20. Transforming Growth Factor-β2 Induces Bronchial Epithelial Mucin Expression in Asthma

    PubMed Central

    Chu, Hong Wei; Balzar, Silvana; Seedorf, Gregory J.; Westcott, Jay Y.; Trudeau, John B.; Silkoff, Phil; Wenzel, Sally E.

    2004-01-01

    The transforming growth factor (TGF)-β family is important for tissue repair in pathological conditions including asthma. However, little is known about the impact of either TGF-β1 or TGF-β2 on asthmatic airway epithelial mucin expression. We evaluated bronchial epithelial TGF-β1 and TGF-β2 expression and their effects on mucin expression, and the role of TGF-β1 or TGF-β2 in interleukin (IL)-13-induced mucin expression. Epithelial TGF-β1, TGF-β2, and mucin expression were evaluated in endobronchial biopsies from asthmatics and normal subjects. The effects of TGF-β1 or TGF-β2 on mucin MUC5AC protein and mRNA expression, and the impact of IL-13 on epithelial TGF-β1, TGF-β2, and MUC5AC were determined in cultured bronchial epithelial cells from endobronchial brushings of both subject groups. In biopsy tissue, epithelial TGF-β2 expression levels were higher than TGF-β1 in both asthmatics and normals. TGF-β2, but not TGF-β1, was increased in asthmatics compared with normals, and significantly correlated with mucin expression. TGF-β2, but not TGF-β1, increased mucin expression in cultured epithelial cells from both subject groups. IL-13 increased the release of TGF-β2, but not TGF-β1, from epithelial cells. A neutralizing TGF-β2 antibody partially inhibited IL-13-induced mucin expression. These data suggest that TGF-β2 production by asthmatic bronchial epithelial cells may increase airway mucin expression. IL-13-induced mucin expression may occur in part through TGF-β2 up-regulation. PMID:15466377

  1. Transforming growth factor-beta2 induces bronchial epithelial mucin expression in asthma.

    PubMed

    Chu, Hong Wei; Balzar, Silvana; Seedorf, Gregory J; Westcott, Jay Y; Trudeau, John B; Silkoff, Phil; Wenzel, Sally E

    2004-10-01

    The transforming growth factor (TGF)-beta family is important for tissue repair in pathological conditions including asthma. However, little is known about the impact of either TGF-beta1 or TGF-beta2 on asthmatic airway epithelial mucin expression. We evaluated bronchial epithelial TGF-beta1 and TGF-beta2 expression and their effects on mucin expression, and the role of TGF-beta1 or TGF-beta2 in interleukin (IL)-13-induced mucin expression. Epithelial TGF-beta1, TGF-beta2, and mucin expression were evaluated in endobronchial biopsies from asthmatics and normal subjects. The effects of TGF-beta1 or TGF-beta2 on mucin MUC5AC protein and mRNA expression, and the impact of IL-13 on epithelial TGF-beta1, TGF-beta2, and MUC5AC were determined in cultured bronchial epithelial cells from endobronchial brushings of both subject groups. In biopsy tissue, epithelial TGF-beta2 expression levels were higher than TGF-beta1 in both asthmatics and normals. TGF-beta2, but not TGF-beta1, was increased in asthmatics compared with normals, and significantly correlated with mucin expression. TGF-beta2, but not TGF-beta1, increased mucin expression in cultured epithelial cells from both subject groups. IL-13 increased the release of TGF-beta2, but not TGF-beta1, from epithelial cells. A neutralizing TGF-beta2 antibody partially inhibited IL-13-induced mucin expression. These data suggest that TGF-beta2 production by asthmatic bronchial epithelial cells may increase airway mucin expression. IL-13-induced mucin expression may occur in part through TGF-beta2 up-regulation. PMID:15466377

  2. Thymoquinone inhibits cancer metastasis by downregulating TWIST1 expression to reduce epithelial to mesenchymal transition

    PubMed Central

    Khan, Md. Asaduzzaman; Tania, Mousumi; Wei, Chunli; Mei, Zhiqiang; Fu, Shelly; Cheng, Jingliang; Xu, Jianming; Fu, Junjiang

    2015-01-01

    Proteins that promote epithelial to mesenchymal transition (EMT) are associated with cancer metastasis. Inhibition of EMT regulators may be a promising approach in cancer therapy. In this study, Thymoquinone (TQ) was used to treat cancer cell lines to investigate its effects on EMT-regulatory proteins and cancer metastasis. We show that TQ inhibited cancer cell growth, migration and invasion in a dose-dependent manner. At the molecular level, TQ treatment decreased the transcriptional activity of the TWIST1 promoter and the mRNA expression of TWIST1, an EMT-promoting transcription factor. Accordingly, TQ treatment also decreased the expression of TWIST1-upregulated genes such as N-Cadherin and increased the expression of TWIST1-repressed genes such as E-Cadherin, resulting in a reduction of cell migration and invasion. TQ treatment also inhibited the growth and metastasis of cancer cell-derived xenograft tumors in mice but partially attenuated the migration and invasion in TWIST1-overexpressed cell lines. Furthermore, we found that TQ treatment enhanced the promoter DNA methylation of the TWIST1 gene in BT 549 cells. Together, these results demonstrate that TQ treatment inhibits TWIST1 promoter activity and decreases its expression, leading to the inhibition of cancer cell migration, invasion and metastasis. These findings suggest TQ as a potential small molecular inhibitor of cancer growth and metastasis. PMID:26023736

  3. Effect of proton and gamma irradiation on human lung carcinoma cells: Gene expression, cell cycle, cell death, epithelial-mesenchymal transition and cancer-stem cell trait as biological end points.

    PubMed

    Narang, Himanshi; Kumar, Amit; Bhat, Nagesh; Pandey, Badri N; Ghosh, Anu

    2015-10-01

    Proton beam therapy is a cutting edge modality over conventional gamma radiotherapy because of its physical dose deposition advantage. However, not much is known about its biological effects vis-a-vis gamma irradiation. Here we investigated the effect of proton- and gamma- irradiation on cell cycle, death, epithelial-mesenchymal transition (EMT) and "stemness" in human non-small cell lung carcinoma cells (A549). Proton beam (3MeV) was two times more cytotoxic than gamma radiation and induced higher and longer cell cycle arrest. At equivalent doses, numbers of genes responsive to proton irradiation were ten times higher than those responsive to gamma irradiation. At equitoxic doses, the proton-irradiated cells had reduced cell adhesion and migration ability as compared to the gamma-irradiated cells. It was also more effective in reducing population of Cancer Stem Cell (CSC) like cells as revealed by aldehyde dehydrogenase activity and surface phenotyping by CD44(+), a CSC marker. These results can have significant implications for proton therapy in the context of suppression of molecular and cellular processes that are fundamental to tumor expansion. PMID:26278043

  4. Surface expression of CD74 by type II alveolar epithelial cells: a potential mechanism for macrophage migration inhibitory factor-induced epithelial repair.

    PubMed

    Marsh, Leigh M; Cakarova, Lidija; Kwapiszewska, Grazyna; von Wulffen, Werner; Herold, Susanne; Seeger, Werner; Lohmeyer, Juergen

    2009-03-01

    Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine involved in acute lung injury and other processes such as wound repair and tumor growth. MIF exerts pro-proliferative effects on a variety of cell types including monocytes/macrophages, B cells, and gastric epithelial cell lines through binding to the major histocompatibility complex type II-associated invariant chain, CD74. In acute lung injury, inflammatory damage of the alveolar epithelium leads to loss of type I alveolar epithelial cells (AEC-I), which are replaced by proliferation and differentiation of type II alveolar epithelial cells (AEC-II). In this study we have investigated the potential of MIF to contribute to alveolar repair by stimulating alveolar epithelial cell proliferation. We show that murine AEC-II, but not AEC-I, express high surface levels of CD74 in vivo. Culture of AEC-II in vitro resulted in decreased mRNA levels for CD74 and loss of surface CD74 expression, which correlated with a transition of AEC-II to an AEC-I-like phenotype. MIF stimulation of AEC-II induced rapid and prolonged phosphorylation of ERK1/2 and Akt, increased expression of cyclins D1 and E, as well as AEC-II proliferation. Corresponding MIF signaling and enhanced thymidine incorporation was observed after MIF stimulation of MLE-12 cells transfected to overexpress CD74. In contrast, MIF did not induce MAPK activation, gene transcription, or increased proliferation in differentiated AEC-I-like cells that lack CD74. These data suggest a previously unidentified role of MIF-CD74 interaction by inducing proliferation of AEC-II, which may contribute to alveolar repair. PMID:19136583

  5. DNA conformation driven by AP-1 triggers cell-specific expression via a strong epithelial enhancer.

    PubMed

    Virolle, T; Djabari, Z; Ortonne, J P; Aberdam, D

    2000-10-01

    We report here the characterization of the regulatory region of the human LAMA3 gene, coding for the alpha3A chain of laminin-5. A 202 bp fragment is sufficient to confer epithelial-specific expression to a thymidine kinase promoter through the cooperative effect of three AP-1 binding sites. Remarkably, removal of the sequences located between the AP-1 sites does not modify the promoter activity in keratinocytes but allows strong expression in fibroblasts. Replacement of the deleted sequences by non-homologous ones fully restores the restricted enhancement in keratinocytes. Functional analysis and mutagenesis experiments demonstrate that a minimal distance between the AP-1 sites is required for the enhancer DNA fragment to adopt a particular conformation driven by the binding of Jun-Fos heterodimers. In non-permissive cells, this conformation leads to the anchorage of non-DNA-binding fibroblastic cofactors to form an inhibitory ternary complex. Therefore, our results describe for the first time an unusual conformation-dependent epithelial-specific enhancer. PMID:11269498

  6. Methylation Status and Expression of BRCA2 in Epithelial Ovarian Cancers in Indonesia.

    PubMed

    Pradjatmo, Heru

    2015-01-01

    Ovarian cancer is the main cause of mortality in gynecological malignancy and extensive studies have been conducted to study the underlying molecular mechanisms. The BRCA2 gene is known to be an important tumor suppressor in ovarian cancer, thereby BRCA2 alterations may lead to cancer progression. However, the BRCA2 gene is rarely mutated, and loss of function is suspected to be mediated by epigenetic regulation. In this study we investigated the methylation status and gene expression of BRCA2 in ovarian cancer patients. Ovarian cancer pateints (n=69) were recruited and monitored for 54 months in this prospective cohort study. Clinical specimens were used to study the in situ expression of aberrant BRCA2 proteins and the methylation status of BRCA2. These parameters were then compared with clinical parameters and overall survival rate. We found that BRCA2 methylation was found in the majority of cases (98.7%). However, the methylation status was not associated with protein level expression of BRCA2 (49.3%). Therefore in addition to DNA methylation, other epigenetic mechanisms may regulate BRCA2 expresison. Our findings may become evidence of BRCA2 inactivation mechanism through DNA methylation in the Indonesian population. More importantly, from multivariate analysis, BRCA2 expression was correlated with better overall survival (HR 0.32; p=0.05). High percentage of BRCA2 methylation and correlation of BRCA2 expression with overall survival in epithelial ovarian cancer cases may lead to development of treatment modalities specifically to target methylation of BRCA genes. PMID:26745123

  7. Differential DNA methylation profile of key genes in malignant prostate epithelial cells transformed by inorganic arsenic or cadmium

    SciTech Connect

    Pelch, Katherine E.; Tokar, Erik J.; Merrick, B. Alex; Waalkes, Michael P.

    2015-08-01

    Previous work shows altered methylation patterns in inorganic arsenic (iAs)- or cadmium (Cd)-transformed epithelial cells. Here, the methylation status near the transcriptional start site was assessed in the normal human prostate epithelial cell line (RWPE-1) that was malignantly transformed by 10 μM Cd for 11 weeks (CTPE) or 5 μM iAs for 29 weeks (CAsE-PE), at which time cells showed multiple markers of acquired cancer phenotype. Next generation sequencing of the transcriptome of CAsE-PE cells identified multiple dysregulated genes. Of the most highly dysregulated genes, five genes that can be relevant to the carcinogenic process (S100P, HYAL1, NTM, NES, ALDH1A1) were chosen for an in-depth analysis of the DNA methylation profile. DNA was isolated, bisulfite converted, and combined bisulfite restriction analysis was used to identify differentially methylated CpG sites, which was confirmed with bisulfite sequencing. Four of the five genes showed differential methylation in transformants relative to control cells that was inversely related to altered gene expression. Increased expression of HYAL1 (> 25-fold) and S100P (> 40-fold) in transformants was correlated with hypomethylation near the transcriptional start site. Decreased expression of NES (> 15-fold) and NTM (> 1000-fold) in transformants was correlated with hypermethylation near the transcriptional start site. ALDH1A1 expression was differentially expressed in transformed cells but was not differentially methylated relative to control. In conclusion, altered gene expression observed in Cd and iAs transformed cells may result from altered DNA methylation status. - Highlights: • Cd and iAs are known human carcinogens, yet neither appears directly mutagenic. • Prior data suggest epigenetic modification plays a role in Cd or iAs induced cancer. • Altered methylation of four misregulated genes was found in Cd or iAs transformants. • The resulting altered gene expression may be relevant to cellular

  8. Nuclear AXIN2 represses MYC gene expression.

    PubMed

    Rennoll, Sherri A; Konsavage, Wesley M; Yochum, Gregory S

    2014-01-01

    The β-catenin transcriptional coactivator is the key mediator of the canonical Wnt signaling pathway. In the absence of Wnt, β-catenin associates with a cytosolic and multi-protein destruction complex where it is phosphorylated and targeted for proteasomal degradation. In the presence of Wnt, the destruction complex is inactivated and β-catenin translocates into the nucleus. In the nucleus, β-catenin binds T-cell factor (TCF) transcription factors to activate expression of c-MYC (MYC) and Axis inhibition protein 2 (AXIN2). AXIN2 is a member of the destruction complex and, thus, serves in a negative feedback loop to control Wnt/β-catenin signaling. AXIN2 is also present in the nucleus, but its function within this compartment is unknown. Here, we demonstrate that AXIN2 localizes to the nuclei of epithelial cells within normal and colonic tumor tissues as well as colorectal cancer cell lines. In the nucleus, AXIN2 represses expression of Wnt/β-catenin-responsive luciferase reporters and forms a complex with β-catenin and TCF. We demonstrate that AXIN2 co-occupies β-catenin/TCF complexes at the MYC promoter region. When constitutively localized to the nucleus, AXIN2 alters the chromatin structure at the MYC promoter and directly represses MYC gene expression. These findings suggest that nuclear AXIN2 functions as a rheostat to control MYC expression in response to Wnt/β-catenin signaling. PMID:24299953

  9. YAP Regulates the Expression of Hoxa1 and Hoxc13 in Mouse and Human Oral and Skin Epithelial Tissues

    PubMed Central

    Liu, Ming; Zhao, Shuangyun; Lin, Qingjie

    2015-01-01

    Yes-associated protein (YAP) is a Hippo signaling transcriptional coactivator that plays pivotal roles in stem cell proliferation, organ size control, and tumor development. The downstream targets of YAP have been shown to be highly context dependent. In this study, we used the embryonic mouse tooth germ as a tool to search for the downstream targets of YAP in ectoderm-derived tissues. Yap deficiency in the dental epithelium resulted in a small tooth germ with reduced epithelial cell proliferation. We compared the gene expression profiles of embryonic day 14.5 (E14.5) Yap conditional knockout and YAP transgenic mouse tooth germs using transcriptome sequencing (RNA-Seq) and further confirmed the differentially expressed genes using real-time PCR and in situ hybridization. We found that YAP regulates the expression of Hoxa1 and Hoxc13 in oral and dental epithelial tissues as well as in the epidermis of skin during embryonic and adult stages. Sphere formation assay suggested that Hoxa1 and Hoxc13 are functionally involved in YAP-regulated epithelial progenitor cell proliferation, and chromatin immunoprecipitation (ChIP) assay implies that YAP may regulate Hoxa1 and Hoxc13 expression through TEAD transcription factors. These results provide mechanistic insights into abnormal YAP activities in mice and humans. PMID:25691658

  10. Nuclear Neighborhoods and Gene Expression

    PubMed Central

    Zhao, Rui; Bodnar, Megan S.; Spector, David L.

    2009-01-01

    Summary The eukaryotic nucleus is a highly compartmentalized and dynamic environment. Chromosome territories are arranged non-randomly within the nucleus and numerous studies have indicated that a gene’s position in the nucleus can impact its transcriptional activity. Here, we focus on recent advances in our understanding of the influence of specific nuclear neighborhoods on gene expression or repression. Nuclear neighborhoods associated with transcriptional repression include the inner nuclear membrane/nuclear lamina and peri-nucleolar chromatin, whereas neighborhoods surrounding the nuclear pore complex, PML nuclear bodies, and nuclear speckles seem to be transcriptionally permissive. While nuclear position appears to play an important role in gene expression, it is likely to be only one piece of a flexible puzzle that incorporates numerous parameters. We are still at a very early, yet exciting stage in our journey toward deciphering the mechanism(s) that govern the permissiveness of gene expression/repression within different nuclear neighborhoods. PMID:19339170

  11. Differential Gene Expression in Glaucoma

    PubMed Central

    Jakobs, Tatjana C.

    2014-01-01

    In glaucoma, regardless of its etiology, retinal ganglion cells degenerate and eventually die. Although age and elevated intraocular pressure (IOP) are the main risk factors, there are still many mysteries in the pathogenesis of glaucoma. The advent of genome-wide microarray expression screening together with the availability of animal models of the disease has allowed analysis of differential gene expression in all parts of the eye in glaucoma. This review will outline the findings of recent genome-wide expression studies and discuss their commonalities and differences. A common finding was the differential regulation of genes involved in inflammation and immunity, including the complement system and the cytokines transforming growth factor β (TGFβ) and tumor necrosis factor α (TNFα). Other genes of interest have roles in the extracellular matrix, cell–matrix interactions and adhesion, the cell cycle, and the endothelin system. PMID:24985133

  12. Posttraumatic temporal TGF-β mRNA expression in lens epithelial cells of paediatric patients.

    PubMed

    Berezowski, P; Strzalka-Mrozik, B; Forminska-Kapuscik, M; Mazurek, U; Filipek, E; Nawrocka, L; Pieczara, E; Banasiak, P; Kimsa, M

    2012-01-01

    The aim of the study was to determine temporal TGFB1, TGFB2 and TGFB3 gene expression profiles in the anterior lens capsule of paediatric patients with posttraumatic cataract. The patient group comprised 22 children selected with a fragment of anterior lens capsule obtained during elective cataract surgery and sampled for molecular analysis. The levels of TGF-β isoforms in the anterior lens capsule were determined based on the number of mRNA copies per 1 μg total RNA by real-time qRTPCR. Three time-related result clusters were identified based on hierarchical cluster analysis: 2.2, 4.4 and 15.0 months (time span from injury to anterior capsule sampling during surgery) and compared with regard to temporal gene expression profile and quantitative relations of TGF-β1, 2 and 3 mRNAs. TGF-β1, TGF-β2, and TGF-β3 mRNAs were detected in all anterior lens capsule samples. A comparative analysis revealed: TGF-β1>TGF-β2>TGF-β3 during the entire observation period. The TGF-β mRNA levels continued to increase up to four months after injury, then returning close to the base levels after around 15 months. The expression patterns of TGF-β isoforms showed a similar tendency. Differences in the expression levels of TGF-β1 and TGF-β2 between the particular clusters were statistically significant. Posttraumatic transcriptional activities of TGF-β1 and TGF-β2 in the anterior lens capsule of paediatric patients depend on the time elapsing from injury. Our findings indicate that the transcriptional activities of TGFB family genes show a transient period of over-expression during the months after injury. TGF-β1 is a dominant isoform expressed in lens epithelial cells following injury. PMID:22464821

  13. Transgenic Arabidopsis Gene Expression System

    NASA Technical Reports Server (NTRS)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  14. Hyperglycemia-Suppressed Expression of Serpine1 Contributes to Delayed Epithelial Wound Healing in Diabetic Mouse Corneas

    PubMed Central

    Sun, Haijing; Mi, Xiaofan; Gao, Nan; Yan, Chenxi; Yu, Fu-Shin

    2015-01-01

    Purpose. Patients with diabetes mellitus (DM) are at an increased risk for developing corneal complications, including delayed wound healing. The purpose of this study was to characterize the expression and the function of Serpine1 and other components of urokinase plasminogen activator (uPA)–proteolytic system in delayed epithelial wound healing in diabetic mouse corneas. Methods. Mice of the strain C57BL/6 were induced to develop diabetes by streptozotocin, and wound-healing assays were performed 10 weeks afterward. Gene expression and/or distribution were assessed by real-time PCR, Western blotting, and/or immunohistochemistry. The role of Serpine1 in mediating epithelial wound closure was determined by subconjunctival injections of neutralizing antibodies in either normal or recombinant protein in diabetic corneas. Enzyme assay for matrix metalloproteinase (MMP)-3 was also performed. Results. The expressions of Serpine1 (PAI-1), Plau (uPA), and Plaur (uPA receptor) were upregulated in response to wounding, and these upregulations were significantly suppressed by hyperglycemia. In healing epithelia, Plau and Serpine1 were abundantly expressed at the leading edge of the healing epithelia of normal and, to a lesser extent, diabetic corneas. Inhibition of Serpine1 delayed epithelial wound closure in normal corneas, whereas recombinant Serpine1 accelerated it in diabetic corneas. The Plau and MMP-3 mRNA levels and MMP-3 enzymatic activities were correlated to Serpine1 levels and/or the rates of epithelial wound closure. Conclusions. Serpine1 plays a role in mediating epithelial wound healing and its impaired expression may contribute to delayed wound healing in DM corneas. Hence, modulating uPA proteolytic pathway may represent a new approach for treating diabetic keratopathy. PMID:26024123

  15. Nuclear AXIN2 represses MYC gene expression

    SciTech Connect

    Rennoll, Sherri A.; Konsavage, Wesley M.; Yochum, Gregory S.

    2014-01-03

    Highlights: •AXIN2 localizes to cytoplasmic and nuclear compartments in colorectal cancer cells. •Nuclear AXIN2 represses the activity of Wnt-responsive luciferase reporters. •β-Catenin bridges AXIN2 to TCF transcription factors. •AXIN2 binds the MYC promoter and represses MYC gene expression. -- Abstract: The β-catenin transcriptional coactivator is the key mediator of the canonical Wnt signaling pathway. In the absence of Wnt, β-catenin associates with a cytosolic and multi-protein destruction complex where it is phosphorylated and targeted for proteasomal degradation. In the presence of Wnt, the destruction complex is inactivated and β-catenin translocates into the nucleus. In the nucleus, β-catenin binds T-cell factor (TCF) transcription factors to activate expression of c-MYC (MYC) and Axis inhibition protein 2 (AXIN2). AXIN2 is a member of the destruction complex and, thus, serves in a negative feedback loop to control Wnt/β-catenin signaling. AXIN2 is also present in the nucleus, but its function within this compartment is unknown. Here, we demonstrate that AXIN2 localizes to the nuclei of epithelial cells within normal and colonic tumor tissues as well as colorectal cancer cell lines. In the nucleus, AXIN2 represses expression of Wnt/β-catenin-responsive luciferase reporters and forms a complex with β-catenin and TCF. We demonstrate that AXIN2 co-occupies β-catenin/TCF complexes at the MYC promoter region. When constitutively localized to the nucleus, AXIN2 alters the chromatin structure at the MYC promoter and directly represses MYC gene expression. These findings suggest that nuclear AXIN2 functions as a rheostat to control MYC expression in response to Wnt/β-catenin signaling.

  16. Increased programmed death-ligand-1 expression in human gastric epithelial cells in Helicobacter pylori infection

    PubMed Central

    Wu, Y-Y; Lin, C-W; Cheng, K-S; Lin, C; Wang, Y-M; Lin, I-T; Chou, Y-H; Hsu, P-N

    2010-01-01

    B7-H1 [programmed death-ligand-1 (PD-L1)] is a B7-family member that binds to programmed death-1 (PD-1). Recently, deficiency of PD-L1 has been demonstrated to result in accelerated gastric epithelial cell damage in gastritis, and PD-L1 is suggested to play a critical role in regulating T cell homeostasis. Here, we aimed to gain more insight into gastric PD-L1 expression, regulation and function during Helicobacter pylori infection. PD-L1 expression in human gastric epithelial cells was analysed using Western blotting, quantitative polymerase chain reaction and fluorescence activated cell sorter analysis. Furthermore, co-culture experiments of human gastric epithelial cells with primary human T cells or Jurkat T cells were conducted. PD-L1 expression in primary human gastric epithelial cells was strongly enhanced by H. pylori infection and activated T cells, and augmented markedly by further stimulation with interferon-γ or tumour necrosis factor-α. Moreover, PD-L1 expression in gastric epithelial cells significantly induced apoptosis of T cells. Our results indicate that a novel bidirectional interaction between human gastric epithelial cells and lymphocytes modulates PD-L1 expression in human gastric epithelial cells, contributing to the unique immunological properties of the stomach. PMID:20646001

  17. Developmental expression profiles of Celsr (Flamingo) genes in the mouse.

    PubMed

    Tissir, F; De-Backer, O; Goffinet, A M; Lambert de Rouvroit, C

    2002-03-01

    Celsr, also called Flamingo (Fmi) genes encode proteins of the cadherin superfamily. Celsr cadherins are seven-pass transmembrane proteins with nine cadherin repeats in the extracellular domain, and an anonymous intracellular C-terminus. The Drosophila Fmi gene regulates epithelial planar cell polarity and dendritic field deployment. The three Flamingo gene orthologs in man and rodents are named, respectively, CELSR1-3 and Celsr1-3. Celsr1 and 2 are expressed during early development, in the brain and epithelia. In this report, we characterized further Celsr genes in the mouse, and examined their developmental pattern of expression. Each Celsr is expressed prominently in the developing brain following a specific pattern, suggesting that they serve distinct functions. PMID:11850187

  18. Transcriptional Modulation of Enterotoxigenic Escherichia coli Virulence Genes in Response to Epithelial Cell Interactions

    PubMed Central

    Kansal, Rita; Rasko, David A.; Sahl, Jason W.; Munson, George P.; Roy, Koushik; Luo, Qingwei; Sheikh, Alaullah; Kuhne, Kurt J.

    2013-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of morbidity and mortality due to diarrheal illness in developing countries. There is currently no effective vaccine against these important pathogens. Because genes modulated by pathogen-host interactions potentially encode putative vaccine targets, we investigated changes in gene expression and surface morphology of ETEC upon interaction with intestinal epithelial cells in vitro. Pan-genome microarrays, quantitative reverse transcriptase PCR (qRT-PCR), and transcriptional reporter fusions of selected promoters were used to study changes in ETEC transcriptomes. Flow cytometry, immunofluorescence microscopy, and scanning electron microscopy were used to investigate alterations in surface antigen expression and morphology following pathogen-host interactions. Following host cell contact, genes for motility, adhesion, toxin production, immunodominant peptides, and key regulatory molecules, including cyclic AMP (cAMP) receptor protein (CRP) and c-di-GMP, were substantially modulated. These changes were accompanied by visible changes in both ETEC architecture and the expression of surface antigens, including a novel highly conserved adhesin molecule, EaeH. The studies reported here suggest that pathogen-host interactions are finely orchestrated by ETEC and are characterized by coordinated responses involving the sequential deployment of multiple virulence molecules. Elucidation of the molecular details of these interactions could highlight novel strategies for development of vaccines for these important pathogens. PMID:23115039

  19. Transcriptional modulation of enterotoxigenic Escherichia coli virulence genes in response to epithelial cell interactions.

    PubMed

    Kansal, Rita; Rasko, David A; Sahl, Jason W; Munson, George P; Roy, Koushik; Luo, Qingwei; Sheikh, Alaullah; Kuhne, Kurt J; Fleckenstein, James M

    2013-01-01

    Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of morbidity and mortality due to diarrheal illness in developing countries. There is currently no effective vaccine against these important pathogens. Because genes modulated by pathogen-host interactions potentially encode putative vaccine targets, we investigated changes in gene expression and surface morphology of ETEC upon interaction with intestinal epithelial cells in vitro. Pan-genome microarrays, quantitative reverse transcriptase PCR (qRT-PCR), and transcriptional reporter fusions of selected promoters were used to study changes in ETEC transcriptomes. Flow cytometry, immunofluorescence microscopy, and scanning electron microscopy were used to investigate alterations in surface antigen expression and morphology following pathogen-host interactions. Following host cell contact, genes for motility, adhesion, toxin production, immunodominant peptides, and key regulatory molecules, including cyclic AMP (cAMP) receptor protein (CRP) and c-di-GMP, were substantially modulated. These changes were accompanied by visible changes in both ETEC architecture and the expression of surface antigens, including a novel highly conserved adhesin molecule, EaeH. The studies reported here suggest that pathogen-host interactions are finely orchestrated by ETEC and are characterized by coordinated responses involving the sequential deployment of multiple virulence molecules. Elucidation of the molecular details of these interactions could highlight novel strategies for development of vaccines for these important pathogens. PMID:23115039

  20. Expression and function of the epithelial sodium channel δ-subunit in human respiratory epithelial cells in vitro.

    PubMed

    Schwagerus, Elena; Sladek, Svenja; Buckley, Stephen T; Armas-Capote, Natalia; Alvarez de la Rosa, Diego; Harvey, Brian J; Fischer, Horst; Illek, Beate; Huwer, Hanno; Schneider-Daum, Nicole; Lehr, Claus-Michael; Ehrhardt, Carsten

    2015-11-01

    Using human airway epithelial cell lines (i.e. NCI-H441 and Calu-3) as well as human alveolar epithelial type I-like (ATI) cells in primary culture, we studied the contribution of the epithelial sodium channel δ-subunit (δ-ENaC) to transepithelial sodium transport in human lung in vitro. Endogenous δ-ENaC protein was present in all three cell types tested; however, protein abundance was low, and no expression was detected in the apical cell membrane of these cells. Similarly, known modulators of δ-ENaC activity, such as capsazepine and icilin (activators) and Evans blue (inhibitor), did not show effects on short-circuit current (I SC), suggesting that δ-ENaC is not involved in the modulation of transcellular sodium absorption in NCI-H441 cell monolayers. Over-expression of δ-ENaC in NCI-H441 cells resulted in detectable protein expression in the apical cell membrane, as well as capsazepine and icilin-stimulated increases in I SC that were effectively blocked by Evans blue and that were consistent with δ-ENaC activation and inhibition, respectively. Consequently, these observations suggest that δ-ENaC expression is low in NCI-H441, Calu-3, and ATI cells and does not contribute to transepithelial sodium absorption. PMID:25677639

  1. Molecular mechanisms controlling CFTR gene expression in the airway

    PubMed Central

    Zhang, Zhaolin; Ott, Christopher J; Lewandowska, Marzena A; Leir, Shih-Hsing; Harris, Ann

    2012-01-01

    Abstract The low levels of CFTR gene expression and paucity of CFTR protein in human airway epithelial cells are not easily reconciled with the pivotal role of the lung in cystic fibrosis pathology. Previous data suggested that the regulatory mechanisms controlling CFTR gene expression might be different in airway epithelium in comparison to intestinal epithelium where CFTR mRNA and protein is much more abundant. Here we examine chromatin structure and modification across the CFTR locus in primary human tracheal (HTE) and bronchial (NHBE) epithelial cells and airway cell lines including 16HBE14o- and Calu3. We identify regions of open chromatin that appear selective for primary airway epithelial cells and show that several of these are enriched for a histone modification (H3K4me1) that is characteristic of enhancers. Consistent with these observations, three of these sites encompass elements that have cooperative enhancer function in reporter gene assays in 16HBE14o- cells. Finally, we use chromosome conformation capture (3C) to examine the three-dimensional structure of nearly 800 kb of chromosome 7 encompassing CFTR and observe long-range interactions between the CFTR promoter and regions far outside the locus in cell types that express high levels of CFTR. PMID:21895967

  2. Endothelial and Epithelial Cell Transition to a Mesenchymal Phenotype Was Delineated by Nestin Expression.

    PubMed

    Chabot, Andréanne; Hertig, Vanessa; Boscher, Elena; Nguyen, Quang Trinh; Boivin, Benoît; Chebli, Jasmine; Bissonnette, Lyse; Villeneuve, Louis; Brochiero, Emmanuelle; Dupuis, Jocelyn; Calderone, Angelino

    2016-07-01

    Cover: The cover image, by Angelino Calderone et al., is based on the Original Research Article Endothelial and Epithelial Cell Transition to a Mesenchymal Phenotype Was Delineated by Nestin Expression, DOI: 10.1002/jcp.25257. PMID:26995059

  3. Neighboring Genes Show Correlated Evolution in Gene Expression

    PubMed Central

    Ghanbarian, Avazeh T.; Hurst, Laurence D.

    2015-01-01

    When considering the evolution of a gene’s expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (<100 kb) but extends much further. Sex-specific expression change is also genomically clustered. As genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. PMID:25743543

  4. Gene cloning and expression of an aquaporin (AQP-h3BL) in the basolateral membrane of water-permeable epithelial cells in osmoregulatory organs of the tree frog.

    PubMed

    Akabane, Gen; Ogushi, Yuji; Hasegawa, Takahiro; Suzuki, Masakazu; Tanaka, Shigeyasu

    2007-06-01

    An aquaporin (Hyla AQP-h3BL), consisting of 292 amino acid residues, has been cloned from the urinary bladder of Hyla japonica. In a swelling assay using Xenopus oocytes, AQP-h3BL cRNA-injected oocytes developed a sevenfold and 2.8-fold higher permeability to water and glycerol, respectively, than the water-injected oocytes. This permeability was inhibited by HgCl2. Immunofluorescence revealed that AQP-h3BL is localized in the basolateral plasma membrane of both granular cells in the ventral pelvic and dorsal skins and the secretory cells in the mucous glands. Immunopositive cells were also observed in the basolateral membrane of principal cells in the collecting ducts and in a portion of the late distal tubules in the kidneys, as well as in the principal cells of the urinary bladder. Sequence homology suggests that AQP-h3BL is a homolog to mammalian AQP3. This conclusion is supported by the observed localization of AQP-h3BL to the basolateral membrane in water- and glycerol-permeable epithelial cells. In ventral pelvic skins and urinary bladders, water enters into the cytoplasm through the apical plasma membrane at sites where AQP-h2, sometimes in association with AQP-h3, responds to stimulation by vasotocin; the water exits throughout AQP-h3BL to extracellular spaces. In the mucous glands, on the other hand, water enters throughout this AQP-h3BL and exits through AQP-x5, which is in the apical membrane of secretory cells. Thus, water homeostasis in the frog body is regulated by AQP-h3BL expressed in the basolateral membrane in concert with arginine vasotocin (AVT)-dependent or AVT-independent AQP. PMID:17332153

  5. Induction of multidrug resistance downregulates the expression of CFTR in colon epithelial cells.

    PubMed

    Breuer, W; Slotki, I N; Ausiello, D A; Cabantchik, I Z

    1993-12-01

    The epithelial cell line HT-29, which constitutively expresses the cystic fibrosis transmembrane conductance regulator (CFTR), was induced to become drug resistant by cultivation in the presence of colchicine. The gradual acquisition of drug resistance was associated with a corresponding increase in the expression of the multidrug resistance P-glycoprotein (P-gp) and a marked (> 80%) decrease in the constitutive levels of CFTR protein, as determined by immunoblotting. The reduction in CFTR content occurred at the onset of acquisition of drug resistance when P-gp expression was still relatively low. Reversal of drug resistance by removal of colchicine from the culture medium led to a 70% decrease in P-gp levels and a concomitant 40% increase in CFTR. The levels of other membrane proteins such as Na(+)-K(+)-ATPase and alkaline phosphatase remained relatively constant (< 26% variation). We propose that a selective downregulation of CFTR is elicited by acquisition of the multidrug resistance (MDR) phenotype and that induction of P-gp expression leads to a reversible repression of CFTR biosynthesis. These findings provide an experimental foundation for the complementary patterns of expression of the CFTR and MDR1 genes observed in vivo. PMID:7506492

  6. SAFETY AND EFFICIENCY OF MODULATING PARACELLULAR PERMEABILITY TO ENHANCE AIRWAY EPITHELIAL GENE TRANSFER IN VIVO

    EPA Science Inventory


    ABSTRACT

    We evaluated the safety of agents that enhance gene transfer by modulating paracellular permeability. Lactate dehydrogenase (LDH) and cytokine release were measured in polarized primary human airway epithelial (HAE) cells after luminal application of vehicle, ...

  7. Epithelial PIK3R1 (p85) and TP53 Regulate Survivin Expression during Adaptation to Ileocecal Resection.

    PubMed

    Cohran, Valeria; Managlia, Elizabeth; Bradford, Emily M; Goretsky, Tatiana; Li, Ting; Katzman, Rebecca B; Cheresh, Paul; Brown, Jeffrey B; Hawkins, Jennifer; Liu, Shirley X L; De Plaen, Isabelle G; Weitkamp, Jörn-Hendrik; Helmrath, Michael; Zhang, Zheng; Barrett, Terrence A

    2016-07-01

    Intestinal adaptation to small-bowel resection (SBR) after necrotizing enterocolitis expands absorptive surface areas and promotes enteral autonomy. Survivin increases proliferation and blunts apoptosis. The current study examines survivin in intestinal epithelial cells after ileocecal resection. Wild-type and epithelial Pik3r1 (p85α)-deficient mice underwent sham surgery or 30% resection. RNA and protein were isolated from small bowel to determine levels of β-catenin target gene expression, activated caspase-3, survivin, p85α, and Trp53. Healthy and post-resection human infant small-bowel sections were analyzed for survivin, Ki-67, and TP53 by immunohistochemistry. Five days after ileocecal resection, epithelial levels of survivin increased relative to sham-operated on mice, which correlated with reduced cleaved caspase-3, p85α, and Trp53. At baseline, p85α-deficient intestinal epithelial cells had less Trp53 and more survivin, and relative responses to resection were blunted compared with wild-type. In infant small bowel, survivin in transit amplifying cells increased 71% after SBR. Resection increased proliferation and decreased numbers of TP53-positive epithelial cells. Data suggest that ileocecal resection reduces p85α, which lowers TP53 activation and releases survivin promoter repression. The subsequent increase in survivin among transit amplifying cells promotes epithelial cell proliferation and lengthens crypts. These findings suggest that SBR reduces p85α and TP53, which increases survivin and intestinal epithelial cell expansion during therapeutic adaptation in patients with short bowel syndrome. PMID:27157990

  8. Platelet-activating factor induces collagenase expression in corneal epithelial cells.

    PubMed Central

    Bazan, H E; Tao, Y; Bazan, N G

    1993-01-01

    Platelet-activating factor (PAF), a potent lipid mediator involved in inflammatory and immune responses, accumulates rapidly in response to injury in a variety of tissues, including the corneal epithelium. However, the precise role of this compound in the cascade of events following insult has not been defined. Here we examined the effect of PAF on gene expression in the epithelial cells of rabbit corneas in organ culture. We found that incubation with 100 nM methylcarbamoyl PAF, a nonhydrolyzable analog of PAF, produced rapid transient 2.8- and 3.5-fold increases in the expression of c-fos and c-jun, respectively, at 1 hr, followed by increased expression of the collagenase type I gene beginning at 3 hr and peaking at 14-fold by 8 hr. Addition of the protein-synthesis-inhibitor cycloheximide superinduced c-fos and c-jun, strongly potentiating the PAF effect, but inhibited the induction of collagenase type I expression, suggesting the existence of a transcriptional factor linking the two events. BN-50730, a selective antagonist of intracellular PAF-binding sites, blocked the expression of the immediate-early genes as well as the increase in collagenase type I mRNA. Our results suggest that one of the functions of PAF may be to enhance the breakdown of the extracellular matrix as a part of the remodeling process during corneal wound healing after injury. Pathologically, a PAF-induced overproduction of collagenase may be a factor in the development of corneal ulcers, as well as other pathophysiological conditions such as cartilage destruction in arthritis. If so, inhibitors of this signal-transduction pathway may be useful as tools for further investigation and, eventually, as therapeutic agents to treat such disorders. Images Fig. 1 Fig. 2 PMID:8378347

  9. Vascular endothelial growth factor polymorphisms and a synchronized examination of plasma and tissue expression in epithelial ovarian cancers.

    PubMed

    Bhaskari, J; Premalata, C S; Shilpa, V; Rahul, B; Pallavi, V R; Ramesh, G; Krishnamoorthy, Lakshmi

    2016-01-01

    In this study, we have analyzed six genetic polymorphisms of the VEGF-A gene and correlated the genetic data with plasma and tissue expression of VEGF-A in epithelial ovarian carcinomas. A total of 130 cases including 95 malignant carcinomas, 17 low malignant potential and 18 benign tumours were studied. rs699947, rs833061, rs1570360, rs2010963, rs1413711 and rs3025039 were studied by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Plasma levels of VEGF-A were estimated by enzyme-linked immunosorbent assay (ELISA) and tissue expression of VEGF-A by immunohistochemistry (IHC). Four polymorphisms of the above excluding rs699947 and rs3025039 showed significant association with malignancy, and we observed the presence of positive correlation between haplotype CCGGCC and increased expression of VEGF-A in both plasma and tissues which also correlated with poor prognosis and recurrence suggesting a probable increase in resistance to treatment in such carriers. Highly upregulated tissue expression of VEGF-A was seen in all epithelial ovarian carcinomas with intensity of expression increasing from benign to malignant cases. ELISA data from our study showed an increase in circulating levels of VEGF-A in malignancies. VEGF-A plasma levels can be employed as a biomarker for high-grade malignancy in epithelial ovarian cancers alongside tissue expression and CA-125 levels. This study is unique due to the fact that a simultaneous analysis of plasma and tissue expression has been demonstrated and is a first such study in epithelial ovarian cancers and representing the Indian population (South-east Asian) synchronized with genetic polymorphism data as well. PMID:26264619

  10. The evolutionary emergence of cell type-specific genes inferred from the gene expression analysis of Hydra

    PubMed Central

    Hwang, Jung Shan; Ohyanagi, Hajime; Hayakawa, Shiho; Osato, Naoki; Nishimiya-Fujisawa, Chiemi; Ikeo, Kazuho; David, Charles N.; Fujisawa, Toshitaka; Gojobori, Takashi

    2007-01-01

    Cell lineages of cnidarians including Hydra represent the fundamental cell types of metazoans and provides us a unique opportunity to study the evolutionary diversification of cell type in the animal kingdom. Hydra contains epithelial cells as well as a multipotent interstitial cell (I-cell) that gives rise to nematocytes, nerve cells, gland cells, and germ-line cells. We used cDNA microarrays to identify cell type-specific genes by comparing gene expression in normal Hydra with animals lacking the I-cell lineage, so-called epithelial Hydra. We then performed in situ hybridization to localize expression to specific cell types. Eighty-six genes were shown to be expressed in specific cell types of the I-cell lineage. An additional 29 genes were expressed in epithelial cells and were down-regulated in epithelial animals lacking I-cells. Based on the above information, we constructed a database (http://hydra.lab.nig.ac.jp/hydra/), which describes the expression patterns of cell type-specific genes in Hydra. Most genes expressed specifically in either I-cells or epithelial cells have homologues in higher metazoans. By comparison, most nematocyte-specific genes and approximately half of the gland cell- and nerve cell-specific genes are unique to the cnidarian lineage. Because nematocytes, gland cells, and nerve cells appeared along with the emergence of cnidarians, this suggests that lineage-specific genes arose in cnidarians in conjunction with the evolution of new cell types required by the cnidarians. PMID:17766437

  11. Neuropilin-2 expression promotes TGF-β1-mediated epithelial to mesenchymal transition in colorectal cancer cells.

    PubMed

    Grandclement, Camille; Pallandre, Jean René; Valmary Degano, Séverine; Viel, Erika; Bouard, Adeline; Balland, Jérémy; Rémy-Martin, Jean-Paul; Simon, Benoit; Rouleau, Alain; Boireau, Wilfrid; Klagsbrun, Michael; Ferrand, Christophe; Borg, Christophe

    2011-01-01

    Neuropilins, initially characterized as neuronal receptors, act as co-receptors for cancer related growth factors and were recently involved in several signaling pathways leading to cytoskeletal organization, angiogenesis and cancer progression. Then, we sought to investigate the ability of neuropilin-2 to orchestrate epithelial-mesenchymal transition in colorectal cancer cells. Using specific siRNA to target neuropilin-2 expression, or gene transfer, we first observed that neuropilin-2 expression endows HT29 and Colo320 for xenograft formation. Moreover, neuropilin-2 conferred a fibroblastic-like shape to cancer cells, suggesting an involvement of neuropilin-2 in epithelial-mesenchymal transition. Indeed, the presence of neuropilin-2 in colorectal carcinoma cell lines was correlated with loss of epithelial markers such as cytokeratin-20 and E-cadherin and with acquisition of mesenchymal molecules such as vimentin. Furthermore, we showed by surface plasmon resonance experiments that neuropilin-2 is a receptor for transforming-growth factor-β1. The expression of neuropilin-2 on colon cancer cell lines was indeed shown to promote transforming-growth factor-β1 signaling, leading to a constitutive phosphorylation of the Smad2/3 complex. Treatment with specific TGFβ-type1 receptor kinase inhibitors restored E-cadherin levels and inhibited in part neuropilin-2-induced vimentin expression, suggesting that neuropilin-2 cooperates with TGFβ-type1 receptor to promote epithelial-mesenchymal transition in colorectal cancer cells. Our results suggest a direct role of NRP2 in epithelial-mesenchymal transition and highlight a cross-talk between neuropilin-2 and TGF-β1 signaling to promote cancer progression. These results suggest that neuropilin-2 fulfills all the criteria of a therapeutic target to disrupt multiple oncogenic functions in solid tumors. PMID:21747928

  12. Neuropilin-2 Expression Promotes TGF-β1-Mediated Epithelial to Mesenchymal Transition in Colorectal Cancer Cells

    PubMed Central

    Grandclement, Camille; Pallandre, Jean René; Valmary Degano, Séverine; Viel, Erika; Bouard, Adeline; Balland, Jérémy; Rémy-Martin, Jean-Paul; Simon, Benoit; Rouleau, Alain; Boireau, Wilfrid; Klagsbrun, Michael; Ferrand, Christophe; Borg, Christophe

    2011-01-01

    Neuropilins, initially characterized as neuronal receptors, act as co-receptors for cancer related growth factors and were recently involved in several signaling pathways leading to cytoskeletal organization, angiogenesis and cancer progression. Then, we sought to investigate the ability of neuropilin-2 to orchestrate epithelial-mesenchymal transition in colorectal cancer cells. Using specific siRNA to target neuropilin-2 expression, or gene transfer, we first observed that neuropilin-2 expression endows HT29 and Colo320 for xenograft formation. Moreover, neuropilin-2 conferred a fibroblastic-like shape to cancer cells, suggesting an involvement of neuropilin-2 in epithelial-mesenchymal transition. Indeed, the presence of neuropilin-2 in colorectal carcinoma cell lines was correlated with loss of epithelial markers such as cytokeratin-20 and E-cadherin and with acquisition of mesenchymal molecules such as vimentin. Furthermore, we showed by surface plasmon resonance experiments that neuropilin-2 is a receptor for transforming-growth factor-β1. The expression of neuropilin-2 on colon cancer cell lines was indeed shown to promote transforming-growth factor-β1 signaling, leading to a constitutive phosphorylation of the Smad2/3 complex. Treatment with specific TGFβ-type1 receptor kinase inhibitors restored E-cadherin levels and inhibited in part neuropilin-2-induced vimentin expression, suggesting that neuropilin-2 cooperates with TGFβ-type1 receptor to promote epithelial-mesenchymal transition in colorectal cancer cells. Our results suggest a direct role of NRP2 in epithelial-mesenchymal transition and highlight a cross-talk between neuropilin-2 and TGF-β1 signaling to promote cancer progression. These results suggest that neuropilin-2 fulfills all the criteria of a therapeutic target to disrupt multiple oncogenic functions in solid tumors. PMID:21747928

  13. Cofactors of LIM Domains Associate with Estrogen Receptor α to Regulate the Expression of Noncoding RNA H19 and Corneal Epithelial Progenitor Cell Function.

    PubMed

    Klein, Rachel Herndon; Stephens, Denise N; Ho, Hsiang; Chen, Jefferson K; Salmans, Michael L; Wang, Winnie; Yu, Zhengquan; Andersen, Bogi

    2016-06-17

    Cofactors of LIM domain proteins, CLIM1 and CLIM2, are widely expressed transcriptional cofactors that are recruited to gene regulatory regions by DNA-binding proteins, including LIM domain transcription factors. In the cornea, epithelium-specific expression of a dominant negative (DN) CLIM under the keratin 14 (K14) promoter causes blistering, wounding, inflammation, epithelial hyperplasia, and neovascularization followed by epithelial thinning and subsequent epidermal-like differentiation of the corneal epithelium. The defects in corneal epithelial differentiation and cell fate determination suggest that CLIM may regulate corneal progenitor cells and the transition to differentiation. Consistent with this notion, the K14-DN-Clim corneal epithelium first exhibits increased proliferation followed by fewer progenitor cells with decreased proliferative potential. In vivo ChIP-sequencing experiments with corneal epithelium show that CLIM binds to and regulates numerous genes involved in cell adhesion and proliferation, including limbally enriched genes. Intriguingly, CLIM associates primarily with non-LIM homeodomain motifs in corneal epithelial cells, including that of estrogen receptor α. Among CLIM targets is the noncoding RNA H19 whose deregulation is associated with Silver-Russell and Beckwith-Wiedemann syndromes. We demonstrate here that H19 negatively regulates corneal epithelial proliferation. In addition to cell cycle regulators, H19 affects the expression of multiple cell adhesion genes. CLIM interacts with estrogen receptor α at the H19 locus, potentially explaining the higher expression of H19 in female than male corneas. Together, our results demonstrate an important role for CLIM in regulating the proliferative potential of corneal epithelial progenitors and identify CLIM downstream target H19 as a regulator of corneal epithelial proliferation and adhesion. PMID:27129775

  14. Inverse relationship between human papillomavirus (HPV) type 16 early gene expression and cell differentiation in nude mouse epithelial cysts and tumors induced by HPV-positive human cell lines.

    PubMed Central

    Dürst, M; Bosch, F X; Glitz, D; Schneider, A; zur Hausen, H

    1991-01-01

    Two human papillomavirus type 16 (HPV 16)-immortalized human keratinocyte cell lines (HPK) were shown to have retained the ability for differentiation after subcutaneous injection into nude mice. These properties were maintained even at late passage. HPK cells gave rise to transiently growing cysts which exhibited an epitheliumlike architecture. Moreover, differentiation-specific markers such as cytokeratin 10, involucrin, and filaggrin were shown to be expressed in an ordered succession. RNA-RNA in situ hybridization revealed heterogeneous and low levels of HPV 16 E6-E7 RNA in the basal layer of the cysts. In contrast, in progressively growing tumors induced by HPK cells containing an activated ras oncogene (EJ-ras) or in tumors induced by the cervical carcinoma cell line CaSki, high levels of E6-E7-specific RNA could be detected. Irrespective of the growth potential of these cell lines in nude mice, viral transcription was always more evident in the basal layer and in proliferatively active cells rather than in differentiated cells. This contrasts with viral gene expression in HPV 16 positive low-grade cervical dysplasia, in which abundant viral transcriptional activity was mapped to the upper third of the epithelium. It is suggested that the physical state of the viral DNA, i.e., integrated viral DNA in the cell lines as opposed to extrachromosomal DNA in low-grade cervical dysplasia, may influence viral gene regulation. Images PMID:1846200

  15. Fra-1/AP-1 induces EMT in mammary epithelial cells by modulating Zeb1/2 and TGFβ expression.

    PubMed

    Bakiri, L; Macho-Maschler, S; Custic, I; Niemiec, J; Guío-Carrión, A; Hasenfuss, S C; Eger, A; Müller, M; Beug, H; Wagner, E F

    2015-02-01

    Epithelial-to-mesenchymal transition (EMT) is essential for embryonic morphogenesis and wound healing and critical for tumour cell invasion and dissemination. The AP-1 transcription factor Fra-1 has been implicated in tumorigenesis and in tumour-associated EMT in human breast cancer. We observed a significant inverse correlation between Fra-1 mRNA expression and distant-metastasis-free survival in a large cohort of breast cancer patients derived from multiple array data sets. This unique correlation among Fos genes prompted us to assess the evolutionary conservation between Fra-1 functions in EMT of human and mouse cells. Ectopic expression of Fra-1 in fully polarized, non-tumourigenic, mouse mammary epithelial EpH4 cells induced a mesenchymal phenotype, characterized by a loss of epithelial and gain of mesenchymal markers. Proliferation, motility and invasiveness were also increased in the resulting EpFra1 cells, and the cells were tumourigenic and efficiently colonized the lung upon transplantation. Molecular analyses revealed increased expression of Tgfβ1 and the EMT-inducing transcription factors Zeb1, Zeb2 and Slug. Mechanistically, Fra-1 binds to the tgfb1 and zeb2 promoters and to an evolutionarily conserved region in the first intron of zeb1. Furthermore, increased activity of a zeb2 promoter reporter was detected in EpFra1 cells and shown to depend on AP-1-binding sites. Inhibiting TGFβ signalling in EpFra1 cells moderately increased the expression of epithelial markers, whereas silencing of zeb1 or zeb2 restored the epithelial phenotype and decreased migration in vitro and tumorigenesis in vivo. Thus Fra-1 induces changes in the expression of genes encoding EMT-related transcription factors leading to the acquisition of mesenchymal, invasive and tumorigenic capacities by epithelial cells. This study defines a novel function of Fra-1/AP-1 in modulating tgfb1, zeb1 and zeb2 expression through direct binding to genomic regulatory regions, which establishes

  16. Fra-1/AP-1 induces EMT in mammary epithelial cells by modulating Zeb1/2 and TGFβ expression

    PubMed Central

    Bakiri, L; Macho-Maschler, S; Custic, I; Niemiec, J; Guío-Carrión, A; Hasenfuss, S C; Eger, A; Müller, M; Beug, H; Wagner, E F

    2015-01-01

    Epithelial-to-mesenchymal transition (EMT) is essential for embryonic morphogenesis and wound healing and critical for tumour cell invasion and dissemination. The AP-1 transcription factor Fra-1 has been implicated in tumorigenesis and in tumour-associated EMT in human breast cancer. We observed a significant inverse correlation between Fra-1 mRNA expression and distant-metastasis-free survival in a large cohort of breast cancer patients derived from multiple array data sets. This unique correlation among Fos genes prompted us to assess the evolutionary conservation between Fra-1 functions in EMT of human and mouse cells. Ectopic expression of Fra-1 in fully polarized, non-tumourigenic, mouse mammary epithelial EpH4 cells induced a mesenchymal phenotype, characterized by a loss of epithelial and gain of mesenchymal markers. Proliferation, motility and invasiveness were also increased in the resulting EpFra1 cells, and the cells were tumourigenic and efficiently colonized the lung upon transplantation. Molecular analyses revealed increased expression of Tgfβ1 and the EMT-inducing transcription factors Zeb1, Zeb2 and Slug. Mechanistically, Fra-1 binds to the tgfb1 and zeb2 promoters and to an evolutionarily conserved region in the first intron of zeb1. Furthermore, increased activity of a zeb2 promoter reporter was detected in EpFra1 cells and shown to depend on AP-1-binding sites. Inhibiting TGFβ signalling in EpFra1 cells moderately increased the expression of epithelial markers, whereas silencing of zeb1 or zeb2 restored the epithelial phenotype and decreased migration in vitro and tumorigenesis in vivo. Thus Fra-1 induces changes in the expression of genes encoding EMT-related transcription factors leading to the acquisition of mesenchymal, invasive and tumorigenic capacities by epithelial cells. This study defines a novel function of Fra-1/AP-1 in modulating tgfb1, zeb1 and zeb2 expression through direct binding to genomic regulatory regions, which establishes

  17. Laminin-binding integrin gene copy number alterations in distinct epithelial-type cancers

    PubMed Central

    Harryman, William L; Pond, Erika; Singh, Parminder; Little, Andrew S; Eschbacher, Jennifer M; Nagle, Raymond B; Cress, Anne E

    2016-01-01

    Background: The laminin-binding integrin (LBI) family are cell adhesion molecules that are essential for invasion and metastasis of human epithelial cancers and cell adhesion mediated drug resistance. We investigated whether copy number alteration (CNA) or mutations of a five-gene signature (ITGB4, ITGA3, LAMB3, PLEC, and SYNE3), representing essential genes for LBI adhesion, would correlate with patient outcomes within human epithelial-type tumor data sets currently available in an open access format. Methods: We investigated the relative alteration frequency of an LBI signature panel (integrin β4 (ITGB4), integrin α3 (ITGA3), laminin β3 chain (LAMB3), plectin (PLEC), and nesprin 3 (SYNE3)), independent of the epithelial cancer type, within publically available and published data using cBioPortal and Oncomine software. We rank ordered the results using a 20% alteration frequency cut-off and limited the analysis to studies containing at least 100 samples. Kaplan-Meier survival curves were analyzed to determine if alterations in the LBI signature correlated with patient survival. The Oncomine data mining tool was used to compare the heat map expression of the LBI signature without SYNE3 (as this was not included in the Oncomine database) to drug resistance patterns. Results: Twelve different cancer types, representing 5,647 samples, contained at least a 20% alteration frequency of the five-gene LBI signature. The frequency of alteration ranged from 38.3% to 19.8%. Within the LBI signature, PLEC was the most commonly altered followed by LAMB3, ITGB4, ITGA3, and SYNE3 across all twelve cancer types. Within cancer types, there was little overlap of the individual amplified genes from each sample, suggesting different specific amplicons may alter the LBI adhesion structures. Of the twelve cancer types, overall survival was altered by CNA presence in bladder urothelial carcinoma (p=0.0143*) and cervical squamous cell carcinoma and endocervical adenocarcinoma (p=0

  18. Gene expression during memory formation.

    PubMed

    Igaz, Lionel Muller; Bekinschtein, Pedro; Vianna, Monica M R; Izquierdo, Ivan; Medina, Jorge H

    2004-01-01

    For several decades, neuroscientists have provided many clues that point out the involvement of de novo gene expression during the formation of long-lasting forms of memory. However, information regarding the transcriptional response networks involved in memory formation has been scarce and fragmented. With the advent of genome-based technologies, combined with more classical approaches (i.e., pharmacology and biochemistry), it is now feasible to address those relevant questions--which gene products are modulated, and when that processes are necessary for the proper storage of memories--with unprecedented resolution and scale. Using one-trial inhibitory (passive) avoidance training of rats, one of the most studied tasks so far, we found two time windows of sensitivity to transcriptional and translational inhibitors infused into the hippocampus: around the time of training and 3-6 h after training. Remarkably, these periods perfectly overlap with the involvement of hippocampal cAMP/PKA (protein kinase A) signaling pathways in memory consolidation. Given the complexity of transcriptional responses in the brain, particularly those related to processing of behavioral information, it was clearly necessary to address this issue with a multi-variable, parallel-oriented approach. We used cDNA arrays to screen for candidate inhibitory avoidance learning-related genes and analyze the dynamic pattern of gene expression that emerges during memory consolidation. These include genes involved in intracellular kinase networks, synaptic function, DNA-binding and chromatin modification, transcriptional activation and repression, translation, membrane receptors, and oncogenes, among others. Our findings suggest that differential and orchestrated hippocampal gene expression is necessary in both early and late periods of long-term memory consolidation. Additionally, this kind of studies may lead to the identification and characterization of genes that are relevant for the pathogenesis

  19. Osmotic Induction of Angiogenic Growth Factor Expression in Human Retinal Pigment Epithelial Cells

    PubMed Central

    Reichenbach, Andreas; Wiedemann, Peter; Kohen, Leon; Bringmann, Andreas

    2016-01-01

    Background Although systemic hypertension is a risk factor of age-related macular degeneration, antihypertensive medications do not affect the risk of the disease. One condition that induces hypertension is high intake of dietary salt resulting in increased blood osmolarity. In order to prove the assumption that, in addition to hypertension, high osmolarity may aggravate neovascular retinal diseases, we determined the effect of extracellular hyperosmolarity on the expression of angiogenic cytokines in cultured human retinal pigment epithelial (RPE) cells. Methodology/Principal Findings Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Hypoxia and oxidative stress were induced by the addition of the hypoxia mimetic CoCl2 and H2O2, respectively. Alterations in gene expression were determined with real-time RT-PCR. Secretion of bFGF was evaluated by ELISA. Cell viability was determined by trypan blue exclusion. Nuclear factor of activated T cell 5 (NFAT5) expression was knocked down with siRNA. Hyperosmolarity induced transcriptional activation of bFGF, HB-EGF, and VEGF genes, while the expression of other cytokines such as EGF, PDGF-A, TGF-β1, HGF, and PEDF was not or moderately altered. Hypoxia induced increased expression of the HB-EGF, EGF, PDGF-A, TGF-β1, and VEGF genes, but not of the bFGF gene. Oxidative stress induced gene expression of HB-EGF, but not of bFGF. The hyperosmotic expression of the bFGF gene was dependent on the activation of p38α/β MAPK, JNK, PI3K, and the transcriptional activity of NFAT5. The hyperosmotic expression of the HB-EGF gene was dependent on the activation of p38α/β MAPK, ERK1/2, and JNK. The hyperosmotic expression of bFGF, HB-EGF, and VEGF genes was reduced by inhibitors of TGF-β1 superfamily activin receptor-like kinase receptors and the FGF receptor kinase, respectively. Hyperosmolarity induced secretion of bFGF that was reduced by inhibition of autocrine/paracrine TGF-β1

  20. Transcriptome analysis of genes responding to NNV infection in Asian seabass epithelial cells.

    PubMed

    Liu, Peng; Wang, Le; Kwang, Jimmy; Yue, Gen Hua; Wong, Sek-Man

    2016-07-01

    Asian seabass is an important food fish in Southeast Asia. Viral nervous necrosis (VNN) disease, triggered by nervous necrosis virus (NNV) infection, has caused mass mortality of Asian seabass larvae, resulting in enormous economic losses in the Asian seabass industry. In order to better understand the complex molecular interaction between Asian seabass and NNV, we investigated the transcriptome profiles of Asian seabass epithelial cells, which play an essential role in immune regulation, after NNV infection. Using the next generation sequencing (NGS) technology, we sequenced mRNA from eight samples (6, 12, 24, 48 h post-inoculation) of mock and NNV-infected Asian seabass epithelial cell line, respectively. Clean reads were de novo assembled into a transcriptome consisting of 89026 transcripts with a N50 of 2617 bp. Furthermore, 251 differentially expressed genes (DEGs) in response to NNV infection were identified. Top DEGs include protein asteroid homolog 1-like (ASTE1), receptor-transporting protein 3 (RTP3), heat shock proteins 30 (HSP30) and 70 (HSP70), Viperin, interferon regulatory factor 3 (IRF3) and other genes related to innate immunity. Our data suggest that abundant and diverse genes corresponding to NNV infection. The results of this study could also offer vital information not only for identification of novel genes involved in Asian seabass-NNV interaction, but also for our understanding of the molecular mechanism of Asian seabass' response to viral infection. In addition, 24807 simple sequence repeats (SSRs) were detected in the assembled transcriptome, providing valuable resources for studying genetic variations and accelerating quantitative trait loci (QTL) mapping for disease resistance in Asian seabass in the future. PMID:27109582

  1. Microarray analysis of a Chlamydia pneumoniae-infected human epithelial cell line by use of gene ontology hierarchy.

    PubMed

    Alvesalo, Joni; Greco, Dario; Leinonen, Maija; Raitila, Tuomas; Vuorela, Pia; Auvinen, Petri

    2008-01-01

    Chlamydia pneumoniae, a gram-negative obligate intracellular bacterium, is a common cause of upper and lower respiratory tract infections worldwide. Persistent C. pneumoniae infections have been linked to chronic disease processes, such as atherosclerosis. In the present study, we examined gene expression changes in the human epithelial cell line at different stages of acute C. pneumoniae infection and used gene ontology annotation, along with single-gene analysis, to select a small group of target genes that could possibly play a key role in C. pneumoniae infection. Selected genes were silenced using small interfering RNA, and the effect of silencing on the number of C. pneumoniae inclusions was measured by time-resolved fluorometric immunoassay. The greatest reduction in the number of C. pneumoniae inclusions was due to the silencing of the gene coding for the transcription factor early growth response 1, which decreased the number of inclusions by 38.6%. PMID:18171299

  2. Regulation of angiogenin expression and epithelial-mesenchymal transition by HIF-1α signaling in hypoxic retinal pigment epithelial cells.

    PubMed

    Lai, Kairan; Luo, Chenqi; Zhang, Xiaobo; Ye, Panpan; Zhang, Yidong; He, Jiliang; Yao, Ke

    2016-09-01

    Choroidal neovascularization (CNV) is a major cause of vision loss in many retinal diseases. Hypoxia is determined to be a key inducer of CNV and hypoxia-inducible factor-1 (HIF-1) is an important transcription factor. Epithelial-mesenchymal transition (EMT) and the synthesis of proangiogenic cytokines make great contributions to the development of CNV. In the present study, the role of HIF-1α signaling in the regulation of angiogenin (ANG) expression and EMT in hypoxic retinal pigment epithelial cells was investigated. A significant elevation expression of ANG expression level in a mouse model of laser-induced CNV was demonstrated. In a hypoxic model of ARPE-19, an increased expression level of ANG and induction of EMT accompanied with stabilization and nucleus translocation of HIF-1α. Blockage of HIF-1α signaling resulted in inhibition of high expression of ANG and EMT features. The direct interaction between HIF-1α and ANG promoter region was identified by ChIP-qPCR. The association of RNase 4 mRNA level with HIF-1α signaling was also clarified in APRE-19. Moreover, the exogenous ANG translocated into the nucleus, enhanced 45S rRNA transcription, promoted cell proliferation and tube formation in human retinal microvascular endothelial cells. In conclusion, the hypoxic conditions regulate the expression of ANG and EMT via an activation of HIF-1α signaling. It provides molecular evidence for potential therapy strategies of treating CNV. PMID:27259982

  3. Intracellular insulin-like growth factor-1 induces Bcl-2 expression in airway epithelial cells.

    PubMed

    Chand, Hitendra S; Harris, Jennifer Foster; Mebratu, Yohannes; Chen, Yangde; Wright, Paul S; Randell, Scott H; Tesfaigzi, Yohannes

    2012-05-01

    Bcl-2, a prosurvival protein, regulates programmed cell death during development and repair processes, and it can be oncogenic when cell proliferation is deregulated. The present study investigated what factors modulate Bcl-2 expression in airway epithelial cells and identified the pathways involved. Microarray analysis of mRNA from airway epithelial cells captured by laser microdissection showed that increased expression of IL-1β and insulin-like growth factor-1 (IGF-1) coincided with induced Bcl-2 expression compared with controls. Treatment of cultured airway epithelial cells with IL-1β and IGF-1 induced Bcl-2 expression by increasing Bcl-2 mRNA stability with no discernible changes in promoter activity. Silencing the IGF-1 expression using short hairpin RNA showed that intracellular IGF-1 (IC-IGF-1) was increasing Bcl-2 expression. Blocking epidermal growth factor receptor or IGF-1R activation also suppressed IC-IGF-1 and abolished the Bcl-2 induction. Induced expression and colocalization of IC-IGF-1 and Bcl-2 were observed in airway epithelial cells of mice exposed to LPS or cigarette smoke and of patients with cystic fibrosis and chronic bronchitis but not in the respective controls. These studies demonstrate that IC-IGF-1 induces Bcl-2 expression in epithelial cells via IGF-1R and epidermal growth factor receptor pathways, and targeting IC-IGF-1 could be beneficial to treat chronic airway diseases. PMID:22461702

  4. Gene expression profile of pulpitis.

    PubMed

    Galicia, J C; Henson, B R; Parker, J S; Khan, A A

    2016-06-01

    The cost, prevalence and pain associated with endodontic disease necessitate an understanding of the fundamental molecular aspects of its pathogenesis. This study was aimed to identify the genetic contributors to pulpal pain and inflammation. Inflamed pulps were collected from patients diagnosed with irreversible pulpitis (n=20). Normal pulps from teeth extracted for various reasons served as controls (n=20). Pain level was assessed using a visual analog scale (VAS). Genome-wide microarray analysis was performed using Affymetrix GeneTitan Multichannel Instrument. The difference in gene expression levels were determined by the significance analysis of microarray program using a false discovery rate (q-value) of 5%. Genes involved in immune response, cytokine-cytokine receptor interaction and signaling, integrin cell surface interactions, and others were expressed at relatively higher levels in the pulpitis group. Moreover, several genes known to modulate pain and inflammation showed differential expression in asymptomatic and mild pain patients (⩾30 mm on VAS) compared with those with moderate to severe pain. This exploratory study provides a molecular basis for the clinical diagnosis of pulpitis. With an enhanced understanding of pulpal inflammation, future studies on treatment and management of pulpitis and on pain associated with it can have a biological reference to bridge treatment strategies with pulpal biology. PMID:27052691

  5. Carbohydrate Availability Regulates Virulence Gene Expression in Streptococcus suis

    PubMed Central

    Ferrando, M. Laura; van Baarlen, Peter; Orrù, Germano; Piga, Rosaria; Bongers, Roger S.; Wels, Michiel; De Greeff, Astrid; Smith, Hilde E.; Wells, Jerry M.

    2014-01-01

    Streptococcus suis is a major bacterial pathogen of young pigs causing worldwide economic problems for the pig industry. S. suis is also an emerging pathogen of humans. Colonization of porcine oropharynx by S. suis is considered to be a high risk factor for invasive disease. In the oropharyngeal cavity, where glucose is rapidly absorbed but dietary α-glucans persist, there is a profound effect of carbohydrate availability on the expression of virulence genes. Nineteen predicted or confirmed S. suis virulence genes that promote adhesion to and invasion of epithelial cells were expressed at higher levels when S. suis was supplied with the α-glucan starch/pullulan compared to glucose as the single carbon source. Additionally the production of suilysin, a toxin that damages epithelial cells, was increased more than ten-fold when glucose levels were low and S. suis was growing on pullulan. Based on biochemical, bioinformatics and in vitro and in vivo gene expression studies, we developed a biological model that postulates the effect of carbon catabolite repression on expression of virulence genes in the mucosa, organs and blood. This research increases our understanding of S. suis virulence mechanisms and has important implications for the design of future control strategies including the development of anti-infective strategies by modulating animal feed composition. PMID:24642967

  6. Carbohydrate availability regulates virulence gene expression in Streptococcus suis.

    PubMed

    Ferrando, M Laura; van Baarlen, Peter; Orrù, Germano; Piga, Rosaria; Bongers, Roger S; Wels, Michiel; De Greeff, Astrid; Smith, Hilde E; Wells, Jerry M

    2014-01-01

    Streptococcus suis is a major bacterial pathogen of young pigs causing worldwide economic problems for the pig industry. S. suis is also an emerging pathogen of humans. Colonization of porcine oropharynx by S. suis is considered to be a high risk factor for invasive disease. In the oropharyngeal cavity, where glucose is rapidly absorbed but dietary α-glucans persist, there is a profound effect of carbohydrate availability on the expression of virulence genes. Nineteen predicted or confirmed S. suis virulence genes that promote adhesion to and invasion of epithelial cells were expressed at higher levels when S. suis was supplied with the α-glucan starch/pullulan compared to glucose as the single carbon source. Additionally the production of suilysin, a toxin that damages epithelial cells, was increased more than ten-fold when glucose levels were low and S. suis was growing on pullulan. Based on biochemical, bioinformatics and in vitro and in vivo gene expression studies, we developed a biological model that postulates the effect of carbon catabolite repression on expression of virulence genes in the mucosa, organs and blood. This research increases our understanding of S. suis virulence mechanisms and has important implications for the design of future control strategies including the development of anti-infective strategies by modulating animal feed composition. PMID:24642967

  7. MicroRNA-99 Family Members Suppress Homeobox A1 Expression in Epithelial Cells

    PubMed Central

    Chen, Dan; Chen, Zujian; Jin, Yi; Dragas, Dragan; Zhang, Leitao; Adjei, Barima S.; Wang, Anxun; Dai, Yang; Zhou, Xiaofeng

    2013-01-01

    The miR-99 family is one of the evolutionarily most ancient microRNA families, and it plays a critical role in developmental timing and the maintenance of tissue identity. Recent studies, including reports from our group, suggested that the miR-99 family regulates various physiological processes in adult tissues, such as dermal wound healing, and a number of disease processes, including cancer. By combining 5 independent genome-wide expression profiling experiments, we identified a panel of 266 unique transcripts that were down-regulated in epithelial cells transfected with miR-99 family members. A comprehensive bioinformatics analysis using 12 different sequence-based microRNA target prediction algorithms revealed that 81 out of these 266 down-regulated transcripts are potential direct targets for the miR-99 family. Confirmation experiments and functional analyses were performed to further assess 6 selected miR-99 target genes, including mammalian Target of rapamycin (mTOR), Homeobox A1 (HOXA1), CTD small phosphatase-like (CTDSPL), N-myristoyltransferase 1 (NMT1), Transmembrane protein 30A (TMEM30A), and SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (SMARCA5). HOXA1 is a known proto-oncogene, and it also plays an important role in embryonic development. The direct targeting of the miR-99 family to two candidate binding sequences located in the HOXA1 mRNA was confirmed using a luciferase reporter gene assay and a ribonucleoprotein-immunoprecipitation (RIP-IP) assay. Ectopic transfection of miR-99 family reduced the expression of HOXA1, which, in consequence, down-regulated the expression of its downstream gene (i.e., Bcl-2) and led to reduced proliferation and cell migration, as well as enhanced apoptosis. In summary, we identified a number of high-confidence miR-99 family target genes, including proto-oncogene HOXA1, which may play an important role in regulating epithelial cell proliferation and migration during

  8. Heme activates the heme oxygenase-1 gene in renal epithelial cells by stabilizing Nrf2.

    PubMed

    Alam, Jawed; Killeen, Erin; Gong, Pengfei; Naquin, Ryan; Hu, Bin; Stewart, Daniel; Ingelfinger, Julie R; Nath, Karl A

    2003-04-01

    The mechanism of heme oxygenase-1 gene (ho-1) activation by heme in immortalized rat proximal tubular epithelial cells was examined. Analysis of the ho-1 promoter identified the heme-responsive sequences as the stress-response element (StRE), multiple copies of which are present in two enhancer regions, E1 and E2. Electrophoretic mobility shift assays identified Nrf2, MafG, ATF3, and Jun and Fos family members as StRE-binding proteins; binding of Nrf2, MafG, and ATF3 was increased in response to heme. Dominant-negative mutants of Nrf2 and Maf, but not of c-Fos and c-Jun, inhibited basal and heme-induced expression of an E1-controlled luciferase gene. Heme did not affect the transcription activity of Nrf2, dimerization between Nrf2 and MafG, or the level of MafG, but did stimulate expression of Nrf2. Heme did not influence the level of Nrf2 mRNA but increased the half-life of Nrf2 protein from approximately 10 min to nearly 110 min. These results indicate that heme promotes stabilization of Nrf2, leading to accumulation of Nrf2. MafG dimers that bind to StREs to activate the ho-1 gene. PMID:12453873

  9. Systems Biophysics of Gene Expression

    PubMed Central

    Vilar, Jose M.G.; Saiz, Leonor

    2013-01-01

    Gene expression is a process central to any form of life. It involves multiple temporal and functional scales that extend from specific protein-DNA interactions to the coordinated regulation of multiple genes in response to intracellular and extracellular changes. This diversity in scales poses fundamental challenges to the use of traditional approaches to fully understand even the simplest gene expression systems. Recent advances in computational systems biophysics have provided promising avenues to reliably integrate the molecular detail of biophysical process into the system behavior. Here, we review recent advances in the description of gene regulation as a system of biophysical processes that extend from specific protein-DNA interactions to the combinatorial assembly of nucleoprotein complexes. There is now basic mechanistic understanding on how promoters controlled by multiple, local and distal, DNA binding sites for transcription factors can actively control transcriptional noise, cell-to-cell variability, and other properties of gene regulation, including precision and flexibility of the transcriptional responses. PMID:23790365

  10. Control of Renin Gene Expression

    PubMed Central

    Glenn, Sean T.; Jones, Craig A.; Gross, Kenneth W.; Pan, Li

    2015-01-01

    Renin, as part of the renin-angiotensin system, plays a critical role in the regulation of blood pressure, electrolyte homeostasis, mammalian renal development and progression of fibrotic/hypertrophic diseases. Renin gene transcription is subject to complex developmental and tissue-specific regulation. Initial studies using the mouse As4.1 cell line, which has many characteristics of the renin-expressing juxtaglomerular cells of the kidney, have identified a proximal promoter region (−197 to −50 bp) and an enhancer (−2866 to −2625 bp) upstream of the Ren-1c gene, which are critical for renin gene expression. The proximal promoter region contains several transcription factor-binding sites including a binding site for the products of the developmental control genes Hox. The enhancer consists of at least 11 transcription factor-binding sites and is responsive to various signal transduction pathways including cAMP, retinoic acid, endothelin-1, and cytokines, all of which are known to alter renin mRNA levels. Furthermore, in vivo models have validated several of these key components found within the proximal promoter region and the enhancer as well as other key sites necessary for renin gene transcription. PMID:22576577

  11. Expression of serum amyloid a in human ovarian epithelial tumors: implication for a role in ovarian tumorigenesis.

    PubMed

    Urieli-Shoval, Simcha; Finci-Yeheskel, Zvezdana; Dishon, Shira; Galinsky, Daliah; Linke, Reinhold P; Ariel, Ilana; Levin, Mark; Ben-Shachar, Inbar; Prus, Diana

    2010-11-01

    Serum amyloid A (SAA) is an acute phase protein which is expressed primarily in the liver as a part of the systemic response to various injuries and inflammatory stimuli; its expression in ovarian tumors has not been described. Here, we investigated the expression of SAA in human benign and malignant ovarian epithelial tumors. Non-radioactive in situ hybridization applied on ovarian paraffin tissue sections revealed mostly negative SAA mRNA expression in normal surface epithelium. Expression was increased gradually as epithelial cells progressed through benign and borderline adenomas to primary and metastatic adenocarcinomas. Similar expression pattern of the SAA protein was observed by immunohistochemical staining. RT-PCR analysis confirmed the overexpression of the SAA1 and SAA4 genes in ovarian carcinomas compared with normal ovarian tissues. In addition, strong expression of SAA mRNA and protein was found in the ovarian carcinoma cell line OVCAR-3. Finally, patients with ovarian carcinoma had high SAA serum levels, which strongly correlated with high levels of CA-125 and C-reactive protein. Enhanced expression of SAA in ovarian carcinomas may play a role in ovarian tumorigenesis and may have therapeutic application. PMID:20713982

  12. An immortalized goat mammary epithelial cell line induced with human telomerase reverse transcriptase (hTERT) gene transfer.

    PubMed

    He, Y L; Wu, Y H; He, X N; Liu, F J; He, X Y; Zhang, Y

    2009-06-01

    Although mammary epithelial cell lines can provide a rapid and reliable indicator of gene expression efficiency of transgenic animals, their short lifespan greatly limits this application. To provide stable and long lifespan cells, goat mammary epithelial cells (GMECs) were transduced with pLNCX2-hTERT by retrovirus-mediated gene transfer. Transduced GMECs were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR), proliferation assays, karyotype analysis, telomerase activity assay, western blotting, soft agar assay, and injection into nude mice. Non-transduced GMECs were used as a control. The hTERT-GMECs had higher telomerase activity and extended proliferative lifespan compared to non-transfected GMECs; even after Passage 50, hTERT-GMECs had a near diploid complement of chromosomes. Furthermore, they did not gain the anchorage-independent growth property and were not associated with a malignant phenotype in vitro or in vivo. PMID:19303628

  13. Dynamic and Physical Clustering of Gene Expression during Epidermal Barrier Formation in Differentiating Keratinocytes

    PubMed Central

    Copley, Richard; Taylor, Martin S.; Hayden, Patrick J.; Stolper, Gina; Mott, Richard; Hein, Jotun; Moffatt, Miriam F.; Cookson, William O. C. M.

    2009-01-01

    The mammalian epidermis is a continually renewing structure that provides the interface between the organism and an innately hostile environment. The keratinocyte is its principal cell. Keratinocyte proteins form a physical epithelial barrier, protect against microbial damage, and prepare immune responses to danger. Epithelial immunity is disordered in many common diseases and disordered epithelial differentiation underlies many cancers. In order to identify the genes that mediate epithelial development we used a tissue model of the skin derived from primary human keratinocytes. We measured global gene expression in triplicate at five times over the ten days that the keratinocytes took to fully differentiate. We identified 1282 gene transcripts that significantly changed during differentiation (false discovery rate <0.01%). We robustly grouped these transcripts by K-means clustering into modules with distinct temporal expression patterns, shared regulatory motifs, and biological functions. We found a striking cluster of late expressed genes that form the structural and innate immune defences of the epithelial barrier. Gene Ontology analyses showed that undifferentiated keratinocytes were characterised by genes for motility and the adaptive immune response. We systematically identified calcium-binding genes, which may operate with the epidermal calcium gradient to control keratinocyte division during skin repair. The results provide multiple novel insights into keratinocyte biology, in particular providing a comprehensive list of known and previously unrecognised major components of the epidermal barrier. The findings provide a reference for subsequent understanding of how the barrier functions in health and disease. PMID:19888454

  14. Laminin Mediates Tissue-specific Gene Expression in Mammary Epithelia

    SciTech Connect

    Streuli, Charles H; Schmidhauser, Christian; Bailey, Nina; Yurchenco, Peter; Skubitz, Amy P. N.; Roskelley, Calvin; Bissell, Mina J

    1995-04-01

    Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating beta-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the beta-casein gene. Mouse mammary epithelial cells plated on gels of native laminin or laminin-entactin undergo functional differentiation. On tissue culture plastic, mammary cells respond to soluble basement membrane or purified laminin, but not other extracellular matrix components, by synthesizing beta-casein. In mammary cells transfected with chloramphenicol acetyl transferase reporter constructs, laminin activates transcription from the beta-casein promoter through a specific enhancer element. The inductive effect of laminin on casein expression was specifically blocked by the E3 fragment of the carboxy terminal region of the alpha 1 chain of laminin, by antisera raised against the E3 fragment, and by a peptide corresponding to a sequence within this region. Our results demonstrate that laminin can direct tissue-specific gene expression in epithelial cells through its globular domain.

  15. Functional Gene Correction for Cystic Fibrosis in Lung Epithelial Cells Generated from Patient iPSCs.

    PubMed

    Firth, Amy L; Menon, Tushar; Parker, Gregory S; Qualls, Susan J; Lewis, Benjamin M; Ke, Eugene; Dargitz, Carl T; Wright, Rebecca; Khanna, Ajai; Gage, Fred H; Verma, Inder M

    2015-09-01

    Lung disease is a major cause of death in the United States, with current therapeutic approaches serving only to manage symptoms. The most common chronic and life-threatening genetic disease of the lung is cystic fibrosis (CF) caused by mutations in the cystic fibrosis transmembrane regulator (CFTR). We have generated induced pluripotent stem cells (iPSCs) from CF patients carrying a homozygous deletion of F508 in the CFTR gene, which results in defective processing of CFTR to the cell membrane. This mutation was precisely corrected using CRISPR to target corrective sequences to the endogenous CFTR genomic locus, in combination with a completely excisable selection system, which significantly improved the efficiency of this correction. The corrected iPSCs were subsequently differentiated to mature airway epithelial cells where recovery of normal CFTR expression and function was demonstrated. This isogenic iPSC-based model system for CF could be adapted for the development of new therapeutic approaches. PMID:26299960

  16. Characterization of isoform expression and subcellular distribution of MYPT1 in intestinal epithelial cells.

    PubMed

    Zha, Juan-Min; Li, Hua-Shan; Wang, Yi-Tang; Lin, Qian; Tao, Min; He, Wei-Qi

    2016-08-15

    The regulation of intestinal epithelial permeability requires phosphorylation of myosin regulatory light chain (MLC). The phosphorylation status of MLC is regulated by myosin light chain phosphatase (MLCP) activities. The activity of the catalytic subunit of MLCP (PP1cδ) toward MLC depends on its regulatory subunit (MYPT1). In this study, we revealed the presence of two MYPT1 isoforms, full length and variant 2 in human intestinal (Caco-2) epithelial cells and isolated intestinal epithelial cells (IECs) from mice. In confluent Caco-2 cells, MYPT1 was distributed at cell-cell contacts and colocalized with F-actin. These results suggest that MYPT1 isoforms are expressed in intestinal epithelial cells and MYPT1 may be involved in the regulation of intestinal epithelial barrier function. PMID:27129938

  17. Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells.

    PubMed

    Ryan, M C; Lee, K; Miyashita, Y; Carter, W G

    1999-06-14

    Laminin 5 regulates anchorage and motility of epithelial cells through integrins alpha6beta4 and alpha3beta1, respectively. We used targeted disruption of the LAMA3 gene, which encodes the alpha3 subunit of laminin 5 and other isoforms, to examine developmental functions that are regulated by adhesion to the basement membrane (BM). In homozygous null animals, profound epithelial abnormalities were detected that resulted in neonatal lethality, consistent with removal of all alpha3-laminin isoforms from epithelial BMs. Alterations in three different cellular functions were identified. First, using a novel tissue adhesion assay, we found that the mutant BM could not induce stable adhesion by integrin alpha6beta4, consistent with the presence of junctional blisters and abnormal hemidesmosomes. In the absence of laminin 5 function, we were able to detect a new ligand for integrin alpha3beta1 in the epidermal BM, suggesting that basal keratinocytes can utilize integrin alpha3beta1 to interact with an alternative ligand. Second, we identified a survival defect in mutant epithelial cells that could be rescued by exogenous laminin 5, collagen, or an antibody against integrin alpha6beta4, suggesting that signaling through beta1 or beta4 integrins is sufficient for survival. Third, we detected abnormalities in ameloblast differentiation in developing mutant incisors indicating that events downstream of adhesion are affected in mutant animals. These results indicate that laminin 5 has an important role in regulating tissue organization, gene expression, and survival of epithelium. PMID:10366601

  18. Nidogen-1 regulates laminin-1-dependent mammary-specific gene expression

    SciTech Connect

    Pujuguet, Philippe; Simian, Marina; Liaw, Jane; Timpl, Rupert; Werb, Zena; Bissell, Mina J..

    2000-02-01

    Nidogen-1 (entactin) acts as a bridge between the extracellular matrix molecules laminin-1 and type IV collagen, and thus participates in the assembly of basement membranes. To investigate the role of nidogen-1 in regulating cell-type-specific gene expression in mammary epithelium, we designed a culture microecosystem in which each component, including epithelial cells, mesenchymal cells, lactogenic hormones and extracellular matrix, could be controlled. We found that primary and established mesenchymal and myoepithelial cells synthesized and secreted nidogen-1, whereas expression was absent in primary and established epithelial cells. In an epithelial cell line containing mesenchymal cells, nidogen-1 was produced by the mesenchymal cells but deposited between the epithelial cells. In this mixed culture, mammary epithelial cells express b-casein in the presence of lactogenic hormones. Addition of either laminin-1 plus nidogen-1, or laminin-1 alone to mammary epithelial cells induced b- casein production. We asked whether recombinant nidogen-1 alone could signal directly for b-casein. Nidogen-1 did not induce b-casein synthesis in epithelial cells, but it augmented the inductive capacity of laminin-1. These data suggest that nidogen-1 can cooperate with laminin-1 to regulate b-casein expression. Addition of full length nidogen-1 to the mixed cultures had no effect on b-casein gene expression; however, a nidogen-1 fragment containing the laminin-1 binding domain, but lacking the type IV collagen-binding domain, had a dominant negative effect on b-casein expression. These data point to a physiological role for nidogen-1 in the basement membrane-induced gene expression by epithelial cells.

  19. Regulation of tyrosinase expression and activity in cultured human retinal pigment epithelial cells.

    PubMed

    Abul-Hassan, K; Walmsley, R; Tombran-Tink, J; Boulton, M

    2000-12-01

    The purpose of this study was to investigate the regulation of tyrosinase gene expression and activity in cultured human retinal pigment epithelial (RPE) cells. The tyrosinase promoter (Ty.prom) region (400 bp) was PCR amplified and cloned into a modified mammalian expression vector (pcDNA3.1) upstream of a firefly luciferase (Luc) cDNA and was designated 'pcDNA3.1-Ty.prom.Luc'. The plasmid was co-transfected into RPE cells with a second mammalian expression plasmid (pRL-TK) containing a herpes simplex virus thymidine kinase promoter region upstream of Renilla Luc in a protocol designated the 'dual luciferase assay' (DLA). After co-transfection, cells were treated with a range of potential melanogenic agents; basic fibroblast growth factor (bFGF), methyl methane sulphonate, alpha-melanocyte stimulating hormone, verapamil, phorbol myristate acetate, cholera toxin (CT), pigment epithelium derived factor (PEDF), and L-tyrosine. The expression of tyrosinase promoter and enzymatic activities were determined 48 hr post-transfection using the DLA and DOPA oxidase assays, respectively. Tyrosinase activity could not be detected in RPE cells with any of the treatments. Tyrosinase promoter activity was significantly up-regulated in RPE cells treated with bFGF, PEDF, verapamil, CT and tyrosine compared with control cells. In conclusion, the tyrosinase gene is not only expressed but can be regulated in response to different chemicals in cultured human RPE cells. However, it appears that RPE cells in culture lack a post-transcriptional and/or translational modification point(s), which are necessary for tyrosinase enzymic activity. PMID:11153695

  20. FOXA2 regulates a network of genes involved in critical functions of human intestinal epithelial cells.

    PubMed

    Gosalia, Nehal; Yang, Rui; Kerschner, Jenny L; Harris, Ann

    2015-07-01

    The forkhead box A (FOXA) family of pioneer transcription factors is critical for the development of many endoderm-derived tissues. Their importance in regulating biological processes in the lung and liver is extensively characterized, though much less is known about their role in intestine. Here we investigate the contribution of FOXA2 to coordinating intestinal epithelial cell function using postconfluent Caco2 cells, differentiated into an enterocyte-like model. FOXA2 binding sites genome-wide were determined by ChIP-seq and direct targets of the factor were validated by ChIP-qPCR and siRNA-mediated depletion of FOXA1/2 followed by RT-qPCR. Peaks of FOXA2 occupancy were frequent at loci contributing to gene ontology pathways of regulation of cell migration, cell motion, and plasma membrane function. Depletion of both FOXA1 and FOXA2 led to a significant reduction in the expression of multiple transmembrane proteins including ion channels and transporters, which form a network that is essential for maintaining normal ion and solute transport. One of the targets was the adenosine A2B receptor, and reduced receptor mRNA levels were associated with a functional decrease in intracellular cyclic AMP. We also observed that 30% of FOXA2 binding sites contained a GATA motif and that FOXA1/A2 depletion reduced GATA-4, but not GATA-6 protein levels. These data show that FOXA2 plays a pivotal role in regulating intestinal epithelial cell function. Moreover, that the FOXA and GATA families of transcription factors may work cooperatively to regulate gene expression genome-wide in the intestinal epithelium. PMID:25921584

  1. Gene expression throughout a vertebrate's embryogenesis

    PubMed Central

    2011-01-01

    Background Describing the patterns of gene expression during embryonic development has broadened our understanding of the processes and patterns that define morphogenesis. Yet gene expression patterns have not been described throughout vertebrate embryogenesis. This study presents statistical analyses of gene expression during all 40 developmental stages in the teleost Fundulus heteroclitus using four biological replicates per stage. Results Patterns of gene expression for 7,000 genes appear to be important as they recapitulate developmental timing. Among the 45% of genes with significant expression differences between pairs of temporally adjacent stages, significant differences in gene expression vary from as few as five to more than 660. Five adjacent stages have disproportionately more significant changes in gene expression (> 200 genes) relative to other stages: four to eight and eight to sixteen cell stages, onset of circulation, pre and post-hatch, and during complete yolk absorption. The fewest differences among adjacent stages occur during gastrulation. Yet, at stage 16, (pre-mid-gastrulation) the largest number of genes has peak expression. This stage has an over representation of genes in oxidative respiration and protein expression (ribosomes, translational genes and proteases). Unexpectedly, among all ribosomal genes, both strong positive and negative correlations occur. Similar correlated patterns of expression occur among all significant genes. Conclusions These data provide statistical support for the temporal dynamics of developmental gene expression during all stages of vertebrate development. PMID:21356103

  2. SNAIL gene inhibited by hypoxia-inducible factor 1α (HIF-1α) in epithelial ovarian cancer.

    PubMed

    Zhang, Pengnan; Liu, Yanmei; Feng, Youji; Gao, Shujun

    2016-09-01

    The aim of this study was to investigate the relationship between HIF-1α and SNAIL gene expression in the epithelial ovarian cancer (EOC) cell line. EOC cells were treated with hypoxia, hypoxia combined with rapamycin, and control. The expression of HIF-1α and E-cad were assessed by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. The gene expression of SNAIL was studied by RT-PCR and real-time PCR. RNA interference technology was used to determine the relationship between HIF-1α and SNAIL. The present study indicated that the HIF-1α protein was expressed and increased in EOC cell line. SNAIL mRNA was found to increase and E-cad expression decreased with the time of hypoxia prolonged. Hypoxia increased invasion abilities of EOC cell line, but compared with cells exposed to hypoxia, the change of invasive ability of cells with rapamycin had no effect. The expression of HIF-1α protein and SNAIL mRNA could be inhibited gradually by rapamycin. siRNA of HIF-1α could suppress the expression of SNAIL while siRNA of SNAIL had no influence on HIF-1α protein expression. HIF-1α may be the upstream of the SNAIL gene in EOC. Our data suggested that HIF-1α might be an upregulator of the SNAIL gene and HIF-1α-SNAIL-E-cad pathway may play an important role in EOC invasion and metastasis. PMID:27044634

  3. Neutrophil Interactions with Epithelial Expressed ICAM-1 Enhances Intestinal Mucosal Wound Healing

    PubMed Central

    Sumagin, R; Brazil, JC; Nava, P; Nishio, H; Alam, A; Luissint, AC; Weber, DA; Neish, AS; Nusrat, A; Parkos, CA

    2015-01-01

    A characteristic feature of gastrointestinal tract inflammatory disorders, such as inflammatory bowel disease, is polymorphonuclear neutrophil (PMN) transepithelial migration (TEM) and accumulation in the gut lumen. PMN accumulation within the intestinal mucosa contributes to tissue injury. While epithelial infiltration by large numbers of PMNs results in mucosal injury, we found that PMN interactions with luminal epithelial membrane receptors may also play a role in wound healing. Intercellular adhesion molecule-1 (ICAM-1) is a PMN ligand that is upregulated on apical surfaces of intestinal epithelial cells under inflammatory conditions. In our study, increased expression of ICAM-1 resulted in enhanced PMN binding to the apical epithelium, which was associated with reduced PMN apoptosis. Following TEM, PMN adhesion to ICAM-1 resulted in activation of Akt and β-catenin signaling, increased epithelial-cell proliferation, and wound healing. Such responses were ICAM-1 dependent as engagement of epithelial ICAM-1 by antibody-mediated cross-linking yielded similar results. Furthermore, using an in-vivo biopsy-based, colonic-mucosal-injury model, we demonstrated epithelial ICAM-1 plays an important role in activation of epithelial Akt and β-catenin signaling and wound healing. These findings suggest that post-migrated PMNs within the intestinal lumen can regulate epithelial homeostasis, thereby identifying ICAM-1 as a potential therapeutic target for promoting mucosal wound healing. PMID:26732677

  4. Neutrophil interactions with epithelial-expressed ICAM-1 enhances intestinal mucosal wound healing.

    PubMed

    Sumagin, R; Brazil, J C; Nava, P; Nishio, H; Alam, A; Luissint, A C; Weber, D A; Neish, A S; Nusrat, A; Parkos, C A

    2016-09-01

    A characteristic feature of gastrointestinal tract inflammatory disorders, such as inflammatory bowel disease, is polymorphonuclear neutrophil (PMN) transepithelial migration (TEM) and accumulation in the gut lumen. PMN accumulation within the intestinal mucosa contributes to tissue injury. Although epithelial infiltration by large numbers of PMNs results in mucosal injury, we found that PMN interactions with luminal epithelial membrane receptors may also play a role in wound healing. Intercellular adhesion molecule-1 (ICAM-1) is a PMN ligand that is upregulated on apical surfaces of intestinal epithelial cells under inflammatory conditions. In our study, increased expression of ICAM-1 resulted in enhanced PMN binding to the apical epithelium, which was associated with reduced PMN apoptosis. Following TEM, PMN adhesion to ICAM-1 resulted in activation of Akt and β-catenin signaling, increased epithelial-cell proliferation, and wound healing. Such responses were ICAM-1 dependent as engagement of epithelial ICAM-1 by antibody-mediated cross-linking yielded similar results. Furthermore, using an in-vivo biopsy-based, colonic-mucosal-injury model, we demonstrated epithelial ICAM-1 has an important role in activation of epithelial Akt and β-catenin signaling and wound healing. These findings suggest that post-migrated PMNs within the intestinal lumen can regulate epithelial homeostasis, thereby identifying ICAM-1 as a potential therapeutic target for promoting mucosal wound healing. PMID:26732677

  5. Gene Expression Studies in Mosquitoes

    PubMed Central

    Chen, Xlao-Guang; Mathur, Geetika; James, Anthony A.

    2009-01-01

    Research on gene expression in mosquitoes is motivated by both basic and applied interests. Studies of genes involved in hematophagy, reproduction, olfaction, and immune responses reveal an exquisite confluence of biological adaptations that result in these highly-successful life forms. The requirement of female mosquitoes for a bloodmeal for propagation has been exploited by a wide diversity of viral, protozoan and metazoan pathogens as part of their life cycles. Identifying genes involved in host-seeking, blood feeding and digestion, reproduction, insecticide resistance and susceptibility/refractoriness to pathogen development is expected to provide the bases for the development of novel methods to control mosquito-borne diseases. Advances in mosquito transgenesis technologies, the availability of whole genome sequence information, mass sequencing and analyses of transcriptomes and RNAi techniques will assist development of these tools as well as deepen the understanding of the underlying genetic components for biological phenomena characteristic of these insect species. PMID:19161831

  6. The Gene Expression Omnibus database

    PubMed Central

    Clough, Emily; Barrett, Tanya

    2016-01-01

    The Gene Expression Omnibus (GEO) database is an international public repository that archives and freely distributes high-throughput gene expression and other functional genomics data sets. Created in 2000 as a worldwide resource for gene expression studies, GEO has evolved with rapidly changing technologies and now accepts high-throughput data for many other data applications, including those that examine genome methylation, chromatin structure, and genome–protein interactions. GEO supports community-derived reporting standards that specify provision of several critical study elements including raw data, processed data, and descriptive metadata. The database not only provides access to data for tens of thousands of studies, but also offers various Web-based tools and strategies that enable users to locate data relevant to their specific interests, as well as to visualize and analyze the data. This chapter includes detailed descriptions of methods to query and download GEO data and use the analysis and visualization tools. The GEO homepage is at http://www.ncbi.nlm.nih.gov/geo/. PMID:27008011

  7. The Gene Expression Omnibus Database.

    PubMed

    Clough, Emily; Barrett, Tanya

    2016-01-01

    The Gene Expression Omnibus (GEO) database is an international public repository that archives and freely distributes high-throughput gene expression and other functional genomics data sets. Created in 2000 as a worldwide resource for gene expression studies, GEO has evolved with rapidly changing technologies and now accepts high-throughput data for many other data applications, including those that examine genome methylation, chromatin structure, and genome-protein interactions. GEO supports community-derived reporting standards that specify provision of several critical study elements including raw data, processed data, and descriptive metadata. The database not only provides access to data for tens of thousands of studies, but also offers various Web-based tools and strategies that enable users to locate data relevant to their specific interests, as well as to visualize and analyze the data. This chapter includes detailed descriptions of methods to query and download GEO data and use the analysis and visualization tools. The GEO homepage is at http://www.ncbi.nlm.nih.gov/geo/. PMID:27008011

  8. A Core Invasiveness Gene Signature Reflects Epithelial-to-Mesenchymal Transition but Not Metastatic Potential in Breast Cancer Cell Lines and Tissue Samples

    PubMed Central

    Marsan, Melike; Van den Eynden, Gert; Limame, Ridha; Neven, Patrick; Hauspy, Jan; Van Dam, Peter A.; Vergote, Ignace; Dirix, Luc Y.; Vermeulen, Peter B.; Van Laere, Steven J.

    2014-01-01

    Introduction Metastases remain the primary cause of cancer-related death. The acquisition of invasive tumour cell behaviour is thought to be a cornerstone of the metastatic cascade. Therefore, gene signatures related to invasiveness could aid in stratifying patients according to their prognostic profile. In the present study we aimed at identifying an invasiveness gene signature and investigated its biological relevance in breast cancer. Methods & Results We collected a set of published gene signatures related to cell motility and invasion. Using this collection, we identified 16 genes that were represented at a higher frequency than observed by coincidence, hereafter named the core invasiveness gene signature. Principal component analysis showed that these overrepresented genes were able to segregate invasive and non-invasive breast cancer cell lines, outperforming sets of 16 randomly selected genes (all P<0.001). When applied onto additional data sets, the expression of the core invasiveness gene signature was significantly elevated in cell lines forced to undergo epithelial-mesenchymal transition. The link between core invasiveness gene expression and epithelial-mesenchymal transition was also confirmed in a dataset consisting of 2420 human breast cancer samples. Univariate and multivariate Cox regression analysis demonstrated that CIG expression is not associated with a shorter distant metastasis free survival interval (HR = 0.956, 95%C.I. = 0.896–1.019, P = 0.186). Discussion These data demonstrate that we have identified a set of core invasiveness genes, the expression of which is associated with epithelial-mesenchymal transition in breast cancer cell lines and in human tissue samples. Despite the connection between epithelial-mesenchymal transition and invasive tumour cell behaviour, we were unable to demonstrate a link between the core invasiveness gene signature and enhanced metastatic potential. PMID:24586640

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

    PubMed Central

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

    2013-01-01

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

  10. Classification of genes based on gene expression analysis

    NASA Astrophysics Data System (ADS)

    Angelova, M.; Myers, C.; Faith, J.

    2008-05-01

    Systems biology and bioinformatics are now major fields for productive research. DNA microarrays and other array technologies and genome sequencing have advanced to the point that it is now possible to monitor gene expression on a genomic scale. Gene expression analysis is discussed and some important clustering techniques are considered. The patterns identified in the data suggest similarities in the gene behavior, which provides useful information for the gene functionalities. We discuss measures for investigating the homogeneity of gene expression data in order to optimize the clustering process. We contribute to the knowledge of functional roles and regulation of E. coli genes by proposing a classification of these genes based on consistently correlated genes in expression data and similarities of gene expression patterns. A new visualization tool for targeted projection pursuit and dimensionality reduction of gene expression data is demonstrated.

  11. Classification of genes based on gene expression analysis

    SciTech Connect

    Angelova, M. Myers, C. Faith, J.

    2008-05-15

    Systems biology and bioinformatics are now major fields for productive research. DNA microarrays and other array technologies and genome sequencing have advanced to the point that it is now possible to monitor gene expression on a genomic scale. Gene expression analysis is discussed and some important clustering techniques are considered. The patterns identified in the data suggest similarities in the gene behavior, which provides useful information for the gene functionalities. We discuss measures for investigating the homogeneity of gene expression data in order to optimize the clustering process. We contribute to the knowledge of functional roles and regulation of E. coli genes by proposing a classification of these genes based on consistently correlated genes in expression data and similarities of gene expression patterns. A new visualization tool for targeted projection pursuit and dimensionality reduction of gene expression data is demonstrated.

  12. Effects of Phonation Time and Magnitude Dose on Vocal Fold Epithelial Genes, Barrier Integrity, and Function

    PubMed Central

    Kojima, Tsuyoshi; Valenzuela, Carla V.; Novaleski, Carolyn K.; Van Deusen, Mark; Mitchell, Joshua R.; Garrett, C. Gaelyn; Sivasankar, M. Preeti; Rousseau, Bernard

    2014-01-01

    Objective To investigate the effects of increasing time and magnitude doses of vibration exposure on transcription of the vocal fold's junctional proteins, structural alterations, and functional tissue outcomes. Study Design Animal study. Methods 100 New Zealand White breeder rabbits were studied. Dependent variables were measured in response to increasing time doses (30, 60, or 120 minutes) and magnitude doses (control, modal intensity, and raised intensity) of vibration exposure. Messenger RNA expression of occludin, zonula occluden-1 (ZO-1), E-cadherin, β-catenin, interleukin 1β (IL-1β), cyclooxygenase-2 (COX-2), transforming growth factor β-1 (TGFβ1), and fibronectin were measured. Tissue structural alterations were assessed using transmission electron microscopy (TEM). Transepithelial resistance was used to measure functional tissue outcomes. Results Occludin gene expression was downregulated in vocal folds exposed to 120 minute time doses of raised intensity phonation, relative to control, and modal intensity phonation. ZO-1 gene expression was upregulated following a 120 minute time dose of modal intensity phonation, compared to control, and downregulated after a 120 minute time dose of raised intensity phonation, compared to modal intensity phonation. E-cadherin gene expression was downregulated after a120 minute time dose of raised intensity phonation, compared to control and modal intensity phonation. TEM revealed extensive desquamation of the stratified squamous epithelial cells with increasing time and magnitude doses of vibration exposure. A general observation of lower transepithelial resistance measures was made in tissues exposed to raised intensity phonation, compared to all other groups. Conclusions This study provides evidence of vocal fold tissue responses to varying time and magnitude doses of vibration exposure. Level of Evidence N/A PMID:25073715

  13. Chromium(VI) stimulates Fyn to initiate innate immune gene induction in human airway epithelial cells

    PubMed Central

    Nemec, Antonia A.; Zubritsky, Lindsey M.; Barchowsky, Aaron

    2009-01-01

    Mechanisms for pathogenic metal signaling in airway injury or disease promotion are poorly understood. It is widely believed that one mechanism for pathogenic and possible carcinogenic effects of inhaled chromium (Cr(VI)) is inhibition of inducible gene transactivation. However, we recently reported that Cr(VI) inhibition of Sp1-dependent transactivation required signal transducer and activator of transcription 1 (STAT1)-dependent expression of an inhibitory protein in airway epithelium. Thus, Cr(VI) exposures can induce genes and we hypothesized this induction resulted from Cr(VI) signaling through an innate immune-like STAT1-dependent pathway initiated by Fyn. Exposure of human airway epithelial (BEAS-2B) cells to Cr(VI) selectively transactivated STAT-responsive interferon-stimulated response element (ISRE) and induced ISRE-driven transactivation of interferon regulatory factor 7 (IRF7), without affecting the gamma interferon-activated site (GAS)-driven IRF1 expression. Cr(VI)-induced IRF7 was absent or greatly reduced in cells that lacked STAT1, were treated with the Src family kinase inhibitor, PP2, or lacked Fyn. Expressing Fyn, but not Src, in mouse embryonic fibroblasts cells null for Src, Yes, and Fyn restored Cr(VI)-stimulated STAT1 tyrosine phosphorylation and IRF7 expression. Finally, shRNA knockdown of Fyn in BEAS-2B cells prevented Cr(VI)-activated STAT1 transactivation of IRF7. These data support a novel mechanism through which Cr(VI) stimulates Fyn to initiate interferon-like signaling for STAT1-dependent gene transactivation. PMID:19994902

  14. Protein Expression Profile of Rat Type Two Alveolar Epithelial Cells During Hyperoxic Stress and Recovery

    NASA Astrophysics Data System (ADS)

    Bhargava, Maneesh

    Rationale: In rodent model systems, the sequential changes in lung morphology resulting from hyperoxic injury are well characterized, and are similar to changes in human acute respiratory distress syndrome (ARDS). In the injured lung, alveolar type two (AT2) epithelial cells play a critical role restoring the normal alveolar structure. Thus characterizing the changes in AT2 cells will provide insights into the mechanisms underpinning the recovery from lung injury. Methods: We applied an unbiased systems level proteomics approach to elucidate molecular mechanisms contributing to lung repair in a rat hyperoxic lung injury model. AT2 cells were isolated from rat lungs at predetermined intervals during hyperoxic injury and recovery. Protein expression profiles were determined by using iTRAQRTM with tandem mass spectrometry. Results: Of 959 distinct proteins identified, 183 significantly changed in abundance during the injury-recovery cycle. Gene Ontology enrichment analysis identified cell cycle, cell differentiation, cell metabolism, ion homeostasis, programmed cell death, ubiquitination, and cell migration to be significantly enriched by these proteins. Gene Set Enrichment Analysis of data acquired during lung repair revealed differential expression of gene sets that control multicellular organismal development, systems development, organ development, and chemical homeostasis. More detailed analysis identified activity in two regulatory pathways, JNK and miR 374. A Short Time-series Expression Miner (STEM) algorithm identified protein clusters with coherent changes during injury and repair. Conclusion: Coherent changes occur in the AT2 cell proteome in response to hyperoxic stress. These findings offer guidance regarding the specific molecular mechanisms governing repair of the injured lung.

  15. Common variants at the CHEK2 gene locus and risk of epithelial ovarian cancer.

    PubMed

    Lawrenson, Kate; Iversen, Edwin S; Tyrer, Jonathan; Weber, Rachel Palmieri; Concannon, Patrick; Hazelett, Dennis J; Li, Qiyuan; Marks, Jeffrey R; Berchuck, Andrew; Lee, Janet M; Aben, Katja K H; Anton-Culver, Hoda; Antonenkova, Natalia; Bandera, Elisa V; Bean, Yukie; Beckmann, Matthias W; Bisogna, Maria; Bjorge, Line; Bogdanova, Natalia; Brinton, Louise A; Brooks-Wilson, Angela; Bruinsma, Fiona; Butzow, Ralf; Campbell, Ian G; Carty, Karen; Chang-Claude, Jenny; Chenevix-Trench, Georgia; Chen, Ann; Chen, Zhihua; Cook, Linda S; Cramer, Daniel W; Cunningham, Julie M; Cybulski, Cezary; Plisiecka-Halasa, Joanna; Dennis, Joe; Dicks, Ed; Doherty, Jennifer A; Dörk, Thilo; du Bois, Andreas; Eccles, Diana; Easton, Douglas T; Edwards, Robert P; Eilber, Ursula; Ekici, Arif B; Fasching, Peter A; Fridley, Brooke L; Gao, Yu-Tang; Gentry-Maharaj, Aleksandra; Giles, Graham G; Glasspool, Rosalind; Goode, Ellen L; Goodman, Marc T; Gronwald, Jacek; Harter, Philipp; Hasmad, Hanis Nazihah; Hein, Alexander; Heitz, Florian; Hildebrandt, Michelle A T; Hillemanns, Peter; Hogdall, Estrid; Hogdall, Claus; Hosono, Satoyo; Jakubowska, Anna; Paul, James; Jensen, Allan; Karlan, Beth Y; Kjaer, Susanne Kruger; Kelemen, Linda E; Kellar, Melissa; Kelley, Joseph L; Kiemeney, Lambertus A; Krakstad, Camilla; Lambrechts, Diether; Lambrechts, Sandrina; Le, Nhu D; Lee, Alice W; Cannioto, Rikki; Leminen, Arto; Lester, Jenny; Levine, Douglas A; Liang, Dong; Lissowska, Jolanta; Lu, Karen; Lubinski, Jan; Lundvall, Lene; Massuger, Leon F A G; Matsuo, Keitaro; McGuire, Valerie; McLaughlin, John R; Nevanlinna, Heli; McNeish, Iain; Menon, Usha; Modugno, Francesmary; Moysich, Kirsten B; Narod, Steven A; Nedergaard, Lotte; Ness, Roberta B; Noor Azmi, Mat Adenan; Odunsi, Kunle; Olson, Sara H; Orlow, Irene; Orsulic, Sandra; Pearce, Celeste L; Pejovic, Tanja; Pelttari, Liisa M; Permuth-Wey, Jennifer; Phelan, Catherine M; Pike, Malcolm C; Poole, Elizabeth M; Ramus, Susan J; Risch, Harvey A; Rosen, Barry; Rossing, Mary Anne; Rothstein, Joseph H; Rudolph, Anja; Runnebaum, Ingo B; Rzepecka, Iwona K; Salvesen, Helga B; Budzilowska, Agnieszka; Sellers, Thomas A; Shu, Xiao-Ou; Shvetsov, Yurii B; Siddiqui, Nadeem; Sieh, Weiva; Song, Honglin; Southey, Melissa C; Sucheston, Lara; Tangen, Ingvild L; Teo, Soo-Hwang; Terry, Kathryn L; Thompson, Pamela J; Timorek, Agnieszka; Tworoger, Shelley S; Van Nieuwenhuysen, Els; Vergote, Ignace; Vierkant, Robert A; Wang-Gohrke, Shan; Walsh, Christine; Wentzensen, Nicolas; Whittemore, Alice S; Wicklund, Kristine G; Wilkens, Lynne R; Woo, Yin-Ling; Wu, Xifeng; Wu, Anna H; Yang, Hannah; Zheng, Wei; Ziogas, Argyrios; Coetzee, Gerhard A; Freedman, Matthew L; Monteiro, Alvaro N A; Moes-Sosnowska, Joanna; Kupryjanczyk, Jolanta; Pharoah, Paul D; Gayther, Simon A; Schildkraut, Joellen M

    2015-11-01

    Genome-wide association studies have identified 20 genomic regions associated with risk of epithelial ovarian cancer (EOC), but many additional risk variants may exist. Here, we evaluated associations between common genetic variants [single nucleotide polymorphisms (SNPs) and indels] in DNA repair genes and EOC risk. We genotyped 2896 common variants at 143 gene loci in DNA samples from 15 397 patients with invasive EOC and controls. We found evidence of associations with EOC risk for variants at FANCA, EXO1, E2F4, E2F2, CREB5 and CHEK2 genes (P ≤ 0.001). The strongest risk association was for CHEK2 SNP rs17507066 with serous EOC (P = 4.74 x 10(-7)). Additional genotyping and imputation of genotypes from the 1000 genomes project identified a slightly more significant association for CHEK2 SNP rs6005807 (r (2) with rs17507066 = 0.84, odds ratio (OR) 1.17, 95% CI 1.11-1.24, P = 1.1×10(-7)). We identified 293 variants in the region with likelihood ratios of less than 1:100 for representing the causal variant. Functional annotation identified 25 candidate SNPs that alter transcription factor binding sites within regulatory elements active in EOC precursor tissues. In The Cancer Genome Atlas dataset, CHEK2 gene expression was significantly higher in primary EOCs compared to normal fallopian tube tissues (P = 3.72×10(-8)). We also identified an association between genotypes of the candidate causal SNP rs12166475 (r (2) = 0.99 with rs6005807) and CHEK2 expression (P = 2.70×10(-8)). These data suggest that common variants at 22q12.1 are associated with risk of serous EOC and CHEK2 as a plausible target susceptibility gene. PMID:26424751

  16. Impairment of the ubiquitin-proteasome pathway in RPE alters the expression of inflammation related genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin-proteasome pathway (UPP) plays an important role in regulating gene expression. Retinal pigment epithelial cells (RPE) are a major source of ocular inflammatory cytokines. In this work we determined the relationship between impairment of the UPP and expression of inflammation-related f...

  17. Thyroid hormone regulates stromelysin expression, protease secretion and the morphogenetic potential of normal polarized mammary epithelial cells.

    PubMed Central

    López-Barahona, M; Fialka, I; González-Sancho, J M; Asunción, M; González, M; Iglesias, T; Bernal, J; Beug, H; Muñoz, A

    1995-01-01

    Stromelysins are a group of proteases which degrade the extracellular matrix and activate other secreted proteases. Stromelysin (ST)-1 and ST-2 genes are induced by tumor promoters, oncogenes and growth factors, and have been involved in acquisition of the malignant phenotype. We show here that the thyroid hormone (T3) increases ST-1 and ST-2 expression in a non-transformed mouse mammary epithelial cell line (EpH4) in a way that is dependent on the level of thyroid receptor/c-erbA (TR alpha-1) expression. In agreement with this, T3 increases the secreted stromelysin activity and enhances the gelatinolytic activity of type IV collagenase. We have also demonstrated that T3 affects the epithelial polarity of EpH4 cells, diminishing the transepithelial electrical resistance of monolayers cultured on permeable filters, causing an abnormal distribution of polarization markers and the disruption of the organized 3-D structures formed by these cells in type I collagen gels. These results indicate that the ligand-activated TR alpha-1 plays an important role in regulating the morphogenetic and invasive capacities of mammary epithelial cells. Because the c-erbA locus is altered in several types of carcinoma, an altered or deregulated TR alpha-1 expression may also be important for breast cancer development and metastasis. Images PMID:7720705

  18. Transcriptome meta-analysis reveals common differential and global gene expression profiles in cystic fibrosis and other respiratory disorders and identifies CFTR regulators.

    PubMed

    Clarke, Luka A; Botelho, Hugo M; Sousa, Lisete; Falcao, Andre O; Amaral, Margarida D

    2015-11-01

    A meta-analysis of 13 independent microarray data sets was performed and gene expression profiles from cystic fibrosis (CF), similar disorders (COPD: chronic obstructive pulmonary disease, IPF: idiopathic pulmonary fibrosis, asthma), environmental conditions (smoking, epithelial injury), related cellular processes (epithelial differentiation/regeneration), and non-respiratory "control" conditions (schizophrenia, dieting), were compared. Similarity among differentially expressed (DE) gene lists was assessed using a permutation test, and a clustergram was constructed, identifying common gene markers. Global gene expression values were standardized using a novel approach, revealing that similarities between independent data sets run deeper than shared DE genes. Correlation of gene expression values identified putative gene regulators of the CF transmembrane conductance regulator (CFTR) gene, of potential therapeutic significance. Our study provides a novel perspective on CF epithelial gene expression in the context of other lung disorders and conditions, and highlights the contribution of differentiation/EMT and injury to gene signatures of respiratory disease. PMID:26225835

  19. CAP-D3 Promotes Bacterial Clearance in Human Intestinal Epithelial Cells by Repressing Expression of Amino Acid Transporters

    PubMed Central

    Kemp, Jacqueline R.; Nickerson, Kourtney P.; Deutschman, Emily; Kim, Yeojung; West, Gail; Sadler, Tammy; Stylianou, Eleni; Krokowski, Dawid; Hatzoglou, Maria; de la Motte, Carol; Rubin, Brian P.; Fiocchi, Claudio

    2015-01-01

    BACKGROUND & AIMS Defects in colonic epithelial barrier defenses are associated with ulcerative colitis (UC). The proteins that regulate bacterial clearance in the colonic epithelium have not been completely identified. The chromosome-associated protein D3 (dCAP-D3), regulates responses to bacterial infection. We examined whether CAP-D3 promotes bacterial clearance in human colonic epithelium. METHODS Clearance of Salmonella or adherent-invasive Escherichia coli LF82 was assessed by gentamycin protection assays in HT-29 and Caco-2 cells expressing small hairpin RNAs against CAP-D3. We used immunoblot assays to measure levels of CAP-D3 in colonic epithelial cells from patients with UC and healthy individuals (controls). RNA sequencing identified genes activated by CAP-D3. We analyzed the roles of CAP-D3 target genes in bacterial clearance using gentamycin protection and immunofluorescence assays and studies with pharmacologic inhibitors. RESULTS CAP-D3 expression was reduced in colonic epithelial cells from patients with active UC. Reduced CAP-D3 expression decreased autophagy and impaired intracellular bacterial clearance by HT-29 and Caco-2 colonic epithelial cells. Lower levels of CAP-D3 increased transcription of genes encoding SLC7A5 and SLC3A2, whose products heterodimerize to form an amino acid transporter in HT-29 cells following bacterial infection; levels of SLC7A5–SLC3A2 were increased in tissues from patients with UC, compared with controls. Reduced CAP-D3 in HT-29 cells resulted in earlier recruitment of SLC7A5 to Salmonella-containing vacuoles, increased activity of mTORC1, and increased survival of bacteria. Inhibition of SLC7A5–SLC3A2 or mTORC1 activity rescued the bacterial clearance defects of CAP-D3– deficient cells. CONCLUSIONS CAP-D3 downregulates transcription of genes that encode amino acid transporters (SLC7A5 and SLC3A2) to promote bacterial autophagy by colon epithelial cells. Levels of CAP-D3 protein are reduced in patients with

  20. Survivin expression is associated with lens epithelial cell proliferation and fiber cell differentiation

    PubMed Central

    Mansergh, Fiona C.; Boulton, Michael E.; Gunhaga, Lena

    2012-01-01

    Purpose Survivin (Birc5) is the smallest member of the inhibitor of apoptosis (IAP) protein family, which regulates the cell cycle/apoptosis balance. The purpose of this study was to examine Survivin expression in the embryonic chick lens, in chick lens epithelial cell cultures, and in the postnatal mouse lens. Methods Survivin expression was examined using a combination of quantitative real-time polymerase chain reaction, western blotting, and immunocytochemistry. To correlate Survivin expression with the timing of proliferation, we determined the profile of cell proliferation in the developing lens using the cell cycle marker proliferating cell nuclear antigen (PCNA) in quantitative western blotting and immunocytochemistry studies. We also examined the expression of PCNA and the extent of denucleation using terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick-end labeling (TUNEL) of lentoids (lens fiber-like cells) during chick lens epithelial cell differentiation in vitro. Results At embryonic day (ED) 4, Survivin immunostaining was present in two pools in lens epithelial cells and fiber cells: cytoplasmic and nuclear. The nuclear staining became more pronounced as the lens epithelial cells differentiated into lens fiber cells. At ED12, Survivin staining was observed in lens fiber cell nuclei containing marginalized chromatin, indicative of early denucleation events. Using western blotting, Survivin expression peaked at ED6, diminishing thereafter. This profile of expression correlated with the events in chick lens epithelial cell cultures: i) increased Survivin expression was associated with an increase in PCNA staining up to day 6 of culture and ii) downregulation of Survivin expression at day 8 of culture was coincident with a dramatic decrease in PCNA staining and an increase in TdT-mediated biotin-dUTP nick-end labeling in lentoids. In early postnatal mouse lenses, Survivin and PCNA were highly expressed and decreased thereafter during

  1. Selective gene expression by rat gastric corpus epithelium

    PubMed Central

    Goebel, M.; Stengel, A.; Sachs, G.

    2011-01-01

    The gastrointestinal (GI) tract is divided into several segments that have distinct functional properties, largely absorptive. The gastric corpus is the only segment thought of as largely secretory. Microarray hybridization of the gastric corpus mucosal epithelial cells was used to compare gene expression with other segments of the columnar GI tract followed by statistical data subtraction to identify genes selectively expressed by the rat gastric corpus mucosa. This provides a means of identifying less obvious specific functions of the corpus in addition to its secretion-related genes. For example, important properties found by this GI tract comparative transcriptome reflect the energy demand of acid secretion, a role in lipid metabolism, the large variety of resident neuroendocrine cells, responses to damaging agents and transcription factors defining differentiation of its epithelium. In terms of overlap of gastric corpus genes with the rest of the GI tract, the distal small bowel appears to express many of the gastric corpus genes in contrast to proximal small and large bowel. This differential map of gene expression by the gastric corpus epithelium will allow a more detailed description of major properties of the gastric corpus and may lead to the discovery of gastric corpus cell differentiation genes and those mis-regulated in gastric carcinomas. PMID:21177383

  2. Identification of four soybean reference genes for gene expression normalization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gene expression analysis requires the use of reference genes stably expressed independently of specific tissues or environmental conditions. Housekeeping genes (e.g., actin, tubulin, ribosomal, polyubiquitin and elongation factor 1-alpha) are commonly used as reference genes with the assumption tha...

  3. Mitochondrial RNA granules: Compartmentalizing mitochondrial gene expression.

    PubMed

    Jourdain, Alexis A; Boehm, Erik; Maundrell, Kinsey; Martinou, Jean-Claude

    2016-03-14

    In mitochondria, DNA replication, gene expression, and RNA degradation machineries coexist within a common nondelimited space, raising the question of how functional compartmentalization of gene expression is achieved. Here, we discuss the recently characterized "mitochondrial RNA granules," mitochondrial subdomains with an emerging role in the regulation of gene expression. PMID:26953349

  4. Characterization of gene expression and activated signaling pathways in solid-pseudopapillary neoplasm of pancreas.

    PubMed

    Park, Minhee; Kim, Minhyung; Hwang, Daehee; Park, Misun; Kim, Won Kyu; Kim, Sang Kyum; Shin, Jihye; Park, Eun Sung; Kang, Chang Moo; Paik, Young-Ki; Kim, Hoguen

    2014-04-01

    Solid-pseudopapillary neoplasm is an uncommon pancreatic tumor with distinct clinicopathologic features. Solid-pseudopapillary neoplasms are characterized by mutations in exon 3 of CTNNB1. However, little is known about the gene and microRNA expression profiles of solid-pseudopapillary neoplasms. Thus, we sought to characterize solid-pseudopapillary neoplasm-specific gene expression and identify the signaling pathways activated in these tumors. Comparisons of gene expression in solid-pseudopapillary neoplasm to pancreatic ductal carcinomas, neuroendocrine tumors, and non-neoplastic pancreatic tissues identified solid-pseudopapillary neoplasm-specific mRNA and microRNA profiles. By analyzing 1686 (1119 upregulated and 567 downregulated) genes differentially expressed in solid-pseudopapillary neoplasm, we found that the Wnt/β-catenin, Hedgehog, and androgen receptor signaling pathways, as well as genes involved in epithelial mesenchymal transition, are activated in solid-pseudopapillary neoplasms. We validated these results experimentally by assessing the expression of β-catenin, WIF-1, GLI2, androgen receptor, and epithelial-mesenchymal transition-related markers with western blotting and immunohistochemistry. Our analysis also revealed 17 microRNAs, especially the miR-200 family and miR-192/215, closely associated with the upregulated genes associated with the three pathways activated in solid-pseudopapillary neoplasm and epithelial mesenchymal transition. Our results provide insight into the molecular mechanisms underlying solid-pseudopapillary neoplasm tumorigenesis and its characteristic less epithelial cell differentiation than the other common pancreatic tumors. PMID:24072181

  5. Expression of mitogen-activated protein kinase phosphatase-1 in the early phases of human epithelial carcinogenesis.

    PubMed Central

    Loda, M.; Capodieci, P.; Mishra, R.; Yao, H.; Corless, C.; Grigioni, W.; Wang, Y.; Magi-Galluzzi, C.; Stork, P. J.

    1996-01-01

    Many mitogens and human oncogenes activate extracellular regulated kinases (ERKs), which in turn convey proliferation signals. ERKs or mitogen-activated protein (MAP) kinases are inactivated in vitro by MAP kinase phosphatases (MKPs). The gene encoding one of these MKPs, MKP-1, is a serum-inducible gene and is transcriptionally activated by mitogenic signals in cultured cells. As MKP-1 has been shown to block DNA synthesis by inhibiting ERKs when expressed at elevated levels in cultured cells, it has been suggested that it may act as a tumor suppressor. MKP-1 mRNA and MAP kinase (ERK-1 and -2) protein expression was assessed in 164 human epithelial tumors of diverse tissue origin by in situ hybridization and immunohistochemistry. MKP-1 was overexpressed in the early phases of prostate, colon, and bladder carcinogenesis, with progressive loss of expression with higher histological grade and in metastases. In contrast, breast carcinomas showed significant MKP-1 expression even when poorly differentiated or in late stages of the disease. MKP-1, ERK-1, and ERK-2 were co-expressed in most tumors examined. In a subset of 15 tumors, ERK-1 enzymatic activity as well as structural alterations that might be responsible for loss of function of MKP-1 during tumor progression, were examined. ERK-1 enzymatic activity was found to be elevated despite MKP-1 overexpression. No loss of 5q35-ter (containing the MKP-1 locus) was detected by polymerase chain reaction in metastases compared with primary tumors. Finally, no mutations were found in the catalytic domain of MKP-1. These data indicate that MKP-1 is an early marker for a wide range of human epithelial tumors and suggest that MKP-1 does not behave as a tumor suppressor in epithelial tumors. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8909245

  6. Manganese Superoxide Dismutase Expression Regulates the Switch Between an Epithelial and a Mesenchymal-Like Phenotype in Breast Carcinoma

    PubMed Central

    Loo, Ser Yue; Hirpara, Jayshree L.; Pandey, Vijay; Tan, Tuan Zea; Yap, Celestial T.; Lobie, Peter E.; Thiery, Jean Paul; Goh, Boon Cher

    2016-01-01

    Abstract Aim: Epithelial–mesenchymal transition (EMT) is characterized by the acquisition of invasive fibroblast-like morphology by epithelial cells that are highly polarized. EMT is recognized as a crucial mechanism in cancer progression and metastasis. In this study, we sought to assess the involvement of manganese superoxide dismutase (MnSOD) during the switch between epithelial-like and mesenchymal-like phenotypes in breast carcinoma. Results: Analysis of breast carcinomas from The Cancer Genome Atlas database revealed strong positive correlation between tumors' EMT score and the expression of MnSOD. This positive correlation between MnSOD and EMT score was significant and consistent across all breast cancer subtypes. Similarly, a positive correlation of EMT score and MnSOD expression was observed in established cell lines derived from breast cancers exhibiting phenotypes ranging from the most epithelial to the most mesenchymal. Interestingly, using phenotypically distinct breast cancer cell lines, we provide evidence that constitutively high or induced expression of MnSOD promotes the EMT-like phenotype by way of a redox milieu predominantly driven by hydrogen peroxide (H2O2). Conversely, gene knockdown of MnSOD results in the reversal of EMT to a mesenchymal–epithelial transition (MET)-like program, which appears to be a function of superoxide (O2−•)-directed signaling. Innovation and Conclusion: These data underscore the involvement of MnSOD in regulating the switch between the EMT and MET-associated phenotype by influencing cellular redox environment via its effect on the intracellular ratio between O2−• and H2O2. Strategies to manipulate MnSOD expression and/or the cellular redox milieu vis-a-vis O2−•:H2O2 could have potential therapeutic implications. Antioxid. Redox Signal. 25, 283–299. PMID:27400860

  7. Expression of K6W-ubiquitin inhibits proliferation of human lens epithelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin-proteasome pathway plays an important role in controlling the cell cycle. The purpose of this study was to examine if expression of a dominant negative form of ubiquitin can inhibit the proliferation of lens epithelial cells. Dominant negative K6W-ubiquitin was expressed in cultured hu...

  8. Expression of AQP6 and AQP8 in epithelial ovarian tumor.

    PubMed

    Ma, Jiong; Zhou, Chunxia; Yang, Jianhua; Ding, Xiaoyan; Zhu, Yunshan; Chen, Xuejun

    2016-04-01

    Aquaporins (AQPs), the rapid transition pores for water molecules, play an important role in maintenance of intracellular water balance. Studies showed that AQPs were also involved in occurrence, development, invasion and metastasis of tumors. In this study, we aimed to explore the distribution and expression differences of aquaporin 6 (AQP6) and aquaporin 8 (AQP8) in epithelial ovarian tumors. The expression of AQP6 and AQP8 in 47 cases of epithelial ovarian tumors were measured by immunochemical technique and Western blotting. AQP6 was strongly expressed in benign ovarian tumors, but weak signal was shown in malignant tumors. The difference was not statistically significant (P > 0.05). Compared with serous adenoma and normal tissues, AQP6 expression in serous carcinoma was obviously decreased (P < 0.05). AQP8 expressions were both identified in benign and malignant tumors, but there was no significantly statistical difference (P > 0.05). For patients with large volume of malignant ascites (>1000 ml), AQP8 expression was increased (P < 0.05). AQP8 expression in malignant tumors was not related to different clinical stages, presence of lymphatic metastasis, and differentiation degrees (P > 0.05). These data showed that AQP6 and AQP8 had different expression degrees in epithelial ovarian tissues, which suggests that AQP6 and AQP8 may play certain roles in epithelial ovarian tumors. PMID:26779650

  9. Infant formula alters surfactant protein A (SP-A) and SP-B expression in pulmonary epithelial cells.

    PubMed

    Chen, Maurice G; Atkins, Constance L; Bruce, Shirley R; Khan, Amir M; Liu, Yuying; Alcorn, Joseph L

    2011-09-01

    Surfactant proteins A (SP-A) and SP-B are critical in the ability of pulmonary surfactant to reduce alveolar surface tension and provide innate immunity. Aspiration of infant milk formula can lead to lung dysfunction, but direct effects of aspirated formula on surfactant protein expression in pulmonary cells have not been described. The hypothesis that infant formula alters surfactant protein homeostasis was tested in vitro by assessing surfactant protein gene expression in cultured pulmonary epithelial cell lines expressing SP-A and SP-B that were transiently exposed (6 hr) to infant formula. Steady-state levels of SP-A protein and mRNA and SP-B mRNA in human bronchiolar (NCI-H441) and mouse alveolar (MLE15) epithelial cells were reduced in a dose-dependent manner 18 hr after exposure to infant formula. SP-A mRNA levels remained reduced 42 hr after exposure, but SP-B mRNA levels increased 10-fold. Neither soy formula nor non-fat dry milk affected steady-state SP-A and SP-B mRNA levels; suggesting a role of a component of infant formula derived from cow milk. These results indicate that infant formula has a direct, dose-dependent effect to reduce surfactant protein gene expression. Ultimately, milk aspiration may potentially result in a reduced capacity of the lung to defend against environmental insults. PMID:21520433

  10. Construction of p66Shc gene interfering lentivirus vectors and its effects on alveolar epithelial cells apoptosis induced by hyperoxia

    PubMed Central

    Zhang, Chan; Dong, Wen-Bin; Zhao, Shuai; Li, Qing-Ping; Kang, Lan; Lei, Xiao-Ping; Guo, Lin; Zhai, Xue-Song

    2016-01-01

    Background The aim of this study is to observe the inhibitive effects of p66Shc gene interfering lentivirus vectors on the expression of p66Shc, and to explore its effects on alveolar epithelial cells apoptosis induced by hyperoxia. Methods The gene sequences were cloned into the pLenR-GPH-shRNA lentiviral vector, which was selected by Genebank searches. The pLenR-GPH-shRNA and lentiviral vector packaging plasmid mix were cotransfected into 293T cells to package lentiviral particles. Culture virus supernatant was harvested, and then the virus titer was determined by serial dilution assay. A549 cells were transduced with the constructed lentiviral vectors, and real-time polymerase chain reaction (RT-PCR) and Western blot were used to evaluate p66Shc expression. This study is divided into a control group, a hyperoxia group, an A549-p66ShcshRNA hyperoxia group, and a negative lentivirus group. Cell apoptosis was detected by flow cytometry after 24 hours; the expression of X-linked inhibitor of apoptosis protein (XIAP) and caspase-9 were detected by immunohistochemistry assay. The production of reactive oxygen species and cellular mitochondria membrane potential (ΔΨm) were determined by fluorescence microscopy. Results We successfully established the p66Shc gene interfering lentivirus vectors, A549-p66ShcshRNA. The A549-p66ShcshRNA was transfected into alveolar epithelial cells, and the inhibitive effects on the expression of p66Shc were observed. Both RT-PCR and Western blot demonstrated downregulation of p66Shc expression in A549 cells. In the A549-p66ShcshRNA hyperoxia group, we found dampened oxidative stress. A549-p66ShcshRNA can cause p66Shc gene silencing, reduce mitochondrial reactive oxygen species generation, reduce membrane potential decrease, reduce the apoptosis of A549 cells, and reduce alveolar epithelial cell injury, while the lentiviral empty vector group had no such changes. Conclusion p66Shc gene interfering lentivirus vector can affect the

  11. Consequences of Reduction of Klebsiella pneumoniae Capsule Expression on Interactions of This Bacterium with Epithelial Cells

    PubMed Central

    Favre-Bonte, Sabine; Joly, Bernard; Forestier, Christiane

    1999-01-01

    Most Klebsiella pneumoniae clinical isolates are fully encapsulated and adhere in vitro to intestinal cell lines with an aggregative pattern. In this study, the influence of the capsule on interactions with epithelial cells was investigated by creating an isogenic mutant defective in the synthesis of the capsule. Determination of the uronic acid content of bacterial extracts confirmed that the mutant did not produce capsular polysaccharides whereas, with the wild-type strain, the level of encapsulation was growth phase dependent and reached a maximum during the lag and early log phases. Assays performed with different epithelial cell lines, Int-407, A-549, and HEp-2, showed that the capsule-defective mutant demonstrated greater adhesion than did the wild-type strain and that the aggregative pattern was maintained, indicating that the capsule was not related to the adhesion phenotype. In contrast, when the mucus-producing HT-29-MTX cells were used, the encapsulated wild-type strain adhered more strongly than did the capsule-defective mutant. No invasion properties were observed with any of the capsular phenotypes or cell lines used. The K. pneumoniae adhesin CF29K was detected by Western blot analysis and enzyme-linked immunosorbent assay on the surface of transconjugants obtained after transfer of a conjugative plasmid harboring the CF29K-encoding genes into both the wild-type and the capsule-defective strains. The amounts of adhesin detected were greater in the capsule-defective background strain than in the wild-type encapsulated strain and were associated with an increase in the level of adhesion to Caco-2 cells. Moreover, RNA slot blot experiments showed that transcription of the adhesin-encoding gene was markedly increased in the capsule-defective mutant compared to the wild-type encapsulated background. These results suggest (i) that the capsule plays an active role during the initial steps of the pathogenesis by interacting with mucus-producing cells but is

  12. Glycogene Expression Alterations Associated with Pancreatic Cancer Epithelial-Mesenchymal Transition in Complementary Model Systems

    PubMed Central

    Maupin, Kevin A.; Sinha, Arkadeep; Eugster, Emily; Miller, Jeremy; Ross, Julianna; Paulino, Vincent; Keshamouni, Venkateshwar G.; Tran, Nhan; Berens, Michael; Webb, Craig; Haab, Brian B.

    2010-01-01

    Background The ability to selectively detect and target cancer cells that have undergone an epithelial-mesenchymal transition (EMT) may lead to improved methods to treat cancers such as pancreatic cancer. The remodeling of cellular glycosylation previously has been associated with cell differentiation and may represent a valuable class of molecular targets for EMT. Methodology/Principal Findings As a first step toward investigating the nature of glycosylation alterations in EMT, we characterized the expression of glycan-related genes in three in-vitro model systems that each represented a complementary aspect of pancreatic cancer EMT. These models included: 1) TGFβ-induced EMT, which provided a look at the active transition between states; 2) a panel of 22 pancreatic cancer cell lines, which represented terminal differentiation states of either epithelial-like or mesenchymal-like; and 3) actively-migrating and stationary cells, which provided a look at the mechanism of migration. We analyzed expression data from a list of 587 genes involved in glycosylation (biosynthesis, sugar transport, glycan-binding, etc.) or EMT. Glycogenes were altered at a higher prevalence than all other genes in the first two models (p<0.05 and <0.005, respectively) but not in the migration model. Several functional themes were shared between the induced-EMT model and the cell line panel, including alterations to matrix components and proteoglycans, the sulfation of glycosaminoglycans; mannose receptor family members; initiation of O-glycosylation; and certain forms of sialylation. Protein-level changes were confirmed by Western blot for the mannose receptor MRC2 and the O-glycosylation enzyme GALNT3, and cell-surface sulfation changes were confirmed using Alcian Blue staining. Conclusions/Significance Alterations to glycogenes are a major component of cancer EMT and are characterized by changes to matrix components, the sulfation of GAGs, mannose receptors, O-glycosylation, and specific

  13. Does inbreeding affect gene expression in birds?

    PubMed Central

    Hansson, Bengt; Naurin, Sara; Hasselquist, Dennis

    2014-01-01

    Inbreeding increases homozygosity, exposes genome-wide recessive deleterious alleles and often reduces fitness. The physiological and reproductive consequences of inbreeding may be manifested already during gene regulation, but the degree to which inbreeding influences gene expression is unknown in most organisms, including in birds. To evaluate the pattern of inbreeding-affected gene expression over the genome and in relation to sex, we performed a transcriptome-wide gene expression (10 695 genes) study of brain tissue of 10-day-old inbred and outbred, male and female zebra finches. We found significantly lower gene expression in females compared with males at Z-linked genes, confirming that dosage compensation is incomplete in female birds. However, inbreeding did not affect gene expression at autosomal or sex-linked genes, neither in males nor in females. Analyses of single genes again found a clear sex-biased expression at Z-linked genes, whereas only a single gene was significantly affected by inbreeding. The weak effect of inbreeding on gene expression in zebra finches contrasts to the situation, for example, in Drosophila where inbreeding has been found to influence gene expression more generally and at stress-related genes in particular. PMID:25232028

  14. Seasonal Effects on Gene Expression

    PubMed Central

    Goldinger, Anita; Shakhbazov, Konstantin; Henders, Anjali K.; McRae, Allan F.; Montgomery, Grant W.; Powell, Joseph E.

    2015-01-01

    Many health conditions, ranging from psychiatric disorders to cardiovascular disease, display notable seasonal variation in severity and onset. In order to understand the molecular processes underlying this phenomenon, we have examined seasonal variation in the transcriptome of 606 healthy individuals. We show that 74 transcripts associated with a 12-month seasonal cycle were enriched for processes involved in DNA repair and binding. An additional 94 transcripts demonstrated significant seasonal variability that was largely influenced by blood cell count levels. These transcripts were enriched for immune function, protein production, and specific cellular markers for lymphocytes. Accordingly, cell counts for erythrocytes, platelets, neutrophils, monocytes, and CD19 cells demonstrated significant association with a 12-month seasonal cycle. These results demonstrate that seasonal variation is an important environmental regulator of gene expression and blood cell composition. Notable changes in leukocyte counts and genes involved in immune function indicate that immune cell physiology varies throughout the year in healthy individuals. PMID:26023781

  15. Romo1 expression contributes to oxidative stress-induced death of lung epithelial cells

    SciTech Connect

    Shin, Jung Ar; Chung, Jin Sil; Cho, Sang-Ho; Kim, Hyung Jung; Yoo, Young Do

    2013-09-20

    Highlights: •Romo1 mediates oxidative stress-induced mitochondrial ROS production. •Romo1 induction by oxidative stress plays an important role in oxidative stress-induced apoptosis. •Romo1 overexpression correlates with epithelial cell death in patients with IPF. -- Abstract: Oxidant-mediated death of lung epithelial cells due to cigarette smoking plays an important role in pathogenesis in lung diseases such as idiopathic pulmonary fibrosis (IPF). However, the exact mechanism by which oxidants induce epithelial cell death is not fully understood. Reactive oxygen species (ROS) modulator 1 (Romo1) is localized in the mitochondria and mediates mitochondrial ROS production through complex III of the mitochondrial electron transport chain. Here, we show that Romo1 mediates mitochondrial ROS production and apoptosis induced by oxidative stress in lung epithelial cells. Hydrogen peroxide (H{sub 2}O{sub 2}) treatment increased Romo1 expression, and Romo1 knockdown suppressed the cellular ROS levels and cell death triggered by H{sub 2}O{sub 2} treatment. In immunohistochemical staining of lung tissues from patients with IPF, Romo1 was mainly localized in hyperplastic alveolar and bronchial epithelial cells. Romo1 overexpression was detected in 14 of 18 patients with IPF. TUNEL-positive alveolar epithelial cells were also detected in most patients with IPF but not in normal controls. These findings suggest that Romo1 mediates apoptosis induced by oxidative stress in lung epithelial cells.

  16. MRI of Transgene Expression: Correlation to Therapeutic Gene Expression

    PubMed Central

    Ichikawa, Tomotsugu; Högemanny, Dagmar; Saeki, Yoshinaga; Tyminski, Edyta; Terada, Kinya; Weissleder, Ralph; Chiocca, E Antonio; Basilion, James P

    2002-01-01

    Abstract Magnetic resonance imaging (MRI) can provide highresolution 3D maps of structural and functional information, yet its use of mapping in vivo gene expression has only recently been explored. A potential application for this technology is to noninvasively image transgene expression. The current study explores the latter using a nonregulatable internalizing engineered transferrin receptor (ETR) whose expression can be probed for with a superparamagnetic Tf-CLIO probe. Using an HSV-based amplicon vector system for transgene delivery, we demonstrate that: 1) ETR is a sensitive MR marker gene; 2) several transgenes can be efficiently expressed from a single amplicon; 3) expression of each transgene results in functional gene product; and 4) ETR gene expression correlates with expression of therapeutic genes when the latter are contained within the same amplicon. These data, taken together, suggest that MRI of ETR expression can serve as a surrogate for measuring therapeutic transgene expression. PMID:12407446

  17. Expression and regulation of normal and polymorphic epithelial sodium channel by human lymphocytes.

    PubMed

    Bubien, J K; Watson, B; Khan, M A; Langloh, A L; Fuller, C M; Berdiev, B; Tousson, A; Benos, D J

    2001-03-16

    Gene expression, protein expression, and function of amiloride-sensitive sodium channels were examined in human lymphocytes from normal individuals and individuals with Liddle's disease. Using reverse transcriptase polymerase chain reactions, expression of all three cloned epithelial sodium channel (ENaC) subunits was detected in lymphocytes. Polyclonal antibodies to bovine alpha-ENaC bound to the plasma membrane of normal and Liddle's lymphocytes. A quantitative analysis of fluorescence-tagged ENaC antibodies indicated a 2.5-fold greater surface binding of the antibodies to Liddle's lymphocytes compared with normal lymphocytes. The relative binding intensity increased significantly (25%; p < 0.001) for both normal and Liddle's cells after treatment with 40 microM 8-CPT-cAMP. Amiloride-sensitive whole cell currents were recorded under basal and cAMP-treated conditions for both cell types. Liddle's cells had a 4.5-fold larger inward sodium conductance compared with normal cells. A specific 25% increase in the inward sodium current was observed in normal cells in response to cAMP treatment. Outside-out patches from both cell types under both treatment conditions revealed no obvious differences in the single channel conductance. The P(open) was 4.2 +/- 3.9% for patches from non-Liddle's cells, and 27.7 +/- 5.4% in patches from Liddle's lymphocytes. Biochemical purification of a protein complex, using the same antibodies used for the immunohistochemistry, yielded a functional sodium channel complex that was inhibited by amiloride when reconstituted into lipid vesicles and incorporated into planar lipid bilayers. These four independent methodologies yielded findings consistent with the hypotheses that human lymphocytes express functional, regulatable ENaC and that the mutation responsible for Liddle's disease induces excessive channel expression. PMID:11113130

  18. Protection of photoreceptor cells from phototoxicity by transplanted retinal pigment epithelial cells expressing different neurotrophic factors.

    PubMed

    Abe, Toshiaki; Saigo, Yoko; Hojo, Masayoshi; Kano, Tetsuya; Wakusawa, Ryosuke; Tokita, Yumi; Tamai, Makoto

    2005-01-01

    Transplantation of cells or tissues and the intravitreal injection of neurotrophic factors are two methods that have been used to treat retinal diseases. The purpose of this study was to examine the effects of combining both methods: the transplantation of retinal pigment epithelial (RPE) cells expressing different neurotrophic factors. The neutrophic factors were Axokine, brain derived-neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF). The enhanced green fluorescence protein (eGFP) gene was used as a reporter gene. These genes were transduced into RPE cells by lipofection, selected by antibiotics, and transplanted into the subretinal space of 108 rats. The rats were examined at 1 week and 3 months after the transplantation to determine whether the transduced cells were present, were expressing the protein, and were able to protect photoreceptors against phototoxicity. The survival of the transplanted cells was monitored by the presence of eGFP. The degree of protection was determined by the thickness of the outer nuclear layer. Our results showed that the degree of photoreceptor protection was different for the different types of neurotrophic factors at 1 week. After 3 months, the number of surviving transplanted cell was markedly reduced, and protection was observed only with the BDNF-transduced RPE cells. A significant degree of rescue was also observed by BDNF-transduced RPE cells in the nontransplanted area of the retina at both the early and late times. Lymphocytic infiltration was not detected in the vitreous, retina, and choroid at any time. We conclude that the transplantation of BDNF-transduced RPE cells can reduce the photoreceptor damage induced by phototoxicity in the transplanted area and weakly in the nontransplanted area. PMID:16454354

  19. CRISPR-Cas9-mediated gene knockout in primary human airway epithelial cells reveals a proinflammatory role for MUC18.

    PubMed

    Chu, H W; Rios, C; Huang, C; Wesolowska-Andersen, A; Burchard, E G; O'Connor, B P; Fingerlin, T E; Nichols, D; Reynolds, S D; Seibold, M A

    2015-10-01

    Targeted knockout of genes in primary human cells using CRISPR-Cas9-mediated genome-editing represents a powerful approach to study gene function and to discern molecular mechanisms underlying complex human diseases. We used lentiviral delivery of CRISPR-Cas9 machinery and conditional reprogramming culture methods to knockout the MUC18 gene in human primary nasal airway epithelial cells (AECs). Massively parallel sequencing technology was used to confirm that the genome of essentially all cells in the edited AEC populations contained coding region insertions and deletions (indels). Correspondingly, we found mRNA expression of MUC18 was greatly reduced and protein expression was absent. Characterization of MUC18 knockout cell populations stimulated with TLR2, 3 and 4 agonists revealed that IL-8 (a proinflammatory chemokine) responses of AECs were greatly reduced in the absence of functional MUC18 protein. Our results show the feasibility of CRISPR-Cas9-mediated gene knockouts in AEC culture (both submerged and polarized), and suggest a proinflammatory role for MUC18 in airway epithelial response to bacterial and viral stimuli. PMID:26043872

  20. Interleukin-13 promotes expression of Alix to compromise renal tubular epithelial barrier function.

    PubMed

    Xu, Chen; Sun, Guangdong; Yang, Jie; Sun, Qianmei; Tong, Zhaohui

    2015-05-01

    The epithelial barrier dysfunction plays a critical role in a number of kidney diseases. The mechanism is unclear. Alix is a protein involving in protein degradation in epithelial cells. This study aims to investigate that interleukin (IL)-13 inhibits Alix to compromise the kidney epithelial barrier function. In this study, the murine collecting duct cell line (M-1) was cultured in Transwell inserts to investigate the significance of Alix in compromising the epithelial barrier functions. T cell (Teff cells) proliferation assay was employed to assess the antigenicity of ovalbumin (OVA) that was transported across the M-1 monolayer barrier. The results showed that M-1 cells express Alix. Exposure to interleukin (IL)-13 markedly decreased the expression of Alix in M-1 cells, which compromised the M-1 monolayer barrier functions by showing the increases in the permeability to OVA. Over-expression of Alix abolished the IL-13-induced M-1 monolayer barrier dysfunction. Knockdown of Alix significantly increased M-1 monolayer permeability. The OVA collected from the Transwell basal chambers induced the OVA-specific T cell proliferation. We conclude that IL-13 compromises M-1 epithelial barrier functions via inhibiting Alix expression. PMID:25597757

  1. Syntaxin 1A is expressed in airway epithelial cells, where it modulates CFTR Cl– currents

    PubMed Central

    Naren, Anjaparavanda P.; Di, Anke; Cormet-Boyaka, Estelle; Boyaka, Prosper N.; McGhee, Jerry R.; Zhou, Weihong; Akagawa, Kimio; Fujiwara, Tomonori; Thome, Ulrich; Engelhardt, John F.; Nelson, Deborah J.; Kirk, Kevin L.

    2000-01-01

    The CFTR Cl– channel controls salt and water transport across epithelial tissues. Previously, we showed that CFTR-mediated Cl– currents in the Xenopus oocyte expression system are inhibited by syntaxin 1A, a component of the membrane trafficking machinery. This negative modulation of CFTR function can be reversed by soluble syntaxin 1A peptides and by the syntaxin 1A binding protein, Munc-18. In the present study, we determined whether syntaxin 1A is expressed in native epithelial tissues that normally express CFTR and whether it modulates CFTR currents in these tissues. Using immunoblotting and immunofluorescence, we observed syntaxin 1A in native gut and airway epithelial tissues and showed that epithelial cells from these tissues express syntaxin 1A at >10-fold molar excess over CFTR. Syntaxin 1A is seen near the apical cell surfaces of human bronchial airway epithelium. Reagents that disrupt the CFTR-syntaxin 1A interaction, including soluble syntaxin 1A cytosolic domain and recombinant Munc-18, augmented cAMP-dependent CFTR Cl– currents by more than 2- to 4-fold in mouse tracheal epithelial cells and cells derived from human nasal polyps, but these reagents did not affect CaMK II–activated Cl– currents in these cells. PMID:10675364

  2. Effect of insoluble extracellular matrix molecules on Fas expression in epithelial cells.

    PubMed

    Fine, A; Miranda, K; Farmer, S R; Anderson, N L

    1998-03-01

    Fas, which functions to initiate a signal causing apoptosis, is expressed in epithelia, thus, suggesting a role in controlling cell number during states of cell and matrix turnover. In view of this, we hypothesized that cell-matrix interactions may be an important determinant of Fas expression in epithelial cells. To investigate this, we examined the effect of insoluble extracellular matrix molecules on Fas expression in murine lung epithelial (MLE) cells, a transformed mouse lung epithelial cell line. We report that 1) insoluble extracellular matrices increased Fas mRNA in a time and concentration-dependent manner; 2) induced increases in Fas mRNA were associated with concomitantly increased Fas protein; and 3) nonspecific adherence to a polylysine substrate did not induce Fas mRNA. Consistent with these findings, Fas-induced apoptosis was significantly enhanced in cultures plated on type IV collagen. Employing rat hepatocytes, we confirmed that the insoluble extracellular matrix also increases Fas expression in primary epithelial cells. By amplifying Fas-mediated apoptosis, these data suggest a mechanism whereby the extracellular matrix regulates the fate of specific epithelial cell populations. PMID:9462690

  3. Lumican induces human corneal epithelial cell migration and integrin expression via ERK 1/2 signaling

    SciTech Connect

    Seomun, Young; Joo, Choun-Ki

    2008-07-18

    Lumican is a major proteoglycans of the human cornea. Lumican knock-out mice have been shown to lose corneal transparency and to display delayed wound healing. The purpose of this study was to define the role of lumican in corneal epithelial cell migration. Over-expression of lumican in human corneal epithelial (HCE-T) cells increased both cell migration and proliferation, and increased levels of integrins {alpha}2 and {beta}1. ERK 1/2 was also activated in lumican over-expressed cells. When we treated HCE-T cells with the ERK-specific inhibitor U0126, cell migration and the expression of integrin {beta}1 were completely blocked. These data provide evidence that lumican stimulates cell migration in the corneal epithelium by activating ERK 1/2, and point to a novel signaling pathway implicated in corneal epithelial cell migration.

  4. Epithelial DLD-1 Cells with Disrupted E-cadherin Gene Retain the Ability to Form Cell Junctions and Apico-basal Polarity.

    PubMed

    Fujiwara, Miwako; Fujimura, Kihito; Obata, Shuichi; Yanagibashi, Ryo; Sakuma, Tetsushi; Yamamoto, Takashi; Suzuki, Shintaro T

    2015-01-01

    Gene editing methods were applied to the study of E-cadherin function in epithelial cells. The E-cadherin gene in epithelial DLD-1 cells was ablated using TALEN. The resultant cells showed round fibroblast-like morphology and had almost no Ca(2+)-dependent cell aggregation activity. E-cadherin re-expression in the knockout cells restored epithelial cell morphology and strong Ca(2+)-dependent cell-cell adhesion activity, indicating that the knockout cells retained the ability to support cadherin function. The knockout cells showed partial localization of desmoplakin and ZO-1 at intercellular contact sites. The transfectants expressing mutant E-cadherin lacking the cytoplasmic domain showed clear localization of desmoplakin and ZO-1 at cell-cell contact sites, although the cells had only weak Ca(2+)-dependent cell adhesion activity. Electron microscopy revealed the formation of intercellular junctions and apico-basal polarity in these cells. A portion of these cells occasionally formed an epithelial-like structure after prolonged culture. When the cells were treated with blebbistatin, the localization was enhanced. However, the localization was incomplete and contained defects. Double-knockout MDCK cells for the E-cadherin and cadherin-6 genes showed similar results, suggesting that the above properties were general. The present results showed that an epithelial-like structure could be formed without E-cadherin, but that the construction of mature epithelia requires E-cadherin. PMID:26289297

  5. 293 cells express both epithelial as well as mesenchymal cell adhesion molecules

    PubMed Central

    INADA, MASAKAZU; IZAWA, GENYA; KOBAYASHI, WAKAKO; OZAWA, MASAYUKI

    2016-01-01

    The 293 cell line, used extensively in various types of studies due to the ease with which these cells can be transfected, was thought to be derived by the transformation of primary cultures of human embryonic kidney cells with sheared adenovirus type 5 DNA. Although the 293 cells were assumed to originate from epithelial cells, the exact origin of these cells remains unknown. Previous attempts to characterize these cells combined immunostaining, immunoblot analysis and microarray analysis to demonstrate that 293 cells express neurofilament subunits, α-internexin, and several other proteins typically found in neurons. These findings raised the possibility that the 293 cell line may have originated from human neuronal lineage cells. Contrary to this suggestion, in this study, we found that the 293 cells expressed N-cadherin and vimentin, which are marker proteins expressed in mesenchymal cells. Furthermore, the 293 cells also expressed E-cadherin, cytokeratins 5/8 and desmoglein 2, which are epithelial cell markers. When the cells, primarily cultured from the kidneys of Clawn miniature swine and passaged 10–15 generations [termed porcine kidney epithelial (PKE) cells] were examined, they were found to be positive for the expression of both mesenchymal and epithelial markers. Thus, transformation by adenovirus was not necessary for the cells to express N-cadherin. Occludin and zonula occludens (ZO)-1, two components of tight junctions in epithelial and endothelial cells, were detected in the 293 and the PKE cells. Thus, the findings of the present study demonstrate that 293 cells retain several characteristics of epithelial cells. PMID:27121032

  6. Alveolar epithelial cells express mesenchymal proteins in patients with idiopathic pulmonary fibrosis.

    PubMed

    Marmai, Cecilia; Sutherland, Rachel E; Kim, Kevin K; Dolganov, Gregory M; Fang, Xiaohui; Kim, Sophia S; Jiang, Shuwei; Golden, Jeffery A; Hoopes, Charles W; Matthay, Michael A; Chapman, Harold A; Wolters, Paul J

    2011-07-01

    Prior work has shown that transforming growth factor-β (TGF-β) can mediate transition of alveolar type II cells into mesenchymal cells in mice. Evidence this occurs in humans is limited to immunohistochemical studies colocalizing epithelial and mesenchymal proteins in sections of fibrotic lungs. To acquire further evidence that epithelial-to-mesenchymal transition occurs in the lungs of patients with idiopathic pulmonary fibrosis (IPF), we studied alveolar type II cells isolated from fibrotic and normal human lung. Unlike normal type II cells, type II cells isolated from the lungs of patients with IPF express higher levels of mRNA for the mesenchymal proteins type I collagen, α-smooth muscle actin (α-SMA), and calponin. When cultured on Matrigel/collagen, human alveolar type II cells maintain a cellular morphology consistent with epithelial cells and expression of surfactant protein C (SPC) and E-cadherin. In contrast, when cultured on fibronectin, the human type II cells flatten, spread, lose expression of pro- SPC, and increase expression of vimentin, N-cadherin, and α-SMA; markers of mesenchymal cells. Addition of a TGF-β receptor kinase inhibitor (SB431542) to cells cultured on fibronectin inhibited vimentin expression and maintained pro-SPC expression, indicating persistence of an epithelial phenotype. These data suggest that alveolar type II cells can acquire features of mesenchymal cells in IPF lungs and that TGF-β can mediate this process. PMID:21498628

  7. Alveolar epithelial cells express mesenchymal proteins in patients with idiopathic pulmonary fibrosis

    PubMed Central

    Marmai, Cecilia; Sutherland, Rachel E.; Kim, Kevin K.; Dolganov, Gregory M.; Fang, Xiaohui; Kim, Sophia S.; Jiang, Shuwei; Golden, Jeffery A.; Hoopes, Charles W.; Matthay, Michael A.; Chapman, Harold A.

    2011-01-01

    Prior work has shown that transforming growth factor-β (TGF-β) can mediate transition of alveolar type II cells into mesenchymal cells in mice. Evidence this occurs in humans is limited to immunohistochemical studies colocalizing epithelial and mesenchymal proteins in sections of fibrotic lungs. To acquire further evidence that epithelial-to-mesenchymal transition occurs in the lungs of patients with idiopathic pulmonary fibrosis (IPF), we studied alveolar type II cells isolated from fibrotic and normal human lung. Unlike normal type II cells, type II cells isolated from the lungs of patients with IPF express higher levels of mRNA for the mesenchymal proteins type I collagen, α-smooth muscle actin (α-SMA), and calponin. When cultured on Matrigel/collagen, human alveolar type II cells maintain a cellular morphology consistent with epithelial cells and expression of surfactant protein C (SPC) and E-cadherin. In contrast, when cultured on fibronectin, the human type II cells flatten, spread, lose expression of pro- SPC, and increase expression of vimentin, N-cadherin, and α-SMA; markers of mesenchymal cells. Addition of a TGF-β receptor kinase inhibitor (SB431542) to cells cultured on fibronectin inhibited vimentin expression and maintained pro-SPC expression, indicating persistence of an epithelial phenotype. These data suggest that alveolar type II cells can acquire features of mesenchymal cells in IPF lungs and that TGF-β can mediate this process. PMID:21498628

  8. Stem cell and neurogenic gene-expression profiles link prostate basal cells to aggressive prostate cancer

    PubMed Central

    Zhang, Dingxiao; Park, Daechan; Zhong, Yi; Lu, Yue; Rycaj, Kiera; Gong, Shuai; Chen, Xin; Liu, Xin; Chao, Hsueh-Ping; Whitney, Pamela; Calhoun-Davis, Tammy; Takata, Yoko; Shen, Jianjun; Iyer, Vishwanath R.; Tang, Dean G.

    2016-01-01

    The prostate gland mainly contains basal and luminal cells constructed as a pseudostratified epithelium. Annotation of prostate epithelial transcriptomes provides a foundation for discoveries that can impact disease understanding and treatment. Here we describe a genome-wide transcriptome analysis of human benign prostatic basal and luminal epithelial populations using deep RNA sequencing. Through molecular and biological characterizations, we show that the differential gene-expression profiles account for their distinct functional properties. Strikingly, basal cells preferentially express gene categories associated with stem cells, neurogenesis and ribosomal RNA (rRNA) biogenesis. Consistent with this profile, basal cells functionally exhibit intrinsic stem-like and neurogenic properties with enhanced rRNA transcription activity. Of clinical relevance, the basal cell gene-expression profile is enriched in advanced, anaplastic, castration-resistant and metastatic prostate cancers. Therefore, we link the cell-type-specific gene signatures to aggressive subtypes of prostate cancer and identify gene signatures associated with adverse clinical features. PMID:26924072

  9. Isolation of a sesquiterpene synthase expressing in specialized epithelial cells surrounding the secretory cavities in rough lemon (Citrus jambhiri).

    PubMed

    Uji, Yuya; Ozawa, Rika; Shishido, Hodaka; Taniguchi, Shiduku; Takabayashi, Junji; Akimitsu, Kazuya; Gomi, Kenji

    2015-05-15

    Volatile terpenoids such as monoterpenes and sesquiterpenes play multiple roles in plant responses and are synthesized by terpene synthases (TPSs). We have previously isolated a partial TPS gene, RlemTPS4, that responds to microbial attack in rough lemon. In this study, we isolated a full length RlemTPS4 cDNA from rough lemon. RlemTPS4 localized in the cytosol. The recombinant RlemTPS4 protein was obtained using a prokaryotic expression system and GC-MS analysis of the terpenes produced by the RlemTPS4 enzymatic reaction determined that RlemTPS4 produces some sesquiterpenes such as δ-elemene. The RlemTPS4 gene was specifically expressed in specialized epithelial cells surrounding the oil secretory cavities in rough lemon leaf tissue. PMID:25899729

  10. PDX1 regulation of FABP1 and novel target genes in human intestinal epithelial Caco-2 cells

    PubMed Central

    Chen, Chin; Fang, Rixun; Chou, Lin-Chiang; Lowe, Anson W.; Sibley, Eric

    2012-01-01

    The transcription factor pancreatic and duodenal homeobox 1 (PDX1) plays an essential role in pancreatic development and in maintaining proper islet function via target gene regulation. Few intestinal PDX1 targets, however, have been described. We sought to define novel PDX1-regulated intestinal genes. Caco-2 human intestinal epithelial cells were engineered to overexpress PDX1 and gene expression profiles relative to control cells were assessed. Expression of 80 genes significantly increased while that of 49 genes significantly decreased more than 4-fold following PDX1 overexpression in differentiated Caco-2 cells. Analysis of the differentially regulated genes with known functional annotations revealed genes encoding transcription factors, growth factors, kinases, digestive glycosidases, nutrient transporters, nutrient binding proteins, and structural components. The gene for fatty acid binding protein 1, liver, FABP1, is repressed by PDX1 in Caco-2 cells. PDX1 overexpression in Caco-2 cells also results in repression of promoter activity driven by the 0.6 kb FABP1 promoter. PDX1 regulation of promoter activity is consistent with the decrease in FABP1 RNA abundance resulting from PDX1 overexpression and identifies FABP1 as a candidate PDX1 target. PDX1 repression of FABP1, LCT, and SI suggests a role for PDX1 in patterning anterior intestinal development. PMID:22640736

  11. Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms

    PubMed Central

    2009-01-01

    Background Aspergillus fumigatus, a saprophytic mould, is responsible for life-threatening, invasive pulmonary diseases in immunocompromised hosts. The role of the airway epithelium involves a complex interaction with the inhaled pathogen. Antimicrobial peptides with direct antifungal and chemotactic activities may boost antifungal immune response. Results The inducible expression of defensins by human bronchial epithelial 16HBE cells and A549 pneumocyte cells exposed to A. fumigatus was investigated. Using RT-PCR and real time PCR, we showed an activation of hBD2 and hBD9 defensin genes: the expression was higher in cells exposed to swollen conidia (SC), compared to resting conidia (RC) or hyphal fragments (HF). The kinetics of defensin expression was different for each one, evoking a putative distinct function for each investigated defensin. The decrease of defensin expression in the presence of heat-inactivated serum indicated a possible link between defensins and the proteins of the host complement system. The presence of defensin peptide hBD2 was revealed using immunofluorescence that showed a punctual cytoplasmic and perinuclear staining. Quantification of the cells stained with anti hBD2 antibody demonstrated that SC induced a greater number of cells that synthesized hBD2, compared to RC or HF. Labelling of the cells with anti-hBD-2 antibody showed a positive immunofluorescence signal around RC or SC in contrast to HF. This suggests co-localisation of hBD2 and digested conidia. The HBD2 level was highest in the supernatants of cells exposed to SC, as was determined by sandwich ELISA. Experiments using neutralising anti-interleukine-1β antibody reflect the autocrine mechanism of defensin expression induced by SC. Investigation of defensin expression at transcriptional and post-transcriptional levels demonstrated the requirement of transcription as well as new protein synthesis during A. fumigatus defensin induction. Finally, induced defensin expression in

  12. Hsa-miRNA-31 regulates epithelial cell barrier function by inhibiting TNFSF15 expression.

    PubMed

    Nan, X; Qin, S; Yuan, Z; Li, Y; Zhang, J; Li, C; Tan, X; Yan, Y

    2016-01-01

    Ulcerative colitis (UC) is characterized by epithelial barrier disruption and alterations in immune regulation but with the etiology unknown. MicroRNA-31 is the most consistent differentially expressed miRNA in ulcerative colitis tissue. The aim of this project is to study the important roles of miRNA-31 in regulation of intestinal epithelial barrier function. We found that expression of miRNA-31 is proportional to the proliferation of Caco2-BBE cells and overexpression of miRNA-31 can increase its trans-epithelial resistance (TER) by decreasing the transepithelial permeability. miRNA-31 can directly bind to the 3-UTR of TNFSF15, thereafter negatively regulating its expression in Caco2-BBE cells. BrdU and TUNEL analysis demonstrated that transfection of miRNA-31 stimulates proliferation or apoptosis-resistance. Taken together, these results revealed a novel mecha-nism underlying the regulation of epithelial barrier function by miRNA-31 during its regulation on proliferation of epithelial cells. PMID:27188743

  13. Endothelial and Epithelial Cell Transition to a Mesenchymal Phenotype Was Delineated by Nestin Expression.

    PubMed

    Chabot, Andréanne; Hertig, Vanessa; Boscher, Elena; Nguyen, Quang Trinh; Boivin, Benoît; Chebli, Jasmine; Bissonnette, Lyse; Villeneuve, Louis; Brochiero, Emmanuelle; Dupuis, Jocelyn; Calderone, Angelino

    2016-07-01

    Endothelial and epithelial cell transition to a mesenchymal phenotype was identified as cellular paradigms implicated in the appearance of fibroblasts and development of reactive fibrosis in interstitial lung disease. The intermediate filament protein nestin was highly expressed in fibrotic tissue, detected in fibroblasts and participated in proliferation and migration. The present study tested the hypothesis that the transition of endothelial and epithelial cells to a mesenchymal phenotype was delineated by nestin expression. Three weeks following hypobaric hypoxia, adult male Sprague-Dawley rats characterized by alveolar and perivascular lung fibrosis were associated with increased nestin protein and mRNA levels and marked appearance of nestin/collagen type I((+))-fibroblasts. In the perivascular region of hypobaric hypoxic rats, displaced CD31((+))-endothelial cells were detected, exhibited a mesenchymal phenotype and co-expressed nestin. Likewise, epithelial cells in the lungs of hypobaric hypoxic rats transitioned to a mesenchymal phenotype distinguished by the co-expression of E-cadherin and collagen. Following the removal of FBS from primary passage rat alveolar epithelial cells, TGF-β1 was detected in the media and a subpopulation acquired a mesenchymal phenotype characterized by E-cadherin downregulation and concomitant induction of collagen and nestin. Bone morphogenic protein-7 treatment of alveolar epithelial cells prevented E-cadherin downregulation, suppressed collagen induction but partially inhibited nestin expression. These data support the premise that the transition of endothelial and epithelial cells to a mesenchymal cell may have contributed in part to the appearance nestin/collagen type I((+))-fibroblasts and the reactive fibrotic response in the lungs of hypobaric hypoxic rats. PMID:26574905

  14. Cryptosporidium parvum induces SIRT1 expression in host epithelial cells through downregulating let-7i

    PubMed Central

    Xie, Hongguan; Lei, Ningfei; Gong, Ai-Yu; Chen, Xian-Ming; Hu, Guoku

    2015-01-01

    Epithelial cells along human gastrointestinal mucosal surface express pathogen-recognizing receptors and actively participate in the regulation of inflammatory reactions in response to microbial infection. The NAD-dependent deacetylase sirtuin-1 (SIRT1), one member of the sirtuin family of proteins and an NAD-dependent deacetylase has been implicated in the regulation of multiple cellular processes, including inflammation, longevity, and metabolism. In this study, we demonstrated that infection of cultured human biliary epithelial cells (H69 cholangiocytes) with a parasitic protozoan, Cryptosporidium parvum, induced SIRT1 expression at the protein level without a change in SIRT1 mRNA content. Using real-time PCR and Northern blot analyses, we found that C. parvum infection decreased the expression of let-7i in infected H69 cells. Down-regulation of let-7i caused relief of miRNA-mediated translational suppression of SIRT1 and consequently, resulted in an increased SIRT1 protein level in infected H69 cell cultures. Moreover, gain- and loss-of-function studies revealed that let-7i could modulate NF-κB activation through modification of SIRT1 protein expression. Thus, our data suggest that let-7i regulates SIRT1 expression in human biliary epithelial cells in response to microbial challenge, suggesting a new role of let-7i in the regulation of NF-κB-mediated epithelial innate immune response. PMID:24862934

  15. Loss of neurogenesis in Hydra leads to compensatory regulation of neurogenic and neurotransmission genes in epithelial cells.

    PubMed

    Wenger, Y; Buzgariu, W; Galliot, B

    2016-01-01

    Hydra continuously differentiates a sophisticated nervous system made of mechanosensory cells (nematocytes) and sensory-motor and ganglionic neurons from interstitial stem cells. However, this dynamic adult neurogenesis is dispensable for morphogenesis. Indeed animals depleted of their interstitial stem cells and interstitial progenitors lose their active behaviours but maintain their developmental fitness, and regenerate and bud when force-fed. To characterize the impact of the loss of neurogenesis in Hydra, we first performed transcriptomic profiling at five positions along the body axis. We found neurogenic genes predominantly expressed along the central body column, which contains stem cells and progenitors, and neurotransmission genes predominantly expressed at the extremities, where the nervous system is dense. Next, we performed transcriptomics on animals depleted of their interstitial cells by hydroxyurea, colchicine or heat-shock treatment. By crossing these results with cell-type-specific transcriptomics, we identified epithelial genes up-regulated upon loss of neurogenesis: transcription factors (Dlx, Dlx1, DMBX1/Manacle, Ets1, Gli3, KLF11, LMX1A, ZNF436, Shox1), epitheliopeptides (Arminins, PW peptide), neurosignalling components (CAMK1D, DDCl2, Inx1), ligand-ion channel receptors (CHRNA1, NaC7), G-Protein Coupled Receptors and FMRFRL. Hence epitheliomuscular cells seemingly enhance their sensing ability when neurogenesis is compromised. This unsuspected plasticity might reflect the extended multifunctionality of epithelial-like cells in early eumetazoan evolution. PMID:26598723

  16. Loss of neurogenesis in Hydra leads to compensatory regulation of neurogenic and neurotransmission genes in epithelial cells

    PubMed Central

    2016-01-01

    Hydra continuously differentiates a sophisticated nervous system made of mechanosensory cells (nematocytes) and sensory–motor and ganglionic neurons from interstitial stem cells. However, this dynamic adult neurogenesis is dispensable for morphogenesis. Indeed animals depleted of their interstitial stem cells and interstitial progenitors lose their active behaviours but maintain their developmental fitness, and regenerate and bud when force-fed. To characterize the impact of the loss of neurogenesis in Hydra, we first performed transcriptomic profiling at five positions along the body axis. We found neurogenic genes predominantly expressed along the central body column, which contains stem cells and progenitors, and neurotransmission genes predominantly expressed at the extremities, where the nervous system is dense. Next, we performed transcriptomics on animals depleted of their interstitial cells by hydroxyurea, colchicine or heat-shock treatment. By crossing these results with cell-type-specific transcriptomics, we identified epithelial genes up-regulated upon loss of neurogenesis: transcription factors (Dlx, Dlx1, DMBX1/Manacle, Ets1, Gli3, KLF11, LMX1A, ZNF436, Shox1), epitheliopeptides (Arminins, PW peptide), neurosignalling components (CAMK1D, DDCl2, Inx1), ligand-ion channel receptors (CHRNA1, NaC7), G-Protein Coupled Receptors and FMRFRL. Hence epitheliomuscular cells seemingly enhance their sensing ability when neurogenesis is compromised. This unsuspected plasticity might reflect the extended multifunctionality of epithelial-like cells in early eumetazoan evolution. PMID:26598723

  17. Effect of SIRT1 Gene on Epithelial-Mesenchymal Transition of Human Prostate Cancer PC-3 Cells

    PubMed Central

    Cui, Ying; Li, Jiang; Zheng, Fei; Ouyang, Yongri; Chen, Xi; Zhang, Lei; Chen, Yang; Wang, Lin; Mu, Shijie; Zhang, Huizhong

    2016-01-01

    Background The epithelial-mesenchymal transition (EMT) has been shown to be involved in the process of invasion and metastasis of prostate cancer. SIRT1 is the mammalian homologue of the silent information regulator 2 (Sir2) gene, and is abnormally expressed in prostate cancer cells. Therefore, it is hypothesized that SIRT1 mediates the invasion/metastatic ability of prostate cancer via EMT regulation. This study thus investigated the effect of SIRT1 gene on the invasion and migration of prostate cancer cell line PC-3 via the small interference RNA (siRNA) against SIRT1. Material/Methods SiRNA construct was transfected into PC-3 cells, which were tested for the cell migration and invasion ability by scratch assay and Transwell migration assay, respectively. Expression levels of vimentin, E-cadherin, and N-cadherin were further quantified by Western blotting and RT-PCR. Results Both mRNA and protein levels of SIRT1 were depressed after siRNA transfection, along with weakened migration and invasion ability of PC-3 cells. Elevated E-cadherin and suppressed N-cadherin and vimentin were observed in those transfected cells. Conclusions The silencing of SIRT1 gene in PC-3 cells can suppress the movement, migration, and invasion functions of prostate cancer cells, possibly via the down-regulation of mesenchymal markers vimentin and N-cadherin accompanied with up-regulation of epithelial marker N-cadherin, thus reversing the EMT process. PMID:26847404

  18. [Differential gene expression in the jellyfish Aurelia aurita].

    PubMed

    Matveev, I V

    2005-01-01

    The body of Aurelia aurita, as well as other diploblasts, consists of two epithelial layers: ectodermal and gastral epithelium. These two tissues are separated by mesoglea, or extracellular matrix. In most coelenterates mesoglea is acellular. In A. aurita mesogleal cells are scattered in mesoglea. Differential display PCR was used to compare mRNA pools from ectodermal epithelium, gastral epithelium and mesoglea. 4 novel gene fragments were cloned and sequenced. According to RTPCR results, one of these fragments is differentially expressed in the ectodermal epithelium. PMID:16706147

  19. Gene Expression: Sizing it all up

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genomic architecture appears to be a largely unexplored component of gene expression. Although surely not the end of the story, we are learning that when it comes to gene expression, size is important. We have been surprised to find that certain patterns of expression, tissue-specific versus constit...

  20. Quantitative single-cell gene expression measurements of multiple genes in response to hypoxia treatment.

    PubMed

    Zeng, Jia; Wang, Jiangxin; Gao, Weimin; Mohammadreza, Aida; Kelbauskas, Laimonas; Zhang, Weiwen; Johnson, Roger H; Meldrum, Deirdre R

    2011-07-01

    Cell-to-cell heterogeneity in gene transcription plays a central role in a variety of vital cell processes. To quantify gene expression heterogeneity patterns among cells and to determine their biological significance, methods to measure gene expression levels at the single-cell level are highly needed. We report an experimental technique based on the DNA-intercalating fluorescent dye SYBR green for quantitative expression level analysis of up to ten selected genes in single mammalian cells. The method features a two-step procedure consisting of a step to isolate RNA from a single mammalian cell, synthesize cDNA from it, and a qPCR step. We applied the method to cell populations exposed to hypoxia, quantifying expression levels of seven different genes spanning a wide dynamic range of expression in randomly picked single cells. In the experiment, 72 single Barrett's esophageal epithelial (CP-A) cells, 36 grown under normal physiological conditions (controls) and 36 exposed to hypoxia for 30 min, were randomly collected and used for measuring the expression levels of 28S rRNA, PRKAA1, GAPDH, Angptl4, MT3, PTGES, and VEGFA genes. The results demonstrate that the method is sensitive enough to measure alterations in gene expression at the single-cell level, clearly showing heterogeneity within a cell population. We present technical details of the method development and implementation, and experimental results obtained by use of the procedure. We expect the advantages of this technique will facilitate further developments and advances in the field of single-cell gene expression profiling on a nanotechnological scale, and eventually as a tool for future point-of-care medical applications. PMID:21614642

  1. Common Genetic Variation In Cellular Transport Genes and Epithelial Ovarian Cancer (EOC) Risk

    PubMed Central

    Chornokur, Ganna; Lin, Hui-Yi; Tyrer, Jonathan P.; Lawrenson, Kate; Dennis, Joe; Amankwah, Ernest K.; Qu, Xiaotao; Tsai, Ya-Yu; Jim, Heather S. L.; Chen, Zhihua; Chen, Ann Y.; Permuth-Wey, Jennifer; Aben, Katja KH.; Anton-Culver, Hoda; Antonenkova, Natalia; Bruinsma, Fiona; Bandera, Elisa V.; Bean, Yukie T.; Beckmann, Matthias W.; Bisogna, Maria; Bjorge, Line; Bogdanova, Natalia; Brinton, Louise A.; Brooks-Wilson, Angela; Bunker, Clareann H.; Butzow, Ralf; Campbell, Ian G.; Carty, Karen; Chang-Claude, Jenny; Cook, Linda S.; Cramer, Daniel W.; Cunningham, Julie M.; Cybulski, Cezary; Dansonka-Mieszkowska, Agnieszka; du Bois, Andreas; Despierre, Evelyn; Dicks, Ed; Doherty, Jennifer A.; Dörk, Thilo; Dürst, Matthias; Easton, Douglas F.; Eccles, Diana M.; Edwards, Robert P.; Ekici, Arif B.; Fasching, Peter A.; Fridley, Brooke L.; Gao, Yu-Tang; Gentry-Maharaj, Aleksandra; Giles, Graham G.; Glasspool, Rosalind; Goodman, Marc T.; Gronwald, Jacek; Harrington, Patricia; Harter, Philipp; Hein, Alexander; Heitz, Florian; Hildebrandt, Michelle A. T.; Hillemanns, Peter; Hogdall, Claus K.; Hogdall, Estrid; Hosono, Satoyo; Jakubowska, Anna; Jensen, Allan; Ji, Bu-Tian; Karlan, Beth Y.; Kelemen, Linda E.; Kellar, Mellissa; Kiemeney, Lambertus A.; Krakstad, Camilla; Kjaer, Susanne K.; Kupryjanczyk, Jolanta; Lambrechts, Diether; Lambrechts, Sandrina; Le, Nhu D.; Lee, Alice W.; Lele, Shashi; Leminen, Arto; Lester, Jenny; Levine, Douglas A.; Liang, Dong; Lim, Boon Kiong; Lissowska, Jolanta; Lu, Karen; Lubinski, Jan; Lundvall, Lene; Massuger, Leon F. A. G.; Matsuo, Keitaro; McGuire, Valerie; McLaughlin, John R.; McNeish, Iain; Menon, Usha; Milne, Roger L.; Modugno, Francesmary; Moysich, Kirsten B.; Ness, Roberta B.; Nevanlinna, Heli; Eilber, Ursula; Odunsi, Kunle; Olson, Sara H.; Orlow, Irene; Orsulic, Sandra; Weber, Rachel Palmieri; Paul, James; Pearce, Celeste L.; Pejovic, Tanja; Pelttari, Liisa M.; Pike, Malcolm C.; Poole, Elizabeth M.; Risch, Harvey A.; Rosen, Barry; Rossing, Mary Anne; Rothstein, Joseph H.; Rudolph, Anja; Runnebaum, Ingo B.; Rzepecka, Iwona K.; Salvesen, Helga B.; Schernhammer, Eva; Schwaab, Ira; Shu, Xiao-Ou; Shvetsov, Yurii B.; Siddiqui, Nadeem; Sieh, Weiva; Song, Honglin; Southey, Melissa C.; Spiewankiewicz, Beata; Sucheston, Lara; Teo, Soo-Hwang; Terry, Kathryn L.; Thompson, Pamela J.; Thomsen, Lotte; Tangen, Ingvild L.; Tworoger, Shelley S.; van Altena, Anne M.; Vierkant, Robert A.; Vergote, Ignace; Walsh, Christine S.; Wang-Gohrke, Shan; Wentzensen, Nicolas; Whittemore, Alice S.; Wicklund, Kristine G.; Wilkens, Lynne R.; Wu, Anna H.; Wu, Xifeng; Woo, Yin-Ling; Yang, Hannah; Zheng, Wei; Ziogas, Argyrios; Hasmad, Hanis N.; Berchuck, Andrew; Iversen, Edwin S.; Schildkraut, Joellen M.; Ramus, Susan J.; Goode, Ellen L.; Monteiro, Alvaro N. A.; Gayther, Simon A.; Narod, Steven A.; Pharoah, Paul D. P.; Sellers, Thomas A.; Phelan, Catherine M.

    2015-01-01

    Background Defective cellular transport processes can lead to aberrant accumulation of trace elements, iron, small molecules and hormones in the cell, which in turn may promote the formation of reactive oxygen species, promoting DNA damage and aberrant expression of key regulatory cancer genes. As DNA damage and uncontrolled proliferation are hallmarks of cancer, including epithelial ovarian cancer (EOC), we hypothesized that inherited variation in the cellular transport genes contributes to EOC risk. Methods In total, DNA samples were obtained from 14,525 case subjects with invasive EOC and from 23,447 controls from 43 sites in the Ovarian Cancer Association Consortium (OCAC). Two hundred seventy nine SNPs, representing 131 genes, were genotyped using an Illumina Infinium iSelect BeadChip as part of the Collaborative Oncological Gene-environment Study (COGS). SNP analyses were conducted using unconditional logistic regression under a log-additive model, and the FDR q<0.2 was applied to adjust for multiple comparisons. Results The most significant evidence of an association for all invasive cancers combined and for the serous subtype was observed for SNP rs17216603 in the iron transporter gene HEPH (invasive: OR = 0.85, P = 0.00026; serous: OR = 0.81, P = 0.00020); this SNP was also associated with the borderline/low malignant potential (LMP) tumors (P = 0.021). Other genes significantly associated with EOC histological subtypes (p<0.05) included the UGT1A (endometrioid), SLC25A45 (mucinous), SLC39A11 (low malignant potential), and SERPINA7 (clear cell carcinoma). In addition, 1785 SNPs in six genes (HEPH, MGST1, SERPINA, SLC25A45, SLC39A11 and UGT1A) were imputed from the 1000 Genomes Project and examined for association with INV EOC in white-European subjects. The most significant imputed SNP was rs117729793 in SLC39A11 (per allele, OR = 2.55, 95% CI = 1.5-4.35, p = 5.66x10-4). Conclusion These results, generated on a large cohort of women, revealed associations

  2. BRCA1 185delAG Mutation Enhances Interleukin-1β Expression in Ovarian Surface Epithelial Cells

    PubMed Central

    Woolery, Kamisha T.; Mohamed, Mai; Linger, Rebecca J.; Dobrinski, Kimberly P.; Roman, Jesse; Kruk, Patricia A.

    2015-01-01

    Familial history remains the strongest risk factor for developing ovarian cancer (OC) and is associated with germline BRCA1 mutations, such as the 185delAG founder mutation. We sought to determine whether normal human ovarian surface epithelial (OSE) cells expressing the BRCA1 185delAG mutant, BRAT, could promote an inflammatory phenotype by investigating its impact on expression of the proinflammatory cytokine, Interleukin-1β (IL-1β). Cultured OSE cells with and without BRAT were analyzed for differential target gene expression by real-time PCR, western blot, ELISA, luciferase reporter, and siRNA assays. We found that BRAT cells expressed increased cellular and secreted levels of active IL-1β. BRAT-expressing OSE cells exhibited 3-fold enhanced IL-1β mRNA expression, transcriptionally regulated, in part, through CREB sites within the (−1800) to (−900) region of its promoter. In addition to transcriptional regulation, BRAT-mediated IL-1β expression appears dualistic through enhanced inflammasome-mediated caspase-1 cleavage and activation of IL-1β. Further investigation is warranted to elucidate the molecular mechanism(s) of BRAT-mediated IL-1β expression since increased IL-1β expression may represent an early step contributing to OC. PMID:26357657

  3. BRCA1 185delAG Mutation Enhances Interleukin-1β Expression in Ovarian Surface Epithelial Cells.

    PubMed

    Woolery, Kamisha T; Mohamed, Mai; Linger, Rebecca J; Dobrinski, Kimberly P; Roman, Jesse; Kruk, Patricia A

    2015-01-01

    Familial history remains the strongest risk factor for developing ovarian cancer (OC) and is associated with germline BRCA1 mutations, such as the 185delAG founder mutation. We sought to determine whether normal human ovarian surface epithelial (OSE) cells expressing the BRCA1 185delAG mutant, BRAT, could promote an inflammatory phenotype by investigating its impact on expression of the proinflammatory cytokine, Interleukin-1β (IL-1β). Cultured OSE cells with and without BRAT were analyzed for differential target gene expression by real-time PCR, western blot, ELISA, luciferase reporter, and siRNA assays. We found that BRAT cells expressed increased cellular and secreted levels of active IL-1β. BRAT-expressing OSE cells exhibited 3-fold enhanced IL-1β mRNA expression, transcriptionally regulated, in part, through CREB sites within the (-1800) to (-900) region of its promoter. In addition to transcriptional regulation, BRAT-mediated IL-1β expression appears dualistic through enhanced inflammasome-mediated caspase-1 cleavage and activation of IL-1β. Further investigation is warranted to elucidate the molecular mechanism(s) of BRAT-mediated IL-1β expression since increased IL-1β expression may represent an early step contributing to OC. PMID:26357657

  4. Gene Transfer between Salmonella enterica Serovar Typhimurium inside Epithelial Cells

    PubMed Central

    Ferguson, Gayle C.; Heinemann, Jack A.; Kennedy, Martin A.

    2002-01-01

    Virulence and antibiotic resistance genes transfer between bacteria by bacterial conjugation. Conjugation also mediates gene transfer from bacteria to eukaryotic organisms, including yeast and human cells. Predicting when and where genes transfer by conjugation could enhance our understanding of the risks involved in the release of genetically modified organisms, including those being developed for use as vaccines. We report here that Salmonella enterica serovar Typhimurium conjugated inside cultured human cells. The DNA transfer from donor to recipient bacteria was proportional to the probability that the two types of bacteria occupied the same cell, which was dependent on viable and invasive bacteria and on plasmid tra genes. Based on the high frequencies of gene transfer between bacteria inside human cells, we suggest that such gene transfers occur in situ. The implications of gene transfer between bacteria inside human cells, particularly in the context of antibiotic resistance, are discussed. PMID:11914355

  5. Control of RANKL Gene Expression

    PubMed Central

    O'Brien, Charles A.

    2009-01-01

    Osteoclasts are highly specialized cells capable of degrading mineralized tissue and form at different regions of bone to meet different physiological needs, such as mobilization of calcium, modeling of bone structure, and remodeling of bone matrix. Osteoclast production is elevated in a number of pathological conditions, many of which lead to loss of bone mass. Whether normal or pathological, osteoclastogenesis strictly depends upon support from accessory cells which supply cytokines required for osteoclast differentiation. Only one of these cytokines, receptor activator of NFκB ligand (RANKL), is absolutely essential for osteoclast formation throughout life and is thus expressed by all cell types that support osteoclast differentiation. The central role of RANKL in bone resorption is highlighted by the fact that it is the basis for a new therapy to inhibit bone loss. This review will discuss mechanisms that control RANKL gene expression in different osteoclast-support cells and how the study of such mechanisms may lead to a better understanding of the cellular interactions that drive normal and pathological bone resorption. PMID:19716455

  6. Tissue- and age-dependent expression of the bovine DEFB103 gene and protein.

    PubMed

    Mirabzadeh-Ardakani, Ali; Solie, Jay; Gonzalez-Cano, Patricia; Schmutz, Sheila M; Griebel, Philip J

    2016-02-01

    Beta-defensin 103 (DEFB103) shares little homology with 8 other members of the bovine beta-defensin family and in other species DEFB103 protein has diverse functions, including antimicrobial activity, a chemoattractant for dendritic cells, enhancing epithelial wound repair and regulating hair colour. Expression of the bovine DEFB103 gene was surveyed in 27 tissues and transcript was most abundant in tissues with stratified squamous epithelium. Oral cavity epithelial tissues and nictitating membrane consistently expressed high levels of DEFB103 gene transcript. An age-dependent decrease (P < 0.05) in DEFB103 gene expression was only observed for buccal epithelium when comparing healthy 10- to 14-day-old and 10- to 12-month-old calves. A bovine herpesvirus-1 respiratory infection did, however, significantly (P < 0.05) up-regulate DEFB103 gene expression in the buccal epithelium of 6- to 8-month-old calves. Finally, DEFB103 transcript was low in lymph nodes draining the skin and at the limit of detection in other internal organs such as lung, intestine and kidney. Affinity-purified rabbit antisera to bovine DEFB103 was used to identify cells expressing DEFB103 protein within tissues with stratified squamous epitheliums. DEFB103 protein was most abundant in basal epithelial cells and was present in these cells prior to birth. Beta-defensins have been identified as regulators of dendritic cell (DC) chemokine responses and we observed a close association between DCs and epithelial cells expressing DEFB103 in both the fetus and newborn calf. In conclusion, bovine DEFB103 gene expression is most abundant in stratified squamous epithelium with DEFB103 protein localised to basal epithelial cells. These observations are consistent with proposed roles for DEFB103 in DC recruitment and repair of stratified squamous epithelium. PMID:26299200

  7. Expression of Beta-Defensin 131 Promotes an Innate Immune Response in Human Prostate Epithelial Cells

    PubMed Central

    Kim, Hae Jong; Lee, Jaehyouk; Myung, Soon Chul

    2015-01-01

    Previously, using the Illumina HumanHT-12 microarray we found that β-defensin 131 (DEFB131), an antimicrobial peptide, is upregulated in the human prostate epithelial cell line RWPE-1 upon stimulation with lipoteichoic acid (LTA; a gram-positive bacterial component), than that in the untreated RWPE-1 cells. In the current study, we aimed to investigate the role of DEFB131 in RWPE-1 cells during bacterial infection. We examined the intracellular signaling pathways and nuclear responses in RWPE-1 cells that contribute to DEFB131 gene induction upon stimulation with LTA. Chromatin immunoprecipitation was performed to determine whether NF-κB directly binds to the DEFB131 promoter after LTA stimulation in RWPE-1 cells. We found that DEFB131 expression was induced by LTA stimulation through TLR2 and p38MAPK/NF-κB activation, which was evident in the phosphorylation of both p38MAPK and IκBα. We also found that SB203580 and Bay11-7082, inhibitors of p38MAPK and NF-κB, respectively, suppressed LTA-induced DEFB131 expression. The chromatin immunoprecipitation assay showed that NF-κB directly binds to the DEFB131 promoter, suggesting that NF-κB is a direct regulator, and is necessary for LTA-induced DEFB131 expression in RWPE-1 cells. Interestingly, with DEFB131 overexpression in RWPE-1 cells, the accumulation of mRNA and protein secretion of cytokines (IL-1α, IL-1β, IL-6, and IL-12α) and chemokines (CCL20, CCL22, and CXCL8) were significantly enhanced. In addition, DEFB131-transfected RWPE-1 cells markedly induced chemotactic activity in THP-1 monocytes. We concluded that DEFB131 induces cytokine and chemokine upregulation through the TLR2/NF-κB signaling pathway in RWPE-1 cells during bacterial infection and promotes an innate immune response. PMID:26649771

  8. Monocarboxylate transporters 1 and 4: expression and regulation by PPARα in ovine ruminal epithelial cells.

    PubMed

    Benesch, Franziska; Dengler, Franziska; Masur, Franziska; Pfannkuche, Helga; Gäbel, Gotthold

    2014-12-15

    In the intact rumen epithelium, isoforms 1 and 4 of the monocarboxylate transporter (MCT1 and MCT4) are thought to play key roles in mediating transcellular and intracellular permeation of short-chain fatty acids and their metabolites and in maintaining intracellular pH. We examined whether both MCT1 and MCT4 are expressed at mRNA and protein levels in ovine ruminal epithelial cells (REC) maintained in primary culture and whether they are regulated by peroxisome proliferator-activated receptor-α (PPARα). Because both transporters have been characterized to function coupled to protons, the influence of PPARα on the recovery of intracellular pH after l-lactate exposure was evaluated by spectrofluorometry. MCT1 and MCT4 were detected using immunocytochemistry both at the cell margins and intracellularly in cultured REC. To test regulation by PPARα, cells were exposed to WY 14.643, a selective ligand of PPARα, for 48 h. The subsequent qPCR analysis resulted in a dose-dependent upregulation of MCT1 and PPARα target genes, whereas response of MCT4 was not uniform. Protein expression of MCT1 and MCT4 quantified by Western blot analysis was not altered by WY 14.643 treatment. l-Lactate-dependent proton export was blocked almost completely by pHMB, a specific inhibitor of MCT1 and MCT4. However, l-lactate-dependent, pHMB-inhibited proton export in WY 14.643-treated cells was not significantly altered compared with cells not treated with WY 14.643. These data suggest that PPARα is particularly regulating MCT1 but not MCT4 expression. Extent of lactate-coupled proton export indicates that MCT1 is already working on a high level even under unstimulated conditions. PMID:25320343

  9. Methods for monitoring multiple gene expression

    DOEpatents

    Berka, Randy; Bachkirova, Elena; Rey, Michael

    2008-06-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  10. Methods for monitoring multiple gene expression

    DOEpatents

    Berka, Randy; Bachkirova, Elena; Rey, Michael

    2012-05-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  11. Methods for monitoring multiple gene expression

    DOEpatents

    Berka, Randy; Bachkirova, Elena; Rey, Michael

    2013-10-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  12. Gene 33/Mig6 inhibits hexavalent chromium-induced DNA damage and cell transformation in human lung epithelial cells

    PubMed Central

    Park, Soyoung; Li, Cen; Zhao, Hong; Darzynkiewicz, Zbigniew; Xu, Dazhong

    2016-01-01

    Hexavalent Chromium [Cr(VI)] compounds are human lung carcinogens and environmental/occupational hazards. The molecular mechanisms of Cr(VI) carcinogenesis appear to be complex and are poorly defined. In this study, we investigated the potential role of Gene 33 (ERRFI1, Mig6), a multifunctional adaptor protein, in Cr(VI)-mediated lung carcinogenesis. We show that the level of Gene 33 protein is suppressed by both acute and chronic Cr(VI) treatments in a dose- and time-dependent fashion in BEAS-2B lung epithelial cells. The inhibition also occurs in A549 lung bronchial carcinoma cells. Cr(VI) suppresses Gene 33 expression mainly through post-transcriptional mechanisms, although the mRNA level of gene 33 also tends to be lower upon Cr(VI) treatments. Cr(VI)-induced DNA damage appears primarily in the S phases of the cell cycle despite the high basal DNA damage signals at the G2M phase. Knockdown of Gene 33 with siRNA significantly elevates Cr(VI)-induced DNA damage in both BEAS-2B and A549 cells. Depletion of Gene 33 also promotes Cr(VI)-induced micronucleus (MN) formation and cell transformation in BEAS-2B cells. Our results reveal a novel function of Gene 33 in Cr(VI)-induced DNA damage and lung epithelial cell transformation. We propose that in addition to its role in the canonical EGFR signaling pathway and other signaling pathways, Gene 33 may also inhibit Cr(VI)-induced lung carcinogenesis by reducing DNA damage triggered by Cr(VI). PMID:26760771

  13. The RUNX1 transcription factor is expressed in serous epithelial ovarian carcinoma and contributes to cell proliferation, migration and invasion

    PubMed Central

    Keita, Mamadou; Bachvarova, Magdalena; Morin, Chantale; Plante, Marie; Gregoire, Jean; Renaud, Marie-Claude; Sebastianelli, Alexandra; Trinh, Xuan Bich; Bachvarov, Dimcho

    2013-01-01

    Previously, we have identified the RUNX1 gene as hypomethylated and overexpressed in post-chemotherapy (CT) primary cultures derived from epithelial ovarian cancer (EOC) patients, when compared with primary cultures derived from matched primary (prior to CT) tumors. Here we show that RUNX1 displays a trend of hypomethylation, although not significant, in omental metastases compared with primary EOC tumors. Surprisingly, RUNX1 displayed significantly higher expression not only in metastatic tissue, but also in high-grade primary tumors and even in low malignant potential tumors. The RUNX1 expression levels were almost identical in primary tumors and omental metastases, suggesting that RUNX1 hypomethylation might have a limited impact on its overexpression in advanced (metastatic) stage of the disease. Knockdown of the RUNX1 expression in EOC cells led to sharp decrease of cell proliferation and induced G1 cell cycle arrest. Moreover, RUNX1 suppression significantly inhibited EOC cell migration and invasion. Gene expression profiling and consecutive network and pathway analyses confirmed these findings, as numerous genes and pathways known previously to be implicated in ovarian tumorigenesis, including EOC tumor invasion and metastasis, were found to be downregulated upon RUNX1 suppression, while a number of pro-apoptotic genes and some EOC tumor suppressor genes were induced. Taken together, our data are indicative for a strong oncogenic potential of the RUNX1 gene in EOC progression and suggest that RUNX1 might be a novel EOC therapeutic target. Further studies are needed to more completely elucidate the functional implications of RUNX1 and other members of the RUNX gene family in ovarian tumorigenesis. PMID:23442798

  14. Factors Influencing Adeno-Associated Virus-Mediated Gene Transfer to Human Cystic Fibrosis Airway Epithelial Cells: Comparison with Adenovirus Vectors

    PubMed Central

    Teramoto, S.; Bartlett, J. S.; McCarty, D.; Xiao, X.; Samulski, R. J.; Boucher, R. C.

    1998-01-01

    Adeno-associated virus (AAV) vectors appear promising for use in gene therapy in cystic fibrosis (CF) patients, yet many features of AAV-mediated gene transfer to airway epithelial cells are not well understood. We compared the transduction efficiencies of AAV vectors and adenovirus (Ad) vectors in immortalized cell lines from CF patients and in nasal epithelial primary cultures from normal humans and CF patients. Similar dose-dependent relationships between the vector multiplicities of infection and the efficiencies of lacZ gene transfer were observed. However, levels of transduction for both Ad and recombinant AAV (rAAV) were significantly lower in the airway epithelial cell than in the control cell lines HeLa and HEK 293. Transduction efficiencies differed among cultured epithelial cell types, with poorly differentiated cells transducing more efficiently than well-differentiated cells. A time-dependent increase in gene expression was observed after infection for both vectors. For Ad, but not for AAV, this increase was dependent on prolonged incubation of cells with the vector. Furthermore, for rAAV (but not for rAd), the delay in maximal transduction could be abrogated by wild-type Ad helper infection. Thus, although helper virus is not required for maximal transduction, it increases the kinetics by which this is achieved. Expression of Ad E4 open reading frame 6 or addition of either hydroxyurea or camptothecin resulted in increased AAV transduction, as previously demonstrated for nonairway cells (albeit to lower final levels), suggesting that second-strand synthesis may not be the sole cause of inefficient transduction. Finally, the efficiency of AAV-mediated ex vivo gene transfer to lung cells was similar to that previously described for Ad vectors in that transduction was limited to regions of epithelial injury and preferentially targeted basal-like cells. These studies address the primary factors influencing rAAV infection of human airway cells and should

  15. Factors influencing adeno-associated virus-mediated gene transfer to human cystic fibrosis airway epithelial cells: comparison with adenovirus vectors.

    PubMed

    Teramoto, S; Bartlett, J S; McCarty, D; Xiao, X; Samulski, R J; Boucher, R C

    1998-11-01

    Adeno-associated virus (AAV) vectors appear promising for use in gene therapy in cystic fibrosis (CF) patients, yet many features of AAV-mediated gene transfer to airway epithelial cells are not well understood. We compared the transduction efficiencies of AAV vectors and adenovirus (Ad) vectors in immortalized cell lines from CF patients and in nasal epithelial primary cultures from normal humans and CF patients. Similar dose-dependent relationships between the vector multiplicities of infection and the efficiencies of lacZ gene transfer were observed. However, levels of transduction for both Ad and recombinant AAV (rAAV) were significantly lower in the airway epithelial cell than in the control cell lines HeLa and HEK 293. Transduction efficiencies differed among cultured epithelial cell types, with poorly differentiated cells transducing more efficiently than well-differentiated cells. A time-dependent increase in gene expression was observed after infection for both vectors. For Ad, but not for AAV, this increase was dependent on prolonged incubation of cells with the vector. Furthermore, for rAAV (but not for rAd), the delay in maximal transduction could be abrogated by wild-type Ad helper infection. Thus, although helper virus is not required for maximal transduction, it increases the kinetics by which this is achieved. Expression of Ad E4 open reading frame 6 or addition of either hydroxyurea or camptothecin resulted in increased AAV transduction, as previously demonstrated for nonairway cells (albeit to lower final levels), suggesting that second-strand synthesis may not be the sole cause of inefficient transduction. Finally, the efficiency of AAV-mediated ex vivo gene transfer to lung cells was similar to that previously described for Ad vectors in that transduction was limited to regions of epithelial injury and preferentially targeted basal-like cells. These studies address the primary factors influencing rAAV infection of human airway cells and should

  16. Epithelial-mesenchymal transition-related genes are linked to aggressive local recurrence of hepatocellular carcinoma after radiofrequency ablation.

    PubMed

    Iwahashi, Shuichi; Shimada, Mitsuo; Utsunomiya, Tohru; Imura, Satoru; Morine, Yuji; Ikemoto, Tetsuya; Takasu, Chie; Saito, Yu; Yamada, Shinichiro

    2016-05-28

    We reported that poor prognoses of hepatocellular carcinoma (HCC) patients after radiofrequency ablation (RFA) are owing to up-regulation of expression of hypoxia-inducible factor-1 and epithelial cell adhesion molecule. We investigated aggressive progression in residual liver tumors (RLTs) after RFA to focus on expression of epithelial-mesenchymal transition (EMT)-related genes and miRNAs. Ten patients with recurrent HCC post-RFA who underwent hepatectomy (RFA group) and 78 patients with HCC without prior RFA (non-RFA group) were enrolled. We examined expression of transforming growth factor (TGF)-β, Twist, vimentin, and Snail-1 mRNAs in tumor tissues, and expression of miR-34a and miR-200c. Expression of TGF-β, Twist and Snail-1 in the RFA group was significantly higher than that in the non-RFA group (P < 0.05); vimentin expression in the RFA group was higher than that in the non-RFA group (P = 0.07). Expression of miR-200c and miR-34a in the RFA group was significantly lower than that in the non-RFA group (miR-200c: P = 0.04; miR-34a: P < 0.01). Increased expression of EMT markers through down-regulation of miRNA expression in RLTs after RFA may be related to poor prognoses of HCC patients with aggressive local recurrence after RFA. PMID:26940140

  17. Gene expression in major depressive disorder.

    PubMed

    Jansen, R; Penninx, B W J H; Madar, V; Xia, K; Milaneschi, Y; Hottenga, J J; Hammerschlag, A R; Beekman, A; van der Wee, N; Smit, J H; Brooks, A I; Tischfield, J; Posthuma, D; Schoevers, R; van Grootheest, G; Willemsen, G; de Geus, E J; Boomsma, D I; Wright, F A; Zou, F; Sun, W; Sullivan, P F

    2016-03-01

    The search for genetic variants underlying major depressive disorder (MDD) has not yet provided firm leads to its underlying molecular biology. A complementary approach is to study gene expression in relation to MDD. We measured gene expression in peripheral blood from 1848 subjects from The Netherlands Study of Depression and Anxiety. Subjects were divided into current MDD (N=882), remitted MDD (N=635) and control (N=331) groups. MDD status and gene expression were measured again 2 years later in 414 subjects. The strongest gene expression differences were between the current MDD and control groups (129 genes at false-discovery rate, FDR<0.1). Gene expression differences across MDD status were largely unrelated to antidepressant use, inflammatory status and blood cell counts. Genes associated with MDD were enriched for interleukin-6 (IL-6)-signaling and natural killer (NK) cell pathways. We identified 13 gene expression clusters with specific clusters enriched for genes involved in NK cell activation (downregulated in current MDD, FDR=5.8 × 10(-5)) and IL-6 pathways (upregulated in current MDD, FDR=3.2 × 10(-3)). Longitudinal analyses largely confirmed results observed in the cross-sectional data. Comparisons of gene expression results to the Psychiatric Genomics Consortium (PGC) MDD genome-wide association study results revealed overlap with DVL3. In conclusion, multiple gene expression associations with MDD were identified and suggest a measurable impact of current MDD state on gene expression. Identified genes and gene clusters are enriched with immune pathways previously associated with the etiology of MDD, in line with the immune suppression and immune activation hypothesis of MDD. PMID:26008736

  18. Analysis of Gene Expression Patterns Using Biclustering.

    PubMed

    Roy, Swarup; Bhattacharyya, Dhruba K; Kalita, Jugal K

    2016-01-01

    Mining microarray data to unearth interesting expression profile patterns for discovery of in silico biological knowledge is an emerging area of research in computational biology. A group of functionally related genes may have similar expression patterns under a set of conditions or at some time points. Biclustering is an important data mining tool that has been successfully used to analyze gene expression data for biologically significant cluster discovery. The purpose of this chapter is to introduce interesting patterns that may be observed in expression data and discuss the role of biclustering techniques in detecting interesting functional gene groups with similar expression patterns. PMID:26350227

  19. Effect of Hangeshashinto on calprotectin expression in human oral epithelial cells.

    PubMed

    Hiroshima, Yuka; Bando, Mika; Inagaki, Yuji; Kido, Reiko; Kataoka, Masatoshi; Nagata, Toshihiko; Kido, Jun-Ichi

    2016-05-01

    Oral epithelial cells produce antimicrobial peptides (AMPs) to prevent microbial infection. Calprotectin (S100A8/S100A9) is one of these AMPs in oral epithelial cells, the expression of which is up-regulated by interleukin-1α (IL-1α). Hangeshashinto (HST) is a traditional Japanese herbal medicine that has anti-inflammatory effects. The purpose of this study was to investigate the effect of HST on the expression of calprotectin through the regulation of IL-1α in oral epithelial cells. Human oral epithelial cells (TR146) were cultured with HST in the presence or absence of anti-IL-1α antibody or IL-1 receptor antagonist, or with six major components of HST (3,4-dihydroxybenzaldehyde, baicalin, ginsenoside Rb1, glycyrrhizin, oleanolic acid and berberine). The expression of S100A8, S100A9, other AMPs and cytokine mRNAs was examined by RT-PCR and quantitative real-time PCR. Calprotectin expression and IL-1α secretion were investigated by ELISA. HST (6 μg/ml) increased the expression of S100A8/S100A9 mRNAs and calprotectin protein, and also up-regulated β-defensin 2 (DEFB4) and S100A7 expression. The expression of IL-1α mRNA and its protein was slightly but significantly increased by HST. A neutralizing antibody against IL-1α and IL-1 receptor antagonist inhibited HST-up-regulated S100A8/S100A9 mRNA expression. Although 3,4-dihydroxybenzaldehyde, baicalin and ginsenoside Rb1 as HST components increased S100A8/S100A9 expression, oleanolic acid and berberine decreased their expression. These results suggest that HST increases the expression of calprotectin, DEFB4 and S100A7 in oral epithelial cells. In response to HST, up-regulation of calprotectin expression may be partially induced via IL-1α. PMID:25649126

  20. IL-13 Augments Compressive Stress-Induced Tissue Factor Expression in Human Airway Epithelial Cells.

    PubMed

    Mitchel, Jennifer A; Antoniak, Silvio; Lee, Joo-Hyeon; Kim, Sae-Hoon; McGill, Maureen; Kasahara, David I; Randell, Scott H; Israel, Elliot; Shore, Stephanie A; Mackman, Nigel; Park, Jin-Ah

    2016-04-01

    Tissue factor (TF) is best known as a cellular initiator of coagulation, but it is also a multifunctional protein that has been implicated in multiple pathophysiologic conditions, including asthma. In the lung, airway epithelial cells express TF, but it is unknown how TF expression is regulated by asthma-associated mediators. We investigated the role of IL-13, a type 2 cytokine, alone and in combination with compressive stress, which mimics asthmatic bronchoconstriction, on TF expression and release of TF-positive extracellular vesicles from primary normal human bronchial epithelial cells. Well-differentiated normal human bronchial epithelial cells were treated with IL-13 and compressive stress, alone and in combination. TF mRNA, protein and activity were measured in the cells and conditioned media. TF was also measured in the bronchoalveolar lavage (BAL) fluid of allergen-challenged mice and patients with asthma. IL-13 and compressive stress increased TF expression, but only compressive stress induced TF-positive extracellular vesicle release. Pretreatment with IL-13 augmented compressive stress-induced TF expression and release. TF protein and activity in BAL fluid were increased in allergen-sensitized and -challenged mice. TF was elevated in the BAL fluid of patients with mild asthma after an allergen challenge. Our in vitro and in vivo data indicate close cooperation between mechanical and inflammatory stimuli on TF expression and release of TF-positive extracellular vesicles in the lungs, which may contribute to pathophysiology of asthma. PMID:26407210

  1. REGULATION OF GENE EXPRESSION IN THE BOVINE MAMMARY GLAND BY OVARIAN STEROIDS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    It is well established that estrogen is required for mammary epithelial cell proliferation and ductal development in the growing animal, and that lobuloalveolar development during gestation is dependent upon progesterone. Effects of these steroid hormones on gene expression in the mammary gland are ...

  2. The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples.

    PubMed

    Cai, Jing; Li, Tao; Huang, Bangxing; Cheng, Henghui; Ding, Hui; Dong, Weihong; Xiao, Man; Liu, Ling; Wang, Zehua

    2014-01-01

    Quantitative real-time PCR (qPCR) is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M) were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD), an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA) and nonparametric (Kruskal-Wallis) tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP) and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene. PMID:24776823

  3. Xenbase: gene expression and improved integration.

    PubMed

    Bowes, Jeff B; Snyder, Kevin A; Segerdell, Erik; Jarabek, Chris J; Azam, Kenan; Zorn, Aaron M; Vize, Peter D

    2010-01-01

    Xenbase (www.xenbase.org), the model organism database for Xenopus laevis and X. (Silurana) tropicalis, is the principal centralized resource of genomic, development data and community information for Xenopus research. Recent improvements include the addition of the literature and interaction tabs to gene catalog pages. New content has been added including a section on gene expression patterns that incorporates image data from the literature, large scale screens and community submissions. Gene expression data are integrated into the gene catalog via an expression tab and is also searchable by multiple criteria using an expression search interface. The gene catalog has grown to contain over 15,000 genes. Collaboration with the European Xenopus Research Center (EXRC) has resulted in a stock center section with data on frog lines supplied by the EXRC. Numerous improvements have also been made to search and navigation. Xenbase is also the source of the Xenopus Anatomical Ontology and the clearinghouse for Xenopus gene nomenclature. PMID:19884130

  4. Identification of Liver Epithelial Cell-derived Ig Expression in μ chain-deficient mice

    PubMed Central

    Shao, Wenwei; Zhang, Chi; Liu, Enyang; Zhang, Long; Ma, Junfan; Zhu, Zhu; Gong, Xiaoting; Qin, Zhihai; Qiu, Xiaoyan

    2016-01-01

    Growing evidence indicates that B cells are not the only source of immunoglobulin (Ig). To investigate this discovery further, we used μMT mice, which have a disruption of the first transmembrane exon of the μ heavy chain and do not express the membrane form of IgM. These mice lack mature B cells and thus serve as a good model to explore Ig expression by liver epithelial cells. We found that Ig heavy chains (μ, δ, γ and α) and light chains (κ and λ) were expressed in sorted liver epithelial cells of μMT mice. Surprisingly, each heavy chain class showed its respective variable region sequence characteristics in their variable region, instead of sharing the same VDJ usage, which suggests that class switching does not occur in liver epithelial cells. Moreover, the γ and α chains, but not the μ and δ chains, showed mutations in the variable region, thus indicating that different classes of Ig have different activities. Our findings support the concept that non-B cells, liver epithelial cells here, can produce different classes of Ig. PMID:27020674

  5. Interferon-γ Suppresses Intestinal Epithelial Aquaporin-1 Expression via Janus Kinase and STAT3 Activation

    PubMed Central

    Dicay, Michael S.; Hirota, Christina L.; Ronaghan, Natalie J.; Peplowski, Michael A.; Zaheer, Raza S.; Carati, Colin A.; MacNaughton, Wallace K.

    2015-01-01

    Inflammatory bowel diseases are associated with dysregulated electrolyte and water transport and resultant diarrhea. Aquaporins are transmembrane proteins that function as water channels in intestinal epithelial cells. We investigated the effect of the inflammatory cytokine, interferon-γ, which is a major player in inflammatory bowel diseases, on aquaporin-1 expression in a mouse colonic epithelial cell line, CMT93. CMT93 monolayers were exposed to 10 ng/mL interferon-γ and aquaporin-1 mRNA and protein expressions were measured by real-time PCR and western blot, respectively. In other experiments, CMT93 cells were pretreated with inhibitors or were transfected with siRNA to block the effects of Janus kinases, STATs 1 and 3, or interferon regulatory factor 2, prior to treatment with interferon-γ. Interferon-γ decreased aquaporin-1 expression in mouse intestinal epithelial cells in a manner that did not depend on the classical STAT1/JAK2/IRF-1 pathway, but rather, on an alternate Janus kinase (likely JAK1) as well as on STAT3. The pro-inflammatory cytokine, interferon-γ may contribute to diarrhea associated with intestinal inflammation in part through regulation of the epithelial aquaporin-1 water channel via a non-classical JAK/STAT receptor signalling pathway. PMID:25793528

  6. Identification of Liver Epithelial Cell-derived Ig Expression in μ chain-deficient mice.

    PubMed

    Shao, Wenwei; Zhang, Chi; Liu, Enyang; Zhang, Long; Ma, Junfan; Zhu, Zhu; Gong, Xiaoting; Qin, Zhihai; Qiu, Xiaoyan

    2016-01-01

    Growing evidence indicates that B cells are not the only source of immunoglobulin (Ig). To investigate this discovery further, we used μMT mice, which have a disruption of the first transmembrane exon of the μ heavy chain and do not express the membrane form of IgM. These mice lack mature B cells and thus serve as a good model to explore Ig expression by liver epithelial cells. We found that Ig heavy chains (μ, δ, γ and α) and light chains (κ and λ) were expressed in sorted liver epithelial cells of μMT mice. Surprisingly, each heavy chain class showed its respective variable region sequence characteristics in their variable region, instead of sharing the same VDJ usage, which suggests that class switching does not occur in liver epithelial cells. Moreover, the γ and α chains, but not the μ and δ chains, showed mutations in the variable region, thus indicating that different classes of Ig have different activities. Our findings support the concept that non-B cells, liver epithelial cells here, can produce different classes of Ig. PMID:27020674

  7. Ciliary beating recovery in deficient human airway epithelial cells after lentivirus ex vivo gene therapy.

    PubMed

    Chhin, Brigitte; Negre, Didier; Merrot, Olivier; Pham, Jacqueline; Tourneur, Yves; Ressnikoff, Denis; Jaspers, Martine; Jorissen, Mark; Cosset, François-Loïc; Bouvagnet, Patrice

    2009-03-01

    Primary Ciliary Dyskinesia is a heterogeneous genetic disease that is characterized by cilia dysfunction of the epithelial cells lining the respiratory tracts, resulting in recurrent respiratory tract infections. Despite lifelong physiological therapy and antibiotics, the lungs of affected patients are progressively destroyed, leading to respiratory insufficiency. Recessive mutations in Dynein Axonemal Intermediate chain type 1 (DNAI1) gene have been described in 10% of cases of Primary Ciliary Dyskinesia. Our goal was to restore normal ciliary beating in DNAI1-deficient human airway epithelial cells. A lentiviral vector based on Simian Immunodeficiency Virus pseudotyped with Vesicular Stomatitis Virus Glycoprotein was used to transduce cultured human airway epithelial cells with a cDNA of DNAI1 driven by the Elongation Factor 1 promoter. Transcription and translation of the transduced gene were tested by RT-PCR and western blot, respectively. Human airway epithelial cells that were DNAI1-deficient due to compound heterozygous mutations, and consequently had immotile cilia and no outer dynein arm, were transduced by the lentivirus. Cilia beating was recorded and electron microscopy of the cilia was performed. Transcription and translation of the transduced DNAI1 gene were detected in human cells treated with the lentivirus. In addition, immotile cilia recovered a normal beat and outer dynein arms reappeared. We demonstrated that it is possible to obtain a normalization of ciliary beat frequency of deficient human airway epithelial cells by using a lentivirus to transduce cells with the therapeutic gene. This preliminary step constitutes a conceptual proof that is indispensable in the perspective of Primary Ciliary Dyskinesia's in vivo gene therapy. This is the first time that recovery of cilia beating is demonstrated in this disease. PMID:19300481

  8. Ciliary Beating Recovery in Deficient Human Airway Epithelial Cells after Lentivirus Ex Vivo Gene Therapy

    PubMed Central

    Chhin, Brigitte; Negre, Didier; Merrot, Olivier; Pham, Jacqueline; Tourneur, Yves; Ressnikoff, Denis; Jaspers, Martine; Jorissen, Mark; Cosset, François-Loïc; Bouvagnet, Patrice

    2009-01-01

    Primary Ciliary Dyskinesia is a heterogeneous genetic disease that is characterized by cilia dysfunction of the epithelial cells lining the respiratory tracts, resulting in recurrent respiratory tract infections. Despite lifelong physiological therapy and antibiotics, the lungs of affected patients are progressively destroyed, leading to respiratory insufficiency. Recessive mutations in Dynein Axonemal Intermediate chain type 1 (DNAI1) gene have been described in 10% of cases of Primary Ciliary Dyskinesia. Our goal was to restore normal ciliary beating in DNAI1–deficient human airway epithelial cells. A lentiviral vector based on Simian Immunodeficiency Virus pseudotyped with Vesicular Stomatitis Virus Glycoprotein was used to transduce cultured human airway epithelial cells with a cDNA of DNAI1 driven by the Elongation Factor 1 promoter. Transcription and translation of the transduced gene were tested by RT–PCR and western blot, respectively. Human airway epithelial cells that were DNAI1–deficient due to compound heterozygous mutations, and consequently had immotile cilia and no outer dynein arm, were transduced by the lentivirus. Cilia beating was recorded and electron microscopy of the cilia was performed. Transcription and translation of the transduced DNAI1 gene were detected in human cells treated with the lentivirus. In addition, immotile cilia recovered a normal beat and outer dynein arms reappeared. We demonstrated that it is possible to obtain a normalization of ciliary beat frequency of deficient human airway epithelial cells by using a lentivirus to transduce cells with the therapeutic gene. This preliminary step constitutes a conceptual proof that is indispensable in the perspective of Primary Ciliary Dyskinesia's in vivo gene therapy. This is the first time that recovery